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- Creators: Haydn, Joseph, 1732-1809
Biological and immunological characterization of plant-produced HIV-1 Gag/dgp41 virus-like particles
Description
Anti-retroviral drugs and AIDS prevention programs have helped to decrease the rate of new HIV-1 infections in some communities, however, a prophylactic vaccine is still needed to control the epidemic world-wide. Despite over two decades of research, a vaccine against HIV-1 remains elusive, although recent clinical trials have shown promising results. Recent successes have focused on highly conserved, mucosally-targeted antigens within HIV-1 such as the membrane proximal external region (MPER) of the envelope protein, gp41. MPER has been shown to play critical roles in the viral mucosal transmission, though this peptide is not immunogenic on its own. Gag is a structural protein configuring the enveloped virus particles, and has been suggested to constitute a target of the cellular immunity potentially controlling the viral load. It was hypothesized that HIV-1 enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of gp41 comprising the MPER, transmembrane, and cytoplasmic domains (dgp41) could be expressed in plants. Plant-optimized HIV-1 genes were constructed and expressed in Nicotiana benthamiana by stable transformation, or transiently using a tobacco mosaic virus-based expression system or a combination of both. Results of biophysical, biochemical and electron microscopy characterization demonstrated that plant cells could support not only the formation of HIV-1 Gag VLPs, but also the accumulation of VLPs that incorporated dgp41. These particles were purified and utilized in mice immunization experiments. Prime-boost strategies combining systemic and mucosal priming with systemic boosting using two different vaccine candidates (VLPs and CTB-MPR - a fusion of MPER and the B-subunit of cholera toxin) were administered to BALB/c mice. Serum antibody responses against both the Gag and gp41 antigens could be elicited in mice systemically primed with VLPs and these responses could be recalled following systemic boosting with VLPs. In addition, mucosal priming with VLPs allowed for a robust boosting response against Gag and gp41 when boosted with either candidate. Functional assays of these antibodies are in progress to test the antibodies' effectiveness in neutralizing and preventing mucosal transmission of HIV-1. This immunogenicity of plant-based Gag/dgp41 VLPs represents an important milestone on the road towards a broadly-efficacious and inexpensive subunit vaccine against HIV-1.
ContributorsKessans, Sarah (Author) / Mor, Tsafrir S (Thesis advisor) / Matoba, Nobuyuki (Committee member) / Mason, Hugh (Committee member) / Hogue, Brenda (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
Infections caused by the Hepatitis C Virus (HCV) are very common worldwide, affecting up to 3% of the population. Chronic infection of HCV may develop into liver cirrhosis and liver cancer which is among the top five of the most common cancers. Therefore, vaccines against HCV are under intense study in order to prevent HCV from harming people's health. The envelope protein 2 (E2) of HCV is thought to be a promising vaccine candidate because it can directly bind to a human cell receptor and plays a role in viral entry. However, the E2 protein production in cells is inefficient due to its complicated matured structure. Folding of E2 in the endoplasmic reticulum (ER) is often error-prone, resulting in production of aggregates and misfolded proteins. These incorrect forms of E2 are not functional because they are not able to bind to human cells and stimulate antibody response to inhibit this binding. This study is aimed to overcome the difficulties of HCV E2 production in plant system. Protein folding in the ER requires great assistance from molecular chaperones. Thus, in this study, two molecular chaperones in the ER, calreticulin and calnexin, were transiently overexpressed in plant leaves in order to facilitate E2 folding and production. Both of them showed benefits in increasing the yield of E2 and improving the quality of E2. In addition, poorly folded E2 accumulated in the ER may cause stress in the ER and trigger transcriptional activation of ER molecular chaperones. Therefore, a transcription factor involved in this pathway, named bZIP60, was also overexpressed in plant leaves, aiming at up-regulating a major family of molecular chaperones called BiP to assist protein folding. However, our results showed that BiP mRNA levels were not up-regulated by bZIP60, but they increased in response to E2 expression. The Western blot analysis also showed that overexpression of bZIP60 had a small effect on promoting E2 folding. Overall, this study suggested that increasing the level of specific ER molecular chaperones was an effective way to promote HCV E2 protein production and maturation.
ContributorsHong, Fan (Author) / Mason, Hugh (Thesis advisor) / Gaxiola, Roberto (Committee member) / Chang, Yung (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2011
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
ContributorsHaydn, Joseph, 1732-1809 (Composer)
ContributorsHaydn, Joseph, 1732-1809 (Composer)
Description
Space microbiology, or the study of microorganisms in space, has significant applications for both human spaceflight and Earth-based medicine. This thesis traces the evolution of the field of space microbiology since its creation in 1935. Beginning with simple studies to determine if terrestrial life could survive spaceflight, the field of space microbiology has grown to encompass a substantial body of work that is now recognized as an essential component of NASA' research endeavors. Part one provides an overview of the early period of space microbiology, from high-altitude balloon and rocket studies to work conducted during the Apollo program. Part two summarizes the current state of the field, with a specific focus on the revolutionary contributions made by the Nickerson lab at the Biodesign Institute at ASU using the NASA-designed Rotating Wall Vessel (RWV) Bioreactor. Finally, part three highlights the research I've conducted in the Nickerson lab, as well as continuing studies within the field of space microbiology.
ContributorsMcCarthy, Breanne E. (Author) / Lynch, John (Thesis director) / Foy, Joseph (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Orbiting space debris is an active issue that affects the capability of space launch for future satellites, probes, and space shuttles, and it will become a nearly insurmountable problem without action. Debris of varying sizes and speeds orbit the Earth at a range of heights above the atmosphere and need to be removed to avoid damage to crucial equipment of active orbiting satellites including the International Space Station. Finding a feasible solution to space debris removal requires that several facets be covered to become a reality; these include being aware of the problem in magnitude and source. This literature assessment covers the magnitude of space debris in low-earth and geosynchronous orbit as well as collision events which have increased the amount of space debris. There have been efforts made by several space agencies to control the amount of space debris added to orbit by current and future launches over the last decade \u2014 serving as a temporary fix before removal can be executed. This paper explores known removal efforts through mitigation, projects conceived and tested by DARPA, related space policies and laws, CubeSat technology, and the cataloguing of known space debris. To make space debris removal a reality, roadblocks need to be removed to acquire permission from states or countries for space missions. For example, these restrictions are in place to protect the assets of several countries and organizations. Guidelines set to curb the growth of space debris fail to prevent the growth due to the restrictions for ownership rights making them not as effective. This paper covers space policy and laws, the economy, satellite ownership, international conflict, status of space debris, and the overall feasibility of space debris removal. It will then discuss currently proposed solutions for the removal of space debris. Finally, this paper attempts to weight the advantages and disadvantages of the idea that space debris removal should include the opportunity to recycle materials. For example, defunct satellites and other discarded space crafts could be used for future launches. It will conclude with a personal exploration of what materials can be recycled, what chemical processes can be used to break down materials, and how to combine recycling and chemical processes for space-based recycling stations between Earth and the moon. The overall question that drives the search for making space debris removal a reality is whether it is feasible in multiple areas including technologically, legally, monetarily, and physically.
ContributorsBreden, Elizabeth Catherine (Author) / Foy, Joseph (Thesis director) / Thoesen, Andrew (Committee member) / Maximon, Leonard (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore receptors that allow extracellular siderophores bound to iron to enter the cells to power various biological processes. Previous studies have shown that in E. coli cells that expressed a mutant allele of envZ, called envZ11, which led to altered expression of various iron genes including down regulation of fepA::lacZ. The wild type EnvZ/OmpR system is not considered to regulate iron genes, but because these envz11 strains had downregulated fepA::lacZ, this study was undertaken to understand the connection and mechanisms of this downregulation. A large number of Lac+ revertants were obtained from the B32-2483 strain (envz11 and fepA::lacZ) and 7 Lac+ revertants that had reversion mutations not directly correcting the envZ11 allele were further characterized. With P1 phage transduction genetic mapping that involved moving a kanamycin resistance marker linked to fepA::lacZ, two Lac+ revertants were found to have their reversion mutations in the fepA promoter region, while the other five revertants had their mutations mapping outside the fepA region. These two revertants underwent DNA sequencing and found to carry two different single base pair mutations in two different locations of the fepA promoter region. Each one is in the Fur repressor binding region, but one also may have affected the Shine-Dalgarno region involved in translation initiation. All 7 reveratants underwent beta-galactosidase assays to measure fepA::lacZ expression. The two revertants that had mutations in the fepA promoter region had significantly increased fepA activity, with the revertant with the Shine-Dalgarno mutation having the most elevated fepA expression. The other 5 revertants that did not map in the fepA region had fepA expression elevated to the same level as that found in the wild type EnvZ/OmpR background. The data suggest that the negative effect of envZ11 can be overcome by multiple mechanisms, including directly correcting the envZ11 allele or changing the fepA promoter region.
ContributorsKalinkin, Victor Arkady (Co-author) / Misra, Rajeev (Co-author, Thesis director) / Mason, Hugh (Committee member) / Foy, Joseph (Committee member) / Biomedical Informatics Program (Contributor) / School of Life Sciences (Contributor) / W. P. Carey School of Business (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Einstein's theory of special relativity has been used by accomplished science fiction authors since its discovery in 1905, allowing intrepid adventurers to reach far away worlds without having to fear time's passage. By traveling near light speed, these fictional travelers experience a different passage of time as the universe ensures the commonality of the speed of light in all reference frames. In the here and now, this method of travel has been proposed to assist in interstellar and interplanetary exploration. This paper will investigate the practicality of this method of travel by proposing a mission utilizing a craft with this type of velocity.
ContributorsWaaler, Mason Duran (Author) / Jacob, Richard (Thesis director) / Covatto, Carl (Committee member) / Foy, Joseph (Committee member) / School of Life Sciences (Contributor) / School of Earth and Space Exploration (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The objective for Under the Camper Shell was to build a prototype of a full living environment within the confines of a pickup truck bed and camper shell. The total volume available to work with is approximately 85ft3. This full living environment entails functioning systems for essential modern living, providing shelter and spaces for cooking, sleeping, eating, and sanitation. The project proved to be very challenging from the start. First, the livable space is extremely small, being only tall enough for one to sit up straight. The truck and camper shell were both borrowed items, so no modifications were allowed for either, e.g. drilling holes for mounting. The idea was to create a system that could be easily removed, transforming it from a camper to a utility truck. The systems developed for the living environment would be modular and transformative so to accommodate for different necessities when packing. The goal was to create a low-water system with sustainability in mind. Insulating the space was the largest challenge and the most rewarding, using body heat to warm the space and insulate from the elements. Comfort systems were made of high density foam cushions in sections to allow folding and stacking for different functions (sleeping, lounging, and sitting). Sanitation is necessary for healthy living and regular human function. A composting toilet was used for the design, lending to low-water usage and is sustainable over time. Saw dust would be necessary for its function, but upon composting, the unit will generate sufficient amounts of heat to act as a space heater. Showering serves the functions of exfoliation and ridding of bacteria, both of which bath wipes can accomplish, limiting massive volumes of water storage and waste. Storage systems were also designed for modularity. Hooks were installed the length of the bed for hanging or securing items as necessary. Some are available for hanging bags. A cabinetry rail also runs the length of the bed to allow movement of hard storage to accommodate different scenarios. The cooking method is called "sous-vide", a method of cooking food in air-tight bags submerged in hot water. The water is reusable for cooking and no dishes are necessary for serving. Overall, the prototype fulfilled its function as a full living environment with few improvements necessary for future use.
ContributorsLimsirichai, Pimwadee (Author) / Foy, Joseph (Thesis director) / Parrish, Kristen (Committee member) / Barrett, The Honors College (Contributor) / Materials Science and Engineering Program (Contributor) / School of Sustainability (Contributor)
Created2014-12