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Further characterization of metacaspase expression and activity in marine phytoplankton
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Title
Further characterization of metacaspase expression and activity in marine phytoplankton
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ETD_2391
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http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.000052130
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eng
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theses
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Oceanography
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(authority = ETD-LCSH)
Topic
Cell death
Subject
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(authority = ETD-LCSH)
Topic
Marine phytoplankton
Abstract
Metacaspases are considered to be ancestral cysteinyl aspartate specific proteases involved in the initiation and execution of apoptotic programmed cell death (PCD). The widespread presence of metacaspases genes among a variety of phytoplankton, suggests that they play a fundamental role in cell turnover, aquatic food webs, and biogeochemical cycles. Yet, there are still fundamental questions that exist about these enzymes: How does metacaspase diversity relate to different evolutionary plastid lineages? What is their relationship to cell physiology? Are metacaspases associated with caspase activity?
Using hydrolysis of a fluorogenic canonical tetrapeptide substrate and western blot analysis, we report on the induction of caspase-like activity and metacaspases-like protein expression from four ecologically and evolutionarily diverse phytoplankton species, including a chlorophyte (Dunaliella tertiolecta), a haptophyte (Isochrysis galbana), a diatom (Thalassiosira weissflogii), and a dinoflagellate (Amphidinium carterae). These derived eukaryotic lineages represented phytoplankton from primary (D. tertiolecta), secondary (I. galbana and T. weissflogii), and tertiary endosymbiotic (A. carterae) events. Immunohybridization to polyclonal antisera raised against a coccolithophore metacaspase indicated high conservation of caspase-like proteins and an accumulation of metacaspase complexity with evolutionary complexity.
An E. huxleyi CCMP1516 (Ehux1516) and EhV86 host-virus model system was used to link dramatic increases in caspase specific activity with protein signatures via a genome-enabled, proteomic approach. Both the host and virus have sequenced genomes and EhV86 strongly triggered caspase activity. Caspase activation was measured through in vitro cleavage of fluorogenic peptide substrates with up to ~170-fold increase during infection. Subsets of partially purified proteins were associated with enhanced caspase specific activity and displayed hybridization to metacaspase antibodies. Pooled subsets of caspase active fractions from size exclusion chromatography were subjected to both 1D SDS-PAGE and 2D gel electrophoresis (GE) followed by mass spectrometry analysis. SYPRO-Ruby staining of 2D gels yielded approximately 10-17 clearly definable protein spots for caspase active fractions. Seventeen proteins hits, including four from the EhV86 proteome, were homologues to proteases or death related proteins, suggesting these proteins may be responsible for observed caspase activities and some may be virally derived. Further work should focus on validating the activity and function of these gene products.
Using hydrolysis of a fluorogenic canonical tetrapeptide substrate and western blot analysis, we report on the induction of caspase-like activity and metacaspases-like protein expression from four ecologically and evolutionarily diverse phytoplankton species, including a chlorophyte (Dunaliella tertiolecta), a haptophyte (Isochrysis galbana), a diatom (Thalassiosira weissflogii), and a dinoflagellate (Amphidinium carterae). These derived eukaryotic lineages represented phytoplankton from primary (D. tertiolecta), secondary (I. galbana and T. weissflogii), and tertiary endosymbiotic (A. carterae) events. Immunohybridization to polyclonal antisera raised against a coccolithophore metacaspase indicated high conservation of caspase-like proteins and an accumulation of metacaspase complexity with evolutionary complexity.
An E. huxleyi CCMP1516 (Ehux1516) and EhV86 host-virus model system was used to link dramatic increases in caspase specific activity with protein signatures via a genome-enabled, proteomic approach. Both the host and virus have sequenced genomes and EhV86 strongly triggered caspase activity. Caspase activation was measured through in vitro cleavage of fluorogenic peptide substrates with up to ~170-fold increase during infection. Subsets of partially purified proteins were associated with enhanced caspase specific activity and displayed hybridization to metacaspase antibodies. Pooled subsets of caspase active fractions from size exclusion chromatography were subjected to both 1D SDS-PAGE and 2D gel electrophoresis (GE) followed by mass spectrometry analysis. SYPRO-Ruby staining of 2D gels yielded approximately 10-17 clearly definable protein spots for caspase active fractions. Seventeen proteins hits, including four from the EhV86 proteome, were homologues to proteases or death related proteins, suggesting these proteins may be responsible for observed caspase activities and some may be virally derived. Further work should focus on validating the activity and function of these gene products.
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electronic resource
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xii, 77p : ill.
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M.S.
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Includes bibliographical references
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by Wanjing Liao
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Liao
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Wanjing
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1984-
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Wanjing Liao
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Kay
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Kay Bidle
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Falkowski
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Paul
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Paul Falkowski
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Schofield
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Oscar
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Oscar Schofield
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Rutgers University
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Graduate School - New Brunswick
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2010
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2010-01
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xx
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Rutgers University Electronic Theses and Dissertations
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doi:10.7282/T3MG7PNZ
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ETD graduate
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The author owns the copyright to this work.
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Open
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2010-01-05 13:33:29
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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Rutgers University. Graduate School - New Brunswick
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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180 days
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