Tumor associated macrophages (TAMs) play an important role in establishing a pro-tumor environment in many tumor types. Produce low levels of inflammatory cytokines which creates pro-tumorigenic smoldering inflammation and also TAMs break down the surrounding extracellular matrix, secrete growth factors, and inhibit the adaptive immune response. TAMs are an attractive therapeutic target in cancer treatment, and strategically ablating these cells has been shown to remove their trophic effects. A more elegant solution would be to target these pro-tumor macrophages with a therapeutic agent that can alter their behavior to a strongly immunogenic phenotype capable of stimulating tumor immunity. The NF-κB pathways control macrophage phenotype and the inflammatory response. By selectively manipulating NF-κB in TAMs with siRNA, it should be possible to eradicate the TAM phenotype and recapitulate the normal immune response. I have developed a strategy to activate a classical immunogenic phenotype in TAMs. This strategy would stimulate tumor immunity by specifically activating the classical NF-κB pathway by knocking down the inhibitor of the pathway (IκBα). Using mannosylated endosomal escape nanoparticles MnNP, I have transfected macrophages with siRNA targeting key NF-κB proteins. In vitro experiments show that MnNP rapidly and strongly transfect a small population of cells, as opposed to hydroxyl capped nanoparticles (OHNP) and commercial agents, which transfect many cells to a lesser degree and with less specificity. Analysis of IκBα knockdown macrophages shows a decrease in immunosuppressive cytokines, and an increase in a potent CD8+ T-cell recruiting cytokine, indicating a shift from a TAM-like phenotype, to an immunocompetent phenotype. In in vivo mouse models of human cancer, MnNP delivered nucleotides are taken up by mammary tumor TAMs to a greater extent than phagocytized, free siRNA and are protected within the core of the particle. Similarly, OHNP are not taken up by TAMs in solid ovarian tumors, while MnNP are able to evade non-specific uptake by cells in the ascites fluid and are taken up by tumor TAMs. In an intratracheal delivery model, MnNP have enhanced delivery to lung metastasis TAMs while OHNP are taken up nonspecifically by phagocytic granulocytes and monocytes.

In this study, parameters for the clinical application of optical neural stimulation were developed. Two pulsed diode lasers, with wavelengths centered at 1875 nm and 1450 nm respectively, were used to elicit compound motor action potentials (CMAPs) in Sprague-Dawley rats via irradiation of the sciatic nerve. Comparisons were made between flat-top and Gaussian beam profiles, laser spot size, and damage margins. An unpaired parametric analysis revealed that 1450 nm light required a statistically significant lower radiant exposure than 1875 nm to evoke CMAPs. These findings revealed a 5:1 safety ratio of stimulation via 1450 nm light. There was no statistically significant difference in the radiant exposure needed to evoke action potentials between flat-top and Gaussian beam profiles. Spot size showed a minor correlation with stimulation thresholds at 1875 nm light, but not 1450 nm light. However no trends were observed when comparing the two wavelengths. These results were used to compare INS with electrical stimulation methods for nerve monitoring in healthy and damaged nerves. This comparison revealed that optically evoked signals did not propagate beyond the site of damage in injured nerves, whereas electrically evoked signals did. We thus argue that the use of 1450 nm light will lead to safer and more repeatable stimulations of neurological sites of interest, enabling use as an adjunct to electrical methods in nerve monitoring.

Subarachnoid hemorrhage (SAH) affects approximately 30,000 people each year and accounts for 1-7% of all strokes. Spontaneous SAH occurs due to the rupture of a cerebral aneurysm. SAH is characterized primarily by loss of cerebral autoregulation, development of delayed cerebral vasospasm and subsequent ischemia. Neurological deficits that are direct sequelae of vasospastic events are the most common cause of morbidity and mortality in patients with SAH. Current treatments for the ensuing vasospasm do not successfully reverse or prevent the spasm, as cellular mechanisms are not fully understood. The purpose of this thesis is to better understand thin and thick filaments that govern smooth muscle function in order to develop effective therapeutics for spastic arteries by uncoupling calcium signaling and myosin light chain phosphorylation with force generation. This novel ability to uncouple the two processes suggests that there are indeed calcium-independent mechanisms of relaxation. This work provides insight into mechanisms that regulate smooth muscle tone and may provide therapeutic angles to treat SAH-induced vasospasm. Recent findings have shown that there are biochemical changes in the vascular tissue that undergoes vasospasm. Among them are the down regulation of the actin associated proteins heat shock protein 20 (HSP20) and increased phosphorylation of heat shock protein 27 (HSP27). By down-regulating HSP20 and increasing the amount of HSP27 this study modeled the vasoactive changes that occur in the spastic vessels. This approach is direct and specific for the treatment of SAH-induced vasospasm. Hence targeting phosphorylated HSP20 and HSP27 should specifically modulate SAH-induced vasospastic vessels without causing ensuing hypotension. Engineered delivery techniques were used to enhance the uptake of siRNA and peptides to smooth muscle, in order to modulate HSP20. Taken together, this work provides novel insight into mechanisms that regulate smooth muscle tone and may provide therapeutic angles to treat SAH-induced vasospasm.

Although extracellular matrix (ECM) composition and organization are considered important regulators of cardiomyocyte phenotype in vitro and in vivo, a causal relationship between the ECM structure and effect on maintenance of cardiomyocyte (CM) phenotype has not been established. In this study, we investigated how key structural characteristics of electrospun scaffolds (fiber alignment, spacing, and diameter) regulated CM phenotype specifically cell morphology, actin/myosin patterning, and cardiac gene expression. We found that aligned fiber scaffolds resulted in a longer, more rod shaped morphology in both neonatal and adult CMs. Along with better morphology, CMs were also found to have better-organized cell actin/myosin bands and cardiac specific gene expressions of β-MYH7 and SCN5A.1 and SCN5A.2 in both neonatal and adult cells. Additionally, upon exposure to variously spaced aligned fibers, adult CM action/myosin structure and morphology did not change but the overall orientation of the cells, relative to the fibers, increased from 45° to parallel as fiber spacing increased from 2 µm to 15µm. These findings provide critical insights into ECM-CM interactions that are responsible for maintenance/loss of neonatal and adult cardiomyocyte phenotype, and highlight the importance of developing more relevant cardiomyocyte culture substrates for in vitro studies.

HER2-amplified (HER2+) breast cancers are treated with the anti-HER2 monoclonal antibody trastuzumab. Although trastuzumab reduces production of the angiogenic factor VEGF in HER2+ tumors, the acute and sustained effects of trastuzumab on the tumor vasculature are not understood fully in trastuzumab-resistant tumors. Hyperspectral imaging is used to quantify microvessel density and hemoglobin oxygenation (sO2) of trastuzumab responsive and resistant breast cancers after antibody treatment in vivo. Microvessel dynamics are monitored over a 14 day time-course. Immunohistochemistry validates complementary markers of tumor cell and vascular response to treatment. Trastuzumab treatment in both responsive and resistant tumors resulted in decreased sO2 5 days after initial treatment when compared to IgG-treated controls (p<0.05). Responsive tumors showed significantly higher vessel density and significantly lower sO2 than all other groups at 5 days post-treatment (p<0.05). Distribution analysis of vessel sO2 showed a significant (p<0.05) shift of highly oxygenated vessels towards lower oxygenation over the time-course in both trastuzumab-treated responsive and resistant tumors. This study suggests that longitudinal hyperspectral imaging of microvessel sO2 and density could distinguish trastuzumab-responsive from trastuzumab-resistant tumors.

Clinical translation of RNA interference has been impeded by the lack of safe and effective technologies for cytoplasmic delivery to target cells. Regenerative medicine is a potentially high impact but relatively underexplored application area for short interfering RNA (siRNA) therapies. We have developed a versatile, biodegradable, and injectable polyester urethane (PEUR) tissue scaffold-based approach for sustained and tunable gene silencing at local tissue sites. Tissue regenerative PEUR scaffold-based delivery of endosomolytic nanoparticles (si-NPs) achieved over 90% in vivo gene silencing sustained for 35 days at a low dose of 200 µg/kg siRNA/mouse. Addition of the excipient trehalose was used to control the rate of si-NP release, which enabled modulation of the temporal gene silencing profile in vivo. Finally, prolyl hydroxylase domain 2 (PHD2) silencing studies demonstrated that this platform can promote functional tissue regenerative responses in vivo by increasing vascular volume 3-fold. This versatile system provides an efficient platform to investigate loss of function phenotypes in basic tissue engineering studies and can be therapeutically applied to silence genes that are deleterious to tissue regeneration.

To apply MWI techniques to excised, fixed rodent brains, multi-exponential T2 (MET2) data were acquired at high (7T) and ultra-high (15.2T) fields with and without tissue doping with Gadolinium to increase SNR efficiency. From relaxivity measurements, optimal scan parameters are found based on the minimal Cramér-Rao lower bounds (CRLB) of variance of myelin water fraction (MWF). Subsequently acquired MET2 data were analyzed to obtain myelin water fraction (MWF) maps with and without contrast agent at each field strength with various techniques, displaying the potential for MWI in rodent brain in the study of myelin content across whole brain.

This thesis involves the development of an overnight high-resolution 3D diffusion kurtosis imaging protocol for ex vivo whole rat brain imaging. MRI acquisition parameters were optimized in order to obtain high-resolution, high-precision diffusion kurtosis imaging data. This included the programming and development of a 3D diffusion-weighted fast spin-echo pulse sequence. Image data from ex vivo rat brains were collected to verify the efficacy of the imaging protocol. Values of diffusion kurtosis parameters in normal rat brains were similar to those reported in previous imaging studies. Using this protocol, preliminary data were collected from a model of schizophrenia in rats. These data indicated the ability of the protocol to detect changes in diffusion kurtosis parameters that could correlate to changes in white matter microstructure.

Backscattering Interferometry (BSI) has been used to detect bio-molecular interactions by measuring shifts in the bulk refractive index of a sample due to conformational changes of the interacting species. BSI has the advantage over other biosensors because it can detect interactions in a free solution format, as well as a tethered format. In some applications, non-specific surface adsorption introduces noise to the BSI signal, negatively affecting its limit of detection. We hypothesize that there are regions within the spatial and frequency domain of the BSI signal, which are only sensitive to changes in the bulk fluid, and are thus unaffected by interactions on the surface of the channel. To test this hypothesis, model systems were designed in which the surface conditions of a glass microchannel, as well as the bulk fluid, were changed in a controlled manner. Step changes in glycerol concentration in the bulk fluid were made and successive chemical layers were coupled to the surface to produce independent bulk and surface signals. Regions of the fringe pattern produced by BSI, which have been previously uninvestigated, were analyzed to determine if there are distinct characteristics of the signal that change in response to bulk refractive index but are insensitive to surface binding. We developed an algorithm to identify these regions, effectively eliminating the unwanted signal contributions from surface adhesion while maintaining the signal produced by the bulk fluid. Multiple regions, within the parameter space of each model, were found that are insensitive to surface binding molecules.

Image guided transorbital endoscopic procedures have the potential to introduce new forms of therapy to the optic nerve, such as direct delivery of neuroprotective drugs for glaucoma treatment. This technique was confirmed to be possible and accurate in live animal studies, though featuring long procedure times. Navigation was enhanced by incorporating endoscopic video augmentation, which was demonstrated to significantly reduce procedure times while also improving accuracy in a series of phantom experiments performed by a diverse group of surgeons. In order to further the case for human application, procedure safety was assessed by characterizing the possible forces inflicted on the optic nerve in a phantom simulation. Measured forces implied reasonably low risk when considered in the context of human conditions, and surgeon experience and target nerve location were demonstrated to be significant factors. This research has advanced the case for application of image guided transorbital endoscopy to human therapy.