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
- Creators: Mills, Jeremy H
- Creators: Mangone, Marco
Description
Based on past studies, urinary glycan biomarkers have the potential to be used as diagnostic and prognostic markers for treatment purposes. This study brought into play the bottom-up glycan node analysis approach to analyze 39 urine samples from COVID-19 positive and negative individuals using gas chromatography-mass spectrometry (GC-MS) to determine potential urinary glycan biomarkers of COVID-19. Glycan node analysis involves chemically breaking down glycans in whole biospecimens in a way that conserves both monosaccharide identity and linkage information that facilitates the capture of unique glycan features as single analytical signals. Following data acquisition, the student t-test was done on all the nodes, but only four prominent nodes (t-Deoxyhexopyranose, 2,3-Gal, t-GlcNAc, and 3,6-GalNAc with respective p-values 0.03027, 0.03973, 0.0224, and 0.0004) were below the threshold p-value of 0.05 and showed some differences in the mean between both groups. To eliminate the probability of having false positive p-values, Bonferroni correction was done on the four nodes but only the 3,6-GalNAc node emerged as the only node that was below the newly adjusted p-value. Because sample analyses were done in batches, the Kruskal Wallis test was done to know if the batch effect was responsible for the observed lower relative concentration of 3,6-GalNAc in COVID-19 positive patients than in negative patients. A receiver operating characteristic curve (ROC) was plotted for the 3,6-GalNAc node and the area under the curve (AUC) was calculated to be 0.84, casting the 3,6-GalNAc node was a potential biomarker of COVID-19. 3,6-GalNAc largely arises from branched O-glycan core structures, which are abundant in mucin glycoproteins that line the urogenital tract. Lowered relative concentrations of 3,6-GalNAc in the urine of COVID-19 positive patients may be explained by compromised kidney function that allows non-mucinous glycoproteins from the blood to contribute a greater proportion of the relative glycan node signals than in COVID-19 negative patients. Future prospective clinical studies will be needed to validate both the biomarker findings and this hypothesis.
ContributorsEyonghebi Tanyi, Agbor (Author) / Borges, Chad R (Thesis advisor) / Mills, Jeremy H (Committee member) / Guo, Jia (Committee member) / Arizona State University (Publisher)
Created2023
Description
Mutation is the source of heritable variation of genotype and phenotype, on which selection may act. Mutation rates describe a fundamental parameter of living things, which influence the rate at which evolution may occur, from viral pathogens to human crops and even to aging cells and the emergence of cancer. An understanding of the variables which impact mutation rates and their estimation is necessary to place mutation rate estimates in their proper contexts. To better understand mutation rate estimates, this research investigates the impact of temperature upon transcription rate error estimates; the impact of growing cells in liquid culture vs. on agar plates; the impact of many in vitro variables upon the estimation of deoxyribonucleic acid (DNA) mutation rates from a single sample; and the mutational hazard induced by expressing clustered regularly interspaced short palindromic repeat (CRISPR) proteins in yeast. This research finds that many of the variables tested did not significantly alter the estimation of mutation rates, strengthening the claims of previous mutation rate estimates across the tree of life by diverse experimental approaches. However, it is clear that sonication is a mutagen of DNA, part of an effort which has reduced the sequencing error rate of circle-seq by over 1,000-fold. This research also demonstrates that growth in liquid culture modestly skews the mutation spectrum of MMR- Escherichia coli, though it does not significantly impact the overall mutation rate. Finally, this research demonstrates a modest mutational hazard of expressing Cas9 and similar CRISPR proteins in yeast cells at an un-targeted genomic locus, though it is possible the indel rate has been increased by an order of magnitude.
ContributorsBaehr, Stephan (Author) / Lynch, Michael (Thesis advisor) / Geiler-Samerotte, Kerry (Committee member) / Mangone, Marco (Committee member) / Wilson, Melissa (Committee member) / Arizona State University (Publisher)
Created2023
Description
Enzymes keep life nicely humming along by catalyzing important reactions at relevant timescales. Despite their immediate importance, how enzymes recognize and bind their substrate in a sea of cytosolic small molecules, carry out the reaction, and release their product in microseconds is still relatively opaque. Methods to elucidate enzyme substrate specificity indicate that the shape of the active site and the amino acid residues therein play a major role. However, lessons from Directed Evolution experiments reveal the importance of residues far from the active site in modulating substrate specificity. Enzymes are dynamic macromolecules composed of networks of interactions integrating the active site, where the chemistry occurs, to the rest of the protein. The objective of this work is to develop computational methods to modify enzyme ligand specificity, either through molding the active site to accommodate a novel ligand, or by identifying distal mutations that can allosterically alter specificity. To this end, two homologues in the β-lactamase family of enzymes, TEM-1, and an ancestrally reconstructed variant, GNCA, were studied to identify whether the modulation of position-specific distal-residue flexibility could modify ligand specificity. RosettaDesign was used to create TEM-1 variants with altered dynamic patterns. Experimental characterization of ten designed proteins indicated that mutations to residues surrounding rigid, highly coupled residues substantially affected both enzymatic activity and stability. In contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Five of the TEM-1 variants were crystallized to see if the changes in function observed were due to architectural changes to the active site. In a second project, a computational platform using RosettaDesign was developed to remodel the firefly luciferase active site to accommodate novel luciferins. This platform resulted in the development of five luciferin-luciferase pairs with red-shifted emission maxima, ready for multicomponent bioluminescent imaging applications in tissues. Although the projects from this work focus on two classes of proteins, they provide insight into the structure-function relationship of ligand specificity in enzymes and are broadly applicable to other systems.
ContributorsKolbaba Kartchner, Bethany (Author) / Mills, Jeremy H (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Van Horn, Wade D (Committee member) / Arizona State University (Publisher)
Created2023
Description
The physiological phenomenon of sensing temperature is detected by transient
receptor (TRP) ion channels, which are pore forming proteins that reside in the
membrane bilayer. The cold and hot sensing TRP channels named TRPV1 and TRPM8
respectively, can be modulated by diverse stimuli and are finely tuned by proteins and
lipids. PIRT (phosphoinositide interacting regulator of TRP channels) is a small
membrane protein that modifies TRPV1 responses to heat and TRPM8 responses to cold.
In this dissertation, the first direct measurements between PIRT and TRPM8 are
quantified with nuclear magnetic resonance and microscale thermophoresis. Using
Rosetta computational biology, TRPM8 is modeled with a regulatory, and functionally
essential, lipid named PIP2. Furthermore, a PIRT ligand screen identified several novel
small molecular binders for PIRT as well a protein named calmodulin. The ligand
screening results implicate PIRT in diverse physiological functions. Additionally, sparse
NMR data and state of the art Rosetta protocols were used to experimentally guide PIRT
structure predictions. Finally, the mechanism of thermosensing from the evolutionarily
conserved sensing domain of TRPV1 was investigated using NMR. The body of work
presented herein advances the understanding of thermosensing and TRP channel function
with TRP channel regulatory implications for PIRT.
receptor (TRP) ion channels, which are pore forming proteins that reside in the
membrane bilayer. The cold and hot sensing TRP channels named TRPV1 and TRPM8
respectively, can be modulated by diverse stimuli and are finely tuned by proteins and
lipids. PIRT (phosphoinositide interacting regulator of TRP channels) is a small
membrane protein that modifies TRPV1 responses to heat and TRPM8 responses to cold.
In this dissertation, the first direct measurements between PIRT and TRPM8 are
quantified with nuclear magnetic resonance and microscale thermophoresis. Using
Rosetta computational biology, TRPM8 is modeled with a regulatory, and functionally
essential, lipid named PIP2. Furthermore, a PIRT ligand screen identified several novel
small molecular binders for PIRT as well a protein named calmodulin. The ligand
screening results implicate PIRT in diverse physiological functions. Additionally, sparse
NMR data and state of the art Rosetta protocols were used to experimentally guide PIRT
structure predictions. Finally, the mechanism of thermosensing from the evolutionarily
conserved sensing domain of TRPV1 was investigated using NMR. The body of work
presented herein advances the understanding of thermosensing and TRP channel function
with TRP channel regulatory implications for PIRT.
ContributorsSisco, Nicholas John (Author) / Van Horn, Wade D (Thesis advisor) / Mills, Jeremy H (Committee member) / Wang, Xu (Committee member) / Yarger, Jeff L (Committee member) / Arizona State University (Publisher)
Created2018
Description
The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications.
ContributorsMA, DUO (Author) / Green, Alexander (Thesis advisor) / Mangone, Marco (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2019
Description
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in vivo tumor growth study utilizing the pancreatic tumor cell line MIAPaCa-2 containing a QSOX1-silencing shRNA construct revealed that QSOX1 expression supports a proliferative phenotype. These preliminary studies suggest that suppressing the enzymatic activity of QSOX1 could represent a novel therapeutic strategy to inhibit proliferation and invasion of malignant neoplasms.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
The goal of this research was to identify and characterize biologically active small molecule inhibitors for QSOX1. Chemical inhibition of QSOX1 enzymatic activity was hypothesized to reduce growth and invasion of tumor cells. Recombinant QSOX1 was screened against libraries of small molecules using an enzymatic activity assay to identify potential QSOX1 inhibitors. Two lead QSOX1 inhibitors were confirmed, 2-phenyl-1, 2-benzisoselenazol-3-one (ebselen), and 3-methoxy-n-[4-(1 pyrrolidinyl)phenyl]benzamide. The biological activity of these compounds is consistent with QSOX1 knockdown in tumor cell lines, reducing growth and invasion in vitro. Treatment of tumor cells with these compounds also resulted in specific ECM defects, a phenotype associated with QSOX1 knockdown. Additionally, these compounds were shown to be active in pancreatic and renal cancer xenografts, reducing tumor growth with daily treatment. For ebselen, the molecular mechanism of inhibition was determined using a combination of biochemical and mass spectrometric techniques. The results obtained in these studies provide proof-of-principle that targeting QSOX1 enzymatic activity with chemical compounds represents a novel potential therapeutic avenue worthy of further investigation in cancer. Additionally, the utility of these small molecules as chemical probes will yield future insight into the general biology of QSOX1, including the identification of novel substrates of QSOX1.
ContributorsHanavan, Paul D (Author) / Lake, Douglas (Thesis advisor) / LaBaer, Joshua (Committee member) / Mangone, Marco (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2015
Description
Advances in chemical synthesis have enabled new lines of research with unnatural genetic polymers whose modified bases or sugar-phosphate backbones have potential therapeutic and biotechnological applications. Maximizing the potential of these synthetic genetic systems requires inventing new molecular biology tools that can both generate and faithfully replicate unnatural polymers of significant length. Threose nucleic acid (TNA) has received significant attention as a complete replication system has been developed by engineering natural polymerases to broaden their substrate specificity. The system, however, suffers from a high mutational load reducing its utility. This thesis will cover the development of two new polymerases capable of transcribing and reverse transcribing TNA polymers with high efficiency and fidelity. The polymerases are identified using a new strategy wherein gain-of-function mutations are sampled in homologous protein architectures leading to subtle optimization of protein function. The new replication system has a fidelity that supports the propagation of genetic information enabling in vitro selection of functional TNA molecules. TNA aptamers to human alpha-thrombin are identified and demonstrated to have superior stability compared to DNA and RNA in biologically relevant conditions. This is the first demonstration that functional TNA molecules have potential in biotechnology and molecular medicine.
ContributorsDunn, Matthew Ryan (Author) / Chaput, John C (Thesis advisor) / LaBaer, Joshua (Committee member) / Lake, Douglas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2015
Description
Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment and computational screening methods were employed for the discovery of numerous telomerase RNAs from the poorly characterized echinoderm lineage. This resulted in the revelation that--while closely related to the vertebrate lineage and grossly resembling vertebrate telomerase RNA--the echinoderm telomerase RNA central domain varies extensively in structure and sequence, diverging even within echinoderms amongst sea urchins and brittle stars. Furthermore, the origins of telomerase RNA within the eukaryotic lineage have remained a persistent mystery. The ancient Trypanosoma telomerase RNA was previously identified, however, a functionally verified secondary structure remained elusive. Synthetic Trypanosoma telomerase was generated for molecular dissection of Trypanosoma telomerase RNA revealing two RNA domains functionally equivalent to those found in known telomerase RNAs, yet structurally distinct. This work demonstrates that telomerase RNA is uncommonly divergent in gross architecture, while retaining critical universal elements.
ContributorsPodlevsky, Joshua (Author) / Chen, Julian (Thesis advisor) / Mangone, Marco (Committee member) / Kusumi, Kenro (Committee member) / Wilson-Rawls, Norma (Committee member) / Arizona State University (Publisher)
Created2015
Description
Extracellular vesicles (EVs) represent a heterogeneous population of small vesicles, consisting of a phospholipidic bilayer surrounding a soluble interior cargo. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. Peripheral blood is a rich source of circulating EVs. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic, prognostic as well as therapeutic value. In the current study, a plasma EV enrichment method based on pluronic co-polymer was first established and characterized. Plasma EVs from breast cancer patients were then enriched, profiled and compared to non-cancer controls. Proteins signatures that contributed to the prediction of cancer samples from non-cancer controls were created by a random-forest based cross-validation approach. We found that a large portion of these signatures were related to breast cancer aggression. To verify such findings, KIAA0100, one of the features identified, was chosen for in vitro molecular and cellular studies in the breast cancer cell line MDA-MB-231. We found that KIAA0100 regulates cancer cell aggression in MDA-MB-231 in an anchorage-independent manner and is particularly associated with anoikis resistance through its interaction with HSPA1A. Lastly, plasma EVs contain not only individual proteins, but also numerous molecular complexes. In order to measure millions of proteins, isoforms, and complexes simultaneously, Adaptive Dynamic Artificial Poly-ligand Targeting (ADAPT) platform was applied. ADAPT employs an enriched library of single-stranded oligodeoxynucleotides to profile complex biological samples, thus achieving a deep coverage of system-wide, native biomolecules. Profiling of EVs from breast cancer patients was able to obtain a prediction AUC performance of 0.73 when compared biopsy-positive cancer patient to healthy controls and 0.64 compared to biopsy-negative controls and such performance was not associated with the physical breast condition indicated by BIRAD scores. Taken together, current research demonstrated the potential of profiling plasma EVs in searching for therapeutic targets as well as diagnostic signatures.
ContributorsZhong, Zhenyu (Author) / Spetzler, David (Thesis advisor) / Yan, Hao (Thesis advisor) / Lake, Douglas (Committee member) / Mangone, Marco (Committee member) / Arizona State University (Publisher)
Created2018
Description
Continuing and increasing reliance on fossil fuels to satisfy our population’s energy demands has encouraged the search for renewable carbon-free and carbon-neutral sources, such as hydrogen gas or CO2 reduction products. Inspired by nature, one of the objectives of this dissertation was to develop protein-based strategies that can be applied in the production of green fuels. The first project of this dissertation aimed at developing a controllable strategy to incorporate domains with different functions (e. g. catalytic sites, electron transfer modules, light absorbing subunits) into a single multicomponent system. This was accomplished through the rational design of 2,2’-bipyridine modified dimeric peptides that allowed their metal-directed oligomerization by forming tris(bipyridine) complexes, thus resulting in the formation of a hexameric assembly.
Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction.
Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies.
Additionally, two different approaches to incorporate non-natural organometallic catalysts into protein matrix are discussed. First, cobalt protoporphyrin IX was incorporated into cytochrome b562 to produce a water-soluble proton and CO2 reduction catalyst that is active upon irradiation in the presence of a photosensitizer. The effect of the porphyrin axial ligands provided by the protein environment has been investigated by introducing mutations into the native scaffold, indicating that catalytic activity of proton reduction is dependent on axial coordination to the porphyrin. It is also shown that effects of the protein environment are not directly transferred when applied to other reactions, such as CO2 reduction.
Inspired by the active site of [FeFe]-hydrogenases, the second approach is based on the stereoselective preparation of a novel amino acid bearing a 1,2-benzenedithiol side chain. This moiety can serve as an anchoring point for the introduction of metal complexes into protein matrices. By doing so, this strategy enables the study of protein interactions with non-natural cofactors and the effects that it may have on catalysis. The work developed herein lays a foundation for furthering the study of the use of proteins as suitable environments for tuning the activity of organometallic catalysts in aqueous conditions, and interfacing these systems with other supporting units into supramolecular assemblies.
ContributorsAlcala-Torano, Rafael de Jesus (Author) / Ghirlanda, Giovanna (Thesis advisor) / Moore, Ana L (Committee member) / Mills, Jeremy H (Committee member) / Arizona State University (Publisher)
Created2019