Cultures of the ciliate <i>Tetrahymena</i> grow into dense colonies overnight in chemically defined medium, when seeded above a ‘critical’ density. Below this density, the cells all die within hours. The diverging pathways leading to survival and death are investigated in this thesis. Rescuing activity is demonstrated in conditioned medium (and buffer) and a novel, cell-produced survival factor, TCSF1, is isolated from this, by means of PAGE and HPLC with commercially available and custom made columns. The active factor is investigated through mass spectrometry, amino acid analysis and Edman degradation. It is shown to have a mass of ~10kDa, but the rescuing activity can be located to a ~1.5kDa motif of 15 amino acid residues. The peptide, cellular or synthetic, rescues the cells from LDD. Changes to the primary structure of the synthetic motif result in partial or total loss of activity, suggesting that the motif may be conserved. The intact cellular factor stimulates DNA synthesis as measured by incorporation of labelled Thymidine; an activity not observed in the synthetic peptide, and it is suggested that the cellular protein may comprise active sites lacking in the synthetic peptide. The peptide is demonstrated to exert a rescuing effect on other types of <i>Tetrahymena, Colpidium </i>and fibroblasts. The regulation of cultures entering the stationary phase is investigated, with focus on a hypothesised inhibitory factor. Results indicate that entry into the stationary phase is not caused by depletion of any nutrients, yet do not suggest that any cell released inhibitory component is readily obtainable. The possible role of TCSF1 on the regulation of lipolysis and protein kinase activity is discussed. Another component from the extracellular medium of ~23kDa is observed, but not purified. Its relation to TCSF1 is discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:445151 |
| Date | January 2007 |
| Creators | Rasmussen, Morten Ib |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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