A methodology has been developed for automatically determining inter-image correspondences between cliques of features extracted from a reference and a query image. Cliques consist of up to three features and correspondences between them are determined via a hierarchy of similarity metrics based on the inherent properties of the features and geometric relationships between those features. As opposed to approaches that determine correspondences solely by voxel intensity, features that also include shape description are used. Specifically, medial-based features are employed because they are sparse compared to the number of image voxels and can be automatically extracted from the image. The correspondence framework has been extended to automatically estimate the location of anatomical landmarks in the query image by adding landmarks to the cliques. Anatomical landmark locations are then inferred from the reference image by maximizing landmark correspondences. The ability to infer landmark locations has provided a means to validate the correspondence framework in the presence of structural variation between images. Moreover, automated landmark estimation imparts the user with anatomical information and can hypothetically be used to initialize and constrain the search space of segmentation and registration methods. Methods developed in this dissertation were applied to simulated MRI brain images, synthetic images, and images constructed from several variations of a parametric model. Results indicate that the methods are invariant to global translation and rotation and can operate in the presence of structure variation between images. The automated landmark placement method was shown to be accurate as compared to ground-truth that was established both parametrically and manually. It is envisioned that these automated methods could prove useful for alleviating time-consuming and tedious tasks in applications that currently require manual input, and eliminate intra-user subjectivity.

This thesis presents sodium magnetic resonance as an in vivo means for non-invasively visualizing the changes in cell sodium ion homeostasis that occur in ischemic tissue during acute stroke. Single quantum sodium magnetic resonance imaging (MRI) was used to determine the time course of tissue sodium concentration (TSC) in a non-human primate model of reversible focal brain ischemia. In each animal, TSC increased slowly and linearly as a function of time after the onset of focal brain ischemia. Changes in the TSC accumulation were seen upon reperfusion. The results demonstrate that the increase in TSC in ischemic tissue is readily measurable using sodium MRI at clinical magnetic field strengths (3.0 T) in acceptable imaging times (5 minutes). The results also indicate that sodium MRI could predict the stroke onset time in patients that are unsure when their symptoms began, potentially extending the use of thrombolytic therapy to patients that would otherwise not receive treatment. Many studies have hypothesized that the best means for the in vivo study of the changes in cell sodium ion homeostasis that occur during brain ischemia is to use imaging schemes that isolate the sodium NMR signal from the intracellular compartment. This thesis investigates the contribution of the extracellular sodium pool to the brains triple quantum (TQ) sodium MR signal in the rat using the thulium shift reagent, TmDOTP5-. Within the SNR of the experiment, there was no evidence of any contribution to the TQ sodium MR signal from the sodium in the extracellular brain, vascular, and muscle spaces in the head. Finally, TQ sodium MR images in the in vivo non-human primate are presented for the first time. Moreover, these images were obtained in clinically acceptable 18 minute data acquisition times. TQ sodium MRI during non-human primate focal brain ischemia identified large changes in the ischemic region as early as 34 minutes after the onset of ischemia. The increase in the TQ sodium MRI signal intensity observed in the ischemic hemisphere is hypothesized to be due to an increase in the intracellular sodium concentration as a result of impaired ion homeostasis during evolving brain ischemia.

This thesis describes analysis methods and results from slip-perturbed gait experiments. The risk for falls was related both to the conditions present at heel strike and to the nature of the response. Gait analysis was performed using the Human Movement and Balance Laboratory (HMBL) model, a fifteen segment, fourteen joint model of the human body that was developed as part of this thesis effort. Resulting kinematics and kinetics included three-dimensional angles describing relative segment rotations, segmental and whole-body centers-of-mass, and joint actuation torques for the entire body. The relationship between pre-slip gait characteristics and the magnitude of slips was explored for both younger and older adults. Slip severity, either hazardous or non-hazardous, was determined using a 1.0 m/s peak slip velocity threshold. Hazardous slips were associated with greater step lengths normalized by leg length, larger and more rapidly changing foot-floor angles at heel strike, and increased cadence across the two subject groups. These results suggest that gait characteristics play an important role in the severity of slips. Older adults were found to walk with shorter step lengths and with smaller and more slowly changing foot-floor angles at heel strike compared to younger subjects, suggesting that age effects also impact slip severity. The effects of slipping and trailing leg response on slip outcome (falls or recoveries) were explored. Slip severity was found to be the most significant parameter related to outcome. Response strategies were classified, based on trailing leg dynamics, as either minimal, foot-flat, mid-flight, or toe-down. Slipping and trailing leg hip and knee torques were determined using the HMBL model and timing and magnitude parameters from these torques were then identified. Relationships between these parameters, age group (younger/older), response strategy, and outcome were then explored. Age was not found to be significantly related to response strategy or outcome, nor was response strategy found to be related to outcome. Slipping leg knee torque timing and magnitude parameters were related to slip severity and to outcome for hazardous slips. These results suggest that slip responses, coupled with slip severity, determine fall or recovery outcomes.

Current strategies designed to assess cells in the peripheral blood are limited to evaluation of phenotype or delayed measurement [>6 hours] of function, usually quantifying cytokine production, cytolytic activity, or response to antigens. Furthermore, these assays often do not assess the individual cell types comprising peripheral blood, instead providing a bulk-readout for the total population. We reasoned that functional abnormalities in distinct peripheral blood cells, which act as immune sentinels capable of rapidly reacting to various injurious agents while circulating throughout the body, could serve as migratory biomarkers, reflecting pathological environs that cells experience in the setting of inflammatory states, including cancer. Two major pathways regulating immune responses are the JAK/STAT and MAPK pathways. These pathways are initiated by ligand-receptor binding, and are rapidly propagated by subsequent protein phosphorylation cascades. Additionally, these pathways are often abnormally activated within cancer cells themselves. We applied flow cytometric strategies, evaluating the brief application of cytokines in vitro to interrogate the early phosphorylation events of these signaling pathways in normal peripheral blood mononuclear cells (PBMC). Individual cytokine doses and time intervals of treatment were assessed to identify conditions useful in a clinical laboratory and as an initial goal, to induce maximal phosphorylation. Surprisingly, all of the STAT proteins assessed and ERK1/2 are maximally phosphorylated within 15 minutes in human PBMC, simply following addition of cytokines alone. Within two hours, cells typically return to their basal phosphorylation states. Confirmation of these results was achieved by Western blotting. Increased phosphorylation usually correlated with increased concentrations of individual cytokines. Furthermore, we developed conditions to enable simultaneous staining of cell surface proteins, identifiable with distinct PBMC subpopulations: CD4, CD8, CD14, CD19, and CD56, together with intracellular phosphorylated proteins. Of the permeabilizing conditions tested, 75% methanol enabled superior simultaneous detection of both cell surface and intracellular epitopes. Using this technique, we were able to identify differential responsiveness between individual cell types. These strategies will enable robust development of simple blood analyses to identify normal activation as well as impairments in STAT and MAPK signaling pathways associated with various human disease states including acute and chronic inflammatory conditions throughout clinical immunology.

Breast cancer, one of the most common types of cancer among women, is now the second leading cause of cancer deaths after lung cancer. Since a majority of cancer deaths are due to metastasis of breast tumor cells to distant organs, understanding tumor cell invasion and metastasis at a molecular level will help us in developing therapeutics that will improve the quality of life of breast cancer patients. Cell migration, an integral component of tumor invasion and metastasis, is regulated by the assembly and disassembly of actin cytoskeleton, which involves the coordinated action of several classes of actin binding proteins. It has been shown previously that there is reduced expression of profilin 1 (Pfn1- an ubiquitously expressed actin binding protein) in invasive breast cancer cells. Pfn1 is now considered as a tumor-suppressor protein based on its ability to restrict the growth and tumorigenesis of breast cancer cells when overexpressed. Besides actin, Pfn1 also binds to several families of proline-rich ligands and these interactions have been implicated in several cellular processes including actin assembly, endocytosis and gene transcription. We have previously shown that overexpression of Pfn1 reduces the migration of BT474, a ductal carcinoma breast cancer cell line. The aim of the present work is to determine whether overexpression or selective inhibition of ligand binding of Pfn1 alter the migration and invasion of metastatic breast cancer cells. Specifically, we have studied how stable overexpression of Pfn1 and its mutant forms that are selectively impaired in binding to either actin or proline-rich ligands affect the migration and invasion of MDA-MB-231, a highly metastatic breast cancer cell line. We show that functional perturbation of Pfn1 affects the F-actin content in MDA-MB-231 cells. Specifically, Pfn1 overexpression stimulates actin polymerization, whereas expression of an actin-binding deficient mutant of Pfn1 decreases the overall level of polymerized actin in MDA-MB-231 cells. Increased focal adhesion formation in MDA-MB-231 cells as a result of Pfn1 overexpression appears to require a functional actin-binding site of Pfn1. We show that cell migration and invasion in response to chemotactic stimulus are inhibited when either fully functional or mutant forms of Pfn1 are expressed in these cells. Finally, we demonstrate that perturbation of Pfn1 affects the secretion of matrix-metalloproteinases (enzymes that are important for matrix degradation during cell invasion) by MDA-MB-231 cells.

Therapy for patients suffering from acute respiratory distress syndrome (ARDS) is substantially inadequate, resulting in a 40% mortality rate. A biohybrid lung prototype consisting of a rotating endothelialized microporous hollow fiber (MHF) bundle was studied as an alternative solution for improved patient outcome. It is hypothesized that endothelialized MHFs could present a surface mimicking the native vascular lining to reduce thrombotic deposition on underlying MHF. Such an approach might thus allow blood oxygenation and CO2 removal for extended periods with reduced anticoagulation requirements. Development of the biohybrid lung prototype, evaluation of endothelial cell (EC) response to shear stress, influence of endothelialization on gas transfer, and impact of bundle rotational speed on gas transfer and alterations in EC phenotype were studied. MHFs were surface modified to promote EC attachment and proliferation. Endothelialized MHF bundles were rotated in the biohybrid lung prototype up to 1500 RPM (26.4 dynes/cm2). Blood-surface biocompatibility testing was performed on MHFs and MHF bundles in the biohybrid lung prototype, with or without ECs. Partial O2 pressures were recorded for blood samples to measure oxygen buildup within the biohybrid lung. Scanning electron micrographs (SEMs) of thrombotic deposition were taken. Upregulation of e-selectin and p-selectin on ECs were assessed for indication of an inflammatory EC phenotype. ECs maintained near confluent coverage on MHFs under rotation at the tested speeds, and showed minimal p-selectin expression subsequent to rotation. It was observed that even low to moderate levels of EC coverage greatly reduced thrombotic deposition on MHFs. Statistically significant differences in oxygen accumulation between MHF bundles with or without endothelialization in the presence of 95% O2 were not found. Thrombotic deposition on endothelialized MHF bundles was less than or equivalent to thrombotic deposition on non-endothelialized MHF bundles following rotation. Low levels of e-selectin and p-selectin expression were observed following 24 hr hyperoxia. These results suggest that endothelialized MHFs may serve to improve blood-surface biocompatibility in the presence of hyperoxia. Although further development and testing is required, a biohybrid lung employing endothelialized MHFs and a rotating fiber bundle may provide an alternative therapy for patients suffering from ARDS.

The detrimental results of whole-body vibration (WBV) and their effect on humans in the seated position have been documented. Although wheelchair users are subjected to WBV little research has been conducted to assess these vibrations or attempt to reduce them. Sixteen able bodied subjects tested two powered wheelchairs: a Quickie S-626 and an Invacare 3G Torque SP. Each subject tested all of the configurations of the suspension wheelchairs and solid inserts to represent a non-suspension wheelchair. In each of the configurations of the wheelchairs, the subjects traversed an Activities of Daily Living (ADL) course. Vibrations were collected from a tri-axial accelerometer mounted to a ¼ inch aluminum seat plate during driving over the activities course. Root Mean Square Values and Vibration Dose Values were analyzed Statistical analyses of the RMS and VDV data revealed significant differences between the six different suspensions over each of the obstacles in the activities of daily living course. Post-hoc analyses revealed that for each of the obstacles, significant differences existed between the Invacare suspension and the Invacare solid insert. For the Quickie power wheelchair the solid insert setting was not significantly different from the most-stiff setting for each of the obstacles except the smooth surface. The solid insert setting was significantly different than the lowest and mid stiffness settings for all of the obstacles except the smooth surface and the deck surface. Without significant periods of rest the effects of WBV are cumulative throughout the course of the day, and the longer the exposure time the lower the threshold of non-harmful vibrations. Although most of the suspension systems are capable of reducing the amounts of vibration transmitted to the users the results of the vibration dose values seem to indicate that they may not reduce them enough to reduce probability of injury in powered wheelchair users. Future work should move towards examining these vibrations to evaluate what actual levels of WBV wheelchair users are experiencing over the course of an entire day and examining relationships between suspension and user weight to make data available to clinicians and wheelchair companies for suspension tuning.

The glenohumeral joint is the most dislocated major joint in the body; however despite such a high rate of injury, the proper treatment protocol remains unclear. Rehabilitation has proved to be insufficient with an 80% chance of redislocation in teenagers and 10-15% chance after the age of 40. Following surgical repair, nearly 25% of patients still experience redislocation and complain of joint stiffness and osteoarthritis. In an attempt to improve these results, the normal function of the glenohumeral capsule has been evaluated using both experimental and computational methods. Recent data (strain and force patterns) suggests that the capsule functions multiaxially. Therefore, simple uniaxial methods may not be sufficient to fully characterize the tissue and identify the appropriate constitutive model of the tissue. Inconclusive data has been presented in the literature regarding the collagen fiber architecture and the mechanical properties of the capsule that make it unclear whether the capsule is an isotropic or a transversely isotropic material. For instance, the collagen fiber architecture has been shown by one researcher to be randomly distributed, while another researcher reported the fibers to be aligned. In addition, the axillary pouch has been shown to be the primary stabilizer of the glenohumeral joint in positions of extreme external rotation while the posterior region of the capsule has been shown to stabilize the joint in positions of extreme internal rotation. The rate of dislocations, however, is more frequent in the position of external rotation. Therefore, the overall objective of this work was to utilize a combined experimental - computational methodology to characterize the mechanical properties of the axillary pouch and posterior region of the glenohumeral capsule. Using an isotropic constitutive model, the stress-stretch relationship of the axillary pouch and posterior regions in response to two perpendicular tensile and finite simple shear elongations showed no statistical difference. Further, the constitutive coefficients of pure tensile and simple finite shear elongations in the direction parallel to the longitudinal axis of the anterior band of the inferior glenohumeral ligament (longitudinal) were able to predict the response of the same tissue sample in the direction perpendicular to the longitudinal axis of the anterior band (transverse). These similarities between the longitudinal and transverse elongations of the tissue imply that the capsule is an isotropic material and functions to resist dislocation the same in all directions, rather than just along the longitudinal axis of the anterior band of the inferior glenohumeral ligament, as previously thought. Further, the coefficients of the axillary pouch and posterior regions of the capsule showed no statistical difference, suggesting that these regions have similar mechanical properties, despite a difference in geometry. Thus, when developing finite element models of the glenohumeral capsule, an isotropic constitutive model should be utilized; and both the axillary pouch and posterior regions could be evaluated using the same coefficients. However, due to discrepancies when comparing the constitutive coefficients of tensile and shear elongations, an update to the constitutive model is required. With the proper representation of the glenohumeral capsule known, finite element models can be developed to pursue the understanding of normal joint function, including the effects of age and gender, as well as injured and surgically repaired joints.

Myoblast transplantation has been investigated as a treatment for Duchenne Muscular Dystrophy and injured myocardial tissue. Multiple groups have isolated an early myogenic precursor population, termed muscle-derived stem cells (MDSCs) that have a superior ability to regenerate dystrophin-positive myofibers and improve cardiac function following an ischemic event as compared to more differentiated myoblasts. The initial local environment of these transplantations involves a high degree of inflammation and its associated major component, oxidative stress. Here, we report that a resistance to these stresses is an important factor determining the regenerative capacity of muscle stem cells in skeletal and cardiac muscle. MDSCs have an increased antioxidant capacity that protects them from intracellular oxidative damage while myoblasts have lower levels of survival and delayed differentiation following exposure to oxidative stress. These experiments were conducted using a series of high throughput assays that combine robotic live-cell microscopy with custom image analysis software. Further, when antioxidant levels in MDSCs are lowered to values comparable to myoblasts, the regenerative capacity of MDSCs decreases to levels comparable to myoblasts in both skeletal muscle and cardiac cell therapies. These findings indicate the important role inflammation plays in cell therapies, and identifies an important new mechanism by which stem cells display a unique regenerative capacity and also a new phenotype in selecting cell populations with enhanced regeneration capacity.

Slip-initiated falls often cause injury and death, especially in older adults. Previous research involving perturbed walking has to a large extent focused on lower extremity reactions, yet arm responses are often part of postural reactions to such perturbations. This research is focused on arm responses to an unexpected slip. In Aim 1, the relationship between slip severity and shoulder biomechanics was examined. In Aim 2, we determined if the vestibular system is involved in the triggering of arm responses. The correlation between shoulder moment magnitude and the extent of the body center of mass (COM) perturbation was examined in Aim 3. Subjects (17 younger and 12 older adults) were exposed to two conditions: (1) baseline dry (subjects knew the floor was dry), and (2) unexpected slip (a diluted glycerol solution was spread on the floor beneath the stance/left foot). Shoulder Euler angles and moments in flexion/extension and abduction/adduction were derived. The spherical elevation angle was used to further describe shoulder kinematics. Slip severity was quantified using measures reported in the literature. Although arm responses were bilateral, only left (side of slipping foot) shoulder biomechanics, specifically moment generation rates, spherical elevation angle, and abduction angle were positively correlated with slip severity. Left shoulder responses were triggered later than left hip and knee responses. Delayed shoulder moment onsets, slower abduction moment generation rate, and reduced range of motion were found in older adults compared to their younger counterparts. Aim 2 results indicated a weak but statistically significant positive relationship between the timing of the slip-initiated downward head acceleration and the onset of the left shoulder flexion/extension moment (true only when slip severity was controlled in the analysis). In Aim 3, increased left shoulder flexion generation rate correlated with decreased COM perturbation. In conclusion, evidence presented in this study implies (1) arm responses play a role in balance recovery, (2) a legs-to-arms response sequence appears to drive the reaction to a slip, although the potential implication of the vestibular system cannot be ruled out, (3) age-related effects on arm responses may aggravate the risk of slips and falls in older adults.