A. James Clark School of Engineering
Permanent URI for this communityhttp://hdl.handle.net/1903/1654
The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item Functionalized Nanoparticles for the Controlled Modulation of Cellular Behavior(2023) Pendragon, Katherine Evelyn; Fisher, John; Delehanty, James; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The ability to control cellular behavior at the single-cell level is of great importance for gaining a nuanced understanding of cellular machinery. This dissertation focuses on the development of novel hard nanoparticle (NP) bioconjugate materials, specifically gold nanoparticles (AuNPs) and quantum dots (QDs), for the controlled modulation of cellular behavior. These hard NPs offer advantages such as small size on the order of 1 – 100 nm, high stability, unique optical properties, and the ability to load cargo on a large surface area to volume ratio, making them ideal tools for understanding and controlling cell behavior. In Aim 1, we demonstrate the use of AuNPs to manipulate cellular biological functions, specifically the modulation of membrane potential. We present the conception of anisotropic-shaped AuNPs, known as gold nanoflowers (AuNFs), which exhibit broad absorption extending into the near-infrared region of the spectrum. We demonstrate the effectiveness of utilizing the plasmonic properties AuNFs for inducing plasma membrane depolarization in rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells. Importantly, this is achieved with temporal control and without negatively impacting cellular viability. Aim 2 explores the use of QDs as an optical, trackable scaffold for the multivalent display of growth factors, specifically erythropoietin (EPO), for the enhanced induction of protein expression of aquaporin-4 (AQPN-4) within human astrocytes. This results in enhanced cellular water transport within human astrocytes, a critical function in the brain's glymphatic system. We show that EPO-QD-induced augmented AQPN-4 expression does not negatively impact astrocyte viability and augments the rate of water efflux from astrocytes by approximately two-fold compared to cells treated with monomeric EPO, demonstrating the potential of EPO-NP conjugates as research tools and prospective therapeutics for modulating glymphatic system function. Overall, the body of work presented in this dissertation develops new NP tools, namely solid anisotropic AuNFs and growth factor-delivering QDs, for the understanding and control of cell function. These new functional nanomaterials pave the way for the continued development of novel NP-based tools for the precise modulation of cellular physiology.Item 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.Item Metabolic Flux Analysis for Metabolic Engineering of Marine Organisms(2018) Quinn, Andrew Higgins; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)We explored the metabolic pathways in two industrially relevant marine microorganisms to understand their core metabolic capabilities. It is necessary to track how an organism distributes organic building blocks throughout its metabolic pathways so that we can devise strategies to alter its metabolism and reroute substantial metabolic flux towards target compound(s). Though we cannot measure intracellular metabolic fluxes directly, we can retro-biosynthetically calculate them by supplying substrates labeled with non-radioactive isotopes to an organism. We then measure the resulting isotope labeling patterns of metabolites and calculate the fluxes that produced them. We addressed three goals with our research, (i) resolving questions surrounding organic carbon metabolism in the diatom Phaeodactylum tricornutum (P. tricornutum), (ii) identifying reactions in a putative photosynthetic carbon concentrating mechanism in P. tricornutum and (iii) mapping central carbon metabolism of the cellulolytic aerobe Saccharophagus degradans (S. degradans). Towards goal (i) we show that P. tricornutum predominantly consumes glucose, as opposed to atmospheric CO2, under mixotrophic conditions using the Entner-Doudoroff (ED) glycolytic pathway instead of the more common Embden-Meyerhof-Parnas pathway (EMP). We utilized metabolic flux analysis (MFA) to discover that acetate is metabolized for energy production instead of for biomass formation during mixotrophic growth on CO2 and acetate. Finally, we developed a method for measuring isotopic labeling in polyunsaturated fatty acids via gas chromatography-mass spectrometry (GC-MS), and demonstrated its utility in resolving outstanding questions about glucose metabolism by P. tricornutum. Towards goal (ii) we utilized isotope labeling and gene silencing in combination to identify pyruvate carboxylase as a key enzyme in a C4 carbon concentrating mechanism in P. tricornutum, while also ruling out phosphoenolpyruvate carboxylase as a key enzyme in the pathway. Towards goal (iii) we present 13C-MFA of aerobic consumption of glucose, xylose, and cellobiose by S. degradans. This is the first reported MFA of cellobiose metabolism and one of only a handful analyzing xylose metabolism in an aerobic microorganism.Item OPTIMIZATION OF RECOMBINANT PROTEIN EXPRESSION FOR CELL-PENETRATING PEPTIDE FUSIONS TO PROTEIN CARGO(2017) Adhikari, Sayanee; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Recombinant production of cell-penetrating peptides (CPPs) as fusions to protein “cargo” leads to low yields for some CPP-cargo fusions; thus, ways to enhance the recombinant expression of peptide-cargo fusions need to be identified. We optimized expression conditions for fusions of five CPPs (NPFSD, pVEC, SynB, histatin-5 and MPG) to the cargo proteins biotin carboxyl carrier protein (BCCP), maltose binding protein (MBP) and green fluorescent protein GFP. Glutathione-S-transferase was incorporated as a fusion partner to improve expression. In general, expression at 37 oC for 6 h and 10 h led 2 to the highest levels of expression for the different CPP-cargo constructs. The fusion of histatin-5 to GFP was purified, and its translocation into the fungal pathogen Candida albicans was studied. The purified protein translocated into the nearly 3% of C. albicans cells. These results provide the foundation for future studies to improve translocation of varied CPP-cargo fusions into C. albicans cells.Item Investigation of Vesicular-Mediated Transport of Intercellular Adhesion Molecule-1-Targeted Carriers for Treatment of Lysosomal Storage Disorders(2017) Manthe, Rachel Lee; Muro, Silvia; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Numerous cellular processes and therapeutic interventions rely on vesicular-mediated endocytosis to gain entry into cells and sub-cellular compartments, as well as for transcellular transport across biological barriers such as found at the blood-brain interface. Yet, endocytic behavior can be altered in disease, representing an additional hurdle in the design of effective therapeutic strategies. Lysosomal storage disorders (LSDs), characterized by lysosomal accumulation of undigested substrates as a result of deficient enzymatic activity, illustrate this paradigm. Currently, intravenous infusion of recombinant lysosomal enzymes to replace those deficient is the standard clinical approach for these disorders. However, clathrin-mediated endocytosis utilized by replacement enzymes for cellular uptake and lysosomal trafficking is altered, thereby impacting treatment efficacy as recently demonstrated in acid sphingomyelinase-deficient type A Niemann-Pick disease (NPD). Therefore, alternative means to bypass defunct routes is warranted. Therapeutic delivery via polymer nanocarriers targeting intercellular adhesion molecule-1 (anti-ICAM NCs), a cell-surface molecule overexpressed in endothelial and subjacent tissue cells during inflammation, such as in LSDs, represents a viable option since it permits uptake, intra- and transcellular transport via a unique endocytic route called the cell adhesion molecule (CAM) pathway. In this dissertation, cell culture and animal models were used to examine the (1) endocytic activity of the CAM pathway and other clathrin-independent routes in type A NPD, (2) role of targeting valency (i.e., density of ICAM-1-targeting molecules on the NC surface) in regulating the CAM pathway, and (3) effects induced via engagement of ICAM-1 on cells by anti-ICAM NCs. The results herein demonstrate the CAM pathway is more active in diseased cells compared to other classical endocytic pathways, making it the most amenable route for therapeutic enzyme replacement. Further, modulating targeting valency of NCs optimized this strategy for enhanced enzyme delivery to the brain, a target organ for type A NPD. Lastly, anti-ICAM NCs attenuated endothelial release of soluble ICAM-1, an inflammatory regulator, representing a secondary benefit of this system. Overall, this work validates utility of anti-ICAM NCs for enzyme replacement to treat NPD and likely other LSDs, and provides insight into biological processes and design parameters that influence the therapeutic efficacy of targeted drug carriers.Item THE ROLE OF THE MECHANICAL ENVIRONMENT ON CANCER CELL TRANSMIGRATION AND MRNA LOCALIZATION(2016) Hamilla, Susan M.; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Most cancer-related deaths are due to metastasis formation, the ability of cancer cells to break away from the primary tumor site, transmigrate through the endothelium, and form secondary tumors in distant areas. Many studies have identified links between the mechanical properties of the cellular microenvironment and the behavior of cancer cells. Cells may experience heterogeneous microenvironments of varying stiffness during tumor progression, transmigration, and invasion into the basement membrane. In addition to mechanical factors, the localization of RNAs to lamellipodial regions has been proposed to play an important part in metastasis. This dissertation provides a quantitative evaluation of the biophysical effects on cancer cell transmigration and RNA localization. In the first part of this dissertation, we sought to compare cancer cell and leukocyte transmigration and investigate the impact of matrix stiffness on transmigration process. We found that cancer cell transmigration includes an additional step, ‘incorporation’, into the endothelial cell (EC) monolayer. During this phase, cancer cells physically displace ECs and spread into the monolayer. Furthermore, the effects of subendothelial matrix stiffness and endothelial activation on cancer cell incorporation are cell-specific, a notable difference from the process by which leukocytes transmigrate. Collectively, our results provide mechanistic insights into tumor cell extravasation and demonstrate that incorporation into the endothelium is one of the earliest steps. In the next part of this work, we investigated how matrix stiffness impacts RNA localization and its relevance to cancer metastasis. In migrating cells, the tumor suppressor protein, adenomatous polyposis coli (APC) targets RNAs to cellular protrusions. We observed that increasing stiffness promotes the peripheral localization of these APC-dependent RNAs and that cellular contractility plays a role in regulating this pathway. We next investigated the mechanism underlying the effect of substrate stiffness and cellular contractility. We found that contractility drives localization of RNAs to protrusions through modulation of detyrosinated microtubules, a network of modified microtubules that associate with, and are required for localization of APC-dependent RNAs. These results raise the possibility that as the matrix environment becomes stiffer during tumor progression, it promotes the localization of RNAs and ultimately induces a metastatic phenotype.Item The Effect of Zinc in Carbon Concentrating Mechanisms in Phaeodactylum tricornutum(2015) Zhou, Nairui; Sriram, Ganesh; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Photosynthesis is crucial for life but is a slow process because the CO2 concentration near the principal carbon-assimilation enzyme RuBisCO is extremely low. Very few plants and algae perform a carbon-concentrating mechanism (CCM) to overcome the insufficiency, which are classified into biophysical and biochemical (C4) mechanism. The enzyme CA catalyzes the reversible dehydration of HCO3- to CO2 in biophysical CCMs and its active site contains a Zn2+. In this study, we hypothesized that Zn2+ availability can impact CCMs and therefore investigated the effect of Zn2+ availability on photosynthetic metabolism in a unicellular marine diatom Phaeodactylum tricornutum. P. tricornutum has a sequenced genome and can conduct both biophysical and C4 CCMs. We observed that Zn2+ has a significant effect on cell growth rate but no significant interference on intracellular metabolism, suggesting no essential compensation of C4 CCMs for biophysical CCMs even at low CA activity anticipated at low Zn2+ concentration.Item A Proposed Mechanical-Metabolic Model of the Human Red Blood Cell(2014) Oursler, Stephen Mark; Solares, Santiago D; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The theoretical modeling and computational simulation of human red blood cells is of interest to researchers for both academic and practical reasons. The red blood cell is one of the simplest in the body, yet its complex behaviors are not fully understood. The ability to perform accurate simulations of the cell will assist efforts to treat disorders of the cell. In this thesis, a computational model of a human red blood cell that combines preexisting mechanical and metabolic models is proposed. The mechanical model is a coarse-grained molecular dynamics model, while the metabolic model considers the set of chemical reactions as a system of first-order ordinary differential equations. The models are coupled via the connectivity of the cytoskeleton with a novel method. A simulation environment is developed in MATLAB® to evaluate the combined model. The combined model and the simulation environment are described in detail and illustrated in this thesis.Item Clinorotation time-lapse microscopy for live-cell assays in simulated microgravity(2013) Yew, Alvin G.; Hsieh, Adam; Atencia, Javier; Mechanical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)To address the health risks associated with long-term manned space exploration, we require an understanding of the cellular processes that drive physiological alterations. Since experiments in spaceflight are expensive, clinorotation is commonly used to simulate the effects of microgravity in ground experiments. However, conventional clinostats prohibit live-cell imaging needed to characterize the time-evolution of cell behavior and they also have limited control of chemical microenvironments in cell cultures. In this dissertation, I present my work in developing Clinorotation Time-lapse Microscopy (CTM), a microscope stage-amenable, lab-on-chip technique that can accommodate a wide range of simulated microgravity investigations. I demonstrate CTM with stem cells and show significant, time-dependent alterations to morphology. Additionally, I derive momentum and mass transport equations for microcavities that can be incorporated into various lab-on-chip designs. Altogether, this work represents a significant step forward in space biology research.Item Cytoskeletal Mechanics and Mobility in the Axons of Sensory Neurons(2011) Chetta, Joshua; Shah, Sameer B; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The axon is a long specialized signaling projection of neurons, whose cytoskeleton is composed of networks of microtubules and actin filaments. The dynamic nature of these networks and the action of their associated motor and cross-linking proteins drives axonal growth. Understanding the mechanisms that control these processes is vitally important to neuroregenerative medicine and in this dissertation, evidence will be presented to support a model of interconnectivity between actin and microtubules in the axons of rat sensory neurons. First, the movement of GFP-actin was evaluated during unimpeded axonal outgrowth and a novel transport mechanism was discovered. Most other cargoes in the axon are actively moved by kinesin and dynein motor proteins along stationary microtubules, or are moved along actin filaments by myosin motor proteins. Actin, however, appears to be collected into short-lived bundles that are either actively carried as cargoes along other actin filaments, or are moved as passive cargoes on short mobile microtubules. Additionally, in response to an applied stretch, the axon does not behave as a uniform visco-elastic solid but rather exhibits local heterogeneity, both in the instantaneous response to stretch and in the remodeling which follows. After stretch, heterogeneity was observed in both the realized strain and long term reorganization along the length of the axon suggesting local variation in the distribution and connectivity of the cytoskeleton. This supports a model of stretch response in which sliding filaments dynamically break and reform connections within and between the actin and microtubule networks. Taken together, these two studies provide evidence for the mechanical and functional connectivity between actin and microtubules in the axonal cytoskeleton and suggest a far more important role for actin in the development of the peripheral nervous system. Moreover this provides a biological framework for the exploration of future regenerative therapies.