Fischell Department of Bioengineering

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13C Metabolic Flux Analysis Indicates Endothelial Cells Attenuate Metabolic Perturbations by Modulating TCA Activity
(MDPI, 2021-04-07) Moiz, Bilal; Garcia, Jonathan; Basehore, Sarah; Sun, Angela; Li, Andrew; Padmanabhan, Surya; Albus, Kaitlyn; Jang, Cholsoon; Sriram, Ganesh; Clyne, Alisa Morss
Disrupted endothelial metabolism is linked to endothelial dysfunction and cardiovascular disease. Targeted metabolic inhibitors are potential therapeutics; however, their systemic impact on endothelial metabolism remains unknown. In this study, we combined stable isotope labeling with 13C metabolic flux analysis (13C MFA) to determine how targeted inhibition of the polyol (fidarestat), pentose phosphate (DHEA), and hexosamine biosynthetic (azaserine) pathways alters endothelial metabolism. Glucose, glutamine, and a four-carbon input to the malate shuttle were important carbon sources in the baseline human umbilical vein endothelial cell (HUVEC) 13C MFA model. We observed two to three times higher glutamine uptake in fidarestat and azaserine-treated cells. Fidarestat and DHEA-treated HUVEC showed decreased 13C enrichment of glycolytic and TCA metabolites and amino acids. Azaserine-treated HUVEC primarily showed 13C enrichment differences in UDP-GlcNAc. 13C MFA estimated decreased pentose phosphate pathway flux and increased TCA activity with reversed malate shuttle direction in fidarestat and DHEA-treated HUVEC. In contrast, 13C MFA estimated increases in both pentose phosphate pathway and TCA activity in azaserine-treated cells. These data show the potential importance of endothelial malate shuttle activity and suggest that inhibiting glycolytic side branch pathways can change the metabolic network, highlighting the need to study systemic metabolic therapeutic effects.
3D ENGINEERING OF VIRUS-BASED PROTEIN NANOTUBES AND RODS: A TOOLKIT FOR GENERATING NOVEL NANOSTRUCTURED MATERIALS
(2018) Brown, Adam Degen; Culver, James N; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Technological innovation at the nanometer scale has the potential to improve a wide range of applications, including energy storage, sensing of environmental and medical signals, and targeted drug delivery. A key challenge in this area is the ability to create complex structures at the nanometer scale. Difficulties in meeting this challenge using traditional fabrication methods have prompted interest in biological processes, which provide inspiration for complex structural organization at nanometer to micrometer length scales from self-assembling components produced inexpensively from common materials. From that perspective, a system of targeted modifications to the primary amino acid structure of Tobacco mosaic virus (TMV) capsid protein (CP) has been developed that induces new self-assembling behaviors to produce nanometer-scale particles with novel architectures. TMV CPs contain several negatively charged carboxylate residues which interact repulsively with those of adjacent CP subunits to destabilize the assembled TMV particle. Here, the replacement of these negatively charged carboxylate residues with neutrally charged or positively charged residues results in the spontaneous assembly of bacterially expressed CP into TMV virus-like particles (VLPs) with a range of environmental stabilities and morphologies and which can be engineered to attach perpendicularly to surfaces and to display functional molecular patterns such as target-binding peptide chains or chemical groups for attachment of functional targets. In addition, the distinct electrostatic surface charges of these CP variants enable the higher-level coassembly of TMV and VLP into continuous rod-shaped nanoparticles with longitudinally segregated distribution of functionalities and surface properties. Furthermore, the unique, novel, environmentally responsive assembly and disassembly behaviors of the modified CPs are shown to act as simple mechanisms to control the fabrication of these hierarchically structured functional nanoparticles.
3D PRINTED MULTILAYERED / INTERFACIAL SCAFFOLDS FOR OSTEOCHONDRAL REGENERATION
(2021) choe, Robert; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Osteoarthritis is a highly prevalent rheumatic musculoskeletal disorder that primarily affects the knee joint. This disease is characterized by the progressive breakdown of the articular cartilage and remodeling of the subchondral bone in the synovial joint. Repetitive overloading perpetuates the damage to the affected cartilage and undermines the structural integrity of the osteochondral unit. Despite much research in osteochondral tissue engineering, no particular strategy has stood out as a potential alternative to conventional treatment options. One major issue that arises during osteochondral regeneration is that the defect site is exposed to a significant physiological load. To overcome these challenges, various tissue engineering strategies have been employed to design multiphasic osteochondral scaffolds that recapitulate layer-specific biomechanical properties. However, multilayered scaffolds have failed to fully satisfy the mechanical requirements to persists within the osteochondral defect. Through the use of extrusion-based bioprinting, we attempt to fabricate a biphasic osteochondral scaffold with improved load-bearing properties and a mechanically strong interface.
Abnormal coordination of upper extremity during target reaching in persons post stroke
(Springer Nature, 2023-08-08) Koh, Kyung; Oppizzi, Giovanni; Kehs, Glenn; Zhang, Li-Qun
Understanding abnormal synergy of the upper extremity (UE) in stroke survivors is critical for better identification of motor impairment. Here, we investigated to what extent stroke survivors retain the ability to coordinate multiple joints of the arm during a reaching task. Using an exoskeleton robot, 37 stroke survivors’ arm joint angles (θ) and torques (τ) during hand reaching in the horizontal plane was compared to that of 13 healthy controls. Kinematic and kinetic coordination patterns were quantified as variances of the multiple-joint angles and multiple-joint torques across trials, respectively, that were partitioned into task-irrelevant variance (TIVθ and TIVτ) and task-relevant variance (TRVθ and TRVτ). TIVθ and TRVθ (or TIVτ and TRVτ) led to consistent and inconsistent hand position (or force), respectively. The index of synergy (ISθ and ISτ) was determined as ISθ = (TIVθ - TRVθ)/(TIVθ + TRVθ) and ISτ = (TIVτ - TRVτ)/(TIVτ + TRVτ) for kinematic and kinetic coordination patterns, respectively. Both kinematic ISθ and kinetic ISτ in the stroke group were significantly lower than that of the control group, indicating stroke survivors had impaired reaching abilities in utilizing the multiple joints of the UE for successful completion of a reaching task. The reduction of kinematic ISθ in the stroke group was mainly attributed to the lower TIVθ as compared to the control group, while the reduction of kinetic ISτ was mainly due to the higher TRVτ as well as lower TIVτ. Our results also indicated that stroke may lead to motor deficits in formation of abnormal kinetic synergistic movement of UE, especially during outward movement. The findings in abnormal synergy patterns provides a better understanding of motor impairment, suggesting that impairment-specific treatment could be identified to help improve UE synergies, focusing on outward movements.
Acquired Platelet And Neutrophil Dysfunction Due To High Mechanical Shear Stress
(2022) Arias, Katherin; Wu, Zhongjun; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Heart failure (HF) is a public health burden. In the next ten years, 8 million Americans are expected to have HF. A subset of these patients will develop advanced HF. They are refractory to medical therapies and have limited treatment options, including heart transplantation or a left ventricular assist device (LVAD). Heart transplants for all advanced HF patients are impractical due to the scarcity of donors. LVAD therapy is the sole viable option for advanced HF patients as a bridge to transplant, a temporary treatment while the heart recovers, and a long-term destination therapy. Over the last two decades, significant progress in LVADs have been made through various iterations. Advances in LVADs have been due to redesign focused on lowering adverse events. However, bleeding and infections are still the most prevalent adverse complications. LVADs and other mechanical circulatory support devices induce damage to blood cells and plasma components due to the high mechanical shear stress (HMSS) generated. Therefore, there must be a link between LVAD-induced cellular damage and the adverse events experienced in LVAD patients. This dissertation aimed to investigate the relationship between cellular blood damage and LVAD-associated complications and qualify the extent of cellular damage/defects and functional alterations.The overall objective of this dissertation was to investigate the acquired cellular defects of platelets and neutrophils in blood after shear stress exposure. This objective was accomplished through in-vivo, in-vitro, and in-silico studies. The in-vivo studies examined the shear stress-induced injury of platelets in LVAD recipients and linked the adverse bleeding events (Chapter 3). The in-vitro studies explored the shear stress-induced injury of leukocytes (Chapter 4). The extent of the structural damage and functional alterations related to shear stress level and the exposure time was quantified (Chapter 5 and Chapter 6). Finally, the in-silico studies developed a simulation of leukocyte function with experimental data that was used to predict the extent of the shear stress-induced leukocyte function change (Chapter 6). The damaging effects of the high shear stress produced by mechanical circulatory support devices such as LVADs were conveyed through an integrated biological and engineering approach.
ACTUATION OF MULTIFUNCTIONAL HARD NANOPARTICLES FOR ACTIVELY CONTROLLED DRUG RELEASE
(2019) Sangtani, Ajmeeta; Delehanty, James B; Stroka, Kimberly M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Systemic drug delivery relies on repeated dosing of large concentrations of poorly targeted drug leading to off-target toxicity. Recently, nanoparticle (NP)-mediated drug delivery (NMDD) has been developed as an approach to overcome the limitations of traditional drug delivery. The unique size-dependent properties of NPs and their ability to augment the activity of attached/loaded cargos makes them attractive drug delivery vectors. NPs are classified into two categories (soft or hard depending on their material composition) and our understanding of how to load and control soft NP materials currently surpasses that of hard NPs. In this dissertation we seek to further our fundamental knowledge of hard NP-based drug delivery systems. In Aim 1 we utilize a quantum dot (QD)-cell uptake peptide complex as a central scaffold to append various responsive peptide-drug constructs in order to modulate the toxicity of one of the most widely used chemotherapeutics, doxorubicin. By doing a comparative study of four chemical linkages, we determine the role played by attachment chemistry in controlling drug release. In Aim 2, we utilize the knowledge gained from Aim 1 to develop a system capable of overcoming multidrug resistance in cancer cells, which is known to severely limit the efficacy of chemotherapeutics. Our hard NP conjugate system is unique as it is one of the few systems reported in the literature to bypass multidrug resistance pumps without the need for exogenous drugs. Finally, in Aim 3 we append a peptide for membrane targeting and a photosensitizing drug capable of generating reactive oxygen species to the QD. This multifunctional system displays augmented therapeutic efficacy of the appended photosensitizer by delivering it to the membrane of cells and controlling its actuation using energy transfer. The work described here details basic concepts for the design of “smart” hard NP materials for internally and externally-triggered, active release of surface-appended drug cargos. Additionally, we hope to elucidate the important design considerations that must be taken into account when designing hard NP systems for controlled drug delivery.
Additive Manufacturing for Recapitulating Biology in vitro and Establishing Cellular & Molecular Communication
(2023) Chen, Chen-Yu; Bentley, William E.; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Recapitulating biological systems within laboratory devices, particularly those with analytical instrumentation, has enhanced our ability to understand biology. Especially useful are systems that provide data at the length and time scales characteristic of the assembled biological systems. In this dissertation, we have employed two advanced technologies — additive manufacturing and electrobiofabrication to create systems that both recapitulate biology and provide ready access to molecular data. First, we utilized two-photon direct laser writing (DLW) and digital light processing (DLP) 3D printing to reconstruct morphologies of human gut villi. Our constructs enable small molecule diffusion through pores and enable epithelial cell growth and differentiation, as in the gastrointestinal (GI) tract. We also developed a cell/particle alignment methodology that applies a vacuum on the underside of a device to rapidly facilitate attachment to 3D printed scaffolds. These simple demonstrations of additive manufacturing show how one can better tailor geometric features of organ-on-a-chip and other in vitro models. We then added electrobiofabrication as a means create functionalized surfaces that rapidly assemble biological components, noted for their labile nature, onto devices with just an applied voltage. In one example, we show how a thiolated polyethylene glycol (PEG) can be electroassembled as a sensor interface that includes antibody binding proteins for both titer and glycan analysis. Rapid assessment of titer and glycan structure is important for biopharmaceuticals development and manufacture. While the interface and sensing methodology was performed using standard laboratory instrumentation, we show that the methodology can be streamlined and operated in parallel by incorporating into a microfluidic sensor platform. Additionally, we show how the combination of optical and electrochemical (redox) based measurements can be combined in a simplified insert that “fits” nearly any microplate reader or other fairly standardized laboratory spectrophotometric unit. We believe that by adapting transformative electrochemical analytical methods so they can augment more traditional optical techniques, we might ultimately generate devices that provide a far more comprehensive picture of the target, promoting better investigation. Specifically, we show how three important biological and chemical systems can be interrogated using both optical measurements and electrochemistry: the oxidation state of proteins including monoclonal antibodies, redox status of hydrogel materials, and electrobiofabrication and electrogenetic induction. Lastly, we demonstrate how electrobiofabrication can be used to create designer communities of bacteria — artificial biofilms — the study of which is important for understanding phenomena from infectious disease to food contamination. That is, we discovered that by varying the applied voltage, surface area, and composition of the to-be-assembled hydrogel solution, we can precisely control the intercellular environment among bacterial populations. In sum, this dissertation integrates advances in assembly, through additive manufacturing, electrobiofabrication, with advances in electrochemical analysis to bring to the fore an electronic understanding of complex biological phenomena. We believe that the capability of translating biological information into a processible digital language opens tremendous opportunities for advancing our understanding of nature’s amazing systems, potentially enabling electronic means to control her subsystems.
Adsorption Kinetic Model Predicts and Improves Reliability of Electrochemical Serotonin Detection
(MDPI, 2023-01-09) Chapin, Ashley Augustiny; Han, Jinjing; Ghodssi, Reza
Serotonin (5-HT) is a neurotransmitter involved in many biophysiological processes in the brain and in the gastrointestinal tract. Electrochemical methods are commonly used to quantify 5-HT, but their reliability may suffer due to the time-dependent nature of adsorption-limited 5-HT detection, as well as electrode fouling over repeated measurements. Mathematical characterization and modeling of adsorption-based electrochemical signal generation would improve reliability of 5-HT measurement. Here, a model was developed to track 5-HT electrode adsorption and resulting current output by combining Langmuir adsorption kinetic equations and adsorption-limited electrochemical equations. 5-HT adsorption binding parameters were experimentally determined at a carbon-nanotube coated Au electrode: KD = 7 × 10−7 M, kon = 130 M−1 s−1, koff = 9.1 × 10−5 s−1. A computational model of 5-HT adsorption was then constructed, which could effectively predict 5-HT fouling over 50 measurements (R2 = 0.9947), as well as predict electrode responses over varying concentrations and measurement times. The model aided in optimizing the measurement of 5-HT secreted from a model enterochromaffin cell line—RIN14B—minimizing measurement time. The presented model simplified and improved the characterization of 5-HT detection at the selected electrode. This could be applied to many other adsorption-limited electrochemical analytes and electrode types, contributing to the improvement of application-specific modeling and optimization processes.
ADVANCED VISION INTELLIGENT METHODS FOR FOOD, AGRICULTURAL, AND HEALTHCARE APPLICATIONS
(2021) Wang, Dongyi; Tao, Yang; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
With fast software and hardware developments, vision intelligence models have attracted great attention and showed unprecedented performance on large-scale datasets. In practice, studies are still needed to design innovative intelligence models for niche applications with limited data accessibilities in uncertain real-world scenarios. This research casts light on cutting edge vision intelligent applications that enhance the essential areas of people’s livelihood, including food, agriculture, and healthcare. First, a 2D/3D imaging system was developed to facilitate the autonomous processing of Chesapeake Bay blue crabs, as an efficient solution to current hand-picking protocols. The system integrates a semantic segmentation model to understand crab 2D morphology. It can detect crab back-fin knuckles with R2 larger than 0.995, which guides movements of a two degree-of-freedom gantry station in removing crab legs and extracting crab body cores with 2mm accuracy. The customized active laser line scanning 3D range imaging system shows high imaging accuracy (0.15mm) and is able to assist a linear actuator in removing crab chamber cartilages. Second, computer aided vision intelligent methods were applied to an emerging ophthalmologic imaging modality known as, erythrocyte mediated angiography. A novel regression-based segmentation model and a Monte Carlo based tracking method were proposed to monitor the erythrocytes in stasis and in movements. Both models displayed comparable performance to human experts. Preliminary clinical results also manifest the potential relationships between paused erythrocyte densities and primary open-angle glaucoma. To better understand retinal vessel and erythrocyte distributions, a novel network architecture, the Hard Attention Net was proposed. This network has achieved state-of-art retinal vessel segmentation performance across different ophthalmologic imaging modalities. Finally, deep learning based qualitative and quantitative analyses were applied to spectral signals for monitoring high-level status and low-level chemical properties of agricultural bioproducts. Experiments include early-stage tomato spotted wilt virus detection as well as nutrition content estimation of plant and corn kernels. By using adversarial training and feature weighting ideas, the two proposed networks were effectively trained with a limited dataset. The results of these studies show great potential for vision intelligence models for promoting applications of advanced imaging modalities and vision-guided automations in food, agricultural, and healthcare fields.
Analysis of recent segmental duplications in the bovine genome
(Springer Nature, 2009-12-01) Liu, George E; Ventura, Mario; Cellamare, Angelo; Chen, Lin; Cheng, Ze; Zhu, Bin; Li, Congjun; Song, Jiuzhou; Eichler, Evan E
Duplicated sequences are an important source of gene innovation and structural variation within mammalian genomes. We performed the first systematic and genome-wide analysis of segmental duplications in the modern domesticated cattle (Bos taurus). Using two distinct computational analyses, we estimated that 3.1% (94.4 Mb) of the bovine genome consists of recently duplicated sequences (≥ 1 kb in length, ≥ 90% sequence identity). Similar to other mammalian draft assemblies, almost half (47% of 94.4 Mb) of these sequences have not been assigned to cattle chromosomes. In this study, we provide the first experimental validation large duplications and briefly compared their distribution on two independent bovine genome assemblies using fluorescent in situ hybridization (FISH). Our analyses suggest that the (75-90%) of segmental duplications are organized into local tandem duplication clusters. Along with rodents and carnivores, these results now confidently establish tandem duplications as the most likely mammalian archetypical organization, in contrast to humans and great ape species which show a preponderance of interspersed duplications. A cross-species survey of duplicated genes and gene families indicated that duplication, positive selection and gene conversion have shaped primates, rodents, carnivores and ruminants to different degrees for their speciation and adaptation. We identified that bovine segmental duplications corresponding to genes are significantly enriched for specific biological functions such as immunity, digestion, lactation and reproduction. Our results suggest that in most mammalian lineages segmental duplications are organized in a tandem configuration. Segmental duplications remain problematic for genome and assembly and we highlight genic regions that require higher quality sequence characterization. This study provides insights into mammalian genome evolution and generates a valuable resource for cattle genomics research.
ARRHYTHMOGENESIS AND CONDUCTION PROPERTIES OF CARDIOMYOCYTES IN RESPONSE TO DYSSYNCHRONOUS MECHANICAL AND ELECTRICAL STIMULATION
(2010) Chan, Dulciana; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Many cardiac therapeutic modalities, including pacemakers, implantable cardioverter defibrillators, and cardiac resynchronization therapy devices, are used to treat abnormalities in cardiac function and conduction. Both electrical and mechanical dyssynchrony can have deleterious effects including reduced cardiac output and an increased susceptibility to cardiac arrhythmias. It is postulated that electro-mechanical dyssynchrony may contribute to the susceptibility of the heart to cardiac arrhythmias. In this study, a novel system was developed to study these effects by altering the electro-mechanical activation sequence in cultured neonatal rat cardiomyocyte monolayers by dyssynchronously stimulating the monolayers with applied electrical fields and pulsatile mechanical strain. Specifically, optical mapping was utilized to compare action potential duration and quantify arrhythmia susceptibility of cardiomyocytes subjected to pulsatile mechanical strain, electrical stimulation, and dyssynchronous electrical and mechanical stimulation. This system provides a method to evaluate changes in cardiomyocyte conduction properties due to altered electro-mechanical coupling and the subsequent impact on arrhythmogenesis.
Assembly complexity of prokaryotic genomes using short reads
(2010-01-12) Kingsford, Carl; Schatz, Michael C; Pop, Mihai
Background: De Bruijn graphs are a theoretical framework underlying several modern genome assembly programs, especially those that deal with very short reads. We describe an application of de Bruijn graphs to analyze the global repeat structure of prokaryotic genomes. Results: We provide the first survey of the repeat structure of a large number of genomes. The analysis gives an upper-bound on the performance of genome assemblers for de novo reconstruction of genomes across a wide range of read lengths. Further, we demonstrate that the majority of genes in prokaryotic genomes can be reconstructed uniquely using very short reads even if the genomes themselves cannot. The non-reconstructible genes are overwhelmingly related to mobile elements (transposons, IS elements, and prophages). Conclusions: Our results improve upon previous studies on the feasibility of assembly with short reads and provide a comprehensive benchmark against which to compare the performance of the short-read assemblers currently being developed.
Assessing the benefits of using mate-pairs to resolve repeats in de novo short-read prokaryotic assemblies
(2011-04-13) Wetzel, Joshua; Kingsford, Carl; Pop, Mihai
Background: Next-generation sequencing technologies allow genomes to be sequenced more quickly and less expensively than ever before. However, as sequencing technology has improved, the difficulty of de novo genome assembly has increased, due in large part to the shorter reads generated by the new technologies. The use of mated sequences (referred to as mate-pairs) is a standard means of disambiguating assemblies to obtain a more complete picture of the genome without resorting to manual finishing. Here, we examine the effectiveness of mate-pair information in resolving repeated sequences in the DNA (a paramount issue to overcome). While it has been empirically accepted that mate-pairs improve assemblies, and a variety of assemblers use mate-pairs in the context of repeat resolution, the effectiveness of mate-pairs in this context has not been systematically evaluated in previous literature. Results: We show that, in high-coverage prokaryotic assemblies, libraries of short mate-pairs (about 4-6 times the read-length) more effectively disambiguate repeat regions than the libraries that are commonly constructed in current genome projects. We also demonstrate that the best assemblies can be obtained by ‘tuning’ mate-pair libraries to accommodate the specific repeat structure of the genome being assembled - information that can be obtained through an initial assembly using unpaired reads. These results are shown across 360 simulations on ‘ideal’ prokaryotic data as well as assembly of 8 bacterial genomes using SOAPdenovo. The simulation results provide an upper-bound on the potential value of mate-pairs for resolving repeated sequences in real prokaryotic data sets. The assembly results show that our method of tuning mate-pairs exploits fundamental properties of these genomes, leading to better assemblies even when using an off -the-shelf assembler in the presence of basecall errors. Conclusions: Our results demonstrate that dramatic improvements in prokaryotic genome assembly quality can be achieved by tuning mate-pair sizes to the actual repeat structure of a genome, suggesting the possible need to change the way sequencing projects are designed. We propose that a two-tiered approach - first generate an assembly of the genome with unpaired reads in order to evaluate the repeat structure of the genome; then generate the mate-pair libraries that provide most information towards the resolution of repeats in the genome being assembled - is not only possible, but likely also more cost-effective as it will significantly reduce downstream manual finishing costs. In future work we intend to address the question of whether this result can be extended to larger eukaryotic genomes, where repeat structure can be quite different.
Assessment of Mechanical Cues to Enhance the Clinical Translation of Extracellular Vesicles
(2022) Kronstadt, Stephanie Marie; Jay, Steven M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Mesenchymal stem cells (MSCs) are a common source for cell-based therapies due to their innate regenerative properties. However, these cells often die shortly after injection and, if they do survive, run the risk of forming tumors. Cell-secreted nanoparticles known as extracellular vesicles (EVs) have been identified as having therapeutic effects similar to those of their parental cells without the safety risks. Specifically, MSC EVs have emerged as a promising therapeutic modality in a multitude of applications, including autoimmune and cardiovascular diseases, cancer, and wound healing. Despite this promise, low levels of naturally occurring EV cargo may necessitate repeated doses to achieve clinical benefit, countering the advantages of EVs over MSCs. The current techniques to combat low EV potency (e.g., loading external molecules or using chemicals) are not agreeable to large-scale manufacturing techniques and would substantially increase the regulatory burden associated with EV translation. Fortunately, mechanical cues within the microenvironment have potential to overcome these translational barriers as they can alter EV therapeutic effects but are also cost-effective and can be precisely manipulated in a reproducible manner. The goal of this project is to understand how these cues impact MSC EV secretion and physiological effects. We showed that flow-derived shear stress applied to MSCs seeded within a 3D-printed scaffold (i.e., the bioreactor) can significantly upregulate EV production (EVs/cell) while maintaining the in vitro pro-angiogenic effects of MSC EVs. Interestingly, we demonstrated that MSC EVs generated using the bioreactor system significantly improved wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Furthermore, for the first time, we showed that mechanical confinement of MSCs within micropillars could augment MSC EV production and bioactivity. Lastly, we demonstrated that soft substrates composed of various polydimethylsiloxane (PDMS) formulations could increase MSC EV production and activity as well. Through the work performed here, we have laid the groundwork to elucidate the relationship between cell mechanobiology and EV activity that will ultimately enable an adaptable and scalable EV therapeutic platform.
Auditory Streaming: Behavior, Physiology, and Modeling
(2011) Ma, Ling; Shamma, Shihab A; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Auditory streaming is a fundamental aspect of auditory perception. It refers to the ability to parse mixed acoustic events into meaningful streams where each stream is assumed to originate from a separate source. Despite wide interest and increasing scientific investigations over the last decade, the neural mechanisms underlying streaming still remain largely unknown. A simple example of this mystery concerns the streaming of simple tone sequences, and the general assumption that separation along the tonotopic axis is sufficient for stream segregation. However, this dissertation research casts doubt on the validity of this assumption. First, behavioral measures of auditory streaming in ferrets prove that they can be used as an animal model to study auditory streaming. Second, responses from neurons in the primary auditory cortex (A1) of ferrets show that spectral components that are well-separated in frequency produce comparably segregated responses along the tonotopic axis, no matter whether presented synchronously or consecutively, despite the substantial differences in their streaming percepts when measured psychoacoustically in humans. These results argue against the notion that tonotopic separation per se is a sufficient neural correlate of stream segregation. Thirdly, comparing responses during behavior to those during the passive condition, the temporal correlations of spiking activity between neurons belonging to the same stream display an increased correlation, while responses among neurons belonging to different streams become less correlated. Rapid task-related plasticity of neural receptive fields shows a pattern that is consistent with the changes in correlation. Taken together these results indicate that temporal coherence is a plausible neural correlate of auditory streaming. Finally, inspired by the above biological findings, we propose a computational model of auditory scene analysis, which uses temporal coherence as the primary criterion for predicting stream formation. The promising results of this dissertation research significantly advance our understanding of auditory streaming and perception.
Automated quantification and classification of human kidney microstructures obtained by optical coherence tomography
(2009) Li, Qian; Chen, Yu; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Optical coherence tomography (OCT) is a rapidly emerging imaging modality that can non-invasively provide cross-sectional, high-resolution images of tissue morphology such as kidney in situ and in real-time. Because the viability of a donor kidney is closely correlated with its tubular morphology, and a large amount of image datasets are expected when using OCT to scan the entire kidney, it is necessary to develop automated image analysis methods to quantify the spatially-resolved morphometric parameters such as tubular diameter, and to classify various microstructures. In this study, we imaged the human kidney in vitro, quantified the diameters of hollow structures such as blood vessels and uriniferous tubules, and classified those structures automatically. The quantification accuracy was validated. This work can enable studies to determine the clinical utility of OCT for kidney imaging, as well as studies to evaluate kidney morphology as a biomarker for assessing kidney's viability prior to transplantation.
The Binding Effect of Proteins on Medications and Its Impact on Electrochemical Sensing: Antipsychotic Clozapine as a Case Study
(Multidisciplinary Digital Publishing Institute (MDPI), 2017-08-01) Banis, George E.; Winkler, Thomas; Barton, Patricia; Chocron, Sheryl E.; Kim, Eunkyoung; Kelly, Deanna L.; Payne, Gregory F.; Ben-Yoav, Hadar; Ghodssi, Reza
Clozapine (CLZ), a dibenzodiazepine, is demonstrated as the optimal antipsychotic for patients suffering from treatment-resistant schizophrenia. Like many other drugs, understanding the concentration of CLZ in a patient’s blood is critical for managing the patients’ symptoms, side effects, and overall treatment efficacy. To that end, various electrochemical techniques have been adapted due to their capabilities in concentration-dependent sensing. An open question associated with electrochemical CLZ monitoring is whether drug–protein complexes (i.e., CLZ bound to native blood proteins, such as serum albumin (SA) or alpha-1 acid-glycoprotein (AAG)) contribute to electrochemical redox signals. Here, we investigate CLZ-sensing performance using fundamental electrochemical methods with respect to the impact of protein binding. Specifically, we test the activity of bound and free fractions of a mixture of CLZ and either bovine SA or human AAG. Results suggest that bound complexes do not significantly contribute to the electrochemical signal for mixtures of CLZ with AAG or SA. Moreover, the fraction of CLZ bound to protein is relatively constant at 31% (AAG) and 73% (SA) in isolation with varying concentrations of CLZ. Thus, electrochemical sensing can enable direct monitoring of only the unbound CLZ, previously only accessible via equilibrium dialysis. The methods utilized in this work offer potential as a blueprint in developing electrochemical sensors for application to other redox-active medications with high protein binding more generally. This demonstrates that electrochemical sensing can be a new tool in accessing information not easily available previously, useful toward optimizing treatment regimens.
Bio-templated Substrates for Biosensor Applications
(2013) Fu, Angela Li-Hui; Kofinas, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Nanopatterning of materials is of particular interest for applications in biosensors, microfluidics, and drug delivery devices. In biosensor applications there is a need for rapid, low cost, and durable system for detection. This dissertation aims to investigate methods to pattern nanostructured surfaces using virus particles as templates. The virus species used in these experiments is a cysteine modified tobacco mosaic virus. The first project utilized the lamellar microphase separation of a block copolymer to pattern the virus particles. Although microphase separation of the poly(styrene-b-2-vinylpyridine) (PS-P2VP) into lamellae was confirmed, specificity of the viruses to the gold doped block of the polymer could not be achieved. Single virus particles lay across multiple lamellae and aggregated in side-to-side and head-to-tail arrangements. The second project studied the effect of a surfactant on virus assembly onto a gold chip. The experiments included placing a gold chip in virus solutions with varying triton concentrations (0-0.15%), then plating the virus particles with a metal. Results showed that as the triton concentration in the virus solution increases, the virus density on the surface decreases. The gold coated virus particles were applied to Surface Enhanced Raman Spectroscopy (SERS) detection in the final project. SERS is of interest for biosensor applications due to its rapid detection, low cost, portability, and label-free characteristics. In recent years, it has shown signal enhancement using gold, silver, and copper nanoparticles in solutions and on roughened surfaces. The gold plated virus surfaces were tested as SERS substrates using R6G dye as the analyte. An enhancement factor (EF) of 10^4 was seen in these samples versus the non-SERS substrate. This corresponded to the sample with 0.05% triton in the virus solution which showed the most intersection points between the virus particles and the most uniform coverage of the viruses on the surface. This value is lower than that of previous studies; however, future work may be performed to optimize conditions to achieve the highest signal possible.
Biodegradable Prussian blue nanoparticles for photothermal immunotherapy of advanced cancers
(2015) Cano-Mejia, Juliana; Fernandes, Rohan; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Multifunctional nanoparticles represent a class of materials with diverse therapy and imaging properties that can be exploited for the treatment of cancers that have significantly progressed or advanced, which are associated with a poor patient prognosis. Here, we describe the use of biodegradable Prussian blue nanoparticles (PBNPs) in combination with anti-CTLA-4 checkpoint blockade immunotherapy for the treatment of advanced cancers. Our nanoparticle synthesis scheme yields PBNPs that possess pH-dependent intratumoral stability and photothermal therapy (PTT) properties, and degrade under mildly alkaline conditions mimicking the blood and lymph. Studies using PBNPs for PTT in a mouse model of neuroblastoma, a hard-to-treat cancer, demonstrate that PTT causes rapid reduction of tumor burden and growth rates, but results in incomplete responses to therapy and tumor relapse. Studies to elucidate the underlying immunological responses demonstrate that PTT causes increased tumor infiltration of lymphocytes and T cells and a systemic activation of T cells against re-exposed tumor cells in a subset of treated mice. PBNP-based PTT in combination with anti-CTLA-4 immunotherapy results in complete tumor regression and long-term survival in 55.5% of neuroblastoma tumor-bearing mice compared to only 12.5% survival in mice treated with anti-CTLA-4 alone and 0% survival both in mice treated with PTT alone, or remaining untreated. Further, all of the combination therapy-treated mice exhibit protection against tumor rechallenge indicating the development of antitumor immunity as a consequence of therapy. Our studies indicate the immense potential of our combination photothermal immunotherapy in improving the prognosis and outlook for patients with advanced cancers.
Bioelectronic Sensor for Cellular Assays Using Polyelectrolyte Multilayer-Modified Electrodes
(2008-04-25) Mijares, Geraldine; DeVoe, Donald L; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Cell-based impedance biosensors provide non-invasive, quantitative, and instantaneous detection of cellular responses to applied stimuli. Extracellular matrix proteins, which degrade over time, are commonly used as cell adhesion promoters on planar electrodes, but decrease the lifetime of biosensors. In this work, the feasibility of using non-biological polyelectrolyte multilayers (PEMs) to facilitate cell attachment on titanium-tungsten alloy/gold electrodes for cell assays is investigated. The PEMs-modified electrode system is modeled as an equivalent electrical circuit and the addition of cells to the system is defined by their electrical properties. Electrode performance is characterized by cyclic voltammetry and impedance spectroscopy. The electrodes are found to have the ability to specifically probe non-faradaic processes and show a 15% increase in impedance due to cell proliferation. This thesis work demonstrates the use of PEMs-modified electrodes for the continuous monitoring of cell proliferation and for the future application of probing cell confluency in microfluidic cytotoxicity assays.

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