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 G<sub>0</sub>/G<sub>1</sub> phase into the S phase, where it peaked in mid-S phase, and decreased in G<sub>2</sub>/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 G<sub>0</sub>/G<sub>1</sub> phase into the S phase, where it peaked in mid-S phase, and decreased in G<sub>2</sub>/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 G<sub>0</sub>/G<sub>1</sub>-phase cells decreased as the S-phase and G<sub>2</sub>/M-phase cells increased. The cell cycle analysis of both tissues also showed the opposite effect, as G<sub>0</sub>/G<sub>1</sub>-phase cells increased the S-phase and G<sub>2</sub>/M-phase cells decreased. Heart and labial palps cells for oysters held between 10°C and 25°C 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 15°C and 20°C 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.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-04152005-095943 |
| Date | 22 April 2005 |
| Creators | Jimenez, Fernando |
| Contributors | Terrence R. Tiersch, Jerome F. La Peyre, Steven G. Hall, Jill A. Jenkins |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-04152005-095943/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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