Matching Items (56)
Filtering by
- All Subjects: Molecular Biology
- All Subjects: Obesity
- Creators: School of Life Sciences
- Creators: Mason, Hugh
- Member of: Theses and Dissertations
- Status: Published
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
Over 40% of adults in the United States are considered obese. Obesity is known to cause abnormal metabolic effects and lead to other negative health consequences. Interestingly, differences in metabolism and contractile performance between obese and healthy weight individuals are associated with differences in skeletal muscle fiber type composition between these groups. Each fiber type is characterized by unique metabolic and contractile properties, which are largely determined by the myosin heavy chain isoform (MHC) or isoform combination that the fiber expresses. In previous studies, SDS-PAGE single fiber analysis has been utilized as a method to determine MHC isoform distribution and single fiber type distribution in skeletal muscle. Herein, a methodological approach to analyze MHC isoform and fiber type distribution in skeletal muscle was fine-tuned for use in human and rodent studies. In the future, this revised methodology will be implemented to evaluate the effects of obesity and exercise on the phenotypic fiber type composition of skeletal muscle.
ContributorsOhr, Jalonna Rose (Author) / Katsanos, Christos (Thesis director) / Tucker, Derek (Committee member) / Serrano, Nathan (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
In this project I explored the relationship between Qigong and Tai Chi Easy meditative practices and cardiometabolic risk factors, specifically looking at obesity and stress. The meditative focus of Qigong and Tai Chi Easy was expected to improve cardiac vagal tone which should lead to decreases in the inflammatory effects of stress. Additionally, due to the decreases in the harmful effects of stress, we expect to see a decrease in obesity through decreases in BMI and in waist circumference.
ContributorsRameshkumar, Ramya (Author) / Larkey, Linda (Thesis director) / James, Dara (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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
Nucleic acids encode the information required to create life, and polymerases are the gatekeepers charged with maintaining the storage and flow of this genetic information. Synthetic biologists utilize this universal property to modify organisms and other systems to create unique traits or improve the function of others. One of the many realms in synthetic biology involves the study of biopolymers that do not exist naturally, which is known as xenobiology. Although life depends on two biopolymers for genetic storage, it may be possible that alternative molecules (xenonucleic acids – XNAs), could be used in their place in either a living or non-living system. However, implementation of an XNA based system requires the development of polymerases that can encode and decode information stored in these artificial polymers. A strategy called directed evolution is used to modify or alter the function of a protein of interest, but identifying mutations that can modify polymerase function is made problematic by their size and overall complexity. To reduce the amount of sequence space that needs to be samples when attempting to identify polymerase variants, we can try to make informed decisions about which amino acid residues may have functional roles in catalysis. An analysis of Family B polymerases has shown that residues which are involved in substrate specificity are often highly conserved both at the sequence and structure level. In order to validate the hypothesis that a strong correlation exists between structural conservation and catalytic activity, we have selected and mutated residues in the 9°N polymerase using a loss of function mutagenesis strategy based on a computational analysis of several homologues from a diverse range of taxa. Improvement of these models will hopefully lead to quicker identification of loci which are ideal engineering targets.
ContributorsHaeberle, Tyler Matthew (Author) / Chaput, John (Thesis director) / Chen, Julian (Committee member) / Larsen, Andrew (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Introduction: Human papillomavirus (HPV) infection is seen in up to 90% of cases of cervical cancer, the third leading cancer cause of death in women. Current HPV screening focuses on only two HPV types and covers roughly 75% of HPV-associated cervical cancers. A protein based assay to test for antibody biomarkers against 98 HPV antigens from both high and low risk types could provide an inexpensive and reliable method to screen for patients at risk of developing invasive cervical cancer. Methods: 98 codon optimized, commercially produced HPV genes were cloned into the pANT7_cGST vector, amplified in a bacterial host, and purified for mammalian expression using in vitro transcription/translation (IVTT) in a luminescence-based RAPID ELISA (RELISA) assay. Monoclonal antibodies were used to determine immune cross-reactivity between phylogenetically similar antigens. Lastly, several protein characteristics were examined to determine if they correlated with protein expression. Results: All genes were successfully moved into the destination vector and 86 of the 98 genes (88%) expressed protein at an adequate level. A difference was noted in expression by gene across HPV types but no correlation was found between protein size, pI, or aliphatic index and expression. Discussion: Further testing is needed to express the remaining 12 HPV genes. Once all genes have been successfully expressed and purified at high concentrations, DNA will be printed on microscope slides to create a protein microarray. This microarray will be used to screen HPV-positive patient sera for antibody biomarkers that may be indicative of cervical cancer and precancerous cervical neoplasias.
ContributorsMeshay, Ian Matthew (Author) / Anderson, Karen (Thesis director) / Magee, Mitch (Committee member) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
The Intercellular Adhesion Molecule-1 (ICAM-1, known as CD54) is a cell surface type I transmembrane glycoprotein with a molecular weight of 85 to 110 kDa. The primary function of ICAM-1 is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 is used as a receptor for various pathogens such as rhinoviruses, coxsackievirus A21 and the malaria parasite Plasmodium falciparum. ICAM-1 contains five immunoglobulin (Ig) domains in its long N-terminal extracellular region, a hydrophobic transmembrane domain, and a small C-terminal cytoplasmic domain. The Ig domains 1-2 and Ig domains 3-4-5 have been crystallized separately and their structure solved, however the full ICAM-1 structure has not been solved. Because ICAM-1 appears to be important for the mediation of cell-to-cell communication in physiological and pathological conditions, gaining a structural understanding of the full-length membrane anchored ICAM-1 is desirable. In this context, we have transiently expressed a plant-optimized gene encoding human ICAM-1 in Nicotiana benthamiana plants using the MagnICON expression system. The plant produced ICAM-1 is forming aggregates according to previous data. Thus, the current extraction and purification protocols have been altered to include TCEP, a reducing agent. The protein was purified using TALON metal affinity resin and partially characterized using various biochemical techniques. Our results show that there is a reduction in aggregation formation with the use of TCEP.
ContributorsPatel, Heeral (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kannan, Latha (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Obesity is a growing issue in the Western world, as well as other international countries. This is leading to increases in complications associated with obesity. One such complication is osteoarthritis (OA) of load bearing joints that requires surgical treatment by total knee arthroplasty (TKA). Obesity is also associated with an increase in surgical complications that may lead to poor TKA outcomes. Additionally, the female gender is also known to be associated with increased rates of severe, clinical OA. This study was designed to determine the comparative efficacy of two knee implants in the obese female population through retrospective chart review and data analysis. The implants differ in their level of constraint, with the total stabilizing (TS) being more constrained than the posterior stabilizing (PS). We hypothesized that the TS implants would be associated with improved functional outcomes in the obese female population. The TS implant was observed to be associated with earlier improvement of both passive and active range of motion. This implant also showed greater improvement from pre-operative condition in stability, rejecting our null hypothesis and supporting our hypothesis.
ContributorsWorhacz, Kellen Michael (Author) / Hinrichs, Richard (Thesis director) / Jacofsky, Marc (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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. 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
Description
There has been an alarming rise in the prevalence of obesity which has been attributed to the paralleled rise in consumption of high-fat foods. It’s commonly accepted that high-fat diets can lead to increased weight gain, however not all fats have the same physiological action. This study primarily focuses on the effect of canola oil, a monounsaturated fat, on energy homeostasis and body composition when it’s given as a supplement to a high-fat diet composed of saturated fatty acid. Rodent models were divided into three dietary groups: 1) low-fat diet (LFD), 2) high-fat diet (HFD) and 3) canola oils supplemented HFD (HF+CAN). After 4 weeks of dietary intervention, samples of epididymal fat, perinephric fat, and liver were analyzed across the three groups to see if the changes in energy homeostasis could be explained by the cellular behavior and composition of these tissues. Interestingly, the supplement of canola oil appeared to reverse the deleterious effects of a saturated fat diet, reverting energy intake, body weight gain and adipose tissue sizes to that (if not lower than that) of the LFD group. The only exception to this effect was the liver: the livers remained larger and fattier than those of the HFD. This occurrence is possibly due to a decrease in free fatty acid uptake in the adipose tissues—resulting in smaller adipose tissue sizes—and increased fatty acid uptake in the liver. The mechanism by which this occurs has yet to be elucidated and will be the primary focus of upcoming studies on the effect of monounsaturated fat on other diets.
ContributorsZuo, Connie Wanda (Author) / Washo-Krupps, Delon (Thesis director) / Deviche, Pierre (Committee member) / Herman, Richard (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2015-05