Thesis (Ph. D.)--Oregon Graduate Institute of Science and Technology, 1990.

Thesis (Ph.D.)--University of Western Australia, 2004.

Thesis (M.S.)--Ohio State University, 2008. / Title from first page of PDF file. Includes bibliographical references (p. 57-62).

Thesis (Ph. D.)--Drexel University, 2004. / Includes abstract and vita. Includes bibliographical references (leaves 135-137).

Thesis (M.Sc.)--Memorial University of Newfoundland, 2004. / Bibliography: leaves 79-90.

Thesis (Ph. D.)--University of Virginia, 2000. / Spine title: Phospholipase A₂ binding. Includes bibliographical references (p. 245-258). Also available online through Digital Dissertations.

Thesis (Ph. D.)--Drexel University, 2007. / Includes abstract and vita. Includes bibliographical references (leaves 101-108).

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 112-125).

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Timothy Durrett / In plants including the members of Brassicaceae family, such as Arabidopsis thaliana and Brassica juncea, seed storage reserves, which include lipids and proteins, accumulate in seeds during development. Triacylglycerols (TAG) are the major storage lipids found in the developing seeds, petals, pollen grains, and fruits of plants. In Arabidopsis seeds, acyl-CoA: diacylglycerol acyltransferase 1 (DGAT1) is the major enzyme contributing to TAG biosynthesis. In Arabidopsis, dgat1-1 mutants retain 60-80% seed TAG content due to the involvement of phospholipid: diacylglycerol acyltransferase (PDAT1) in acyl-CoA independent TAG biosynthesis. My study focuses on the elucidation and functional characterization of novel genes involved in the regulation of the TAG biosynthesis pathway. In developing seeds of the dgat1-1 mutant, altered fatty acid composition was observed with reduced TAG content and increased polar lipid content as compared to wild type. RNA-Seq of developing Arabidopsis seeds was employed to detect differentially expressed genes in dgat1-1. An empirical analysis for differential gene expression revealed a significant number of differentially expressed genes among all developmental stages in dgat1-1. Significant changes in gene expression profile were detected in lipid-related genes such as lipases and desaturases. RT-PCR was used to confirm the differential expression of major lipid-related genes including DGAT1, PDAT, and FAD2. Lipid profiling of T-DNA insertion mutants for differentially expressed genes revealed significant changes in lipid content and composition. Mutations in a member of the α, β-hydrolase family, encoded by gene named PLIP1, resulted in smaller seed and an altered seed oil phenotype. Also, combining the dgat1-1 and plip1-2 mutations resulted in a lethal phenotype, demonstrating the important role of this enzyme in embryo development and TAG biosynthesis. To identify key components in the regulation of storage lipid biosynthesis, correlation analysis using differential transcript abundance and lipid profile during different stages of seed development from dgat1-1 and wild type lines of Arabidopsis was performed. Using clustering analysis with Pearson correlation coefficient and single linkage identified one cluster of genes which included PLIP1, FAD2, FAD3, and PDCT . Similar analysis using combined data from the neutral and polar fractions resulted in clustering of lipids containing polyunsaturated fatty acids. To investigate the reduced seed germination phenotype for mature seeds of dgat1-1 and non-germinating green seed phenotype of dgat1-1 plip1-2 lines, differential expression (DE) analysis for genes involved in hormone metabolism was performed. Upregulation of expression was observed for genes involved in promoting abscisic acid (ABA) response, which led us to specuate the role of altered hormone metabolism in delayed germination of dgat1-1 seeds. Development of allopolyploid Brassica species from its diploid progenitors involves duplication, loss, and reshuffling of genes leading to massive genetic redundancy. It leads to selective expression or newly acquired role for duplicated homeologs. Differential expression (DE) analysis for homoeologous genes from A and B subgenomes of allopolyploid B. juncea implicated in FA synthesis, acyl editing, and TAG biosynthesis and metabolism was performed. Differential expression (DE) analysis identified the transcriptional dominance of A subgenome homoeologs. Identification of these homoeologs will enable their use in breeding programs directed towards improvement of lipid content and composition in seeds.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Lisa R. Wilken / Microalgae are a potential feedstock for renewable and sustainable bioproducts and energy but there are significant scientific and engineering challenges to address before widespread acceptance of this platform. In particular, biorefining microalgae serves to maximize biomass valorization and minimize waste to improve process economics. The overall goal of this dissertation was the development of a biological-based microalgae biorefinery to enhance the economic feasibility of Chlamydomonas reinhardtii as a source of multiple products including native proteins and lipids. Specific objectives included accumulating biomass enriched in target biomolecules and determining processing strategies that eliminated the need to dry biomass, employed mild conditions to maintain extractability and quality, and minimized application of petroleum-derived and toxic solvents during extraction. The microalgae biorefinery developed included biomolecule accumulation, biomass harvesting, and targeted enzymatic degradation of the cell wall and organelles for release of native proteins and lipids. Biomass was cultivated, and kinetic studies indicated that 48 h nitrogen deprivation was adequate for protein and lipid accumulation. Four lytic enzymes were screened for their ability to permeate the C. reinhardtii cell wall and the C. reinhardtii-produced enzyme, autolysin, led to >85% cell disruption. TEM imaging confirmed cell disruption and retention of lipid droplets in organelle remnants indicating that protein, lipids, and starch could be distinctly partitioned and recovered. A design of experiments optimization study determined that incubation of disrupted biomass at pH 12 for 4 h at 45°C resulted in up to 65% of total protein released from disrupted biomass followed by 40-50% protein recovery with isoelectric precipitation. The cell disruption and protein extraction steps were subsequently integrated to minimize unit operations, processing time, and energy inputs. Secondary application of trypsin led to release of ~73% of total lipids (enriched in triacylglycerols) from the disrupted biomass. Characterization by thin layer chromatography and GC-FID of released lipids revealed similar profiles of enzymatically released lipids as compared to those released by conventional extraction procedures. Finally, the composition of released lipids indicated favorable combustion behavior, high oxidation stability, and suitability as biodiesel. The developed biological-based biorefinery is a promising step towards adoption of microalgae as a source of bioproducts to provide energy and food to meet the needs of a growing population. The second focus of the work was mitigation strategies for isolation of critical impurities (or potential co-products) while processing microalgae and plant hosts. Specific emphasis was placed on evaluating the impact of proteases, polysaccharides, phenolic compounds and pigments, phytic acid, and host cell proteins on the processing of microalgae and other plant hosts for extraction, recovery, and purification of therapeutic proteins. This review served as evaluation of the broader implications of application of the biorefinery to transgenic microalgae and other plants.