Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item 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.Item SYNTHESIS AND CHARACTERIZATION OF SURFACTANT-FREE, FLUORESCENT POLY(LACTIC-CO-GLYCOLIC) ACID NANOPARTICLES TARGETED TO INTERCELLULAR ADHESION MOLECULE -1(2015) TSINAS, ZOIS; Muro, Silvia; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Targeted drug delivery to endothelial cells lining the vasculature can improve treatment of many pathologies. Intercellular adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein overexpressed in many diseases, is a good determinant for endothelial targeting of drug nanoparticles (NPs). In this study we synthesized surfactant-free, FITC-labeled poly(lactic-co-glycolic) acid (PLGA) NPs coated with anti-ICAM, and used fluorescence microscopy and radiotracing to study their interaction with endothelial cells in culture and in vivo. These NPs were stable in storage conditions and degraded in conditions mimicking intracellular lysosomes. Furthermore, NPs showed specific ICAM-1 binding, which was enhanced in diseased-like conditions, followed by efficient uptake and lysosomal trafficking via the CAM-mediated pathway. Intravenous administration of NPs in mice resulted in organ-specific accumulation, most prominently the lungs. Hence, surfactant-free, FITC-labeled anti-ICAM PLGA NPs enabled the study of NP interactions with biological systems, which along with their fast degradation profile in physiological-like conditions, will guide future therapeutic applications.Item STUDYING mRNA TRANSPORT AND REGULATION OF RETROGRADE INJURY SIGNALING ASSOCIATED AXONAL TRANSCRIPT LEVELS IN THE CONTEXT OF AXONAL REGENERATION(2014) Pathak, Gunja; Shah, Sameer; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation focuses on a detailed mechanism of mRNA transport during development of hippocampal neurons, and regulation of retrograde injury signaling (RIS)-associated genes in the context of regeneration. Damaged neurons in the CNS are unable to regenerate leading to neuronal degeneration and cell death. Identifying mechanisms that promote axonal regeneration of the damaged fibers is beneficial. First, I set to explore the detailed quantification of mRNA transport during the development of hippocampal neurons. Rigorous quantitative assessment of mRNA transport concluded that mRNA transport is driven by the functional demands of the cell. I measured the velocity, directionality and the duration of mRNA particles. In the axons, net velocity was highest at day 7 in vitro, which coincides with the initial stage of synapse formation. Within dendrites, it continues to increase through day 12 in vitro coinciding with an increased duration of synaptic contact, suggesting role of protein synthesis in context of sustained synaptic connectivity. Next, I set to explore regulations of genes involved in RIS process, a process stimulated upon injury and required for axonal regeneration. Investigation of regulation of RIS associated axonal transcript levels led to development of a whole hippocampal explant culture system. The hippocampal explant culture system enabled examination of axonal gene and protein expression independent of neuronal cell bodies. The study of RIS process suggests a novel biphasic increase in axonal gene expression (1 & 24 hrs post-injury). These genes are tightly and differentially regulated contributing to early synthesis of corresponding axonal proteins in hippocampal neurons. Additionally, importin β-dependent activity at the nucleus then appears to modulate a second wave (24 hrs) of RIS-associated transcripts, which are likely to further support axonal outgrowth. These studies provide insight into a powerful set of axonal processes that may be exploited to enhance CNS regeneration and repair.Item Enzymatic Activity Preservation through Entrapment within Degradable Hydrogel Networks(2012) Mariani, Angela Marie; Kofinas, Peter; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation aimed to design and develop a "biogel;" a reproducible, abiotic, and biocompatible polymer hydrogel matrix, that prolongs enzymatic stability allowing for rapid production of biomolecules. The researched entrapment method preserves enzyme activity within an amicable environment while resisting activity reduction in the presence of increased pH environmental challenges. These biogels can be used in a number of applications including repeated production of small molecules and in biosensors. Five main objectives were accomplished: 1) Biogels capable of maintaining enzymatic functionality post-entrapment procedures were fabricated; 2) Biogel activity dependence on crosslinker type and crosslink density was determined; 3) Biogel composition effects on sustained activity after storage were compared; 4) Biogel activity dependence on charged monomer moieties was evaluated, and 5) Combined optimization knowledge gained from the first four objectives was utilized to determine the protection of enzymes within hydrogels when challenged with an increased pH above 8. Biogels were fabricated by entrapping beta-galactosidase (lactase) enzyme within acrylamide (ACR) gels crosslinked with poly(ethylene glycol) diacrylate (PEGDA, degradable through hydrolysis) or N,N'-methylenebisacrylamide (BIS, non-degradable). Initial hydrogel entrapment reduced activity to 40% in ACR/PEGDA gels, compared to a 75% reduction in initial activity of ACR/BIS biogels. Once entrapped, these enzymes resist activity reduction in the presence of environmental challenges, such as altering the pH from 7 to above 8. When biogels were challenged at a pH of 8, activity retention positively correlated to PEGDA crosslinker density; increasing from 48% to 91% retention in 30 to 40 mole % PEGDA biogels as compared to solution based control which retained only 23%. Retention of activity when perturbed from pH 7 is advantageous for biogel applications including the repeated production of desired small molecules and biosensors. Biogels with positive or negative monomer moiety functionalities were also investigated to increase enzyme-matrix interactions and enzyme stability. The researched entrapment method illustrates the potential to sterically hinder and diffusively impede enzymes from performing their function, potentially enabling the reactivation of the enzyme at a site and time dictated by the user by degrading the crosslinks of the network.Item TUBULAR PERFUSION SYSTEM BIOREACTOR FOR THE DYNAMIC CULTURE OF HUMAN MESENCHYMAL STEM CELLS(2012) Yeatts, Andrew Bryan; Fisher, John P; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In vitro culture techniques must be improved in order to increase the feasibility of cell based tissue engineering strategies. Limitations of current techniques are largely a result of the slow diffusion of molecules such as oxygen into the interior of three dimensional scaffolds in static culture. In order to enhance nutrient transport we have developed a novel bioreactor, the tubular perfusion system (TPS), to culture human mesenchymal stem cells (hMSCs) in three dimensional scaffolds. In our design, hMSCs are cultured on scaffolds tightly packed in a tubular growth chamber. Media is perfused by a peristaltic pump through the growth chamber and around the tightly packed scaffolds. In the first part of the work hMSCs are encapsulated in alginate scaffolds and results demonstrate bioreactor culture enhances late osteoblastic differentiation of hMSCs. An investigation into shear stress in the system revealed that osteogenic markers increase with increasing shear stress and that the differentiation of hMSCs is dependent on cell radial position within scaffolds. In order to enhance the ability to implant these constructs in vivo, a method to create an aggregated cell containing construct in vitro in a bioreactor system was developed. In this part of the work hMSCs are cultured in individual alginate beads in the TPS bioreactor and the beads are aggregated to form one large construct. Following this the TPS bioreactor was investigated to culture synthetic poly-L-lactic acid scaffolds which were fabricated using supercritical carbon dioxide gel drying. In addition to investigating the effects of perfusion on hMSC growth in these scaffolds, the effect of microporosity was investigated. In the final part of the work, a study was completed to determine how TPS culture influenced in vivo bone regeneration. Here alginate beads as well as synthetic PLGA/PCL constructs were used as scaffolds. Results revealed the efficacy of using the tubular perfusion system for bone tissue engineering and demonstrated increased bone formation as a result of hMSC implantation in both alginate and PLGA/PCL scaffolds. These studies highlighted the need for bioreactor culture in vitro as well as scaffolds to support in vivo tissue interaction.Item PLANT PRODUCTIVITY AND COMPETITIVE RESPONSE TO PRESCRIBED FIRE IN MID-ATLANTIC BRACKISH MARSHES(2011) Bickford, Wesley Alan; Needelman, Brian A; Weil, Raymond R; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Prescribed fire may increase productivity of some brackish marsh species. An understanding of the mechanisms behind this stimulatory effect is important for land managers to maximize the benefits to the ecosystem. I found that canopy removal is the dominant mechanism through which fire stimulates biomass production in the marshes at the Blackwater National Wildlife Refuge in Dorchester County, Maryland. The stimulatory effect of canopy removal was stronger in sedge-dominated systems than grass-dominated systems. Faster sprouting sedge species may be able to take fuller advantage of light and soil temperature increases following canopy removal than later-sprouting grass species. Results of a greenhouse study indicated that canopy removal gives sedges a competitive advantage over grasses. These studies have numerous implications for land managers using anthropogenic disturbances as a management technique. Canopy-level disturbances, such as fire may increase productivity in sedge-dominated marshes and may suppress grass species in mixed compositions.Item A DECISION SUPPORT SYSTEM FOR THE SPATIAL CONTROL OF INVASIVE BIOAGENTS(2010) Hebou, Luc; Montas, Hubert J; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A Decision Support System (DSS) is developed and applied to the spatial control of invasive bioagents, exemplified in this study by the resident Canada goose species (Branta Canadensis) in the Anacostia River system of the District of Columbia. The DSS incorporates a model of goose movement that responds to resource distribution; a twocompartment Expert System (ES) that identifies the causes of goose congregation in hotspots (Diagnosis ES) and prescribes strategies for goose population control (Prescription ES); and a Geographic Information System (GIS) that stores, analyzes, and displays geographic data. The DSS runs on an HP xw8600 64-bit Workstation running Window XP Operating System. The mathematical model developed in this study simulates goose-resource dynamics using partial differential equations - solved numerically using the Finite Element Method (FEM). MATLAB software (v. 7.1) performed all simulations. ArcGIS software (v. 9.3) produced by Environmental Systems Research Institute (ESRI) was used to store and manipulate georeferenced data for mapping, image processing, data management, and hotspot analysis. The rule-based Expert Systems (ES) were implemented within the GIS via ModelBuilder, a modular and intuitive Graphical User Interface (GUI) of ArcGIS software. The Diagnosis ES was developed in three steps. The first step was to acquire knowledge about goose biology through a literature search and discussions with human experts. The second step was to formalize the knowledge acquired in step 1 in the form of logical sentences (IF-THEN statements) representing the goose invasion diagnosis rules. Finally, in the third step, the rules were translated into decision trees. The Prescription ES was developed by following the same steps as in the development of the Diagnosis ES, the major difference being that, in this case, knowledge was acquired relative to goose control strategies rather than overpopulation causes; and additionally, knowledge was formalized based on the Diagnosis and on other local factors. Results of the DSS application indicate that high accessibility to food and water resources is the most likely cause of the congregation of geese in the critical areas identified by the model. Other causes include high accessibility to breeding and nesting habitats, and supplementary, artificial food provided by people in urban areas. The DSS prescribed the application of chemical repellents at feeding sites as a goose control strategy (GCS) to reduce the quality of the food resources consumed by resident Canada geese, and therefore the densities of geese in the infested locations. Two other prescribed GCSs are egg destruction and harvest of breeding adult geese, both of which have direct impacts on the goose populations by reducing their densities at hotspots or slowing down their increase. Enclosing small wetlands with fencing and banning the feeding of geese in urban areas are other GCSs recommended by the ES. Model simulations predicted that these strategies would reduce goose densities at hotspots by over 90%. It is suggested that further research is needed to investigate the use of similar systems for the management of other invasive bioagents in ecologically similar environments.