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Description
Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
ContributorsDeb, Arpan (Author) / Johnson, William (Author) / Kline, Alexander (Author) / Scott, Boston (Author) / Meador, Lydia (Author) / Srinivas, Dustin (Author) / Martin Garcia, Jose Manuel (Author) / Dorner, Katerina (Author) / Borges, Chad (Author) / Misra, Rajeev (Author) / Hogue, Brenda (Author) / Fromme, Petra (Author) / Mor, Tsafrir (Author) / ASU Biodesign Center Immunotherapy, Vaccines and Virotherapy (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor) / Biodesign Institute (Contributor) / School of Molecular Sciences (Contributor) / Applied Structural Discovery (Contributor) / Personalized Diagnostics (Contributor)
Created2017-02-22
Description
Across the animal kingdom, communication serves a vital purpose. The transfer of information between and among species is often paramount to many behaviors including mating, collaboration, and defense. While research has provided tremendous insight into animal communication and interaction, there is still much that we have yet to understand. Due to their reliance on interactions that maximize efficiency within their complicated colony structure and array of member roles, eusocial insects serve as an excellent model for animal communication. Among eusocial insects, ants are some of the most heavily researched, with a tremendous amount of literature focused on their cuticular hydrocarbons. Along with serving as a waterproofing agent, cuticular hydrocarbons also play a major role in recognition and communication in these insects. By studying the importance of hydrocarbons in ant social structure, their tremendously specialized olfactory system, and the use of learning assays in its study, parallels between communication in ants and other animals are revealed, demonstrating how ants serve as a relevant model for animal communication as a whole.
ContributorsSpirek, Benton Forest Ensminger (Author) / Liebig, Juergen (Thesis director) / Pratt, Stephen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-12
Description
The 18S ribosomal RNA gene is ubiquitous across eukaryotes as it encodes the RNA component of the ribosomal small subunit. It is the most commonly used marker in molecular studies of unicellular eukaryotes (protists) due to its species specificity and high copy number in the protist genome. Recent studies have revealed the widespread occurrence of intragenomic (intra-individual) polymorphism in many protists, an understudied phenomenon which contradicts the assumed homogeneity of the 18S throughout an individual genome. This thesis quantifies and analyzes the level of intragenomic and intraspecific 18S sequence variability in three Trichonympha species (T. campanula, T. collaris, T. postcylindrica) from Zootermopsis termites. Single-cell DNA extractions, PCR, cloning, and sequencing were performed to obtain 18S rRNA sequence reads, which were then analyzed to determine levels of sequence divergence among individuals and among species. Intragenomic variability was encountered in all three species. However, excluding singleton mutations, sequence divergence was less than 1% in 53 of the 56 compared individuals. T. collaris exhibited the most substantial intragenomic variability, with sequence divergence ranging from 0 to 3.4%. Further studies with more clones per cell are needed to elucidate the true extent of intragenomic variability in Trichonympha.
ContributorsBobbett, Bradley (Author) / Gile, Gillian (Thesis director) / Liebig, Juergen (Committee member) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Adaptation of Camponotus floridanus’ Cuticular Hydrocarbon Profile under High Temperature Conditions
Description
Insects are small creatures highly susceptible to water loss. A major factor in the prevention of water loss through an insect’s cuticle are their cuticular hydrocarbons (CHC), a lipid layer consisting mostly of long-chain hydrocarbons. CHCs consist of different molecules called alkanes, alkenes, and methyl branched hydrocarbons which all have varying levels of hydrophobicity. Ants are a massively abundant family of insects with important roles in the ecosystem that also utilize CHCs. Camponotus floridanus isare athe native ant species of the Florida Keys which areis known to have variable environmental temperature. Being exposed to temperatures as high as 35 °C, these ants are expected to have mechanisms that allow them to adapt to their environment. It was hypothesized that CHCs may change in concentration or composition as a means to combat the changes in cuticular permeability due to the variable temperatures that the ants experience. We therefore used C. floridanus worker ants to learn more about CHC plasticity in insects when exposed to elevated temperatures. We found four CHC componentspeaks that showed a statistically significant increase in concentration when comparing the control to treatment colonies: 3,7 dimethyl C31, an underdetermined methyl branched C31, 3,7,11 trimethyl C31, and an undetermined tetramethylbranched C31. These significant changes in concentration occurred on longer chain hydrocarbons. Under further examination, it was found that there was a strong positive correlation between elution time and the differences in medians of peak area between control and treatment colonies. This shows that there was a shift in the CHC profile resulting in an increased concentration of longer chained methyl-branched hydrocarbons. It also suggests that branched hydrocarbons also play some role in the water proofing mechanism of C. floridanus.
ContributorsOn, Thomas (Co-author) / On, Tyler (Co-author) / Liebig, Juergen (Thesis director) / Harrison, Jon (Committee member) / Murdock, Tyler (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
When ants encounter liquid food, they have two options of transporting that food to their nests. The first is the social bucket method in which liquid is carried in the mandibles of the workers back to the nest. The second is trophallaxis in which liquid is imbibed and then transported inside the ant back to the nest. The liquid is then regurgitated to fellow nestmates. Ectatomma have been observed using the social bucket method of transport and were considered members of the Ponerine family. However, a new phylogeny created by Borowiec and Rabeling places Ectatomma near to Formecinae and Myrmicinae, both know for practicing trophallaxis. This seems to suggest either Ectatomma is able to utilize trophallaxis as well or that the evolutionary practice of trophallaxis is more plastic than previously believed. The ability of Ectatomma ruidum to utilize trophallaxis was examined in two experiments. The first experiment examined E. ruidum’s ability to practice worker to worker trophallaxis and the second examined E. ruidum’s ability to perform worker to larva trophallaxis. The results of both experiments indicated that E. ruidum cannot utilize trophallaxis but the larva of E. ruidum may be able to regurgitate to the workers. These results in turn seem to suggest that trophallaxis is a bit more plastic than originally thought.
ContributorsCunningham, Cassius Alexander (Author) / Pratt, Stephen (Thesis director) / Liebig, Juergen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The rise in community-associated methicillin-resistant Staphylococcus aureus (MRSA) infections and the ability of the organism to develop resistance to antibiotics necessitate new treatment methods for MRSA. Geopolymers (GPs) are cheap, porous materials that have demonstrated adsorptive capabilities. In this study, GPs were investigated for their ability to adsorb whole MRSA cells and MRSA secreted proteins [culture filtrate proteins (CFPs)] as a complementary method of controlling MRSA infections. GPs have been synthesized with variable pore sizes (meso/macro scale) and further modified with stearic acid (SA) to increase surface hydrophobicity. Four GPs (SA-macroGP, macroGP, SA-mesoGP, and mesoGP) were incubated with whole cells and with CFPs to quantify GP adsorption capabilities. Following MRSA culture incubation with GPs, unbound MRSA cells were filtered and plated to determine cell counts. Following CFP incubation with GPs, unbound CFPs were separated via SDS-PAGE, stained with SYPRO Ruby, and analyzed using densitometry. Results indicate that macroGP was the most effective at adsorbing whole MRSA cells. Visual banding patterns and densitometry quantitation indicate that SA-mesoGP was the most effective at adsorbing CFP. Ultimately, GP-based products may be further developed as nonselective or selective adsorbents and integrated into fibrous materials for topical applications.
ContributorsGanser, Collin (Co-author, Co-author) / Haydel, Shelley E. (Thesis director) / Seo, Don (Committee member) / Borges, Chad (Committee member) / School of Earth and Space Exploration (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
An intimate view of the unique architecture of Harpegnathos saltwater nest using aluminum nest casts
Description
Abstract:
Given the incredible variety in ant nest architecture, this experiment sought to evaluate how the nest architecture of Harpegnathos saltator differs from other species’ nests. To achieve the ability to evaluate the structure of H. saltator nest, we created experimental colonies varying in size from 20, 40, 60, 80 workers of Harpegnathos saltator in five-gallon buckets of sand and then allowing the colonies to grow for four months and twelve days. To create the nest casts, we developed a charcoal kiln out of a galvanized trash can and used a ceramic crucible to hold the aluminum being melted. Using molten aluminum to create nest casts of each colony produced, we obtained three poorly developed nests and one decent nest. The decent nest cast, the 80 worker H. saltator nest, was lacking key features of H. saltator nests that have been excavated in the field. However, they do share many of the same structures such as the shaping of the chambers. The ability of the experimental colonies to excavate the soil provided in the buckets to them was likely halted by poor penetration of water into superficial layers of the soil, thus making the soil too difficult to excavate and form the structures that are key elements of the species nest architecture. Despite these key challenges which the colonies faced, the 80-worker colony showed extensive vertical development and did display features associated with natural H. saltator colonies. Thus, given the display of some key features associated with characteristics of the H. saltator nests excavated in the field, it can be said that with some modification to technique that this is a viable avenue for future study of nest architecture and colony structure.
Given the incredible variety in ant nest architecture, this experiment sought to evaluate how the nest architecture of Harpegnathos saltator differs from other species’ nests. To achieve the ability to evaluate the structure of H. saltator nest, we created experimental colonies varying in size from 20, 40, 60, 80 workers of Harpegnathos saltator in five-gallon buckets of sand and then allowing the colonies to grow for four months and twelve days. To create the nest casts, we developed a charcoal kiln out of a galvanized trash can and used a ceramic crucible to hold the aluminum being melted. Using molten aluminum to create nest casts of each colony produced, we obtained three poorly developed nests and one decent nest. The decent nest cast, the 80 worker H. saltator nest, was lacking key features of H. saltator nests that have been excavated in the field. However, they do share many of the same structures such as the shaping of the chambers. The ability of the experimental colonies to excavate the soil provided in the buckets to them was likely halted by poor penetration of water into superficial layers of the soil, thus making the soil too difficult to excavate and form the structures that are key elements of the species nest architecture. Despite these key challenges which the colonies faced, the 80-worker colony showed extensive vertical development and did display features associated with natural H. saltator colonies. Thus, given the display of some key features associated with characteristics of the H. saltator nests excavated in the field, it can be said that with some modification to technique that this is a viable avenue for future study of nest architecture and colony structure.
ContributorsAnderson, Clayton Edward (Author) / Liebig, Juergen (Thesis director) / Pratt, Stephen (Committee member) / School of Politics and Global Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The purpose of this thesis creative project was to create an educational video to present research findings on the increasingly important issue of human biospecimen preanalytic variables. When a human biospecimen, such as blood, urine, or tissue, is removed from the body, it is subjected to a plethora of variables that are not recorded or regulated in a vast majority of cases. Frequently, these samples arrive at the research or pathology lab with an unknown history, then undergo analysis for translational research purposes, or to guide clinical management decisions. Thus, compromised specimen quality caused by preanalytic variables has substantial, and potentially devastating, downstream effects. To identify the preanalytic variables with the greatest impact on blood and tissue specimen quality, 45 articles were gathered using PubMed and Google Scholar databases and cited. Based on the articles, the top five variables with the most detrimental effects were identified for both blood and tissue samples. Multiple sets of parameters ensuring specimen fitness were compared for each of the five variables for each specimen type. Then, specific parameters guaranteeing the fitness of the greatest number of analytes were verified. To present the research findings in greater detail, a paper was written that focused on identifying the top variables and key parameters to ensure analyte fitness. To present the overall issue in an easy-to-digest format, a storyboard and script were created as a guideline for a final video project. Ultimately, two alternate versions of the video were created to pertain to the audience of choice (one version for patients, one version for professionals). It is the hope that these videos will be used as educational tools to continue efforts to standardize and enforce human biospecimen preanalytic variable parameters. This is a necessary step to improve the accuracy of our biomedical research data and the healthcare of patients worldwide.
ContributorsAzcarate, Heather (Author) / Compton, Carolyn (Thesis director) / LaBaer, Joshua (Committee member) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor)
Created2018-12
Description
Complex animal societies consist of a plethora of interactions between members. To successfully thrive they must be able to recognize members and their kin, and to understand how they do this we need sufficient and reliable methods of testing. Eusocial insects are especially good at recognizing their nestmates, but the exact mechanism or how well they can discriminate is unknown. Ants achieve nestmate recognition by identifying varying proportions of cuticular hydrocarbons. Previous studies have shown ants can be trained to discriminate between pairs of hydrocarbons. This study aims to compare two methodologies previously shown to demonstrate odor learning to identify which one is the most promising to use for future odor learning experiments. The two methods tested were adapted from Sharma et al. (2015) and Guerrieri and d’Ettorre (2010). The results showed that the Guerrieri method demonstrated learning better and was more reliable and faster than the Sharma method. The Guerrieri method should be used in future experiments regarding odor learning discrimination
ContributorsDavis, Cole (Author) / Liebig, Juergen (Thesis director) / Stephen, Pratt (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
In the development of personalized medicine and many other clinical studies, biospecimen integrity serves as the prerequisite for not only the accurate derivation of patient- and disease-specific molecular data from biological specimens but the meaningful downstream validation of biomarkers. However, a large number of preanalytical variables may influence the quality of biospecimens in an undesired way and ultimately render the samples unsuitable for molecular analysis. The limited ability to directly reduce discrepancies caused by preanalytical variables gives rise to the need for development and retrospective application of appropriate tests for assessment of biospecimen integrity. Nevertheless, the most standard approaches to assessing biospecimen integrity involve nontrivial procedures. Thus, the need for quality control tools or tests that are readily applicable and can produce results in a straightforward way becomes critical. As one of the major ex vivo biomolecular degradation mechanisms, oxidation that occurs when blood plasma and serum samples are exposed to thawed states during storage and processing is hard to forestall and detect. In an attempt to easily detect and monitor the degree of oxidation, the technique of Fluorescence Resonance Energy Transfer (FRET) was examined to determine whether this concept could be employed to monitor exposure of samples to thawed conditions when controlled by spontaneous oxidative disulfide bonding. The intended mode of usage was envisioned as a fluorescence liquid being stored in a separate compartment but within the same test tube as archived plasma and serum samples. This would allow the assessment of sample integrity by direct visualization of fluorescence under a hand-held black light. The fluorescent dynamic range as well as kinetic control of the reaction were studied. While the addition of Cu(II) proved to facilitate excellent dynamic range with regard to fluorescence quenching, the kinetics of the reaction were too rapid for practical use. Further investigation revealed that the fluorescence quenching mechanism might have actually occurred via Intramolecular Charge Transfer (ICT) rather than FRET mediated by oxidative disulfide bond formation. Introduction of Cu(II) via copper metal slowed fluorescence quenching to the point of practical utility; facilitating demonstration that storing at room temperature, refrigerating or freezing the samples delayed fluorescence quenching to different extents. To establish better kinetic control, future works will focus on establishing controlled, thoroughly understood kinetic release of Cu(II) from copper metal.
ContributorsZhang, Zihan (Author) / Borges, Chad (Thesis director) / Emady, Heather (Committee member) / Williams, Peter (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12