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- Creators: Department of Information Systems
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Description
The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR, residues 649-683) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain (residues 684-705) of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the pre-fusion and post-fusion conformations, most of which could not react with the broadly neutralizing antibodies 2F5 and 4E10. Structural information on the TM domain of gp41 is scant and at low resolution.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
This thesis describes the structural studies of MPR-TM (residues 649-705) of HIV-1 gp41 by X-ray crystallography. MPR-TM was fused with different fusion proteins to improve the membrane protein overexpression. The expression level of MPR-TM was improved by fusion to the C-terminus of the Mistic protein, yielding ∼1 mg of pure MPR-TM protein per liter cell culture. The fusion partner Mistic was removed for final crystallization. The isolated MPR-TM protein was biophysically characterized and is a monodisperse candidate for crystallization. However, no crystal with diffraction quality was obtained even after extensive crystallization screens. A novel construct was designed to overexpress MPR-TM as a maltose binding protein (MBP) fusion. About 60 mg of MBP/MPR-TM recombinant protein was obtained from 1 liter of cell culture. Crystals of MBP/MPR-TM recombinant protein could not be obtained when MBP and MPR-TM were separated by a 42 amino acid (aa)-long linker but were obtained after changing the linker to three alanine residues. The crystals diffracted to 2.5 Å after crystallization optimization. Further analysis of the diffraction data indicated that the crystals are twinned. The final structure demonstrated that MBP crystallized as a dimer of trimers, but the electron density did not extend beyond the linker region. We determined by SDS-PAGE and MALDI-TOF MS that the crystals contained MBP only. The MPR-TM of gp41 might be cleaved during or after the process of crystallization. Comparison of the MBP trimer reported here with published trimeric MBP fusion structures indicated that MBP might form such a trimeric conformation under the effect of MPR-TM.
ContributorsGong, Zhen (Author) / Fromme, Petra (Thesis advisor) / Mor, Tsafrir (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2014
Description
Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012
Description
Plants are a promising upcoming platform for production of vaccine components and other desirable pharmaceutical proteins that can only, at present, be made in living systems. The unique soil microbe Agrobacterium tumefaciens can transfer DNA to plants very efficiently, essentially turning plants into factories capable of producing virtually any gene. While genetically modified bacteria have historically been used for producing useful biopharmaceuticals like human insulin, plants can assemble much more complicated proteins, like human antibodies, that bacterial systems cannot. As plants do not harbor human pathogens, they are also safer alternatives than animal cell cultures. Additionally, plants can be grown very cheaply, in massive quantities.
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
In my research, I have studied the genetic mechanisms that underlie gene expression, in order to improve plant-based biopharmaceutical production. To do this, inspiration was drawn from naturally-occurring gene regulatory mechanisms, especially those from plant viruses, which have evolved mechanisms to co-opt the plant cellular machinery to produce high levels of viral proteins. By testing, modifying, and combining genetic elements from diverse sources, an optimized expression system has been developed that allows very rapid production of vaccine components, monoclonal antibodies, and other biopharmaceuticals. To improve target gene expression while maintaining the health and function of the plants, I identified, studied, and modified 5’ untranslated regions, combined gene terminators, and a nuclear matrix attachment region. The replication mechanisms of a plant geminivirus were also studied, which lead to additional strategies to produce more toxic biopharmaceutical proteins. Finally, the mechanisms employed by a geminivirus to spread between cells were investigated. It was demonstrated that these movement mechanisms can be functionally transplanted into a separate genus of geminivirus, allowing modified virus-based gene expression vectors to be spread between neighboring plant cells. Additionally, my work helps shed light on the basic genetic mechanisms employed by all living organisms to control gene expression.
ContributorsDiamos, Andy (Author) / Mason, Hugh S (Thesis advisor) / Mor, Tsafrir (Committee member) / Hogue, Brenda (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2017
Description
The advertising agency, in its variety of forms, is one of the most powerful forces in the modern world. Its products are seen globally through various multimedia outlets and they strongly impact culture and economy. Since its conception in 1843 by Volney Palmer, the advertising agency has evolved into the recognizable—and unrecognizable—firms scattered around the world today. In the United States alone, there are roughly 13.4 thousand agencies, many of which also have branches in other countries. The evolution of the modern advertising agency coincided with, and even preceded, some of the major inflection points in history. Understanding how and why changes in advertising agencies affected these inflection points provides a glimpse of understanding into the relationship between advertising, business, and societal values.
In the pages ahead we will explore the future of the advertising industry. We will analyze our research to uncover the underlying trends pointing towards what is to come and work to apply those explanations to our understanding of advertising in the future.
In the pages ahead we will explore the future of the advertising industry. We will analyze our research to uncover the underlying trends pointing towards what is to come and work to apply those explanations to our understanding of advertising in the future.
ContributorsHarris, Chase (Co-author) / Potthoff, Zachary (Co-author) / Gray, Nancy (Thesis director) / Samper, Adriana (Committee member) / Department of Information Systems (Contributor) / Department of Marketing (Contributor) / Herberger Institute for Design and the Arts (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The town of Guadalupe, Arizona has a long history of divided residents and high poverty rates. The high levels of poverty in the town can be attributed to numerous factors, most notably high rates of drug abuse, low high school graduation rates, and teen pregnancy. The town has named one of its most pressing issues of today to be youth disengagement. There are currently a handful of residents and community members passionate about finding a solution to this issue. After working with Guadalupe's Ending Hunger Task Force and resident youth, I set out to create a program design for a Guadalupe Youth Council. This council will contribute to combating youth disengagement. The program design will assist the task force in creating a standing youth council and deciding on the structure and role the council has in the town. I will offer learning outcomes and suggestions to the Task Force, youth council staff, and the youth of the youth council. This study contains an analysis of relevant literature, youth focus group results and data, and how the information gathered has contributed to the design of the youth council. The results of this study contain recommendations about four themes within the program design of a youth council: size, recruitment, activities and engagement, and adult support. The results also explore how the youth council will impact the power, policy, and behavior of Guadalupe youth.
ContributorsBalderas, Erica Theresa (Author) / Wang, Lili (Thesis director) / Avalos, Francisco (Committee member) / School of Community Resources and Development (Contributor) / Department of Information Systems (Contributor) / W.P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
This thesis project was conducted to create a practical tool to help micro and small local food enterprises identify potential strategies and sources of finance. Currently, many of these enterprises are unable to obtain the financial capital needed to start-up or maintain operations.
Sources and strategies of finance studied and ultimately included in the tool were Loans, Equity, Membership, Crowdfunding, and Grants. The tool designed was a matrix that takes into account various criteria of the business (e.g. business lifecycle, organizational structure, business performance) and generates a financial plan based on these criteria and how they align with the selected business strategies. After strategies are found, stakeholders can search through an institutional database created in conjunction with the matrix tool to find possible institutional providers of financing that relate to the strategy or strategies found.
The tool has shown promise in identifying sources of finance for micro and small local food enterprises in practical use with hypothetical business cases, however further practical use is necessary to provide further input and revise the tool as needed. Ultimately, the tool will likely become fully user-friendly and stakeholders will not need the assistance of another expert helping them to use it. Finally, despite the promise of the tool itself, the fundamental and underlying problem that many of these businesses face (lack of infrastructure and knowledge) still exists, and while this tool can also help capacity-building efforts towards both those seeking and those providing finance, an institutional attitude adjustment towards social and alternative enterprises is necessary in order to further simplify the process of obtaining finance.
Sources and strategies of finance studied and ultimately included in the tool were Loans, Equity, Membership, Crowdfunding, and Grants. The tool designed was a matrix that takes into account various criteria of the business (e.g. business lifecycle, organizational structure, business performance) and generates a financial plan based on these criteria and how they align with the selected business strategies. After strategies are found, stakeholders can search through an institutional database created in conjunction with the matrix tool to find possible institutional providers of financing that relate to the strategy or strategies found.
The tool has shown promise in identifying sources of finance for micro and small local food enterprises in practical use with hypothetical business cases, however further practical use is necessary to provide further input and revise the tool as needed. Ultimately, the tool will likely become fully user-friendly and stakeholders will not need the assistance of another expert helping them to use it. Finally, despite the promise of the tool itself, the fundamental and underlying problem that many of these businesses face (lack of infrastructure and knowledge) still exists, and while this tool can also help capacity-building efforts towards both those seeking and those providing finance, an institutional attitude adjustment towards social and alternative enterprises is necessary in order to further simplify the process of obtaining finance.
ContributorsDwyer, Robert Francis (Author) / Wiek, Arnim (Thesis director) / Forrest, Nigel (Committee member) / Department of Finance (Contributor) / Department of Information Systems (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
RecyclePlus is an iOS mobile application that allows users to be knowledgeable in the realms of sustainability. It gives encourages users to be environmental responsible by providing them access to recycling information. In particular, it allows users to search up certain materials and learn about its recyclability and how to properly dispose of the material. Some searches will show locations of facilities near users that collect certain materials and dispose of the materials properly. This is a full stack software project that explores open source software and APIs, UI/UX design, and iOS development.
ContributorsTran, Nikki (Author) / Ganesh, Tirupalavanam (Thesis director) / Meuth, Ryan (Committee member) / Watts College of Public Service & Community Solut (Contributor) / Department of Information Systems (Contributor) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
In the past ten years, the United States’ sound recording industries have experienced significant decreases in employment opportunities for aspiring audio engineers from economic imbalances in the music industry’s digital streaming era and reductions in government funding for career and technical education (CTE). The Recording Industry Association of America reports promises of music industry sustainability based on increasing annual revenues in paid streaming services and artists’ high creative demand. The rate of new audio engineer entries in the sound recording subsection of the music industry is not viable to support streaming artists’ high demand to engineer new music recordings. Offering CTE programs in secondary education is rare for aspiring engineers with insufficient accessibility to pursue a post-secondary or vocational education because of financial and academic limitations. These aspiring engineers seek alternatives for receiving an informal education in audio engineering on the Internet using video sharing services like YouTube to search for tutorials and improve their engineering skills. The shortage of accessible educational materials on the Internet restricts engineers from advancing their own audio engineering education, reducing opportunities to enter a desperate job market in need of independent, home studio-based engineers. Content creators on YouTube take advantage of this situation and commercialize their own video tutorial series for free and selling paid subscriptions to exclusive content. This is misleading for newer engineers because these tutorials omit important understandings of fundamental engineering concepts. Instead, content creators teach inflexible engineering methodologies that are mostly beneficial to their own way of thinking. Content creators do not often assess the incompatibility of teaching their own methodologies to potential entrants in a profession that demands critical thinking skills requiring applied fundamental audio engineering concepts and techniques. This project analyzes potential solutions to resolve the deficiencies in online audio engineering education and experiments with structuring simple, deliverable, accessible educational content and materials to new entries in audio engineering. Designing clear, easy to follow material to these new entries in audio engineering is essential for developing a strong understanding for the application of fundamental concepts in future engineers’ careers. Approaches to creating and designing educational content requires translating complex engineering concepts through simplified mediums that reduce limitations in learning for future audio engineers.
ContributorsBurns, Triston Connor (Author) / Tobias, Evan (Thesis director) / Libman, Jeff (Committee member) / Department of Information Systems (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
This paper looks at the growth of influencer marketing in application and how it has shifted the relationship between brands and consumers. Barriers to enter the space and methods of practice are discussed and analyzed to project the accessibility of obtaining influencer status. Best practices for brands and influencers are outlined based on research, and key findings are analyzed from interviewed participants that play an active role in the field. Another component of the paper includes the discussion of the significance of platform dependence regarding influencers and brands using social media channels to reach consumers. The dynamic of the relationship that exists between consumers, brands and platforms is demonstrated through a model to demonstrate the interdependence of the relationship. The final component of the paper involves the exploration of the field as an active participant through an experiment that was conducted by the researcher on behalf of the question: can anyone be an influencer? The answer to this question is explored through personal accounts on the journey during an eight month process of testing content creation and promotion to build awareness and increase engagement. The barriers to enter the space as an influencer and to collaborate with brands is addressed through the process of testing tactics and strategies on social channels, along with travel expeditions across Arizona to contribute to content creation purposed into blog articles. The findings throughout the paper are conclusive that the value of influencer marketing is increasing as more brands validate and utilize this method in their marketing efforts.
ContributorsDavis, Natalie Marie (Author) / Giles, Bret (Thesis director) / Schlacter, John (Committee member) / Department of Information Systems (Contributor) / Department of Marketing (Contributor) / Walter Cronkite School of Journalism and Mass Communication (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Pandora is a play exploring our relationship with gendered technology through the lens of artificial intelligence. Can women be subjective under patriarchy? Do robots who look like women have subjectivity? Hoping to create a better version of ourselves, The Engineer must navigate the loss of her creation, and Pandora must navigate their new world. The original premiere run was March 27-28, 2018, original cast: Caitlin Andelora, Rikki Tremblay, and Michael Tristano Jr.
ContributorsToye, Abigail Elizabeth (Author) / Linde, Jennifer (Thesis director) / Abele, Kelsey (Committee member) / Department of Information Systems (Contributor) / Economics Program in CLAS (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05