Theses and Dissertations from UMD
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New submissions to the thesis/dissertation collections are added automatically as they are received from the Graduate School. Currently, the Graduate School deposits all theses and dissertations from a given semester after the official graduation date. This means that there may be up to a 4 month delay in the appearance of a give thesis/dissertation in DRUM
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Item CO-CULTURE OF BONE MARROW STROMAL CELLS AND CHONDROCYTES FOR BONE TISSUE ENGINEERING: MICROARRAY STUDY OF CHONDROCYTE SECRETED FACTORS(2011) Janardhanan, Sathyanarayana; Fisher, John P; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tissue engineering refers to the assembly of biomaterials, cells and signaling molecules to develop functional tissues based on strategies derived from developmental processes. Cells play a crucial role, in that they can secrete a library of molecules, not entirely characterized in the laboratory, and yet provide repeatable results during in vitro experiments. Under conditions of co-culture with mesenchymal stem cells, the underlying biology of chondrocytes can elucidate the signal expression during the early bone development process called endochondral ossification. This interaction is tightly regulated in chondrocytes and results in the recruitment and differentiation of mesenchymal stem cells (MSCs) into osteoblasts. We executed a co-culture system, to observe the potential of alginate encapsulated bovine articular cartilage chondrocytes to induce osteogenic differentiation of bovine bone marrow stromal cells and to observe the interaction on a global scale by making use of the microarray platform. We identified certain genes expressed by chondrocytes that show substantial activity in co-culture systems such as versican (VCAN), secreted frizzled related protein 1 (SFRP1), matrix metallopeptidase 13 (MMP13), extracellular matrix protein 1 ( ECM1) and collagen type 1 ( Col1A1, Col1A2).Item ALTERING THE AI-2 MEDIATED QUORUM SENSING CIRCUITRY TO QUENCH BACTERIAL COMMUNICATION NETWORKS(2011) Roy, Varnika; Bentley, William E; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The emergence of antibiotic resistant bacteria poses a global threat to human health and has been classified as a clinical super-challenge of the 21st century. This has necessitated research on new antimicrobials that inhibit bacterial virulence by mechanisms other than those that target bacterial growth or viability. Such approaches have been reported to pose less evolutionary pressure on bacteria to evolve and become resistant to antibiotics. Bacterial cell-cell communication, termed quorum sensing (QS), is mediated by signatures of small molecules. QS via these small molecules has been linked to numerous undesirable bacterial phenotypes such as biofilm formation, onset of pathogenicity, triggering of virulence genes etc. The small signaling molecules represent targets for intercepting bacterial communication (and their resultant undesirable phenotypes). We have devised two strategies that interrupt bacterial communication in multispecies bacterial cultures by targeting the interspecies signaling molecule autoinducer-2 (AI-2), which is produced or recognized by over 70 species of bacteria. Our first approach is to bring the native intracellular AI-2 signal processing mechanisms to the extracellular surroundings to quench the QS response of bacteria. Specifically we deliver the Escherichia coli AI-2 kinase, LsrK, to E. coli populations ex vivo and phosphorylate and degrade the extracellular AI-2. This significantly attenuates the native QS response in E. coli. Similar results are obtained in a tri-species synthetic ecosystem comprising E. coli, Salmonella typhimurium and Vibrio harveyi. In our second quenching strategy, we explore a panel of small synthetic molecules that are analogs of AI-2 (C1-alkyl analogs). The analogs are observed to cause species-specific and cross-species quorum quenching in the tri-species synthetic ecosystems of the aforementioned strains. Some of the AI-2 analogs quench pyocyanin (toxin production) in the opportunistic pathogen Pseudomonas aeruginosa. Based on these observations, I used analog cocktails to quench QS en masse in assembled synthetic ecosystems. Finally, I tested the efficiency of the analogs in quenching pathogenic phenotypes such as biofilm formation in E. coli. The analogs inhibit biofilm formation and act in concert with antibiotics to reduce biofilm formation even further. Our results suggest entirely new modalities for interrupting or tailoring the networks of communication among bacteria and identifying drug targets to develop the next generation of antimicrobials based on QS.Item LOCAL AND GLOBAL GENE REGULATION ANALYSIS OF THE AUTOINDUCER-2 MEDIATED QUORUM SENSING MECHANISM IN ESCHERICHIA COLI(2011) Byrd, Christopher Matthew; Bentley, William E; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The term `quorum sensing' (QS) is used to define a population density based communication mechanism which uses chemical signal molecules called autoinducers to trigger unique and varied changes in gene expression. Although several communication methods have been identified in bacteria that are unique to a particular species, one type of signal molecule, autoinducer-2 (AI-2) is linked to interspecies communication, indicating its potential as a universal signal for cueing a QS response among multiple bacterial types. In E. coli, AI-2 acts as an effector by binding to the QS repressor LsrR. As a result, LsrR unbinds and relieves repression of the lsr regulon, stimulating a subsequent QS gene expression cascade. In this dissertation, LsrR structure and in vitro binding activity are examined. Genomic binding and DNA microarray analyses are conducted and three novel sites putatively regulated by LsrR, yegE-udk, mppA and yihF, are revealed. Two cAMP receptor protein (CRP) binding locations in intergenic region of the lsr regulon are also confirmed. The role of each CRP site in divergent expression is qualified, indicating the lsr intergenic region to be a class III CRP-dependent promoter. Also, four specific DNA binding sites for LsrR in the lsr intergenic region are proposed, and reliance upon simultaneous binding to these various sites and the resulting effects on LsrR repression is presented. Finally, a complex model for regulation of the lsr regulon is depicted incorporating LsrR, CRP, DNA looping, and a predicted secondary layer of repression by an integration host factor (IHF)-like protein. Further understanding of this QS genetic mechanism may potentially be used for inhibiting bacterial proliferation and infection, modifying the natural genetic system to elicit alternate desired responses, or extracted and applied to a highly customizable and sensitive in vitro biosensor.Item Oxygen Measurement During Cell Culture: From Multiwell Plates to Microfluidic Devices(2011) Thomas, Peter Chung; Forry, Samuel P; Raghavan, Srinivasa R; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Oxygen is an important regulator of normal cell behavior. Proper supply of oxygen is required to maintain ATP production, while perturbation of oxygen supply alters cell behavior and leads to tissue damage and cell death. In vivo, cells are exposed to a mean partial pressure of oxygen between 0.03 to 0.09 atm that is tissue specific. In contrast, conventional cell cultures are routinely performed at an atmospheric oxygen level of 0.21 atm. The disparity between in vivo and in vitro oxygen levels have been shown to affect cell viability, growth and differentiation. Continuous measurements and control of oxygen levels are thus critical to maintaining proper cell behavior. Current methods of oxygen measurement are invasive, difficult to integrate with microscopy and lack imaging capabilities. To improve the current state of measurements, we have developed a new non-invasive oxygen sensor for in vitro cell culture. The sensor was prepared by incorporating a porphyrin dye, Pt(II) meso-Tetra(pentafluoro-phenyl)porphine (PtTFPP), into gas permeable poly(dimethylsiloxane) (PDMS) thin films. The response of the sensor to oxygen followed the linear Stern-Volmer equation and demonstrated an order of magnitude higher sensitivity compared to other sensors (KSV = 548 ± 71 atm-1). A multilayer design created by sandwiching the PtTFPP-PDMS with a thin film of Teflon AF followed by a second layer of PDMS effectively mitigated against cytotoxicity effects and provided a suitable substrate for cell attachment. To demonstrate the utility of the sensor, oxygen measurements were made continuously with NIH 3T3 mouse fibroblast cells. The oxygen levels were found to decrease as a result of oxygen consumption by the cells. Using Fick's law, the data was analyzed and a per-cell oxygen consumption rate for the 3T3 fibroblasts was calculated. In addition, cells were clearly visualized on the sensor demonstrating the ability to integrate with phase-contrast and fluorescence microscopy. Next, human hepatocellular carcinoma HepG2 were cultured on the oxygen sensor and continuous oxygen measurements showed a drastic decrease in oxygen level such that the cells were exposed to hypoxic conditions within 24 h. The per-cell oxygen consumption rate for HepG2 was determined to be 30 times higher than the 3T3 fibroblasts, confirming the high metabolic nature of these cells. At high densities, oxygen flux measurements showed an asymptotic behavior reaching the theoretical maximum of the culture condition. When the oxygen diffusion barrier was reduced, the oxygen flux increased, demonstrating insufficient oxygenation for HepG2 at these densities. In routine culture, HepG2 adhere to their neighboring cells which results in formation of cell clusters. Oxygen measurement confirmed the presence of oxygen gradient across the cell clusters with the lowest oxygen levels observed in the middle. Finally, we successfully integrated the oxygen sensor into microfluidic systems. The sensor provided real-time non-invasive measurements of oxygen levels on-chip. To regulate the oxygen levels in the device, water with different dissolved oxygen concentrations was used instead of gas. This method successfully mitigated the problems of pervaporation associated with previous devices. Physiologically relevant oxygen levels and oxygen gradients were easily generated on the device and the results showed excellent agreement with numerical simulations.Item 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.Item Desing of Click Hydrogels for Cell Encapsulation(2011) Breger, Joyce; Wang, Nam Sun; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The long-term stability of ionically crosslinked alginate hinders the development of a bioartificial pancreas for the treatment of Type I Diabetes. Ionically crosslinked alginate with divalent cations is traditionally utilized to encapsulate islets of Langerhans serving as a protective barrier between the host's immune system and the donor islets of Langerhans. However, due to ion exchange with monovalent ions from the surrounding serum, alginate degrades exposing donor tissue to the host's immune system. The overall goal of this dissertation was to explore the possibility of utilizing `click' chemistry to introduce covalent crosslinking in alginate for therapeutic cell encapsulation. `Click' chemistry is customarily defined as the Cu (I) catalyzed reaction between an azide and alkyne to form a 1,2,3 triazole ring. To achieve the goal of covalently crosslinked polysaccharides, the following aims were determined: (1) synthesis and characterization of functionalized polysaccharides (alginate and/or hyaluronic acid) with alkyne or azide end groups; (2) measurement and comparison of the stability and transport properties of covalently crosslinked alginate hydrogels to that of ionically crosslinked alginate hydrogels; (3) determination of the inflammatory potential and cytotoxicity of these functionalized polysaccharides and `click' reagents by employing RAW264.7, a murine macrophage cell line under various simulated inflammatory states (with or without endotoxin, with or with out the inflammatory cytokine gamma-interferon); (4) optimization of the `click' reaction for therapeutic cell encapsulation utilizing RIN-5F, a rat insulinoma cell line, while minimizing cytotoxicity and maintaining insulin production; (5) encapsulation of primary porcine islets of Langerhans in either ionically and/or covalently crosslinked alginate capsulation and comparing insulin response to a glucose challenge. The results of these experiments demonstrate the utility of employing `click' chemistry to increase the overall stability of alginate hydrogels while maintaining therapeutic cell function.Item The Regulation Of Intervertebral Disc Cell Interactions With Their Surrounding Microenvironment(2010) Rastogi, Anshu; Hsieh, Adam H; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Intervertebral disc degeneration is the major cause of back pain in the US, which can be both physically debilitating and costly to treat. Current treatments include invasive surgeries, which can be effective in ameliorating pain, but also contain the risk of complications. Additionally, these strategies target clinical manifestations of disc degeneration, rather than examine the cause of degenerative changes. Therefore, current research focuses on finding minimally invasive treatments for disc disease such as gene therapy. Regulating intervertebral disc cell interactions with their immediate environment can be a useful tool in the development of therapeutic strategies. This was explored through environmental changes to assess shifts in cell phenotype as well as genetic modulation to elucidate alterations in cell function. Biochemical, nutritional, and physical factors were examined in immature nucleus pulposus cells to assess changes in gene expression, attachment, and proliferation. It was found that nutritional and physical factors can alter gene expression levels of NP cells, thereby altering cell phenotype. In addition, down-regulation of the proteolytic enzyme MMP-2 was explored through RNAi interference. Five shRNA lentiviral vectors were designed and validated for the sustained gene silencing of MMP-2. Silencing MMP-2 activity resulted in the inability of disc cells to focally degrade gelatin films as well as reduced ability of disc cells to remodel fibers in type I collagen gels, resulting in weakened gel architecture. These functional consequences were further explored in an in vivo study utilizing an annular needle-puncture model of disc degeneration. Injection of the shMMP lentiviral construct lead to decreased expression of MMP-2 in the disc, as well as improved disc height and morphology. Thus, the functional consequences of silencing MMP-2 were examined, elucidating its role in the degradative pathway leading to degenerative disc disease. The results of these studies can lay the foundation for developing therapeutic treatments for intervertebral disc degeneration.Item A dual modality, DCE-MRI and x-ray, physical phantom for quantitative evaluation of breast imaging protocols(2010) Freed, Melanie; Badano, Aldo; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The current clinical standard for breast cancer screening is mammography. However, this technique has a low sensitivity which results in missed cancers. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) has recently emerged as a promising technique for breast cancer diagnosis and has been reported as being superior to mammography for screening of high-risk women and evaluation of extent of disease. At the same time, low and variable specificity has been documented in the literature as well as a rising number of mastectomies possibly due to the increasing use of DCE-MRI. In this study, we developed and characterized a dual-modality, x-ray and DCE-MRI, anthropomorphic breast phantom for the quantitative assessment of breast imaging protocols. X-ray properties of the phantom were quantitatively compared with patient data, including attenuation coefficients, which matched human values to within the measurement error, and tissue structure using spatial covariance matrices of image data, which were found to be similar in size to patient data. Simulations of the phantom scatter-to-primary ratio (SPR) were produced and experimentally validated then compared with published SPR predictions for homogeneous phantoms. SPR values were as high as 85% in some areas and were heavily influenced by the heterogeneous tissue structure. MRI properties of the phantom, T1 and T2 relaxation values and tissue structure, were also quantitatively compared with patient data and found to match within two error bars. Finally, a dynamic lesion that mimics lesion border shape and washout curve shape was included in the phantom. High spatial and temporal resolution x-ray measurements of the washout curve shape were performed to determine the true contrast agent concentration as a function of time. DCE-MRI phantom measurements using a clinical imaging protocol were compared against the x-ray truth measurements. MRI signal intensity curves were shown to be less specific to lesion type than the x-ray derived contrast agent concentration curves. This phantom allows, for the first time, for quantitative evaluation of and direct comparisons between x-ray and MRI breast imaging modalities in the context of lesion detection and characterization.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.Item POLY (AMIDO AMINE) DENDRIMERS: TRANSEPITHELIAL TRANSPORT MECHANISMS AND APPLICATIONS IN ORAL DRUG DELIVERY(2010) Goldberg, Deborah Sweet; Ghandehari, Hamidreza; Bentley, William; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Small molecule chemotherapy drugs used in clinical practice are plagued by dose-limiting side effects due to off-target toxicities. In addition, because of their low water solubility and poor bioavailability, they must be administered intravenously, leading to high treatment costs and recurring hospital visits. There is a significant need for therapies that improve the bioavailability of chemotherapy agents and enhance specific drug release in the tumor environment. Dendrimers, a class of highly-branched, nanoscale polymers, share many characteristics with traditional polymeric carriers, including water solubility, high capacity of drug loading and improved biodistribution. Poly (amido amine) (PAMAM) dendrimers have shown promise as oral drug carriers due to their compact size, high surface charge density and permeation across the intestinal epithelial barrier. Attachment of chemotherapy drugs to PAMAM dendrimers has the potential to make them orally administrable and reduce off-target toxicities. In this dissertation we investigate the transport mechanisms of PAMAM dendrimers and their potential in oral drug delivery. We demonstrate that anionic G3.5 dendrimers are endocytosed by dynamin-dependent mechanisms and their transport is governed by clathrin-mediated pathways. We show that dendrimer cellular internalization may be a requisite step for tight junction opening. We also demonstrate that conjugation of small poly (ethylene glycol) chains to anionic dendrimers decreases their transport and tight junction opening due to reduction in surface charge, illustrating that small changes in surface chemistry can significantly impact transepithelial transport. Knowledge of transport mechanisms and the impact of surface chemistry will aid in rational design of dendrimer oral drug delivery systems. The potential of dendrimers as oral drug delivery carriers is demonstrated by the evaluation of G3.5 PAMAM dendrimer-SN38 conjugates for oral therapy of hepatic colorectal cancer metastases, a pathology present in over 50% of colorectal cancer cases that is responsible for two-thirds of deaths. Conjugation of SN38, a potent chemotherapy drug with poor solubility and low bioavailability, to PAMAM dendrimers via a glycine linker increased intestinal permeability, decreased intestinal toxicity and showed selective release in the presence of liver carboxylesterase, illustrating that PAMAM dendrimers have the potential to improve the oral bioavailability of potent anti-cancer therapeutics.