Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references (p. 95-101). / This thesis describes results from two projects related to the efferent innervation of the cochlea. First, we investigated peripheral olivocochlear effects of sustained contralateral broadband noise in anesthetized guinea pig. We found evidence of medial olivocochlear (MOC) effects on two timescales: the classic MOC 'fast effect', followed by a gradually increasing suppression, which we call the MOC 'delayed effect'. Delayed suppression typically takes 2-3 minutes to build up, occurs at all frequencies of guinea pig hearing, and suppresses distortion product otoacoustic emissions (DPOAEs), compound action potentials (CAPs), and round window noise. In contrast to the MOC slow effect, which has been reported for sustained shockevoked MOC activity, MOC delayed suppression does not build up in the outer hair cells but is due to a central modulation (enhancement) of MOC responsiveness and can thus be viewed as a gradual increase in the strength of the MOC fast effect. We found that, on average, the magnitude of the delayed suppression is comparable to that of the MOC fast effect, but that there is an overall negative correlation between fast and delayed effect magnitudes. Thus, it may have significant implications for the functional roles of the MOC system, such as protection against acoustic trauma, anti-masking, and dynamic range extension. Second, we investigated the LOC bilateral balancing model, which proposes that the LOC system acts to balance long-term average neural output from both cochleae, which would be important for binaural processing of sounds. For this, we tested various cohorts of mice by repeatedly measuring bilateral auditory brainstem responses (ABR) and "DPOAE growth functions across a wide range of frequencies and levels for periods of about 1-2 months. About halfway through the period, a unilateral reduction in neural output was created, either by acoustic overexposure or conductive impairment. Although the LOC balancing model predicts that the unilateral reduction in neural output should be matched contralaterally, we found no evidence for short-term or long-term efferent-induced contralateral response changes in any of the cohorts, either for DPOAE or ABR metrics. / (cont.) In view of these results, a revision of the LOC bilateral balancing model is called for. / by Erik Larson. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student submitted PDF version of thesis. / Includes bibliographical references. / The human genome sequence can be thought of as an instruction manual for our species, written and rewritten over more than a billion of years of evolution. Taking a complete inventory of our genome, dissecting its genes and their functional components, and elucidating how these genes are selectively used to establish and maintain cell types with markedly different behaviors, are key challenges of modern biology. In this thesis we present contributions to our understanding of the structure, function and evolution of the human genome. We rely on two complementary approaches. First, we study signatures of evolutionary processes that have acted on the genome using comparative sequence analysis. We generate high quality draft genome sequences of the chimpanzee, the dog and the opossum. These species share a last common ancestor with humans approximately 6 million, 80 million and 140 million years ago, respectively, and therefore provide distinct perspectives on our evolutionary history. We apply computational methods to explore the functional organization of the genome and to identify genes that contribute to shared and species-specific traits. Second, we study how the genome is bound by proteins and packaged into chromatin in distinct cell types. We develop new methods to map protein-DNA interactions and DNA methylation using single-molecule based sequencing technology. We apply these methods to identify new functional sequence elements based on characteristic chromatin signatures, and to explore the relationship between DNA sequence, chromatin and cellular state. / by Tarjei Sigurd Mikkelsen. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Pages 185-186 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (p. 171-184). / This thesis focuses on the use of ultrathin therapeutic protein delivery films to control host tissue/medical device implant interactions, thereby reducing complications that lead to implant failure. The Layer by Layer (LbL) deposition platform was used to fabricate conformal, tunable, micron scale reservoirs for the controlled release of a wide variety of proteins including enzymes, growth factors, and antibodies that were shown to be capable of directing cells in vitro to desired outcomes including proliferation, differentiation, and quiescence. Film release profiles were controlled through rational polymer design, tuning film composition, and varying film architecture. In studies with a model protein lysozyme, 100% retention of protein function was observed, underscoring gentle process conditions. In vitro experiments with Fibroblast Growth Factor-2 (FGF-2) and Bone Morphogenetic Protein -2 (BMP-2) showed that released growth factors are more active than growth factors supplemented in medium, suggesting a surface concentration mechanism and/or specific growth factor interactions with LbL film components. Anti-VEGF releasing LbL films afforded new opportunities to modify cancer therapy nanoparticles for multi therapeutic release, and provided an important switch to turn off the cellular response to growth factors. Using an orthopedic hip implant model as a test case, the first LbL film with enough growth factor load to direct in vivo host cell response was demonstrated. BMP-2 releasing LbL films were used to direct MC3T3 pre-osteoblast differentiation in vitro, and the differentiation of host mesenchymal stem cells in a rat quadriceps model in vivo to form bone tissue in a first generation model for remediating orthopedic hip implant complications. Preliminary data on second generation, multifunctional drug delivery films are promising. These studies contribute to the mechanistic design of protein LbL films and show promise for a wide variety of clinical applications, opening avenues for multifunctional drug delivery from LbL films. / by Mara Lee Macdonald. / Ph.D.

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. / Advances in imaging, such as coronary angiography, intravascular ultrasound, and optical coherence tomography, can improve procedural success and outcomes for endovascular catheter intervention, such as stent implantation. Yet, these imaging modalities are not universally embraced; and thus, optimization of stent implantation and management of the adverse outcomes remain challenging. This is partially because full adoption of complex imaging awaits methods to reconstruct precise 3D structure of lumen and implanted stent, and to track vascular responses to stent implantation over time in 3D. This thesis creates new methods for reconstruction and registration in 3D by melding disparate imaging modalities, coronary angiography and optical coherence tomography (OCT), that provide different 2D-plane information (longitudinal and cross-sectional) using widely-varied experimental models (static phantom models, preclinical swine model with controlled scenarios of stent implantation in coronary arteries, and clinical unbiased model of stent implantation). A 3D vessel centerline from coronary angiography serves as a fusion path for OCT to reconstruct 3D structures and as a registration path for the reconstructed 3D structures across time. The developed vessel centerline reconstruction method overcame current spatial and temporal alignment challenges, and demonstrated high reproducibility across imaging angles and throughout the cardiac cycle. Structural reconstruction by angiography-OCT fusion was established and improved to account for the cardiac motion, reducing error in estimation of the stent length from 5.5% ± 4.5% with standard fusion to 2.4% ± 2.0%. Time-point registration was accomplished by detecting landmarks that are least affected by the vascular responses - its error, i.e., stent-strut shift from post-implantation to follow-up, was 1.6 mm ± 0.5 mm (9.2% ± 3.0% of the stent length). These methods were validated in a clinical setting and the errors of all methods were within those in the preclinical setting, suggesting potential for clinical applicability. / by Mie Kunio. / Ph. D. in Medical Engineering and Medical Physics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 105-109). / Few empirically-derived guidelines exist for optimizing the assessment of vocal function in children with voice disorders. The goal of this investigation was to identify a minimal set of speech tasks and associated acoustic analysis methods that are most salient in characterizing the impact of vocal nodules on vocal function in children. Hence, a pediatric assessment protocol was developed based on the standardized Consensus Auditory Perceptual Evaluation of Voice (CAPE-V) used to evaluate adult voices. Adult and pediatric versions of the CAPE-V protocols were used to gather recordings of vowels and sentences from adult females and children (4-6 and 8-10 year olds) with normal voices and vocal nodules, and these recordings were subjected to perceptual and acoustic analyses. Results showed that perceptual ratings for breathiness best characterized the presence of nodules in children's voices, and ratings for the production of sentences best differentiated normal voices and voices with nodules for both children and adults. Selected voice quality-related acoustic algorithms designed to quantitatively evaluate acoustic measures of vowels and sentences, were modified to be pitch-independent for use in analyzing children's voices. Synthesized vowels for children and adults were used to validate the modified algorithms by systematically assessing the effects of manipulating the periodicity and spectral characteristics of the synthesizer's voicing source. / (cont.) In applying the validated algorithms to the recordings of subjects with normal voices and vocal nodules, the acoustic measure tended to differentiate normal voices and voices with nodules in children and adults, and some displayed significant correlations with the perceptual attributes of overall severity of dysphonia, roughness, and/or breathiness. None of the acoustic measures correlated significantly with the perceptual attribute of strain. Limitations in the strength of the correlations between acoustic measures and perceptual attributes were attributed to factors that can be addressed in future investigations, which can now utilize the algorithms that were developed in this investigation for children's voices. Preliminary recommendations are made for the clinical assessment of pediatric voice disorders. / by Asako Masaki. / Ph.D.

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, September 2018. / Page 399 blank. Cataloged from PDF version of thesis. / Includes bibliographical references (pages 210-229). / RNA plays important and diverse roles in biology, yet molecular tools to measure and manipulate RNA are limited. Recently, the bacterial adaptive immune system, CRISPR, has revolutionized our ability to manipulate DNA, but no known RNA-targeting versions exist. To discover parallel bacterial RNA-targeting systems that could be used for transcriptome engineering, we developed a computational pipeline to mine for novel Class 2 CRISPR systems across more than 25,000 bacterial genomes. Among the many novel CRISPR systems, we found a programmable RNA-targeting CRISPR system, CRISPR-Cas 13, that could provide immunity to E. coli against the ssRNA MS2 phage and biochemically characterized the enzyme. We adapted CRISPR-Casl3 for modulating the transcriptome in mammalian and plant cells by heterologously expressing Casl 3 and engineering the enzyme to precisely knockdown, bind, and edit RNA. Cas 13 knockdown was as efficient as RNA interference, but much more specific, across many transcripts tested. RNA editing with Cas 13 was also highly efficient, with up to 90% base editing rates, and as low as 20 off-targets with engineered specificity versions. Lastly, we combined Cas13 with isothermal amplification to develop a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with single-molecule sensitivity and singlebase mismatch specificity. We used this Casl3a-based molecular detection platform, termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing), to specifically detect pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Our results establish CRISPR-Cas13 as a flexible platform for RNA targeting with wide applications in RNA biology, diagnostics, and therapeutics. / by Omar O. Abudayyeh. / Ph. D. in Medical Engineering and Medical Physics

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 169-175). / This thesis is motivated by the gaps between speech science and technology in analyzing dialects. In speech science, investigating phonetic rules is usually manually laborious and time consuming, limiting the amount of data analyzed. Without sufficient data, the analysis could potentially overlook or over-specify certain phonetic rules. On the other hand, in speech technology such as automatic dialect recognition, phonetic rules are rarely modeled explicitly. While many applications do not require such knowledge to obtain good performance, it is beneficial to specifically model pronunciation patterns in certain applications. For example, users of language learning software can benefit from explicit and intuitive feedback from the computer to alter their pronunciation; in forensic phonetics, it is important that results of automated systems are justifiable on phonetic grounds. In this work, we propose a mathematical framework to analyze dialects in terms of (1) phonetic transformations and (2) acoustic differences. The proposed Phonetic based Pronunciation Model (PPM) uses a hidden Markov model to characterize when and how often substitutions, insertions, and deletions occur. In particular, clustering methods are compared to better model deletion transformations. In addition, an acoustic counterpart of PPM, Acoustic-based Pronunciation Model (APM), is proposed to characterize and locate fine-grained acoustic differences such as formant transitions and nasalization across dialects. We used three data sets to empirically compare the proposed models in Arabic and English dialects. Results in automatic dialect recognition demonstrate that the proposed models complement standard baseline systems. Results in pronunciation generation and rule retrieval experiments indicate that the proposed models learn underlying phonetic rules across dialects. Our proposed system postulates pronunciation rules to a phonetician who interprets and refines them to discover new rules or quantify known rules. This can be done on large corpora to develop rules of greater statistical significance than has previously been possible. Potential applications of this work include speaker characterization and recognition, automatic dialect recognition, automatic speech recognition and synthesis, forensic phonetics, language learning or accent training education, and assistive diagnosis tools for speech and voice disorders. / by Nancy Fang-Yih Chen. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 156-175). / Organ transplantation is a valuable treatment for organ failure; however, it is limited by an increasing shortage of donor organs. Because of this, mechanical support has emerged as an effective therapy to promote recovery of native organ function, especially in the setting of acute heart failure. Acute heart failure is increasingly prominent and inadequately treated by traditional medical therapy. Mechanical circulatory support (MCS) devices unload the heart by offering a range of support that reduces mortality and promotes cardiac recovery when correctly used. The challenge in use of these devices is the lack of metric-driven control for the level of support currently manually determined by a clinician. We hypothesize that optimization of device use requires novel insights in physiology and definition of organ state through an understanding of device-organ interconnectivity in support devices that are coupled with residual organ function. Thus, the goals of this work are to leverage the interaction between support device and organ to assess the state of the organ and then use this information towards improved device control and understanding of organ pathophysiology. The research program used an integrated approach of bench-top testing, animal models, and retrospective patient data to determine advanced markers of cardiac function using the Abiomed Impella as a paradigmatic device. We developed a mock circulatory loop to identify how MCS devices operate over the cardiac cycle during changing cardiovascular states. Parametric analysis revealed a hysteretic state-responsive relationship between the device and subject physiology. Since device operation is characterized using the MCL, unaccounted hysteresis changes can be attributed to variation in the cardiac state. We utilized this model to predict novel metrics of cardiac dynamics and easily-validated parameters of cardiac state in both acute animal models and retrospective patient data in which we accurately differentiated disease states and clinical outcomes. Finally, we investigated how MCS can affect downstream vascular response in animals and patients by analyzing arterial pressure waveforms with known device performance to quantify vascular state and device-vascular coupling. / by Brian Yale Chang. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 157-162). / Epilepsy is a chronic disorder of the central nervous system that predisposes individuals to experiencing recurrent seizures. It affects 3 million Americans and 50 million people world-wide. A seizure is a transient aberration in the brain's electrical activity that produces disruptive physical symptoms such as a lapse in attention and memory, a sensory hallucination, or a whole-body convulsion. Approximately 1 out of every 3 individuals with epilepsy continues to experience frequent seizures despite treatment with multiple anti-epileptic drugs. These intractable seizures pose a serious risk of injury, limit the independence and mobility of an individual, and result in both social isolation and economic hardship. This thesis presents novel technology intended to ease the burden of intractable seizures. At its heart is a method for computerized detection of seizure onset. The method uses machine learning to construct patient-specific classifiers that are capable of rapid, sensitive, and specific detection of seizure onset. The algorithm detects the onset of a seizure through analysis of the brain's electrical activity alone or in concert with other physiologic signals. When trained on 2 or more seizures and tested on 844 hours of continuous scalp EEG from 23 pediatric epilepsy patients, our algorithm detected 96% of 163 test seizures with a median detection delay of 3 seconds and a median false detection rate of 2 false detections per 24 hour period. / (cont.) In this thesis we also discuss how our detector can be embedded within a low power, implantable medical device to enable the delivery of just-in-time therapy that has the potential to either eliminate or attenuate the clinical symptoms associated with seizures. Finally, we report on the in-hospital use of our detector to enable delay-sensitive therapeutic and diagnostic applications. We demonstrate the feasibility of using the algorithm to control the Vagus Nerve Stimulator (an implantable neuro stimulator for the treatment of intractable seizures), and to initiate ictal SPECT (a functional neuroimaging modality useful for localizing the cerebral site of origin of a seizure). / by Ali Hossam Shoeb. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Program in Health Sciences and Technology. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 63-66). / Acoustic overexposure can cause death of cochlear nerve fibers, even if elevations in cochlear sensitivity are reversible and even if there is no hair cell loss. We hypothesized that this primary neural degeneration does not raise cochlear thresholds because it is selective for cochlear neurons with high thresholds and low spontaneous rates (SRs). We tested this hypothesis by recording from single cochlear neurons in guinea pigs and evaluating neuronal degeneration via confocal microscopy 14 days after exposure to noise (4-8kHz, 106dB SPL, 2 hrs). Suprathreshold amplitudes of otoacoustic emissions recovered post-exposure, suggesting complete hair cell recovery; suprathreshold amplitudes of the auditory brainstem response remained attenuated for high-frequency stimuli, consistent with primary neural degeneration in the basal half of the cochlea. Single-fiber recordings from exposed animals showed that all sound-evoked response properties, including threshold, sharpness of tuning, maximum discharge rate, dynamic range, response adaptation, and recovery from forward masking, were indistinguishable from responses recorded from unexposed controls. The only physiological abnormalities were revealed in the population statistics: in high-frequency regions where low- SR (<20sp/sec) fibers are 47% of the control ear, that number was reduced to 29% in the exposed ear. Cochlear afferent synapses were counted after immunostaining for juxtaposed pre-synaptic ribbons and post-synaptic glutamate receptor patches in the inner hair cell area. The observed 20% synapse loss throughout the basal half of the cochlea was consistent with the physiological data. Analysis of synaptic morphology suggested remaining pre-synaptic ribbons were hypertrophied and membrane localization of post-synaptic glutamate receptors was reduced. The significance of the ribbon hypertrophy is unclear, but reduced receptor expression may represent receptor internalization, suggested as a mechanism to minimize the glutamate excitotoxicity that underlies this noise-induced degeneration. In the normal ear, fibers with high thresholds and low SRs are relatively insensitive to masking, thus are important for hearing in a noisy environment. We speculate that over a lifetime of repeated moderate noise exposure, the typical human ear may selectively lose these low-SR fibers and that this selective neuropathy may be an important contributor to the well-known problems with hearing-in-noise in the aging ear. / by Adam C. Furman. / Ph.D.