Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 114-118). / Spacesuit Assemblies (SSAs) provide life support for human operators performing extravehicular activities (EVAs). The overall goal of this research was to investigate three research questions to address gaps in the field of spacesuit assembly (SSA) evaluations: [1] What are the mobility and agility limitations causing operators to experience performance decrements when wearing a SSA?; [2] What is causing operators to experience increased joint torques?; and [3] How does the distributed weight of an SSA, transferred to the operator, affect performance? This research leveraged both experimental and computational modeling capabilities to evaluate SSAs with a human-centered focus, in ways previously unachievable. The space suit evaluated for this research was NASA's Mark III (MkIII) Planetary Technology Demonstrator SSA, built to test the next generation in planetary exploration capabilities, improving upon Apollo era technology. The hip brief assembly (HBA) is built with three nested bearings, each with a single rotational degree of freedom that together provide the range of motion, walking efficiency, and kneeling capabilities. An initial investigation, combining a pilot study and supporting modeling, revealed limitations in the current human-SSA system that may impair the operator's mobility/stability and agility. Limitations identified and investigated in this thesis include SSA degrees of freedom (DOFs), the SSA range of motion (ROM) envelope, the bearing resistances, the SSA component's inertial effects, the SSA mass load transfer dynamics, and suit fit. The SSA architecture was modeled as part of the thesis, creating a tool that was useful in the investigation of the human-suit system. The model relied on SSA component geometries and inherent mass/inertia and bearing resistance characteristics to output joint dynamics, rather than requiring those dynamics as an input (which would require extensive experimental setups). The model was used to isolate components that contribute to the measured operator performance degradations and to quantify the extent of their contributions. These investigations lead to suggestions for design requirements and evaluation techniques that can guide future SSA development and evaluations. / by Conor R. Cullinane. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 143-144). / Hepatitis C virus (HCV) infection accounts for approximately 40% of chronic liver disease in the United States and results in an estimated 8,000-10,000 deaths annually. Simulations suggest that in the next decade morbidity and mortality associated with HCV infections will result in approximately 200,000 deaths and direct medical expenditures of over $10 billion. Furthermore, recent WHO estimates of worldwide prevalence suggest that up to 2% of the world population is infected with HCV, representing between 120 and 200 million people. For reasons that are still poorly understood, the current standard of care is effective only in a subset of patients, and depends on both patient-related and disease-related characteristics. Sustained virological response (SVR) - HCV RNA in patient plasma drops below detectable levels at week 24 following completion of treatment, which is thought to be indicative of curing the disease - is attainable in only ~50% of patients. In recent years, HCV production has been shown to be inextricably linked to lipid metabolism and to the secretion of very low density lipoproteins (VLDL) from hepatocytes. This suggests that by modulating lipid metabolism in the cell, viral production may be reduced in a clinically relevant manner. / (cont.) This work begins by characterizing the link between VLDL secretion and HCV production in the Huh7.5.1/JFH-1 system. We proceed to examine the effects of naringenin, a grapefruit flavonoid, on the production of HCV. Naringenin has been shown previously to reduce VLDL secretion from hepatocytes, and we demonstrate its ability to block HCV production, as well. We explore the mechanism of naringenin's effect on HCV, and show that the flavonoid prevents the assembly of infectious viruses in the cell. Despite previous success by several groups in describing the mechanisms involved in naringenin's effect on VLDL secretion, these mechanisms are thought to account only for ~50% of the observed inhibition, suggesting a deeper understanding of the underlying principals is still lacking. We suggest that naringenin exerts its metabolic, and consequently, antiviral effects through modulation of nuclear recepetor (NR) activity. NRs are a superfamily of ligand-regulated transcription factors known to have an important role in maintaining the homeostasis of metabolites. We show that naringenin activates peroxisome proliferator activated receptors (PPARs), NRs known to drive [beta]-oxidation; and inhibits the liver X receptor (LXR), known to drive lipogenesis and cholesterol synthesis. Despite naringenin's promise as a possible treatment for HCV, its low bioavailability, limits its clinical potential. We conclude this work by showing that naringenin's solubility and bioavailability - and thus, clinical relevance - can be greatly enhanced by complexation with [beta]-cyclodextrins. / by Jonathan Goldwasser. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references (leaves 172-183). / In embryonic stem (ES) cell tumors, the hypoxia-inducible transcription factor, HIF- 1[alpha], has been shown to be a tumor suppressor, and HIF-1[alpha]-expressing cells have been shown to localize preferentially in vivo to regions near tumor vasculature. These differences were proposed to be due to increased hypoxia-induced apoptosis and growth arrest of HIF-1[alpha]-expressing ES cells. This thesis presents a careful investigation into the localization of ES cells in vitro and in vivo with respect to hypoxia. A sandwich culture system was utilized in which controlled gradients of oxygen and nutrients are developed in the vicinity of the tumor cells. A diffusion-consumption model was utilized to predict the oxygen and glucose concentration profiles within the system. Oxygen and glucose consumption rates were measured and used as inputs into the model, and the concentration profiles were found to depend on a single experimental parameter, the cell density within the system. The optimum cell density was found in which stable, measurable oxygen gradients develop over 2-3 mm. The model demonstrated excellent agreement between the predicted oxygen concentration profiles and experimentally determined oxygen gradients. In vitro, there was no difference in localization with respect to hypoxia between tumor cells expressing or lacking HIF-1[alpha]. / (cont.) In addition, there was no difference in apoptosis, proliferation, or migration of the tumor cells in vitro based on HIF-1[alpha] expression. Likewise, a quantitative study on localization of tumor cells within tumors in vivo demonstrated no difference between localization of HIF-1[alpha]-expressing vs. HIF-1[alpha]-lacking ES cells within tumors with respect to blood vessels or hypoxia. These results differ from previous studies, perhaps due to clonal variation of the cell phenotype or the interplay of other complex environmental factors that were not considered in this study. Interestingly, the HIF-1[alpha]-lacking cells were found to exhibit increased tumor growth relative to the HIF-1[alpha]-expressing cells, perhaps due to a normalization of the blood vessels within the HIF-1[alpha]-lacking tumors. These studies reveal the complex role of HIF-1[alpha] in tumor growth and tumor cell localization, as well as develop a useful quantitative experimental model for studying the role of the microenvironment in tumors or in embryonic stem cell biology. / by David M. Cochran. / Ph.D.

Thesis (Ph. D. in Biomedical Engineering)--Harvard-MIT Program in Health Sciences and Technology, 2012. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 114-144). / Afflicting almost 200 million worldwide, hepatitis C virus (HCV) mounts a chronic infection of liver hepatocytes that causes substantial morbidity and mortality. An understanding of host-virus interactions will drive the development of therapeutics, but research is restrained by available experimental tools. Due to the cost and unreliability of existing humanized mouse and primate in vivo models, HCV research is almost exclusively performed using in vitro platforms which suffer from three major limitations. First, challenges in primary hepatocyte culture and the general non-permissiveness of liver cell lines have necessitated the use of a uniquely permissive hepatoma line derived from a single donor, questioning the generalizability of findings to the broader population. Second, this cell line deviates appreciably from native liver in functions central to HCV infection, including innate immune signaling, polarization, and proliferation. Third, infection is typically studied using bulk assays with suboptimal specificity, sensitivity, and content. Here, we describe three technologies for overcoming these limitations in the study of host-virus interactions. We demonstrate their utility in exploring innate immune signaling, a clinically significant component of HCV pathogenesis. Section I describes an in vitro platform for investigating inter-host variations in the natural history of infection and treatment response. We show that directed differentiation of induced pluripotent stem cells (iPSCs) yields patient-specific liver tissue that is permissive to HCV and responds to infection with a robust innate immune response, opening the door to "personalizing" the study and treatment of infection. In Section II, we demonstrate that tissue-engineered, micropatterned co-cultures (MPCCs) of primary hepatocytes and supportive stroma are permissive to HCV, enabling investigations in a more natural host. We then show that innate immune signaling curtails infection in this model, and that its inhibition enhances infection 2-3 orders of magnitude. Lastly, we use MPCCs to uncover a novel liver immunoregulatory mechanism whereby innate immune surveillance is depressed, permitting efficient replication of hepatotropic pathogens. Finally, Section III details a high-content imaging assay that enables visualization and enumeration of single viral genomes in individual cells. We demonstrate that single-cell, multiplexed quantification of viral genomes and host gene transcripts can be used to dissect host-virus interactions, yielding an unexpected positive correlation between stage of infection and response to an innate immune cytokine. The solutions described here will enable the pursuit of previously intractable research questions for HCV and other viruses, accelerating progress towards the development of antivirals and vaccines. Further, the insights gained regarding the interplay between HCV and innate immunity have important clinical ramifications, including a novel therapeutic strategy. / by Kartik Trehan. / Ph.D.in Biomedical Engineering

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Vita. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 49-51). / This study investigates the effect of age, gender, medical condition, and daily free text input on classification accuracy for resuscitation code status. Data was extracted from the MIMICII database. Natural language processing (NLP) was used to evaluate the social section of the nurses' progress notes. BoosTexter was used to predict the code-status using text, age, gender, and SAPS scoring. The relative impact of features was analyzed by feature ablation. Social text was the greatest single indicator of code status. The addition of text to medical condition features increased classification accuracy significantly (p<0.001.) N-gram frequency was analyzed. Gender differences were noted across all code-statuses, with women always more frequent (e.g. wife>husband.) Visitors and contact were more common in the less aggressive resuscitation codes. Logistic regression on medical, age, and gender features was used to determine gender bias or ageism. Evidence of bias was found; both females (OR=1.47) and patients over age 70 (OR=3.72) were more likely to be DNR. Feature ablation was also applied to the social section of physician discharge summaries, as well as to annotated features. The addition of annotated features increased classification accuracy, but the nursing social text remained the most individually predictive. The annotated features included: children; living situation; marital status; and working status. Having zero to one child; living alone or in an institution; being divorced or widow or widower; and working, working in white collar job, or being retired, were all associated with higher rates of DNR status, and lower rates of FC status. Contrarily, living with family; being married; and being unemployed, were all associated with lower rates of DNR status, and higher rates of FC status. Some of these findings were gender and/or age dependent. / 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 163-182). / Two different hemodynamic-based neuroimaging techniques were studied in this work. Near-Infrared Spectroscopy (NIRS) is a promising technique to measure cerebral hemodynamics in a clinical setting due to its potential for continuous monitoring. However, the presence of strong systemic interference in the signal significantly limits our ability to recover the hemodynamic response without averaging tens of trials. Developing a new methodology to clean the NIRS signal from systemic interference and isolate the cortical signal would therefore significantly increase our ability to recover the hemodynamic response opening the door for clinical NIRS studies such as epilepsy. Toward this goal, a new method based on multi-distance measurements and state-space modeling was developed and further optimized to remove systemic physiological oscillations contaminating the NIRS signal. Furthermore, the cortical and pial contributions to the NIRS signal were quantified using a new multimodal regression analysis. Functional Magnetic Resonance Imaging (fMRI) based on the Blood Oxygenation Level Dependent (BOLD) response has become the method of choice for exploring brain function, and yet the physiological basis of this technique is still poorly understood. Despite the effort, a detailed and validated model relating the signal measured to the physiological changes occurring in the cortical tissue is still lacking. Modeling the BOLD signal is challenging because of the difficulty to take into account the complex morphology of the cortical microvasculature, the distribution of oxygen in those microvessels and its dynamics during neuronal activation. Here, we overcome this difficulty by performing Monte Carlo simulations over real microvascular networks and oxygen distributions measured in vivo on rodents, at rest and during forepaw stimulation, using two-photon microscopy. Our model reveals for the first time the specific contribution of individual vascular compartment to the BOLD signal, for different field strengths and different cortical orientations. Our model makes a new prediction: the amplitude of the BOLD signal produced by a given physiological change during neuronal activation depends on the spatial orientation of the cortical region in the MRI scanner. This occurs because veins are preferentially oriented either perpendicular or parallel to the cortical surface in the gray matter. / by Louis Gagnon. / Ph.D.in Medical Engineering and Medical Physics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007. / Includes bibliographical references (p. 117-135). / Children with dyslexia struggle with learning to read despite adequate intelligence, motivation, and schooling. Over the years, there has been a growing consensus about the role of phonological processing in reading disability. Poor readers typically do worse than their normal reading peers on tasks that require phonological processing which has been linked, directly or indirectly, to their speech perception abilities. The work in this thesis combined behavioral, MEG, and EEG methods to examine how normal and reading-impaired children, 7-13 years of age, perceive speech under varying degrees of phonological contrast (1 vs. 3 phonetic features). In a series of auditory word perception experiments, good and poor readers were found to do worse in accuracy and/or reaction times in phonologically similar (i.e., 1-feature contrast) than phonologically dissimilar (i.e., 2 or 3-feature contrast) conditions. Despite the similar behavioral performance and EEG responses for the two groups, a region of interest (ROI) based MEG approach revealed differences in the brain activation of the two groups in superior temporal regions at 140 to 300 ms. / (cont.) In the auditory word discrimination task, differences in activation were found in good readers but not poor readers, as a function of the degree of phonological contrast, reflecting poor readers' lack of sensitivity to the phonological characteristics of the word stimuli. In the sentence plausibility judgment task, the impaired phonological processing abilities of the poor readers may have led them to rely more on top-down sentence context to perceptually disambiguate phonologically confusing terminal words, thereby deceiving them into accepting the phonologically similar incongruent sentences as being congruent. This may account for the poor reader group's reduced brain activation in the phonologically demanding condition in the sentence task. The results of the experiments are consistent with a phonological view of reading disability according to which children with reading impairments have poorly defined phonological representations. / by Daniel T. Wehner. / Ph.D.

Thesis (Ph. D. in Speech and Hearing Bioscience and Technology)--Harvard-MIT Program in Health Sciences and Technology, 2013. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 46-57). / Perceptual abnormalities such as hyperacusis and tinnitus often occur following acoustic overexposure. Although such exposure can also result in permanent threshold elevation, some individuals with noise-induced hyperacusis or tinnitus show clinically normal thresholds. Recent work in animals has shown that noise exposure can cause permanent degeneration of the cochlear nerve despite complete threshold recovery and lack of hair cell damage (Kujawa and Liberman, J Neurosci 29:14077-14085, 2009). Here we ask whether this noise-induced primary neuronal degeneration results in abnormal auditory behavior, indexed by the acoustic startle response and prepulse inhibition (PPI) of startle. Responses to tones and to broadband noise were measured in mice exposed either to a neuropathic exposure causing primary neuronal degeneration, or to a lower intensity, nonneuropathic noise, and in unexposed controls. Mice with cochlear neuronal loss displayed hyper-responsivity to sound, as evidenced by lower startle thresholds and enhanced PPI, while exposed mice without neuronal loss showed control-like responses. Gap PPI tests, often used to assess tinnitus, revealed spectrally restricted, as well as broadband, gap-detection deficits in mice with primary neuronal degeneration, but not in exposed mice without neuropathy. Crossmodal PPI tests and behavioral assays of anxiety revealed no significant differences among groups, suggesting that the changes in startle-based auditory behavior reflect a neuropathyrelated alteration specifically of auditory neural pathways. Despite significantly reduced cochlear nerve response, seen as reduced wave 1 of the auditory brainstem response, later peaks were unchanged or enhanced, suggesting neural hyperactivity in the auditory brainstem that could underlie the abnormal behavior on the startle tests. Taken together, the results suggest a role for cochlear primary neuronal degeneration in central neural excitability and, by extension, in the generation of tinnitus and hyperacusis. / by Ann E. Hickox. / Ph.D.in Speech and Hearing Bioscience and Technology

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 111-121). / More than 14,000 women in the United States die from ovarian cancer each year. The standard of care is tumor-debulking surgery followed by adjuvant chemotherapy. Combination intraperitoneal (IP) and intravenous (IV) chemotherapy has been shown to lengthen survival over IV therapy alone. Large-volume infusions, drug-associated toxicity, and catheter-associated complications, however, increase morbidity and limit patient adherence, often resulting in discontinuation of IP therapy. The technical skill required for catheter implantation and IP chemotherapy administration has also limited its clinical adoption. The proposed solution is an implantable IP device capable of localized drug delivery that maintains the efficacy of the standard of care and overcomes current clinical challenges. A reservoir-based device was developed to release cisplatin at a constant rate. In vivo studies demonstrated that continuous dosing reduces tumor burden to the same extent as weekly IP injections. The implanted device induced significantly less systemic toxicity compared to IP injections, despite administration of higher cumulative doses. A subsequent in vitro study revealed that greater tumor shrinkage following continuous cisplatin exposure was achieved with smaller tumor nodules. These results support that an implanted device would be maximally effective against microscopic residual disease. In vitro results also illustrated that a human-scale device fabricated from orifice-lined silicone can be designed to release cisplatin continuously at the desired rate. The promising preclinical results in this thesis highlight the potential for this novel IP dosing regimen to improve the treatment of late-stage ovarian cancer and set the stage for development of the proposed human device. / by Laura Melanie Tanenbaum. / Ph. D. in Medical Engineering and Medical Physics

Thesis (M. Eng.)--Harvard-MIT Division of Health Sciences and Technology, June 2011. / "June 2011." Cataloged from PDF version of thesis. / Includes bibliographical references (p. 75-79). / For this project, N-terminal and C-terminal peptide library fusions were designed,bconstructed, and screened in order to improve the repression achievable with a novel gene regulatory system. This system, based on the interaction between proteins and proteinbinding RNA aptamers, takes advantage of the reversible interaction between TetR and its RNA aptamer binding partner 5-1.2 to modulate gene expression. With no tetracyclines present, TetR preferentially binds to aptamer 5-1.2 in the mRNA of a gene of interest with low nanomolar affinity and represses translation. Tetracyclines such as aTc induce a conformation change in TetR, prevent TetR binding to aptamer 5-1.2, and induce gene expression. Therefore, TetR binds aptamer 5-1.2 in an aTc-dependent manner, allowing inducible control of gene expression through the TetR-aptamer system. Initial characterization showed a regulatory range of 78% or approximately 5 fold in S. cerevisiae. The aim of this project is to improve repression levels achievable with the TetR-aptamer system by creating libraries of N-terminal and C-terminal peptide fusions to TetR and screening for increased repression in S. cerevisiae. The N-terminal and C-terminal library fusions were constructed from synthesized oligonucleotide fragments and a baseline TetR vector containing library insertions sites at both the N-terminal and C-terminal ends. The library fragments contain 20 random amino acids and a standard SSG linker peptide flanked by both single-cutting restriction enzyme sites and 40 bases of homology to the library insertion sites on the baseline TetR vector, allowing for construction by both restriction/ligation cloning in bacteria and yeast homologous recombination. Both libraries were constructed using restriction/ligation cloning after initial experiments determined optimized conditions for PCR, digest, purification, ligation, and electrocompetent bacterial transformation to achieve a maximum efficiency, fidelity, and purity. The N-terminal and C-terminal libraries produced have a combined diversity of 2.5x 105 variants. These library variants were screened using a plate-based assay with URA3 as a reporter gene. A selection with 5-fluoroorotic acid (5-FOA) was performed to identify library variants with improved repression. Since 5-FOA is a competitive inhibitor of URA3, cells that have URA3 expression cannot live on media containing 5-FOA. Preliminary experiments determined that 0.035% 5-FOA is the threshold for growth for the baseline / (cont.) TetR-aptamer system. Library variants containing 5-1.2-URA3 were grown on media containing a gradient of 5-FOA concentrations between 0.03% and 0.06% and compared to baseline TetR growth. Thirty-one library variants grew at a 5-FOA concentration greater than the baseline threshold for growth. These library hits underwent testing to further characterize their repression, inducibility, and library sequence. The 31 colony hits were streaked on discrete concentrations of 5- FOA to determine colony-specific 5-FOA thresholds and on uracil dropout media in the presence and absence of aTc to screen for preserved inducibility. Of the original 31 colonies, 25 passed both plate assays, growing on 0.045% 5-FOA and in a Tc-dependent manner on uracil dropout media. These 25 colonies were sequenced and analyzed using MEME to detect any conserved motifs. Twenty of the 25 sequenced colonies contained correct and unique libraries, 3 with N-terminal libraries and 17 with C-terminal libraries. Both the N-terminal and C-terminal library sequences had significant motifs. For the Nterminal sequences, all three contained the same 14 nucleotide motif, and 16 of the 17 Cterminal sequences also contained a 21 nucleotide motif. However, the unbiased selection for improved TetR functionality likely returned hits that use multiple mechanisms of action to enhance repression. Therefore, out of a diverse library pool containing 2.5x 105 variants, 20 unique library variants conferred increased repression on the TetR-aptamer system while maintaining inducibility in the presence of tetracycline. / by Jessica Karen Wong. / M.Eng.