Fischell Department of Bioengineering Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/6628

Browse

Search Results

Now showing 1 - 10 of 87

ENGINEERING THE LYMPH NODE MICROENVIRONMENT TO MODULATE ANTIGEN-SPECIFIC T CELL RESPONSE
(2019) Gammon, Joshua Marvin; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Vaccines and immunotherapies have provided enormous benefit to human health. However, the development of effective vaccines and immunotherapies for many diseases is hindered by challenges created by the complex pathologies of these targets. For example, in cancer the tumor microenvironment suppresses the function of tumor-specific T cells. In autoimmune diseases, lymphocytes specific for self-antigens attack self-tissue. New technologies providing more sophisticated control over immune response are needed to address these challenges. Lymph nodes (LNs) are tissues where adaptive immune responses develop. Therefore, local delivery of combinations of immune signals is a potential strategy to modulate antigen-specific T cell response for pro-immune or regulatory function. However, application of this idea is hindered since traditional administration routes provide little control over the kinetics, combinations and concentrations with which immune signals are delivered to LNs. Biomaterials have emerged as important tools to overcome these challenges as they provide unique capabilities, including co-delivery, targeting, and controlled release. The research presented here harnesses biomaterials to control immune signals present in LNs to modulate antigen-specific T cell response. In one area, intra-LN injection (i.LN) was used to deposit microparticles (MPs) encapsulating tumor-antigens, adjuvants and immunomodulators to promote tumor-specific central memory T cells. These cells display increased proliferative capacity and resistance to tumor-mediated immunosuppression. MPs encapsulating CpG, an inflammatory adjuvant, and a melanoma antigen potently expanded tumor-specific T cells. MPs delivering low doses of rapamycin – a regulatory immune signal – promoted tumor-specific central memory T cells when co-delivered with the melanoma vaccine. Another important aspect of T cell phenotype which can be modulated for therapeutic benefit is regulatory immune response to control autoimmunity. In this second area, biomaterial-based strategies were used to deliver regulatory immune signals to expand regulatory T cells (TREG) and promote immune tolerance. In one direction, liposomes were designed to deliver regulatory metabolic modulators to bias T cells. In a parallel direction, MPs encapsulating rapamycin and islet self-antigens were designed to promote tolerance in T1D. i.LN delivery of MPs expanded islet-specific TREG and inhibited disease in a mouse model of T1D. Together this work demonstrates potent and modular strategies to therapeutically modulate T cell response.
ENABLING RAPID PHENOTYPIC DETECTION OF CEPHALOSPORIN RESISTANCE BEYOND THE CENTRAL LABORATORY
(2019) Nguyen, Hieu Thuong; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The so-called bacterial “superbugs” are largely resistant to some of the most commonly prescribed antibiotics, including a drug class known as cephalosporins used to treat many hospital and community-acquired infections. This major public health threat has been acknowledged for decades by the Centers for Disease Control (CDC) as a major concern; yet, the detection of superbugs has not been made routine since standard testing practices have been limited to specialized “central” laboratories with sophisticated yet bulky and expensive equipment and highly trained personnel. As a result, the lack of simpler testing methods that can be used in everyday clinics and doctor’s offices can be viewed as a source of error contributing to incorrect antibiotic treatment and poorer patient outcomes, factors that drive even more advanced resistance, depleting our drugs or last resort. In this dissertation, we explore new strategies for simplified methods to test for cephalosporin resistance in order to give higher accessibility in the timely detection of superbugs to support the improvement of patient care. To do this, we take an organic chemistry and biochemical approach to develop new detection molecules that report resistance activity in bacteria expressing extended-spectrum β-lactamase (ESBL) enzymes, one of the most prolific resistance strategies used by superbugs. Next, we describe methods of integrating these detection molecules into practical testing methods, and detail the engineering of simpler assays that allow for rapid readout of ESBL phenotypes using commonplace laboratory plate readers, portable Raman devices, and even handheld personal glucose meters (used for diabetes monitoring) purchased from the drugstore.
Dual-Chambered Membrane Bioreactor for the Dynamic Co-Culture of Dermal Stratified Tissues
(2019) Navarro Rueda, Javier; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Every year over 11 million patients suffer severe burns worldwide. Facial burn statistics include victims of violence (warfare, acid attacks, scalding) and trauma (flame, electrical, chemical). Skin is the first barrier against external mechanical and biochemical factors, such as burning agents, and is composed of the epidermis, dermis, and hypodermis layers. When burned, skin cannot regulate temperature or fluid transport, or stop bacterial infection. Due to the importance of the skin barrier, natural healing and grafting treatments aim to quickly close the wounds with fast proliferation of fibroblasts and collagen deposition, a process that results in scarring, loss of function, and disfigurement. Tissue engineering has produced epidermis-dermis skin scaffolds for clinical use and in vitro dermal models. Throughout this work we studied 3D printing and bioreactor strategies for the simultaneous physiologic and topographic reconstruction of burnt facial skin tissues. First, we formulated a keratin-based bioink that can be used for 3D printing on a lithography-based 3D printer. Second, we implemented the keratin bioink in the production of Halofuginone-laden face masks for the improvement of contracture, scarring, and aesthetics in severe skin wound healing in an animal model. Due to lack of collagen organization and microstructural development, we introduced a novel dual-chambered (DCB) bioreactor system to study stratified tissues. For this, crosslinking density of the keratin-based hydrogels was used to fine tune the transport properties of membranes for potential use in guided tissue regeneration applications. Then, we assessed the viability of our novel DCB for co-culturing adjacent cell populations with the inclusion of a regulatory keratin membrane. Last, having studied the DCB with flat interfaces, we assessed its viability for perfusing curved interfaces. The integration of both curvature and cell populations allowed to assess the synergistic development of adjacent dermis fibroblasts and hypodermis stem-cell-derived adipocytes and evaluate whether including topography parameters would alter cell viability in the DCB. The strategies developed here elucidate on tissue stratification and aesthetic reconstruction. Furthermore, the keratin-based bioink, the engineered membranes, and the DCBs can be extended to study other stratified or gradient tissues and to fine-tune communication between cell populations in complex 3D constructs.
DEVELOPMENT OF AN ENDOTHELIAL CELL/MESENCHYMAL STEM CELL COCULTURE STRATEGY FOR THE VASCULARIZATION OF ENGINEERED BONE TISSUE.
(2019) Piard, Charlotte Marianne; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In the past two decades, remarkable progress has been made in the development of surgical techniques for bone reconstruction, significantly improving clinical outcomes. However, major reconstruction after trauma or cancer is still limited by the paucity of autologous material and donor site morbidity. Recent advances in the field of tissue engineering have generated new approaches for restoring bone defects. In spite of this progress, the necessity of suitable blood supply to ensure cell function is a major challenge in the development of more complex and functional grafts. Many investigators have successfully demonstrated the use of different strategies including growth factor delivery and in vitro coculture of ECs and MSCs to develop vascular structures. MSCs have the ability to secrete a wide range of bioactive cytokines and growth factors that can influence nearby cells via paracrine signaling. This crosstalk between ECs and MSCs is mutually beneficial, as ECs enhance osteogenic differentiation of hMSCs through direct cell-cell contact and paracrine signaling. In the native environment of cortical bone, both cell populations, osteogenic and vasculogenic, follow a unique well-defined pattern, called osteons. The goal of this proposed study was to develop a novel bio-inspired and vascularized bone construct, harvesting the synergistic effects of pro-angiogenic growth factor delivery and coculture of ECs and MSCs. To address this goal, we first developed mesoporous calcium deficient hydroxyapatite apatite microparticles, with biological properties closer to bone than commercially available hydroxyapatite, and capable of efficiently loading and sustainably releasing pro-angiogenic growth factors. We then demonstrated the successful fabrication of a novel bio-inspired 3DP fibrin-PCL composite scaffold, with mechanical strength comparable to bone. The utilization of these scaffolds in constructing osteons for bone regeneration demonstrated the promising capacity of the construct to improve neovascularization. In light of these results, we hypothesized that cell placement or patterning could play a critical role in neovascularization. Which lead us to investigate the role of distance between cell populations, introduced via 3D printing, in ECs/MSCs crosstalk. Our results suggested that controlling the distance between ECs and MSCs in coculture, using 3D printing, could influence angiogenesis.
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.
VIMENTIN AND CYTOKERATIN INTERMEDIATE FILAMENTS IN THE MECHANOBIOLOGY AND MALIGNANT BEHAVIORS OF CHORDOMA CELLS
(2018) Resutek, Lauren; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Chordoma, an aggressive tumor derived from notochordal remnants, is difficult to treat due to its proximity to the spinal cord and brain stem and its resistance to conventional treatments, such as radiation and chemotherapy. The development of effective treatments requires research at the molecular level, which presumably due to its rare diagnosis, is lacking for chordoma. Recent studies have identified potential targets for systemic therapy; however, there are currently no drugs approved by the Food and Drug Administration (FDA) to treat chordoma. One promising approach is to target the cytoskeleton, in order to stall progression and sensitize cells to chemotherapeutics. Similar to other cancers, chordoma cells co-express vimentin and cytokeratin intermediate filaments (IFs), which have both been found to play roles in cell mechanical properties and behaviors and their expression has been associated with cancer metastasis, chemoresistance, and poor prognosis. Therefore, we investigated the functional roles of vimentin and cytokeratin IFs in chordoma cells using RNA interference (RNAi). First, we examined whether cytoskeletal disruption by siRNA-mediated silencing of vimentin or cytokeratin-8 altered the chordoma phenotype. We determined that the vacuolated cytoplasm, a distinguishing feature of chordoma, was dependent on cytokeratin-8 IFs. Next, we examined the effects of vimentin and cytokeratin-8 knockdown on chordoma cell mechanics. We found that chordoma cell stiffness, traction forces, and mechanosensitivity to substrate stiffness were all dependent on vimentin IFs. These results suggest that vimentin, rather than cytokeratin, IFs play a predominant role in chordoma cell mechanobiology. Finally, we analyzed the roles of vimentin and cytokeratin-8 IFs in cellular behaviors associated with cancer progression. We demonstrated that chordoma cell invasion and expression of the biomarker sonic hedgehog were dependent on vimentin. Further, we found that decreasing vimentin expression in chordoma cells may increase their sensitivity to chemotherapeutics. Because mechanical cues are important determinants of cell function, we hypothesize this correlation is in part due to the newly discovered role of vimentin IFs in chordoma cell mechanobiology. These results elucidate novel roles of vimentin and cytokeratin-8 IFs in chordoma cells, which may assist in the development of effective treatments for chordoma.
MAPPING LANGUAGE FUNCTION AND PREDICTING CORTICAL STIMULATION RESULTS WITH INTRACRANIAL ELECTROENCEPHALOGRAPHY
(2018) Wang, Yujing; Chen, Yu; Crone, Nathan E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
To avoid post-operative language impairments after surgery for drug-resistant epilepsy, clinicians rely primarily on electrocortical stimulation mapping (ESM), but this can trigger afterdischarges, clinical seizures, or cause uncomfortable sensations. Moreover, ESM can be time-consuming and the results are usually all-or-none, complicating their interpretation. These practical limitations have long motivated spatial-temporal analysis of passive intracranial electroencephalographic (iEEG) recordings as an alternative or complementary technique that can map cortical function at all sites simultaneously, resulting in significant time savings without adverse side-effects. However, there has not yet been widespread clinical adoption of passive iEEG for pre-operative language mapping, largely because of a failure to realize the potential advantages of iEEG over ESM and other methods for language mapping. The overall goals of this dissertation were to improve and validate passive iEEG as a method for mapping human language function prior to surgical resection for epilepsy and other brain disorders. This was accomplished through three separate aims. First, a spatial-temporal functional mapping (STFM) system was developed and tested for online passive iEEG mapping, providing immediate mapping feedback to both clinicians and researchers. The system output was compared to ESM and to canonical regions of interest in the human language network. In the second aim, the STFM system was used to study the fine temporal dynamics by which Broca’s area is activated and interacts with other areas of language network during a sentence completion task. This study showed that Broca’s area plays a pivotal role in the coordination of language networks responsible for lexical selection. Finally, the third aim sought to reconcile inconsistencies between the results of STFM and ESM. Agreement between these methods has not been as good for language mapping as it has been for motor mapping, which may be due to propagation of ESM effects to cortical areas connected to the site of stimulation. We used cortico-cortical evoked potentials to estimate the effective connectivity of stimulation sites to other sites in the language network. We found that this method improved the accuracy of STFM in predicting ESM results and helped explain similarities and differences between STFM and ESM language maps.
THE APPLICATION OF MICRODEVICES FOR INVESTIGATING BIOLOGICAL SYSTEMS
(2018) Shang, Wu; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The gastrointestinal (GI) tract is a complex ecosystem with cells from different kingdoms organized within dynamically-changing structures and engaged in complex communication through a network of molecular signaling pathways. One challenge for researchers is that the GI tract is largely inaccessible to experimental investigation. Even animal models have limited capabilities for revealing the rich spatiotemporal variation in the intestine and fail to predict human responses due to genetic variation. Exciting recent advances in in vitro organ model (i.e., organ-on- chips (OOC)) based on microfluidics are offering new hope that these experimental systems may be capable of recapitulating the complexities in structure and context inherent to the intestine. A current limitation to OOC systems is that while they can recapitulate structure and context, they do not yet offer capabilities to observe or engage in the molecular based signaling integral to the functioning of this complex biological system. This dissertation focuses on developing microfluidic tools that provide access to interrogating signaling events amongst populations in the GI tract (e.g., microbes and enterocytes). First, a membrane-based gradient generator is built to establish linear and stable chemical gradients for investigating gradient-mediated behaviors of bacteria. Specifically, this platform enables the study of bacterial chemotaxis and potentially facilitates the development of genetically rewired lesion-targeted probiotics. Second, “electrobiofabrication” is coupled with microelectronics, for the first time, to create molecular-to-electronic (i.e., “molectronic”) sensors to observe and report the dynamic exchange of biochemical information in OOC systems. Last, to address the issue of poor compatibility between OOCs and sensors, we assemble OOCs with molectronic sensors in a modular format. The concept of modularity greatly reduces the system complexity and enables sensors to be built immediately before applications, avoiding functional decay of active biorecognition components after long-term device storage and use. We envision this work will “open” OOC systems for molecular measurement and interrogation, which, in turn, will expand the in vitro toolbox that researchers can use to design, build and test for the investigation of GI disease and drug discovery.
Design of Self-Assembling Nanostructures to Promote Immune Tolerance
(2018) Hess, Krystina; Jewell, Christopher M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
In autoimmune diseases, which affect more than 23 million Americans, the immune system mistakenly attacks healthy tissue. This occurs when the process that normally controls self-reactive inflammatory cells (i.e. tolerance) fails. In multiple sclerosis (MS), the myelin sheath, which insulates nerves, is recognized as a foreign antigen. Demyelination by immune cells results in serious symptoms of neurodegeneration. Current treatments for MS are not curative, but rather manage symptoms by broadly suppressing the immune system, leaving patients unable to fight infection. New therapies that are more specific and effective could greatly improve the quality of life for patients. Biomaterials offer specific advantages for generating antigen-specific tolerance, such as cargo protection, targeted delivery, and controlled release of signals. Additionally, recent reports demonstrate that materials themselves can be intrinsically immunogenic. Two promising biomaterials-based strategies for combating autoimmunity involve: 1) delivery of self-antigen with a regulatory molecule or 2) delivery of self-antigen alone. Aim 1 of this dissertation focuses on the first strategy, creating a novel delivery system for myelin peptide and GpG, an immunomodulatory oligonucleotide. This approach involves electrostatic self-assembly of the two immune signals, eliminating the need for a carrier that could exacerbate inflammation, while still offering attractive features of biomaterials, such as co-delivery. The goal is for immune cells to encounter both signals simultaneously, biasing the response towards tolerance. This work represents the first studies using self-assembled materials to target toll-like receptor signaling, recently shown to be implicated in many autoimmune diseases. Aim 2 of this dissertation is based on the second strategy above, which relies on evidence that changing the trafficking and processing of a self-antigen can impact the development of inflammation or tolerance. Quantum dots, NPs that are intrinsically fluorescent and rapidly drain to lymph nodes, can be decorated with a large and controllable number of myelin peptides. These key features of QDs were exploited to reveal that parameters of self-antigen display (i.e. dose, density) impact biodistribution and immune cell uptake, and are directly correlated to the level of tolerance induced. Together, the described nanotechnologies offer opportunities to probe important questions towards the design of antigen-specific therapies.
Optical and Thermal Systems for Automation of Point-of-Care Assays
(2018) Goertz, John; White, Ian M; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Modern medicine has detailed 70,000 different diagnoses; the 21st century challenge is bringing those diagnoses to over 7 billion people. This phenomenal feat requires precision biosensing strategies that minimize necessary training and manual effort while maximizing portability and affordability. Microfluidic strategies, both fabricated chips and paper-based devices, held the promise to facilitate point-of-care diagnostics but have been inadequate for many applications due to the trade-off between bulky pumps or limited control and complexity. This dissertation details novel strategies that control the progression of biochemical reactions with high functionality, portability, and ease-of-use. First, I will describe an amplified signaling reaction that leverages both positive and negative feedback loops to achieve optically-regulated control. This assay, termed “Peroxidyme-Amplified Radical Chain Reaction” enables naked-eye detection of catalytic reporter DNA structures at concentrations across five orders of magnitude down to 100 pM while eliminating the need for manual addition of hydrogen peroxide common to other such detection reactions. Next, I will describe the development of a platform for thermal regulation of generic reactions. To address the need for a broadly capable automation platform that provides equal utility in the lab and field alike, we recently developed “phase-change partitions”. In our system, purified waxes segregate reagents until incremental heating melts the partitions one by one, causing the now-liquid alkane to float and allowing the desired reagents to interact with the sample on demand. This tight control over reaction progression enabled us to construct hands-free detection systems for isothermal DNA amplification, heavy metal contamination, and antibiotic resistance profiling. My work has demonstrated a broadly capable suite of assay control systems with the potential to enable simple, inexpensive automation of a broad array of chemical and biological analysis across human medicine, environmental surveillance, and industrial chemical synthesis.

Fischell Department of Bioengineering Theses and Dissertations

Browse

Filters

Settings

Sort By

Results per page

Search Results