Date of Award

Fall 11-21-2014

Degree Type

Dissertation

Degree Name

PhD Molecular Bioscience

Department

Biology

Advisor

Carolyn S. Bentivegna, Ph.D.

Committee Member

Constantine Bitsaktsis, Ph.D.

Committee Member

Tin-Chun Chu, Ph.D.

Committee Member

Wyatt R. Murphy Jr., Ph.D.

Committee Member

Daniel B. Nichols, Ph.D.

Keywords

Chironomidae, Hemoglobin protein, Biomarker, SDS-PAGE, Cadmium, Environmental quality

Abstract

Studying environmental quality is a challenging task. It is a complicated exercise since the environment is constantly influenced by numerous variables such as climate change, anthropogenic activities, and unexpected natural disasters. Traditionally, exposure to chemical pollutants depended on chemical and physical analysis of environmental media. Unfortunately, this approach has not taken into consideration bioavailability of the chemical(s) of interest to exposed organisms and/or modification of the chemical (bioactivation/detoxification) by the organism. Benthic macroinvertebrates (BMIs) have been chosen as bioindicators for numerous environmental biomonitoring programs geared towards the assessment of aquatic ecosystems. Biomonitoring requires a more subtle measure – a biomarker – which should be dependable, reliable, and specific for assessing various ecological issues and human health risks. A wide array of biomarkers has focused on sublethal changes at the cellular and molecular levels. Good cellular and molecular biomarkers can respond quickly to low concentrations of contaminants with some specificity for particular types of contaminants. It is important that the mechanism by which they respond to contaminants be understood. This improves their reproducibility and provides an understanding of their limitations. In this study, various molecular responses exhibited by hemoglobin (Hb) protein from hemolymph of larvae of Chironomidae, or chironomids were characterized, in order to develop a biomarker for evaluating environmental quality. Hb proteins in the hemolymph of wild chironomids were separated by SDS-PAGE and compared to head capsule morphology. Results showed unique profiles for different genera and particular bands that identified species. However, some species had multiple profiles. The source of Hb polymorphisms observed among wild species was investigated by determining the effect of proteases – chymotrypsin, trypsin, and pepsin – and an environmental stressor, cadmium (Cd), on Hb profiles. Results showed that individual and/or combination of proteases could account for intraspecies Hb protein profiles. However, 3.0 µM Cd generated its own distinct profile with upper bands similar to early 4th instar larvae and a loss of lower bands. Cd’s mechanism of action was investigated by measuring endpoints associated with Hb biosynthesis pathways. Endpoints included porphobilinogen (PBG) synthase activity and expressions of genes: hemoglobin IV and VII (Hb IV & VII), ubiquitin (Ub), and metallothionein (MT). Results showed that the effect of Cd on Hb protein profiles could be explained by inhibition of PBG synthase, up-regulation of Ub and down-regulation of Hb IV and VII. Overall, Hb protein profiles could be used to identify different genera and some species of wild chironomids. Hb profile polymorphisms could be explained by larval stage of development, levels of protease activity and modulation of Hb biosynthesis pathways.

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