ingest cdrApp 2018-06-13T19:28:20.981Z 51cd2fe2-3fd7-401f-a923-a97bc3db68a2 modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-06-13T20:45:52.302Z Setting exclusive relation addDatastream MD_TECHNICAL fedoraAdmin 2018-06-13T20:46:03.634Z Adding technical metadata derived by FITS addDatastream MD_FULL_TEXT fedoraAdmin 2018-06-13T20:46:15.483Z Adding full text metadata extracted by Apache Tika modifyDatastreamByValue RELS-EXT fedoraAdmin 2018-06-13T20:46:37.708Z Setting exclusive relation modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-07-11T11:48:13.791Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-07-18T07:42:49.322Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-08-17T13:38:16.612Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-08-21T16:31:46.680Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-09-27T16:38:41.199Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-10-12T07:48:29.481Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2018-10-17T13:05:09.095Z modifyDatastreamByValue MD_DESCRIPTIVE cdrApp 2019-03-21T17:42:22.969Z Emily Werth Author Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Spring 2018 2018 Analytical chemistry Chlamydomonas, mass spectrometry, phosphoproteomics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text Emily Werth Author Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Spring 2018 2018 Analytical chemistry Chlamydomonas, mass spectrometry, phosphoproteomics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text Emily Werth Author Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Spring 2018 2018 Analytical chemistry Chlamydomonas, mass spectrometry, phosphoproteomics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text Emily Werth Author Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Spring 2018 2018 Analytical chemistry Chlamydomonas, mass spectrometry, phosphoproteomics eng Doctor of Philosophy Dissertation Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text University of North Carolina at Chapel Hill Degree granting institution Emily Werth Author Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Spring 2018 2018 Analytical chemistry Chlamydomonas, mass spectrometry, phosphoproteomics eng Doctor of Philosophy Dissertation Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text University of North Carolina at Chapel Hill Degree granting institution Emily Werth Creator Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Analytical chemistry Chlamydomonas; mass spectrometry; phosphoproteomics eng Doctor of Philosophy Dissertation Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text University of North Carolina at Chapel Hill Degree granting institution 2018 2018-05 Emily Werth Author Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Spring 2018 2018 Analytical chemistry Chlamydomonas, mass spectrometry, phosphoproteomics eng Doctor of Philosophy Dissertation University of North Carolina at Chapel Hill Graduate School Degree granting institution Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text Emily Werth Author Department of Chemistry College of Arts and Sciences EXPLORATION OF KINASE-REGULATED CELLULAR SIGNALING IN CHLAMYDOMONAS REINHARDTII Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. Spring 2018 2018 Analytical chemistry Chlamydomonas, mass spectrometry, phosphoproteomics eng Doctor of Philosophy Dissertation Chemistry Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text University of North Carolina at Chapel Hill Degree granting institution Emily Werth Creator Department of Chemistry College of Arts and Sciences Post-translational modifications (PTMs) on proteins to form functional protein products are a key level of cellular signaling regulation. Because of this, there has been an immense effort in the proteomics community to improve quantitative enrichment, acquisition, and bioinformatics strategies for the analysis of PTMs to probe metabolic pathways. The identification of dynamic protein phosphorylation events, a vital PTM, is especially important for understanding kinase/ phosphatase-regulated signaling pathways, and is the focus of this dissertation. The aim of this dissertation is to develop and apply phosphoproteomic strategies in the alga Chlamydomonas reinhardtii to characterize the role of protein phosphorylation on cellular regulation in a diverse array of signaling networks. Techniques for algal cell culturing, protein extraction, quantitative enrichment, acquisition and bioinformatics processing developed and adapted for Chlamydomonas are discussed (Chapter 2). Using these techniques, a quantitative workflow for a dual enrichment strategy to target intact protein kinases via capture on immobilized multiplexed inhibitor beads with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment was developed (Chapter 3). This workflow obtained quantitative coverage on 115 protein kinases and 2,304 phosphopeptides. Application of the quantitative phosphoproteomic pipeline was employed to study the effect of Target of Rapamycin (TOR) kinase inhibition on the Chlamydomonas phosphoproteome in wild-type (Chapter 4) and extension into a rapamycin hypersensitive mutant line (Chapter 5). From the wild-type study, three TOR inhibitors with varying mechanisms of inhibition were used to obtain quantitative coverage on 2,547 unique phosphosites with 258 phosphosites differentially changing following inhibition. This approach identified Chlamydomonas homologs of TOR signaling-related proteins such as RPS6 and LARP1 that had decreased phosphorylation upon TORC1 inhibition. Additionally, this led to follow-up experiments guided by our phosphoproteomic findings showing that carotenoid levels are affected by TORC1 inhibition, the first evidence that carotenoid production is under TOR control. From the rapamycin hypersensitive mutant study, the workflow obtained quantitative coverage on 2,699 phosphosites with 316 sites changing following rapamycin treatment. This study showed similarities with the sites modulated in the wild-type study described in Chapter 4 while also providing another distinct group of phosphosites not previously interrogated. 2018-05 2018 Analytical chemistry Chlamydomonas; mass spectrometry; phosphoproteomics eng Doctor of Philosophy Dissertation Leslie Hicks Thesis advisor James Jorgenson Thesis advisor Gary Glish Thesis advisor Lee Graves Thesis advisor Brandie Ehrmann Thesis advisor text University of North Carolina at Chapel Hill Degree granting institution Werth_unc_0153D_17685.pdf uuid:384ea518-67f9-406c-964e-18443788418d 2020-06-13T00:00:00 2018-04-25T20:39:40Z proquest application/pdf 3643194