Fischell Department of Bioengineering Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/6628
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Item Brillouin confocal microscopy in off-axis configuration(2021) Fiore, Antonio; Scarcelli, Giuliano; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Three-dimensional Brillouin confocal microscopy is an imaging modality that correlates with mechanical properties in biological media from subcellular to tissue level. Over the years we developed new approaches to this technique that improve the spectral performance and can measure directly the local refractive index as well as the complex modulus of the sample; to achieve this goal, we probed two co-localized Brillouin scattering geometries. The confocal microscopy setting ensures three-dimensional mapping with high resolution, while the back scattering configuration allows access to the sample from the same side. For these reasons, such an instrument constitutes a new approach in investigating biological phenomena providing both local index of refraction and mechanical information with a single measurement. This technique has been improved in speed and spatial resolution in order to be applied to some specific challenging material characterization such as liquid-liquid phase separation.Item INTRACELLULAR REGULATION OF ATRIAL EXCITATION CONTRACTION COUPLING IN NORMAL AND ARRHYTHMOGENIC HEARTS(2017) Garber, Libet; Lederer, Jonathan W; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Atrial fibrillation (AF) is the most common arrhythmia with a prevalence of 1-2% of the US population and it is the most important single risk factor for an ischemic stroke. Despite decades of research, successful termination of the arrhythmia remains difficult. The challenge is in part due to our incomplete understanding of atrial myocyte Ca2+ signaling and underlying disease mechanisms. In the atria, like all cardiac tissue, the conducted action potential (AP) underlies triggering of the [Ca2+]i transient, which is responsible for activating contraction. The process that links electrical activity to Ca2+ signaling and contraction is known as excitation-contraction coupling (ECC). The objective of this dissertation is to understand the mechanism of excitation contraction coupling in atrial myocytes. To achieve this goal, we (1) developed tools to specifically study atrial cell biology, (2) we studied the role of altered Ca2+ buffering on ionic membrane currents and Ca2+ signaling, (3) we investigated the role that reactive oxygen species (ROS) plays in altered Ca2+ signaling and the morphology of the AP and (4) we measured intracellular sodium concentration ([Na+]i ) and studied Na+ and Ca2+ signaling in a transgenic murine model of AF. This work includes mathematical modeling of atrial cell electrical and Ca2+ signaling to define our quantitative understanding of the processes involved. Our results indicate that increased Ca2+ buffering plays a major role in speeding the inactivation of the L type Ca2+ current (ICa,L ). This work also shows that low concentrations of H2O2 for a brief period increases atrial Ca2+ spark rate, changes spark characteristics and decreases the duration of the AP. We quantified for the first time the [Na+]i in murine atrial cells both at rest and during field stimulation in control and transgenic mice. Our results indicate that [Na+]i is significantly lower in atrial myocytes in comparison to their ventricular counterparts, which reveal important differences in how [Na+]i is regulated in atrial cells. Moreover, our work demonstrates that [Na+]i and [Ca2+]i homeostasis are profoundly affected during AF. The results further our understanding of mechanisms that modulate excitation-contraction coupling in atrial myocytes in normal and pathophysiological conditions.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 Coarse Graining to Invesitigate Peptide-Lipid Interactions(2016) Ganesan, Sai Janani; Matysiak, Silvina; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Experimental characterization of molecular details is challenging, and although single molecule experiments have gained prominence, oligomer characterization remains largely unexplored. The ability to monitor the time evolution of individual molecules while they self assemble is essential in providing mechanistic insights about biological events. Molecular dynamics (MD) simulations can fill the gap in knowledge between single molecule experiments and ensemble studies like NMR, and are increasingly used to gain a better understanding of microscopic properties. Coarse-grained (CG) models aid in both exploring longer length and time scale molecular phenomena, and narrowing down the key interactions responsible for significant system characteristics. Over the past decade, CG techniques have made a significant impact in understanding physicochemical processes. However, the realm of peptide-lipid interfacial interactions, primarily binding, partitioning and folding of amphipathic peptides, remains largely unexplored compared to peptide folding in solution. The main drawback of existing CG models is the inability to capture environmentally sensitive changes in dipolar interactions, which are indigenous to protein folding, and lipid dynamics. We have used the Drude oscillator approach to incorporate structural polarization and dipolar interactions in CG beads to develop a minimalistic peptide model, WEPPROM (Water Explicit Polarizable PROtein Model), and a lipid model WEPMEM (Water Explicit Polarizable MEmbrane Model). The addition of backbone dipolar interactions in a CG model for peptides enabled us to achieve alpha-beta secondary structure content de novo, without any added bias. As a prelude to studying amphipathic peptide-lipid membrane interactions, the balance between hydrophobicity and backbone dipolar interactions in driving ordered peptide aggregation in water and at a hydrophobic-hydrophilic interface, was explored. We found that backbone dipole interactions play a crucial role in driving ordered peptide aggregation, both in water and at hydrophobic-hydrophilic interfaces; while hydrophobicity is more relevant for aggregation in water. A zwitterionic (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and an anionic lipid (POPS: 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine) are used as model lipids for WEPMEM. The addition of head group dipolar interactions in lipids significantly improved structural, dynamic and dielectric properties of the model bilayer. Using WEPMEM and WEPPROM, we studied membrane-induced peptide folding of a cationic antimicrobial peptide with anticancer activity, SVS-1. We found that membrane-induced peptide folding is driven by both (a) cooperativity in peptide self interaction and (b) cooperativity in membrane-peptide interactions. The dipolar interactions between the peptide and the lipid head-groups contribute to stabilizing folded conformations. The role of monovalent ion size and peptide concentration in driving lipid domain formation in anionic/zwitterionic lipid mixtures was also investigated. Our study suggest monovalent ion size to be a crucial determinant of interaction with lipid head groups, and hence domain formation in lipid mixtures. This study reinforces the role of dipole interactions in protein folding, lipid membrane properties, membrane induced peptide folding and lipid domain formation. Therefore, the models developed in this thesis can be used to explore a multitude of biomolecular processes, both at longer time-scales and larger system sizes.Item Role of BK channels in cardiac function(2015) Lai, Michael; Meredith, Andrea L; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Large-conductance voltage- and Ca2+-activated potassium (BK) channels are critical modulators of cellular excitability throughout the cardiovascular and nervous systems. The first aim of this work focuses on a novel role for BK channels in regulating cardiac pacing. Recently, BK channels were implicated in heart rate regulation, but the underlying mechanism was unclear. We hypothesized that BK channels regulate heart rate by modulating the intrinsic excitability of sinoatrial node cells (SANCs), the predominant cardiac pacemaking cells. We found that BK channel protein was expressed in SANCs, and that elimination of BK currents via pharmacological inhibition and genetic ablation reduces SANC excitability. Additionally, we characterized the properties of BK currents from SANCs. Our results indicate that BK channels are novel regulators of SANC function, and suggest that BK channels can serve as a novel therapeutic target for treating heart rate disorders. The second aim of this work focuses on the effect of single-nucleotide polymorphisms (SNPs) on BK current properties. There are approximately 100 known non-synonymous SNPs in human KCNMA1, the gene that encodes BK channels, but few have been characterized or linked with disease. We hypothesized that SNPs in KCNMA1 associated with disease, or located in domains of the BK channel gating ring that mediate Ca2+-dependent activation would alter BK current properties. We determined that the effects of SNPs on BK current properties were Ca2+ concentration-dependent. Also, we found that SNP-induced alterations in current kinetics influenced the amplitude of BK currents evoked by action potential waveforms. These results indicate that SNPs in KCNMA1 can modulate BK current properties and could contribute to the diversity of BK currents evoked by physiological stimuli.Item Boron neutron capture therapy for the treatment of prostate cancer using a boron-containing cholesteryl ester compound(2013) Gifford, Ian; Al-Sheikhly, Mohamad; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Boron neutron capture therapy (BNCT) for the treatment of prostate cancer using a boron-containing cholesteryl ester compound (BCH) was investigated. BNCT is a binary radiation therapy that relies on targeted delivery of 10B to cancer cells followed by irradiation with thermal neutrons. High-linear energy transfer (LET) α particles and 7Li nuclei released from the 10B neutron capture event result in lethal double-strand DNA breaks within a 9 μm range. Given the high density energy deposition and short range, neighboring cells without 10B remain unharmed. To evaluate the efficacy of BCH as a BNCT compound, a sample chamber within the thermal column experimental facility of the Maryland University Training Reactor (MUTR) was designed to provide a means of irradiating samples in vitro in a thermal neutron field. The thermal neutron fluence rate at 250 kW within the sample chamber is 8.7 x108 n/cm2/s with the < 3 eV neutron energy region representing 94.6% of the total neutron field. The hydrophobic BCH compound was embedded in the lipid bilayer of DPPC:cholesterol liposomes for delivery to PC-3 prostate cancer cells. Liposomes were synthesized by the thin film layer technique with high-pressure homogenization size reduction. Dynamic light scattering analysis of the liposomes yielded a mean diameter of 111.5 nm and 0.113 relative variance. Cytotoxicity of the BCH-containing liposomes was evaluated by neutral red, MTS, LDH, and colony formation assays. Boron uptake by PC-3 cells was analyzed with high-performance liquid chromatography (HPLC) and inductively coupled plasma-mass spectrometry (ICP-MS). Drug delivery conditions that minimized cytotoxic effects yielded a boron uptake of 35.2 + 4.3 μg/g cell. PC-3 cells were irradiated in the MUTR thermal column sample chamber to quantify the enhanced cell killing of the high-LET thermal neutron capture 10B reactions. PC-3 cells treated with BCH and exposed to a 9.4 x 1011 n/cm2/s thermal neutron fluence yielded a 20-25% increase in cell death compared to the untreated control.Item Biophysical Aspects of Leukocyte Transmigration through the Vascular Endothelium(2011) Stroka, Kimberly Murley; Aranda-Espinoza, Helim; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Leukocyte transmigration through the vascular endothelium is a key step in the immune response, and also in progression of the cardiovascular disease atherosclerosis. Much work has previously focused on the biological aspects of leukocyte transmigration, such as cytokine exposure, junctional protein organization in the endothelium, and signaling pathways. However, in recent years, many studies have identified links between the mechanical properties of the cellular microenvironment and cell behavior. This is relevant to the cardiovascular system in two ways: (1) it is likely that the mechanical properties of vasculature depend on both vessel size (large vessels versus microvasculature) and tissue type (soft brain versus stiffer muscle or tumor), and (2) both large vessels and microvasculature stiffen in atherosclerosis. For the first time, this dissertation provides a quantitative evaluation of the biophysical effects of vasculature stiffening on endothelial cell (EC) biomechanical properties, as well as leukocyte migration and transmigration. A novel in vitro model of the vascular endothelium was created. This model mimics physiological conditions more closely than previous models, by taking into account the flexibility of the subendothelial matrix; previous models have mostly utilized glass or plastic substrates that are much stiffer than physiological. EC monolayers were formed on extracellular matrix (ECM) protein-coated hydrogels and activated with tumor necrosis factor-α or oxidized low density lipoprotein to induce an inflammatory response. We determined that three important components of the in vitro model (cell-cell adhesion, cytokine exposure, and subendothelial matrix stiffness) have significant effects on EC biomechanical properties. Next, we showed that neutrophils are mechanosensitive, as their migration is biphasic with substrate stiffness and depends on an interplay between substrate stiffness and ECM protein amount; these results suggest that any biomechanical changes which occur in vasculature may also affect the immune response. Finally, we discovered that neutrophil transmigration increases with subendothelial matrix stiffness, and we demonstrated that this effect is due to substrate stiffness-dependent EC contractile forces. These results indicate, for the first time, that the biophysical states of the endothelium and subendothelial matrix, which likely vary depending on size, location, and health of vasculature, are important regulators of the immune response.Item 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.