Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.361390
Title: An investigation into placental protein 14, a modulator of the immune response associated with human reproduction
Author: Dalton, Caroline Frances
Awarding Body: Sheffield Hallam University
Current Institution: Sheffield Hallam University
Date of Award: 1994
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Abstract:
This thesis describes investigations into Placental Protein 14 (PP14), a immunomodulator involved in human reproduction. The studies included the development of a purification procedure and an investigation of the activity of the protein. In addition the cDNA coding for the protein was cloned and expressed as a recombinant fusion protein and the molecular structure of the protein was predicted and analysed using computer-assisted modelling. Finally the clinical significance of the protein was studied in a range of patient groups. The purification scheme consisted of ion exchange, hydrophobic interaction and gel filtration chromatography, and the pure protein obtained was analysed by SDS-PAGE and Western blotting. The results demonstrate that the purification procedure is a suitable method to obtain PP14 in large quantity and with high purity. PP14 purified by this method retained its activity and was shown to suppress, in a dose-dependent manner, the uptake of 3H-Thymidine by peripheral blood mononuclear cells stimulated with interleukin-2. Purified PP14 was also shown to suppress the uptake of 3H-Thymidine by the cell line U937, also in a dose-dependent manner. This suppression could be removed by the incubation of the PP14 sample with an immunoabsorbent gel linked to monoclonal antibodies against PP14, demonstrating that PP14 was the molecule responsible for the observed activity. Based on the suppression by PP14 of U937 cell growth a bioassay for PP14 was developed, this assay was used to express the specific activity of PPM in Units/ml. To obtain recombinant PP14, mRNA was purified from a tissue sample and reverse transcription used to prepare cDNA. Specific primers were used to amplify the portion of cDNA coding for PP14 which was then ligated into the plasmids pUC 18 and pGEX-KG. Recombinant PP14 was then expressed as a fusion protein with glutathione-S-transferase. The expression conditions were optimised and the fusion protein was purified using affinity chromatography. The structure of PPM was investigated using computer assisted modelling. PPM is a member of the lipocalin family of proteins which share the feature of binding small hydrophobic molecules. The X-ray coordinates of two lipocalins known to share sequence homology with PP14 were used as a basis to model a predicted structure for PP14. An analysis of the structural motifs of the protein was carried out, and it was established that PP14 shares many of the characteristic features of this family of proteins including the presence of a binding pocket. The model was then used to predict potential ligands for PP14.PP14 was measured by radioimmunoassay in uterine flushings from fertile women, women with unexplained infertility and women suffering from recurrent miscarriages, and in plasma samples from fertile and infertile women. The results from the uterine flushings from fertile women showed that PP14 levels rose during the second half of the menstrual cycle reaching ug/ml levels by the end of the cycle. These physiological concentrations are in the same range as the concentrations at which the immunomodulatory activity of PP14 was observed in vitro. The levels of PP14 measured in uterine flushings were lower in infertile women than in fertile women, indicating that a deficiency in PP14 may be associated with infertility. The levels measured in plasma samples from these two groups of women did not pick up this difference. These results suggests that the measurement of proteins such as PP14 in uterine flushings instead of plasma samples may be a more sensitive indicator of local uterine function. In women suffering from recurrent miscarriage a significant lack of secretion of PP14 was observed around the time of implantation. This may be conected with the failure of implantation in these patients. A correlation was observed between the PP14 levels measured in uterine flushings from recurrent miscarriage patients and the level of endometrial development.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.361390  DOI: Not available
Keywords: Pregnancy; Implantation; Fetal protection
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