Dissertations, Theses, and Capstone Projects

Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Jean Gaffney

Committee Members

David Gruber

Kevin Gardner

Michael Tessler

Kate Buettner

Subject Categories

Cell Biology | Marine Biology | Molecular Biology

Keywords

Fluorescence, Biofluorescence, Marine, Fluorescent Proteins

Abstract

Fluorescence is a phenomenon in which a wavelength of light excites a fluorophore and then emits light at a higher, longer wavelength. This occurs throughout the biological world, in both the terrestrial and marine realms. Encompassing a diverse set of mechanisms from small molecules to large protein complexes; fluorescence has been used as a biological tool with a wide range of applications to study molecular and cellular biology for decades. This dissertation looks to identify and characterize novel fluorescent proteins and small molecules to allow for more efficient translation in developing new biotechnologies. Using a combination of RNA-Seq, Mass-Spec, protein expression and purification, novel fluorescent proteins and small molecules can be identified and characterized.

There are several well-described fluorescent fatty-acid binding proteins present throughout the Anguilliformes order, common-name eels. Chlopsid FP is one novel fluorescent fatty acid-binding protein, which has been isolated from the Kaupichthys hyoproroides eel. In this study Chlopsid FP is further characterized by looking at its ability to bind its ligand bilirubin, through a point mutation study. By expanding the characterization of Chlopsid FP the significance of a glycine-proline-proline loop in fluorophore formation is confirmed. Furthermore, several specimens of eels are identified as being fluorescent, including a moray eel, Gymnothorax zonipectis. In doing so fluorescent proteins are implicated as the cause of fluorescence in several new eel specimens, expanding on the current understanding of fluorescent FABPs.

In addition, Synodontidae, commonly called Lizardfish, a family of fish displaying both red and green fluorescence, are examined further. Until recently Lizardfish have been overlooked as potential keys to mechanistically understanding fluorescence. Using RNA-Seq technology and analyses, including transcriptomic annotation and differential expression, the Lizardfish family of fish is examined. Specifically, Saurida gracilis and Synodus lucioceps, and their associated fluorescent moieties are studied. The Saurida gracilis is examined due its dual fluorescence, as it expresses both red and green fluorescence. The Synodus lucioceps was selected as a vividly green fluorescent sample. Through examining both species a greater understanding of the Synodontidae family of fish is gained. Furthermore, using different approaches to examine each fish has allowed for the development of an efficient pathway to isolate and identify novel fluorescent moieties from marine organisms.

Finally, two shark species, Cephaloscyllium ventriosum and Scyliorhinus retifer, are examined and characterized as cartilaginous fish expressing green fluorescence. A series of novel fluorescent metabolites are identified as small molecules responsible for fluorescence in both species. Additionally, the fluorescent properties of these metabolites, including excitation and emission spectra, are characterized. Ultimately, leading to the identification of a bromo-kynurenine metabolic pathway, furthering the current understanding of fluorescent small molecules.

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