Doctor of Philosophy

dissertationIntrinsically unstructured protein (IUP) regions and conformational disorder are increasingly recognized for their prevalence in the eukaryotic genome and functional importance in disease-related proteins and biological regulatory processes. The dynamic molecular motions inherent in these regions, as well as those found in structured protein regions, are proving to be important targets of regulation. The autoregulation of DNA binding in the transcription factor Ets-1 provides an example of how a flexible, unstructured region can modulate the activity of a structured, regulatable unit by affecting the dynamic character of the protein. This thesis explores the mechanism of phosphorylation-dependent regulation of Ets-1 DNA binding. The serine-rich region (SRR) of Ets-1 is shown to be predominantly unstructured before and after Ca2+-dependent phosphorylation. Phosphorylation of the SRR is shown to stabilize the regulatable unit, which is composed of domains responsible for DNA binding and autoinhibition, and to reduce the DNA-binding affinity of an Ets-1 fragment that is amenable to spectroscopic analysis. NMR-based experiments further determine that phosphorylation partially dampens the fast timescale mobility of the SRR and enhances its localization to the regulatable unit. Aromatic residues adjacent to the phosphor-acceptor sites are found to be required for the reported 100-fold decrease in binding affinity and inhibitory structural alterations. The juxtaposition of phosphoacceptor site and aromatic residue are shown to form a functional unit within the SRR which can be artificially amplified to further increase aromatic residue-dependent, phosphorylation-induced inhibition of Ets-1. We conclude the discovery of a new mechanism whereby phosphorylation of an IUP region enhances a transient hydrophobic interaction, modulating the activity of a structured unit without adopting a structured conformation

Time-resolved and steady-state studies of biologically and chemically relevant systems using laser, absorption, and fluorescence spectroscopy

In Chapter 2 several experimental and data analysis methods used in this thesis are described. In Chapter 3 steady-state fluorescence spectroscopy was used to determine the concentration of the efflux pump inhibitors (EPIs), pheophorbide a and pyropheophorbide a, in the feces of animals and it was found that their levels far exceed those reported to be inhibitory to efflux pumps. In Chapter 4 the solvation dynamics of 6-Propionyl-2-(N,N-dimethyl) aminonaphthalene (PRODAN) was studied in reverse micelles. The two fluorescent states of PRODAN solvate on different time scales and as such care must be exercised in solvation dynamic studies involving it and its analogs. In Chapter 5 we studied the experimental and theoretical solvation dynamics of coumarin 153 (C153) in wild-type (WT) and modified myoglobins. Based on the nuclear magnetic resonance (NMR) spectroscopy and time-resolved fluorescence studies, we have concluded that it is important to thoroughly characterize the structure of a protein and probe system before comparing the theoretical and experimental results. In Chapter 6 the photophysical and spectral properties of a derivative of the medically relevant compound curcumin called cyclocurcumin was studied. Based on NMR, fluorescence, and absorption studies, the ground- and excited-states of cyclocurcumin are complicated by the existence of multiple structural isomers. In Chapter 7 the hydrolysis of cellulose by a pure form of cellulase in an ionic liquid, HEMA, and its aqueous mixtures at various temperatures were studied with the goal of increasing the cellulose to glucose conversion for biofuel production. It was found that HEMA imparts an additional stability to cellulase and can allow for faster conversion of cellulose to glucose using a pre-treatment step in comparison to only buffer

Does choice of drought index influence estimates of drought-induced rice losses in India?

Drought events have critical impacts on agricultural production yet there is little consensus on how these should be measured and defined, with implications for drought research and policy. We develop a flexible rainfall-temperature drought index that captures all dry events and we classify these as Type 1 (above-average cooling degree days) and Type 2 droughts (below-average cooling degree days). Applied to a panel dataset of Indian districts over 1966-2009, Type 2 droughts are found to have negative marginal impacts comparable to those of Type 1 droughts. Irrigation more effectively reduces Type 2 drought-induced yield losses than Type 1 yield losses. Over time, Type 1 drought losses have declined while Type 2 losses have risen. Estimates of average yield losses due to Type 1 droughts are reduced by up to 27 per cent when Type 2 droughts are omitted. The associated ex-post economic costs in terms of rice production are underestimated by up to 124 per cent

Exploring the Role of Spirituality and Spiritual Beliefs in the Pursuit of Excellence and Attainment of Peak Performance in Professional Athletes

Little research has been found which examines the assessment of spirituality in sport performers and its possible role in performance enhancement. The goal of the current study was to understand if and how spirituality impacts athleticism. The subjective experiences of Christian spirituality in an American World Sprinter (male), a British Olympic Sprint Canoe Racer (female), and a retired American Minor League Baseball player (male) were analyzed through Interpretative Phenomenological Analysis (IPA) (Smith & Osborn, 2003). Semi-structured interviews were used to gather data leading to three major themes: “The Belief and Confidence in God”, “Individual and Communal Prayer”, and “The Influential Role of Scripture”. These findings support previous research into athletic excellence and optimal performance. Moreover, this research provides insight into how spirituality can be acknowledged and used in athlete-centered models to enhance performance while providing holistic care for the entirety the athlete: mind, body, and spirit

Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA.

High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples