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Description
Cancer claims hundreds of thousands of lives every year in US alone. Finding ways for early detection of cancer onset is crucial for better management and treatment of cancer. Thus, biomarkers especially protein biomarkers, being the functional units which reflect dynamic physiological changes, need to be discovered. Though important, there are only a few approved protein cancer biomarkers till date. To accelerate this process, fast, comprehensive and affordable assays are required which can be applied to large population studies. For this, these assays should be able to comprehensively characterize and explore the molecular diversity of nominally "single" proteins across populations. This information is usually unavailable with commonly used immunoassays such as ELISA (enzyme linked immunosorbent assay) which either ignore protein microheterogeneity, or are confounded by it. To this end, mass spectrometric immuno assays (MSIA) for three different human plasma proteins have been developed. These proteins viz. IGF-1, hemopexin and tetranectin have been found in reported literature to show correlations with many diseases along with several carcinomas. Developed assays were used to extract entire proteins from plasma samples and subsequently analyzed on mass spectrometric platforms. Matrix assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) mass spectrometric techniques where used due to their availability and suitability for the analysis. This resulted in visibility of different structural forms of these proteins showing their structural micro-heterogeneity which is invisible to commonly used immunoassays. These assays are fast, comprehensive and can be applied in large sample studies to analyze proteins for biomarker discovery.
ContributorsRai, Samita (Author) / Nelson, Randall (Thesis advisor) / Hayes, Mark (Thesis advisor) / Borges, Chad (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2012
Description
It has been well established that mitochondria play a critical role in the pathology of Friedreich's Ataxia. This disease is believed to be caused by a deficiency of frataxin, which research suggests is responsible for iron sulfur cluster assembly. This incomplete assembly of iron sulfur clusters is believed to be linked with dysfunctional complexes in the mitochondrial respiratory chain, increased oxidative stress, and potential cell death. Increased understanding of the pathophysiology of this disease has enabled the development of various therapeutic strategies aimed at restoring mitochondrial respiration. This thesis contains an analysis of the biological activity of several classes of antioxidants against oxidative stress induced by diethyl maleate in Friedreich's Ataxia lymphocytes and CEM leukemia cells. Analogues of vitamin E α-tocopherol have been shown to protect cells under oxidative stress. However, these same analogues show various levels of inhibition towards the electron transport chain complex I. Bicyclic pyridinols containing a ten carbon substituent provided favorable cytoprotection. N-hydroxy-4-pyridone compounds were observed to provide little protection. Similarly, analogues of CoQ10 in the form of pyridinol and pyrimidinol compounds also preserved cell viability at low concentrations.
ContributorsJaruvangsanti, Jennifer (Author) / Hecht, Sidney (Thesis advisor) / Woodbury, Neal (Committee member) / Skibo, Edward (Committee member) / Arizona State University (Publisher)
Created2012
Description
The field of biomedical research relies on the knowledge of binding interactions between various proteins of interest to create novel molecular targets for therapeutic purposes. While many of these interactions remain a mystery, knowledge of these properties and interactions could have significant medical applications in terms of understanding cell signaling and immunological defenses. Furthermore, there is evidence that machine learning and peptide microarrays can be used to make reliable predictions of where proteins could interact with each other without the definitive knowledge of the interactions. In this case, a neural network was used to predict the unknown binding interactions of TNFR2 onto LT-ɑ and TRAF2, and PD-L1 onto CD80, based off of the binding data from a sampling of protein-peptide interactions on a microarray. The accuracy and reliability of these predictions would rely on future research to confirm the interactions of these proteins, but the knowledge from these methods and predictions could have a future impact with regards to rational and structure-based drug design.
ContributorsPoweleit, Andrew Michael (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Chiu, Po-Lin (Committee member) / School of Molecular Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
Lung cancer is the leading cause of cancer-related deaths in the US. Low-dose computed tomography (LDCT) scans are speculated to reduce lung cancer mortality. However LDCT scans impose multiple risks including false-negative results, false- positive results, overdiagnosis, and cancer due to repeated exposure to radiation. Immunosignaturing is a new method proposed to screen and detect lung cancer, eliminating the risks associated with LDCT scans. Known and blinded primary blood sera from participants with lung cancer and no cancer were run on peptide microarrays and analyzed. Immunosignatures for each known sample collectively indicated 120 peptides unique to lung cancer and non-cancer participants. These 120 peptides were used to determine the status of the blinded samples. Verification of the results from Vanderbilt is pending.
ContributorsNguyen, Geneva Trieu (Author) / Woodbury, Neal (Thesis director) / Zhao, Zhan-Gong (Committee member) / Stafford, Phillip (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Department of Psychology (Contributor)
Created2015-05
Description
Bacteria play a vital role in the world ecosystem, more importantly human health and disease. The capability to differentiate and identify these microorganisms serves as an important research objective. In past years, separations-based approaches have served as a way to observe and identify bacteria based on their characteristics. Gradient insulator dielectrophoresis (g-iDEP) provides benefits in identifying serotypes of a single species with precise separation. Separation of Staphylococcus epidermidis in a single g-iDEP microchannel is conducted exploiting their electrophoretic and electrokinetic properties. The cells were captured and concentrated at gates with interacting forces within the microchannel to clearly distinguish between the two strains. These results provide support for g-iDEP serving as a separating method and, furthermore, future clinical applications.
ContributorsDavis, Paige Elizabeth (Author) / Hayes, Mark (Thesis director) / Borges, Chad (Committee member) / Jones, Paul (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / T. Denny Sanford School of Social and Family Dynamics (Contributor)
Created2015-05
Description
With a quantum efficiency of nearly 100%, the electron transfer process that occurs within the reaction center protein of the photosynthetic bacteria Rhodobacter (Rh.) sphaeroides is a paragon for understanding the complexities, intricacies, and overall systemization of energy conversion and storage in natural systems. To better understand the way in which photons of light are captured, converted into chemically useful forms, and stored for biological use, an investigation into the reaction center protein, specifically into its cascade of cofactors, was undertaken. The purpose of this experimentation was to advance our knowledge and understanding of how differing protein environments and variant cofactors affect the spectroscopic aspects of and electron transfer kinetics within the reaction of Rh. sphaeroides. The native quinone, ubiquinone, was extracted from its pocket within the reaction center protein and replaced by non-native quinones having different reduction/oxidation potentials. It was determined that, of the two non-native quinones tested—1,2-naphthaquinone and 9,10- anthraquinone—the substitution of the anthraquinone (lower redox potential) resulted in an increased rate of recombination from the P+QA- charge-separated state, while the substitution of the napthaquinone (higher redox potential) resulted in a decreased rate of recombination.
ContributorsSussman, Hallie Rebecca (Author) / Woodbury, Neal (Thesis director) / Redding, Kevin (Committee member) / Lin, Su (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
Background: High risk types of human papillomavirus (HPV) are known to cause cancer, including cervical (99%) and oropharyngeal cancer (70%). HPV type 16 is the most common subtype. Three antigens that are critical for integration or tumor progression are E2, E6 and E7. In this study, we developed a systematic approach to identify naturally-processed HPV16-derived HLA class I epitopes for immunotherapy development. Methods: K562 cells, which lack HLA expression, were transduced with each HPV16 antigen using lentivirus and supertransfected with HLA-A2 by nucleofection. Stable cell lines expressing each antigen were selected for and maintained throughout the investigation. In order to establish a Gateway-compatible vector for robust transient gene expression, a Gateway recombination expression cloning cassette was inserted into the commercial Lonza pMAX GFP backbone, which has been experimentally shown to display high transfection expression efficiency. GFP was cloned into the vector and plain K562 cells were transfected with the plasmid by nucleofection. Results: Expression of K562-A2 was tested at various time points by flow cytometry and A2 expression was confirmed. Protein expression was shown for the transduced K562 E7 by Western blot analysis. High transfection efficiency of the pMAX_GFP_Dest vector (up to 97% GFP+ cells) was obtained 48 hours post transfection, comparable to the commercial GFP-plasmid. Conclusion: We have established a rapid system for target viral antigen co-expression with single HLA molecules for analysis of antigen presentation. Using HPV as a model system, our goal is to identify specific antigenic peptide sequences to develop immunotherapeutic treatments for HPV-associated cancers.
ContributorsVarda, Bianca Marie (Author) / Anderson, Karen (Thesis director) / Borges, Chad (Committee member) / Krishna, Sri (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Advances in peptide microarray technology have allowed for the creation of fast-paced and modular experiments within affinity ligand discovery. Previously, low density peptide arrays of 10,000 peptides were used to identify low affinity peptide ligands for a target protein; an approach that can be subsequently improved upon with a number of techniques. VDAP[a] offers more information about the relative affinity of protein-peptide interactions via signal intensity in contrast to high throughput screening (HTS) and display technologies which offer binary data. Now, high density peptide arrays with 130,000 to 330,000 peptides are available that allow screening across peptide libraries of greater diversity. With this increase in scale and diversity, faster analytical tools are needed to adequately characterize array data. Using the statistical power available in the R programming language, we have created a flexible analysis package that efficiently processes high density peptide array data from a variety of layouts, rank existing peptide hits, and utilize signal intensity data to generate new hits. This analysis provides a user-friendly method to efficiently analyze high density peptide array data, generate peptide leads for targeted therapeutic development, and further improve peptide array technologies.
ContributorsMoore, Cody Allen (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Barrett, The Honors College (Contributor)
Created2015-12
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 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
Description
The influenza virus is the main cause of thousands of deaths each year in the United States, and far more hospitalizations. Immunization has helped in protecting people from this virus and there are a number of therapeutics which have proven effective in aiding people infected with the virus. However, these therapeutics are subject to various limitations including increased resistance, limited supply, and significant side effects. A new therapeutic is needed which addresses these problems and protects people from the influenza virus. Synbodies, synthetic antibodies, may provide a means to achieve this goal. Our group has produced a synbody, the 5-5 synbody, which has been shown to bind to and inhibit the influenza virus. The direct pull down and western blot techniques were utilized to investigate how the synbody bound to the influenza virus. Our research showed that the 5-5 synbody bound to the influenza nucleoprotein (NP) with a KD of 102.9 ± 74.48 nM. It also showed that the synbody bound strongly to influenza viral extract from two different strains of the virus, the Puerto Rico (H1N1) and Sydney (H3N2) strains. This research demonstrated that the 5-5 synbody binds with high affinity to NP, which is important because influenza NP is highly conserved between various strains of the virus and plays an important role in the replication of the viral genome. It also demonstrated that this binding is conserved between various strains of the virus, indicating that the 5-5 synbody potentially could bind many different influenza strains. This synbody may have potential as a therapeutic in the future if it is able to demonstrate similar binding in vivo.
ContributorsKombe, Albert E. (Author) / Diehnelt, Chris (Thesis director) / Woodbury, Neal (Committee member) / Legutki, Bart (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of International Letters and Cultures (Contributor)
Created2014-05