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Description
Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction.

Variants of human butyrylcholinesterase (BChE) have been designed to have high cocaine hydrolytic activity. These variants have potential pharmacological applications toward treating cocaine overdose and addiction. These enzymes must be stable in the human body over fairly long periods of time in order to be effective at treating cocaine addiction. Recombinantly expressed BChE, however, tends to be in monomer or dimer oligomeric forms, which are far less stable than the tetramer form of the enzyme. When BChE is transiently expressed in Nicotiana benthamiana, it is produced mainly as monomers and dimers. However, when the protein is expressed through stable transformation, it produces much greater proportions of tetramers. Tetramerization of WT human plasma derived BChE is facilitated by the binding of a proline rich peptide. In this thesis, I investigated if a putative plant-derived analog of the mammalian proline-rich attachment domain caused stably expressed cocaine hydrolase variants of human BChE to undergo tetramerization. I also examined if co-expression of peptides with known proline-rich attachment domains further shifted the monomer-tetramer ratio toward the tetramer.
ContributorsKendle, Robert Player (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Larrimore, Kathy (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description
Electrical stimulation has previously been effective in neural cells activation within retinas affected by degenerative retinal disease. However current technology has at most allowed blind individuals to perceive light without significant resolution, as implants are limited by the spatial constraints of the eye. Photoreactive nanoparticles may provide a solution to

Electrical stimulation has previously been effective in neural cells activation within retinas affected by degenerative retinal disease. However current technology has at most allowed blind individuals to perceive light without significant resolution, as implants are limited by the spatial constraints of the eye. Photoreactive nanoparticles may provide a solution to this issue, as their small size would allow for the incorporation of higher numbers of stimulatory elements, thus increasing visual resolution. Semiconductive nanocrystal quantum dots (QDs) and gold nanoparticles (AuNPs) both exhibit photoreactive properties which may result in sufficient electrical stimulation to activate neural cells in the retina. This study investigated the electrochemistry and photoreactivity of QDs and AuNPs encapsulated within the hydrophobic region of small unilamellar lipid vesicles (SUVs) to evaluate their potential for application in retinal stimulation. Absorbance of the constructs was evaluated on the day of fabrication and 24 hours later to determine the ability of the particles to react to light while encapsulated, as well as to evaluate stability of the construct over time. Electrical impedance spectroscopy (EIS) was conducted at both time points to determine the electrochemical character of the bilayer and further evaluate construct stability. Although quantum dots may increase the stability of the bilayer over time and improve its capacitative properties, lipid encapsulation appears to obscure the photoreactive properties of the quantum dots. In the case of gold nanoparticles, the construct is initially stabilized but deteriorates more quickly than those SUVs containing quantum dots, as evidenced by an increase in substrate diffusion. Additionally, although these constructs are more photoreactive than those containing QDs, the increase in absorbance is observed primarily in a range below that of the visible spectrum, a feature which is of limited use for the proposed application. Further studies should investigate alternative methods of nanoparticle capping to improve stability and absorbance in this system.
ContributorsReidell, Olivia Rose (Author) / La Belle, Jeffrey T (Thesis director) / Coursen, Jerry (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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Description
Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high

Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.
ContributorsFavre, Brandon Chetan (Author) / Mason, Hugh (Thesis director) / Mor, Tsafrir (Committee member) / Diamos, Andrew (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Description
Photosynthesis is the process by which plants, algae, and bacteria use light energy to synthesize organic compounds to use as energy. Among these organisms are a kind of purple photosynthetic bacteria called Rhodobacter sphaeroides, a non-sulfur purple bacteria that grows aerobically in the dark by respiration. There have been many

Photosynthesis is the process by which plants, algae, and bacteria use light energy to synthesize organic compounds to use as energy. Among these organisms are a kind of purple photosynthetic bacteria called Rhodobacter sphaeroides, a non-sulfur purple bacteria that grows aerobically in the dark by respiration. There have been many contributions throughout the history of this group of bacteria. Rhodobacter sphaeroides is metabolically very diverse as it has many different ways to obtain energy--aerobic respiration and anoxygenic photosynthesis being just a couple of the ways to do so. This project is part of a larger ongoing project to study different mutant strains of Rhodobacter and the different ways in which carries out electron transfer/photosynthesis. This thesis focused on the improvements made to protocol (standard procedure of site directed mutagenesis) through a more efficient technique known as infusion.
ContributorsNucuta, Diana Ileana (Author) / Woodbury, Neal (Thesis director) / Lin, Su (Committee member) / Loskutov, Andrey (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2014-05
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Description
Fermentative bioproduction is an efficient production avenue for many small organic acids with less greenhouse gas emissions than petrochemical conversion. Export of these organic acids from the cell is proposed to be mediated by networks of transmembrane transport proteins. However characterization of full transporter networks or the substrate promiscuity of

Fermentative bioproduction is an efficient production avenue for many small organic acids with less greenhouse gas emissions than petrochemical conversion. Export of these organic acids from the cell is proposed to be mediated by networks of transmembrane transport proteins. However characterization of full transporter networks or the substrate promiscuity of individual transporters is often incomplete. Here, we used a cheminformatic approach to predict previously unknown native activity of E. coli transporters based on substrate promiscuity. Experimental validation in characterized several major putative malate exporters, whereas others were characterized as weak putative lactate exporters. The lactate export network remains incompletely characterized and might be mediated by a large, evolved network of promiscuous transporters.
ContributorsSchneider, Aidan (Author) / Wang, Xuan (Thesis director) / Varman, Arul (Committee member) / Nielsen, David (Committee member) / Department of Finance (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-12
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Description

Women’s roles in society have changed significantly throughout the years. The movement to support the rights of women has been ongoing throughout the evolution of society but has been especially prevalent in the last century. The 1960s are when women began to enter the workforce instead of being limited to

Women’s roles in society have changed significantly throughout the years. The movement to support the rights of women has been ongoing throughout the evolution of society but has been especially prevalent in the last century. The 1960s are when women began to enter the workforce instead of being limited to presuming roles as homemakers. Since that point in time, women have continued to thrive in the workforce and have pursued a larger variety of positions in various fields. Even though the opportunities for women continue to grow, there still seems to be an underrepresentation of women in science, technology, engineering, and mathematics (STEM) related fields. The underrepresentation of women pursuing physician and entrepreneurship roles in STEM will be analyzed and the challenges this group of people specifically encounter will be examined. Our first proposal to encourage women to enter STEM focuses on middle-school initiatives and incubator programs. The second proposal, based on commonalities females face within the workforce, is finding a better work/home life balance with the development of new maternity/paternity leave policies. Through these initiatives, we believe that the gender gap in STEM can be bridged.

ContributorsWillbrandt, Mary Madison (Co-author) / Torres, Julianna (Co-author) / Martin, Thomas (Thesis director) / Fette, Donald (Committee member) / Tech Entrepreneurship & Mgmt (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Description

Women’s roles in society have changed significantly throughout the years. The movement to support the rights of women has been ongoing throughout the evolution of society but has been especially prevalent in the last century. The 1960s are when women began to enter the workforce instead of being limited to

Women’s roles in society have changed significantly throughout the years. The movement to support the rights of women has been ongoing throughout the evolution of society but has been especially prevalent in the last century. The 1960s are when women began to enter the workforce instead of being limited to presuming roles as homemakers. Since that point in time, women have continued to thrive in the workforce and have pursued a larger variety of positions in various fields. Even though the opportunities for women continue to grow, there still seems to be an underrepresentation of women in science, technology, engineering, and mathematics (STEM) related fields. The underrepresentation of women pursuing physician and entrepreneurship roles in STEM will be analyzed and the challenges this group of people specifically encounter will be examined. Our first proposal to encourage women to enter STEM focuses on middle-school initiatives and incubator programs. The second proposal, based on commonalities females face within the workforce, is finding a better work/home life balance with the development of new maternity/paternity leave policies. Through these initiatives, we believe that the gender gap in STEM can be bridged.

ContributorsTorres, Julianna M (Co-author) / Willbrandt, Maddie (Co-author) / Martin, Thomas (Thesis director) / Fette, Donald (Committee member) / School of Social Transformation (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description

The debate around genetic engineering has permeated society for decades. A crucial aspect of this debate is the containment of genetically engineered organisms. This project outlines the three types of biocontainment and the conclusions drawn about each in the form of policy briefs. These briefs utilize case studies to sketch

The debate around genetic engineering has permeated society for decades. A crucial aspect of this debate is the containment of genetically engineered organisms. This project outlines the three types of biocontainment and the conclusions drawn about each in the form of policy briefs. These briefs utilize case studies to sketch an overview of the current biocontainment paradigm in the United States. In addition, there is a brief discussing the major conclusions drawn from the case studies, as well as a brief containing useful definitions.

ContributorsDanciu, Mark (Author) / Frow, Emma (Thesis director) / Vogel, Kathleen (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Historical, Philosophical & Religious Studies, Sch (Contributor)
Created2023-05
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Description
HIV continues to remain a global health issue, in particular in many low and middle-income countries. The World Health Organization (WHO) estimates that of the nearly 38 million HIV-1 positive individuals, 25% are unaware they are infected. Despite decades of research, a safe and effective preventative vaccine has yet to

HIV continues to remain a global health issue, in particular in many low and middle-income countries. The World Health Organization (WHO) estimates that of the nearly 38 million HIV-1 positive individuals, 25% are unaware they are infected. Despite decades of research, a safe and effective preventative vaccine has yet to be produced. The HIV-1 envelope glycoprotein41 and the Gag structural protein have been identified to be particularly important in HIV-1 transcytosis and cytotoxic lymphocyte response, respectively. Enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of glycoprotein (dgp41) comprising the membrane proximal external region (MPER), transmembrane domain and cytoplasmic tail may present a unique and safe way of presenting these proteins in a state mimicking their natural formation. Another form of presenting the immunogenic glycoprotein41, particularly the MPER component, is by presenting it onto the N-terminal of an IgG molecule, thereby creating an IgG fusion molecule. In our lab, both VLPs and IgG fusion molecules are highly expressed and purified within GnGn Nicotiana benthamiana. The results indicated that these recombinant proteins can be assembled properly within plants and can elicit an immune response in mice. This provides a preliminary step in using such Gag/dpg41 VLPs and RIC as present a safe, effective, and inexpensive HIV vaccine.
ContributorsGarcia, Izamar (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kamzina, Aigerim (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
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Description

Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with

Plant-made virus-like particles (VLPs), composed of HIV-1 Gag and deconstructed gp41 proteins, have been shown to be safe and immunogenic in mice. Here, we report the successful production of HIV-1 Gag/dgp41 VLPs in Nicotiana benthamiana, using an enhanced geminivirus-based expression vector. This novel vector results in unique expression kinetics, with peak protein accumulation and minimal necrosis achieved on day 4 post-infiltration. In comparing various purification strategies, it was determined that a 20% ammonium sulfate precipitation is an effective and efficient method for removing plant proteins and purifying the recombinant VLPs of interest. If further purification is required, this may be achieved through ultracentrifugation. VLPs are a useful platform for a variety of biomedical applications and developing the technology to efficiently produce VLPs in the plant expression system is of critical importance.

ContributorsFleming, Claire (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kamzina, Aigerim (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2022-05