<p>Microscopic objects such as biological cells produce only minor modulation in the intensity of transmitted light, leading many researchers to add exogenous contrast agents for image enhancement. However, cells and other semitransparent objects that have not been chemically modified impart phase delays to the transmitted electromagnetic fields, which can be measured using interferometric microscopy methods. In this dissertation, instrumentation and methods are developed to investigate the spatiotemporal dynamics and spectral signatures of individual cells and semitransparent polymer film samples.</p><p>An off-axis quantitative phase microscope is applied to (1) quantitatively image the two-dimensional refractive index distributions of microbicide films undergoing hydration and compare effects of thickness and composition on dissolution dynamics, and (2) investigate the morphological and volumetric changes of individual RBCs undergoing mechanical flow stresses in <italic>in vitro</italic> models of capillaries. The quantitative phase microscope is further modified to capture high-resolution hyperspectral holographic phase and amplitude images. This novel hyperspectral imaging system is applied to compare the sensitivity of phase-based and amplitude-based spectral quantification of optically-absorbing molecules, and then used to measure spectroscopic changes in RBCs that take place during infection by <italic>P. falciparum</italic> parasites.</p><p>Measurements of an object's optical volume, which is defined as a novel metric for characterizing objects whose refractive index and thickness profiles are not known a priori. The composition and thickness of microbicide films are both found to impact spatiotemporal dissolution kinetics. A comparison of fluorophore concentration determination by amplitude and phase spectra indicates that both methods of quantification have comparable sensitivity, and that the two may be combined to improve the precision of quantity determination. Both optical volume and hemoglobin mass measurements are seen to decrease in cells infected by <italic>P. falciparum</italic>, although the two metrics are only loosely correlated. Finally, RBCs flowing through in vitro capillary models exhibit large changes in optical volume when deforming in response to mechanical stresses, which is attributed to a combination of cytosolic volume changes as well as conformational changes in the intracellular protein configuration.</p><p>These results demonstrate the applicability of QPM as a tool for evaluating (1) microbicide film performance, (2) spectroscopic changes in infected individual RBCs, and (3) novel biophysical changes observed in RBCs under mechanical stresses.</p> / Dissertation

Gold nanoparticles (AuNPs) were functionalized for rapid binding of Acinetobacter baumannii (A. baumannii), a Gram-negative bacterium. AuNPs were functionalized with colistin (Col), a polycationic antibiotic, using a two-step self-assembly process, in which heterobifunctional polyethylene glycol (PEG) was used as a linker. Colistin was successfully conjugated to the AuNPs (Col-PEG-AuNP), as validated by dynamic light scattering (DLS) and proton nuclear magnetic resonance (H1 NMR). Images taken using a scanning transmission electron microscope (STEM), in combination with x-ray energy dispersive spectroscopy (EDS), confirmed binding of Col-PEG-AuNPs to the cell wall of A. baumannii. Results generated from a rapid binding assay indicated that the reaction rate of Col-PEG-AuNP complexation with A. baumannii reached half-maximum saturation in approximately 7 minutes. Quantitative measurement of the kinetics of Col-PEG-AuNP binding to A. baumannii is essential to inform the design of colistin-functionalized magnetic nanoparticles for magnetic separation of nanoparticle-bound A. baumannii.

The goal of this research is to develop image reconstruction techniques to automatically correct for errors in Magnetic Resonance Imaging (MRI) data due to errors in the trajectory used in the data acquisition. A method is presented to perform an iterative image reconstruction correcting for these trajectory errors in non-Cartesian acquisitions, without additional scans or measurements. The Trajectory Auto-Corrected image Reconstruction (TrACR) method jointly estimates k-space trajectory errors and images, based on SENSE and SPIRiT parallel imaging reconstruction. The underlying idea is that parallel imaging and oversampling in the center of k-space provides data redundancy that can be exploited to simultaneously reconstruct images and correct trajectory errors. Trajectory errors are represented as weighted sums of trajectory-dependent error basis functions, the coefficients of which are estimated using gradient-based optimization. TrACR was applied to reconstruct images and errors in golden angle radial, center-out radial, and spiral in vivo 7 Tesla brain acquisitions in 5 subjects. Compared to reconstructions using nominal trajectories, TrACR reconstructions contained considerably less blurring and streaking, and were of similar quality to images reconstructed using measured k-space trajectories in the center-out radial and spiral cases. Reconstruction cost function reductions and improvements in normalized image gradient squared were also similar to those for images reconstructed using measured trajectories. TrACR enables non-Cartesian image reconstructions free from trajectory errors without the need for separate gradient calibrations or trajectory measurements.

Small interfering RNAs (siRNAs) have emerged as a potent new class of therapeutics that regulate gene expression through sequence-specific inhibition of mRNA translation. Clinical trials of siRNAs have highlighted the need for robust delivery and detection techniques that would enable the application of these therapeutics to increasingly complex diseases and organ systems. Here we detail the generation and evaluation of siRNA-based optical and magnetic resonance imaging (MRI) contrast agents for the treatment of neurological diseases, including ischemic stroke and glioblastoma multiforme brain cancer. First, we designed and tested a fluorescent probe for neuroprotection in the setting of stroke that consists of siRNA complexed with myristoylated poly-arginine peptide (MPAP). MPAP, a peptide shown to cross cell membranes and the blood brain barrier, promoted robust internalization of siRNA by neurons in vitro and in mouse brain after intracerebral injection. Cellular uptake of MPAP-siRNA probes directed against a protein implicated in stroke pathology, c-Src, led to statistically-significant reductions of endogenous mRNA expression. The neuroprotective potential of probes was tested in a mouse model of ischemic stroke. Second, superparamagnetic iron oxide nanoparticles were investigated as vectors for siRNA delivery to glioblastoma multiforme brain tumors. Nanoparticles were designed to enhance chemotherapeutic treatment of tumors through siRNA-mediated knockdown of O6-methylguanine–DNA methyltransferase (MGMT), a protein implicated in glioblastoma chemotherapy resistance. The iron oxide core of nanoparticles rendered them detectable by MRI while fluorescent labeling was used for optical imaging. Functionalizing nanoparticles with the peptide chlorotoxin enabled tumor targeting and cellular accumulation of probe. Probe uptake was accompanied by reductions in MGMT activity and enhanced cellular responses to the chemotherapeutic temozolomide. Nanoparticles were tested in an orthotopic glioblastoma mouse model, where intratumoral administration proved effective in suppressing MGMT expression and tumor volume. These studies serve as proof-of-principle that siRNA-based imaging agents can be used as therapeutic tools for diseases of the central nervous system. / Engineering and Applied Sciences

Atherosclerosis is initiated by the adhesion of leukocytes to the endothelial surface of arteries followed by migration beneath the intima. Current therapies to combat atherosclerotic plaque, such as statins or antihypertensive drugs, treat atherosclerosis indirectly; they do not specifically target inflamed vasculature or improve the vascular condition. Few studies have focused on antibody mobility or membrane fluidity as an approach to improve drug delivery vehicle binding and uptake. / Engineering and Applied Sciences

Endothelial progenitor cells are potentially useful as a cell therapy for the treatment of ischemic cardiovascular diseases, but clinical outcomes have been limited likely because very few systemically delivered cells reach the target tissue. Biomaterials may improve outcomes of endothelial progenitor-based therapies because they can generate well-defined microenvironments capable of directing cell behavior. The hypothesis guiding this thesis is that local, sustained delivery of exogenous Vascular Endothelial Growth Factor (VEGF) and Stromal Cell-Derived Factor (SDF) from alginate hydrogels can enhance recruitment of endothelial progenitors to ischemic sites and also promote their contribution to new blood vessel growth. / Engineering and Applied Sciences

BIOMEDICAL ENGINEERING CHARACTERIZATION OF A PASSIVE DIFFUSION DEVICE FOR ASSAYS OF CHEMOTAXIS AND MORPHOGENESIS IPSHITA CHAKRABORTY Thesis under the direction of Professor John Wikswo Cell migration plays an important role in a wide variety of physiological phenomena, such as cancer, wound healing, and embryonic development. Microfluidic devices have become a highly useful platform for studying how cell migration influences these processes. An effort initiated in 2005 at the Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE) has resulted in the development and fabrication of a class of multi-chambered implantable cell trap devices that can be used to examine the combinatorial effects of the gradients of diffusive substances. We have developed a Computational Flow Dynamics (CFD) model of one of these devices to visualize its function and derive information regarding significant parameters, such as concentration at different points in space at varying time points, flux entering the device chamber, and the gradient of substances entering the device at different time points. An important feature of this device is a narrow restriction channel that limits the amount of substance entering the device chamber. In this thesis, we present a set of studies conducted with the CFD model on the effect of the width of this channel on the diffusion rate in the device chamber. This thesis also investigates how CFD packages like Fluent and GAMBIT calculate small numbers such as those derived in microscale flows and the extent to which a microfluidic device can be approximated with a CFD model. Our results indicate that Fluent is a valuable tool for modeling these kinds of microfluidic devices; however, phenomena such as numerical diffusion and contour algorithms influence the final data values obtained, and future work on this model should be aimed at a more detailed study of these effects and further ways to circumvent them. Approved________________________________ Date______________

Window chamber models have served as a tool for optically visualizing a tissue environment over time. Their use throughout the years has furthered the study of cancer. However, optical imaging techniques utilized with the model are limited in the depth from which light can penetrate and signal can be received. Further, ectopic placement of a xenograft in a model animal may modify the relevancy of findings by altering the normal environmental conditions. In the first section of the dissertation, improvements on the traditional window chamber model are described in the context of enabling multiple imaging modalities (optical, MR and nuclear) to be complementarily applied. The developed model, specifically geared toward breast cancer, is orthotopic and supports uninhibited tumor growth into the body of the animal. The three main imaging modalities applied provide unique strengths in obtaining information from the model system. Optical imaging allows for use of targeted fluorescent contrast agents, as well as sufficient resolution to visualize individual cells and capillaries. Magnetic resonance imaging provides the possibility of acquiring quantitative information about tumor morphology as well as a variety of physiological processes. This can be accomplished over the entire 3D volume of the tumor. Nuclear imaging provides functional and/or metabolic information using radiolabeled agents. The MWC model provides a platform for more specifically focused cancer imaging approaches to be applied and tested. The presence of hypoxia in tumors has a broad impact on cancer development and treatment. Current oxygenation assessment methods for longitudinally following spatially resolved oxygen changes over time are lacking. The development and testing of an oxygen sensitive porphyrin coating used in conjunction with the mammary window chamber model is detailed in the second section of the dissertation. Three different modulations were applied to induce physiologic oxygenation changes. All were capable of being detected over time utilizing a phosphorescence lifetime approach. An assessment of the stability of the coating found the coating remained suitable for a minimum of one week. The oxygen dependent phosphorescence lifetime of the coating was determined to be worthwhile for temporally and spatially monitoring oxygen changes of the tissue in contact with the surface of the coating. The third section of this dissertation work utilized the developed window chamber and oxygen measurement technique to investigate a novel oxygen modulator. The effectiveness of radiation therapy is reduced in tumors with low oxygen. The drug, NVX-108, is under investigation as a means to increase oxygenation prior to radiation treatment. NVX-108 is given while the patient breaths carbogen and has not been thoroughly tested when the patient is breathing oxygen or air. The study described herein focused on measuring the increase in oxygenation when NVX-108 was delivered while an anesthetized mouse breathed carbogen, oxygen or air. A similar average increase was measured under carbogen and oxygen breathing at two dosage levels of NVX-108. The increase was higher than with air breathing conditions. Additional animal experiments are needed in order to obtain a statistically significant finding.

Ovarian cancer is a deadly disease owing to the non-specific symptoms and suspected rapid progression, leading to frequent late stage detection and poor prognosis. Medical imaging methods such as CT, MRI and ultrasound as well as serum testing for cancer markers have had extremely poor performance for early disease detection. Due to the poor performance of available screening methods, and the impracticality and ineffectiveness of taking tissue biopsies from the ovary, women at high risk for developing ovarian cancer are often advised to undergo prophylactic salpingo-oophorectomy. This surgery results in many side effects and is most often unnecessary since only a fraction of high risk women go on to develop ovarian cancer. Better understanding of the early development of ovarian cancer and characterization of morphological changes associated with early disease could lead to the development of an effective screening test for women at high risk. Optical imaging methods including optical coherence tomography (OCT) and multiphoton microscopy (MPM) are excellent tools for studying disease progression owing to the high resolution and depth sectioning capabilities. Further, these techniques are excellent for optical biopsy because they can image in situ non-destructively. In the studies described in this dissertation OCT and MPM are used to identify cellular and tissue morphological changes associated with early tumor development in a mouse model of ovarian cancer. This work is organized into three specific aims. The first aim is to use the images from the MPM phenomenon of second harmonic generation to quantitatively examine the morphological differences in collagen structure in normal mouse ovarian tissue and mouse ovarian tumors. The second aim is to examine the differences in endogenous two-photon excited fluorescence in normal mouse ovarian tissue and mouse ovarian tumors. The third and final aim is to identify changes in ovarian microstructure resulting from early disease development by imaging animals in vivo at three time points during a long-term survival study.

Accurate and reliable in vivo measurement systems for orthopedic applications will allow a better understanding of native joint loading, gait patterns, and changes during healing and tissue regeneration. Robust and reliable telemetry units with an implantable transmitter and data acquisition software are necessary to insure long-term measurements. It was the goal of this study to update the current implantable telemetry system. Updates included using a new tablet computer for increased rates of data acquisition and encasing transmitters in a new waterproof casing. Software was developed using Labview on a Windows based Acer Iconia Tab. The Labview program allowed the user to save data to a measurement file and view the data in real time. The increased processing power of the tablet resulted in an increase in data collection rates from 29Hz to 87Hz. Interfacing the tablet computer with the telemetry system required the use of a RS-232 protocol to USB adapter. The newly developed tablet computer system was also used for load measurement collection during the most recent in vivo study. In order to insure transmitter function in vivo it was necessary to characterize the factors affecting transmission in vivo and develop transmitter and power coil designs that operated reliably. In the past implantable transmitters were noted to operate properly during bench top testing, but often failed after being placed in vivo. The two factors studied that limited power transfer to the transmitters were immersion in an aqueous environment and exposure to elevated temperatures. An aqueous environment significantly decreased power transfer by 11.9% (p-value = 0.014) relative to testing on the bench top. Additionally, a temperature increase to 40°C decreased power transfer by 6.2% (p-value = 0.017) when compared to power transfer in room temperature air. A solution that restored transmitter function required encasing transmitters in a new waterproof casing. Different casing designs made of silicone and semi-solid triglycerides were developed and tested on the bench top. Two different casing designs were used during in vivo testing and implanted into test animals. One casing design insured transmission while the other separated in vivo and did not facilitate transmission.