Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / This work investigates the fundamental processes used by mammalian cells and organisms to make decisions during embryonic development. Current technologies that evaluate biological phenomenon often force a compromise between quantification of gene expression via bulk assays and qualitative imaging of cell and tissue heterogeneity. There are few options that allow for quantitative, high-resolution, single-cell analysis that is robust but not associated with a high degree of technical difficulty or obscured by amplification. Here, we address these issues using two model systems, the developing mammalian inner ear and single mouse embryonic stem cells (mESCs) during the process of X inactivation, to demonstrate our ability to perform single-cell, single-molecule assays that reveal both highly quantitative and spatial information. Accordingly, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression in the inner ear and perform allele-specific detection of the X chromosome in mESCs. We used previously-published smFISH procedures as our initial template for investigating biological signaling phenomena in these two systems. To study gene expression in the mouse inner ear, we developed a modified smFISH strategy to investigate mRNA transcript expression patterns in the cochlea during auditory hair cell development. The mammalian cochlea, a highly specialized and complex organ, beautifully demonstrates both the depth and breadth of the smFISH technique. To assay signaling behavior and topological changes of the X chromosome prior to X inactivation, we incorporated a novel allele-specific modification into the smFISH technique. We investigate the allele-specific expression patterns of eight genes that tile the X chromosome, which were chosen for their varied putative roles before, during and after X chromosome inactivation. Taken together, these two systems recapitulate the strength of the smFISH technique and its adaptations. The goals of this thesis were twofold: (1) expand the smFISH technique to work in specialized mammalian systems such as the cochlea and (2) demonstrate allele-specific DNA topological changes and expression patterns in mESCs. Elucidating high-resolution, single-molecule quantifiable imaging methods for application to complex tissues or allele-specific probing will have profound impacts on future investigations and promote a deeper comprehension of these systems. / by Annalisa M. Pawlosky. / Ph. D.

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Complex hierarchical structures are a hallmark of life. Within multicellular organisms, the building blocks of these structures are cells; within cells, they are genes. The interdependence of these building blocks is difficult to measure but is integral to the biological processes of health and disease, which emerge from the dynamism of thousands of interacting genes. This cooperativity manifests in particular mutations which accumulate over the course of cancer progression, gender-specific medical conditions, and transcription factor cocktails used to reprogram differentiated cells into stem cells. However, it is experimentally intractable to test the significance of perturbing every unique combination of genes. Instead, we explore gross features of this interaction space to determine how prevalent these synergies are. We take a top-down approach, creating new methods to measure the effects of removing genes from the full set. In the first, we develop a method to measure the transcriptional response to genetic perturbations across hundreds of thousands of cells revealing opposing classes of transcription factors regulating the immune response of dendritic cells. In the second, we create a method to measure how millions of combinations of genetic perturbations impact the growth rate of cancer cell lines. / by Atray Dixit. / Ph. D. in Medical Engineering and Medical Physics

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 243-263). / Two parallel research efforts were pursued. First, we conducted a systematic exploration of how the genomic landscape of cancer shapes and is shaped by anti-tumor immunity. Using large-scale genomic data sets of solid tissue tumor biopsies, we quantified the cytolytic activity of the local immune infiltrate and identified associated properties across 18 tumor types. The number of predicted MHC Class I-associated neoantigens was correlated with cytolytic activity and was lower than expected in colorectal and other tumors, suggesting immune-mediated elimination. We identified recurrently mutated genes that showed positive association with cytolytic activity, including beta-2- microglobulin (B2M), HLA-A, -B and -C and Caspase 8 (CASP8), highlighting loss of antigen presentation and blockade of extrinsic apoptosis as key strategies of resistance to cytolytic activity. Genetic amplifications were also associated with high cytolytic activity, including immunosuppressive factors such as PDL1/2 and ALOX12B/15B. Our genetic findings thus provide evidence for immunoediting in tumors and uncover mechanisms of tumor-intrinsic resistance to cytolytic activity. Second, we combined measurements of protein production and degradation and mRNA dynamics so as to build a quantitative genomic model of the differential regulation of gene expression in lipopolysaccharide-stimulated mouse dendritic cells. Changes in mRNA abundance play a dominant role in determining most dynamic fold changes in protein levels. Conversely, the preexisting proteome of proteins performing basic cellular functions is remodeled primarily through changes in protein production or degradation, accounting for more than half of the absolute change in protein molecules in the cell. Thus, the proteome is regulated by transcriptional induction for newly activated cellular functions and by protein lifecycle changes for remodeling of preexisting functions. / by Michael Steven Rooney. / Ph. D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Page 283 blank. Cataloged from PDF version of thesis. / Includes bibliographical references. / RNA interference (RNAi) is a powerful technology that provides a means to alter the expression of a specific protein based on a targeted RNA sequence. This is done by taking advantage of existing cellular machinery present within all eukaryotic cells which use short double-stranded RNA sequences as guides for RNA induced silencing. The potential for RNAi in medicine is enormous, providing a new approach to treat the complex biological dysregulation underlying many diseases. This promise of a new branch of therapeutics however has been mired with difficulties. RNA is quickly degraded by nucleases that are prevalent in the blood and throughout the body, it is highly immunogenic, and systemic delivery is complicated by high clearance rates. As such, developing formulations for the effective delivery of short RNAs presents significant hurdles. Local delivery can limit numerous unwanted systemic side effects of therapies and it maintains the highest therapeutic index possible in a targeted area before clearance. As such, the local delivery of siRNA may hold just as much potential as systemic delivery with significantly reduced complications. Layer-by-layer (LbL) assembly is a robust method that has been successfully demonstrated for the localized and sustained delivery of many biologic therapeutics and biomolecules. Developing an LbL film capable of delivering siRNA locally would offer a powerful new approach to the treatment of local disorders. This approach could be combined with existing medical devices to improve patient outcomes by directly addressing pathologic dysregulation in the area of interest. One field where the local treatment of dysregulation could be of particular interest is that of wound healing. Wound healing is a complex and highly synchronized process of multiple biological pathways, consisting of an assortment of cytokines, growth factors, and varied cell types which evolves over time. The development of a drug delivery system that can locally modify cell behavior on the basic level of gene transcription would be a powerful tool to alter the dynamics of wound healing. There are many known complications of wound healing, ranging from chronic ulcerative wounds to hypertrophic contractile scars, which dramatically affect the lives of tens of millions of patients every year. Through RNA interference, one could specifically target the key mediators of these complications, providing a means to more effectively regulate the wound healing process in vivo. The capability to deliver siRNA locally to address these complications is a significant advance in the current state of wound treatment. As such, this work presents an opportunity to substantially improve the current standard of treatment for patients and their wound healing outcomes. Herein, we present the design and preclinical evaluation of a number of strategies to develop ultra-thin polymer coatings for the controlled delivery of RNA both in vitro and in vivo. We used Layer-by-Layer assembly to create siRNA containing polymer-based films that can sustain the release of complexed siRNA over physiologically relevant timescales for local delivery into tissues. We describe the development of the first high-throughput approach for LbL assembly and screening and its use to identify lead candidate film architectures for RNA delivery. We then apply these findings to treat dysregulation in two distinct animal models; a chronic diabetic mouse wound model and a third-degree bum model in rats, targeting three different genes of interest independently. These coatings were demonstrated to effectively coat a number of medically relevant substrates including bandages, sutures, surgical staples, nanoparticles, microparticles, and microneedles. This body of work provides insight into how siRNA can be incorporated into thin film assemblies and the design criteria to achieve successful gene knockdown in vitro and in vivo. / by Steven Andrew Castleberry. / Ph. D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / As profiling the molecular states of cellular subpopulations has become increasingly important to understand complex systems in biology and medicine, considerable efforts are being made to develop multiplexed techniques. While current fluorescent probes play indispensable roles, their broad emission spectra (about 30-100 nm) limit multiplexing capability. Recently, optical probes emitting narrowband laser spectra (about 0.1-1 nm), called 'laser particles', has drawn attention. Semiconductor microdisk lasers fabricated by top-down lithography have shown potential for massive multiplexing of thousands to millions of samples. In the thesis, I investigated lead halide perovskites (LHP) as a novel material for scalable production of laser particles in a lab flask. I discovered a sonochemical method to produce a large quantity (10 billions/L) of high-quality LHP micro- and sub-micron particles in a polar solvent within minutes. This method enabled me to coat the surface of individual CsPbBr3 laser particles using poly-catecholamine and thereby to improve optical properties and material stability against moisture. With CsPbBr3 microparticles coated with nano-scatterers, I realized disordered lasing based on Anderson localization. In addition, by incorporating plasmonic materials, I demonstrated plasmonic-lasing particles as small as 580 nm. This work paves the way for highly multiplexable laser particles for biomedical applications. / by Sangyeon Cho. / Ph. D. / Ph.D. Harvard-MIT Program in Health Sciences and Technology

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2019 / Cataloged from PDF version of thesis. / Includes bibliographical references. / An intricate interplay of signaling molecules underlies brain activity, yet studying these molecular events in living whole organisms remains a challenge. Magnetic resonance imaging (MRI) is the most promising imaging modality for development of molecular signaling sensors with deeper tissue penetration than optical imaging, and better spatial resolution and more dynamic potential in sensor design, compared to radioactive probes. MRI molecular sensors, however, have largely required micromolar concentrations to achieve detectable signals. In order to detect signaling molecules in the brain at their native low nanomolar concentrations, an improvement in MRI molecular sensors is necessary. Here we introduce a new in vivo imaging paradigm that uses vasoactive probes (vasoprobes) that couple molecular signals to vascular responses. We apply the vasoprobes to detect molecular targets at nanomolar concentrations in living rodent brains, thus satisfying the sensitivity requirement for imaging endogenous signaling events. Even with more sensitive probes, molecular imaging of the brain is further complicated by the presence of the blood-brain barrier (BBB), designed by nature to protect this most vital of organs. We have therefore implemented a means to permit noninvasive delivery of imaging agents following ultrasonic BBB opening. We use the ultrasound technique to deliver another potent class of contrast agents, superparamagnetic iron oxides, and we show that effective permeation of brain tissue is achieved using this approach. We have also designed ultrasensitive vasoprobe variants designed to permeate the brain completely noninvasively, using endogenous transporter-mediated mechanisms. We present preliminary results based on this approach and discuss future directions. / by Agata E. Wiśniowska. / Ph. D. in Medical Engineering and Medical Physics / Ph.D.inMedicalEngineeringandMedicalPhysics Harvard-MIT Program in Health Sciences and Technology

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / Spinal cord injury (SCI) is a devastating condition drastically reducing the quality of life that affects about 300,000 patients in the USA. As a result of the injury, sensory perception and motor functions are lost. Current treatments do not address the root cause - degeneration and loss of neural tissue. The overall goal of this pre-clinical work was to evaluate a novel gelatin-based conjugate (gelatin-hydroxyphenyl propionic acid; Gtn-HPA) capable of undergoing covalent cross-linking in vivo after being injected as a liquid. Gtn-HPA incorporating epidermal growth factor (EGF) and/or stromal cell-derived factor - 1ɑ (SDF-1ɑ) was evaluated for promoting tissue healing and functional recovery using a standardized 2-mm hemi-resection SCI rat model, four weeks after injection. Injection of Gtn-HPA/EGF immediately after the surgical excision injury significantly improved motor functional recovery, compared to gel alone and non-treated controls. / Bladder function was also improved in Gtn-HPA/EGF-treated animals. Functional improvement correlated with the amount of spared tissue. The volume of gel in the defects was quantified by a newly developed MRI-based method employing T1-weighted inversion recovery to unambiguously image Gtn-HPA in the injury site in a non-destructive manner. Histological analysis showed the presence of multiple islands of Gtn-HPA in the injury site after four weeks. There was a significantly greater number of cells migrating into the Gtn-HPA/EGF, compared to the gel alone, and these cells displayed neural progenitor cell markers: nestin, vimentin, and Musashi. The cells infiltrating Gtn- HPA were negative for glial fibrillary acidic protein (GFAP), a marker for astrocytes. Injection of the gel reduced the reactive astrocytic presence at the border outlining the injury site indicating the reduction of glial scar. / There was no notable inflammatory response to the Gtn-HPA gel, reflected in the number of CD68-positive cells, including macrophages. Of note was the demonstration by immunohistochemistry that the Gtn-HPA remaining at 4 weeks post-injection contained EGF. MMP2 was found to be playing a role in in vivo degradation of the Gtn-HPA gel. Additional behavioral and histological results were acquired injecting Gtn-HPA/EGF in 2-mm complete resection SCI rat model. Collectively, the findings signaled that injury sites injected with Gtn-HPA/EGF had greater potential for regeneration. In summary, this work commends an injectable, covalently cross-linkable formulation of Gtn-HPA incorporating EGF for further investigation in promoting functional recovery and potential regeneration for treatment of SCI and thereby improve the quality of life of SCI patients. / by Adhvait M. Shah. / Ph. D. in Medical Engineering and Medical Physics / Ph.D.inMedicalEngineeringandMedicalPhysics Harvard-MIT Program in Health Sciences and Technology

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages 201-222). / Diffusion magnetic resonance imaging (dMRI) tractography is the only non-invasive tool for studying the connectional architecture of the brain in vivo. By measuring the diffusion of water molecules dMRI provides unique information about white matter pathways and their integrity, making it an invaluable neuroimaging tool that has improved our understanding of the human brain and how it is affected by disease. A major roadblock to its acceptance into clinical practice has been the difficulty in assessing its anatomical accuracy and reliability. In fact, obtaining a map of brain pathways is a multi-step process with numerous variables, assumptions and approximations that can influence the veracity of the generated pathways. Validation is, thus, necessary and yet challenging because there is no gold standard which dMRI can be compared to, since the configuration of human brain connections is largely unknown. Which aspects of tractography processing have the greatest effect on its performance? How do mapping methods compare? Which one is the most anatomically accurate? We tackle these questions with a multi-modal approach that capitalizes on the complementary strengths of available validation strategies to probe dMRI performance on different scales and across a wide range of acquisition and analysis parameters. The outcome is a multi-layered validation of dMRI tractography that 1) quantifies dMRI tractography accuracy both on the level of brain connections and tissue microstructure; 2) highlights the strengths and weaknesses of different modeling and tractography approaches, offering guidance on the issues that need to be resolved to achieve a more accurate mapping of the human brain. / by Giorgia Grisot. / Ph. D. / Ph.D. Harvard-MIT Program in Health Sciences and Technology

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Thesis: Ph. D. in Medical Engineering and Bioastronautics, Harvard-MIT Program in Health Sciences and Technology, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references. / In spaceflight, the loss of mechanical loading has detrimental effects on the musculoskeletal system. These muscular changes will likely affect spinal loading, a key aspect of vertebral fracture risk, but no prior studies have examined how spinal loading is affected by long duration spaceflight. Moreover, the effect of spaceflight on vertebral strength has not been determined, despite reports of significant vertebral trabecular bone loss in long-duration astronauts. Thus trunk muscle and vertebral bone changes and their impact on risk of injury following long-duration spaceflight remain unknown. This is of particular concern for NASA's planned Mars missions and return to Earth after prolonged deconditioning. Our lab has developed a musculoskeletal model of the thoracolumbar spine that has been validated for spinal loading, but has not yet been extended to maximal effort activities or full-body simulations. / Thus, the overall goal of this work consisted of two main sections: 1) address the knowledge gap regarding spaceflight and post-flight recovery effects on trunk muscle properties, vertebral strength, compressive spine loading and vertebral fracture risk, and 2) extend our musculoskeletal modeling work into maximal effort simulations in an elderly population and create a full-body scaled model to investigate reproducibility of spine loading estimates using opto-electronic motion capture data. Whereas deficits in trunk muscle area returned to normal during on-Earth recovery, spaceflight-induced increases in intramuscular fat persisted in some muscles even years after landing. Similarly, spaceflight led to a decrease in lumbar vertebral strength that did not recover even after multiple years on Earth. / To gain insight into the effect of spaceflight on vertebral fracture risk, we created subject-specific musculoskeletal models using an individual's height, weight, sex, muscle measurements, and spine curvature. We found that compressive spine loading was minimally affected by spaceflight and that vertebral fracture risk, calculated as a ratio of vertebral load to strength, was slightly elevated post-flight and remained elevated during readaptation on Earth. Additionally, we focused on the development of additional musculoskeletal modeling tools. Using maximal effort model simulations, we estimated trunk maximum muscle stress in an elderly population, and this critical parameter in musculoskeletal modeling will assist with more detailed model creation. Lastly, we found excellent reliability of spine loading estimations from opto-electronic marker data. / by Katelyn A. Burkhart. / Ph. D. in Medical Engineering and Bioastronautics / Ph.D.inMedicalEngineeringandBioastronautics Harvard-MIT Program in Health Sciences and Technology

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2019 / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages 105-110). / Targeted patch clamp recording is a powerful method for characterizing visually identified cells in intact neural circuits, but it requires skill to perform. We found that a closed-loop real-time imaging strategy, which continuously compensates for cell movement while approaching the cell with a pipette tip, allows for the development of an algorithm amenable to automation. We built a robotic system that can implement this algorithm and validated that our system can automatically patch fluorophore-expressing neurons of multiple types in the living mouse cortex, with yields comparable to skilled human experimenters. By facilitating targeted patch clamp recordings in vivo, our robot may enable scalable characterization of identified cell types in intact neural circuits. Activities of individual neurons in neural circuits give rise to network oscillations, whose frequencies are closely related to specific brain states. / For example, network oscillations in the 30 - 90 Hz range, observed using electroencephalogram (EEG), are called gamma oscillations and increase during attention, memory formation, and recall. In Alzheimer's disease (AD), gamma oscillations are disrupted compared to healthy individuals. Recently, noninvasive visual and auditory stimulations at 40 Hz, called Gamma ENtrainment Using Sensory stimulus ("GENUS"), have been shown to positively impact pathology and improve memory in AD mouse models, with concurrent visual and auditory GENUS leading to a more widespread effect in the AD mouse brain compared to visual or auditory stimulation alone. However, it is unclear what effect such sensory stimulations would have on the human brain. To test for the safety and feasibility of GENUS in humans, we developed a device that can deliver 40 Hz light and sound stimulations at intensity levels tolerable to humans. / We found that our device can safely lead to steady 40 Hz entrainment in cognitively normal young (20 - 33 years old) and older (55 - 75 years old) subjects, with concurrent visual and auditory stimulation leading to stronger and more widespread entrainment than visual or auditory stimulation alone. These findings suggest that GENUS can be a safe and effective method for widespread 40 Hz entrainment, which may have therapeutic effects in people suffering from AD. / by Ho-Jun Suk. / Ph. D. / Ph.D. Harvard-MIT Program in Health Sciences and Technology