ASU Electronic Theses and Dissertations
This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.
In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.
Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.
Displaying 1 - 10 of 10
Filtering by
- All Subjects: Biophysics
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
Deoxyribonucleic acid (DNA), a biopolymer well known for its role in preserving genetic information in biology, is now drawing great deal of interest from material scientists. Ease of synthesis, predictable molecular recognition via Watson-Crick base pairing, vast numbers of available chemical modifications, and intrinsic nanoscale size makes DNA a suitable material for the construction of a plethora of nanostructures that can be used as scaffold to organize functional molecules with nanometer precision. This dissertation focuses on DNA-directed organization of metallic nanoparticles into well-defined, discrete structures and using them to study photonic interaction between fluorophore and metal particle. Presented here are a series of studies toward this goal. First, a novel and robust strategy of DNA functionalized silver nanoparticles (AgNPs) was developed and DNA functionalized AgNPs were employed for the organization of discrete well-defined dimeric and trimeric structures using a DNA triangular origami scaffold. Assembly of 1:1 silver nanoparticle and gold nanoparticle heterodimer has also been demonstrated using the same approach. Next, the triangular origami structures were used to co-assemble gold nanoparticles (AuNPs) and fluorophores to study the distance dependent and nanogap dependencies of the photonic interactions between them. These interactions were found to be consistent with the full electrodynamic simulations. Further, a gold nanorod (AuNR), an anisotropic nanoparticle was assembled into well-defined dimeric structures with predefined inter-rod angles. These dimeric structures exhibited unique optical properties compared to single AuNR that was consistent with the theoretical calculations. Fabrication of otherwise difficult to achieve 1:1 AuNP- AuNR hetero dimer, where the AuNP can be selectively placed at the end-on or side-on positions of anisotropic AuNR has also been shown. Finally, a click chemistry based approach was developed to organize sugar modified DNA on a particular arm of a DNA origami triangle and used them for site-selective immobilization of small AgNPs.
ContributorsPal, Suchetan (Author) / Liu, Yan (Thesis advisor) / Yan, Hao (Thesis advisor) / Lindsay, Stuart (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2012
Description
The response of living cells to electric field (EF) has been observed for more than a hundred years, but the mechanism of how cells interact with EF is not entirely ascertained. Although many efforts have been devoted to the application of EF stimulation in tissue engineering and regeneration, the fundamental scientific principle of such practice remains unveiled and keeps drawing attention during the pursuit of consistent outcomes. In this regard, my research focuses on the underlying mechanism by which EF stimulation evokes cellular responses and the EF modulation of cell signaling pathways to physiological behaviors. The first part of my research focuses on developing the platform for controlled EF stimulation and real-time imaging/analysis. High-k dielectric passivated microelectrodes are fabricated to send capacitively coupled alternating current electric field (AC EF) stimulation to cells. I have developed two generations of EF stimulation devices with environmental control chambers: the first one is used to study cell signaling pathway dynamics; the second one is upgraded with long-term culture capability to study cell physiological behaviors. The second part of my research focuses on the quantification and mechanistic study of AC EF perturbation of the extracellular signal-related kinase (ERK) signaling pathway. I demonstrate that AC EF stimulation can induce both inhibition and activation of the ERK pathway, with different AC EF amplitude thresholds and time and magnitude scales. The mechanistic study shows that the ERK activation is initiated by AC EF-induced epidermal growth factor receptor (EGFR) phosphorylation, and the ERK inhibition is related to AC EF-induced change of Ras activities. In addition, these ERK responses show high sensitivity to AC EF waveform and timing, indicating electrostatic coupling mechanism and providing new parameter spaces for further investigation on the modulation of the ERK signaling pathway via AC EF stimulation. The last part of my research steers to cell physiological behaviors under prolonged AC EF stimulation. I report that AC EF stimulation can clearly inhibit cell proliferation and migration, and the inhibition in cell proliferation is sensitive to AC EF amplitude, stimulation pattern, and pulse rising time. These findings can benefit the AC EF application in medical treatment.
ContributorsHu, Minxi (Author) / Qing, Quan (Thesis advisor) / Lindsay, Stuart (Committee member) / Guo, Jia (Committee member) / Arizona State University (Publisher)
Created2023
Description
Mechanical properties, in particular elasticity, of cancer cells and their microenvironment are important in governing cancer cell fate, for example function, mobility, adhesion, and invasion. Among all tools to measure the mechanical properties, the precision and ease of atomic force microscopy (AFM) to directly apply force—in the range of Pico to micronewtons—onto samples—with length scales from nanometers to tens of micrometers—has made it a powerful tool to investigate the mechanics of materials. AFM is widely used to measure deformability and stiffness of soft biological samples. Principally, these samples are indented by the AFM probe and the forces and indentation depths are recorded. The generated force-indentation curves are fitted with an elastic contact model to quantify the elasticity (e.g. stiffness). AFM is a precise tool; however, the results are as accurate as the contact model used to analyze them. A new contact model was introduced to analyze force-indentation curves generated by spherical AFM probes for deep indentations. The experimental and finite element analysis results demonstrated that the new contact model provides more accurate mechanical properties throughout the indentation depth up to radius of the indenter, while the Hertz model underestimates the mechanical properties. In the classical contact models, it is assumed that the sample is vertically homogenous; however, many biological samples—for example cells—are heterogeneous. A novel two-layer model was utilized to probe Polydimethylsiloxane hydrogel (PDMS) layers on PDMS substrates with stiffness mismatch. In this experiment the stiffness of the substrate was deconvoluted from the AFM measurements to obtain the stiffness of the layer. AFM and confocal reflectance microscopy were utilized along with a novel 3D microengineered breast cancer tumor model to study the crosstalk between cancer tumor and the stromal cells (CAFs) and the ECM remodeling caused by their interplay. The results showed that as the cancer cells invade into the extracellular matrix (ECM), they release PDGF ligands which enable Cafes to remodel the ECM and this remodeling increased the invasion rate of the cancer cells. Next, the effect of the ECM remodeling on anti-cancer drug resistant was investigated within the 3D microengineered cancer model. It was demonstrated that the combinatory treatment by anti-cancer and-anti-fibrotic drugs enhance the efficiency of the cancer treatment. A novel DNA-based 3D hydrogel model with tunable stiffness was investigated by AFM. The results showed the hydrogel stiffness can be enhanced by adding DNA crosslinkers. In addition, the stiffness was reduced to the control sample level by introducing the displacement DNA. Biophysical quantifications along with the in vitro microengineered tumor models provide a unique frame work to study cancer in more detail.
ContributorsRahmani Eliato, Kiarash (Author) / Ros, Robert (Thesis advisor) / Nikkhah, Mehdi (Committee member) / Ozkan, Sefika (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2019
Description
Mechanical properties of cells are important in maintaining physiological functions of biological systems. Quantitative measurement and analysis of mechanical properties can help understand cellular mechanics and its functional relevance and discover physical biomarkers for diseases monitoring and therapeutics.
This dissertation presents a work to develop optical methods for studying cell mechanics which encompasses four applications. Surface plasmon resonance microscopy based optical method has been applied to image intracellular motions and cell mechanical motion. This label-free technique enables ultrafast imaging with extremely high sensitivity in detecting cell deformation. The technique was first applied to study intracellular transportation. Organelle transportation process and displacement steps of motor protein can be tracked using this method. The second application is to study heterogeneous subcellular membrane displacement induced by membrane potential (de)polarization. The application can map the amplitude and direction of cell deformation. The electromechanical coupling of mammalian cells was also observed. The third application is for imaging electrical activity in single cells with sub-millisecond resolution. This technique can fast record actions potentials and also resolve the fast initiation and propagation of electromechanical signals within single neurons. Bright-field optical imaging approach has been applied to the mechanical wave visualization that associated with action potential in the fourth application. Neuron-to-neuron viability of membrane displacement was revealed and heterogeneous subcellular response was observed.
All these works shed light on the possibility of using optical approaches to study millisecond-scale and sub-nanometer-scale mechanical motions. These studies revealed ultrafast and ultra-small mechanical motions at the cellular level, including motor protein-driven motions and electromechanical coupled motions. The observations will help understand cell mechanics and its biological functions. These optical approaches will also become powerful tools for elucidating the interplay between biological and physical functions.
This dissertation presents a work to develop optical methods for studying cell mechanics which encompasses four applications. Surface plasmon resonance microscopy based optical method has been applied to image intracellular motions and cell mechanical motion. This label-free technique enables ultrafast imaging with extremely high sensitivity in detecting cell deformation. The technique was first applied to study intracellular transportation. Organelle transportation process and displacement steps of motor protein can be tracked using this method. The second application is to study heterogeneous subcellular membrane displacement induced by membrane potential (de)polarization. The application can map the amplitude and direction of cell deformation. The electromechanical coupling of mammalian cells was also observed. The third application is for imaging electrical activity in single cells with sub-millisecond resolution. This technique can fast record actions potentials and also resolve the fast initiation and propagation of electromechanical signals within single neurons. Bright-field optical imaging approach has been applied to the mechanical wave visualization that associated with action potential in the fourth application. Neuron-to-neuron viability of membrane displacement was revealed and heterogeneous subcellular response was observed.
All these works shed light on the possibility of using optical approaches to study millisecond-scale and sub-nanometer-scale mechanical motions. These studies revealed ultrafast and ultra-small mechanical motions at the cellular level, including motor protein-driven motions and electromechanical coupled motions. The observations will help understand cell mechanics and its biological functions. These optical approaches will also become powerful tools for elucidating the interplay between biological and physical functions.
ContributorsYang, Yunze (Author) / Tao, Nongjian (Thesis advisor) / Wang, Shaopeng (Committee member) / Goryll, Michael (Committee member) / Si, Jennie (Committee member) / Arizona State University (Publisher)
Created2016
Description
Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is αMβ2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering quantitative insights.
ContributorsChristenson, Wayne B (Author) / Ros, Robert (Thesis advisor) / Beckstein, Oliver (Committee member) / Lindsay, Stuart (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2016
Description
Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal of the tightly bound sugar phosphates from the active sites of RuBisCO. In this work, we investigated the ATP/ADP dependent oligomerization equilibrium of fluorescently tagged Rca for a wide range of concentrations using fluorescence correlation spectroscopy. Results show that in the presence of ADP-Mg2+, the oligomerization state of Rca gradually changes in steps of two subunits. The most probable association model supports the dissociation constants (K_d) of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equlibria, respectively. Rca continues to assemble at higher concentrations which are indicative of the formation of aggregates. In the presence of ATP-Mg2+, a similar stepwise assembly is observed. However, at higher concentrations (30-75 µM), the average oligomeric size remains relatively unchanged around six subunits per oligomer. This is in sharp contrast with observations in ADP-Mg2+, where a marked decrease in the diffusion coefficient of Rca was observed, consistent with the formation of aggregates. The estimated K_d values obtained from the analysis of the FCS decays were similar for the first steps of the assembly process in both ADP-Mg2+ and ATP-Mg2+. However, the formation of the hexamer from the tetramer is much more favored in ATP-Mg2+, as evidenced from 20 fold lower K_d associated with this assembly step. This suggests that the formation of a hexameric ring in the presence of ATP-Mg2+. In addition to that, Rca aggregation is largely suppressed in the presence of ATP-Mg2+, as evidenced from the 1000 fold larger K_d value for the hexamer-24 mer association step. In essence, a fluorescence-based method was developed to monitor in vitro protein oligomerization and was successfully applied with Rca. The results provide a strong hint at the active oligomeric structure of Rca, and this information will hopefully help the ongoing research on the mechanistic enzymology of Rca.
ContributorsChakraborty, Manas (Author) / Levitus, Marcia (Thesis advisor) / Angell, Charles (Committee member) / Lindsay, Stuart (Committee member) / Arizona State University (Publisher)
Created2014
Description
Calcitonin Gene-Related Peptide (CGRP) is an intrinsically disordered protein
that has no regular secondary structure, but plays an important role in vasodilation and pain transmission in migraine. Little is known about the structure and dynamics of the monomeric state of CGRP or how CGRP is able to function in the cell, despite the lack of regular secondary structure. This work focuses characterizing the non-local structural and dynamical properties of the CGRP monomer in solution, and understanding how these are affected by the sequence and the solution environment. The unbound, free state of CGRP is measured using a nanosecond laser-pump spectrophotometer, which allows measuring the end-to-end distance (a non-local structural property) and the rate of end-to-end contact formation (intra-chain diffusional dynamics). The data presented in this work show that electrostatic interactions strongly modulate the structure of CGRP, and that peptide-solvent interactions are sequence and charge dependent and can have a significant effect on the internal dynamics of the peptide. In the last few years migraine research has shifted focus to disrupting the CGRP-receptor pathway through the design of pharmacological drugs that bind to either CGRP or its receptor, inhibiting receptor activation and therefore preventing or reducing the frequency of migraine attacks. Understanding what types of intra- and inter-chain interactions dominate in CGRP can help better design drugs that disrupt the binding of CGRP to its receptor.
that has no regular secondary structure, but plays an important role in vasodilation and pain transmission in migraine. Little is known about the structure and dynamics of the monomeric state of CGRP or how CGRP is able to function in the cell, despite the lack of regular secondary structure. This work focuses characterizing the non-local structural and dynamical properties of the CGRP monomer in solution, and understanding how these are affected by the sequence and the solution environment. The unbound, free state of CGRP is measured using a nanosecond laser-pump spectrophotometer, which allows measuring the end-to-end distance (a non-local structural property) and the rate of end-to-end contact formation (intra-chain diffusional dynamics). The data presented in this work show that electrostatic interactions strongly modulate the structure of CGRP, and that peptide-solvent interactions are sequence and charge dependent and can have a significant effect on the internal dynamics of the peptide. In the last few years migraine research has shifted focus to disrupting the CGRP-receptor pathway through the design of pharmacological drugs that bind to either CGRP or its receptor, inhibiting receptor activation and therefore preventing or reducing the frequency of migraine attacks. Understanding what types of intra- and inter-chain interactions dominate in CGRP can help better design drugs that disrupt the binding of CGRP to its receptor.
ContributorsSizemore, Sara (Author) / Vaiana, Sara (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Lindsay, Stuart (Committee member) / Ozkan, Sefika (Committee member) / Arizona State University (Publisher)
Created2015
Description
The atomic force microscope (AFM) is capable of directly probing the mechanics of samples with length scales from single molecules to tissues and force scales from pico to micronewtons. In particular, AFM is widely used as a tool to measure the elastic modulus of soft biological samples by collecting force-indentation relationships and fitting these to classic elastic contact models. However, the analysis of raw force-indentation data may be complicated by mechanical heterogeneity present in biological systems. An analytical model of an elastic indentation on a bonded two-layer sample was solved. This may be used to account for substrate effects and more generally address experimental design for samples with varying elasticity. This model was applied to two mechanobiology systems of interest. First, AFM was combined with confocal laser scanning fluorescence microscopy and finite element analysis to examine stiffness changes during the initial stages of invasion of MDA-MB-231 metastatic breast cells into bovine collagen I matrices. It was determined that the cells stiffen significantly as they invade, the amount of stiffening is correlated with the elastic modulus of the collagen gel, and inhibition of Rho-associated protein kinase reduces the elastic modulus of the invading cells. Second, the elastic modulus of cancer cell nuclei was investigated ex situ and in situ. It was observed that inhibition of histone deacetylation to facilitate chromatin decondenstation result in significantly more morphological and stiffness changes in cancerous cells compared to normal cells. The methods and results presented here offer novel strategies for approaching biological systems with AFM and demonstrate its applicability and necessity in studying cellular function in physiologically relevant environments.
ContributorsDoss, Bryant Lee (Author) / Ros, Robert (Thesis advisor) / Lindsay, Stuart (Committee member) / Nikkhah, Mehdi (Committee member) / Beckstein, Oliver (Committee member) / Arizona State University (Publisher)
Created2015
Description
My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale.
My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule.
My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.
My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule.
My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.
ContributorsSadar, Joshua Stephen (Author) / Qing, Quan (Thesis advisor) / Lindsay, Stuart (Committee member) / Vaiana, Sara (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2019