Professor Deborah Power, Comparative and Molecular Endocrinology group, CCMAR 2009 Universidade do Algarve Report – Society for Endocrinology 2009 Summer Studentship The reasons for the choice of student The studentship was awarded to the Comparative and Molecular Endocrinology Group which is located in the Universidade do Algarve which has recently developed the area of Biomedicine and Medicine. The objective of the studentship was to stimulate interest in the area of Endocrinology. For this reason an advertisement was prepared which was placed on the University webpage and circulated to other Universities. Three applications were received and the best ranked student, based on the results of their undergraduate course was offered the position. The student selected was completing the 3 year degree in Biomedicine at the Universidade do Algarve and was called Emanuel Esgaio (average mark 17/20 (equivalent to a first class degree). The aims of the project Recently a family of cellular transporters, the organic anion‐transporting polypeptide (OATP) family and monocarboxylate transporter (MCT) family which regulate cellular TH uptake and efflux were identified in mammals. The scientific objectives of the present study were: 1) explore if TH cellular transporters exist in other vertebrates using in silico approaches, 2) clone TH cellular transporters and establish the kinetics of TH transport in teleosts and 3) determine the tissue distribution of transcripts for TH transporters and how their expression is modified during TH driven post‐embryonic development of teleosts. The training objectives included: 1) tutoring of the appointed student in endocrinology focusing on the thyroid axis, 2) provide laboratory training in the area of bioinformatics, molecular biology and biochemistry and 3) provide training in presentation skills. Description of work The model chosen for the study was the zebrafish (Danio rerio) which has a well defined TH driven larval to juvenile transition. The zebrafish has numerous molecular resources and in silico database searches will be used to identify monocarboxylate transporter 8 (MCT8) and 10 (MCT10) which are both specific TH transporters in humans. If cellular transporters of TH are identified in zebrafish the in silico analysis will be extended to other teleost fish and vertebrates. Phylogenetic studies will be carried out to establish the evolutionary relationship between MCT8 and 10 and gene structure and linkage analysis in zebrafish will contribute to this analysis. If available MCT8 and MCT10 cDNA will be obtained from zebrafish cDNA collection and cloned into an expression vector and used to transfect CHO cells and study the kinetics of TH uptake and release using radiotracers for T3 and T4 (5 weeks). RT‐PCR of MCT8 and MCT10 will be used to evaluate transcript distribution in a range of adult tissue (brain, skin, kidney, muscle, bone, liver, gills) and also to establish if changes in tissue distribution are associated with tissue maturation during the larval/juvenile transition (3 weeks). If feasible quantitative RT‐PCR will be developed to permit quantification of MCT8 and 10 and also for future experimental studies ongoing in the host laboratory. Assessment of results and outcomes of studentship In silico searches in public databases retrieved MCT8 from zebrafish, stickleback (Gasterosteus aculeatus) and Chinese rare minnow (Gobiocypris rarus) and MCT10 from zebrafish, catfish, medaka (Oryzias latipes), Atlantic salmon (Salmo salar) and fathead minnow (Pimephales promelas). MCT8 and MCT10 share respectively, 49% and 73% similarity with the human homologue. Phylogenetic analysis using MCT1, MCT5, MCT8 and MCT10 from all vertebrates’ sequences deposited in public databases revealed the different forms of MCT clustered in Professor Deborah Power, Comparative and Molecular Endocrinology group, CCMAR 2009 Universidade do Algarve distinct groups and the tree branching indicated MCT8 and 10 probably shared an ancestral gene. Comparison of the gene organization of MCT10 in human and teleost revealed it is conserved although the gene is 135kb in human and 19.8kb in teleosts as a result of differences in the intron size. MCT8 also shared a conserved gene organization between teleosts and humans and was also much bigger in human. The tissue distribution of MCT8 and 10 was more restricted in the zebrafish than the human and transcripts of the former gene were detected in liver, heart and brain, while the latter gene was detected in liver only. A clone for the full length transcript was obtained and MCT8 and 10 cloned into the expression vector pcDNA3.1/V5‐His‐TOPO (Invitrogen) and verified by RT‐PCR and sequencing. Adherent epithelial cells (HEK‐293, Sigma‐Aldrich) were transfected with the expression vectors and several stable cell lines produced for each cloned transcript. It was not possible in the time frame of the studentship to carry out the evaluation of TH transporting activity as a consequence of some technical problems associated with cloning of inserts into the expression vector. Scientific training involved daily lab meetings and supervision of work by senior scientists in the host laboratory. Weekly meetings provided the opportunity for tutorials about the endocrine system and the thyroid axis in general. The student gave two oral presentation of his work during laboratory meetings and submitted an abstract and was selected for a poster presentation at the Iberian meeting of comparative Endocrinology (September 2009). The overall outcome of the project was excellent: 1) the results will form the basis of a publication; 2) the student advanced their knowledge about endocrinology and their capacities in the area of molecular biology; 3) the studentship elicited interest within the University in the Department of Biomedicine and Medicine and 4) the studentship and its outcome were divulged at the meeting of Iberian Comparative Endocrinologists. Future directions in which the project could be taken The work will be continued in order to carry out the functional characterisation of teleost MCT8 and MCT10. The work will form the basis of a project proposal to investigate the role of cellular TH transporters in controlling differential responsiveness of tissue organs during fish metamorphosis. Departures from original proposal On the whole the planned work was carried out and MCT8 and MCT10 which were identified in teleosts and they were selected for analysis and to determine if they are functional homologues of the forms found in humans. Additional work included the characterisation of MCT8 and MCT10 gene organisation and linkage analysis. It was not possible to characterise the functional characteristics of the cell transporters because insufficient time was available. Value of studentship to the student and to the lab The studentship has heightened the visibility of the host laboratory in the Universidade do Algarve and drawn attention to the area of Endocrinology which is largely overlooked by the Biomedicine and medicine degrees. The work executed by the student allowed a speculative project to be carried out and the positive results will “pump prime” a project application. The inclusion of a students in the host laboratory stimulated training activities an interest in including more students for short‐term training activities in the future. Details of any publications resulting from the grant The work formed the basis of the student’s final thesis for his degree an resulted in a 30 page report (in Portuguese). See Annex I for the abstract accepted for the Vth meeting of the Iberian Association for Comparative Endocrinologists. A publication is in preparation. Professor Deborah Power, Comparative and Molecular Endocrinology group, CCMAR 2009 Universidade do Algarve Annex I Abstract for the Vth meeting of the Iberian Association for Comparative Endocrinology, Porto, September 2009 Thyroid hormone cell transporters: Do they exist in fish? Emanuel Esgaio, João CR Cardoso & Deborah M Power Comparative Molecular Endocrinology, Centre of Marine Sciences, Universidade do Algarve, 8005‐139 Faro The T3 (Triiodothyronine) and T4 (Thyroxine) are important vertebrate thyroid hormones (TH) produced by the follicular cells and involved in many physiological functions, such as growth and development. They bring about their action by binding to nuclear thyroid receptors (TRs) by modulating gene expression and despite their lipophilic nature, T3 and T4 actions have recently been associated with the existence of highly specific membrane cell transporters. In humans, three transporters: MCT8 and MCT10 (monocarboxylate transporters) and the OATP1C1 (an organic anion‐transporting polypeptide) have been identified and an unbalance of T3 and T4 transport by these receptors was associated with the development of important human syndromes. Despite their functional importance their mechanism/regulation of action are poorly understood and evidences of their existence in other vertebrates such as teleosts, a basal vertebrate group is scarce. The aim of the present study was to characterise the fish thyroid hormones transporters using a series of in silico and functional approaches and to compare the homologue system with human. Data mining the teleost EST and genome databases with the human homologues identified a single MCT8 and MCT10 genes and in silico comparisons revealed they are highly similar and share an identical gene organisation with the human gene and are composed by 6 coding exons but are at least 10 times smaller. Tissue expression indicates the zebrafish transporters have a similar distribution with the humans and were found in liver, brain and heart, suggesting similar functions. Currently mammalian cell assays are under development and their role in the T3 and T4 transport will be further elucidated. Acknowledgments: EE was funded by a summer studentship from the British Endocrine Society. The work was supported by CCMAR Pluriannual project.