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- All Subjects: Biochemistry
- Creators: Borges, Chad
- Creators: LaBaer, Joshua
Description
Recombinant protein expression is essential to biotechnology and molecular medicine, but facile methods for obtaining significant quantities of folded and functional protein in mammalian cell culture have been lacking. Here I describe a novel 37-nucleotide in vitro selected sequence that promotes unusually high transgene expression in a vaccinia driven cytoplasmic expression system. Vectors carrying this sequence in a monocistronic reporter plasmid produce >1,000-fold more protein than equivalent vectors with conventional vaccinia promoters. Initial mechanistic studies indicate that high protein expression results from dual activity that impacts both transcription and translation. I suggest that this motif represents a powerful new tool in vaccinia-based protein expression and vaccine development technology.
ContributorsFlores, Julia Anne (Author) / Chaput, John C (Thesis advisor) / Jacobs, Bertram (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2012
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
This study was conducted to observe the effects of vitamin C supplementation upon the expression of sICAM-1 in asthmatic subject. Two groups were created, each with a sample size of 4 subjects. One group was the vitamin C group (VC) and the other was the placebo group (PL). The study was analyzed through observing concentrations of biomolecules present within samples of blood plasma and nasal lavages. These included vitamin C, sICAM-1 expression, and histamine. The following P-values calculated from the data collected from this study. The plasma vitamin C screening was p=0.3, and after 18 days of supplementation, p=0.03. For Nasal ICAM p=0.5 at Day 0, p=0.4 at Day 4, and p=0.9 at Day 18. For the Histamine samples p=0.9 at Day 0 and p=0.9 at Day 18. The following P-values calculated from the data collected from both studies. The plasma vitamin C screening was p=0.8, and after 18 days of supplementation, p=0.03. The change of vitamin C at the end of this study and the combined data both had a P-value that was calculated to be lower than 0.05, which meant that this change was significant because it was due to the intervention and not chance. For Nasal ICAM samples p=0.7 at Day 0, p=0.7 at Day 4, and p=1 at Day 18. For the Histamine p=0.7 at Day 0 and p=0.9 at Day 18. This study carries various implications although the study data was unable to show much significance. This was the second study to test this, and as more research is done, and the sample size grows, one will be able to observe whether this really is the mechanism through which vitamin C plays a role in immunological functions.
ContributorsKapadia, Chirag Vinay (Author) / Johnston, Carol (Thesis director) / LaBaer, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
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
In this thesis, glycan nodes, the basic subunits of complex biological sugars, were studied to determine the reproducibility of gas chromatography-mass spectrometry (GC/MS) based methylation analysis of whole blood plasma by normalization using an internal standard of heavy permethylated glycans. Glycans are complex biological sugars that have a variety of applications in the human body and will display aberrant compositions when produced by cancerous cells. Thus an assay to determine their composition can be used as a diagnostic tool. It was shown that the assay may have potential use, but needs further refinement to become an improvement over current methods by analyzing the results of ratio-determination and replicate experiments.
ContributorsMiyasaki, Tyler Takeo (Author) / Borges, Chad (Thesis director) / Van Horn, Wade (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
Description
In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method.
ContributorsFu, Qiang (Author) / Lindsay, Stuart M (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010
Description
Dielectrophoresis is a separations strategy that has the potential to separate small amounts of different proteins from each other. The forces at play in the channel used for dielectrophoresis are electroosmotic flow (EOF), electrophoresis (EP), and dielectrophoresis (DEP). EOF is the force exerted on liquid from an applied potential (1). EP is the force exerted on charged particles in a uniform electric field (2). DEP is the force exerted on particles (charged and uncharged) in a non-uniform electric field (3). This experiment was focused on the testing of a new microfluidic device to see if it could improve the focusing of proteins in dielectrophoresis. It was predicted that the addition of a salt bridge would improve focusing by preventing the ions created by the electrolysis of water around the electrodes from interacting with the proteins and causing aggregation, among other problems. Control trials using the old device showed that electrolysis was likely occurring and was the causal agent for poor outcomes. After applying the electric potential for some time a pH front traveled through the channel causing aggregation of proteins and the current in the channel decreased rapidly, even while the voltage was held constant. The resistance in the channels of the control trials also slightly decreased over time, until the pH shift occurred, at which time it increased rapidly. Experimental trials with a new device that included salt bridges eliminated this pH front and had a roughly linear increase of current in the channel with the voltage applied. This device can now be used in future research with protein dielectrophoresis, including in the potential differentiation of different proteins. References: 1) Electroosmosis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 2) Electrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006. 3) Dielectrophoresis. Oxford Dictionary of Biochemistry and Molecular Biology. 2. Oxford University Press: Oxford, England. 2006.
ContributorsHayes, Katelyn Donna (Author) / Hayes, Mark (Thesis director) / Borges, Chad (Committee member) / School of Life Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Plasma and serum are the most commonly used liquid biospecimens in biomarker research. These samples may be subjected to several pre-analytical variables (PAVs) during collection, processing and storage. Exposure to thawed conditions (temperatures above -30 °C) is a PAV that is hard to control, and track and could provide misleading information, that fail to accurately reveal the in vivo biological reality, when unaccounted for. Hence, assays that can empirically check the integrity of plasma and serum samples are crucial. As a solution to this issue, an assay titled ΔS-Cys-Albumin was developed and validated. The reference range of ΔS-Cys-Albumin in cardio vascular patients was determined and the change in ΔS-Cys-Albumin values in different samples over time course incubations at room temperature, 4 °C and -20 °C were evaluated. In blind challenges, this assay proved to be successful in identifying improperly stored samples individually and as groups. Then, the correlation between the instability of several clinically important proteins in plasma from healthy and cancer patients at room temperature, 4 °C and -20 °C was assessed. Results showed a linear inverse relationship between the percentage of proteins destabilized and ΔS-Cys-Albumin regardless of the specific time or temperature of exposure, proving ΔS-Cys-Albumin as an effective surrogate marker to track the stability of clinically relevant analytes in plasma. The stability of oxidized LDL in serum at different temperatures was assessed in serum samples and it stayed stable at all temperatures evaluated. The ΔS-Cys-Albumin requires the use of an LC-ESI-MS instrument which limits its availability to most clinical research laboratories. To overcome this hurdle, an absorbance-based assay that can be measured using a plate reader was developed as an alternative to the ΔS-Cys-Albumin assay. Assay development and analytical validation procedures are reported herein. After that, the range of absorbance in plasma and serum from control and cancer patients were determined and the change in absorbance over a time course incubation at room temperature, 4 °C and -20 °C was assessed. The results showed that the absorbance assay would act as a good alternative to the ΔS-Cys-Albumin assay.
ContributorsJehanathan, Nilojan (Author) / Borges, Chad (Thesis advisor) / Guo, Jia (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2022
Description
Transient Receptor Potential Vanilloid-1 (TRPV1) is an integral membrane polymodal cation channel involved in various essential biological functions, including thermosensing, thermoregulation, and nociception. Discrete TRPV1 activation modes such as ligand, heat, and proton have been challenging to disentangle. However, dissecting the polymodal nature of TRPV1 is essential for therapeutic development. The human TRPV1 (hTRPV1) voltage-sensing like domain (VSLD; transmembrane helices S1-S4) contains the canonical vanilloid ligand binding site and significantly contributes to thermosensing. Nuclear magnetic resonance (NMR)-detected studies probe the role of the hTRPV1-VSLD in TRPV1 polymodal function. The hTRPV1-VSLD is identified as an allosteric hub for all three primary TRPV1 activation modes and demonstrates plasticity in chemical ligand modulation. The presented results underscore molecular features in the VSLD that dictate TRPV1 function, highlighting important considerations for future therapeutic design.
ContributorsOwens, Aerial M. (Author) / Van Horn, Wade D. (Thesis advisor) / Levitus, Marcia (Committee member) / LaBaer, Joshua (Committee member) / Arizona State University (Publisher)
Created2023
Description
Redox homeostasis is described as the net physiologic balance between inter-convertible oxidized and reduced equivalents within subcellular compartments that remain in a dynamic equilibrium. This equilibrium is impacted by reactive oxygen species (ROS), which are natural by-products of normal cellular activity. Studies have shown that cancer cells have high ROS levels and altered redox homeostasis due to increased basal metabolic activity, mitochondrial dysfunction, peroxisome activity, as well as the enhanced activity of NADPH oxidase, cyclooxygenases, and lipoxygenases. Glioblastoma (GBM) is the most prevalent primary brain tumor in adults with a median survival of 15 months. GBM is characterized by its extreme resistance to therapeutic interventions as well as an elevated metabolic rate that results in the exacerbated production of ROS. Therefore, many agents with either antioxidant or pro-oxidant mechanisms of action have been rigorously employed in preclinical as well as clinical settings for treating GBM by inducing oxidative stress within the tumor. Among those agents are well-known antioxidant vitamin C and small molecular weight SOD mimic BMX-001, both of which are presently in clinical trials on GBM patients. Despite the wealth of investigations, limited data is available on the response of normal brain vs glioblastoma tissue to these therapeutic interventions. Currently, a sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of a panel of oxidative stress biomarkers: glutathione (GSH), cysteine (Cys), glutathione disulfide (GSSG), and cysteine disulfide in human-derived brain tumor and mouse brain samples; this method will be enriched with additional oxidative stress biomarkers homocysteine (Hcy), methionine (Met), and cystathionine (Cyst). Using this enriched method, we propose to evaluate the thiol homeostasis and the redox state of both normal brain and GBM in mice after exposure with redox-active therapeutics. Our results showed that, compared to normal brain (in intact mice), GBM tissue has significantly lower GSH/GSSG and Cys/CySS ratios indicating much higher oxidative stress levels. Contralateral “normal” brain tissue collected from the mice with intracranial GBM were also under significant oxidative stress compared to normal brains collected from the intact mice. Importantly, normal brain tissue in both studies retained the ability to restore redox homeostasis after treatment with a redox-active therapeutic within 24 hours while glioblastoma tissue does not. Ultimately, elucidating the differential redox response of normal vs tumor tissue will allow for the development of more redox-active agents with therapeutic benefit.
ContributorsShaik, Kamal (Author) / LaBaer, Joshua (Thesis director) / Tovmasyan, Artak (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Historical, Philosophical & Religious Studies, Sch (Contributor) / Dean, W.P. Carey School of Business (Contributor)
Created2022-12