Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2004. / Includes bibliographical references. / Non-muscle cell shape change and motility depend primarily on the dynamics and distributions of cytoplasmic actin. In cells, actin cycles between monomeric and polymeric phases tightly regulated by actin binding proteins that control cellular architecture and movement. Here, we characterize actin remodeling in shear stress stimulated endothelial cells and in actin networks reconstituted with purified proteins. Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Alignment requires 24 h of exposure to flow, but the cells respond within minutes to flow by diminishing their movements by 50%. Although movement slows, actin filament turnover times and the amount of polymerized actin in cells decreases, increasing actin filament remodeling in individual cells composing a confluent endothelial monolayer to levels used by disperse, non-confluent cells for rapid movement. Hours later, motility returns to pre-shear stress levels, but actin remodeling remains highly dynamic in many cells. We conclude that shear stress initiates a cytoplasmic actin remodeling response that is used to modify endothelial cell shape instead of bulk cell translocation. We determine the steady state dynamics of purified actin filament networks in the entangled state and after orthogonal cross-linking with filamins using a novel, non-perturbing fluorescence system. Human filamin A or Dictyosteliun discoidium filamin slow actin filament turnover by [approximately] 50% and recruit much of a significant population of actin oligomers that we measure are present in polymerized purified actin solutions into the immobile filament fraction. Surprisingly, these observations occur at very low stoichiometry to actin, approximately requiring only one / (cont.) filamin molecule bound per actin filament, similar to the amount required for actin filament gelation in vitro. Networks formed with filamin truncates localize this activity to the actin binding domain and reveal that dimerization and orthogonal cross-linking are not required for dynamic stabilization. Re-expression of filamin A with or without the actin binding domain in human melanoma cells that naturally lack this protein support the findings in purified actin networks. These results indicate that filamin cross-linking stabilizes filament dynamics by, slowing filament subunit cycling rates and by either decreasing spontaneous filament fragmentation or promoting filament annealing. / by Eric A. Osborn. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008. / Includes bibliographical references. / At the present time, there are many hurdles to overcome in order to create a long-lasting and engineered tissue for tissue transplant in patients. The challenges include the isolation and expansion of appropriate cells, the arrangement of assorted cells into correct spatial organization, and the development of proper growth conditions. Furthermore, the creation of a three dimensional engineered tissue is limited by the fact that tissue assemblies greater than 100-200 micrometers, the limit of oxygen diffusion, require a perfused vascular bed to supply nutrients and to remove waste products and metabolic intermediates. To overcome this limitation, this thesis aims to pre-seed a tissue engineered construct with vascular cells (both endothelial and perivascular cells), so the vascular cells could readily form functional vessels in situ. Previous work in the laboratory had successfully demonstrated the formation of functional microvascular network by co-implantation of human umbilical cord vein endothelial cells (HUVECs) and 10 T 1/2 cells, a line of mouse embryonic fibroblasts. To translate this concept to the clinic, we need to utilize cells that can be secured and used in clinic. To this end, we systematically replace each individual vascular cell type with a readily available source of cells. First, we investigated human embryonic stem cells (hESCs) derived endothelial cells. We demonstrated that when hESCs derived endothelial cells were implanted into SCID mice, they formed blood vessels that integrated into the host circulatory system and served as blood conduits. Second, we compared the formation and function of engineered blood vessels generated from circulating endothelial progenitor cells (EPCs) derived from either adult peripheral blood or umbilical cord blood. / (cont.) We found that adult peripheral blood EPCs formed blood vessels that were unstable and regressed within three weeks. In contrast, umbilical cord blood EPCs formed normal-functioning blood vessels that lasted for more than four months. These vessels exhibited normal blood flow, perm-selectivity to macromolecules and induction of leukocyte-endothelial interactions in response to cytokine activation similar to normal vessels. Third, we evaluated human bone marrow-derived mesenchymal stem cells (hMSCs) as a source of vascular progenitor cells. hMSCs expressed a panel of smooth muscle markers in vitro and cell-cell contact between endothelial cells and hMSCs up-regulated the transcription of smooth muscle markers. hMSCs efficiently stabilized nascent blood vessels in vivo by functioning as perivascular precursor cells. The engineered blood vessels derived from HUVECs and hMSCs remained stable and functional for more than 130 days in vivo. On the other hand, we could not detect differentiation of hMSCs to endothelial cell in vitro and hMSCs by themselves could not form conduit for blood flow in vivo. Similar to normal perivascular cells, hMSCs-derived perivascular cells contracted in response to endothelin-1 in vivo. Thus, our work demonstrates the potential to generate a patent and functional microvascular network by pre-seeding vascular cells in a tissue-engineered construct. It serves as a platform for the addition of parenchymal cells to create a functional and vascularized engineered tissue. / by Pakwai (Patrick) Au. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Includes bibliographical references (p. 141-152). / Disrupted blood-brain barrier after an ischemic attack can cause vasogenic edema and increase the risk of hemorrhagic transformation. Therefore, early detection and monitoring of BBB damage is important in the pathological understanding and therapeutic treatment of stroke. Currently, MR contrast agents have been widely used in clinics for disease diagnosis and treatment evaluation, and in basic research to achieve better anatomical structure visualization and to understand pathological mechanisms of various human diseases in animal models. Thus, the central theme of this thesis to exploit the use of MR contrast agents in the study of ischemic stroke using both in vivo and ex vivo MR techniques. Specifically, the overall goals of this thesis are twofold: (1) to exploit the multiple relaxation mechanisms and varying tissue-dependent affinities of different MR contrast agents for better structure delineation, tissue differentiation, and image contrast manipulation in magnetic resonance microscopy (MRM) staining, and (2) to develop an MRI technique that employs intrinsic water as a biomarker for qualitative and quantitative monitoring of blood-brain barrier (BBB) integrity alteration in a mouse model of stroke using an intravascular long-circulating MRI contrast agent. Despite the great success of MRM in anatomical studies, MRM images based on intrinsic tissue contrast lack the flexibility and target-specificity offered by conventional histological staining. Therefore, the first focus of this thesis was on the development of MRM staining method by utilizing the different tissue relaxation ability and tissue biophysical/biochemical properties of different MR contrast agents. Two common MR contrast agents, Gd-DTPA and MnCl2 were used in this thesis. The ability of MR contrast agents to increase SNR and enhance image contrast was first tested in a relatively simple in vitro glioma spheroid (diameter ' 400 um) system. / (cont.) We then fully characterized the relaxation mechanisms and tissue-dependent staining properties of these contrast agents in the brain tissue, and demonstrated that their unique relaxation and tissue properties led to differentiated MR staining in the ex vivo mouse brains, which greatly enhanced the ability of MRM to delineate tissue structures in addition to providing improved SNR. This MRM staining method was then applied to the Kif2la knockout mouse model for the anatomical phenotyping of the new born Kif2la knockout mice. The BBB damage is usually detected through the spatial leakage profiles of extrinsically administrated markers such as staining dyes, fluoresceins, radiolabeled compounds, or gadolinium based compound, which are only possible when BBB is compromised to the extent that allows extravasation of these markers. It is therefore desirable to develop a technique that allows the early detection of BBB damage. In the second part of thesis, we first presented the theoretical background of measuring trans vascular water exchange based on a two-compartment water exchange model. Parameters affecting the quantitative BBB water exchange measurement were initially characterized using computer simulations. We then performed graded hypercapnia and Mannitol-induced BBB-opening experiments to test the ability of this novel MRI technique to detect and monitor the changes of BBB integrity and cerebral blood volume (CBV). Upon the characterization of this MRI technique, we measured baseline BBB water exchange and other MRI-derived cerebrovascular parameters in the eNOS knockout mice, and showed that there is basal increase of trans vascular water exchange in addition to the morphological changes in the vasculature of eNOS knockout mice. / (cont.) After developing and characterizing these ex vivo and in vivo MR techniques, we applied the in vivo MRI BBB water exchange detection technique and the ex vivo MRM staining method to a mouse model of transient stroke. We demonstrated the importance of CBV restoration in the BBB integrity change at acute stage after reperfusion, and showed that MRM staining may have a great potential in histopathological studies of ischemic brain injury. / by Shuning Huang. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Includes bibliographical references. / Clinical decision support systems (CDSS) are developed primarily from knowledge gleaned from evidence-based research, guidelines, trusted resources and domain experts. While these resources generally represent information that is research proven, time-tested and consistent with current medical knowledge, they lack some qualities that would be desirable in a CDSS. For instance, the information is presented as generalized recommendations that are not specific to particular patients and may not consider certain subpopulations. In addition, the knowledge base that produces the guidelines may be outdated and may not reflect real-world practice. Ideally, resources for decision support should be timely, patient-specific, and represent current practice. Patient-oriented clinical decision support is particularly important in the practice of pediatrics because it addresses a population in constant flux. Every age represents a different set of physiological and developmental concerns and considerations, especially in medication dosing patterns. Patient clinical data warehouses (CDW) may be able to bridge the knowledge gap. CDWs contain the collective intelligence of various contributors (i.e. clinicians, administrators, etc.) where each data entry provides information regarding medical care for a patient in the real world. CDWs have the potential to provide information as current as the latest upload, be focused to specific subpopulations and reflect current clinical practice. In this paper, I study the potential of a well-known patient clinical data warehouse to provide information regarding pediatric levothyroxine dosing as a form of clinical decision support. I study the state of the stored data, the necessary data transformations and options for representing the data to effectively summarize and communicate the findings. / (cont.) I also compare the resulting transformed data, representing actual practice within this population, against established dosing recommendations. Of the transformed records, 728 of the 854 (85.2%, [95% confidence interval 82.7:87.6]) medication records contained doses that were under the published recommended range for levothyroxine. As demonstrated by these results, real world practice can diverge from established recommendations. Delivering this information on real-world peer practice medication dosing to clinicians in real-time offers the potential to provide a valuable supplement to established dosing guidelines, enhancing the general and sometimes static dosing recommendations. / by Elisabeth Lee Scheufele. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Vita. / Includes bibliographical references (leaves 149-150). / The overall goal of this dissertation was to study the auditory component of feedback control in speech production. The first study investigated auditory sensorimotor adaptation (SA) as it relates to speech production: the process by which speakers alter their speech production in order to compensate for perturbations of normal auditory feedback. Specifically, the first formant frequency (F1) was shifted in the auditory feedback heard by naive adult subjects as they produced vowels in single syllable words. These results indicated that subjects demonstrate compensatory formant shifts in their speech. This compensation was maintained when auditory feedback was masked by noise. The second study investigated perceptual discrimination of vowel stimuli differing in F frequency, using the same subjects as in the SA studies. This study showed that the extent of adaptation was positively correlated with subject auditory acuity. The last study consisted of a series of simulations of SA experiments using a model which describes the motor planning and control of human speech by the brain; these simulations showed that the model can account for several properties of adaptation as measured from the human subjects. / (cont.) The findings in this dissertation support the idea that phonemic speech movements are planned as goal regions in an auditory space, and that mappings between this auditory space and the speech motor plan are adaptable. Moreover, the size of these goal regions-as reflected in speaker auditory acuity-influences the degree to which speakers adapt to errors in auditory feedback. / by Virgilio Mangubat Villacorta. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 87-99). / Synaptic ribbons are electron-dense structures surrounded by vesicles and anchored to the presynaptic membrane of photoreceptors, retinal bipolar cells and hair cells. Ribbon synapses are characterized by sustained exocytosis that is graded with stimulus intensity and can achieve high release rates. Leading hypotheses implicate the ribbon in maintenance of a large readily releasable pool (RRP) of presynaptic vesicles which enables rapid and precisely-timed exocytosis that supports instantaneous discharge rates of well over 1000 spikes per second. To gain insight into the function of this specialized presynaptic molecular machinery, we characterized the response properties of single auditory nerve (AN) fibers in a mouse with targeted deletion of a presynaptic scaffolding gene, bassoon, in which ribbons are no longer anchored to the active zone. Since each mammalian AN fiber usually receives input from a single inner hair cell active zone to which a single ribbon is typically anchored, single-fiber recordings from bassoon mutants and control mice offer a sensitive functional metric of the contribution of individual ribbons to neural function. Response properties of mutant AN fibers were similar, in many respects, to wild-type. Spike intervals remained irregular, thresholds were unaffected, dynamic range was unchanged, spike synchronization to / (cont.) stimulus phase was unimpaired, the time course of post-onset adaptation and recovery from adaptation were normal, and the ability to sustain discharge throughout a long-duration stimulus was unaffected. These data indicate that the presynaptic mechanisms which regulate precise timing of exocytosis, graded release rates and sustained neurotransmitter release were not impaired by loss of the ribbon. However, reductions were seen in spontaneous and sound-evoked AN fiber discharge rates, coinciding with an increased variance of first spike timing to stimulus onset. Unlike fibers from wild-type mice, mutants failed to show increased peak rate as stimulus onset became more abrupt. The reduction of peak rates and increased first spike variance likely result from degraded reliability of discharge to stimulus onset via a mechanism such as reduced RRP size. Thus, the ribbon appears to support a large RRP that enables the rapid onset rates necessary for the auditory system to resolve stimulus features key for many perceptual tasks. / by Bradley N. Buran. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 192-204). / Recovery from central nervous system (CNS) injuries is hindered by a lack of spontaneous regeneration. In injuries such as stroke and traumatic brain injury, loss of viable tissue can lead to cavitation as necrotic debris is cleared. Using a rat model of penetrating brain injury, this thesis investigated the use of collagen biomaterials to fill a cavitary brain defect and deliver therapeutic agents. Characterization of the untreated injury revealed lesion volume expansion of 29% between weeks 1 and 5 post-injury. The cavity occupied parts of the striatum and cortex in the left hemisphere, and was surrounded by glial scarring. Implantation of a collagen scaffold one week after injury resulted in a modest cellular infiltrate four weeks later consisting of macrophages, astrocytes, and endothelial cells. The scaffold was able to fill the cavity and provide a substrate for cellular migration into the defect. Incorporation of a Nogo receptor molecule aimed at binding inhibitory myelin proteins did not appear to promote axonal regeneration, but resulted in increased infiltration of macrophages and endothelial cells. The increased vascularization observed within the scaffolds represents a modified environment that might be more suitable for regenerative therapies. A scaffold was also used to investigate the delivery of neural progenitor cells one week after injury. After four weeks, viable implanted cells were found to have differentiated into astrocytes, oligodendrocytes, endothelial cells, neurons, and possibly macrophages/microglia. These results demonstrate the potential utility of combinatorial therapies involving collagen biomaterials, myelin protein antagonists, and neural progenitors for treatment of CNS injuries. / by Paul Ziad Elias. / Ph.D.

Thesis (S.M.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references (leaves 33-35). / Location tracking systems in healthcare produce a wealth of data applicable across many aspects of care and management. However, since dedicated location tracking systems, such as the oft mentioned RFID tracking system, are still sparsely deployed, a number of other data sources may be utilized to serve as a proxy for physical location, such as barcodes and manual timestamp entry, and may be better suited to indicate progress through clinical workflows. INCOMING!, a web-based platform that monitors and tracks patient progress from the operating room to the post-anesthesia care unit (PACU), is one such system that utilizes manual timestamps routinely entered as standard process of care in the operating room in order to track a patient's progress through the post-operative period. This integrated real time system facilitates patient flow between the PACU and the surgical ward and eases PACU workload by reducing the effort of discharging patients. / (cont.) We have also developed a larger-scale integrated system for perioperative processes that integrates perioperative data from anesthesia and surgical devices and operating room (OR) / hospital information systems, and projects the real-time integrated data as a single, unified, easy to visualize display. The need to optimize perioperative throughput creates a demand for integration of the datastreams and for timely data presentation. The system provides improved context-sensitive information display, improved real-time monitoring of physiological data, real-time access to readiness information, and improved workflow management. These systems provide improved data access and utilization, providing context-aware applications in healthcare that are aware of a user's location, environment, needs, and goals. / by Mark A. Meyer. / S.M.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2006. / Includes bibliographical references. / Endothelial cells and basement membrane interact as a biochemical and mechanical co-regulatory unit. The wide spectrum of manifestations of diabetic vascular disease could be related to altered kinetics of vasoactive compounds within this regulatory unit. We hypothesized that hyperglycemic stress mediates storage, release, and function of fibroblast growth factor-2 (FGF-2) through changes in interaction between endothelial cells and basement membrane. We discovered that basement membrane associated FGF-2 increased linearly with culture glucose concentration. Using novel assays, we demonstrated that FGF-2 binding kinetics were surprisingly unchanged over a range of basement membrane culture glucose. Instead, the combination of increased endothelial cell apoptosis-associated FGF-2 release and enhanced endothelial cell permeability allowed more FGF-2 to bind into the basement membrane. Such high levels of basement membrane FGF-2 abrogated the effects of hyperglycemia on proliferation but not apoptosis. An FGF-2 stimulus returned endothelial cell proliferation close to euglycemic levels, but increased apoptosis was still evident as FGF-2 signaling down an intracellular survival pathway was inhibited by glucose. / (cont.) These same findings were confirmed in vivo where FGF-2 levels were elevated in the aortic subendothelial space of diabetic animals. This thesis suggests a new paradigm for active cellular control of basement membrane and indicates the complexities of growth factor signaling in endothelial cells. Characterization of the interaction between endothelial cells and basement membrane in health and disease may advance our understanding of diabetic vascular disease and lead to development of novel biomimetic materials for therapeutic intervention. / by Alisa Sharon Morss. / Ph.D.

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2009. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 137-154). / Because there is little knowledge in the areas of stereocilia development, maintenance, and function in the hearing system, I decided to pursue a proteomics-based approach to discover proteins that play a role in stereocilia function. I employed a modified "twist-off" technique to isolate hair bundle proteins, and I developed a method to purify proteins and to process them for analysis using multi-dimensional protein identification technology (MudPIT). The MudPIT analysis yielded a substantial list of proteins. I verified the presence of 21 out of 34 (62%) existing proteins known to be present in stereocilia. This provided strong evidence that my proteomics approach was efficient in identifying hair bundle proteins. Next, I selected three proteins and localized them to murine cochlear stereocilia. StarD10, a putative phospholipid binding protein, was detectable along the shaft of stereocilia. Nebulin, a putative F-actin regulator, was located toward the base of stereocilia. Finally, twinfilin 2, a putative modulator of actin polymerization, was found at the tips of stereocilia. In order to determine the function of twinfilin 2, I localized the protein predominately to the tips of shorter stereocilia where it is up-regulated during the final phase of elongation. When overexpressed, I found that twinfilin 2 causes a shortening of microvilli in LLC-PK1/CL4 cells and in native cochlear stereocilia. The main result of this thesis was determining the sub-cellular localization of three interesting proteins and functionally characterizing one protein. My thesis also confirmed the proteomics screen I developed as an efficient method for identifying proteins in stereocilia. / by Anthony Wei Peng. / Ph.D.