A clinical need exists for improved methods to identify cancer patients with tumors prone to drug resistance, and prescribe effective alternative therapies. Cellular metabolism is a powerful marker of tumor response to treatment, because the oncogenic drivers targeted by anti-cancer drugs often regulate cellular metabolism. In this dissertation, optical metabolic imaging (OMI) is developed to detect drug-induced metabolism changes in breast cancer cells, in vivo xenograft tumors, and primary-tumor derived organoids. OMI probes the auto-fluorescence intensity and lifetime of NAD(P)H and FAD, coenzymes of metabolism, to quantify protein concentrations and protein-binding dynamics. A composite endpoint, the OMI index, provides a robust, dynamic readout of cellular metabolism. OMI endpoints were evaluated and characterized in a panel of breast cancer cell lines and three xenograft models in vivo with and without drug treatment. For optimal clinical utility, protocols were developed to generate 3D organoid cultures of primary tumor biopsies for high-throughput testing of potential drugs. In xenograft tumor derived organoids, the OMI index decreased in responsive organoids treated with anti-cancer drugs, corroborating with in vivo tumor growth curves. Feasibility experiments on organoids derived from human patient biopsies demonstrated viability of organoids and drug-induced changes in OMI index. The high-resolution images of OMI allow cellular-level analysis of heterogeneity. Heterogeneity profiles of resistant cell populations within organoids were identified and tracked over 72 hours. With these findings, OMI shows potential for development into a high-throughput organoid screen to test the efficacy of a panel of drugs and drug combinations to direct clinical therapy selection and expedite pre-clinical studies.

Polyhemoglobin (polyHb) is one of the most promising blood substitutes in development It is awaiting approval from the FDA and has already been approved for routine surgical use in South Africa. Though it has been successful as an oxygen carrier, we have shown in the present study that with the lack of platelets and coagulation factors in the current polyHb formulations, there could be a risk of coagulation problems when large volumes ofpolyHb are administered. We therefore develop a novel blood substitute that would solve potential coagulation problems in a hemodiluted setting, while still being able to support oxygen transport. We developed polyhemoglobin-fibrinogen (polyHb-Fg), by crosslinking hemoglobin and fibrinogen with glutaraldehyde. Our in vitro tests showed that platelet aggregation was not potentiated by the addition ofpolyHb or polyHb-Fg. Using in vitro whole blood coagulation tests, hemodilution with polyHb adversely delayed the clotting mechanism. On the other hand a formulation ofpolyHb-Fg was able to achieve similar clotting times as whole blood, even with hemodilution. Thus, this formulation of polyHb-Fg has the potential for applications in the infusion of large volumes of blood substitute without interfering with coagulation. / La polyhémoglobine (polyHb) est l'un des substituts du sang les plus prometteurs à l'étude. Ce composé attend l'approbation de la FDA et a déjà été approuvé pour l'usage chirurgical courant en Afrique du Sud. Bien que la polyHb ait connu un succès comme porteur de l'oxygène, nous avons montré dans la présente étude que le manque de plaquettes et des facteurs de coagulation dans les formulations courantes de polyHb, il pourrait y avoir un risque de problèmes de coagulation quand de grands olumes de polyHb sont administrés. Nous développons donc un nouveau substitut du sang qui résoudrait les problèmes potentiels de coagulation dans une situation d'hémodilution, tout en continuant d'assurer le transport de l'oxygène. Nous avons développé le polyhémoglobine-fibrinogène (polyHb-Fg), en réticulant l'hémoglobine et le fibrinogène avec du glutaraldéhyde. Nos essais in vitro ont prouvé que l'agrégation de plaquette n'a pas été augmentée par l'addition du polyHb ou du polyHb-Fg. Lors d'essais in vitro de coagulation de sang entier, l'hémodilution avec le polyHb retarde défavorablement le mécanisme de coagulation. D'autre part une formulation de polyHb-Fg permet d'obtenir un temp de coagulation semblable à celui du sang entier, même en cas d'hémodilution. Ainsi, cette formulation de polyHb-Fg a le potentiel requis pour des applications dans l'injection de grands volumes de produits de remplacement du sang sans interférer avec la coagulation.

Thesis (Ph.D.)--Syracuse University, 2008. / "Publication number: AAT 3333592."

<p>Cell differentiation is the foundation for tissue development and regeneration, disease modeling, and cell-based therapies. The differentiation of skeletal myoblasts has been well-studied, and expression of the transcription factor myogenin is recognized as an early indicator of a cell committed to the myogenic differentiation. Not as much is known regarding how individual cells activate myogenin, the dynamics with which this happens, or the genomic regions that regulate this activation beyond the promoter. This research tested the following hypotheses: 1) The single-cell expression profile of myogenin could reveal distinct subpopulations of myogenic cells and indicate unique cell states. 2) The dynamic evolution of myogenin expression is indicative of the processes regulating differentiation. 3) There exist other genomic loci beyond the immediate promoter of myogenin that regulate the expression of myogenin. The primary conclusions of this dissertation are as follows: 1) Differentiating or reprogrammed cells activating myogenin show a bimodal distribution, with cells either expressing low or high levels of myogenin, and there is a critical dose of MyoD required to transition to differentiate. 2) Myogenic lineage commitment can be delayed but not prevented by serum, and myogenic reprogramming can be accelerated by increasing the forced expression of MyoD. 3) In addition to the promoter, there are additional enhancer sites that regulate the expression of myogenin in differentiating myoblasts.</p> / Dissertation

<p>This dissertation focuses on enhancing our understanding of microbicide transport mechanisms from gel delivery vehicles into vaginal mucosal tissues through the development and application of novel transport assays. We demonstrated the capabilities of Raman spectroscopy to measure four leading microbicide drugs, Tenofovir, IQP-0528, MIV 150, and Dapivirine, in fluids, microbicide gels, and biological tissues. Strong linear dependences of spectral Raman intensities on drug concentrations suggested that Raman spectrometry could be used in practice for quantitative analysis of drug concentrations in biologically-relevant matrices. Concentration measurements in fluids and tissues demonstrated good correlation with gold standard LC-MS/MS data.</p><p>Next, we investigated the accuracy of confocal Raman measurements of uniform and non-uniform (linear) concentration distributions of Vitamin K in a tissue phantom. A chemometric method was used to perform the quantitative analyses of concentration distributions and to correct for signal attenuation due to elastic scattering that increased with depth into the tissue. The measured concentration distributions demonstrated an expected trend of the linear profile up to a depth of 180 to 200 μm, thereby validating the confocal Raman approach for concentration profiling in scattering matrices.</p><p>We developed a label-free assay using Raman spectroscopy to quantify the drug diffusion coefficient within a gel. The concentration distributions of Tenofovir in gel over time and space were measured and the concentration profiles were fitted to PDE diffusion models to obtain diffusion coefficients. The gel was tested undiluted, and in 10-50% serial dilutions with vaginal fluid simulant, to capture the range of conditions that likely occur in vivo. Tenofovir exhibited diffusion-like behavior that could be fitted to the diffusion model reasonably well (R2: 0.86 – 0.99). The dilutions with vaginal fluid simulant increased diffusion coefficients of Tenofovir in the test gel by up to 50%. </p><p>Finally, we developed a real-time transport assay using a combined CRS-OCT instrument constructed by our team to study and distinguish the microbicide transport mechanisms between epithelial and stromal layers of vaginal tissue. Measurements were corrected by taking into account the effects of out-of-focus light contributions from the Tenofovir overlying gel layer. Data were translated to fundamental transport properties, the diffusion and partition coefficients. The diffusion coefficient in stroma was found to be 2 to 15 times larger than that in epithelium, suggesting that epithelium could present a diffusion barrier to Tenofovir transport in vaginal tissue.</p><p>The series of transport assays developed here can provide useful information of microbicide transport mechanisms. The transport parameters can be used as salient measures to investigate the drug delivery performance of different drugs and gel vehicles. The parameters are important inputs to the deterministic transport models that predict the gels’ dosing regimen in PK studies. Collectively, this thesis will contribute to a better understanding of anti-HIV drug delivery and pharmacokinetics, and be of significant help in creation of products for prevention of HIV/AIDS.</p> / Dissertation

<p>X-ray CT is widely used, both clinically and preclinically, for fast, high-resolution, anatomic imaging; however, compelling opportunities exist to expand its use in functional imaging applications. For instance, spectral information combined with nanoparticle contrast agents enables quantification of tissue perfusion levels, while temporal information details cardiac and respiratory dynamics. Common implementations of spectral and temporal (spectro-temporal) CT discretely sample the time points and energies to be reconstructed, proportionally increasing acquisition time and ionizing radiation dose with data dimensionality. Here, we propose and develop an integrated framework for spectro-temporal CT data acquisition, reconstruction, and analysis which drastically reduces the sampling time and radiation dose associated with spectro-temporal CT imaging. Specifically, we exploit the latent, gradient sparse and low rank structure of spectro-temporal CT data sets to recover their full dimensionality from highly undersampled projection measurements. We achieve reliable, high fidelity results through a novel combination of hierarchical projection sampling, the split Bregman optimization method, and piecewise-constant kernel regression. The integrated framework generalizes to arbitrary spectral and temporal CT reconstruction problems, while maintaining or even improving upon the sampling time and radiation dose associated with anatomic imaging protocols. We believe that this integrated framework will serve as the basis for a new generation of routine, functional CT imaging protocols.</p> / Dissertation

<p> Humans use their upper limbs for interacting with many objects, tools and loads. It is crucial to maintain arm stability during these interactions, which can be done by regulating the impedance of the arm through a combination of feedforward and feedback neural mechanisms. Feedback tuning, in particular, provides an efficient and task-specific means for impedance control. However, the factors that drive feedback tuning remain unclear. In addition, it is uncertain how feedback mechanisms may contribute differently to impedance control in the dominant versus nondominant arm, which frequently perform different types of tasks. The goal of this dissertation was to examine mechanisms of impedance control in either arm, using various experimental methods to examine neuromuscular control during single-joint posture and movement. Firstly, we examined attention as a factor that may be crucial for feedback tuning in either arm. By comparing feedback responses evoked under different attention conditions, we determined that attention alone is not sufficient for feedback tuning, indicating that interaction with destabilizing dynamics is key for task-specific feedback tuning. Secondly, we examined differences between the dominant and nondominant arm in terms of feedback tuning during a postural task. By comparing feedback responses evoked when postural stability in each arm was challenged, we showed that feedback tuning contributes equally to either arm during posture. Finally, we investigated the idea that inter-arm differences in impedance control may only manifest when switching between movement and posture. This was accomplished by comparing the contributions of feedforward and feedback mechanisms during unperturbed, point-to-point reaching in either arm. The results indicate that the nondominant arm demonstrates greater reliance on impedance control and feedback mechanisms during reaching, but that prior training with the dominant arm can reduce this bias.</p>

Acoustic cavitation (i.e. acoustically stimulated microbubble activity) has gained interest in the biomedical community due to its ability to locally concentrate mechanical forces inside the body. Biological structures in close proximity experience stresses that temporally disrupt their normal function and allow passage of material that would otherwise be impermeable. Examples include blood-brain barrier disruption, enhanced penetration of drugs into tumors, disruption of the blood vessel endothelium, and permeabilization of cell membranes (i.e. sonoporation). The goal of this thesis was to investigate a new class of acoustic cavitation nuclei for sonoporation called phase-shift nanoemulsions (PSNE). Ultrasound can be used to nucleate, or phase-shift PSNE into microbubbles with a process termed acoustic droplet vaporization (ADV). Specifically, the focus was to use PSNE for delivery of small interfering RNA (siRNA) to an in vitro cell suspension using sonoporation. Small interfering RNA is an exogenous RNA molecule and has gained increased attention due to its ability to knockdown specific proteins central to disease progression. Results showed that siRNA delivery with PSNE is possible with high uptake efficiency (i.e. ratio of the number of cells with uptake to the number of cells originally). Uptake was highly dependent on the amount of acoustic cavitation activity generated from PSNE. The acoustic emissions from individual PSNE were explored to understand the microbubble dynamics following ADV. Results showed that PSNE immediately undergo an explosive growth and collapse at the ADV threshold, and the maximum size of the microbubble depends on the ultrasound frequency. This led to the hypothesis that the sonoporation efficiency with PSNE is governed by the choice of frequency. Lower frequencies were shown to expand microbubbles to larger maximum radii, which in turn caused more energetic collapses leading to cell death. This explains the lower uptake efficiencies at lower frequencies (39.45% at 1 MHz and 46.62% at 2.5 MHz), compared to the relatively high uptake efficiency at 5 MHz (66.81%). In general, uptake efficiencies > 50% have rarely been achieved with current sonoporation methods and these results are a significant improvement. PSNE could also serve as a unique platform for numerous other therapeutic ultrasound applications that utilize the mechanical effects of acoustic cavitation. The frequency-dependent control over the microbubble dynamics following ADV could provide a way to tune the level of stress experienced by biological structures. / 2017-02-17T00:00:00Z

There is a compelling body of evidence implicating continuous propagation (reentry) sustained by multiple meandering wavelets in the pathology of advanced human atrial fibrillation (AF). This forms the basis for many current therapies such as the Cox MAZE procedure and its derivatives, which aim to create non-conducting lesions in order to "transect" these circuits before they form. Nevertheless, our ability to successfully treat persistent and permanent AF using catheter ablation remains inadequate due to current limitations of clinical mapping technology as well as an incomplete understanding of how to place lesions in order to maximize circuit transection and, more importantly, minimize AF burden. Here, we used a hybrid cellular automaton model to study the dynamics of chaotic, multi-wavelet reentry (MWR) in excitable tissue. First, we used reentry as an exemplar to investigate a hysteretic disease mechanism in a multistable nonlinear system. We found that certain interactions with the environment can cause persistent changes to system behavior without altering its structure or properties, thus leading to a disconnect between clinical symptoms and the underlying state of disease. Second, we developed a novel analytical method to characterize the spatiotemporal dynamics of MWR. We identified a heterogeneous spatial distribution of reentrant pathways that correlated with the spatial distribution of cell activation frequencies. Third, we investigated the impact of topological and geometrical substrate alterations on the dynamics of MWR. We demonstrated a multi-phasic relationship between obstacle size and the fate of individual episodes. Notably, for a narrow range of sizes, obstacles appeared to play an active role in rapidly converting MWR to stable structural reentry. Our studies indicate that reentrant-pathway distributions are non-uniform in heterogeneous media (such as the atrial myocardium) and suggest a clinically measurable correlate for identifying regions of high circuit density, supporting the feasibility of patient-specific targeted ablation. Moreover, we have elucidated the key mechanisms of interaction between focal obstacles and MWR, which has implications for the use of spot ablation to treat AF as some recent studies have suggested.

After myocardial infarction, the stressed environment may cause negative cardiac remodeling. An emerging treatment option, engineered cardiac patches can be mechanically conditioned to increase alignment or electrically stimulated to enable anisotropic conduction. While proper integration with native tissue may require both stimuli, very few studies have applied both simultaneously, and only to extracted tissues. To demonstrate feasibility, a rigid electrode prototype was constructed to incorporate electrical stimulation into a commercially available mechanical conditioning system. Electrodes were assembled to fit the system’s geometry, and parameters were optimized to mimic the human heart rate. Previously, a study used 5-Azacytidine (5-Aza) to differentiate mesenchymal stem cells (MSCs) toward cardiac lineage, which was used here for proof-of-concept testing. Unexpectedly, MSCs treated with 5-Aza and electrically stimulated showed a decrease in cardiac marker troponin and an increase in MSC surface marker gene expression. In this setup, current from rigid electrodes passes through the media; however, under physiologically relevant conditions, electrical signals should propagate directly through cardiomyocytes. Therefore, a method to apply electromechanical stimulation to individual cells was explored in a point source stimulation platform. Electroconductive adhesive (ECA), a composite of silver and polydimethylsiloxane, was used to fabricate flexible elastic microelectrode arrays that provided positive and negative voltage sources to individual cells. Devices were not cytotoxic before applying an electric field; however, applied current caused electrolysis of media and cytotoxicity, even using current stimulation parameters lower than those in published studies. These findings suggest ECA electrochemical properties need more characterization and alternative materials for microelectrodes.