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- All Subjects: Biology
- Creators: Arizona State University
Description
Cryogenic Electron Microscopy (Cryo-EM) is a method that can be used for studying the structure of biological systems. Biological samples are frozen to cryogenic temperatures and embedded in a vitreous ice when they are imaged by electrons. Due to its ability to preserve biological specimens in near-native conditions, cryo-EM has a significant contribution to the field of structural biology.Single-particle cryo-EM technique was utilized to investigate the dynamical characteristics of various protein complexes such as the Nogo receptor complex, polymerase ζ (Polζ) in yeast and human integrin ⍺vβ8-pro-TGFβ1-GARP complex. Furthermore, I proposed a new method that can potentially improve the sample preparation for cryo-EM. The Nogo receptor complex was expressed using baculovirus expression system in sf9 insect cells and isolated for structural studies. Nogo receptor complex was found to have various stoichiometries and interactions between individual proteins. A structural investigation of the yeast apo polymerase ζ holoenzyme was also carried out. The apo Polζ displays a concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. Furthermore, a lysine residue that obstructs the DNA-binding channel in apo Polζ was found and suggested a gating mechanism. In addition, cryo-EM studies of the human integrin ⍺vβ8-pro-TGFβ1-GARP complex was conducted to assess its dynamic interactions. The 2D classifications showed the ⍺vβ8-pro-TGFβ1-GARP complex is highly flexible and required several sample preparation techniques such as crosslinking and graphene oxide coating to improve protein homogeneity on the EM grid. To overcome challenges within the cryo-EM technique such as particle adsorption on air-water interface, I have documented a collaborative work on the development and application of lipid monolayer sandwich on cryo-EM grid. Cryogenic electron tomography (cryo-ET) along with cryo-EM were used to study the characteristics of lipid monolayer sandwich as a potential protective layer for EM grid. The cryo-ET results demonstrated that the thickness of lipid monolayer is adequate for single-particle cryo-EM processing. Furthermore, there was no appearance of preferred orientations in cryo-EM and cryo-ET images. To establish that this method is actually beneficial, more data must be collected, and high-resolution structures of protein samples must be obtained using this methodology.
ContributorsTruong, Chloe Du (Author) / Chiu, Po-Lin (Thesis advisor) / Liu, Wei (Committee member) / Mazor, Yuval (Committee member) / Arizona State University (Publisher)
Created2021
Climate Sensitivity in Mature Versus Pre-Reproductive Saguaro Determined from Spine Isotopic Signals
Description
The saguaro cactus is vital to the unique biodiversity of the Sonoran Desert. Many animals rely on it for survival and shelter. Due to its thick waxy exterior, it is able to retain a massive amount of water in its succulent stems, making it an ideal habitat for many birds that are native to the region. It also holds a large cultural significance for many native tribes of this region. Thus, it is important to understand how these magnificent plants are affected by changing environmental conditions in order to protect them in the future. Measurements of carbon isotope (13C) signals in saguaros spine tissues have been shown to be a robust approach for evaluating plant responses to climate variation over multiple seasons and years. This study investigated growth and 13C signals in young saguaros (< 2.5 m tall) at a location north of Phoenix to compare with large saguaros (> 4 m tall) that actively flower in the spring. Adolescent saguaros were examined to determine how they responded to variation in moisture between the years 2012 through 2016. As with a previous study by Hultine et al. (2018), this research demonstrated a strong relationship between height growth and precipitation. Stem growth was also highly correlated with the number of areoles produced across both young and old plants. Winter and late summer moisture from the previous year had a significant effect on stem growth, and a legacy effect could be seen in the low growth rates in 2014 which may be a result of low precipitation during the previous fall and winter. This study also established that the adolescent saguaro may be less sensitive to seasonal changes than the mature saguaro. When comparing the large plants with the adolescent plants, spine 13C values were similar between mature and adolescent plants, indicating that perhaps flower does not impose a significant added cost to saguaro water budgets. However, identifying the cost of flowering requires more investigation than that from only spine carbon isotope measurements and coarse measurements of plant productivity.
ContributorsDe Leon, Isabella (Author) / Hultine, Kevin (Thesis advisor) / Pigg, Kathleen (Thesis advisor) / Maienschein, Jane (Committee member) / Arizona State University (Publisher)
Created2021
Description
Acetylcholinesterase (AChE) inhibition by chemical toxicants such as organophosphates, nerve agents, and carbamates can lead to a series of adverse health outcomes including seizures, coma, and death. An adverse outcome pathway (AOP) is a framework that describes a series of biologically measurable key events (KEs) leading from some molecular initiating event (MIE) to an adverse outcome (AO) of regulatory significance, all developed and hosted in the AOP Wiki. A quantitative AOP (qAOP) is a mathematical model that predicts how perturbations in the MIE affect KEs based on the key event relationships (KERs) that define the AOP. The purpose of this thesis was to expand upon the KERs that define the AOP for AChE inhibition leading to neurodegeneration in order to better understand the effects of AChE inhibitors and the risks they pose to ecosystems, wildlife, and human health. In order to reduce the resources and time spent for chemical toxicity testing, a qAOP was developed based on the available quantitative data and models that supported the AOP. A literature review for the collection of qualitative evidence and quantitative data in support of the AOP was performed resulting in further expansion of the relationships between key events (KERs) through construction of additional KER description pages. A model evaluation was performed by comparing the qAOP model predictions with experimental data, with a subsequent sensitivity analysis of unknown parameters. The qAOP model simulates the MIE through its fifth KE (KE 5) and KE 7. Model predictions compared to experimentally measured data either under- or overpredicting multiple KEs warranting additional refinement such as a formal parameter optimization. Overall, more data amenable to qAOP model development are needed. To aid qAOP model development, the presentation of data in the AOPWiki may be improved by presenting the quantitative data in the AOP Wiki in a tabular format and allowing for the hosting of mathematical models or raw data. With these recommendations in mind, and through continued AOP construction in the AOP Wiki, new qAOP models will be developed, ultimately supporting chemical risk assessment and the mitigation of effects upon exposed individuals and wildlife populations.
ContributorsSinitsyn, Dennis (Author) / Watanabe, Karen (Thesis advisor) / Vinas, Natalia (Committee member) / Wirkus, Stephen (Committee member) / Arizona State University (Publisher)
Created2023
Description
Mycobacterium leprae, the causative agent of Hansen’s disease (leprosy), has plagued humans and other animal species for millennia and remains of concern to public health throughout the world today. Recent research into the expanded use of medical tissues preserved as formalin-fixed, paraffin-embedded samples (FFPE), opened the door for the study of M. leprae DNA from preserved skin samples. However, problems persist with damage to the DNA including fragmentation and cross linkage. This study evaluated two methods commonly used for the recovery of host DNA from FFPE samples for their efficacy in extracting pathogen DNA (hot alkaline lysis protocol and QIAGEN QIAamp FFPE DNA kit). Twenty FFPE skin samples collected from 1995-2015 from human subjects in the Pacific Islands suffering from M. leprae infection, each exhibiting a range of bacillary loads, were analyzed to determine which extraction method was most successful in terms of ability to consistently yield reliable, robust traces of M. leprae infection. This study further examined these samples to understand the phylogeny of leprosy in the region, where gaps in the evolutionary history of M. leprae persist. DNA recovery from paired samples was similar using either method. However, by extending the incubation time of post-paraffin removal sample lysis, both protocols were more likely to yield positive traces of M. leprae, with this enhancement being especially evident in paucibacillary samples with low bacterial presence. The qPCR assay findings suggest that the hot alkaline procedure is most likely to yield positive identification of infection in these traditionally challenging samples.
ContributorsKing, Felicia Clarice (Author) / Stone, Anne (Thesis advisor) / Wilson, Melissa (Committee member) / Buetow, Ken (Committee member) / Arizona State University (Publisher)
Created2023
Description
Genome-wide, single nucleotide polymorphisms (SNPs) and germination data were analyzed to better understand species delimitation and salt-tolerance within the legume genus Medicago. Molecular phylogenies revealed that the widely-used, genomic model line R108 and two deeply divergent accessions of Medicago truncatula are in fact more closely related to Medicago littoralis than to other accessions representing Medicago truncatula. This result was supported by germination data wherein the two accessions representing deeply divergent Medicago truncatula demonstrated salt-tolerance that was more similar to Medicago littoralis than to other accessions of Medicago truncatula. Molecular phylogenies revealed that two additional accessions representing deeply divergent Medicago truncatula appear to be more closely related to Medicago italica than to other accessions representing Medicago truncatula. The results of the present study elucidate complex evolutionary relationships and contribute to the present understanding of existing salt-tolerance within Medicago.
ContributorsHopkins, Andrew David (Author) / Wojciechowski, Martin (Thesis advisor) / Park, Yujin (Committee member) / Steele, Kelly (Committee member) / Arizona State University (Publisher)
Created2023
Description
As the planet is rapidly urbanizing, understanding the ecological effects of urbanization is a grand challenge for modern biology. For example, increased city temperatures known as the urban heat island effect, disproportionately impact nocturnal taxa and this consideration is widely overlooked. Slight shifts in the thermal microclimate have a cascade of ramifications that directly impact species density and distribution. Animal behavior is a trait that may explain why some species thrive after urbanization when others go locally extinct. In this study I followed 22 adult females of the western black widow, Latrodectus hesperus, from both urban and undisturbed Sonoran Desert habitats. First, I began looking for differences between urban and desert spiders under field conditions: boldness, voracity, web size and body condition. Both urban and desert spiders were then brought to the laboratory to see how their behavior changed. I found no behavioral differences between urban and desert spiders in the field or the laboratory. I did find that spider behavior differed between the field and the laboratory. Specifically, boldness in the laboratory was significantly lower compared to the field. Voracity was more repeatable in the laboratory versus the field, and boldness was strongly positively correlated with voracity in the laboratory, but not in the field. These behavioral shifts from the field to the laboratory favor the conclusion that black widow behavior is highly plastic and context dependent. Lastly, I monitored web temperature of black widow microhabitat continuously for an entire year using iButton data loggers. I found microhabitat temperatures differences between urban and desert sites were greatest at night and absent during the daytime. I uncovered a seasonal effect with the highest magnitude temperature difference occurring during the springtime. Additionally, behavior was significantly correlated with field temperatures; the boldest spiders come from the warmest webs. However, I found little evidence that temperature predicts spider body condition or voracity, and body condition does not predict its behavioral expression. My results highlight the importance of studying animal behavior to increase understanding of the factors that shape distribution and density in a lethal pest species.
ContributorsClark, Ryan Carter (Author) / Johnson, James C (Thesis advisor) / Bang, Christofer (Thesis advisor) / Sullivan, Brian (Committee member) / Arizona State University (Publisher)
Created2021
Description
Dryland ecosystems are integral to the global agricultural system and play an important role in soil carbon (C) storage. Despite their importance, drylands are currently facing many challenges including climate-change induced rainfall variability and soil degradation. These challenges are predicted to have effects on the soil microbial communities in drylands. Compost, an organic soil amendment, is a land management strategy that has been proposed to increase soil C storage as well as improve soil conditions in drylands, specifically in restoration and agricultural sites where degradation has affected soil properties like microbial biomass and respiration. Compost additions and rainfall variability may interact to affect soil moisture, an important catalyst for microbial activity. Assessing microbial activity responses under compost applications and variable moisture will aid in understanding how land management strategies will be affected by climate change in the future. This study investigates how soil microbial activity from a degraded dryland restoration site is affected by different compost applications amounts and variable soil moistures. A laboratory incubation study was conducted in a controlled environmental chamber for 60 days. Soils were amended with different treatments of compost (0, 0.35, and 0.70 g cm -2) and water pulses (5, 10, and 15 mm) in a full factorial design. Each treatment received the same cumulative amount of water throughout the incubation, but pulses were administered in different frequencies (every 5, 10, and 15 days). Soil respiration and soil water content were measured daily, and microbial biomass was measured at the end of the incubation to assess treatment effects on microbial activity. Microbial respiration and soil water content increased with increasing compost additions and water pulse sizes. Microbial biomass did not have consistent increases with compost additions or water pulse size. Cumulative microbial respiration was highest with the large-infrequent pulse size and smallest with the small-frequent pulse size. These results suggest that microbial activity and carbon dynamics in soils where compost amendments are used will respond to future changes in precipitation variability. The results of this study can aid in understanding how microbial activity is influenced by compost applications, which will be critical in making informed management decisions in the context of climate change.
ContributorsAmari, Katherine Nicole (Author) / Throop, Heather L (Thesis advisor) / Ball, Becky A (Committee member) / Blankinship, Joseph C (Committee member) / Gherardi, Laureano A (Committee member) / Cueva Rodriguez, Alejandro H (Committee member) / Arizona State University (Publisher)
Created2021
Description
Cardiovascular disease (CVD) remains the leading cause of mortality, resulting in 1 out of 4 deaths in the United States at the alarming rate of 1 death every 36 seconds, despite great efforts in ongoing research. In vitro research to study CVDs has had limited success, due to lack of biomimicry and structural complexity of 2D models. As such, there is a critical need to develop a 3D, biomimetic human cardiac tissue within precisely engineered in vitro platforms. This PhD dissertation involved development of an innovative anisotropic 3D human stem cell-derived cardiac tissue on-a-chip model (i.e., heart on-a-chip), with an enhanced maturation tissue state, as demonstrated through extensive biological assessments. To demonstrate the potential of the platform to study cardiac-specific diseases, the developed heart on-a-chip was used to model myocardial infarction (MI) due to exposure to hypoxia. The successful induction of MI on-a-chip (heart attack-on-a-chip) was evidenced through fibrotic tissue response, contractile dysregulation, and transcriptomic regulation of key pathways.This dissertation also described incorporation of CRISPR/Cas9 gene-editing to create a human induced pluripotent stem cell line (hiPSC) with a mutation in KCNH2, the gene implicated in Long QT Syndrome Type 2 (LQTS2). This novel stem cell line, combined with the developed heart on-a-chip technology, led to creation of a 3D human cardiac on-chip tissue model of LQTS2 disease.. Extensive mechanistic biological and electrophysiological characterizations were performed to elucidate the mechanism of R531W mutation in KCNH2, significantly adding to existing knowledge about LQTS2. In summary, this thesis described creation of a LQTS2 cardiac on-a-chip model, incorporated with gene-edited hiPSC-cardiomyocytes and hiPSC-cardiac fibroblasts, to study mechanisms of LQTS2.
Overall, this dissertation provides broad impact for fundamental studies toward cardiac biological studies as well as drug screening applications. Specifically, the developed heart on-a-chip from this dissertation provides a unique alternative platform to animal testing and 2D studies that recapitulates the human myocardium, with capabilities to model critical CVDs to study disease mechanisms, and/or ultimately lead to development of future therapeutic strategies.
ContributorsVeldhuizen, Jaimeson (Author) / Nikkhah, Mehdi (Thesis advisor) / Brafman, David (Committee member) / Ebrahimkhani, Mo (Committee member) / Migrino, Raymond Q (Committee member) / Plaisier, Christopher (Committee member) / Arizona State University (Publisher)
Created2021
Description
The built environment increases radiant heat exchange in urban areas by several degrees hotter compared to non-urban areas. Research has investigated how urbanization and heat affect human health; but there is scant literature on the effects of urban heat on wildlife. Animal body condition can be used to assess overall health. This parameter estimates the storage of energy-rich fat, which is important for growth, survival, and reproduction. The purpose of my research was to examine the Urban Heat Island effect on wild rodents across urban field sites spanning three strata of land surface temperature. Site level surface temperatures were measured using temperature data loggers and I captured 116 adult pocket mice (Chaetodipus spp. and Perognathus spp.) and Merriam’s kangaroo rats (Dipodomys merriami) to measure their body condition using accurate and noninvasive quantitative magnetic resonance. I used baited Sherman live traps from mid-May to early September during 2019 and 2020 in mountainous urban parks and open spaces over two summers. Rodents were captured at seven sites near the Phoenix metropolitan area; an ideal area for examining the effect of extreme heat experienced by urban wildlife. Results supported the prediction that rodent body condition was greatest in the cooler temperature stratas compared to the hottest temperature strata. I related rodent body condition to environmental predictors to dispute to environmental predictors to dispute alternative hypotheses; such as vegetation cover and degree of urbanization. Results based on measures of body fat and environmental predictors show pocket mice have more fat where vegetation is higher, nighttime temperatures are lower, surface temperatures are lower, and urbanization is greater. Kangaroo rats have more fat where surface temperature is lower. My results contribute to understanding the negative effects of extreme heat on body condition and generalized health experienced by urban wildlife because of the built environment. This research shows a need to investigate further impacts of urban heat on wildlife. Management suggestions for urban parks and open spaces include increasing vegetation cover, reducing impervious surface, and building with materials that reduce radiant heat.
ContributorsAllen, Brittany D'Ann (Author) / Bateman, Heather L (Thesis advisor) / Moore, Marianne S (Committee member) / Hondula, David M (Committee member) / Arizona State University (Publisher)
Created2021
Description
In Hawaiʻi, native macroalgae or “limu” are of ecological, cultural, and economic value. Invasive algae threaten native algae and coral that serve a key role in the reef ecosystem. Spectroscopy can be a valuable tool for species discrimination, while simultaneously providing insight into chemical processes occurring within photosynthetic organisms. The spectral identity and separability of Hawaiian macroalgal taxonomic groups and invasive and native macroalgae are poorly known and thus were the focus of this study. A macroalgal spectroscopic library of 30 species and species complexes found in Hawaiʻi was created. Spectral reflectance signatures were aligned with known absorption bands of division-specific photosynthetic pigments. Discriminant analysis was used to explore if taxonomic groups of algae and native versus invasive algae were separable. Discriminant analyses resulted in high overall classification accuracies. Algae were correctly classified based on taxonomic divisions 96.5% of the time and by species 83.2% of the time. Invasive versus native algae was correctly classified at a rate of 93% and higher. Analyses suggest there is spectral separability of algal taxonomic divisions and native-invasive status, which could have significant implications for coastal management. This study lays the groundwork for testing spectral mapping of native and invasive algal species using current airborne and forthcoming spaceborne imaging spectroscopy.
ContributorsFuller, Kimberly (Author) / Asner, Gregory P (Thesis advisor) / Vaughn, Nicholas (Committee member) / Martin, Roberta E (Committee member) / Arizona State University (Publisher)
Created2022