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 14
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- All Subjects: Biochemistry
- Creators: Allen, James
- Creators: Chaput, John
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
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
The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. Chapter 1 describes bleomycin and related DNA targeting antitumor agents as well as the specific structural domains of bleomycin. Various structural analogues of pyrimidoblamic acid were synthesized and subsequently incorporated into their corresponding full deglycoBLM A6 derivatives by utilizing a solid support. Their activity was measured using a pSP64 DNA plasmid relaxation assay and is summarized in Chapter 2. The specifics of bleomycin—DNA interaction and kinetics were studied via surface plasmon resonance and are presented in Chapter 3. By utilizing carefully selected 64-nucleotide DNA hairpins with variable 16-mer regions whose sequences showed strong binding in past selection studies, a kinetic profile was obtained for several BLMs for the first time since bleomycin was discovered in 1966. The disaccharide moiety of bleomycin has been previously shown to be a specific tumor cell targeting element comprised of L-gulose-D-mannose, especially between MCF-7 (breast cancer cells) and MCF-10A ("normal" breast cells). This phenomenon was further investigated via fluorescence microscopy using multiple cancerous cell lines with matched "normal" counterparts and is fully described in Chapter 4.
ContributorsBozeman, Trevor C (Author) / Hecht, Sidney M. (Thesis advisor) / Chaput, John (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2013
Description
Cyanovirin-N (CV-N) is a naturally occurring lectin originally isolated from the cyanobacteria Nostoc ellipsosporum. This 11 kDa lectin is 101 amino acids long with two binding sites, one at each end of the protein. CV-N specifically binds to terminal Manα1-2Manα motifs on the branched, high mannose Man9 and Man8 glycosylations found on enveloped viruses including Ebola, Influenza, and HIV. wt-CVN has micromolar binding to soluble Manα1-2Manα and also inhibits HIV entry at low nanomolar concentrations. CV-N's high affinity and specificity for Manα1-2Manα makes it an excellent lectin to study for its glycan-specific properties. The long-term aim of this project is to make a variety of mutant CV-Ns to specifically bind other glycan targets. Such a set of lectins may be used as screening reagents to identify biomarkers and other glycan motifs of interest. As proof of concept, a T7 phage display library was constructed using P51G-m4-CVN genes mutated at positions 41, 44, 52, 53, 56, 74, and 76 in binding Domain B. Five CV-N mutants were selected from the library and expressed in BL21(DE3) E. coli. Two of the mutants, SSDGLQQ-P51Gm4-CVN and AAGRLSK-P51Gm4-CVN, were sufficiently stable for characterization and were examined by CD, Tm, ELISA, and glycan array. Both proteins have CD minima at approximately 213 nm, indicating largely β-sheet structure, and have Tm values greater than 40°C. ELISA against gp120 and RNase B demonstrate both proteins' ability to bind high mannose glycans. To more specifically determine the binding specificity of each protein, AAGRLSK-P51Gm4-CVN, SSDGLQQ-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN were sent to the Consortium for Functional Glycomics (CFG) for glycan array analysis. AAGRLSK-P51Gm4-CVN, wt-CVN, and P51G-m4-CVN, have identical specificities for high mannose glycans containing terminal Manα1-2Manα. SSDGLQQ-P51Gm4-CVN binds to terminal GlcNAcα1-4Gal motifs and a subgroup of high mannose glycans bound by P51G-m4-CVN. SSDGLQQ-wt-CVN was produced to restore anti-HIV activity and has a high nanomolar EC50 value compared to wt-CVN's low nanomolar activity. Overall, these experiments show that CV-N Domain B can be mutated and retain specificity identical to wt-CVN or acquire new glycan specificities. This first generation information can be used to produce glycan-specific lectins for a variety of applications.
ContributorsRuben, Melissa (Author) / Ghirlanda, Giovanna (Thesis advisor) / Allen, James (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2013
Description
DNA nanotechnology has been a rapidly growing research field in the recent decades, and there have been extensive efforts to construct various types of highly programmable and robust DNA nanostructures. Due to the advantage that DNA nanostructure can be used to organize biochemical molecules with precisely controlled spatial resolution, herein we used DNA nanostructure as a scaffold for biological applications. Targeted cell-cell interaction was reconstituted through a DNA scaffolded multivalent bispecific aptamer, which may lead to promising potentials in tumor therapeutics. In addition a synthetic vaccine was constructed using DNA nanostructure as a platform to assemble both model antigen and immunoadjuvant together, and strong antibody response was demonstrated in vivo, highlighting the potential of DNA nanostructures to serve as a new platform for vaccine construction, and therefore a DNA scaffolded hapten vaccine is further constructed and tested for its antibody response. Taken together, my research demonstrated the potential of DNA nanostructure to serve as a general platform for immunological applications.
ContributorsLiu, Xiaowei (Author) / Liu, Yan (Thesis advisor) / Chang, Yung (Thesis advisor) / Yan, Hao (Committee member) / Allen, James (Committee member) / Zhang, Peiming (Committee member) / Arizona State University (Publisher)
Created2012
Description
The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type BsQueD, each Mn(II) is ligated by three histidines (His) and one glutamate (Glu). All efforts to mutate His residues to non-ligating residues resulted in insoluble protein or completely inactive enzyme. A soluble mutant was expressed that replaced the Glu residue with a fourth His at the N-terminal domain. This mutant (E69H) has a specific activity of 0.00572 &mumol;/min/mg, which is nearly 3000-fold lower than the rate of wild type BsQueD (15.9 &mumol;/min/mg). Further analysis of E69H by inductively couple plasma mass spectrometry revealed that this mutant contains only 0.062 mol of Mn(II) per mol of enzyme. This is evidence that disabling metal-ligation at one domain influences metal-incorporation at the other. During the course of the mutagenic study, a second, faster purification method was developed. A hexahistidine tag and an enterokinase cleavage site were fused to the N-terminus of BsQueD (6xHis-BsQueD). Active enzyme was successfully expressed and purified with a nickel column in 3 hours. This is much faster than the previous multi-column purification, which took two full days to complete. However, the concentration of soluble, purified enzyme (1.8 mg/mL) was much lower than concentrations achieved with the traditional method (30 mg/mL). While the concentration of 6xHis-BsQueD is sufficient for some analyses, there are several characterization techniques that must be conducted at higher concentrations. Therefore, it will be advantageous to continue using both purification methods in the future.
ContributorsBowen, Sara (Author) / Francisco, Wilson A (Thesis advisor) / Allen, James (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2010
Description
A novel small metal-binding protein (SmbP), with only 93 residues and no similarity to other known proteins, has been isolated from the periplasm of Nitrosomonas europaea. It is characterized by its high percentage (17%) of histidines, a motif of ten repeats of seven residues, a four α-helix bundle structure, and a high binding affinity to about six equivalents of Cu2+. The goal of this study is to investigate the Cu2+ binding sites in SmbP and to understand how Cu2+ stabilizes the protein. Preliminary folding experiments indicated that Cu2+ greatly stabilizes SmbP. In this study, protein folding data from circular dichroism (CD) spectroscopy was used to elucidate the role of Cu2+ in stabilizing SmbP structure against unfolding induced by decreased pH, increased temperature, and chemical denaturants. The significant stabilization effects of Cu2+ were demonstrated by the observation that Cu2+-SmbP remained fully folded under extreme environmental conditions, such as acidic pH, 96 °C, and 8 M urea. Also, it was shown that Cu2+ is able to induce the refolding of unfolded SmbP in acidic solutions. These findings imply that the coordination of Cu2+ to histidine residues is responsible for the stabilization effects. The crystal structure of SmbP without Cu2+ has been determined. However, attempts to crystallize Cu2+-SmbP have not been successful. In this study, multidimensional NMR experiments were conducted in order to gain additional information regarding the Cu2+-SmbP structure, in particular its metal binding sites. Unambiguous resonance assignments were successfully made. Cα secondary chemical shifts confirmed that SmbP has a four α-helical structure. A Cu2+-protein titration experiment monitored by NMR indicated a top-to-bottom, sequential metal binding pattern for SmbP. In addition, several bioinformatics tools were used to complement the experimental approach and identity of the ligands in Cu2+-binding sites in SmbP is proposed.
ContributorsYan, Qin (Author) / Francisco, Wilson A (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
The ability to manipulate the interaction between small molecules and biological macromolecules towards the study of disease pathogenesis has become a very important part of research towards treatment options for various diseases. The work described here shows both the use of DNA oligonucleotides as carriers for a nicotine hapten small molecule, and the use of microsomes to study the stability of compounds derived to treat mitochondrial diseases.
Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A new vaccine conjugate against nicotine held within a DNA tetrahedron delivery system has been studied. For this purpose, several strands of DNA, conjugated with a modified dTpT having three or six carbon atom alkynyl linkers, have been synthesized. These strands have later been conjugated to three separate hapten small molecules to analyze which conjugates formed would be optimal for further testing in vivo.
Mitochondrial diseases are hard to treat, given that there are so many different variations to treat. There is no one compound that can treat all mitochondrial and neurodegenerative diseases; however, improvements can be made to compounds currently under study to improve the conditions of those afflicted. A significant issue leading to compounds failing in clinical trials is insufficient metabolic stability. Many compounds have good biological activity, but once introduced to an animal, are not stable enough to have any effect. Here, several synthesized compounds have been evaluated for metabolic stability, and several showed improved stability, while maintaining biological activity.
Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A new vaccine conjugate against nicotine held within a DNA tetrahedron delivery system has been studied. For this purpose, several strands of DNA, conjugated with a modified dTpT having three or six carbon atom alkynyl linkers, have been synthesized. These strands have later been conjugated to three separate hapten small molecules to analyze which conjugates formed would be optimal for further testing in vivo.
Mitochondrial diseases are hard to treat, given that there are so many different variations to treat. There is no one compound that can treat all mitochondrial and neurodegenerative diseases; however, improvements can be made to compounds currently under study to improve the conditions of those afflicted. A significant issue leading to compounds failing in clinical trials is insufficient metabolic stability. Many compounds have good biological activity, but once introduced to an animal, are not stable enough to have any effect. Here, several synthesized compounds have been evaluated for metabolic stability, and several showed improved stability, while maintaining biological activity.
ContributorsSchmierer, Margaret (Author) / Hecht, Sidney M. (Thesis advisor) / Allen, James (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2016
Description
Glycosaminoglycans (GAGs) are a class of complex biomolecules comprised of linear, sulfated polysaccharides whose presence on cell surfaces and in the extracellular matrix involve them in many physiological phenomena as well as in interactions with pathogenic microbes. Decorin binding protein A (DBPA), a Borrelia surface lipoprotein involved in the infectivity of Lyme disease, is responsible for binding GAGs found on decorin, a small proteoglycan present in the extracellular matrix. Different DBPA strains have notable sequence heterogeneity that results in varying levels of GAG-binding affinity. In this dissertation, the structures and GAG-binding mechanisms for three strains of DBPA (B31 and N40 DBPAs from B. burgdorferi and PBr DBPA from B. garinii) are studied to determine why each strain has a different affinity for GAGs. These three strains have similar topologies consisting of five α-helices held together by a hydrophobic core as well as two long flexible segments: a linker between helices one and two and a C-terminal tail. This structural arrangement facilitates the formation of a basic pocket below the flexible linker which is the primary GAG-binding epitope. However, this GAG-binding site can be occluded by the flexible linker, which makes the linker a negative regulator of GAG-binding. ITC and NMR titrations provide KD values that show PBr DBPA binds GAGs with higher affinity than B31 and N40 DBPAs, while N40 binds with the lowest affinity of the three. Work in this thesis demonstrates that much of the discrepancies seen in GAG affinities of the three DBPAs can be explained by the amino acid composition and conformation of the linker. Mutagenesis studies show that B31 DBPA overcomes the pocket obstruction with the BXBB motif in its linker while PBr DBPA has a retracted linker that exposes the basic pocket as well as a secondary GAG-binding site. N40 DBPA, however, does not have any evolutionary modifications to its structure to enhance GAG binding which explains its lower affinity for GAGs. GMSA and ELISA assays, along with NMR PRE experiments, confirm that structural changes in the linker do affect GAG-binding and, as a result, the linker is responsible for regulating GAG affinity.
ContributorsMorgan, Ashli M (Author) / Wang, Xu (Thesis advisor) / Allen, James (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2015
Description
Protein affinity reagents have aptly gained profound importance as capture reagents and
drugs in basic research, biotechnology, diagnostics and therapeutics. However, due to the
cost, labor and time associated with production of antibodies focus has recently changed
towards potential of peptides to act as protein affinity reagents. Affinity peptides are easy
to work with, non-immunogenic, cost effective and amenable to scale up. Even though
researchers have developed several affinity peptides, we are far from compiling library of
peptides that encompasses entire human proteome. My thesis describes high throughput
pipeline that can be used to develop and characterize affinity peptides that bind several
discrete sites on target proteins.
Chapter 2 describes optimization of cell-free protein expression using commercially
available translation systems and well-known leader sequences. Presence of internal
ribosome entry site upstream of coding region allows maximal expression in HeLa cell
lysate whereas translation enhancing elements are best suited for expression in rabbit
reticulocyte lysate and wheat germ extract. Use of optimal vector and cell lysate
combination ensures maximum protein expression of DNA libraries.
Chapter 3 describes mRNA display selection methodology for developing affinity peptides
for target proteins using large diversity DNA libraries. I demonstrate that mild denaturant
is not sufficient to increase selection pressure for up to three rounds of selection and
increasing number of selection rounds increases probability of finding affinity peptide s.
These studies enhance fundamental understanding of mRNA display and pave the way
for future optimizations to accelerate convergence of in vitro selections.
Chapter 4 describes a high throughput double membrane dot blot system to rapidly
screen, identify and characterize affinity peptides obtained from selection output. I used
dot blot to screen potential affinity peptides from large diversity of previously
ii
uncharacterized mRNA display selection output. Further characterization of potential
peptides allowed determination of several high affinity peptides from having Kd range 150-
450 nM. Double membrane dot blot is automation amenable, easy and affordable solution
for analyzing selection output and characterizing peptides without ne ed for much
instrumentation.
Together these projects serve as guideline for evolution of cost effective high throughput
pipeline for identification and characterization of affinity peptides.
drugs in basic research, biotechnology, diagnostics and therapeutics. However, due to the
cost, labor and time associated with production of antibodies focus has recently changed
towards potential of peptides to act as protein affinity reagents. Affinity peptides are easy
to work with, non-immunogenic, cost effective and amenable to scale up. Even though
researchers have developed several affinity peptides, we are far from compiling library of
peptides that encompasses entire human proteome. My thesis describes high throughput
pipeline that can be used to develop and characterize affinity peptides that bind several
discrete sites on target proteins.
Chapter 2 describes optimization of cell-free protein expression using commercially
available translation systems and well-known leader sequences. Presence of internal
ribosome entry site upstream of coding region allows maximal expression in HeLa cell
lysate whereas translation enhancing elements are best suited for expression in rabbit
reticulocyte lysate and wheat germ extract. Use of optimal vector and cell lysate
combination ensures maximum protein expression of DNA libraries.
Chapter 3 describes mRNA display selection methodology for developing affinity peptides
for target proteins using large diversity DNA libraries. I demonstrate that mild denaturant
is not sufficient to increase selection pressure for up to three rounds of selection and
increasing number of selection rounds increases probability of finding affinity peptide s.
These studies enhance fundamental understanding of mRNA display and pave the way
for future optimizations to accelerate convergence of in vitro selections.
Chapter 4 describes a high throughput double membrane dot blot system to rapidly
screen, identify and characterize affinity peptides obtained from selection output. I used
dot blot to screen potential affinity peptides from large diversity of previously
ii
uncharacterized mRNA display selection output. Further characterization of potential
peptides allowed determination of several high affinity peptides from having Kd range 150-
450 nM. Double membrane dot blot is automation amenable, easy and affordable solution
for analyzing selection output and characterizing peptides without ne ed for much
instrumentation.
Together these projects serve as guideline for evolution of cost effective high throughput
pipeline for identification and characterization of affinity peptides.
ContributorsShah, Pankti (Author) / Chaput, John (Thesis advisor) / Hecht, Sidney (Committee member) / Wachter, Rebekka (Committee member) / Arizona State University (Publisher)
Created2014