Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2005. / Vita. / Includes bibliographical references. / The goal of this thesis study was to understand the role of TGF-[beta] in skin photoaging, especially in solar elastosis. Solar elastosis, the accumulation of elastotic material in the dermal extracelluar matrix, is a major hallmark of photoaging. However, the mechanisms by which UV radiation causes solar elastosis are poorly understood. TGF-[beta] is a multifunctional cytokine involved in the regulation of extracelluar matrix and is known to be up-regulated by UVR. Involvement of reactive oxygen species (ROS) in the development of solar elastosis has been demonstrated by many studies using antioxidants and anti-inflammatory agents in the mouse skin in vivo. We hypothesized that ROS produced by TGF-[beta] are key components in the tropoelastin (TE, a soluble precursor of elastin) up-regulation in dermal fibroblasts, and that TGF-[beta] is a major regulator in the photoaging processes. Using human skin fibroblasts system in vitro, we found that ROS generated from NADPH oxidase and mitochondria are involved in the TGF-[beta] induced elastin production, and intracellular sources of ROS vary with time. We showed that both Smad and non-Smad pathways, e.g. MAPK and PKC pathways, are required for TE mRNA up-regulation by TGF-[beta]. / (cont.) However, ROS were not involved in some of the important steps in these pathways, such as phosphorylations of p38 or ERK or Smad2, suggesting that ROS acts downstream of these pathways. The in vivo chronic UVB irradiation study using a Skh- 1 hairless mouse model with a small molecule inhibitor for the TGF-[beta] type I receptor showed that the TGF-[beta] receptor inhibitor increased the number of mast cells, but decreased the levels of active TGF-[beta] protein, and mRNA levels for collagen III and IV, MMP-2 and 9, and TE in the chronically UVB irradiated mouse skin. However, those responses did not result in the changes in the collagen and elastin content, or the wrinkle formation. Overall, this work indicates that TGF-[beta] contributes to the solar elastosis, through the effects on the TE mRNA level in skin. Implication of this role of TGF-[beta] in the elastin fiber deposition or visible changes of photoaged skin requires further investigation. / by Won Seon Choi. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2010. / Page 104 blank. Cataloged from PDF version of thesis. / Includes bibliographical references. / The monitoring of physiological biomarkers is fundamental to the diagnosis and treatment of disease. We describe here the development of molecular sensors which can be read by magnetic resonance (MR) relaxometry. MR is an advantageous bio-sensor readout because it can be determined from opaque samples and through intervening layers of matter. Wash steps can therefore be avoided in in vitro MR assays and non-invasive interrogation achieved for in vivo MR sensing. Functionalized magnetic nanoparticles originally developed as in vivo contrast agents have recently been adapted for use in magnetic relaxometry assays. The first half of this thesis describes a simple particle functionalization strategy and its application to the detection of myocardial infarction ("heart attack") associated biomarkers. The particles were subcutaneously implanted in the form of small discrete sensors and shown to be efficacious in measuring the physiological release of three protein biomarkers. Alternative contrast mechanisms may also be employed by MR readable sensors. The second half of this thesis introduces the novel use of 'smart' polymers which produce analyte-responsive changes in MR relaxivity. We show that MR responsive calcium-crosslinked and pH-swelling hydrogels can be incorporated within discrete sensors. / by Yibo Ling. / Ph.D.

Thesis (Ph.D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, February 2002. / Includes bibliographical references (p. 99-103). / A mechanotransduction model aims to explain how a cell senses mechanical forces and translates them into molecular events, such as changes in intracellular ion concentrations, gene expression, post-translational protein modification, or cytoskeletal redistribution. Many studies of mechanotransduction explore the responses of cell populations to large and, occasionally, poorly characterized deformation. More recent techniques provide the means for characterizing the physical properties of the cell, with the potential for uniting the molecular responses with the material properties and deformation characteristics of the cell. These recent techniques have an additional advantage - they can be used to probe cells at the single cell level. Single cell studies have the potential to elucidate more precisely the specific mechanisms by which a cell senses and transmits signals, because the information gathered from each cell is not averaged over thousands or millions of cells. Over the past decade, several approaches to studying mechanical properties at the single cell level were developed. Atomic force microscopy, optical and magnetic tweezers, micropipette aspiration and other techniques have been used to explore and develop physical models of the cell. By using these techniques, the basic physical response of a cell to mechanical forces has been analyzed, yet the reported cell moduli in the literature, when taken in aggregate, span up to 6 orders of magnitude. It is not clear whether this wide distribution is due to the disparity in different cell types being examined, the methods being used to stress the cell, or the models used to analyze the responses. / (cont.) No study to date has addressed this variability, nor have many studies used the same physical model to test the variability of cellular mechanical properties across different cell types. To help clarify this broad variability in cellular mechanical properties, and to understand factors that may influence the responses of cells, two magnetic traps were designed to study the mechanical and molecular responses of different cell types. It was found that different cells may in fact have different mechanical properties, but the shear moduli are distributed over only one or two orders of magnitude, and not the five or six reported in the literature. Thus, the remaining variance in results is likely due to differences in modeling or technique. It is likely that differences in technique contribute substantially to the remaining variance, since molecular studies indicate that cellular responses are sensitive to environmental conditions such as temperature and to cellular conditions such as confluency. / by Hayden Huang. / Ph.D.

Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2015. / 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 (pages 174-199). / The human microbiota consists of 100 trillion microbial cells that naturally inhabit the body and harbors a rich reservoir of genetic elements collectively called the microbiome. Efforts based on metagenomic sequencing of microbiomes associated with healthy and diseased individuals have revealed vast effects of microbiota on human health. However, compared to the expanding amount of sequence data, little is known about the function of these microbes and their genes. Furthermore, current clinical approaches to modify the microbiota face several challenges, including colonization resistance in competitive environments such as the gut, and imprecise ecological perturbations using antibiotics and fecal transplants. The fundamental objective of this research is to develop safe methods to genetically edit the microbiome in vivo to promote human health. The abilities to introduce commensally fit strains and to control specificity of microbial modulations are critical steps towards ecological engineering of healthy microbiota. This thesis describes strategies to investigate, propagate, and ultimately engineer desired functions in microbiota. In particular, we developed a temporal functional metagenomics method to identify genes that improved microbial fitness in the mammalian gut in vivo. We also built foundational tools for delivering genetic elements and immunizing endogenous microbiota against acquiring antibiotic resistance and toxins. In addition to leveraging bacterial conjugation and the prokaryotic defense system CRISPR-Cas9, we employed bacteriophages for depleting native strains to empty the niche for an engineered version. Our work enables applications in engineering probiotic strains with augmented fitness and anti-pathogenesis properties, tempering host autoimmunity, and combating hospital-acquired infections and enteric diseases. / by Stephanie J. Yaung. / Ph. D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2007. / Includes bibliographical references. / As human spaceflight extends in both duration and scope, it is critical to better understand the physiologic effects of this novel environment. In the weight bearing structures of the body, bone loss and muscle atrophy far in excess of age-related declines are hallmarks of microgravity adaptation. However, while the physiological effects of such disuse unloading are well-described, the effects of partial weight bearing, such as expected on the moon (16% of Earth's gravity) and Mars (38% of Earth's gravity), have yet to be quantified. In these environments, the risks of musculoskeletal atrophy and accompanying orthopedic injury are uncertain, and a means of further investigation is needed. To address this need, we developed a novel model of Partial Weight Suspension (PWS) that supports investigation of the physiologic effects of chronically reduced quadrupedal loading in mice. Validation of the PWS system was conducted using a gait analysis treadmill and high-precision force platform. These studies showed that peak ground reaction forces were significantly reduced under conditions of partial weightbeari:ng, and changes in gait dynamics were consistent with previous studies of human locomotion. Using the PWS system, we conducted the first known studies of chronic musculoskeletal adaptation to Mars and lunar levels of weight bearing. Adult female BALB/cByJ mice underwent 21 days of partial weight bearing or control treatment. Relative to controls, suspended animals showed significant bone and muscle loss. In particular, bone formation rate was decreased, leading to deterioration of both cortical and trabecular bone structure in mice exposed to weight bearingtbearing. Although material properties of the bone were largely unaffected, structural and geometric changes resulted in lower bone strength. / weight bearinged weight bearing at Mars and lunar levels led to similar losses of muscle and bone relative to controls. Comparison with previous literature suggests that adaptation to partial weight-bearing associated with both Mars and lunar loading provided some protection relative to the deconditioning seen in full unloading. Although additional studies are needed, the data also indicated that the musculoskeletal deterioration was not linearly related to the degree of unloading. Altogether, this model provides a validated, controlled system for investigaweight bearingof partial weightbearing and countermeasures on musculoskeletal deconditioning. Our initial findings have practical applications for bioastronautics, suggesting that physiological investigations on the surface of the moon may not be fully predictive for future Mars exploration. / by Erika Brown Wagner. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 55-59). / Access to the cerebral ventricle (e.g. ventriculostomy) is required to manage multiple life-threatening ailments. It can be done either in the operating room or at the bedside to relieve increased intracranial pressure or deliver medication. At the bedside, the procedure is normally performed freehand, with the occasional use of a Ghajar guide for guidance support. In the operating room, ventriculostomy may be performed with an image-guidance system, whether optical or electromagnetic. The most common complications of ventriculostomy are hemorrhage and infection. It is unclear whether catheter placement accuracy and the number of passes of the catheter for each placement are correlated with ventriculostomy complications. Our goals are 1) to evaluate the current state of practice, including complications of ventriculostomy, and 2) to evaluate a targeted image guidance system for use with ventriculostomy - the Smart Stylet. To address these goals, an Institutional Review Board-approved retrospective cross-sectional study was conducted at the Brigham and Women's Hospital (BWH) to characterize the practice of external ventricular drain placements using data from the patient electronic medical record. Post-procedure catheter location was measured on post-procedure CT and MRI imaging studies. Most cases were performed in the operating room and the operative reports provided all procedure-related information. Microbiology reports were collected within a four-week interval following catheter placements to evaluate presence of invading pathogens. All imaging studies, microbiology reports, and operative reports were reviewed manually. The rest of the medical records were not reviewed and, therefore, cerebrospinal fluid leak and shunt malfunction were not evaluated. Catheter placement accuracy and the numbers of passes for each placement were assessed. We evaluated whether these metrics were associated with the occurrence of procedure complications. A procedure-targeted image guidance system in development stage, the Smart Stylet, was implemented for use on a ventricular phantom model with a right-sided midline shift. Smart Stylet consists of an electromagnetic tracking system and ventriculostomy catheter connected to a PC and display. The operator of the Smart Stylet can interface with the system via a custom designed module in BWH's 3DSlicer software system. The system was tested for accuracy by calculating targeting error and reporting the precision of catheter placement. Precision was measured using pair-wise distances among experimental groups. The system was reviewed and commented on by three novices and two neurosurgical residents from the Massachusetts General Hospital by using the NASA-TLX grading scale questionnaire and a targeted survey. The phantom model was designed to gauge whether further tests in animals and cadavers are warranted using Smart Stylet. Patients with trauma were more likely to have catheters misplaced (OR = 9.13±2.31; p<0.05). It seems there is an opportunity to improve patient care if catheter placement is made more accurate and reliable. Use of the Smart Stylet system in a phantom study provided improvements in mean pair-wise distance and accuracy for catheter placement at the sub-centimeter level. A blinded operator achieved statistically significant improvement in targeting error using the right frontal approach (p<0.0 5). The operator also significantly improved mean pairwise distances using left and right frontal approaches (p<0.05). Novice operators and neurosurgical residents both showed improvements in targeting accuracy for catheter placement when using the system for the first time. However, the improvements were not statistically significant. Novices' pair-wise distances were significantly better with Smart Stylet guidance using the left frontal approach (p<0.05). Improved guidance techniques, such as the Smart Stylet approach, can potentially decrease ventriculostomy complications if they can be easily integrated into clinical use at low cost. / by Vaibhav Devidas Patil. / S.M.

Thesis (Ph. D.)--Harvard University--MIT Division of Health Sciences and Technology, 2005. / Includes bibliographical references (leaves 91-97). / The ease of targeted genetic modification makes the mouse a valuable model to study hearing and deafness. A powerful window into cochlear function can be obtained by recording from single auditory (AN) fibers, primary afferents connecting cochlear inner hair cells to the brain. This thesis provides the first systematic analysis of fundamental response properties of the mouse AN, including rate and timing of spontaneous and sound-evoked discharge, frequency selectivity, dynamic range, and the relations between spontaneous rate and these other response properties. Three wildtype strains were compared: CBA/CaJ (because it retains normal cochlear sensitivity as it ages); and C57BL/6 and 129/SvJ (because they are used in "knockout" generation). Two lines with targeted deletion of inner ear genes were also studied. The cochlea in mouse responds to frequencies octaves higher than in humans or most other mammals; nevertheless, most other AN characteristics were fundamentally similar to those in well-studied mammals. The only significant differences were 1) smaller dynamic ranges in all three strains; and 2) high-frequency threshold elevation and tuning degradation in C57BL/6 and 129/SvJ, consistent with early-onset degeneration of basal-turn sensory cells in these strains. The mutant lines studied included deletion of 1) Calcitonin-Gene-Related Peptide (CGRP), a neurotransmitter expressed by cochlear efferent fibers, or 2) the a subunit of BK channels, a large-conductance K+ channel, expressed in hair cells and AN fibers, and implicated in cochlear tuning in lower vertebrates. / (cont.) Loss of CGRP had no detectable effect on AN responses, suggesting a minimal role in cochlear processing. Loss of BK channels led to dramatic decreases in sound- evoked discharge rates and spike synchronization, without changes in frequency selectivity. The degraded spike synchronization likely arises from slowing rise-times of inner hair cell receptor potentials observed in vitro; the decreased steady-state rates likely arise from depolarization block in the AN fibers themselves. / by Annette M. Taberner. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Beta band (13-30 Hz) oscillations in sensorimotor cortex are associated with motor performance, but the nature of this relationship is not clear. Recently, excessive beta activity in cortico-basal ganglia circuits has been recognized as a hallmark of Parkinson's disease. Renewed interest in beta oscillations has since led to the suggestion that they might reflect the preservation of the current output or state of a given brain region. To investigate the potential role of beta activity in the brain, we recorded local field potentials in the frontal cortex and striatum of monkeys as they performed single and sequential arm movement tasks. To facilitate these experiments, we developed novel methods for recording simultaneously from independently moveable electrodes implanted chronically at over 100 sites in cortical and subcortical areas of the monkey brain. We found that, across tasks, beta oscillations occurred in brief, spatially localized bursts that were most prominent following task performance. Across brain regions, post-performance bursts were differentially modulated by the preceding task. In motor cortex they tracked the number of movements just performed. In contrast, striatal and prefrontal burst rates were proportional to the number of visual cues, or to a combination of the cues and movements, respectively, and were higher following correct, rewarded, trials than unrewarded errors. Pairs of striatal-prefrontal sites exhibited increased cross-covariance and coherence during post-trial beta bursts, suggesting that these bursts might be involved in communication or coordination across brain regions. Based on our results, we propose that beta oscillations may represent post-performance reinforcement of the network dynamics that led to the desired behavioral outcome obtained immediately prior. / by Joseph Feingold. / Ph.D.

Thesis (Ph. D.)--Harvard--Massachusetts Institute of Technology Division of Health Sciences and Technology, 2002. / Includes bibliographical references. / Magnetic resonance diffusion imaging provides an exquisitely sensitive probe of tissue microstructure. Owing to the microscopic length scale of diffusion in biological tissues, diffusion imaging can reveal histological architecture irresolvable by conventional magnetic resonance imaging methods. However, diffusion imaging methods to date have chiefly been based on analytical models of the underlying diffusion process. For example, diffusion tensor imaging assumes homogeneous Gaussian diffusion within each voxel, an assumption which is clearly invalid for the vast majority of the brain at presently achievable voxel resolutions. In this thesis I developed a diffusion imaging method capable of measuring the microscopic diffusion function within each voxel. In contrast to previous approaches to diffusion imaging, the method presented here does not require any assumptions on the underlying diffusion function. The model-independent approach can resolve complex intravoxel tissue structure including fiber crossing and fiber divergence within a single voxel. The method is capable of resolving not only deep white matter intersections, but also composite tissue structure at the cortical margin, and fiber-specific degeneration in neurodegenerative pathology. In sum, the approach can reveal complex intravoxel tissue structure previously thought to be beyond the scope of diffusion imaging methodology. / by David Solomon Tuch. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2004. / Includes bibliographical references (p. 96-103). / The olivocochlear (OC) efferent innervation of the mammalian inner ear consists of two subdivisions, medial (MOC) and lateral (LOC), with peripheral terminations on outer hair cells and cochlear afferent terminals, respectively. The cochlear effects of electrically activating MOC efferents are well known: the MOC efferents suppress cochlear responses by reducing outer hair cells' contribution to cochlear amplification. In contrast, LOC peripheral effects are unknown, because their unmyelinated axons are difficult to stimulate. To overcome the difficulty of directly activating the LOC system, stimulating electrodes were placed in the inferior colliculus (IC) to activate the LOC system indirectly, while recording cochlear responses bilaterally from anesthetized guinea pigs. Neural potentials and outer hair cell based potentials were recorded before and after IC stimulation for tens of minutes. Stimulation at some IC sites produced novel cochlear effects attributable to activation of the LOC system: long-lasting (1-20 min) enhancement or suppression of cochlear neural responses (compound action potentials (CAP) and round window noise), without changes in cochlear responses dominated by outer hair cells (otoacoustic emissions and cochlear microphonics). These novel effects also differed from classic MOC effects in their laterality and their dependence on level and frequency of the acoustic stimulus. The changes in CAP are well described as "constant %" increases or decreases. These effects disappeared upon sectioning the entire OC bundle, but not after selective lesioning of the MOC tracts or the cochlea's autonomic innervation. Based on this evidence, I conclude that the LOC pathway comprises two functional subdivisions capable of inducing / (cont.) slow increases or decreases in response magnitudes in the auditory nerve. The LOC system originates in the lateral superior olive (LSO), which is the ascending nucleus where sound localization is computed. Such a system may be useful in maintaining accurate binaural comparisons necessary for sound localization in the face of slow changes in interaural sensitivity. / by J. Alan Groff. / Ph.D.