<p>An objective measure of muscular low back pain (LBP) symptoms eludes clinicians. This study assessed efficacy of magnetic resonance imaging (MRI) of the lumbar multifidus using diffusion tensor imaging (DTI), blood oxygen level dependent (BOLD) signal fractal dimension (FD) analysis and muscle cross sectional area (CSA) in LBP assessment. MRI results were compared to two questionnaires, the Oswestry disability index (ODI) and visual analog score (VAS).</p> <p>Right-left asymmetry in both DTI metrics and T2-weighted (T2W) CSA were greater in the injured. Also, asymmetry measures were correlated with body mass index (BMI) but not age, height, or level of physical activity (measured via Godin activity questionnaire). The relationship between asymmetry and LBP symptoms in T2W and DTI scans increased for subjects with BMI below 35kg/m².</p> <p>BOLD FD did not scale with LBP symptoms. However, FD analysis showed promise following therapeutic Swedish massage, hypothesized as being related to local perfusion changes, indicating that FD is sensitive to changes in the lumbar muscle, just not LBP symptoms. Thus the BOLD FD does change with treatment, just not with the symptoms of LBP.</p> <p>When combining data from multiple scan types, the symptoms of LBP correlated best with the unweighted mean of DTI fractional anisotropy (FA) and T2W CSA asymmetry, and the correlation was greatest (R²=0.88) when only <em>symptomatic (not both symptomatic and control)</em> subjects with BMIs from 18-25kg/m² were considered. From these results there appears to be clinical utility in characterizing the symptoms of non-acute LBP using DTI and CSA.</p> / Doctor of Philosophy (PhD)

There is a large body of evidence supporting the theory that cell physiology largely depends on the mechanical properties of its surroundings or micro-environment. More recently studies have shown that changes to intra-cellular mechanical properties can also have a meaningful impact on cell function and in some cases lead to the progression of ailments or disease. For example, small changes to the protein sequence of a structural nuclear envelope protein called lamin-A is known to cause a variety of neurological and musculoskeletal diseases referred to as laminopathies. Currently, there is little incite into how these mutations lead to disease progression due in part to an inability to measure protein-specific mechanical changes and how these alterations may relate to disruptions in intra-cellular signaling or function. \par To improve upon the ability to measure mechanical properties inside living cells, a previously validated, genetically-encoded resonant energy transfer (FRET)-force biosensor was modified to localize to the nuclear envelope. This biosensor integrated into the nuclear envelope protein Nesprin-2G and senses small deformations that are resolved by indirect measurements of spectroscopic fluctuations in the fluorescent emission of the sensor. To accurately measure these changes, a new spectral-imaging technique named SensorFRET was developed which can resolve small changes in the FRET sensor under varying levels of fluorescent intensity and with known absolute precision. Using SensorFRET, the Nesprin-2G biosensor (Nesprin-TS) reported changes in actomyosin contractility, nuclear shape, and nuclear deformation. Using Nesprin-TS, fibroblasts derived from patients with Hutchinson-Gilford progeria syndrome (HGPS) reported less force on Nesprin-2G molecules relative to healthy fibroblasts on average.\par To demonstrate how intra-cellular forces on the nucleus may impact normal cell physiology, bone-marrow derived mesenchymal stem cells (MSCs) were genetically modified such that the cytoskeleton was decoupled from the nucleus by saturating KASH binding proteins with a non-functional truncated protein called DN-KASH. MSCs treated with DN-KASH preferentially differentiated into osteocytes (bone cells) at a higher rate than MSCs exposed to osteogenic growth factors. This osteogenic preference after DN-KASH treatment was independent of the cell substrate topology and did not significantly alter integrin expression. However, this tendency to differentiate into osteocytes was dependent on substrate stiffness. Overall, the data imply that an intra-cellular force-dependent mechanism connected to the cell nucleus strongly influences MSC differentiation.

Positron Emission Tomography (PET) is frequently used for medical imaging. Maturity and flexibility of PET as an imaging technique has expanded its utility beyond the medical domain. It can be used as a tool for fluid flow studies in opaque fluids and for flow within complex geometry where conventional optical flow measurement approaches fail. This study explores the capabilities of PET as flow measurement tool suited to validation of computational fluid dynamic (CFD) predictions. The MicroPET P4 scanner was used to image the diffusion process in flow around a rod bundle geometry similar to that found in a nuclear reactor fuel assembly. The PET data are compared with results from COMSOL CFD simulation and dye injection images. PET image resolution, acquisition speed and sensitivity are also examined in the context of flow measurement.

This study combines noninvasive hybrid diffuse optical spectroscopies [near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS)] with occlusive calibration for continuous measurement of absolute blood flow (BF), tissue blood oxygenation (StO2), and oxygen consumption rate (VO2) in exercising skeletal muscle. Subjects performed rhythmic dynamic handgrip exercise, while an optical probe connected to a hybrid NIRS/DCS flow-oximeter directly monitored oxy-, deoxy-, and total hemoglobin concentrations ([HbO2], [Hb], and [tHb]), StO2, relative BF (rBF), and relative VO2 (rVO2) in the forearm flexor muscles. Absolute baseline BF and VO2 were obtained through venous and arterial occlusions, respectively, and used to calibrate continuous relative parameters. Previously known problems with muscle fiber motion artifact in optical measurements were mitigated with a novel dynamometer-based gating algorithm. Nine healthy young subjects were measured and results validated against previous literature findings. Ten older subjects with fibromyalgia and thirteen age-matched healthy controls were then successfully measured to observe differences in hemodynamic and metabolic response to exercise. This study demonstrates a novel application of NIRS/DCS technology to simultaneously evaluate quantitative hemodynamic and metabolic parameters in exercising skeletal muscle. This method has broad application to research and clinical assessment of disease (e.g. peripheral vascular disease, fibromyalgia), treatment evaluation, and sports medicine.

This research investigates various applications of diffuse correlation spectroscopy (DCS) on in-vivo human muscle tissue, both at rest and during dynamic exercise. Previously suspected muscle tissue relative blood flow (rBF) baseline shift during extended measurement with DCS and DCS-Near infrared spectroscopy (NIRS) hybrid optical systems are verified, quantified, and resolved by redesign of optical probe and alteration in optical probe attachment methodology during 40 minute supine bed rest baseline measurements. We then translate previously developed occlusion techniques, whereby rBF and relative oxygen consumption rV̇O2 are calibrated to initial resting absolute values by use of a venous occlusion (VO) and arterial occlusion (AO) protocol, respectively, to the lower leg (gastrocnemius) and these blood flows are cross validated at rest by strain gauge venous plethysmography (SGVP). Methods used to continuously observe 0.5Hz, 30% maximum voluntary isometric contraction (MVIC) plantar flexion exercise via dynamometer are adapted for our hybrid DCS-Imagent diffuse optical flow-oximeter in the medial gastrocnemius. We obtain healthy control muscle tissue hemodynamic profiles for key parameters BF, V̇O2, oxygen saturation (StO2), deoxyhemoglobin, oxyhemoglobin, and total hemoglobin concentrations ([Hb], [HbO2], and THC respectively), as well as systemic mean arterial pressure (MAP) and pulse rate (PR), at rest, during VO/AO, during dynamic exercise and during 15 minute recovery periods. Next, we began investigation of muscle tissue hemodynamic disease states by performing a feasibility pilot study using limited numbers of controls and peripheral arterial disease (PAD) patients using the translated methods/techniques to determine the ability of our technology to assess differences in these populations.

Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture. The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.

Skin cancer is a prevalent disease that globally affects 2-3 million people per year [1]. This number is expected to grow tenfold as depletion of the ozone layer contributes to harsher rays reaching Earth’s surface [2]. A common way to protect against those ultraviolet waves is to apply sunscreen, however, recent reports call into question the safety of some active ingredients as they can enter through the skin into the bloodstream [3]. This thesis aims to investigate an alternative solution that uses bovine sphingomyelin (BSM) as photoprotective solution against UV irradiation. In order to evaluate the effectiveness of BSM against UV radiation, p21 intensity was measured on a monolayer of keratinocytes, as the intensity directly correlates to cell damage. Additionally, fluorescent sphingomyelin (FSM) was added as a treatment because it was created to be an analog to BSM and allowed for visualization of sphingomyelin within the cell. Differences in p21 intensities were observed with BSM and FSM showing a reduced p21 intensity compared to the no sphingomyelin case. FSM helped locate sphingomyelin within the cell and a mechanism was proposed for how it reduces cell damage. Lastly, high variation was seen between experimental designs. Further measures were needed to reduce this intra-subject standard deviation, so additional experimental parameters were tested such as min/max intensity values, cell count, and nucleus circularity to explain this variation.

A greater understanding of bone materials would be beneficial in creating more accurate computer models and in the making of biomedical products involving bone. This study set out to determine whether cortical and trabecular bone are two separate materials, or whether they are the same material with a variance in porosity. To answer this question, samples were taken from different sections of the equine metacarpus, underwent densitometry analysis and were statistically analyzed. The majority of results suggest that the material is the same between varying densities of bone and thus the same between cortical and trabecular bone. These particular results are consistent with current standard practices. However, in several instances specifically regarding high porosity trabecular bone, a variance in density was found likely indicating a combination of differences in both material and architecture. Further studies should be done with specific focus on material variances to high porosity trabecular bone to improve the accuracy of computer models and general knowledge.

The ultraviolet (UV) spectrum has been known to cause damage to skin in varying degrees. UVB radiation (290-320 nm) in particular, has been proven to be highly mutagenic and carcinogenic in many animal experiments compared to either UVA or UVC. The alarming rate of increase in skin cancer incidence has prompted many investigators to pursue other alternatives to sunscreens including changes in lifestyle habits and dietary consumption in order to boost our efforts in tackling this widespread disease. Previous studies employing confocal reflectance, MTT assay and histology suggest that milk phospholipids may possess protective properties against UVB-mediated damage but the molecular mechanism for this effect remains unclear. This study aims to evaluate changes in cell morphology, apoptosis and p21 expression in tissue engineered epidermis to increase our understanding of the mechanisms behind the potential protective effects of milk phospholipids against UV-induced photodamage. Human skin tissue equivalents were incubated in either 1% milk phospholipid solution or maintenance media then exposed to 120 mJ/cm2 dose of 300 nm UVB after 24 hours. The upregulation of p21 protein in response to DNA damage was measured with Western blot and immunofluorescence microscopy and markers for apoptosis and hyperplasia were examined 24 hours after irradiation. Results revealed that p21 levels and the amount of apoptotic markers such as fragmented DNA and nuclear condensation were significantly reduced in UV-exposed tissues pre-incubated with milk phospholipids compared to levels seen in both the positive control and UV-exposed skin tissue not incubated with milk phospholipids. This decrease in p21 expression may imply a reduction in DNA damage 24 hours after UV exposure or a decrease in acquired photodamage at the outset. Milk phospholipid incubation however, induced an increase in epidermal thickening with or without UV exposure, which may imply induction of a protective mechanism to enhance the barrier properties of skin.

It was found that the measurements captured by confocal microscopy and scanning electron microscopy had a statistically significant difference for bovine tissue. There was not a statistically significant for porcine and poultry tissue. The intent of the study is to perform a comparative study to examine efficacy of two distinct technologies for a singular purpose: tissue surface roughness characterization. The two technologies compared are a confocal reflectance microscope and a scanning electron microscope. The comparison was made by comparing two surface roughness parameters [Ra and Rq] within ImageJ. The study examined three different animal species [porcine, bovine, and poultry] to highlight if different tissues presented alternative conclusions for the efficiency of either technology. Additional analysis was produced comparing two cutting methods [Kleen Kut versus conventional], as well as six different poultry processing technique combinations.