Use this URL to cite or link to this record in EThOS:
Title: Study of sea bass cytokines : molecular characterisation and expression analysis in response to Photobacterium damselae spp. piscicida exposure
Author: Nascimento, Diana Esperança dos Santos
ISNI:       0000 0001 3439 6304
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2007
Availability of Full Text:
Access from EThOS:
Cytokines are key regulators of the immune system and play a central role in the resistance to all infectious diseases. In the present thesis, and with the aim to determine the cytokine expression in response to Photobacterium damsefae ssp. piscicid6. (Phdp) exposure, sea bass TNF-a and IL-12 molecules were isolated. The molecular characterization of sea bass TNF.a showed that the putative protein conserves the TNF-a family signature, as well as the two cysteines usually involved in the formation of a disulfide bond. The mouse TNF-a Thr-Leu cleavage sequence and a potential transmembrane domain were also found, suggesting that sbTNF-a exists as a membrane bound form and a soluble protein. The single copy sbTNF-a gene contains a four exon-three intron structure similar to other known TNF a genes. Homology modelling of sbTNF-a is compatible with the trimeric quaternary architecture of its mammalian counterparts. The two subunits, IL12 p40 and IL-12 p35, that constitute the biological active IL-12 were also cloned and sequenced in sea bass. Sea bass IL-12 p40 and p35 conserve most cysteines involved in the intra-chain disulfide bonds of human IL-12 subunits as well as the important structural residues for human IL-12 heterodimerization. The gene organization of sea bass IL-12 p40 is si~ilar to the human orthologue, whilst the sea bass IL12 p35 gene structure, as reported fur pufferfish, differs from the human one in containing an additional exon and lacking a second copy of a duplicated exon present in the mammalian genes. Three differentially regulated IL-12 p40 genes (IL-12 p40 a, b and c) had been previously reported in bony fishes. In this thesis, the analysis of pufferfish and zebrafish genomes shows that two IL12 p35 genes (IL-12 p35 a and b) are present in these species. Furthermore, the synteny and phylogenetic analysis of pufferfish and zebrafish putative IL12 p40 and p35 genes showed that these are duplicated genes likely generated by the WGO in the ray-finned fish lineage. Sea bass IL-1, TNF-a, IL-10 and IL.12 expression profiles were then investigated in non-stimulated conditions and in response to Phdp or Phdp extracellular products. These studies contributed to the understanding of cytokine expression in sea bass but also to the finding that Phdp is able to inhibit IL-12 p40 expression most probably by a secreted protein. This raises the question about how IL-12 might influence the resistance to pasteurellosis, a disease caused by Phdp, and that induces high mortalities in marine fish farms. The promoters· of sea bass IL-10 and IL12 subunits were also sequenced and the presence of potential cis-elements discussed. The final experimental. chapter of this thesis describes the production of the recombinant sea bass TNF-a and IL-12 proteins, providing goOd tools for further studies on the immunobiology of these cytokines.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available