CHARACTERIZATION OF POLYUBIQUITIN CHAINS BY MASS SPECTROMETRY

The work outlined in this dissertation will allow biochemists and cellular biologists to characterize polyubiquitin chains involved in their cellular environment by following a facile mass spectrometric based workflow. The characterization of polyubiquitin chains has been of interest since their discovery in 1984. The profound effects of ubiquitination on the movement and processing of cellular proteins depend exclusively on the structures of mono and polyubiquitin modifications anchored or unanchored on the protein within the cellular environment. However, structure-function studies have been hindered by the difficulty in identifying complex chain structures due to limited instrument capabilities of the past. Genetic mutations or reiterative immunoprecipitations have been used previously to characterize the polyubiquitin chains, but their tedium makes it difficult to study a broad ubiquitinome. Top-down and middle-out mass spectral based proteomic studies have been reported for polyubiquitin and have had success in characterizing parts of the chain, but no method to date has been successful at differentiating all theoretical ubiquitin chain isomers (ubiquitin chain lengths from dimer to tetramer alone have 1340 possible isomers). The workflow presented here can identify chain length, topology and linkages present using a chromatographic-time-scale compatible, LC-MS/MS based workflow. To accomplish this feat, the strategy had to exploit the most recent advances in top-down mass spectrometry. This included the most advanced electron transfer dissociation (ETD) activation and sensitivity for large masses from the orbitrap Fusion Lumos. The spectral interpretation had to be done manually with the aid of a graphical interface to assign mass shifts because of a lack of software capable to interpret fragmentation across isopeptide linkages. However, the method outlined can be applied to any mass spectral based system granted it results in extensive fragmentation across the polyubiquitin chain; making this method adaptable to future advances in the field.

Flooded with Information from Social Media: Effects of Disaster Information Source and Visuals on Viewers' Cognitive, Affective, and Behavioral Responses

While a variety of crisis types loom as real risks for organizations and communities, and the media landscape continues to evolve, research is needed to help explain and predict how people respond to various kinds of crisis and disaster information. For example, despite the rising prevalence of digital and mobile media centered on still and moving visuals, and stark increases in Americans’ use of visual-based platforms for seeking and sharing disaster information, relatively little is known about how the presence or absence of disaster visuals online might prompt or deter resilience-related feelings, thoughts, and/or behaviors. Yet, with such insights, governmental and other organizational entities as well as communities themselves may best help individuals and communities prepare for, cope with, and recover from adverse events. Thus, this work uses the theoretical lens of the social-mediated crisis communication model (SMCC) coupled with the limited capacity model of motivated mediated message processing (LC4MP) to explore effects of disaster information source and visuals on viewers’ resilience-related responses to an extreme flooding scenario. Results from two experiments are reported. First a preliminary 2 (disaster information source: organization/US National Weather Service vs. news media/USA Today) x 2 (disaster visuals: no visual podcast vs. moving visual video) factorial between-subjects online experiment with a convenience sample of university students probes effects of crisis source and visuals on a variety of cognitive, affective, and behavioral outcomes. A second between-subjects online experiment manipulating still and moving visual pace in online videos (no visual vs. still, slow-pace visual vs. still, medium-pace visual vs. still, fast-pace visual vs. moving, slow-pace visual vs. moving, medium-pace visual vs. moving, fast-pace visual) with a convenience sample recruited from Amazon’s Mechanical Turk (mTurk) similarly probes a variety of potentially resilience-related cognitive, affective, and behavioral outcomes. The role of biological sex as a quasi-experimental variable is also investigated in both studies. Various implications for community resilience and recommendations for risk and disaster communicators are explored. Implications for theory building and future research are also examined. Resulting modifications of the SMCC model (i.e., removing “message strategy” and adding the new category of “message content elements” under organizational considerations) are proposed.