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Phage therapy has been around for more than a century, but has regained interest in the field of medicine and holds significant potential to act as a treatment against a deadly bacterial infection in various cactus species. It was discovered that bacteriophages isolated from soil samples of potato plants were able to suppress Pectobacterium carotovorum, ‘Pectobacterium’ being within the family Pectobacteriaceae which contains the ‘Erwinia’ genus that causes soft rot diseases in various plants (Jones, 2012). The two scientists had co-inoculated “... the phage with the phytobacterium” (Jones, 2012) in order to suppress the growth and prevent the infection from occurring.
ContributorsFry, Danielle Elizabeth (Author) / Geiler-Samerotte, Kerry (Thesis director) / Pfeifer, Susanne (Committee member) / Varsani, Arvind (Committee member) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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
Following the journey through the sewerage system, wastewater is subject to a series of purification procedures, prior to water reuse and disposal of the resultant sewage sludge. Biosolids, also known as treated sewage sludge, deemed fit for application on land, is a nutrient-rich, semisolid byproduct of biological wastewater treatment. Technological progression in metagenomics has allowed for large-scale analysis of complex viral communities in a number of samples, including wastewater. Members of the Microviridae family are non-enveloped, ssDNA bacteriophages, and are known to infect enterobacteria. Members of the Genomoviridae family similarly are non-enveloped, ssDNA viruses, but are presumed to infect fungi rather than eubacteria. As these two families of viruses are not relatively documented and their diversity poorly classified, this study aimed to analyze the presence of genomoviruses and the diversity of microviruses in nine samples representative of wastewater in Arizona and other regions of the United States. Using a metagenomic approach, the nucleic acids of genomoviruses and microviruses were isolated, assembled into complete genomes, and characterized through visual analysis: a heat chart showing percent coverage for genomoviruses and a circular phylogenetic tree showing diversity of microviruses. The heat map results for the genomoviruses showed a large presence of 99 novel sequences in all nine wastewater samples. Additionally, the 535 novel microviruses displayed great diversity in the cladogram, both in terms of sub-family and isolation source. Further research should be conducted in order to classify the taxonomy of microviruses and the diversity of genomoviruses. Finally, this study suggests future exploration of the viral host, prior to entering the wastewater system.
ContributorsSchreck, Joshua Reuben (Author) / Varsani, Arvind (Thesis director) / Rolf, Halden (Committee member) / Misra, Rajeev (Committee member) / School of Film, Dance and Theatre (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Yellow-bellied marmots (Marmota flavivent) are semi-fossorial ground-dwelling sciurid rodents native to the western United States. They are facultatively social and live in colonies that may contain over 50 individuals. Marmot populations are well studied in terms of their diet, life cycle, distribution, and behavior, however, knowledge about viruses associated with marmots is very limited. In this study we aim to identify DNA viruses by non-invasive sampling of their feces. Viral DNA was extracted from fecal material of 35 individual marmots collected in Colorado and subsequently submitted to rolling circle amplification for circular molecule enrichment. Using a viral metagenomics approach which included high-throughput sequencing and verification of viral genomes using PCR, cloning and sequencing, a diverse group of single-stranded (ss) DNA viruses were identified. Diverse ssDNA viruses were identified that belong to two established families, Genomoviridae (n=7) and Anelloviridae (n=1) and several others that belong to unclassified circular replication associated encoding single-stranded (CRESS) DNA virus groups (n=19). There were also circular DNA molecules extracted (n=4) that appear to encode one viral-like gene and are composed of <1545 nt. The viruses that belonged to the family Genomoviridae clustered with those in the Gemycircularvirus genus. The genomoviruses were extracted from 6 samples. These clustered with gemycircularvirus extracted from arachnids and feces. The anellovirus, extracted from one sample, identified here has a genome sequence that is most similar to those from other rodent species, lagomorphs, and mosquitos. The CRESS viruses identified here were extracted from 9 samples and are novel and cluster with others identified from avian species. This study gives a snapshot of viruses associated with marmots based on fecal sampling.
ContributorsKhalifeh, Anthony (Author) / Varsani, Arvind (Thesis director) / Kraberger, Simona (Committee member) / Dolby, Greer (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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
Description
To date, there have been few, if any, studies evaluating the venom toxin levels in dogs that have been naturally envenomated by pit vipers. Understanding venom toxin pharmacokinetics in a clinical setting is important for a variety of reasons, including the potential to better elucidate treatment options, prognosis, and other factors associated with pit viper envenomation. In addition, dogs serve as a comparative species to humans for evaluating pit viper envenomations. This pilot study’s primary objective was to address the question of “What do we see?” in dogs presenting for rattlesnake envenomation. To answer this question, we obtained serum from envenomated dogs presenting at three veterinary clinics, then used enzyme-linked immunosorbent assay (ELISA) and western blot analysis to measure total venom and key toxins in sera. Phospholipase A2, a primary venom toxin, was identified in a few samples by the western blot, and contributed to the positive correlation between percent echinocytes in the blood and venom concentration. Medical data records were compared to venom concentrations measured using ELISA to determine whether there were any significant correlations. First, the hematological results were compared. Clotting times showed a strong positive correlation, clotting times and platelets showed a negative correlation, while echinocytes and platelets showed no correlation. When compared to venom concentration, clotting times showed a negative correlation, while age showed a positive correlation. Weight and platelets were also compared to venom concentration, but no significant correlations were found. The logistics of this study provided a real-world model where time elapsed between envenomation and hospital admission, thus giving a realistic look at what occurs in both animal and human medicine.
ContributorsNelson, Alexis (Co-author, Co-author) / DeNardo, Dale (Thesis director) / Woods, Craig (Thesis director) / Varsani, Arvind (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Human papillomavirus (HPV) is the causative agent of cervical cancer. Persistent infection with high-risk HPV 16, 18 or 45 species is associated with the development and progression of cervical cancer. HPV genotyping and Pap smear tests are the regular methods used to detect pre-invasive cervical lesions, but there is a need for developing a rapid biomarker to profile immunity to these viruses. The viral E7 oncogene is expressed in most HPV-associated cancers and anti-E7 antibodies can be detected in the blood of patients with cervical cancer. This research was focused on viral E7 oncogene expression to be used in development of low-cost point of care tests, enabling patients from low resource settings to detect the asymptotic stage of cervical cancer and be able to seek treatment early. In order to produce the E7 protein in vitro to measure antibody levels, GST tagged E7 genes from HPV 16, 18 and 45 species were inserted into the pDEST15 vector and expressed in E. coli BL21DE3 cells that were induced with 1mM of IPTG. The E7-GST fused expressed protein was then purified using glutathione beads and resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein expression was 5.8 \u03bcg/ml for HPV 16E7 in 500 ml culture and for the 500 ml culture of HPV 18 E7 and 45 E7 were 10.5 \u03bcg/ml and 10.5 \u03bcg/ml for HPV 18E7 and 45E7 respectively. High yield values are showing high expression levels of GST-tagged E7 recombinant protein which can be used for serotyping a number of individuals. This shows that HPV E7 can be produced in large quantities that can potentially be used in point of care tests that can help identify women at risk of cervical cancer. In conclusion, the E7 protein produced in this study can potentially be used to induce humoral responses in patients\u2019 sera for understanding the immune response of cervical cancer.
ContributorsMakuyana, Ntombizodwa (Author) / Anderson, Karen (Thesis director) / Ewaisha, Radwa (Committee member) / Varsani, Arvind (Committee member) / Hou, Ching-Wen (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
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
Description
Biomarkers are the cornerstone of modern-day medicine. They are defined as any biological substance in or outside the body that gives insight to the body's condition. Doctors and researchers can measure specific biomarkers to diagnose and treat patients, such as the concentration of hemoglobin Alc and its connection to diabetes. There are a variety of methods, or assays, to detect biomarkers, but the most common assay is enzyme-linked immunosorbent assay (ELISA). A new-generation assay termed mass spectrometric immunoassay (MSIA) can measure proteoforms, the different chemical variations of proteins, and their relative abundance. ELISA on the other hand measures the overall concentration of protein in the sample. Measuring each of the proteoforms of a protein is important because only one or two variations could be biologically significant and/or cause diseases. However, running MSIA is expensive. For this reason, an alternative plate-based MSIA technique was tested for its ability to detect the proteoforms of a protein called apolipoprotein C-III (ApoC-III). This technique combines the protein capturing procedure of ELISA to isolate the protein with detection in a mass spectrometer. A larger amount of ApoC-III present in the body indicates a considerable risk for coronary heart disease. The precision of the assay is determined on the coefficient of variation (CV). A CV value is the ratio of standard deviation in relation to the mean, represented as a percentage. The smaller the percentage, the less variation the assay has, and therefore the more ability it has to detect subtle changes in the biomarker. An accepted CV would be less than 10% for single-day tests (intra-day) and less than 15% for multi-day tests (inter-day). The plate-based MSIA was started by first coating a 96-well round bottom plate with 2.5 micrograms of ApoC-III antibody. Next, a series of steps were conducted: a buffer wash, then the sample incubation, followed by another buffer wash and two consecutive water washes. After the final wash, the wells were filled with a MALDI matrix, then spotted onto a gold plate to dry. The dry gold target was then placed into a MALDI-TOF mass spectrometer to produce mass spectra for each spot. The mass spectra were calibrated and the area underneath each of the four peaks representing the ApoC-III proteoforms was exported as an Excel file. The intra-day CV values were found by dividing the standard deviation by the average relative abundance of each peak. After repeating the same procedure for three more days, the inter-day CVs were found using the same method. After completing the experiment, the CV values were all within the acceptable guidelines. Therefore, the plate-based MSIA is a viable alternative for finding proteoforms than the more expensive MSIA tips. To further validate this, additional tests will need to be conducted with different proteins and number of samples to determine assay flexibility.
ContributorsTieu, Luc (Author) / Borges, Chad (Thesis director) / Nedelkov, Dobrin (Committee member) / Harrington Bioengineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2017-12
Description
Brown adipose tissue (BAT) is thought to be important in combating obesity as it can expend energy in the form of heat, e.g. thermogenesis. The goal of this study was to study the effect of injected norepinephrine (NE) on the activation of BAT in rats that were fed a high fat diet (HFD). A dose of 0.25 mg/kg NE was used to elicit a temperature response that was measured using transponders inserted subcutaneously over the BAT and lower back and intraperitoneally to measure the core temperature. The results found that the thermic effect of the BAT increased after the transition from low fat diet to a high fat diet (LFD) yet, after prolonged exposure to the HFD, the effects resembled levels found with the LFD. This suggests that while a HFD may stimulate the effect of BAT, long term exposure may have adverse effects on BAT activity. This may be due to internal factors that will need to be examined further.
ContributorsSion, Paul William (Author) / Herman, Richard (Thesis director) / Borges, Chad (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05