<p>A number of significant muscle diseases, such as cachexia, sarcopenia, systemic chronic inflammation, along with inflammatory myopathies share TNFα-mediated inflammation in their pathogenesis. TNFα-based inflammatory stress may directly sensitize skeletal muscle to drug-induced toxicity. The two main difficulties when assessing the role of TNFα on skeletal muscle drug toxicity using in vitro methodology are first creating an experimental model that recapitulates the basal functional responses of human skeletal muscle and second validating that the experimental model accurately represents human skeletal muscle response to TNFα. The aim of this research is to resolve these two difficulties. </p><p>We first assayed the bioenergetic profile of engineered three-dimensional human skeletal muscle myobundles to probe mitochondrial health and compared its result to muscle fatigue. Then engineered a perfusion system to measure respiration under basal conditions and electrical stimulation. Finally, to assess the effect of TNFα-induced inflammation on drug responses, we engineered 3D, human skeletal myobundles, chronically exposed them the TNFα during maturation, and measured the combined response of TNFα and the chemotherapeutic, doxorubicin, on muscle function. We concluded that the human myobundles reproduce normal muscle metabolism under both basal and maximal energy demand conditions enabling the detection of drug-induced mitochondrial toxicity. The bioenergetic health index, which is a measure of normal oxidative mitochondrial function, was inversely correlated with the extent of fatigue. The relationship between mitochondrial function and physiological muscle function suggests that the mitochondrial dysfunction produced the fatigue. The custom perfusion chamber was validated to accurately measure oxygen uptake during basal and electrical stimulation conditions. This in vitro non-invasive tool enables the investigation of human muscle physiology during exercise. Utilizing the myobundle platform, a synergistic effect of the combination of TNFα and 10nM doxorubicin was found on contractile force production. Overall, we demonstrated the feasibility of the in vitro 3D, human muscle inflammatory system to act as a drug-toxicity testing platform and validated assays to quantify drug-induced decrements in mitochondrial function.</p> / Dissertation

<p>The primary barriers to multiplexed point of care immunoassays are: (1) cost; (2) response time; and (3) sample handling. Described here is a self-contained, multiplexed immunoassay platform for point of care detection that leverages a number of enabling technologies to address these barriers. This platform is referred to as the "D4" assay, as it is composed of the following four sequential, concerted events (Figure 1): (1) Dispense (droplet of blood); (2) Dissolve (printed reagents on chip); (3) Diffuse across surface; (4) Detect binding event. </p><p>The D4 assay process begins when a finger-stick is administered and the resulting droplet of blood is applied to the surface of a detector chip. Hydrophobic ink printed onto the surface of the chip confines the blood droplet to a non-fouling region containing soluble, labile spots of detection antibodies and insoluble, non-labile spots of capture antibodies. As the soluble detection antibodies are dissolved from their printed spots by the droplet of blood, three serial events occur to generate signal (Figure 2): (1) the first half of the detection complex is formed by the binding of analytes present in blood to the stable capture agent spots; (2) diffusion of the blood laterally through the polymer brush, resulting in the dissolution and diffusion of soluble detection antibody spots; (3) solubilized detection antibodies bind to their respective analyte-capture agent spots, completing the detection complex and resulting in signal generation at the position of the non-labile capture antibody spots. </p><p>This assay relies upon the ability of labeled detection antibodies, printed into a nonfouling brush as "labile spots", to be carried by blood flow to adjacent rows of stably immobilized capture antibodies by diffusion of the analyte solution (Figure 2). Generation of signal at a given capture spot location provides identification of individual analytes (positives). Quantification of the concentration of the different analytes is carried out identically to a conventional fluorescence immunoassay by pre-calibration of the system using a dilution series of the analyte spiked into whole blood.</p><p>The D4 assay addresses several critical needs in point of care testing as follows: First, the cost of testing is reduced through miniaturization, multiplexing and one-step, on-site processing of undiluted whole blood obtained from a finger stick. Second, in order to simplify the immunoassay process, the D4 relies on diffusion to bring spatially localized reagents together to create a functional assay and thereby eliminate the need for liquid transfer steps, microfluidic manipulation of sample or reagents, and wash steps. Third, this multiplexed platform is capable of screening for a panel of markers in a single drop of blood with no sample preprocessing. Fourth, the assay is fast, which alleviates the difficulties often associated with communicating the outcome of diagnostic tests. A prototype of the D4 assay is shown in Figure 3 below.</p> / Dissertation

Cancer is a multistage, heterogeneous disease that develops through a series of genetic mutations. Early stage cancer is most responsive to treatment but can be the hardest to detect due to its small size, lack of definitive symptoms and potential location deep in the body. Whole body imaging methods, MRI/CT/PET, lack the necessary resolution to detect cellular level abnormalities. Optical methods, which have sufficient resolution, can be miniaturized into endoscopes, which are necessary to overcome limited penetration of light into tissue. By combining optical coherence tomography (OCT) and fluorescence imaging methods it is possible to create endoscopes sensitive to molecular and structural changes. I applied a dual-modality 2mm diameter rigid endoscope to the study of the natural history of colon cancer in a mouse model, and later applied this knowledge to the design and characterization of a 0.8 mm dual-modality flexible probe for use in human fallopian tubes. By using this endoscope, which is introduced through the natural orifice and is compatible with existing hysteroscopes, high-risk women could be screened in a procedure at a similar level of invasiveness as a colonoscopy. Therefore, the endoscope fills this gap in clinical care for women at high-risk for ovarian cancer.

<p> Kinesthesia refers to sensations of limb position and movement, and deficits of upper limb kinesthetic feedback are common after stroke, impairing stroke survivors&rsquo; ability to perform the fundamental reaching and stabilization behaviors needed for daily functions like self-feeding. I attempt to mitigate the negative impact of post-stroke kinesthesia deficits by evaluating the utility of vibrotactile sensory substitution to restore closed-loop kinesthetic feedback of the upper limb.</p><p> As a first step, this study evaluated performance in healthy individuals during fundamental reaching, stabilization, and tracking behaviors while using supplemental vibrotactile feedback encoding either limb state information or goal-aware error information. First, I determined that performance in reaching and stabilization tasks varies systematically with the amount of limb position and velocity information encoded in limb state feedback and that there is an optimal combination. Next, I compared the utility of optimal limb state to goal-aware error feedback. Both types of feedback reduced error in the reaching and stabilization tasks. Random task-irrelevant sham feedback did not reduce error, demonstrating participants could perceive and understand the information contained within the vibrotactile feedback. Error feedback improved performance more than state feedback; however the relative difficulty of using error feedback outside of a laboratory setting means state feedback should not be discounted. The performance while tracking could not be quantified due to issues with the task design.</p><p> As a second step, I performed a series of case studies in five chronic stroke survivors. The stroke survivors all tolerated the vibrotactile feedback well and were able to perceive and understand at least one of the limb state or error feedback encodings. Stroke survivors practiced each information encoding type for one session. During this short period our stroke survivors struggled to integrate visual and vibrotactile inputs and motor control in order to use the vibrotactile information to control the arm. However, two additional practice sessions with error feedback for one participant led to a two thirds reduction in reaching error. These results suggest stroke survivors can learn to use supplemental vibrotactile feedback to enhance control of the contralesional arm.</p>

<p>Cortical representations of rhythmic and discrete movements are analyzed and used to create a novel neural decoding algorithm for brain-machine interfaces. This algorithm is then implemented to decode both cyclic movements and reach-and-hold movements in awake behaving rhesus macaques using their cortical activity alone. Finally, a healthy macaque wears and controls a lower body exoskeleton using the developed BMIas a proof of concept of a brain-controlled neuroprosthetic device for locomotion.</p> / Dissertation

In theory, siRNAs can inhibit every known cancer-causing gene through sequence-specific RNA interference. However, almost twenty years after the discovery of RNA interference, the use of siRNAs as targeted molecular medicines remains challenging due to comprehensively poor pharmacokinetic properties of siRNA. Naked siRNA molecules are rapidly excreted through the urine and cannot inherently enter cells or access the cytosol through endosomal escape, resulting in limited bioavailability within tumor cells after systemic administration. Strategies to complex negatively-charged siRNA into cationic polyion complexes (polyplexes) have been effective for the treatment of diseases in the liver where polyplexes naturally biodistribute. But the same polyplexes have shown limited success in oncology due to rapid disassembly within the kidneys, off-target accumulation within the liver, and limited on-target accumulation within tumor tissue. Thus, a broader set of polyplex physicochemical parameters remain to be optimized in order to improve siRNA delivery to tumors after systemic administration. Here, we show that fine-tuning hydrophobic stabilizing forces of siRNA polyplexes, through altering either the polymer carrier or siRNA molecule, can simply and effectively improve siRNA bioavailability and accumulation within solid breast tumors. In both cases, increased polyplex stability through the optimization of core hydrophobicity decreased rapid renal clearance and led to appreciable increases in blood circulation, tumor accumulation, and intratumoral siRNA bioactivity. Our updated siRNA polyplex technology enabled the first selective, therapeutic silencing of mTORC2 in HER2-amplified breast tumors. Due to the prominent role of mTORC2 within the oncogenic PI3K-Akt-mTOR pathway, selective mTORC2 inhibition slowed tumor growth through the induction of cell death and cooperated with the HER2 receptor tyrosine kinase inhibitor, lapatinib, to kill HER2-amplified tumor cells and halt tumor growth. In sum, this work systematically elucidates the impact of core hydrophobicity on siRNA polyplex performance in vivo, illustrates the broad potential for therapeutically inhibiting currently âundruggableâ cancer-causing oncogenes, and highlights the specific therapeutic potential of selectively inhibiting mTORC2 as a tumor cell killing strategy in HER2-amplified breast cancers.

Osteoarthritis (OA) is a disease characterized by degradation of joints with the development of painful inflammation in the surrounding tissues. Post-traumatic osteoarthritis (PTOA) is OA that develops following a traumatic injury to the joint. Currently, there are a limited number of treatments for this disease and many of these only provide temporary, palliative relief. Here, we discuss polymer drug delivery systems that can provide targeted and sustained delivery of imaging and therapeutic agents to OA-affected sites. Polymer based microparticles were investigated as a therapeutic for PTOA. The inherent antioxidant function of poly(propylene sulfide) (PPS) microspheres (MS) was dissected for different reactive oxygen species (ROS), and therapeutic benefits of PPS-MS were explored in mechanically induced PTOA. PPS-MS scavenged hydrogen peroxide (H2O2), hypochlorite, and peroxynitrite but not superoxide in vitro in cell-free and cell based assays. Elevated ROS levels were confirmed in a mouse model of PTOA. In the PTOA model, PPS-MS reduced matrix metalloproteinase activity. These results suggest that local delivery of PPS-MS to site of PTOA reduces articular cartilage destruction. These results motivate further exploration of PPS as a stand-alone, locally-sustained antioxidant therapy and as a material for microsphere-based, sustained local drug delivery to inflamed tissues at risk of ROS damage.

Duchenne muscular dystrophy (DMD) is a progressive myopathy caused by mutations in the dystrophin gene, leading to contraction-induced damage, inflammation, and necrosis in skeletal and cardiac muscles. Reliable methods of characterizing DMD cardiomyopathy are essential for effective pharmacological therapy. Myocardial circumferential strain (εcc) measured via harmonic phase (HARP) analysis is commonly used to measure cardiomyopathy. Heart rate variability (HRV) data can be used to quantify autonomic compensation in diseased patients, potentially providing an additional method of disease characterization. In this retrospective investigation, we hypothesized that 1) our custom HARP algorithm would be correlated with equivalent results from standard clinical software, and 2) that the εcc results would be correlated with HRV parameters. Twenty-eight boys with DMD were studied (ages 8-21). Cardiac MRI data included spatial modulation of magnetization (SPAMM)-tagged images, acquired throughout the cardiac cycle. HARP analysis was used to calculate peak Lagrangian εcc. Forty-eight-hour Holter monitoring data were acquired. Parasympathetic input-associate power (PIAP) was determined by spectral analysis of R-R intervals observed during sleep to determine the proportion of total power in the high-frequency band. The εcc results from our HARP algorithm were correlated with clinically-used values (r=0.79, p<1.0x10-7). PIAP measurements from two randomly sampled periods did not differ significantly (p=0.51) and had high reliability (intraclass correlation=0.923, p<0.001; n=28). However, PIAP did not prove to be a significant measure of disease characterization. Though promising patterns exist, key study limitations must be addressed in order to conclude that HRV parameters can provide an alternate method of assessing DMD.

Small interfering RNA (siRNA) can potently and specifically suppress translation of any gene, including intracellular targets traditionally considered âundruggableâ. However, emergence of translational siRNA therapies has remained slow, with the primary challenge being the formidable anatomical and physiological barriers that must be overcome to deliver siRNA to its intracellular site of action in target cell types. Polymeric nanoparticle (NP) carriers can protect the siRNA against degradation and transport it into the cell, but these polyelectrolyte systems are hampered by poor in vivo stability and toxic/immunogenic effects. Hydrophobic modification of siRNA is a promising approach to improve the pharmacokinetic properties of siRNA without the complexity and toxicity of traditional NP carriers. siRNA conjugated to the lipid palmitic acid (PA) acts synergistically with two distinct polymer NP carriers, improving carrier stability, pharmacokinetics, and cellular internalization, leading to enhanced gene silencing efficacy at reduced polymer doses. Modification of siRNA with PA is thus a powerful strategy to broaden the therapeutic index of NP-based strategies. Additionally, conjugation of an albumin-binding hydrophobe (termed L2) to siRNA broadly enhances its pharmacokinetic profile, biodistribution to tumors, and tissue penetration capacity. siRNA-L2 consistently outperformed a commercial NP carrier in tumor accumulation and biocompatibility and elicited significant and sustained in vivo tumor gene silencing, highlighting its potential as a translational and potentially transformative approach to improve systemic RNAi cancer therapies. The benefits conferred through PA and L2 conjugation establish a pivotal role for hydrophobic siRNA conjugates in the advance of siRNA to medical application.

With the rapid emergence of antibiotic-resistant bacteria and the lack of antibiotics in the development pipeline, bacteremia and sepsis are becoming an increasing health concern. Rapid diagnosis currently suffers from the need for the amplification of the bacterial signal, which is often only accomplished by overnight blood culture. Without a proper diagnosis, effective treatment may not be administered, resulting in increased mortality. Extracorporeal bacterial separation methods remove bacteria from whole blood, making them a promising avenue for both diagnosis and treatment. Specifically, micromagnetic separation removes bacteria bound to paramagnetic beads using a strong magnetic field. A small-footprint microfluidic device for micromagnetic separation was fabricated and characterized by volumetric flow testing and computational fluid dynamics. The framework for a computational analysis of micromagnetic separation was also developed, incorporating a magnetostatic finite element analysis of the device and a physiologically-based pharmacokinetic model of bacteremia. Small-footprint microfluidic devices were shown to be able to capture bacteria, but at rates and with overall capacities inadequate as an effective treatment for bacteremia. As such, these devices could be considered as means of rapid diagnosis; the development of high-throughput extracorporeal blood-cleansing devices is required for bacteremia treatment.