Photochemical and Logic-Based Regulation over the Cellular Microenvironment

Thesis (Ph.D.)--University of Washington, 2020The biological microenvironment is a complex, constantly changing space featuring varying amounts of many cell-secreted signaling molecules that serve as the communicatory elements of biology. By better understanding how well-defined combinations of individual biochemical signals including proteins presented in this space operate in (a)synchrony to direct cellular and integrated tissue function, we can begin to unravel irregularities within diseased systems and utilize this information to engineer therapies that promote healthy recovery. As photochemical reactions can be uniquely modulated in time and space, this dissertation develops and subsequently exploits novel light-based strategies to spatiotemporally control biochemical microenvironments and cellular signaling. First, we establish the cytocompatibility of near-ultraviolet light treatments common to photochemical manipulation through mammalian cell survival assays and global proteomic analyses. Expanding on existing concepts of photoresponsive drug delivery, we then introduce a generalizable strategy to govern biochemical signal presentation within biomaterials in response to precise combinations of environmental factors following user-programmable Boolean logic. Finally, we introduce the first genetically encoded protein-protein photoligation chemistry, and demonstrate its utility in irreversibly directing protein binding, function, and interactions both intra- and extracellularly. Employing this versatile photoreaction, we demonstrate 3D patterned immobilization of full-length proteins within biomaterials and user-guided intracellular protein activity. Such newly afforded 4D control over biological systems is expected to further basic biological understanding and advance medicine through enhanced tissue engineering and therapeutic delivery approaches

Salt-Gradient Solar Pond and Membrane Distillation System for Water Desalination Powered by Renewable Energy

A salt gradient solar pond (SGSP) coupled with a direct contact membrane distillation (DCMD) system was investigated to desalinate water for reclamation of Walker Lake, a terminal saline lake in Northern Nevada. Two transparent floating elements and a continuous plastic cover were tested to determine their ability to suppress evaporation rates and increase overall solar pond heat content. An aquatic chemistry analysis was performed on Walker Lake water to determine possible scalants when concentrating lake water to fill the SGSP and during DCMD concentration. Membrane cleaning was evaluated to remove membrane scalant and to recover flux.It was found that petri dishes were the best evaporation suppression element due to their transmissivity, low refraction of radiation, and ease of installation. Suppression of evaporative losses from the solar pond surface also resulted in increased temperatures throughout the solar pond and increased overall solar pond heat content. The investigation of transparent covers/elements is unique from previous studies in ponds where increasing temperature and heat content are not desired.Hydromagnesite and calcite were predicted to form during concentration of Walker Lake water to fill the SGSP. Hydromagnesite and halite were found to be the main scalants on the membrane surface. Halite most likely formed due to concentration polarization at the membrane surface; small quantities of calcite were also likely present although not detected. Cleaning cycles were conducted and were able to recover approximately 94% of the original flux using citric acid or EDTA cleaning solutions. The citric acid cleaning solution performed slightly better than the EDTA cleaning solution in terms of flux recovery and removal of scalant (as observed by SEM). SEM, EDS, and XRD analyses were performed in order to determine the magnitude of scale deposit and the type of scalant present.The proposed coupled SGSP/DCMD system was found to be feasible for terminal lake reclamation when utilizing floating elements to suppress evaporation and cleaning cycles to remove membrane scale and restore flux across the membrane. Positive freshwater production rates from the coupled SGSP/DCMD system, which have not been achieved in previous coupled systems, were achieved in this investigation

Light-Activated Proteomic Labeling <i>via</i> Photocaged Bioorthogonal Non-Canonical Amino Acids

This work introduces light-activated bioorthogonal noncanonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging l-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compound remains stable for many hours in culture, but can be photochemically liberated rapidly and on demand with spatial control. Upon directed light exposure, the uncaged amino acid is available for local translation, enabling downstream proteomic interrogation <i>via</i> bioorthogonal conjugation. Exploiting the reactive azide moiety present on Aha’s amino acid side chain, we demonstrate that newly synthesized proteins can be purified for quantitative proteomics or visualized in synthetic tissues with a new level of spatiotemporal control. Shedding light on when and where proteins are translated within living samples, we anticipate that laBONCAT will aid in understanding the progression of complex protein-related disorders