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.
Plasticizers are plastic additives used to enhance the physical properties of plastic and are ubiquitous in the environment. A class of plasticizer compounds called phthalate esters that are not fully eliminated in wastewater treatment facilities are relevant to the ecological health of downstream ecosystems and urban areas due to their ecotoxicity, tendency for soil accumulation, and the emerging concern about their effects on public health. However, plasticizer concentrations in a constructed wetland environment have rarely been studied in the United States, prompting the need for a method of plasticizer quantification in the Tres Rios Constructed Wetlands which are sustained by the effluent of the 91st Avenue Wastewater Treatment Plant in Phoenix, Arizona. The concentrations of four common plasticizer compounds (dimethyl: DMP, diethyl: DEP, di-n-butyl: DnBP, and bis(2-ethylhexyl): DEHP phthalate) at five sites across the wetland surface water were quantified using solid-phase extraction followed by gas chromatography coupled with mass spectrometry (GC/MS). The sampling period included four sample sets taken from March 2022 to September 2022, which gave temporal data in addition to spatial concentration data. Quantification and quality control were performed using internal standard calibration, replicate samples, and laboratory blanks. Higher molecular weight phthalates accumulated in the wetland surface water at significantly higher average concentrations than those of lower molecular weight at a 95% confidence level, ranging from 8 ng/L to 7349 ng/L and 4 ng/L to 27876 ng/L for DnBP and DEHP, respectively. Concentrations for dimethyl phthalate and diethyl phthalate were typically less than 50 ng/L and were often below the method detection limit. Average concentrations of DnBP and DEHP were significantly higher during periods of high temperatures and arid conditions. The spatial distribution of phthalates was analyzed. Most importantly, a method for successful ultra-trace quantification of plasticizers at Tres Rios was established. These results confirm the presence of plasticizers at Tres Rios and a significant seasonal increase in their surface water concentrations. The developed analytical procedure provides a solid foundation for the Wetlands Environmental Ecology Lab at ASU to further investigate plasticizers and contaminants of emerging concern and determine their ultimate fate through volatilization, sorption, photodegradation, hydrolysis, microbial biodegradation, and phytoremediation studies.
There are limited methods and techniques to quantitatively assess protein content in single cells or small cell populations of tissues. The standard protein insulin was used to understand how potential changes in the preparation or co-crystallization process could improve sensitivity and limit of detection through matrix assisted laser desorption ionization (MALDI) mass spectrometry analysis in Bruker’s Microflex LRF using polydimethylsiloxane (PDMS) reservoirs. In addition, initial imaging tests were performed on Bruker’s RapifleX MALDI Tissuetyper to determine the instrument’s imaging capabilities on proteins of interest through the use of a single layer “Christmas tree” microfluidic device, with the aim of applying a similar approach to future tissue samples. Data on 2µM insulin determined that a 95% laser power in the Microflex corresponded to 12-15% laser power in the RapifleX. Based on the experiments with insulin, the process of mixing insulin and saturated ɑ-Cyano-4-hydroxycinnamic acid (HCCA) matrix solvent in a 1:1 ratio using 10mM sodium phosphate buffer under area analysis is most optimized with a limit of detection value of 110 nM. With this information, the future aim is to apply this method to a double layer Christmas tree device in order to hopefully quantitatively analyze and image protein content in single or small cell populations.
The 5-year survival rate for late-stage metastatic melanoma is only ~30%. A major reason for this low survival rate is that one of the most commonly mutated genes in melanoma, NRAS, has no FDA-approved targeted therapies. Because the RAS protein does not have any targeted therapies, patients with RAS mutant tumors have an ongoing need for treatments that indirectly target RAS. This thesis project aims to identify expression and phosphorylation levels of proteins downstream of RAS in melanoma cell lines with the most common driver mutations. By analyzing the protein-level differences between these genetic mutants, we hope to identify additional indirect RAS protein targets for the treatment of NRAS mutant melanoma. RAS has several downstream effector proteins involved in oncogenic signaling pathways including FAK, Paxillin, AKT, and ERK. 5 melanoma cell lines (2 BRAF mutant, 2 NRAS mutant, and 1 designated wildtype) were analyzed using western bloting for FAK, Paxillin, AKT, and ERK phosphorylation and total expression levels. The results of western blot analysis showed that NRAS mutant cell lines had increased expression of phosphorylated Paxillin. Increased Paxillin phosphorylation corresponds to increased Paxillin binding at the FAT domain of FAK. Therefore, cell lines with increased FAK FAT – Paxillin interaction would be more sensitive to FAK FAT domain inhibition. The data presented provide an an explanation for the reduction in cell viability in NRAS mutant cell lines infected with Ad-FRNK. This information also has significant clinical relevance as researchers work to develop synthetic FAK FAT domain inhibitors, such as cyclic peptides. Additionally, cell lines with high levels of phosphorylated AKT showed a significant reduction in the amount of phosphorylated ERK. The identification of this inverse relationship may help to explain why BRAF and NRAS mutations are mutually exclusive. To conclude, NRAS mutant cell lines have increased expression of phosphorylated Paxillin and AKT which may explain why NRAS mutant cell lines are more sensitive to FAK FAT domain inhibition.