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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
Geology and its tangential studies, collectively known and referred to in this thesis as geosciences, have been paramount to the transformation and advancement of society, fundamentally changing the way we view, interact and live with the surrounding natural and built environment. It is important to recognize the value and importance of this interdisciplinary scientific field while reconciling its ties to imperial and colonizing extractive systems which have led to harmful and invasive endeavors. This intersection among geosciences, (environmental) justice studies, and decolonization is intended to promote inclusive pedagogical models through just and equitable methodologies and frameworks as to prevent further injustices and promote recognition and healing of old wounds. By utilizing decolonial frameworks and highlighting the voices of peoples from colonized and exploited landscapes, this annotated syllabus tackles the issues previously described while proposing solutions involving place-based education and the recentering of land within geoscience pedagogical models. (abstract)
ContributorsReed, Cameron E (Author) / Richter, Jennifer (Thesis director) / Semken, Steven (Committee member) / School of Earth and Space Exploration (Contributor, Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The ASU COVID-19 testing lab process was developed to operate as the primary testing site for all ASU staff, students, and specified external individuals. Tests are collected at various collection sites, including a walk-in site at the SDFC and various drive-up sites on campus; analysis is conducted on ASU campus and results are distributed virtually to all patients via the Health Services patient portal. The following is a literature review on past implementations of various process improvement techniques and how they can be applied to the ABCTL testing process to achieve laboratory goals. (abstract)
ContributorsKrell, Abby Elizabeth (Co-author) / Bruner, Ashley (Co-author) / Ramesh, Frankincense (Co-author) / Lewis, Gabriel (Co-author) / Barwey, Ishna (Co-author) / Myers, Jack (Co-author) / Hymer, William (Co-author) / Reagan, Sage (Co-author) / Compton, Carolyn (Thesis director) / McCarville, Daniel R. (Committee member) / Industrial, Systems & Operations Engineering Prgm (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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
For as long as humans have been working, they have been looking for ways to get that work done better, faster, and more efficient. Over the course of human history, mankind has created innumerable spectacular inventions, all with the goal of making the economy and daily life more efficient. Today, innovations and technological advancements are happening at a pace like never seen before, and technology like automation and artificial intelligence are poised to once again fundamentally alter the way people live and work in society. Whether society is prepared or not, robots are coming to replace human labor, and they are coming fast. In many areas artificial intelligence has disrupted entire industries of the economy. As people continue to make advancements in artificial intelligence, more industries will be disturbed, more jobs will be lost, and entirely new industries and professions will be created in their wake. The future of the economy and society will be determined by how humans adapt to the rapid innovations that are taking place every single day. In this paper I will examine the extent to which automation will take the place of human labor in the future, project the potential effect of automation to future unemployment, and what individuals and society will need to do to adapt to keep pace with rapidly advancing technology. I will also look at the history of automation in the economy. For centuries humans have been advancing technology to make their everyday work more productive and efficient, and for centuries this has forced humans to adapt to the modern technology through things like training and education. The thesis will additionally examine the ways in which the U.S. education system will have to adapt to meet the demands of the advancing economy, and how job retraining programs must be modernized to prepare workers for the changing economy.
ContributorsCunningham, Reed P. (Author) / DeSerpa, Allan (Thesis director) / Haglin, Brett (Committee member) / School of International Letters and Cultures (Contributor) / Department of Finance (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Businesses stand to face many uncertainties from the moment they start up to every moment in between. A business can try to recognize them and plan ahead, react to them as they occur, or be rocked by a black swan they never saw coming. How a business deals with unforeseen events can increase its potential for success or failure. With this in mind, there is no better bridge between the here and now and the future than planning for change in order to move a company toward preparing for change, adapting to change and achieving optimal results. Interested in taking a step toward the digital age, Alpha Homes Management, Inc. (Alpha Homes) sought our help to explore ideas and options to take their company to a new level. This Barrett Creative Project was centered on designing a system for Alpha Homes that will replace their outdated paper-based system with a more digital one. This aligns with the project also featured as a capstone project as required by the information technology degree expectations. In supplement to the capstone, and for the Barrett Creative Project, the final product was presented to the owners of Alpha Homes Management, Inc. to be utilized by the business. The end goal is to provide a platform which provides a paperless environment for documentation and bring the company a step closer to having a robust internet presence. Now that the web-based application product has been created and presented, the testing phase can now begin to evaluate its efficacy.
ContributorsBrice-Nash, Tristan (Co-author) / Alfawzan, Mohammad (Co-author) / Doheny, Damien (Thesis director) / Rodriguez, Carlos (Committee member) / Information Technology (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
An ethical dilemma is not a matter of “right” versus “wrong,” but rather it is a situation of conflicting values. A common ethical dilemma is that of honesty versus loyalty—is it better to tell the truth, or remain loyal to the company? In the Japanese culture, truth is circumstantial and can vary with different situations. In a way, the Japanese idea of honesty reflects how highly they value loyalty. This overlap of values results in the lack of an ethical dilemma for the Japanese, which creates a new risk for fraud. Without this struggle, a Japanese employee does not have strong justification against committing fraud if it aligns with his values of honesty and loyalty.
This paper looks at the Japanese values relating to honesty and loyalty to show how much these ideas overlap. The lack of a conflict of values creates a risk for fraud, which will be shown through an analysis of the scandals of two Japanese companies, Toshiba and Olympus. These scandals shine light on the complexity of the ethical dilemma for the Japanese employees; since their sense of circumstantial honesty encourages them to lie if it maintains the harmony of the group, there is little stopping them from committing the fraud that their superiors asked them to commit.
In a global economy, understanding the ways that values impact business and decisions is important for both interacting with others and anticipating potential conflicts, including those that may result in or indicate potential red flags for fraud.
This paper looks at the Japanese values relating to honesty and loyalty to show how much these ideas overlap. The lack of a conflict of values creates a risk for fraud, which will be shown through an analysis of the scandals of two Japanese companies, Toshiba and Olympus. These scandals shine light on the complexity of the ethical dilemma for the Japanese employees; since their sense of circumstantial honesty encourages them to lie if it maintains the harmony of the group, there is little stopping them from committing the fraud that their superiors asked them to commit.
In a global economy, understanding the ways that values impact business and decisions is important for both interacting with others and anticipating potential conflicts, including those that may result in or indicate potential red flags for fraud.
ContributorsTabar, Kelly Ann (Author) / Samuelson, Melissa (Thesis director) / Goldman, Alan (Committee member) / WPC Graduate Programs (Contributor) / W.P. Carey School of Business (Contributor) / School of Accountancy (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
This paper will be exploring a marketing plan for a Kpop Fan artist, Jennifer Lee. Kpop is a genre of music originating from South Korea that provides a whole-package entertainment. Fan artists are producers who create produce for the consumption and purchase of other Kpop fans. The paper will consider segmentation and the products and platforms that best target them in order to maximize revenue. A survey was performed with a sample size of 314 participants to find out consumer behavior and preference as well as producer situation. Consumers come from both the United States and abroad. Customers come directly and almost exclusively from followers. Therefore, increasing the number of followers on Instagram is essential to increasing revenue. Jennifer has time, resource, and ability constraints, while the market has limited potential. The conclusion is that Jennifer should become more organized as a business. To grow her following, she should cater more towards the most popular fandoms (BTS), make art tutorials, consider collaborations, and better inform followers of her products/services available for purchase. The social media platforms key to marketing Jennifer's products are Instagram and Twitter. Other platforms to be used to increase exposure are Tumblr, Amino Apps, DeviantArt, Reddit, and YouTube. She must also declutter all of these virtual storefronts of unnecessary content to varying degrees in order to build ease of access and a trustworthy brand image. The best platforms for transaction is a personal store, RedBubble (a website that allows users to sell a variety of products with their uploaded images printed onto them), Patreon, and in-person at conventions.
ContributorsXu, Everest Christine (Author) / Eaton, Kathryn (Thesis director) / Ingram-Waters, Mary (Committee member) / Department of Marketing (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
In recent years, sex trafficking awareness and intervention have skyrocketed in the United States. The 2016 Polaris Hotline Statistics Sheet reports a drastic increase of reported sex trafficking cases over the span of four years, with only 3,409 cases of human trafficking in 2012 and 8,042 in 2016, 73% of which were specifically sex trafficking cases (Polaris Project, 2016). The incidence of sex trafficking has not increased, but rather, attention to sex trafficking and implementation of legislation has increased awareness and reporting (Farrell et al., 2012). While this rise in public awareness of sex trafficking has positively impacted victim identification, there has not been an increase in convicting sex traffickers (Polaris Project, 2016). According to the 2016 Trafficking in Persons Report, 3,000 federal investigations that involved human trafficking, the majority of which specifically involved sex trafficking, were opened in 2015. Of these federal investigations, only 10% led to case prosecutions. Analyzing the relationship of law enforcement, specifically vice units, and victims of sex trafficking is just one of the many ways to address this complex issue. This study consisted of a qualitative analysis of the makeup, training, and policing methods of vice units nationwide. It further aimed to determine the vice officer perceptions regarding the elements that make sex trafficking cases convictable.
ContributorsPradhan, Krishnaa (Author) / Maienschein, Jane (Thesis director) / Kinzig, Ann (Committee member) / Kim, Grace (Committee member) / Barrett, The Honors College (Contributor)
Created2018-05