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Cell-free protein synthesis (CFPS) is becoming an increasingly popular method of in vitro protein expression for biotechnology applications. However, there is still no comprehensive resource that outlines the most effective lysate and template combinations for efficient eukaryotic CFPS. To address this issue, expression vectors were constructed and assayed in order to determine their activity within three commercial eukaryotic CFPS systems: Wheat Germ Extract (WGE), Rabbit Reticulocyte Lysate (RRL), and HeLa Cell Lysate (HCL). Previously in the Chaput lab, a luciferase reporter vector was expressed in each lysate system, testing different template variables impacting protein expression, including the 5' UTR sequence, presence of poly(A) tail, and DNA type. It was found that plasmid DNA templates generally yielded ~500-fold greater amount of protein than linear DNA templates and the inclusion of a poly(A) tail did not significantly increase protein expression in the plasmid systems. Additionally, the incorporation of a viral translation enhancing sequence into the 5' UTR increased translation in a lysate-specific manner. The HCL system had a strong preference for the EMCV sequence, WGE had a preference for the sequences from AMV and TMV, and RRL showed no specific preference. Overall, the HCL-EMCV system generated the greatest amount of protein per volume, producing 10-fold more protein than the second best template-lysate combination tested. Here, four human genes fused with a c-Myc tag were expressed in each lysate using the EMCV 5' UTR sequence in order to test the generality of the previous results. Protein synthesis was assayed using a luciferase construct with a c-Myc tag to recapitulate the previous luminometer data and western blotting of the human proteins. These analyses showed the same EMCV expression trends across all systems, with the HCL system synthesizing the greatest amount of each protein. In the future, when choosing commercial eukaryotic CFPS systems for gene expression, these template variables should be considered when performing cost analysis for cell-free protein production.
ContributorsHartsough, Emily Mae (Author) / Chaput, John (Thesis director) / Chandler, Douglas (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Arts, Media and Engineering (Contributor)
Created2015-05
Description
Birds have unusually high plasma glucose concentrations compared to mammals of similar size despite their high metabolic rate. While birds use lipids as their main source of energy, it is still unclear how and why they maintain high plasma glucose concentrations. To investigate a potential underlying mechanism, this study looks at the role of lipolysis in glucose homeostasis. The purpose of this study is to examine the effects of decreased glycerol availability (through inhibition of lipolysis) on plasma glucose concentrations in mourning doves. The hypothesis is that decreased availability of glycerol will result in decreased production of glucose through gluconeogenesis leading to reduced plasma glucose concentrations. In the morning of each experiment, mourning doves were collected at the Arizona State University Tempe campus, and randomized into either a control group (0.9% saline) or experimental group (acipimox, 50mg/kg BM). Blood samples were collected prior to treatment, and at 1, 2, and 3 hours post-treatment. At 3 hours, doves were euthanized, and tissue samples were collected for analysis. Acipimox treatment resulted in significant increases in blood glucose concentrations at 1 and 2 hours post- treatment as well as renal triglyceride concentrations at 3 hours post-treatment. Change in plasma free glycerol between 0h and 3h followed an increasing trend for the acipimox treated animals, and a decreasing trend in the saline treated animals. These results do not support the hypothesis that inhibition of lipolysis should decrease blood glycerol and blood glucose levels. Rather, the effects of acipimox in glucose homeostasis appear to differ significantly between birds and mammals suggesting differing mechanisms for glucose homeostasis.
ContributorsKouteib, Soukaina (Author) / Sweazea, Karen (Thesis director) / Deviche, Pierre (Committee member) / Chandler, Douglas (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Phosphoinositol-Dependent Kinase 1 (PDK1) acts in conjunction with phosphorylated lipids such as Phosphoinositol-3,4,5-triphosphate (PIP3) to activate a variety of proteins that regulate mechanisms ranging from cell growth and survival to cytoskeletal rearrangement. In this investigation PDK1 was examined in the context of cellular division. The techniques of immunocytochemistry and live cell imaging were used to visualize the effects of the inhibition of PDK1 on division in HeLa cells. Division was impaired at metaphase of mitosis. The inhibited cells were unable to initiate anaphase cell-elongation ultimately leading to the flattening of spherical, metaphase cells. Preliminary studies with imunocytochemistry and live cell imaging suggested that insulin treatment reversed PDK1 inhibition, but the results were not statistically significant. Therefore, the recovery of PDK1 inhibition by insulin treatment could not be confirmed. Based on these observations a possible reason for the inability of the treated cells to complete cytokinesis could be the role of PDK1 in the Rho-kinase pathway that is required for the processes cell-elongation necessary for anaphase of mitosis.
ContributorsMasserano, Benjamin Max (Author) / Capco, David (Thesis director) / Baluch, Debra (Committee member) / Chandler, Douglas (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
Osteosarcoma (OS) is the most prevalent primary tumor of bone in the pediatric age group [1]. The long-term cancer free survival has improved in patients with localized cancer; however, less than 20% of patients diagnosed with metastatic disease survive without relapse [2]. While these findings emphasize the urgent need for new therapeutic agents, the lack of understanding of the factors and the tumor microenvironment that lead to therapy resistance in OS has significantly hampered progress towards improved prognosis. Recent clinical reports have shown a negative correlation between tumor hypoxia and overall survival in OS patients [4]. In addition to the up-regulation of hypoxia inducible factors (HIFs), it has been shown that hypoxia can trigger an adaptive response such as the unfolded protein response (UPR) that allows tumor cells to avoid therapy-induced death [3,4,7,10].
Using in vitro experimental models of both SAOS-2 (non-metastatic) and 143-b (metastatic) osteosarcoma cell lines and Western blot analysis, we have demonstrated that basal levels of molecular chaperone BiP (Binding immunoglobulin protein, or GRP-78) and peIF2α (phospho-eukaryotic initiation factor 2 alpha), both markers of the UPR, were higher in SAOS-2 than 143-b cells. We also show that both these markers were further up-regulated upon exposure to hypoxia, as evidenced by the increase in banding intensity in both SAOS-2 and 143-b cells. Furthermore, analysis of another UPR marker, ATF6 (activating transcription factor 6) showed that basal levels of active nuclear ATF6 were slightly higher in SAOS-2 cells than in 143-b cells. However, unlike the other UPR markers these levels were significantly reduced upon exposure to hypoxia (0.1% O2). In addition to hypoxia, treatment with Cisplatin also had similar effects on the expression of aforementioned UPR markers: BiP and peIF2α. We found that the 143-b OS cells were more sensitive to the Cisplatin treatment than the SAOS-2 OS cells, and thus more prone to cell-mediated death.
Our findings shed light on the unknown mechanisms underlying chemotherapeutic drug resistance in osteosarcoma patients. Our research may lead to novel therapies that seek out and destroy the chemoresistant OS cells within the hypoxia core of tumors, thereby preventing survival and metastasis, and ultimately improving the chances of survival amongst OS patients.
Using in vitro experimental models of both SAOS-2 (non-metastatic) and 143-b (metastatic) osteosarcoma cell lines and Western blot analysis, we have demonstrated that basal levels of molecular chaperone BiP (Binding immunoglobulin protein, or GRP-78) and peIF2α (phospho-eukaryotic initiation factor 2 alpha), both markers of the UPR, were higher in SAOS-2 than 143-b cells. We also show that both these markers were further up-regulated upon exposure to hypoxia, as evidenced by the increase in banding intensity in both SAOS-2 and 143-b cells. Furthermore, analysis of another UPR marker, ATF6 (activating transcription factor 6) showed that basal levels of active nuclear ATF6 were slightly higher in SAOS-2 cells than in 143-b cells. However, unlike the other UPR markers these levels were significantly reduced upon exposure to hypoxia (0.1% O2). In addition to hypoxia, treatment with Cisplatin also had similar effects on the expression of aforementioned UPR markers: BiP and peIF2α. We found that the 143-b OS cells were more sensitive to the Cisplatin treatment than the SAOS-2 OS cells, and thus more prone to cell-mediated death.
Our findings shed light on the unknown mechanisms underlying chemotherapeutic drug resistance in osteosarcoma patients. Our research may lead to novel therapies that seek out and destroy the chemoresistant OS cells within the hypoxia core of tumors, thereby preventing survival and metastasis, and ultimately improving the chances of survival amongst OS patients.
ContributorsFaraj, Janine Jean (Author) / Chandler, Douglas (Thesis director) / Sertil, Aparna (Committee member) / Sweazea, Karen (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Historical, Philosophical and Religious Studies (Contributor)
Created2014-05
Description
DNA is a natural genetic polymer capable of storing and preserving genetic information in biological systems. Due to its natural information storage capacity, recent scientific progress demonstrates that DNA has the potential to exceed standard information storage technologies. However, DNA is limited in its information storage capacities due to its susceptibility to degradation in the presence of naturally occurring nucleases. Threose nucleic acid (TNA), an unnatural genetic polymer with a 3'->2'phosphodiester-linked threose sugar backbone, has promising potential to overcome this limitation. TNA is not a substrate for natural nucleases and thus shows a dramatic increase in stability compared to DNA. However, TNA transcription has a tendency to generate G:G mispairs and lead to a gradual loss of information within the template. It was hypothesized that the mutation occurs through a G:G Hoogsteen base pair that forms preferentially over the canonical G:C Watson-Crick base pair. Incorporation of 7-deaza-dG into a four letter template effectively eliminated G:G mispairings and improved the replication fidelity from 60% to 99.6% with only four errors in a thousand. These results have laid the groundwork for further research to increase the length of the TNA product synthesized and to test TNA's ability to store genetic information.
ContributorsMeyers, Madeline Ashby (Author) / Chaput, John C. (Thesis director) / Chandler, Douglas (Committee member) / Dunn, Matthew R. (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
Art is an inherent concept instilled in human nature, which utilizes the abilities of the creative mind to invent. Art has served many purposes in the history of mankind, including, but not limited to story telling, entertainment, decoration, exploration, propaganda, education, and therapy. The primary aim of this creative project was to explore the importance of the art, as a creative process, as a way to supplement academic endeavors. The idea derived from an observation made by myself that contemporary regard for art has been on a decline, which made me question if I also value art as much as I think I do, having done art in the past and recently added a studio art minor. I thought of ways to again incorporate art and the creative process into my life. I asked myself the question: can the creative process be used as a supplement to schoolwork in order to relieve stress? To explore this, an experiment was designed, which entailed my creation of drawings twice a week, accompanied by journal documentation for a full semester of college. Afterwards, analyses were done between the documented journal entries and the artworks to see if any relationships were apparent between various aspects of my life at the times of the drawings and the drawings themselves. Further research was also conducted in related areas of study and documented in written format, which cited and analyzed numerous journal articles, artworks, artists, and research papers. This included art therapy, art education, and the relationships between art and science. Results from the experiment indicated that art as a creative process allowed for the relief of stress by cleansing my mind from any concern or interferences, therefore offering myself a complete break and relaxation, effectively refreshing my mind and allowing me to resume schoolwork or other tasks more mentally taxing. In addition, the research also showed that art therapy could effectively utilize this palliative effect of art making to ease the problems of people in distress. The findings also concluded that art and science go hand in hand, which explains a lot of the similarities in methodologies utilized by scientists and artists. In conclusion, art is a paramount part of mankind in exercising the creative mind and is ubiquitous; we should learn to actively embrace it to enrich our lives.
ContributorsSun, Sean Yu-Hsiang (Author) / Dove-Viebahn, Aviva (Thesis director) / Chandler, Douglas (Committee member) / School of Art (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05