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.
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- All Subjects: Biochemistry
- All Subjects: Molecular Biology
- Genre: Doctoral Dissertation
Description
The elaborate signals of animals are often costly to produce and maintain, thus communicating reliable information about the quality of an individual to potential mates or competitors. The properties of the sensory systems that receive signals can drive the evolution of these signals and shape their form and function. However, relatively little is known about the ecological and physiological constraints that may influence the development and maintenance of sensory systems. In the house finch (Carpodacus mexicanus) and many other bird species, carotenoid pigments are used to create colorful sexually selected displays, and their expression is limited by health and dietary access to carotenoids. Carotenoids also accumulate in the avian retina, protecting it from photodamage and tuning color vision. Analogous to plumage carotenoid accumulation, I hypothesized that avian vision is subject to environmental and physiological constraints imposed by the acquisition and allocation of carotenoids. To test this hypothesis, I carried out a series of field and captive studies of the house finch to assess natural variation in and correlates of retinal carotenoid accumulation and to experimentally investigate the effects of dietary carotenoid availability, immune activation, and light exposure on retinal carotenoid accumulation. Moreover, through dietary manipulations of retinal carotenoid accumulation, I tested the impacts of carotenoid accumulation on visually mediated foraging and mate choice behaviors. My results indicate that avian retinal carotenoid accumulation is variable and significantly influenced by dietary carotenoid availability and immune system activity. Behavioral studies suggest that retinal carotenoid accumulation influences visual foraging performance and mediates a trade-off between color discrimination and photoreceptor sensitivity under dim-light conditions. Retinal accumulation did not influence female choice for male carotenoid-based coloration, indicating that a direct link between retinal accumulation and sexual selection for coloration is unlikely. However, retinal carotenoid accumulation in males was positively correlated with their plumage coloration. Thus, carotenoid-mediated visual health and performance or may be part of the information encoded in sexually selected coloration.
ContributorsToomey, Matthew (Author) / McGraw, Kevin J. (Thesis advisor) / Deviche, Pierre (Committee member) / Smith, Brian (Committee member) / Rutowski, Ronald (Committee member) / Verrelli, Brian (Committee member) / Arizona State University (Publisher)
Created2011
Description
Molecular dynamics (MD) simulations provide a particularly useful approach to understanding conformational change in biomolecular systems. MD simulations provide an atomistic, physics-based description of the motions accessible to biomolecular systems on the pico- to micro-second timescale, yielding important insight into the free energy of the system, the dynamical stability of contacts and the role of correlated motions in directing the motions of the system. In this thesis, I use molecular dynamics simulations to provide molecular mechanisms that rationalize structural, thermodynamic, and mutation data on the interactions between the lac repressor headpiece and its O1 operator DNA as well as the ERK2 protein kinase. I performed molecular dynamics simulations of the lac repressor headpiece - O1 operator complex at the natural angle as well as at under- and overbent angles to assess the factors that determine the natural DNA bending angle. I find both energetic and entropic factors contribute to recognition of the natural angle. At the natural angle the energy of the system is minimized by optimization of protein-DNA contacts and the entropy of the system is maximized by release of water from the protein-DNA interface and decorrelation of protein motions. To identify the mechanism by which mutations lead to auto-activation of ERK2, I performed a series of molecular dynamics simulations of ERK1/2 in various stages of activation as well as the constitutively active Q103A, I84A, L73P and R65S ERK2 mutants. My simulations indicate the importance of domain closure for auto-activation and activity regulation. My results enable me to predict two loss-of-function mutants of ERK2, G83A and Q64C, that have been confirmed in experiments by collaborators. One of the powerful capabilities of MD simulations in biochemistry is the ability to find low free energy pathways that connect and explain disparate structural data on biomolecular systems. An extention of the targeted molecular dynamics technique using constraints on internal coordinates will be presented and evaluated. The method gives good results for the alanine dipeptide, but breaks down when applied to study conformational changes in GroEL and adenylate kinase.
ContributorsBarr, Daniel Alan (Author) / van der Vaart, Arjan (Thesis advisor) / Matyushov, Dmitry (Committee member) / Wolf, George (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
Synthetic biology is constantly evolving as new ideas are incorporated into this increasingly flexible field. It incorporates the engineering of life with standard genetic parts and methods; new organisms with new genomes; expansion of life to include new components, capabilities, and chemistries; and even completely synthetic organisms that mimic life while being composed of non-living matter. We have introduced a new paradigm of synthetic biology that melds the methods of in vitro evolution with the goals and philosophy of synthetic biology. The Family B proteins represent the first de novo evolved natively folded proteins to be developed with increasingly powerful tools of molecular evolution. These proteins are folded and functional, composed of the 20 canonical amino acids, and in many ways resemble natural proteins. However, their evolutionary history is quite different from natural proteins, as it did not involve a cellular environment. In this study, we examine the properties of DX, one of the Family B proteins that have been evolutionarily optimized for folding stability. Described in chapter 2 is an investigation into the primitive catalytic properties of DX, which seems to have evolved a serendipitous ATPase activity in addition to its selected ATP binding activity. In chapters 3 and 4 we express the DX gene in E. coli cells and observe massive changes in cell morphology, biochemistry, and life cycle. Exposure to DX activates several defense systems in E. coli, including filamentation, cytoplasmic segregation, and reversion to a viable but non-culturable state. We examined these phenotypes in detail and present a model that accounts for how DX causes such a rearrangement of the cell.
ContributorsStomel, Joshua (Author) / Chaput, John C (Thesis advisor) / Korch, Shaleen (Committee member) / Roberson, Robert (Committee member) / Ghirlanda, Gionvanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of telomeres functions as a naturally occurring molecular clock limiting the replicative capacity of cells. Telomerase is over activated in many cancers, while inactivation leads to multiple lifespan limiting human diseases. In order to further study the interaction between telomerase RNA (TR) and telomerase reverse transcriptase protein (TERT), vertebrate TERT fragments were screened for solubility and purity following bacterial expression. Soluble fragments of medaka TERT including the RNA binding domain (TRBD) were identified. Recombinant medaka TRBD binds specifically to telomerase RNA CR4/CR5 region. Ribonucleotide and amino acid pairs in close proximity within the medaka telomerase RNA-protein complex were identified using photo-activated cross-linking in conjunction with mass spectrometry. The identified cross-linking amino acids were mapped on known crystal structures of TERTs to reveal the RNA interaction interface of TRBD. The identification of this RNA TERT interaction interface furthers the understanding of the telomerase complex at a molecular level and could be used for the targeted interruption of the telomerase complex as a potential cancer treatment.
ContributorsBley, Christopher James (Author) / Chen, Julian (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2011
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
Description
Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.
ContributorsKessans, Sarah (Author) / Mor, Tsafrir S (Thesis advisor) / Matoba, Nobuyuki (Committee member) / Mason, Hugh (Committee member) / Hogue, Brenda (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
The concept of vaccination dates back further than Edward Jenner's first vaccine using cowpox pustules to confer immunity against smallpox in 1796. Nevertheless, it was Jenner's success that gave vaccines their name and made vaccinia virus (VACV) of particular interest. More than 200 years later there is still the need to understand vaccination from vaccine design to prediction of vaccine efficacy using mathematical models. Post-exposure vaccination with VACV has been suggested to be effective if administered within four days of smallpox exposure although this has not been definitively studied in humans. The first and second chapters analyze post-exposure prophylaxis of VACV in an animal model using v50ΔB13RMγ, a recombinant VACV expressing murine interferon gamma (IFN-γ) also known as type II IFN. While untreated animals infected with wild type VACV die by 10 days post-infection (dpi), animals treated with v50ΔB13RMγ 1 dpi had decreased morbidity and 100% survival. Despite these differences, the viral load was similar in both groups suggesting that v50ΔB13RMγ acts as an immunoregulator rather than as an antiviral. One of the main characteristics of VACV is its resistance to type I IFN, an effect primarily mediated by the E3L protein, which has a Z-DNA binding domain and a double-stranded RNA (dsRNA) binding domain. In the third chapter a VACV that independently expresses both domains of E3L was engineered and compared to wild type in cells in culture. The dual expression virus was unable to replicate in the JC murine cell line where both domains are needed together for replication. Moreover, phosphorylation of the dsRNA dependent protein kinase (PKR) was observed at late times post-infection which indicates that both domains need to be linked together in order to block the IFN response. Because smallpox has already been eradicated, the utility of mathematical modeling as a tool for predicting disease spread and vaccine efficacy was explored in the last chapter using dengue as a disease model. Current modeling approaches were reviewed and the 2000-2001 dengue outbreak in a Peruvian region was analyzed. This last section highlights the importance of interdisciplinary collaboration and how it benefits research on infectious diseases.
ContributorsHolechek, Susan A (Author) / Jacobs, Bertram L (Thesis advisor) / Castillo-Chavez, Carlos (Committee member) / Frasch, Wayne (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
The cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis. Light energy conversion in photosynthesis takes place in photosystem I (PSI) and photosystem II (PSII) that contain chlorophyll, which absorbs light energy that is utilized as a driving force for photosynthesis. However, excess light energy may lead to formation of reactive oxygen species that cause damage to photosynthetic complexes, which subsequently need repair or replacement. To gain insight in the degradation/biogenesis dynamics of the photosystems, the lifetimes of photosynthetic proteins and chlorophyll were determined by a combined stable-isotope (15N) and mass spectrometry method. The lifetimes of PSII and PSI proteins ranged from 1-33 and 30-75 hours, respectively. Interestingly, chlorophyll had longer lifetimes than the chlorophyll-binding proteins in these photosystems. Therefore, photosynthetic proteins turn over and are replaced independently from each other, and chlorophyll is recycled from the damaged chlorophyll-binding proteins. In Synechocystis, there are five small Cab-like proteins (SCPs: ScpA-E) that share chlorophyll a/b-binding motifs with LHC proteins in plants. SCPs appear to transiently bind chlorophyll and to regulate chlorophyll biosynthesis. In this study, the association of ScpB, ScpC, and ScpD with damaged and repaired PSII was demonstrated. Moreover, in a mutant lacking SCPs, most PSII protein lifetimes were unaffected but the lifetime of chlorophyll was decreased, and one of the nascent PSII complexes was missing. SCPs appear to bind PSII chlorophyll while PSII is repaired, and SCPs stabilize nascent PSII complexes. Furthermore, aminolevulinic acid biosynthesis, an early step of chlorophyll biosynthesis, was impaired in the absence of SCPs, so that the amount of chlorophyll in the cells was reduced. Finally, a deletion mutation was introduced into the sll1906 gene, encoding a member of the putative bacteriochlorophyll delivery (BCD) protein family. The Sll1906 sequence contains possible chlorophyll-binding sites, and its homolog in purple bacteria functions in proper assembly of light-harvesting complexes. However, the sll1906 deletion did not affect chlorophyll degradation/biosynthesis and photosystem assembly. Other (parallel) pathways may exist that may fully compensate for the lack of Sll1906. This study has highlighted the dynamics of photosynthetic complexes in their biogenesis and turnover and the coordination between synthesis of chlorophyll and photosynthetic proteins.
ContributorsYao, Cheng I Daniel (Author) / Vermaas, Wim (Thesis advisor) / Fromme, Petra (Committee member) / Roberson, Robert (Committee member) / Webber, Andrew (Committee member) / Arizona State University (Publisher)
Created2011
Description
HIV/AIDS is the sixth leading cause of death worldwide and the leading cause of death among women of reproductive age living in low-income countries. Clinicians in industrialized nations monitor the efficacy of antiretroviral drugs and HIV disease progression with the HIV-1 viral load assay, which measures the copy number of HIV-1 RNA in blood. However, viral load assays are not widely available in sub-Saharan Africa and cost between 50-$139 USD per test on average where available. To address this problem, a mixed-methods approach was undertaken to design a novel and inexpensive viral load diagnostic for HIV-1 and to evaluate barriers to its adoption in a developing country. The assay was produced based on loop-mediated isothermal amplification (LAMP). Blood samples from twenty-one individuals were spiked with varying concentrations of HIV-1 RNA to evaluate the sensitivity and specificity of LAMP. Under isothermal conditions, LAMP was performed with an initial reverse-transcription step (RT-LAMP) and primers designed for HIV-1 subtype C. Each reaction generated up to a few billion copies of target DNA within an hour. Presence of target was detected through naked-eye observation of a fluorescent indicator and verified by DNA gel electrophoresis and real-time fluorescence. The assay successfully detected the presence of HIV in samples with a broad range of HIV RNA concentration, from over 120,000 copies/reaction to 120 copies/reaction. In order to better understand barriers to adoption of LAMP in developing countries, a feasibility study was undertaken in Tanzania, a low-income country facing significant problems in healthcare. Medical professionals in Northern Tanzania were surveyed for feedback regarding perspectives of current HIV assays, patient treatment strategies, availability of treatment, treatment priorities, HIV transmission, and barriers to adoption of the HIV-1 LAMP assay. The majority of medical providers surveyed indicated that the proposed LAMP assay is too expensive for their patient populations. Significant gender differences were observed in response to some survey questions. Female medical providers were more likely to cite stigma as a source problem of the HIV epidemic than male medical providers while males were more likely to cite lack of education as a source problem than female medical providers.
ContributorsSalamone, Damien Thomas (Author) / Jacobs, Bertram L (Thesis advisor) / Marsiglia, Flavio (Committee member) / Stout, Valerie (Committee member) / Johnson, Crista (Committee member) / Arizona State University (Publisher)
Created2011
Description
Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, which retain function in trans. The in vitro reconstituted Neurospora telomerase is highly processive, synthesizing canonical TTAGGG repeats. Similar to Schizosaccharomycetes pombe, filamentous fungal TERs utilize the spliceosomal splicing machinery for 3' processing. Neurospora telomerase, while associating with the Est1 protein in vivo, does not bind homologous Ku or Sm proteins found in both budding and fission yeast telomerase holoenzyme, suggesting a unique biogenesis pathway. The development of Neurospora as a model organism to study telomeres and telomerase may shed light upon the evolution of the canonical TTAGGG telomeric repeat and telomerase processivity within fungal species.
ContributorsQi, Xiaodong (Author) / Chen, Julian (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Chaput, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein MBP-c1 was purified on an affinity column, and the c1 subunit was subsequently severed by protease cleavage in the presence of detergent. Final purification of the monomeric c1 subunit was accomplished using reversed phase column chromatography with ethanol as an eluent. Circular dichroism spectroscopy data showed clear evidence that the purified c-subunit is folded with the native alpha-helical secondary structure. Recent experiments appear to indicate that this monomeric recombinant c-subunit forms an oligomeric ring that is similar to its native tetradecameric form when reconstituted in liposomes. The F-type ATP synthase c-subunit stoichiometry is currently known to vary from 8 to 15 subunits among different organisms. This has a direct influence on the metabolic requirements of the corresponding organism because each c-subunit binds and transports one H+ across the membrane as the ring makes a complete rotation. The c-ring rotation drives rotation of the gamma-subunit, which in turn drives the synthesis of 3 ATP for every complete rotation. The availability of a recombinantly produced c-ring will lead to new experiments which can be designed to investigate the possible factors that determine the variable c-ring stoichiometry and structure.
ContributorsLawrence, Robert Michael (Author) / Fromme, Petra (Thesis advisor) / Chen, Julian J.L. (Committee member) / Woodbury, Neal W. (Committee member) / Arizona State University (Publisher)
Created2011