Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / A human mission to Mars will be physically demanding and presents a variety of medical risks to crew members. It has been recognized for over a century that loading is fundamental for bone health, and that reduced loading, as in prolonged bed rest or space flight, leads to bone loss. Osteocytes, the most abundant bone cell type, are thought to be key mechanical sensors in bone, yet the molecular mechanism of this action remains poorly understood. Improved understanding of how osteocytes regulate skeletal responses to mechanical loading and unloading could have significant implications for treatment of bone disorders related to disuse or immobilization. Thus, we conducted in vitro and in vivo studies on osteocytes exposed to unloading to investigate their role in disuse and microgravity-induced bone loss. Specifically, we generated and characterized a novel osteocytic cell line that recapitulates the response to hormonal and mechanical stimuli of osteocytes in vivo. This novel cell line provided the first evidence of a cell-autonomous increase in sclerostin, a potent inhibitor of Wntsignaling, following exposure to simulated microgravity. These cells were also used for a spaceflight mission after demonstrating their ability to maintain an osteocytic phenotype when cultured in a fully automated flight-certified system. Finally, we utilized murine models of unloading to show that pharmacologic inhibition of sclerostin induces bone formation and prevents disuse-induced bone loss. / by Jordan Matthew Spatz. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references (leaves 152-160). / Imaging speed in conventional magnetic resonance imaging (MRI) is limited by the performance of magnetic field gradients and the rate of power deposition in tissue. Parallel MRI techniques overcome these constraints by exploiting information stored within the spatial sensitivity patterns of radiofrequency detector arrays to substitute for some of the spatial information that would normally be obtained using magnetic field gradients. Parallel MRI strategies have been applied clinically to increase patient comfort, enhance spatial resolution, expand anatomical coverage, and reduce image artifacts. The effectiveness of parallel MRI techniques is largely determined by the amount of spatial information that is stored in the detector coil sensitivities. This dissertation investigates the spatial encoding properties of coil arrays from three practical and fundamental perspectives. First, a novel array design is presented that enables spatial encoding in multiple directions simultaneously. Second, the impact of inductive coupling between array elements in parallel MRI is investigated theoretically and experimentally. Finally, electromagnetic calculations are described that permit computation of the ultimate intrinsic signal-to-noise ratio available to any physically realizable coil array for parallel MR. These calculations help to establish fundamental limits to the image accelerations that may be achieved using parallel MRI techniques. These limits are intrinsically related to the wavelengths of the electromagnetic fields at MR imaging frequencies. The sensitivity patterns that correspond to the ultimate intrinsic SNR also represent potential starting points for new coil designs. / by Michael A. Ohliger. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007. / Includes bibliographical references (p. 119-133). / High-resolution ex vivo magnetic resonance microscopy (MRM) can be used to delineate prominent architectonic features in the human brain, but increased contrast is required to visualize more subtle distinctions. The goal of this thesis is to employ target-specific MR contrast agents to regionally alter relaxation rates, resulting in increased contrast in ex vivo MRM of the human brain, with the aim of providing richer information about cyto- and/or myelo-architechtonics than is currently achievable. To accomplish this goal, a traditional optical myelin stain, luxol fast blue (LFB) MBSN with a paramagnetic copper core, has been introduced as a white-matter-selective MR contrast agent in ex vivo brain tissue. The solution relaxivity of LFB was measured at high (4.7 Tesla) and ultra-high (14 Tesla) field strengths. A methodology was developed for staining large tissue samples, enabling MR imaging. Longitudinal (R1) and transverse (R.2) relaxation rates in LFB-stained tissue increased proportionally with myelination at both field strengths. Ri changes produced larger contrast-to-noise ratios (CNR), per unit time, on Ti-weighted images between the deeper, more myelinated cortical layers (IV-VI) and adjacent, superficial layers (I-III) at both field strengths. Specifically, CNR for LFB-treated samples increased by 229± 13 per cent at 4.7T and 269± 25 per cent at 14T when compared to controls. Also, additional cortical layers (IVca, IVd, and Va) became resolvable in 14TMR images after en bloc staining with LFB. After imaging was completed, the LFB-stained sample was prepared for light microscopy. / (cont.) Both macroscopic and microscopic distributions of LFB were found to mimic those of traditional histological preparations. Next, the LFB-MR method was employed to investigate microstructure in X-linked adrenoleukodystrophy (ALD), a confluent demyelinating disorder characterized by accumulation of abnormal lipids. LFB-MR revealed an additional zone, unseen in formualin preparations and best visualized in T2*-weighted images, which produced four-fold increases in contrast-to-noise ratio. Immunohistological analysis identified a corresponding area of perivascular macrophages, and ultrastructural examination suggested LFB particulates bound to lipids within these macrophages. We thus conclude that LFB-MR is able to detect the actively demyelinating edge in cerebral ALD. The results presented in this thesis suggest target-specific contrast agents will 1) enable more detailed MR images, permitting the construction of better MR atlases and advancing the field of MR histopathology, and 2) guide the design of future in vivo contrast agents. / by Megan Leticia Hepler Blackwell. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. "Pages 65-70 contain illegible text. This is the best copy available"--P. after t.p. / Includes bibliographical references (p. 63-64). / Investors have difficulty funding the life sciences because of the high risks involved in research and development and commercialization of new products. Risk in the biopharmaceutical industry is the result of scientific, regulatory and economic uncertainty. The nature of the biopharmaceutical industry introduces many challenges. Each of these challenges incorporates a measure of risk into drug development. The level of understanding of technical success interdependencies has not been fully investigated. These interdependencies (correlations) could lead to an overall greater risk to the company's portfolio than previously expected. A better understanding of the risks that lead to success or failure in drug development might encourage more investment in the life sciences and specifically in the biopharmaceutical industry, and a greater awareness of the correlations between risks and products might lead to more informed decision making on a biopharmaceutical portfolio leading increased productivity. A dataset was collected from Thomson Reuters. The dataset is the oncology portfolio from a biopharmaceutical company, Genentech Inc. Logistic regression was used to determine if any of the defined variables contributed to the success or failure of the oncology products. The chi-square value was 7.738 with the degrees of freedom equal to 5 and with a p-value of 0.17. Therefore, none of the variables significantly contributed to the outcome. More research should be performed in this area in order to better understand the risk in a biopharmaceutical portfolio. / by Alice Elizabeth Wagner. / S.M.

Thesis (Ph. D. in Medical Engineering and Medical Physics)--Harvard-MIT Program in Health Sciences and Technology, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 356-386). / Elucidation of molecular mechanisms underlying metastasis is the final frontier in cancer biology research. Identifying individual pathways in the metastatic cascade could lead to development of metastasis-specific therapeutics; however, current in vivo metastasis model systems are not efficient tools for isolating a single molecular event from the network of complex biological pathways. In response to these needs, we have developed a 3D in vitro co-culture system that isolates molecular and physical interactions between metastatic cells and the endothelium, which are prerequisite for invasive spread. We have used this model to identify key mediators of epithelial-endothelial cell interactions, to screen metastasis specific therapeutics, and most significantly, to elucidate a novel form of intercellular communication through thin cytoskeletal projections called nanoChannels (nCs) that is involved in pathological angiogenesis and that may prime metastatic spread. Metastatic cells preferentially form nCs with the endothelium, enabling rapid and directed transfer of intracellular contents. Proteins, small cytoplasmic dyes, nanoparticles, and most interestingly, functional microRNAs (miRNAs) are transported through these structures. Communication of miRNAs through nCs presents a novel mechanism of pathological angiogenesis and the angiogenic switch. NanoChannel-mediated communication introduces a new paradigm of cancer progression in which tumor cells can directly transform surrounding cell populations in order to facilitate cancer pathogenesis. / by Yamicia Doyasi Connor. / Ph.D.in Medical Engineering and Medical Physics

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014. / 203 / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 139-150). / Chronic liver disease affects more than 500 million people worldwide. The only therapy shown to directly prevent mortality is organ transplantation. However, there is growing discrepancy between supply and demand of transplant-grade organs and transplant recipients are subject to a lifetime of immunosuppressive regimens. Therefore, the overall aim of this thesis is to advance alternatives to whole organ transplantation for liver diseases. Treating the failure of organs serving a multitude of biochemical functions, such as the liver, requires cell-based therapies. Such therapies should ideally employ human cells due to immunological concerns and the substantial differences between animal and human hepatocellular functions. Human cell lines, while renewable, lack the full functional capacity of primary adult hepatocytes, and for clinical applications there is a tumorigenic risk. Primary human hepatocytes have exhibited therapeutic potential; however, limited sourcing has been a bottleneck for many fields of research and clinical therapies. Pluripotent human stem cells are an attractive cell source, but to date, the hepatocyte-like cells obtain by directed differentiation continue to exhibit an immature hepatic phenotype, which resembles fetal hepatocytes more than adult hepatocytes. Motivated by these limitations, we report in the first section of this thesis, the development of a 384-well liver platform that incorporates cell-cell interactions to stabilize the hepatocyte phenotype, and enable high-throughput screening using cryopreserved primary human hepatocytes. We also developed attendant assays to assess cell fates in this platform through an automated image-based proliferation assay and an ELISA-based functional assay. In the second section of this thesis, we use the high-throughput liver platform to conduct a chemical screen of 12,480 small molecules. We identified 12 bioactive factors in three classes: small molecules that enhanced proliferation (PH), or function (FH) or both proliferation and function (FPH) of human primmary hepatocytes. 2 FPHs expanded primary human hepatocytes in vitro, resulting in up to 10 fold more cells over 7 days. This proliferation rate is consistent with in vivo liver regeneration kinetics and similar to PGE2-induced Wnt-mediated human hepatocyte proliferation in vitro. To date, we have tested 10 different donors of primary human hepatocytes and found all to expand upon FPH treatment, though kinetics and degrees of response vary. Additionally, FPH1 and FHl were shown to differentiate hepatocyte-like cells derived from induced pluripotent stem cells (iPS) toward a phenotype more mature than what was previously obtainable, causing upregulation of previously low or absent adult markers such as CYP3A4, CYP2A6, PXR and BSEP, concomitant with downregulation of persistent fetal marker AFP. In the final section of this thesis, we explore the therapeutic potential of identified small molecules in vivo. Using zebrafish models from the Goessling/North lab, we observed that both PHIl and FHl enhanced liver sizes in lfabp:GFP reporter zebrafish embryos. In another model of acute liver failure induced by overdose of acetaminophen (APAP), PH1 and FHl acted as hepatoprotectants that increased liver size, and in the case of FHi, rescued zebrafish survival. At sub-lethal concentrations (5mM APAP), the respective therapeutic windows of FHl and PHI both exceed that of N-acetylcystein, the only antidote in use clinically, by at least 12 hours. At lethal concentrations (IOmM APAP), FHl therapy further improved zebrafish survival by up to 63% in both embryonic and adult models of APAP toxicity. The high-throughput liver platform developed in this thesis will enable studies of previously inaccessible aspects of liver biology and small-molecule bioactivity, and have led to the identification of first generation small molecules that have the potential to address cell sourcing challenge impacting many facets of liver disease. / by Jing (Meghan) Shan. / Ph. D.

Thesis (Ph. D.)--Harvard University--MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references. / Breast cancer is the most commonly diagnosed cancer among women in the United States. It is the most common cause of death in women ages 45-55. Optical techniques can potentially play a diagnostic role in several aspects of breast cancer evaluation and treatment. This thesis outlines progress on the use of Raman spectroscopy to diagnose breast cancer. Laboratory studies on fresh-frozen tissues are used to demonstrate that the detailed information provided by Raman spectroscopy yields accurate breast disease diagnosis. A Raman spectroscopic-based diagnostic algorithm was developed which classifies samples into four categories according to specific pathological diagnoses: normal, fibrocystic change, fibroadenoma, and infiltrating carcinoma. Cancerous lesions were separated from non- cancerous tissues with a sensitivity of 94% and a specificity of 95%. Further, use of a spectral model based on the morphological structures that comprise breast tissue allows increased understanding of the relationship between a Raman spectrum and tissue disease state. Based on the excellent results of our laboratory work, two clinical studies were undertaken. These studies translate Raman spectroscopy from a laboratory technique into a clinically useful tool. The first study tests the diagnostic algorithm in a prospective manner on freshly excised tissue. Preliminary results are promising. The second study is the first demonstration of in vivo data acquisition of Raman spectra of breast tissue. The culmination of this research is the demonstration of accurate intra-operative margin status assessment during partial mastectomy surgeries. / (cont.) Application of our previously developed diagnostic algorithm resulted in perfect sensitivity and specificity in this small in vivo data set. These preliminary findings indicate that Raman spectroscopy has the potential to lessen the need for re-excision surgeries resulting from positive margins and thereby reduce the recurrence rate of breast cancer following partial mastectomy surgeries. The experiments and theory presented throughout this thesis demonstrate that Raman spectroscopy is a viable clinical tool that can be used to accurately diagnosis breast cancer and breast disease. / by Abigail Susan Haka. / Ph.D.

Thesis: Ph. D. in Bioinformatics and Integrative Genomics, Harvard-MIT Program in Health Sciences and Technology, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 149-158). / Next-generation DNA sequencing has allowed us to extract vast quantities of functional information from genetic systems. However, natural systems represent only a fraction of all possible DNA sequences. Our understanding of how genomes function is limited by our ability to make modifications and test hypotheses. Multiplexed DNA synthesis now allows us to generate thousands of computationally designed sequences, each representing a physical hypothesis to test. Here, we combine DNA sequencing and synthesis technologies to design, make, and measure the behavior of thousands of new genetic elements in the bacterium E. coli. We begin by quantifying the interactions between regulatory elements that control transcription and translation and show that these interactions create large deviations from the predicted behavior of individual elements. Regulatory elements also interact with the codons of the genes they control. We show that rare codon usage at the beginning of genes unexpectedly leads to a strong increase in protein translation due to the relationship between codon rarity, genomic nucleotide bias, and mRNA structure. We next examine the behavior of regulatory elements that bind transcription factors by designing and synthesizing over 100,000 transcriptional circuits. From each circuit we measure repression, activation, and small-molecule induction, deriving relationships between DNA sequence features and functional properties including cooperativity, sensitivity, and dynamic range of gene expression response. Finally, as the scale and speed of DNA synthesis and functional readout continues to increase, our ability to computationally design and analyze genetic systems has become the bottleneck. We have built software to predict and design individual genetic elements in high throughput (Promuter) as well as software to analyze and compare hundreds of evolved or engineered bacterial whole genomes (Millstone). As generating high dimensional datasets becomes exponentially easier than designing experiments and extracting knowledge, bioinformatics, machine learning, and data science will become the primary tools we use to pose new hypotheses and build models of biology. / by Daniel B. Goodman. / Ph. D. in Bioinformatics and Integrative Genomics

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 181-190). / Optogenetic technologies have been the subject of great excitement within the scientific community for their ability to demystify complex neurophysiological pathways in the central and peripheral nervous systems. Optogenetics refers to the transduction of mammalian cells with a light-sensitive transmembrane protein, called an opsin, such that illumination of the target tissue initiates depolarization; in the case of a neuron, illumination results in the firing of an action potential that can control downstream physiology. The excitement surrounding optogenetics has also extended to the clinic with a human trial using the opsin ChR2 in the treatment of retinitis pigmentosa currently underway and several more trials potentially planned for the near future. In this thesis, we focus on the use of viral techniques to transduce peripheral nerve tissue to be responsive to light. We characterize the properties of optogenetic peripheral nerve transduction, optimizing for variables such as expression strength, wavelength specificity, and time-course of expression. Within the scope of this thesis, three new methods for optogenetic peripheral nerve stimulation are described: (1) a method for optogenetic motor nerve control using transdermal illumination, (2) a method employing unique wavelengths to selectively target optogenetic subsets of motor nerves, and (3) a method for extending optogenetic expression strength and timecourse. The work is important because it lays the foundation for future advancements in optogenetic peripheral nerve stimulation in both a scientific and clinical context. / by Benjamin E. Maimon. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2006. / Includes bibliographical references (leaves 73-85). / Proteomics has been revolutionized in the last couple of years through integration of new mass spectrometry technologies such as -Enhanced Laser Desorption/Ionization (SELDI) mass spectrometry. As data is generated in an increasingly rapid and automated manner, novel and application-specific computational methods will be needed to deal with all of this information. This work seeks to develop a Bayesian framework in mass-based proteomics for protein identification. Using the Bayesian framework in a statistical signal processing manner, mass spectrometry data is filtered and analyzed in order to estimate protein identity. This is done by a multi-stage process which compares probabilistic networks generated from mass spectrometry-based data with a mass-based network of protein interactions. In addition, such models can provide insight on features of existing models by identifying relevant proteins. This work finds that the search space of potential proteins can be reduced such that simple antibody-based tests can be used to validate protein identity. This is done with real proteins as a proof of concept. Regarding protein interaction networks, the largest human protein interaction meta-database was created as part of this project, containing over 162,000 interactions. A further contribution is the implementation of the massome network database of mass-based interactions- which is used in the protein identification process. / (cont.) This network is explored in terms potential usefulness for protein identification. The framework provides an approach to a number of core issues in proteomics. Besides providing these tools, it yields a novel way to approach statistical signal processing problems in this domain in a way that can be adapted as proteomics-based technologies mature. / by Gil Alterovitz. / Ph.D.