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

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / Cataloged from PDF version of thesis. / Includes bibliographical references. / RNA potently regulates gene expression. However, the utility of RNA has been limited by the ability to efficiently deliver it to specific cells in vivo. In vivo RNA delivery is challenging; vehicles must avoid phagocytosis in the bloodstream, reach the target tissue, and get into, and out of, an endosome, all without setting off an unwanted immune response. Despite these challenges, nanoparticles have delivered siRNA to hepatocytes after intravenous injections as low as 0.001 mg/kg. By contrast, efficient, durable, and robust silencing in other cell types has remained challenging. Herein we describe 7C I, a low molecular weight polymeric nanoparticle that delivers siRNA to endothelial cells in vivo at doses as low as 0.017 mg/kg. 7C1 nanoparticles reduced target mRNA expression for more than three weeks after a single injection, and delivered five siRNAs concurrently in vivo. Notably, 7C I transfects endothelial cells at low doses without significantly reducing gene expression in hepatocytes or immune cells. 7C I was optimized for stability and consistency, and used to study inflammation, cardiovascular disease, emphysema, primary tumor growth, and metastasis in labs across the United States. These data demonstrate that 7C I can be used to potently modify the expression of multiple endothelial genes in vivo. / by James E. Dahlman. / Ph. D.

Thesis (D.L.S.)--Columbia University, 1991. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 701-814).

Thesis (D.L.S.)--Columbia University, 1991. / Includes bibliographical references (leaves 701-814).

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, September, 2020 / Cataloged from the official PDF version of thesis. / Includes bibliographical references (pages 107-118). / Engineering small caliber (< 6mm) arterial grafts remains an unsolved problem. Current synthetic and autologous grafts suffer from short and long-term limitations including decreased patency rates, risk of bacterial infection, and compliance mismatching that results in neointimal hyperplasia. Tissue engineering is seen as a solution; however, a true arterial replacement remains elusive. Despite the numerous publications that have appeared over the last three decades, most reported strategies mimic functional and structural arterial properties to a limited extent. Furthermore, these strategies require long maturation times before implantation and carry the risk of failure in patients, who are often elderly with multiple comorbidities. Our central hypothesis was that living arterial substitutes that display normal physiological responses after in vivo implantation can be engineered through the controlled assembly of vascular cells and free-standing collagen sheets of controlled fibril orientation in a manner that recapitulates native vessel microstructure. We first present a scalable and continuous strategy for generating strong, free-standing, ultrathin, and centimeter-wide collagen sheets with controlled anisotropy using a flow-focusing approach. This strategy represents the first of its kind to generate anisotropic collagen sheets with control over nano- and macro-molecular properties. Next, controlled assembly of vascular cells and free-standing collagen sheets allowed us to design living blood vessels that recapitulated the arterial wall microstructure, and through structural, mechanical and biological characterization confirmed mimicry of native physiologic properties. We believe that the scalable fabrication schemes, and thorough characterization techniques, presented here will serve as a strong reference for future blood vessel tissue engineering efforts. / by David Miranda-Nieves. / 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, June, 2018 / Cataloged from the official PDF version of thesis. / Includes bibliographical references. / Antibiotic resistance is a global health emergency that mandates new drug development strategies. Natural antimicrobial peptides (AMPs) have been long-recognized as a potential source of bacteriolytic drugs, but the shortcomings of non-specific membrane toxicity, proteolytic instability, and in vivo toxicity have stymied their clinical translation. Here, we subjected expansive stapled-peptide libraries of the magainin II (Mag2) AMP to structure-function analyses and uncovered the biophysical and mechanistic determinants that allow for the rational design of stapled AMPs (StAMPs) that are bacterial-membrane selective, proteolytically-stable, well tolerated in mice upon intravenous administration, and most importantly, overcome even the most antibiotic-resistant bacteria, including colistin-resistant A. baumannii and mobilized colistin resistance plasmid-bearing E. coli. Specifically, we discovered that the topographic continuity and strength of hydrophobic networks, in the context of alpha-helical amphipathic cationic peptides, dictates both the selectivity and mechanism of membrane lysis. We further harnessed our results to develop an algorithm for the design of a new generation of non-toxic, bacterial-selective StAMPs for clinical development. / by Rida Mourtada. / Ph. D. in Medical Engineering and Medical Physics / Ph.D.inMedicalEngineeringandMedicalPhysics Harvard-MIT Program in Health Sciences and Technology