Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 252-256). / Embryonic Stem Cells (ESCs) are pluripotent and thus are considered the "cell type of choice". ESCs exhibit several phenotypic traits (e.g., proliferation, differentiation, apoptosis, necrosis, etc.) and when differentiated into a particular lineage they can perform an array of functions (e.g., protein secretion, detoxification, energy production). Typically, these cellular objectives compete against each other because of thermodynamic, stoichiometric and mass balance constraints. Analysis of transcriptional regulatory networks and metabolic networks in ESCs thus requires both a nonequilibrium thermodynamic and mass balance framework for designing and understanding complex ESC network approach as well as an optimality approach which can take cellular objectives into account simultaneously. The primary goal of this thesis was to develop an integrated energy and mass balance-based multi objective framework for a transcriptional regulatory network model for ESCs. The secondary goal was to utilize the developed framework for large-scale metabolic flux profiling of hepatic and ESC metabolic networks. Towards the first aim we first developed a complete dynamic pluripotent network model for ESCs which integrates several different master regulators of pluripotency such as transcription factors Oct4, Sox2, Nanog, Klf4, Nacl, Rexl, Daxl, cMyc, and Zfp281, and obtained the dynamic connectivity matrix between various pluripotency related gene promoters and transcription factors. The developed model fully describes the self-renewal state of embryonic stem cells. / (cont.) Next, we developed a transcriptional network model framework for ESCs that incorporates multiobjective optimality-based energy balance analysis. This framework predicts cofactor occupancy, network architecture and feedback memory of ESCs based on energetic cost. The integrated nonequilibrium thermodynamics and multiobjective-optimality network analysis-based approach was further utilized to explain the significance of transcriptional motifs defined as small regulatory interaction patterns that regulate biological functions in highly interacting cellular networks. Our results yield evidence that dissipative energetics is the underlying criteria used during evolution for motif selection and that biological systems during transcription tend towards evolutionary selection of subgraphs which produces minimum specific heat dissipation, thereby explaining the frequency of some motifs. Significantly, the proposed energetic hypothesis uncovers a mechanism for environmental selection of motifs, provides explanation for topological generalization of subgraphs into complex networks and enables identification of new functionalities for rarely occurring motifs. Towards the secondary goal, we have developed a multiobjecive optimization-based approach that couples the normalized constraint with both energy and flux balance-based metabolic flux analysis to explain certain features of metabolic control of hepatocytes, which is relevant to the response of hepatocytes and liver to various physiological stimuli and disease states. We also utilized this approach to obtain an optimal regimen for ESC differentiation into hepatocytes. / (cont.) The presented framework may establish multiobjective optimality-based thermodynamic analysis as a backbone in designing and understanding complex network systems, such as transcriptional, metabolic and protein interaction networks. / by Marco A. Avila. / Ph.D.

Thesis: S.M., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 73-77). / The health of the human respiratory system depends critically on airway clearance via motile hair-like structures (cilia), which transport and eliminate unwanted particles trapped within mucus. Impairment of mucociliary clearance (MCC) can lead to life-threatening airway narrowing and lung infections, and is a major cause of morbidity and mortality in patients with cystic fibrosis, primary ciliary dyskinesia and chronic obstructive lung disease. However, no tool for microscopic in-vivo visualization of ciliary function is currently available, limiting studies of disease pathogenesis, refined diagnosis and phenotyping, and the development of novel therapeutics. In this thesis, a novel, 1-pm resolution, optical interferometric imaging technique termed Micro-OCT was incorporated into miniaturized common-path endoscopes and mucociliary transport was visualized in vivo for the first time. The first-generation Micro-OCT probe had a rigid design with outer diameter of 4 mm and a two-prism configuration providing beam splitting and sample beam shaping into an annular profile. Image quality of the probe allowed visualization of the periodic pattern of ciliary beating, measurement of airway surface liquid depth (ASL) and visualization of mucociliary transport. Unaltered ciliary function was demonstrated in a living, spontaneously breathing swine model. Newer generation common-path endoscope designs were demonstrated that improve, among other limitations, the stability of the reference reflector position and provide greater potential for miniaturization. The presented work opens unprecedented avenues for studying MCC and the effect of novel therapeutics within the complexity of a living organism. Further, it lays the groundwork for the development of a human probe with the potential to revolutionize diagnosis, phenotyping, and therapy management for all patients with respiratory disease involving the mucociliary escalator. / by Carolin Isabella Unglert. / S.M.

Thesis: Ph. D. in Biomedical Engineering, Harvard-MIT Program in Health Sciences and Technology, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Magnetic resonance imaging (MRI) is a remarkably flexible diagnostic platform due to the variety of clinically relevant physical, chemical, and biological phenomena it can detect. In addition to the host of endogenous contrast mechanisms available, MRI functionality can be further extended by incorporating exogenous factors to attain sensitivity to new classes of indicators. Molecular imaging with targeted injectable contrast agents and MR elastography with externally delivered acoustic vibrations are two such advancements with increasing clinical significance. Conventionally employed separately, this work explores how exogenous components can interact cooperatively in imaging disease and may be combined to more accurately stage disease progression and generate novel mechanisms of MR contrast, using contrast agents and acoustic stimulation as model systems. We imaged hepatic fibrosis in a rat model and found that collagen-binding paramagnetic contrast agents and shear wave MR elastography had partially uncorrelated staging abilities, due to the disease condition's differential timing of collagen production and its stiff cross-linking. This complementary feature enabled us to form a composite multivariate model incorporating both methods which exhibited superior diagnostic staging over all stages of fibrosis progression. We then integrated acoustics and molecular-targeting agents at a deeper level in the form of a novel contrast mechanism, Acoustically Induced Rotary Saturation (AIRS), which switches "on" and "off" the image contrast due to the agents by adjusting the resonance of the spin-lock condition. This contrast modulation ability provides unprecedented clarity in identifying contrast agent presence as well as sensitive and quantitative statistical measurements via rapidly modulated block design experiments. Finally, we extend the AIRS method and show preliminary results for Saturation Harmonic Induced Rotary Saturation (SHIRS), which detects the second harmonic time-oscillation of iron oxide nanoparticles' magnetization in response to an oscillating applied field around B0. We also illustrate an exploratory method of selectively imaging iron oxide agents by diffusion kurtosis measures of freely diffusing water in solutions of magnetic nanoparticles. / by Bo Zhu. / Ph. D. in Biomedical Engineering

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 64-66). / The purpose of the research was to identify the clinical and commercial benefits of switching from intravenous (IV) to buccal delivery of antibiotics. then, the research continued to select 3-5 antibiotics that best met the buccal delivery and market requirements. Methods: The research began with the hypothesis that some injectable antibiotics are good candidates for buccal delivery even with the limitations imposed by the buccal tissue. The thesis captures a two-year research period encompassing three critical fronts - the clinical viability of switching from IV to buccal delivery for antibiotics, the market's desire and readiness to switch, and the antibiotic brands available for commercialization. Then the research moved to drug identification and selection in order to assess the antibiotics that would best function in the buccal delivery model. Results: Intravenous (IV) antibiotics are usually reserved for severe infections that require faster treatment. Less aggressive bacterial growths are treated with oral antibiotics, which has fewer side effects and complications. In the past two decades, the understanding of drug transport across different tissues has increased resulting in improved patient adherence to the therapeutic regimen and pharmacologic response. The administration of drugs by transdermal or transmucosal routes are relatively painless, offers patients more choices, and reduces the need to establish intravenous access, which is a particular benefit for children and elderly. These alternative methods also provide clinical care providers with more choices to better manage their patient's course of treatment. In the past, clinicians administered sedatives, narcotics, and a variety of other medications by transdermal, sublingual, nasal, rectal, and even tracheal-mucosal routes. These delivery options have provided flexible practice settings and this paper intends to show that antibiotics could be the next set of drugs to be administered in variety of ways to provide patients and clinicians the best array of choices. Conclusion: A few years ago, the buccal delivery method was fairly unknown. However, advances in nano encapsulation, physiology, toxicity, and the availability of certain drugs make the timing ideal for introducing antibiotics that have undergone a highly selective process for delivering through the buccal tissue. / by Manijeh Nazari Goldberg. / S.M.

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 121-160). / DNA methylation is an important epigenetic mark that is linked to the regulation of gene expression. It is a critical part of controlling cellular identity and is essential for normal development. DNA methylation is generally studied by comparing methylation levels at individual cytosines or computing region-level averages to identify differential methylation. Here we extended this classic viewpoint by capitalizing on a unique feature of next-generation sequencing, which provides the methylation status of CpGs that are located on the same sequencing read and hence originate from the same DNA molecule. When comparing methylation states of CpGs on the same read, we observed different levels of discordant methylation, defined as molecules where the methylation of neighboring cytosines are not correlated. We quantified the proportion of discordantly methylated reads (PDR) in normal and cancer samples, and found that global PDR levels were elevated in cancer, suggesting widespread epigenetic deregulation. While we have not yet established the mechanistic contribution of this feature, we find that discordant methylation is linked to higher genetic diversity, greater cell-to-cell transcriptional heterogeneity, and adverse clinical outcome in chronic lymphocytic leukemia (CLL). Our analytic approach introduces a novel perspective on utilizing epigenomic sequencing data, which we anticipate will be a valuable tool in understanding the regulation of DNA methylation and its contribution to cellular identity. / by Mark Kendell Clement. / Ph. D. in Medical Engineering and Medical Physics

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2004. / Includes bibliographical references (leaves 27-29). / Evolutionarily conserved genomic regions (ecores) are understudied, and yet comprise a very large percentage of the Human Genome. Highly conserved human-mouse non-coding ecores, for example, are more abundant within the Human Genome than those regions, which are currently estimated to encode for proteins. Subsets of these ecores also exhibit conservation that extends across several species. These genomic regions have managed to survive millions of years of evolution despite the fact that they do not appear to directly encode for proteins. The survival of these regions compels us to investigate their potential function. Development of a computational framework for the classification and clustering of these regions may be the first step in understanding their function. The need for a standardized framework is underscored by the explosive growth in the number of publicly available, fully sequenced genomes, and the diverse set of methodologies used to generate cross-species alignments. This project describes the design and implementation of a system for the identification, classification and cataloguing of ecores across multiple species. A key feature of this system is its ability to quickly incorporate new genomes and assemblies as they become available. Additionally, this system provides investigators with a feature rich user interface, which facilitates the retrieval of ecores based on a wide range of parameters. The system returns a dynamically annotated list of evolutionarily conserved regions, which is used as input to several classification schemes, aimed at identifying families of ecores that share similar features, including depth of evolutionary conservation, position relative to known genes, sequence similarity, / (cont.) and content of transcription factor binding sites. Families of ecores have already been retrieved by the system and clustered using this feature space, and are currently awaiting biological validation. / by Sunil K. Saluja. / S.M.

Thesis (S.M.)--Harvard University--MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references (p. 89-92). / Pharmaceutical productivity, as measured by annual output of new molecular entities and new therapeutic biologics, has fallen significantly since reaching a peak in 1996. According to Food and Drug Administration (FDA) data, the number of new drug approvals (new molecular entities and new biologics) fell from 50 in 1996 to 29 in 2003 (FDA-BEP database 2004). Meanwhile, non-inflation adjusted expenditures for research and development have almost doubled (PhRMA 2004). This thesis uses time series analysis to characterize historical trends in new drug introductions. Linear modeling and ARIMA modeling are employed to show that the large increase in new drug approvals in 1996 is inconsistent with previous trends. The hypothesis that the 1996 increase in new drug approvals is the consequence of additional FDA processing capacity pursuant to the implementation of the Prescription Drug User Fee Act (PDUFA) is considered and rejected, based on an analysis of the underlying causes of the increase. Next, approval trends before and after the implementation of PDUFA are compared. Notably, the percentage of new drug applications resulting in approval has increased since the implementation of PDUFA while the number of applications reviewed per year has not changed significantly. The relationship between the success ratio and drug withdrawal rates is examined, with inconclusive results. / (cont.) Finally, seasonal trends in new drug application (NDA) submissions and approvals are described for years preceding and following PDUFA. A significant plurality of NDA approvals occur in the month of December before and after the implementation of PDUFA, while December NDA submissions increase in the post-PDUFA period. The ramifications of these observations on new review guidelines introduced in PDUFA II and PDUFA III, as well as the implications for NDA submission strategy, are discussed. / by James B. Graham. / S.M.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2007. / Includes bibliographical references (leaves 84-85). / Mergers and acquisitions in the Medical Device Industry are the primary mode of exit for early stage companies. The focus of this thesis is to examine factors which influence the value of these M&A transactions from the target and acquiring firm perspectives and to understand the value creation that occurs. Publicly available electronic and published data sources were used to build a database of 674 M&A transactions and 113 IPO events for deals with published deal values and terms between January 1996 and October 2006. In this work, we demonstrate that transaction deal value varies between various medical device industry sectors. Factors that were shown to significantly correlate with M&A transaction deal value included the Sales of the target company, Market Capitalization value of the acquiring company, type of regulatory approval, and whether the company had venture backing prior to acquisition. M&A transactions that involved targets that were Public companies had significantly higher deal values than those that were private. Using 3-day event window analysis, returns of acquiring companies were shown to be slightly negative and significantly less than the S&P composite index returns over the same period. / (cont.) Previous studies suggest that managers in larger firms tend to use overvalued stock and empire building behavior, resulting in overbidding or pay more for acquisitions. No significant difference in deal value was associated with financing terms between cash and stock transactions. The use of earn outs had no effect on the deal value or the acquirer stock returns. Markets that are developing will continue to have increasing deal value as firms attempt to establish market share and or acquire breakthrough technologies. We found that there were positive correlation of M&A or IPO transaction value for companies in less mature markets where companies have opportunities to leap frog other companies in market position and share. Conversely, there is negative correlation of M&A or IPO transaction value for companies in more mature markets. The results are discussed in terms of the specific factors that influence the transaction value and the degree to which target and acquiring firms benefit from M&A transactions. / by Kevin Lee Ohashi. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2010. / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references. / In addition to cancer cells, solid tumors consist of a variety of cell types and tissues defining a complex microenvironment that influences disease progression and response to therapy. To fully characterize and probe the tumor microenvironment, new tools are needed to quantitatively assess microanatomical and physiological changes during tumor growth and treatment. Particularly important, is the metabolic microenvironment defined in tumors by hypoxia (low p02) and acidity (low pH). These parameters have been shown to influence response to radiation therapy and chemotherapy. However, very little is known about spatio-temporal changes in p02 and pH during tumor progression and therapy. By modifying the technique of intravital multiphoton microscopy (MPM) to perform phosphorescence quenching microscopy, I developed a non-invasive method to quantify oxygen tension (p02) in living tissue at high three-dimensional resolution. To probe functional changes in the metabolic microenvironment, I measured in vivo P02 during tumor growth and antiangiogenic (vascular targeted) treatment in preclinical tumor models. Nanotechnology is rapidly emerging as an important source of biocompatible tools that may shape the future of medical practice. Fluorescent semiconductor nanocrystals (NCs), also known as quantum dots, are a powerful tool for biological imaging, cellular targeting and molecular sensing. / (cont.) I adapted novel fluorescence resonance energy transfer (FRET) -based nanocrystal (NC) biosensors for use with MPM to qualitatively measure in vivo extracellular pH in tumors at high-resolution. While intravital multiphoton microscopy demonstrates utility and adaptability in the study of cancer and response to therapy, the requisite high numerical aperture and exogenous contrast agents result in a limited capacity to investigate substantial tissue volumes or probe dynamic changes repeatedly over prolonged periods. By applying optical frequency domain imaging (OFDI) as an intravital microscopic tool, the technical limitations of multiphoton microscopy can be circumvented providing unprecedented access to previously unexplored, critically important aspects of tumor biology. Using entirely intrinsic mechanisms of contrast within murine tumor models, OFDI is able to simultaneously, rapidly, and repeatedly probe the microvasculature, lymphatic vessels, and tissue microstructure and composition over large volumes. Using OFDI-based techniques, measurements of tumor angiogenesis, lymphangiogenesis, tissue viability and both vascular and cellular responses to therapy were demonstrated, thereby highlighting the potential of OFDI to facilitate the exploration of pathophysiological processes and the evaluation of treatment strategies. / by Ryan M. Lanning. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 126-139). / Bone adapts its mass and architecture in response to its mechanical environment. Yet control of this process by mechanical cues is poorly understood, particularly for unloading. Defining the fundamental mechanoregulation of bone adaptation is critical for the better understanding and mitigation of bone loss in astronauts as well as clinical conditions such as spinal cord injury, stroke, muscular dystrophy, and bed rest. The overall goal of this work was to study skeletal adaptation to varying amounts of reduced loading to help delineate the relationship between mechanical stimuli and skeletal adaptation. We first examined the relative contribution of muscle and gravitational forces to the maintenance of skeletal health in mice, using botulinum toxin (BTX) to induce muscle paralysis and hindlimb unloading to eliminate external loading on the hindlimbs, alone and in combination. BTX led to greater bone loss than hindlimb unloading, while the combination of interventions led to the most detrimental effects overall, suggesting that both muscle and gravitational forces play a role in skeletal maintenance, with greater contributions from muscle forces. We then characterized skeletal adaptation to controlled reductions in mechanical loading of varying degrees employing a novel model that enables long-term exposure of mice to partial weightbearing (PWB). We found that declines in bone mass and architecture were linearly related to the degree of unloading. Even mice bearing 70% of their body weight exhibited significant bone loss, suggesting that the gravity of the moon (0.16 G) and Mars (0.38 G) will not be sufficient to prevent bone loss on future exploration missions. Finally, since bone remodeling is highly site-specific, we used gait analysis and inverse dynamics to determine the mechanical environment during PWB, and then developed a finite element model of the tibia to resolve the local strain-related stimulus proposed to drive changes in bone mass. We found modest correlations between cortical bone architecture at different PWB levels and strain energy density. Altogether this work provides a critical foundation and rationale for future studies that incorporate detailed quantification of the mechanical stimuli and longitudinal changes in bone architecture to further advance our understanding of the skeletal response to reduced loading. / by Rachel Eliman. / Ph. D.