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Transient protein-protein and protein-molecule interactions fluctuate between associated and dissociated states. They are widespread in nature and mediate most biological processes. These interactions are complex and are strongly influenced by factors such as concentration, structure, and environment. Understanding and utilizing these types of interactions is useful from both a fundamental and design perspective. In this dissertation, transient protein interactions are used as the sensing element of a biosensor for small molecule detection. This is done by using a transcription factor-small molecule pair that mediates the activation of a CRISPR/Cas12a complex. Activation of the Cas12a enzyme results in an amplified readout mechanism that is either fluorescence or paper based. This biosensor can successfully detect 9 different small molecules including antibiotics with a tuneable detection limit ranging from low µM to low nM. By combining protein and nucleic acid-based systems, this biosensor has the potential to report on almost any protein-molecule interaction, linking this to the intrinsic amplification that is possible when working with nucleic acid-based technologies. The second part of this dissertation focuses on understanding protein-molecule interactions at a more fundamental level, and, in so doing, exploring design rules required to generalize sensors like the ones described above. This is done by training a neural network algorithm with binding data from high density peptide micro arrays incubated with specific protein targets. Because the peptide sequences were chosen simply to evenly, though sparsely, represent all sequence space, the resulting network provides a comprehensive sequence/binding relationship for a given target protein. While past work had shown that this works well on the arrays, here I have explored how well the neural networks thus trained, predict sequence-dependent binding in the context of protein-protein and peptide-protein interactions. Amino acid sequences, either free in solution or embedded in protein structure, will display somewhat different binding properties than sequences affixed to the surface of a high-density array. However, the neural network trained on array sequences was able to both identify binding regions in between proteins and predict surface plasmon resonance-based binding propensities for peptides with statistically significant levels of accuracy.
ContributorsSwingle, Kirstie Lynn (Author) / Woodbury, Neal W (Thesis advisor) / Green, Alexander A (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2022
Description
Bats are a highly diverse mammal species with a dense virome and fascinating immune system. The following project utilizes metagenomics in order to identify DNA viruses present in populations of silver-haired bats and Mexican free-tailed bats from southern Arizona. A significant number of DNA viruses and novel viruses were identified in the Cressdnaviricota phylum and Microvirdae family.
ContributorsHarding, Ciara (Author) / Varsani, Arvind (Thesis director) / Dolby, Greer (Committee member) / Kraberger, Simona (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Watts College of Public Service & Community Solut (Contributor)
Created2022-05
Description
Attitudes and habits are extremely resistant to change, but a disruption of the magnitude of the COVID-19 pandemic has the potential to bring long-term, massive societal changes. During the pandemic, people are being compelled to experience new ways of interacting, working, learning, shopping, traveling, and eating meals. Going forward, a critical question is whether these experiences will result in changed behaviors and preferences in the long term. This paper presents initial findings on the likelihood of long-term changes in telework, daily travel, restaurant patronage, and air travel based on survey data collected from adults in the United States in Spring 2020. These data suggest that a sizable fraction of the increase in telework and decreases in both business air travel and restaurant patronage are likely here to stay. As for daily travel modes, public transit may not fully recover its pre-pandemic ridership levels, but many of our respondents are planning to bike and walk more than they used to. These data reflect the responses of a sample that is higher income and more highly educated than the US population. The response of these particular groups to the COVID-19 pandemic is perhaps especially important to understand, however, because their consumption patterns give them a large influence on many sectors of the economy.
ContributorsConway, Matthew Wigginton (Author) / Salon, Deborah (Author) / da Silva, Denise Capasso (Author) / Mirtich, Laura Christine (Author)
Created2020-09-03
Description
For cold chain tracking systems, precision and versatility across varying time intervals and temperature ranges remain integral to effective application in clinical, commercial, and academic settings. Therefore, while electronic and chemistry/physics based cold chain tracking mechanisms currently exist, both have limitations that affect their application across various biospecimens and commercial products, providing the initiative to develop a time temperature visual indicator system that resolves challenges with current cold chain tracking approaches. As a result, a permanganate/oxalic acid time temperature visual indicator system for cold chain tracking has been proposed. At thawing temperatures, the designed permanganate/oxalic acid reaction system undergoes a pink to colorless transition as permanganate, Mn(VII), is reduced to auto-catalytic Mn(II), while oxalate is oxidized to CO2. Therefore, when properly stored and vitrified or frozen, the proposed visual indicator remains pink, whereas exposure to thawing conditions will result in an eventual, time temperature dependent, designed color transition that characterizes compromised biospecimen integrity. To design visual indicator systems for targeted times at specific temperatures, absorbance spectroscopy was utilized to monitor permanganate kinetic curves by absorbance at 525 nm. As a result, throughout the outlined research, the following aims were demonstrated: (i) Design and functionality of 1x (0.5 mM KMnO4) visual indicator systems across various time intervals at temperatures ranging from 25°C to -20°C, (ii) Design and functionality of high concentration, 5x, visual indicator systems across varying targeted time intervals at temperatures ranging from 25°C to 0°C, (iii) Pre-activation stability and long-term stability of the proposed visual indicator systems.
ContributorsLjungberg, Emil (Author) / Borges, Chad (Thesis advisor) / Levitus, Marcia (Committee member) / Williams, Peter (Committee member) / Arizona State University (Publisher)
Created2024
Description
Wild horses have roamed the Salt River in Mesa, Arizona since the early 1800s and contribute to the great diversity of the region. Conservation of the herd has been a primary focus for many years and a current focus is population stabilization, but little is known about their virome. Circoviridae, Genomoviridae, and Smacoviridae are the three Cressdnaviricota viruses that have been identified in horses to date. Smacoviridae is classified by the rolling circle replication-associated proteins (Rep) and has a small (2.3-2.9kb), circular, single-stranded genome. The goal of this study was to identify DNA viruses within the fecal samples of the Salt River horses. Samples were collected along the lower Salt River and analyzed in the lab using a metagenomics approach. There were 422 full novel genomes of smacoviruses detected across all samples that were grouped into 144 species based on the similarity of the pairwise identity. Phylogenetic analysis shows the smacoviruses from this study fall into 3 classified genera and the rest cluster into 11 new clades. These results expand the viral diversity associated with wild horses and Smacoviridae, and further studies are needed to determine the host of these viruses.
ContributorsMcGraw, Hannah (Author) / Varsani, Arvind (Thesis director) / Murphree, Julie (Committee member) / Kraberger, Simona (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2024-05
Description
Scorpions are predatory arachnids that are among the most ancient terrestrial invertebrates. They are typically found residing in desert and riparian environments. Viruses associated with scorpions have been explored in the past, unveiling partial RNA virus sequences and polyomaviruses, but more research in this area is necessary. Cycloviruses are non-enveloped viruses with circular single-stranded DNA genomes (~1.7 to 1.9 kb). Cycloviruses were initially identified in mammals and have now been detected in samples from a wide range of mammalian and insect species. Polyomaviruses are double-stranded DNA viruses (~4 to 7 kb). They are known for causing tumors in the host it infects, and have previously been identified in a diverse array of organisms, including scorpions. The objective for this study was to identify known and novel viruses in scorpions. Using high-throughput sequencing and traditional molecular techniques we determine the genome sequences of cycloviruses and polyomaviruses. Sixteen of the forty-three scorpion samples were positive for eight different species of cycloviruses. According to ICTV guidelines, seven of the eight species were novel cycloviruses which were found in bark scorpions, stripe-tailed scorpions, yellow ground scorpions, and giant hairy scorpions (Centruroides sculpturatus, Paravaejovis spinigerus, Paravaejovis confusus & Hadrurus arizonensis) from Maricopa, Pinal, and Pima county in Arizona, USA. Additionally, one previously known cyclovirus species was recovered in bark scorpions (Centruroides sculpturatus) in Pima county which had previously been documented in guano from a Mexican free-tailed bat in Arizona. There were ten scorpions out of forty-three for which we recovered polyomavirus scorpion samples that grouped into four different polyomavirus species. Polyomaviruses were only identified in bark scorpions (Centruroides sculpturatus) from Maricopa, Pinal, and Pima county. Of the polyomavirus genomes recovered three belong to previously identified scorpion polyomavirus 1 and five to scorpion polyomavirus 3, and two represent two new species named scorpion polyomavirus 4 and scorpion polyomavirus 5. The implications of the discovery of cycloviruses and polyomaviruses from this study contributes to our understanding of viral diversity associated with Scorpions.
ContributorsGomez, Magali (Author) / Neil, Julia (Co-author) / Varsani, Arvind (Thesis director) / Kraberger, Simona (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2024-05
Description
Scorpions are predatory arachnids that are among the most ancient terrestrial invertebrates. They are typically found residing in desert and riparian environments. Viruses associated with scorpions have been explored in the past, unveiling partial RNA virus sequences and polyomaviruses, but more research in this area is necessary. Cycloviruses are non-enveloped viruses with circular single-stranded DNA genomes (~1.7 to 1.9 kb). Cycloviruses were initially identified in mammals and have now been detected in samples from a wide range of mammalian and insect species. Polyomaviruses are double-stranded DNA viruses (~4 to 7 kb). They are known for causing tumors in the host it infects, and have previously been identified in a diverse array of organisms, including scorpions. The objective for this study was to identify known and novel viruses in scorpions. Using high-throughput sequencing and traditional molecular techniques we determine the genome sequences of cycloviruses and polyomaviruses. Sixteen of the forty-three scorpion samples were positive for eight different species of cycloviruses. According to ICTV guidelines, seven of the eight species were novel cycloviruses which were found in bark scorpions, stripe-tailed scorpions, yellow ground scorpions, and giant hairy scorpions (Centruroides sculpturatus, Paravaejovis spinigerus, Paravaejovis confusus & Hadrurus arizonensis) from Maricopa, Pinal, and Pima county in Arizona, USA. Additionally, one previously known cyclovirus species was recovered in bark scorpions (Centruroides sculpturatus) in Pima county which had previously been documented in guano from a Mexican free-tailed bat in Arizona. There were ten scorpions out of forty-three for which we recovered polyomavirus scorpion samples that grouped into four different polyomavirus species. Polyomaviruses were only identified in bark scorpions (Centruroides sculpturatus) from Maricopa, Pinal, and Pima county. Of the polyomavirus genomes recovered three belong to previously identified scorpion polyomavirus 1 and five to scorpion polyomavirus 3, and two represent two new species named scorpion polyomavirus 4 and scorpion polyomavirus 5. The implications of the discovery of cycloviruses and polyomaviruses from this study contributes to our understanding of viral diversity associated with Scorpions.
ContributorsNeil, Julia (Author) / Gomez, Magali (Co-author) / Varsani, Arvind (Thesis director) / Kraberger, Simona (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Politics and Global Studies (Contributor)
Created2024-05
Description
Insulator-based dielectrophoresis (iDEP) has attracted considerable attention due to its ability to precisely capture and manipulate nanoparticles and biomolecules. A distinctive approach for effective manipulation of nanometer-sized proteins employing iDEP technique by generating higher electric field (E) and gradient (??2) in the iDEP microfluidic devices is delineated. Strategies to generate higher ??2 in the iDEP devices were outlined using numerical simulations. Intriguingly, the numerical simulation results demonstrated that by decreasing the post-to-post gap in the iDEP microfluidic devices, the ??2 was increased by ⁓12 fold. Furthermore, the inclusion of channel constrictions, such as rectangular constriction or curved constriction into the straight channel iDEP microfluidic device led to a significant increase in ??2. In addition, the inclusion of rectangular constrictions in the straight channel iDEP microfluidic device resulted in a greater increase in ??2 compared to the incorporation of curved constrictions in the same device. Moreover, the straight channel device with horizontal post-to-post gap of 20 μm and vertical post-to-post gap of 10 μm generated the lowest ??2 and the ??2 was uniform across the device. The rectangular constriction device with horizontal and vertical post-to-post gap of 5 μm generated the highest ??2 and the ??2 was non-uniform across the device. Subsequently, suitable candidate devices were fabricated using soft lithography as well as high resolution 3D printing and the DEP behavior of ferritin examined under various experimental conditions. Positive streaming DEP could be observed for ferritin at low frequency in the device generating the lowest ??2, whereas at higher frequency of 10 kHz no DEP trapping characteristics were apparent in the same device. Importantly, in the device geometry resulting in the highest ??2 at 10 kHz, labeled ferritin exhibited pDEPtrapping characteristics. This is an indication that the DEP force superseded diffusion and became the dominant force.
ContributorsMAHMUD, SAMIRA (Author) / Ros, Alexandra (Thesis advisor) / Borges, Chad (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2024
Description
Background: Eosinophilic esophagitis (EoE) is an increasingly prevalent allergic disease characterized by eosinophilic inflammation and symptoms of esophageal dysfunction. Diagnosis and monitoring require repeated, invasive endoscopic esophageal biopsies to assess levels of eosinophilic inflammation. Recently, the minimally invasive esophageal string test (EST) has been used collect protein in mucosal secretions as a surrogate for tissue biopsies in monitoring disease activity. From the string, assessment of the eosinophil-associated proteins major basic protein-1 (MBP-1) and eotaxin-3 (Eot3) is used to assess disease activity; however, this requires measurement in a reference laboratory, for which the turnaround time for results exceeds the time required for histopathologic assessment of endoscopic biopsies. In addition, MBP-1 and Eot3 are not markers unique to eosinophils. These obstacles can be overcome by targeting eosinophil peroxidase (EPX), an eosinophil-specific protein, using a rapid point-of-care test. Currently, EPX is measured by a labor-intensive enzyme-linked immunosorbent assay (ELISA), but we sought to optimize a rapid point-of-care test to measure EPX in EST segments.
Methods: We extracted protein from residual EST segments and measured EPX levels by ELISA and a lateral flow assay (LFA).
Results: EPX levels measured by LFA strongly correlated with those quantified by ELISA
(rs = 0.90 {95% CI: 0.8283, 0.9466}). The EPX LFA is comparable to ELISA for measuring EPX levels in ESTs.
Conclusions: The EPX LFA can provide a way to rapidly test EPX levels in ESTs in clinical settings and may serve as a valuable tool to facilitate diagnosis and monitoring of EoE.
ContributorsDao, Adelyn (Author) / Lake, Douglas (Thesis director) / Borges, Chad (Committee member) / Wright, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor)
Created2024-05
Description
Alzheimer’s Disease (AD) is the most common form of dementia affecting the population over the age of 65. AD is characterized clinically by increasing difficulty with memory and language, resulting in a loss of independence. This is due to the presence of two characteristic protein aggregates in the brain: extracellular amyloid plaques and intracellular neurofibrillary tangles (NFTs). Utilizing multiplexed immunofluorescence and dimensional reduction analysis the types of cells present in the hippocampus, the region of the brain most affected by AD, can be explored. Understanding the kinds of cell subtypes present, the mechanism behind how AD develops can be explored. Multiplexed IF was performed on human hippocampus FFPE tissues to detect a total of 37 proteins. Dimensional reduction analysis was performed to identify the four major cell types in the brain: neurons, oligodendrocytes, astrocytes, and microglia. After identifying each cell type, further dimensional reduction analysis was performed within each cell type to identify cell subtypes. A total of 21 neuron, 41 oligodendrocyte, 20 astrocyte, and 22 microglia subtypes were identified. The location of cell subtypes in each region of the hippocampal formation was found to match previous reports, further validating the findings of this project.
ContributorsEllison, Mischa A (Author) / Guo, Jia (Thesis advisor) / Borges, Chad (Committee member) / Mastroeni, Diego (Committee member) / Arizona State University (Publisher)
Created2024