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: Biology
- Creators: Wang, Xuan
- Creators: Wang, Xiao
Description
Random peptide microarrays are a powerful tool for both the treatment and diagnostics of infectious diseases. On the treatment side, selected random peptides on the microarray have either binding or lytic potency against certain pathogens cells, thus they can be synthesized into new antimicrobial agents, denoted as synbodies (synthetic antibodies). On the diagnostic side, serum containing specific infection-related antibodies create unique and distinct "pathogen-immunosignatures" on the random peptide microarray distinct from the healthy control serum, and this different mode of binding can be used as a more precise measurement than traditional ELISA tests. My thesis project is separated into these two parts: the first part falls into the treatment side and the second one focuses on the diagnostic side. My first chapter shows that a substitution amino acid peptide library helps to improve the activity of a recently reported synthetic antimicrobial peptide selected by the random peptide microarray. By substituting one or two amino acids of the original lead peptide, the new substitutes show changed hemolytic effects against mouse red blood cells and changed potency against two pathogens: Staphylococcus aureus and Pseudomonas aeruginosa. Two new substitutes are then combined together to form the synbody, which shows a significantly antimicrobial potency against Staphylococcus aureus (<0.5uM). In the second chapter, I explore the possibility of using the 10K Ver.2 random peptide microarray to monitor the humoral immune response of dengue. Over 2.5 billion people (40% of the world's population) live in dengue transmitting areas. However, currently there is no efficient dengue treatment or vaccine. Here, with limited dengue patient serum samples, we show that the immunosignature has the potential to not only distinguish the dengue infection from non-infected people, but also the primary dengue infection from the secondary dengue infections, dengue infection from West Nile Virus (WNV) infection, and even between different dengue serotypes. By further bioinformatic analysis, we demonstrate that the significant peptides selected to distinguish dengue infected and normal samples may indicate the epitopes responsible for the immune response.
ContributorsWang, Xiao (Author) / Johnston, Stephen Albert (Thesis advisor) / Blattman, Joseph (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2013
Description
The Multiple Antibiotic Resistance Regulator Family (MarR) are transcriptional regulators, many of which forms a dimer. Transcriptional regulation provides bacteria a stabilized responding system to ensure the bacteria is able to efficiently adapt to different environmental conditions. The main function of the MarR family is to create multiple antibiotic resistance from a mutated protein; this process occurs when the MarR regulates an operon. We hypothesized that different transcriptional regulator genes have interactions with each other. It is known that Salmonella pagC transcription is activated by three regulators, i.e., SlyA, MprA, and PhoP. Bacterial Adenylate Cyclase-based Two-Hybrid (BACTH) system was used to research the protein-protein interactions in SlyA, MprA, and PhoP as heterodimers and homodimers in vivo. Two fragments, T25 and T18, that lack endogenous adenylate cyclase activity, were used for construction of chimeric proteins and reconstruction of adenylate cyclase activity was tested. The significant adenylate cyclase activities has proved that SlyA is able to form homodimers. However, weak adenylate cyclase activities in this study has proved that MprA and PhoP are not likely to form homodimers, and no protein-protein interactions were detected in between SlyA, MprA and PhoP, which no heterodimers have formed in between three transcriptional regulators.
ContributorsTao, Zenan (Author) / Shi, Yixin (Thesis advisor) / Wang, Xuan (Committee member) / Bean, Heather (Committee member) / Arizona State University (Publisher)
Created2018
Description
Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.
ContributorsKurgan, Gavin (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2018
Description
Emergence of multidrug resistant (MDR) bacteria is a major concern to global health. One of the major MDR mechanisms bacteria employ is efflux pumps for the expulsion of drugs from the cell. In Escherichia coli, AcrAB-TolC proteins constitute the major chromosomally-encoded drug efflux system. AcrB, a trimeric membrane protein is well-known for its substrate promiscuity. It has the ability to efflux a broad spectrum of substrates alongside compounds such as dyes, detergent, bile salts and metabolites. Newly identified AcrB residues were shown to be functionally relevant in the drug binding and translocation pathway using a positive genetic selection strategy. These residues—Y49, V127, D153, G288, F453, and L486—were identified as the sites of suppressors of an alteration, F610A, that confers a drug hypersensitivity phenotype. Using site-directed mutagenesis (SDM) along with the real-time efflux and the classical minimum inhibitory concentration (MIC) assays, I was able to characterize the mechanism of suppression.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
Three approaches were used for the characterization of these suppressors. The first approach focused on side chain specificity. The results showed that certain suppressor sites prefer a particular side chain property, such as size, to overcome the F610A defect. The second approach focused on the effects of efflux pump inhibitors. The results showed that though the suppressor residues were able to overcome the intrinsic defect of F610A, they were unable to overcome the extrinsic defect caused by the efflux pump inhibitors. This showed that the mechanism by which F610A imposes its effect on AcrB function is different than that of the efflux pump inhibitors. The final approach was to determine whether suppressors mapping in the periplasmic and trans-membrane domains act by the same or different mechanisms. The results showed both overlapping and distinct mechanisms of suppression.
To conclude, these approaches have provided a deeper understanding of the mechanisms by which novel suppressor residues of AcrB overcome the functional defect of the drug binding domain alteration, F610A.
ContributorsBlake, Mellecha (Author) / Misra, Rajeev (Thesis advisor) / Stout, Valerie (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
Description
Synthetic gene networks have evolved from simple proof-of-concept circuits to
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
complex therapy-oriented networks over the past fifteen years. This advancement has
greatly facilitated expansion of the emerging field of synthetic biology. Multistability is a
mechanism that cells use to achieve a discrete number of mutually exclusive states in
response to environmental inputs. However, complex contextual connections of gene
regulatory networks in natural settings often impede the experimental establishment of
the function and dynamics of each specific gene network.
In this work, diverse synthetic gene networks are rationally designed and
constructed using well-characterized biological components to approach the cell fate
determination and state transition dynamics in multistable systems. Results show that
unimodality and bimodality and trimodality can be achieved through manipulation of the
signal and promoter crosstalk in quorum-sensing systems, which enables bacterial cells to
communicate with each other.
Moreover, a synthetic quadrastable circuit is also built and experimentally
demonstrated to have four stable steady states. Experiments, guided by mathematical
modeling predictions, reveal that sequential inductions generate distinct cell fates by
changing the landscape in sequence and hence navigating cells to different final states.
Circuit function depends on the specific protein expression levels in the circuit.
We then establish a protein expression predictor taking into account adjacent
transcriptional regions’ features through construction of ~120 synthetic gene circuits
(operons) in Escherichia coli. The predictor’s utility is further demonstrated in evaluating genes’ relative expression levels in construction of logic gates and tuning gene expressions and nonlinear dynamics of bistable gene networks.
These combined results illustrate applications of synthetic gene networks to
understand the cell fate determination and state transition dynamics in multistable
systems. A protein-expression predictor is also developed to evaluate and tune circuit
dynamics.
ContributorsWu, Fuqing (Author) / Wang, Xiao (Thesis advisor) / Haynes, Karmella (Committee member) / Marshall, Pamela (Committee member) / Nielsen, David (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2017
Characterization and Manipulation of Microbiomes From Arid Landfills for Improved Methane Production
Description
Environmentally harmful byproducts from solid waste’s decomposition, including methane (CH4) emissions, are managed through standardized landfill engineering and gas-capture mechanisms. Yet only a limited number of studies have analyzed the development and composition of Bacteria and Archaea involved in CH4 production from landfills. The objectives of this research were to compare microbiomes and bioactivity from CH4-producing communities in contrasting spatial areas of arid landfills and to tests a new technology to biostimulate CH4 production (methanogenesis) from solid waste under dynamic environmental conditions controlled in the laboratory. My hypothesis was that the diversity and abundance of methanogenic Archaea in municipal solid waste (MSW), or its leachate, play an important role on CH4 production partially attributed to the group’s wide hydrogen (H2) consumption capabilities. I tested this hypothesis by conducting complementary field observations and laboratory experiments. I describe niches of methanogenic Archaea in MSW leachate across defined areas within a single landfill, while demonstrating functional H2-dependent activity. To alleviate limited H2 bioavailability encountered in-situ, I present biostimulant feasibility and proof-of-concepts studies through the amendment of zero valent metals (ZVMs). My results demonstrate that older-aged MSW was minimally biostimulated for greater CH4 production relative to a control when exposed to iron (Fe0) or manganese (Mn0), due to highly discernable traits of soluble carbon, nitrogen, and unidentified fluorophores found in water extracts between young and old aged, starting MSW. Acetate and inhibitory H2 partial pressures accumulated in microcosms containing old-aged MSW. In a final experiment, repeated amendments of ZVMs to MSW in a 600 day mesocosm experiment mediated significantly higher CH4 concentrations and yields during the first of three ZVM injections. Fe0 and Mn0 experimental treatments at mesocosm-scale also highlighted accelerated development of seemingly important, but elusive Archaea including Methanobacteriaceae, a methane-producing family that is found in diverse environments. Also, prokaryotic classes including Candidatus Bathyarchaeota, an uncultured group commonly found in carbon-rich ecosystems, and Clostridia; All three taxa I identified as highly predictive in the time-dependent progression of MSW decomposition. Altogether, my experiments demonstrate the importance of H2 bioavailability on CH4 production and the consistent development of Methanobacteriaceae in productive MSW microbiomes.
ContributorsReynolds, Mark Christian (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Wang, Xuan (Committee member) / Kavazanjian, Edward (Committee member) / Arizona State University (Publisher)
Created2022
Description
Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation.
ContributorsHerschel, Daniel Jordan (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
Description
Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than deoxyribose nucleic acid (DNA) sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. In 2011, Haynes et al. showed that a synthetic regulator called the Polycomb chromatin Transcription Factor (PcTF), a fusion protein that binds methylated histones, reactivated an artificially-silenced luciferase reporter gene. These synthetic transcription activators are derived from the polycomb repressive complex (PRC) and associate with the epigenetic silencing mark H3K27me3 to reactivate the expression of silenced genes. It is demonstrated here that the duration of epigenetic silencing does not perturb reactivation via PcTF fusion proteins. After 96 hours PcTF shows the strongest reactivation activity. A variant called Pc2TF, which has roughly double the affinity for H3K27me3 in vitro, reactivated the silenced luciferase gene by at least 2-fold in living cells.
ContributorsVargas, Daniel A. (Author) / Haynes, Karmella (Thesis advisor) / Wang, Xiao (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2019
Description
A notable challenge when assembling synthetic gene circuits is that modularity often fails to function as intended. A crucial underlying reason for this modularity failure is the existence of competition for shared and limited gene expression resources. By designing a synthetic cascading bistable switches (Syn-CBS) circuit in a single strain with two coupled self-activation modules to achieve successive cell fate transitions, nonlinear resource competition within synthetic gene circuits is unveiled. However, in vivo it can be seen that the transition path was redirected with the activation of one switch always prevailing over that of the other, contradictory to coactivation theoretically expected. This behavior is a result of resource competition between genes and follows a ‘winner-takes-all’ rule, where the winner is determined by the relative connection strength between the two modules. Despite investigation demonstrating that resource competition between gene modules can significantly alter circuit deterministic behaviors, how resource competition contributes to gene expression noise and how this noise can be controlled is still an open issue of fundamental importance in systems biology and biological physics. By utilizing a two-gene circuit, the effects of resource competition on protein expression noise levels can be closely studied. A surprising double-edged role is discovered: the competition for these resources decreases noise while the constraint on resource availability adds its own term of noise into the system, denoted “resource competitive” noise. Noise reduction effects are then studied using orthogonal resources. Results indicate that orthogonal resources are a good strategy for eliminating the contribution of resource competition to gene expression noise.
Noise propagation through a cascading circuit has been considered without resource competition. It has been noted that the noise from upstream genes can be transmitted downstream. However, resource competition’s effects on this cascading noise have yet to be studied. When studied, it is found that resource competition can induce stochastic state
switching and perturb noise propagation. Orthogonal resources can remove some of the resource competitive behavior and allow for a system with less noise.
ContributorsGoetz, Hanah Elizabeth (Author) / Tian, Xiaojun (Thesis advisor) / Wang, Xiao (Committee member) / Lai, Ying-Cheng (Committee member) / Arizona State University (Publisher)
Created2022
Description
Reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and hydroxyl radicals occur naturally as a byproduct of aerobic respiration. To mitigate damages caused by ROS, Escherichia coli employs defenses including two cytosolic superoxide dismutases (SODs), which convert superoxide to hydrogen peroxide. Deletion of both sodA and sodB, the genes coding for the cytosolic SOD enzymes, results in a strain that is unable to grow on minimal medium without amino acid supplementation. Additionally, deletion of both cytosolic SOD enzymes in a background containing the relA1 allele, an inactive version of the relA gene that contributes to activation of stringent response by amino acid starvation, results in a strain that is unable to grow aerobically, even on rich medium. These observations point to a relationship between the stringent response and oxidative stress. To gain insight into this relationship, suppressors were isolated by growing the ∆sodAB relA1 cells aerobically on rich medium, and seven suppressors were further examined to characterize distinct colony sizes and temperature sensitivity phenotypes. In three of these suppressor-containing strains, the relA1 allele was successfully replaced by the wild type relA allele to allow further study in aerobic conditions. None of those three suppressors were found to increase tolerance to exogenous superoxides produced by paraquat, which shows that these mutations only overcome the superoxide buildup that naturally occurs from deletion of SODs. Because each of these suppressors had unique phenotypes, it is likely that they confer tolerance to SOD-dependent superoxide buildup by different mechanisms. Two of these three suppressors have been sent for whole-genome sequencing to identify the location of the suppressor mutation and determine the mechanism by which they confer superoxide tolerance.
ContributorsFlake, Melissa (Author) / Misra, Rajeev (Thesis advisor) / Shah, Dhara (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2024