Two-pore domain potassium channels (K2Ps) are widely expressed ion channels which generate leak-type currents regulated by diverse chemical and physical stimuli including temperature, membrane tension, signaling lipids, and pH, to control the resting membrane potential (RMP) of cells. While these K2Ps are critical to the maintenance of the RMP, the structural mechanisms by which they respond to physiological stimuli remain largely unknown. Additionally, studies of K2Ps in vivo have been hampered by the lack of molecular tools which can be used to probe K2P function and structure.

Here, we describe the structural basis for gating of the K2P TWIK1 and describe a series of molecular tools generated against the K2P TREK1. Utilizing cryo-electron microscopy (cryo-EM), we determine high resolution structure of TWIK1 in lipid nanodiscs at a high and low pH, and in high extracellular K+, and describe the mechanistic basis for pH-gating in this channel. We then describe the generation of a nanobody which specifically binds another K2P, TREK1, with nanomolar affinity. Finally, we describe the computational prediction of a series of novel small-molecule TREK1 agonists. The data and molecular tools presented here further our understanding of the structural and functional properties of the K2P family of ion channels.

Tracking the movement of small organisms is of tremendous importance to understanding the ecology of populations, communities, and ecosystems. However, it remains one of the most difficult challenges facing the field of movement ecology. This dissertation focuses on the movement of an agricultural pest, the navel orangeworm (Amyelois transitella, NOW). I first examined intercrop movement of NOW between two agricultural commodities, almonds and walnuts. By using protein markers and an enzyme-linked immunosorbant assay technique to detect markers on recaptured moths, I demonstrated significant male NOW movement (up to 300 meters) and no significant directional preferences of movement based on crop, season, or wind velocity. However, protein marker contamination of control moths within traps was significant, limiting our ability to detect movement patterns. In addition, the scale of this study may have been too small to capture larger scale directional patterns of movement.

In order to overcome some of the observed challenges of protein marking techniques for small organisms, I developed and tested an intrinsic marking technique for tracking NOW using dietary fatty acid profiles as a biomarker. This was accomplished by analyzing fatty acids from NOW moths raised on two different host plants with significantly different fatty acid profiles. Using this data a linear discriminant analysis model was developed and validated to distinguish NOW based on their larval host plant. Results showed that NOW fatty acid profiles are strikingly similar to those of their host plant. Therefore fatty acids can act as a valuable intrinsic marking technique for tracking small organisms, avoiding many of the drawbacks of external markers, and providing a useful tool for the study of movement ecology.

Fatty acid tracking is effective for small organisms, but does not determine movement paths, direction, or distance of movement in a localized setting. In order to draw meaningful conclusions from localized movement data using intrinsic marking techniques, I developed a Gaussian-based dispersal model. This model was applied to field-caught NOW moths from three sites in the central valley of California. Average movement distance was estimated to be about 50 m per generation at two sites and about 600 m per generation at the third site. The study demonstrates that probability-based dispersal models combined with intrinsic marking techniques provides a useful tool for both tracking and understanding the localized movement capabilities of small organisms.

The goal of precision immunotherapy is to direct a patient’s T cell response against the immunogenic mutations, or neoantigens (NeoAg), expressed by tumor cells. Most immunotherapy approaches to-date have focused on MHC class I-restricted peptide epitopes by which cytotoxic CD8+ T lymphocytes (CTL) can directly recognize tumor cells. This strategy largely overlooks the critical role of major histocompatibility class II (MHC-II)-restricted CD4+ T cells as both positive regulators of CTL and other effector cell types, and as direct effectors of antitumor immunity. To determine the capacity of tumor-specific CD4+ T cells to directly engage and kill tumor cells, we characterized CD4+ T cells recognizing the oncogenes HPV-16 E6 and KRASG12V from two distinct human patients with cancer. In both cases, CD4+ T cells were unable to recognize MHC- II+ antigen-expressing tumor cells, suggesting only CD4+ T cells recognizing a limited range of antigens may directly recognize tumor cells. To determine the antitumor mechanisms of NeoAg- specific CD4+ T cells independent of direct tumor recognition, we sorted single CD4+ T cells specific for a mutated clathrin heavy chain epitope (CltcH129Q) expressed by the MHC-II-negative murine squamous cell carcinoma VII (SCC VII) tumor. T cell receptor (TCR) sequencing analysis revealed the presence of four distinct TCR clonotypes and expression of these TCRs in primary cells was sufficient to confer recognition of CltcH129Q. Both low and high avidity CltcH129Q-specific CD4+ T cells were capable of proliferating to similar degrees in response to tumor antigen in vivo and enhancing primary tumor immunity in a CD8+ T cell- and CD40L-dependent manner. ACT with CltcH129Q-specific CD4+ T cells also synergized with cyclophosphamide treatment to reduce tumor burden in mice with established tumors. The findings herein have important implications for the use of NeoAg-specific CD4+ T cells as next generation cellular therapies for solid tumors.