A known carcinogen, arsenic is elevated in many areas through natural and human causes. Its persistence in the environment and toxicity at low levels impacts both human health and ecosystem integrity. In humans, arsenic has been linked to cancers of the skin, lung, and bladder. Phytoremediation is a cost-effective, eco-friendly technology that uses the natural ability of plants to remediate pollutants, such as arsenic, from contaminated soils. We are investigating whether endophytes, mutualistic microbes colonizing the internal tissues of plants, can improve this process. Endophytes have been shown to increase host-plant tolerance to a wide range of environmental stressors, and we hypothesize that arsenic-tolerant endophytes may improve the tolerance of their host plants to arsenic. Willow (Salix ssp.) are fast growing, high biomass plants, with deep roots, and have demonstrated tolerance to arsenic. In this research, eight willow varieties were screened for arsenic uptake from hydroponic solution in order to establish the best candidates for the phytoremediation of arsenic-contaminated soils. The plants were exposed to arsenic for ten days, then the dried tissues were separated (root/shoot), digested in concentrated nitric acid, and assessed for total arsenic via ICP-OES. Four species, S. scouleriana, S. discolor, S. vimanalis x S. miyabeana, and S. sachalinensis x S. miyabena accumulated the highest level of arsenic in their shoots. Because the purpose of phytoremediation is to remove pollutants from the site, ideal candidates will translocate higher proportions of arsenic into their above ground tissues. These four species will be used in further phytoremediation trials. 

To support the increased production of hemoglobin, the oxygen-binding protein in red blood cells, erythroid progenitor cells need large amounts of its functional constituent biochemical, heme. Despite this increased demand for heme, excessive amounts of free (unutilized) heme form reactive oxygen species which causes damage to cellular components and even apoptosis. To understand the decisions individual cells make to compensate for induced excessive heme, we are analyzing expression data from a pilot single cell RNA-sequencing study. Alongside various exploratory revelations, we identified Tspo (Translocator Protein), a poorly characterized mitochondrial transmembrane protein as one with significant expression changes in cells with heme excess. I have been conducting bioinformatics analyses on the transcriptomic data to classify it's expression pattern and find possible correlations with the expression of other genes. As per my initial findings, it's expression resembles that of a ROS-response gene, which will guide my further molecular/ biochemical experiments into determining causation vs. correlation between Tspo expression and heme excess. I intend to measure TSPO expression alongwith several other genes implicated in pathways related to it under conditions of upregulated heme synthesis. I will also quantify expression patterns through erythroid development in vivo with induced heme overload. I hope to better characterize the role of Tspo, if any, in heme-response pathways with these experimental investigations, together with bioinformatics analysis on large-scale transcriptomic data.

Growth promoting endophytes have been isolated from wild poplar and willow plants in Prof. Sharon Doty’s lab and have been shown to promote growth in several other plant species as broadly diverse as rice, tomato, and Douglas-fir. One of the proposed reasons for this increased growth is that endophytes living within the plant fix nitrogen from the atmosphere and exchange it for a carbon source from the plant. Nitrogen is one of the most important nutrients for plant growth and fertilizers are heavily used worldwide to provide this nitrogen. However, fertilizers are costly to produce and have significant negative impacts on the environment so the goal of our lab is to replace these fertilizers with a consortia of natural endophytes. This study seeks to determine the genes necessary for nitrogen fixation in these endophytes by using random transposon mutagenesis. Endophytes will be selected for their ability to grow on nitrogen-free medium. Then, an E. coli donor strain will transfer a plasmid containing a transposon, a segment of DNA that can transpose into the bacterial genome, with an antibiotic resistance gene to our bacteria through conjugation. The bacteria will then be screened for resistance to that antibiotic to check if the plasmid was taken up by the bacteria, allowing for transposon integration at random areas of the genome. Transposon integration will disrupt the genome of the bacteria, creating mutants. Finally, the conjugated bacteria will be screened for their inability to grow on nitrogen-free medium. Bacteria that lost their ability to grow on this medium will then be sequenced in order to determine the location of the insertion into their genome and therefore the location of genes related to nitrogen fixation. This project will also result in the creation of many mutant endophytes that have lost their ability to fix nitrogen and we will therefore be able to compare their effect on plant growth to wild-type inoculated plants.

TRPV1 is an ion channel of great importance for pain and heat sensing. The EC50 (threshold dose) of the ion channel TRPV1 is highly variable depending on the cell type in which it is expressed. For example, the EC50 of TRPV1 in HEK293T/17 (a cultured mamalian cell line) is ~1 µM whereas the EC50 of TRPV1 in isolated DRG neurons is ~ 100 nM. Numerous explanations have been proposed for this phenomenon, with one possible explanation being the varying lipid contents of the plasma membrane regulating the EC50 of TRPV1. The cholesterol content of the plasma membrane is a candidate for regulating TRPV1, as it is known to vary among types of cells. We measured TRPV1 function with patch-clamp electrophysiology to determine the effect of removing cholesterol from the plasma membrane on channel function. Inside-out excise patches remain stable for tens of minutes, allowing for cholesterol removal and replacement by flowing a Methylated-β-Cyclodextrin, a ring of sugar molecules that bind cholesterol, across the patch. In intact HEK293T/17 cells, we estimated the cholesterol content to be about 30%. Thus, the Methylated-β-Cyclodextrin is expected to produce a very large change in plasma membrane properties as removes cholesterol. Recordings from these patches showed that both the EC50 for activation of TRPV1 by capsaicin and the capsaicin-induced maximum current were unaffected by patch treatment with Methylated-β-Cyclodextrin. These results are significant as they shows that TRPV1 function is unaffected by a large reduction in plasma membrane cholesterol. Because removal of such an abundant component of the plasma membrane likely produces meaningful changes in the bulk properties of the bilayer, these data also indicate that lipid regulation of TRPV1 is caused by lipids which have specific, chemical interactions with the channel, rather than the bulk properties of the plasma membrane.

Transient receptor potential vanilloid receptor 1(TRPV1) is a nonselective cation channel whose activity can be regulated by different kinds of factors including vanilloids such as capsaicin. Nerve growth factor (NGF) has been shown to cause the rapid sensitization of nociceptors neurons expressing TRPV1 under inflammatory hyperalgesia. In other words, TRPV1 is involved the sensation of pain caused by inflammation. It’s been suggested that tropomyosin receptor kinase A (trkA) and its downstream effectors including phosphoinositide 3-kinase (PI3K) play a key role in TRPV1 potentiation and we want to further examine the molecular signaling mechanism. For the purpose of our study, we utilize a system based on a light-sensitive molecule phytochrome B (PhyB) to mimic inflammatory responses in cells. Under 650nm light, PhyB undergoes conformational change and binds to phytochrome interaction factor (PIF). By fusing proteins of interest to PIF, PhyB-PIF interaction controlled by light can be used to manipulate signaling pathways including PI3K pathway. To study the potentiating effect of PI3K on TRPV1, we examine NIH3T3 cells transfected with proteins of interest using calcium imaging. We compare cell responses to capsaicin before and after PI3K translocation activated by 650 nm light. Our experimental data suggest a significant increase of calcium signalling after translocating PI3K to the plasma membrane. In the future, we are hoping to learn more about how the downstream effectors can affect TRPV1 trafficking. We will perform similar procedures as described using dominant-negative Akt and Rac1 cells and investigate the specific mechanisms. Eventually, we hope to utilize what we know about TRPV1 in pain treatment development.

The first member of the vanilloid sensing sub-family of transient receptor potential cation channels (TRPV1) is an ion channel located primarily in the nociceptive (pain-sensing) neurons of the peripheral nervous system. It is most strongly activated by high temperatures, protons, and capsaicin, the molecule responsible for the spice of hot chili peppers. From these examples of TRPV1 activators, it has been deduced that this ion channel plays a key role in the sensory transduction of pain that results from thermal burns, acid burns, and ingestion of noxious chemicals. Thus, the TRPV1 ion channel has potential to be a drug target for innovative pain medications and therapies. Before this is feasible, physiologically relevant details of TRPV1 structure and function must be elucidated. Expanding the understanding of how TRPV1 interacts and changes in various environments is also crucial. My research aims to determine if the lipid environment surrounding TRPV1 has significant impacts on its structural conformation. Electron resonance techniques such as EPR and DEER can be used to detect structural information of proteins; however, a number of steps precede this goal. First, I expressed and purifyied TRPV1 in large quantities. Next, I reconstituted the purified channel into vesicles. Vesicles are spheres of phospholipid bilayer and more accurately resemble the plasma membrane environment in which TRPV1 naturally occurs. Then electron resonance techniques were used to compare the structure of detergent-stabilized TRPV1 and TRPV1 reconstituted into vesicles. The comparison of TRPV1 structure in different lipid environments has a lot of implications in the applicability of structural studies conducted outside of physiologically relevant systems and for the future study of TRPV1.