Title page for ETD etd-04152005-095943


Type of Document Master's Thesis
Author Jimenez, Fernando
Author's Email Address fjimenez@agctr.lsu.edu
URN etd-04152005-095943
Title Detection and Evaluation of Temperature Effects on Cell Proliferation in Somatic Tissues of the Eastern Oyster, Crassostrea virginica, by Flow Cytometry
Degree Master of Science (M.S.)
Department Renewable Natural Resources
Advisory Committee
Advisor Name Title
Jerome F. La Peyre Committee Co-Chair
Terrence R. Tiersch Committee Co-Chair
Jill A. Jenkins Committee Member
Steven G. Hall Committee Member
Keywords
  • cell proliferation
  • eastern oyster
  • pcna
Date of Defense 2005-02-21
Availability unrestricted
Abstract
The goal of this thesis was to evaluate temperature effects on cell proliferation of eastern oyster somatic tissues for the development of an oyster cell line. Understanding the in vivo cell proliferation of an organism is essential for the development of cell culture. Cell proliferation can be measured by identifying nuclear cellular proteins involved in growth regulation and cellular transformation. The primary objectives of this study were to: 1) develop an assay to evaluate cell proliferation, 2) develop an assay to analyze nuclear RNA content, and 3) evaluate temperature effects on cell proliferation in somatic tissues.

Proliferating cell nuclear antigen was detected in all the phases of the cell cycle of the eastern oyster. The concentrations of PCNA increased from G0/G1 phase into the S phase, where it peaked in mid-S phase, and decreased in G2/M phase. The immunohistochemically stained slides were analyzed using image analysis and compared to the flow cytometric detection of PCNA. Overall, flow cytometry was superior to immunohistochemistry in time and cost efficiency, and in sensitivity to the detection of PCNA in oyster somatic tissues. A flow cytometric assay was developed to detect nuclear RNA in oyster somatic tissues. Nuclear RNA was detected in all the phases of the cell cycle of the eastern oyster. The amount of nuclear RNA increased from G0/G1 phase into the S phase, where it peaked in mid-S phase, and decreased in G2/M phase. Temperature effects on cell cycle, proliferation, and cell metabolism of oyster somatic tissues were evaluated. The cell cycle analysis of the heart and labial palps cells showed that the G0/G1-phase cells decreased as the S-phase and G2/M-phase cells increased. The cell cycle analysis of both tissues also showed the opposite effect, as G0/G1-phase cells increased the S-phase and G2/M-phase cells decreased. Heart and labial palps cells for oysters held between 10C and 25C increased in cell proliferation after one week of temperature fluctuation. The nuclear RNA/DNA ratio at all phases of the cell cycle for heart and labial palps cells for oysters between 15C and 20C was higher than at all other temperature regimes. Temperature fluctuations affected the cell cycle, proliferation, and the metabolic condition of oyster somatic cells.These results support the use of flow cytometry for the analysis of cell cycle, cell proliferation, and cell metabolism. The results also suggested that temperature fluctuation has an effect on cell cycle, cell proliferation, and cell metabolism. Future research needs to focus on improving the current in vivo study and adapt the assays for cultured cells.

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