Matching Items (161)
Description
The pathogenesis of type 1 diabetes (T1D) is still not fully understood in the scientific community. Evidence has shown that viral infections are one of the important environmental factors associated with the disease development. Seven of the top T1D related viruses were selected to study the prevalence of viral humoral response in T1D patients using our innovative protein array platform called Nucleic Acid Programmable Protein Array (NAPPA). In this study, each viral gene was individually captured using various PCR based techniques, cloned into a protein expression vector, and assembled as the first version of T1D viral protein array. Humoral responses of IgG, IgA, and IgM were examined. Although each class of immunoglobulin generated a wide-range of reactivity, responses to various viral proteins from different proteins were observed. In summary, we captured most of the T1D related viral genes, established viral protein expression on the protein array, and displayed the serum response on the viral protein array. The successful progress will help to fulfill the long term goal of testing the viral infection hypothesis in T1D development.
ContributorsDavis, Amy Darlene (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Desi, Paul (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
Protein AMPylation is a recently discovered and relatively unstudied post-translational modification (PTM). AMPylation has previously been shown to play an important role in metabolic regulation and host pathogenesis in bacteria, but the recent identification of potential AMPylators across many species in every domain of life has supported the possibility that AMPylation could be a more fundamental and physiologically significant regulatory PTM. For the first time, we characterized the auto-AMPylation capability of the human protein SOS1 through in vitro AMPylation experiments using full-length protein and whole-domain truncation mutants. We found that SOS1 can become AMPylated at a tyrosine residue possibly within the Cdc25 domain of the protein, the Dbl homology domain is vital for efficient auto-AMPylation activity, and the C-terminal proline-rich domain exhibits a complex regulatory function. The proline-rich domain alone also appears to be capable of catalyzing a separate, unidentified covalent self-modification using a fluorescent ATP analogue. Finally, SOS1 was shown to be capable of catalyzing the AMPylation of two endogenous human protein substrates: a ubiquitous, unidentified protein of ~49kDa and another breast-cancer specific, unidentified protein of ~28kDa.
ContributorsOber-Reynolds, Benjamin John (Author) / LaBaer, Joshua (Thesis director) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05
Description
One of the major challenges that were yet to be solved for solid phase peptide synthesis was the lack of an efficient peptide sequencing technique that was less hazardous, easier to perform , and was more cost-effective. Sequencing peptides were held important in the field of Chemistry and Biochemistry because it aided in drug discovery, finding ligands that bind to a specific target protein and finding alternative agents in transporting molecules to its desired location. Therefore, the overall purpose of this experiment was to develop a method of solid phase sequencing technique that was more environmental friendly, sequences at a faster rate, and was more cost-effective.
ContributorsCordovez, Lalaine Anne Ordiz (Author) / Woodbury, Neal (Thesis director) / Zhao, Zhan-Gong (Committee member) / Legutki, Joseph Barten (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2014-05
Description
AMPylation is a post-translation modification that has an important role in the survival of many bacterial pathogens by affecting the host cell's molecular signaling. In the course of studying this intercellular manipulation, there has only been modest progression in the identification of the enzymes with AMPylation capabilities (AMPylators) and their respective targets. The reason for these minimal developments is the inability to analyze a large subset of these proteins. Therefore, to increase the efficiency of the identification and characterization of the proteins, Yu et al developed a high-throughput non-radioactive discovery platform using Human Nucleic Acid Programmable Protein Arrays (NAPPA) and a validation platform using bead-based assays. The large-scale unbiased screening of potential substrates for two bacterial AMPylators containing Fic domain, VopS and IbpAFic2, had been performed and dozens of novel substrates were identified and confirmed. With the efficiency of this method, the platform was extended to the identification of novel substrates for a Legionella virulence factor, SidM, containing a different adenylyl transferase domain. The screening was performed using NAPPA arrays comprising of 10,000 human proteins, the active AMPylator SidM, and its inactive D110/112A mutant as a negative control. Many potential substrates of SidM were found, including Rab GTPases and non-GTPase proteins. Several of which have been confirmed with the bead-based AMPylation assays.
ContributorsGraves, Morgan C. (Author) / LaBaer, Joshua (Thesis director) / Qiu, Ji (Committee member) / Yu, Xiaobo (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2013-05
Description
The purpose of this project was to identify proteins associated with the migration and invasion of non-transformed MCF10A mammary epithelial cells with ectopically expressed missense mutations in p53. Because of the prevalence of TP53 missense mutations in basal-like and triple-negative breast cancer tumors, understanding the effect of TP53 mutations on the phenotypic expression of human mammary epithelial cells may offer new therapeutic targets for those currently lacking in treatment options. As such, MCF10A mammary epithelial cells ectopically overexpressing structural mutations (G245S, H179R, R175H, Y163C, Y220C, and Y234C) and DNA-binding mutations (R248Q, R248W, R273C, and R273H) in the DNA-binding domain were selected for use in this project. Overexpression of p53 in the mutant cell lines was confirmed by western blot and q-PCR analysis targeting the V5 epitope tag present in the pLenti4 vector used to transduce TP53 into the mutant cell lines. Characterization of the invasion and migration phenotypes resulting from the overexpression of p53 in the mutant cell lines was achieved using transwell invasion and migration assays with Boyden chambers. Statistical analysis showed that three cell lines—DNA-contact mutants R248W and R273C and structural mutant Y220C—were consistently more migratory and invasive and demonstrated a relationship between the migration and invasion properties of the mutant cell lines. Two families of proteins were then explored: those involved in the Epithelial-Mesenchymal Transition (EMT) and matrix metalloproteinases (MMPs). Results of q-PCR and immunofluorescence analysis of epithelial marker E-cadherin and mesenchymal proteins Slug and Vimentin did not show a clear relationship between mRNA and protein expression levels with the migration and invasiveness phenotypes observed in the transwell studies. Results of western blotting, q-PCR, and zymography of MMP-2 and MMP-9 also did not show any consistent results indicating a definite relationship between MMPs and the overall invasiveness of the cells. Finally, two drugs were tested as possible treatments inhibiting invasiveness: ebselen and SBI-183. These drugs were tested on only the most invasive of the MCF10A p53 mutant cell lines (R248W, R273C, and Y220C). Results of invasion assay following 30 μM treatment with ebselen and SBI-183 showed that ebselen does not inhibit invasiveness; SBI-183, however, did inhibit invasiveness in all three cell lines tested. As such, SBI-183 will be an important compound to study in the future as a treatment that could potentially serve to benefit triple-negative or basal-like breast cancer patients who currently lack therapeutic treatment options.
ContributorsZhang, Kathie Q (Author) / LaBaer, Joshua (Thesis director) / Anderson, Karen (Committee member) / Gonzalez, Laura (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM).
Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41.
ContributorsGong, Zhen (Author) / Martin Garcia, Jose Manuel (Author) / Daskalova, Sasha (Author) / Craciunescu, Felicia (Author) / Song, Lusheng (Author) / Dorner, Katerina (Author) / Hansen, Debra (Author) / Yang, Jay-How (Author) / LaBaer, Joshua (Author) / Hogue, Brenda (Author) / Mor, Tsafrir (Author) / Fromme, Petra (Author) / Department of Chemistry and Biochemistry (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Innovations in Medicine (Contributor) / Personalized Diagnostics (Contributor) / College of Liberal Arts and Sciences (Contributor) / School of Life Sciences (Contributor)
Created2015-08-21
Description
Dielectrophoresis has been shown in the recent past to successfully separate bioparticles of very subtle differences at high resolutions using biophysical forces. In this study, we test the biophysical differences of methicillin resistant and susceptible Staph. aureus that are known to have very similar genomes by using a modified gradient insulator-based dielectrophoresis device (g-iDEP). MRSA is commonly seen in hospitals and is the leading killer of infectious bacteria, claiming the lives of around 10,000 people annually. G-iDEP improves many capabilities within the DEP field including sample size, cost, ease of use and analysis time. This is a promising foundation to creating a more clinically optimized diagnostic tool for both separation and detection of bacteria in the healthcare field. The capture on-set potential for fluorescently tagged MRSA (801 ± 34V) is higher than fluorescently tagged MSSA (610 ± 32V), resulting in a higher electrokinetic to dielectrophoretic mobility ratio for MRSA. Since the strains have proven to be genomically similar through sequencing, it is reasonable to attribute this significant biophysical difference to the added PBP2a enzyme in MRSA. These results are consistent with other bacterial studied within in this device and have proven to be reproducible.
ContributorsSmithers, Jared (Author) / Hayes, Mark (Thesis director) / Woodbury, Neal (Committee member) / School of Criminology and Criminal Justice (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Spaceflight and spaceflight analogue culture enhance the virulence and pathogenesis-related stress resistance of the foodborne pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). This is an alarming finding as it suggests that astronauts may have an increased risk of infection during spaceflight. This risk is further exacerbated as multiple studies indicate that spaceflight negatively impacts aspects of the immune system. In order to ensure astronaut safety during long term missions, it is important to study the phenotypic effects of the microgravity environment on a range of medically important microbial pathogens that might be encountered by the crew. This ground-based study uses the NASA-engineered Rotating Wall Vessel (RWV) bioreactor as a spaceflight analogue culture system to grow bacteria under low fluid shear forces relative to those encountered in microgravity, and interestingly, in the intestinal tract during infection. The culture environment in the RWV is commonly referred to as low shear modeled microgravity (LSMMG). In this study, we characterized the stationary phase stress response of the enteric pathogen, Salmonella enterica serovar Enteritidis (S. Enteritidis), to LSMMG culture. We showed that LSMMG enhanced the resistance of stationary phase cultures of S. Enteritidis to acid and thermal stressors, which differed from the LSSMG stationary phase response of the closely related pathovar, S. Typhimurium. Interestingly, LSMMG increased the ability of both S. Enteritidis and S. Typhimurium to adhere to, invade into, and survive within an in vitro 3-D intestinal co-culture model containing immune cells. Our results indicate that LSMMG regulates pathogenesis-related characteristics of S. Enteritidis in ways that may present an increased health risk to astronauts during spaceflight missions.
ContributorsKoroli, Sara (Author) / Nickerson, Cheryl (Thesis director) / Barrila, Jennifer (Committee member) / Ott, C. Mark (Committee member) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Diabesity is a global epidemic affecting millions worldwide. Diabesity is the term given to the link between obesity and Type II diabetes. It is estimated that ~90% of patients diagnosed with Type II diabetes are overweight or have struggled with excess body fat in the past. Type II diabetes is characterized by insulin resistance which is an impaired response of the body to insulin that leads to high blood glucose levels. Adipose tissue, previously thought of as an inert tissue, is now recognized as a major endocrine organ with an important role in the body's immune response and the development of chronic inflammation. It is speculated that adipose tissue inflammation is a major contributor to insulin resistance particular to Type II diabetes. This literature review explores the popular therapeutic targets and marketed drugs for the treatment of Type II diabetes and their role in decreasing adipose tissue inflammation. rAGE is currently in pre-clinical studies as a possible target to combat adipose tissue inflammation due to its relation to insulin resistance. Metformin and Pioglitazone are two drugs already being marketed that use unique chemical pathways to increase the production of insulin and/or decrease blood glucose levels. Sulfonylureas is one of the first FDA approved drugs used in the treatment of Type II diabetes, however, it has been discredited due to its life-threatening side effects. Bariatric surgery is a form of invasive surgery to rid the body of excess fat and has shown to normalize blood glucose levels. These treatments are all secondary to lifestyle changes, such as diet and exercise which can help halt the progression of Type II diabetes patients.
ContributorsRobles, Alondra Maria (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Allen, James (Committee member) / Hendrickson, Kirstin (Committee member) / Sanford School of Social and Family Dynamics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The purpose of this thesis creative project was to create an educational video to present research findings on the increasingly important issue of human biospecimen preanalytic variables. When a human biospecimen, such as blood, urine, or tissue, is removed from the body, it is subjected to a plethora of variables that are not recorded or regulated in a vast majority of cases. Frequently, these samples arrive at the research or pathology lab with an unknown history, then undergo analysis for translational research purposes, or to guide clinical management decisions. Thus, compromised specimen quality caused by preanalytic variables has substantial, and potentially devastating, downstream effects. To identify the preanalytic variables with the greatest impact on blood and tissue specimen quality, 45 articles were gathered using PubMed and Google Scholar databases and cited. Based on the articles, the top five variables with the most detrimental effects were identified for both blood and tissue samples. Multiple sets of parameters ensuring specimen fitness were compared for each of the five variables for each specimen type. Then, specific parameters guaranteeing the fitness of the greatest number of analytes were verified. To present the research findings in greater detail, a paper was written that focused on identifying the top variables and key parameters to ensure analyte fitness. To present the overall issue in an easy-to-digest format, a storyboard and script were created as a guideline for a final video project. Ultimately, two alternate versions of the video were created to pertain to the audience of choice (one version for patients, one version for professionals). It is the hope that these videos will be used as educational tools to continue efforts to standardize and enforce human biospecimen preanalytic variable parameters. This is a necessary step to improve the accuracy of our biomedical research data and the healthcare of patients worldwide.
ContributorsAzcarate, Heather (Author) / Compton, Carolyn (Thesis director) / LaBaer, Joshua (Committee member) / Borges, Chad (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor)
Created2018-12