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Cell Biology in Environmental Toxicology OIHANE Department of Zoology and Animal Cell Biology Research Group: Cell Biology in Environmental Toxicology “TRANSCRIPTOMIC TOOLS AND GENE EXPRESSION PROFILES OF TOXIC CHEMICAL EXPOSURE IN AQUATIC POLLUTION SENTINEL ORGANISMS” International PhD Thesis submitted by OIHANE DIAZ DE CERIO ARRUABARRENA for the degree of Philosophiae Doctor Under the supervision of Dr. IBON CANCIO URIARTE Leioa, July 2012 TESIAREN ZUZENDARIAREN BAIMENA TESIA AURKEZTEKO IBON CANCIO URIARTE jaunak, 30616453A I.F.Z. zenbakia duenak “TRANSCRIPTOMIC TOOLS AND GENE EXPRESSION PROFILES OF TOXIC CHEMICAL EXPOSURE IN AQUATIC POLLUTION SENTINEL ORGANISMS” izenburua duen doktorego-tesiaren zuzendari naizenak, tesia aurkezteko baimena ematen dut, defendatua izateko baldintzak betetzen dituelako. OIHANE DIAZ DE CERIO ARRUABARRENA doktoregai andereak egin du aipaturiko tesia, ZOOLOGIA ETA ANIMALIA ZELULEN BIOLOGIA sailean. Leioan, 2012ko uztailaren 26 TESIAREN ZUZENDARIA Iz.: Ibon Cancio Uriarte SAILAREN ADOSTASUNA ZOOLOGIA ETA ANIMALIA ZELULEN BIOLOGIA Saileko Kontseiluak, 2012ko ekaineko 13ko bileran, “TRANSCRIPTOMIC TOOLS AND GENE EXPRESSION PROFILES OF TOXIC CHEMICAL EXPOSURE IN AQUATIC POLLUTION SENTINEL ORGANISMS” izenburua duen doktorego-tesia aurkeztearen alde dagoela adierazi du. IBON CANCIO URIARTE jaunaren zuzendaritzapean egin den tesi hori OIHANE DIAZ DE CERIO ARRUABARRENA andreak aurkeztu du sail honetan. Leioan, 2012ko uzatailaren 23 O. E. SAILEKO ZUZENDARIA SAILEKO IDAZKARIA Iz.: Juan Carlos Iturrondobeitia Bilbao Iz.: Maria del Carmen Barbero González DOKTORE GRADUAREN AKTA DOKTOREGO TESI DEFENTSAREN AKTA Doktoregai and.: OIHANE DIAZ DE CERIO ARRUABARRENA TESIAREN IZENBURUA: “TRANSCRIPTOMIC TOOLS AND GENE EXPRESSION PROFILES OF TOXIC CHEMICAL EXPOSURE IN AQUATIC POLLUTION SENTINEL ORGANISMS” UPV/EHUko Doktoregoko Azpibatzordeak goian adierazitako doktorego tesia kalifikatzeko aukeratutako epaimahaiak, eta adierazitako egunean bilduta, behin doktoregaiak defentsa eginda eta egin zaizkion objekzioak edota iradokizunak erantzunda, ___________________ (Aho batez edo gehiengoaz) eman du honako kalifikazioa: GAI edo EZ GAI Defentsan erabilitako hizkuntzak (hizkuntza bat baino gehiago erabili badira, zehaztu hizkuntza bakoitzean defendatutako atalak edo ehunekoak): INGELESA Leioan, 2012ko irailaren 26an PRESIDENTEA, IDAZKARIA Sin.: Sin.: Dk: ____________________ Dk: ______________________ 1. mahaikidea, 2. mahaikidea, 3. mahaikidea, Sin.: Sin.: Sin.: Dk: Dk: Dk: DOKTOREGAIA, Sin.: _____________________ THIS WORK WAS FUNDED BY A grant to consolidated research groups (GIC07/26–IT–393–07). The University of the Basque Country (UPV/EHU) through a grant to a Unit of Formation and Research (UFI 11/37). The European Commission (Directorate-General Environment) through the PRAGMA project (grant Nº 07.030900/2005/429172/SUB/A5). The Spanish Ministry of Education and Science (project CANCERMAR, CTM2006–06192/MAR). Basque Government (Strategic action ETORTEK–IMPRES 2003–2007 and ETORTEK K-EGOKITZEN-II and research projects SAIOTEK 2006 FISHtoTXIPS S- PR06UN02, SAIOTEK 2007 ITSASonTXIP S-PR07UN01; SAIOTEK 2009 IKERKOSTA S-PC08UN03 and SAIOTEK 2010 GULATOX S-PE09UN32; Special action AE-2008- 1-1 and research project URA 10/02). INDEX INTRODUCTION ................................................................................. 1 A. ECOTOXICOGENOMICS .................................................................................... 3 A.1. Teleost genome and gene information ................................................... 7 A.2. Gene and genome information regarding mollusc ............................... 11 B. HOW IS A GENOME OR A TRANCRIPTOME SEQUENCED? .................. 14 B.1. First sequencing generation (FSG) ........................................................ 14 B.1.1. Application of automated FGS ................................................... 17 B.2. Second (next) generation sequencing (SGS) ......................................... 20 B.3. Third generation sequencing technologies (TGS) ................................. 25 B.4. Sequence banking and classification ..................................................... 32 B.4.1. Fish gene sequence databases ................................................... 33 B.4.2. Mollusc gene sequence database .............................................. 32 C TOOLS FOR THE ANALYSIS AND QUANTIFICATION OF GENE TRANSCRIPTION PROFILES .................................................................. 34 D GENE TR ANSCRIPTION PROFILING IN ECOTOXICOLOGY ...................... 40 E CONCLUDING REMARKS ..................................................................... 49 STATE OF THE ART ........................................................................... 75 HYPOTHESIS ............................................................................................. 79 OBJECTIVES .............................................................................................. 79 RESULTS ........................................................................................... 83 Chapter 1: Construction and characterization of a forward subtracted library of blue mussels Mytilus edulis for the identification of gene transcription signatures and biomarkers of styrene exposure ...................... 85 Chapter 2 : Regulation of xenobiotic transporter and immune/inflammation related genes in liver and brain of juvenile thicklip grey mullets ( Chelon labrosus ) after exposure to Prestige- like fuel oil and to perfluorooctane sulfonate ............................................. 109 Chapter 3 : Toxicology tailored low density microarray for the thicklip grey mullet, Chelon labrosus : development, production and validation ..................................................................................................... 143 Chapter 4 : Gene transcription profiling in caged sentinel thicklip grey mullets ( Chelon labrosus ) and active biomonitoring in a polluted harbour ........................................................................................................ 183 Chapter 5 : Hepatic transcriptional profiles of tributyltin exposure in thicklip grey mullet ( Chelon labrosus ) and the obesogenic theory ............. 219 Chapter 6 : 5S rRNA and accompanying proteins in gonads, powerful markers to identify sex and reproductive endocrine disruption in fish ............................................................................................................... 259 Chapter 7 : Construction of a high throughput gene expression microarray after 454 pyrosequencing of the multitissue transcriptome of the European eel Anguilla anguilla .................................. 299 GENERAL DISCUSSION AND CONCLUSIONS ................................. 323 SUMMARY AND GENERAL DISCUSSION ................................................ 325 CONCLUSIONS ....................................................................................... 331 THESIS .................................................................................................... 333 CONTRIBUTORS AND THANKS .................................................................... 339 INTRODUCTION INTRODUCTION Abbreviations A, Adenine JGI , Joint Genome Institute AGC , Anguilla genomic consortium NCBI , National Centre for Biotechnology bp , Base pair Information C, Cytosine NGS , Next generation sequencing CAT , Clonally amplified template NIG , National institute of Genetics cDNA, Complementary deoxyribonucleic acid PAH , Polycyclic aromatic hydrocarbon CIB , Centre for Bioinformatics PCR , Polymerase chain reaction CRT , cyclic reverse termination Ref-seq, Reference sequence DDBJ , DNA Data Bank of Japan RT-PCR , Reverse transcription Polymerase ddRT-PCR , Differential display chain reaction retrotranscriptase polymerase chain SAGE , Serial Analysis of Gene Expression reaction SBH, Sequencing by hybridization ddRT-PCR , differential display RT-PCR SBL , Sequencing by ligation EBI , European Bioinformatics institute SBS , Sequencing by synthesis emPCR , Emulsion PCR, SGS , Second generation sequencing ENA , European Nucleotide Archive SMS , Single molecule sequencing EST , Expressed sequence tag SMT, Single molecule template FGS , First generation sequencing SNA , Single nucleotide addition method G, Guanine SRA , Sequence read archive GOLD, Genomes Online Database SSH , Suppression subtractive hybridization INSDC, International Nucleotide Sequence T, Thymine Database Collaboration TGS , Third generation sequencing 2 A. ECOTOXICOGENOMICS A. ECOTOXICOGENOMICS Some structures in many chemicals, mainly man made, are estrange to biological structures (xenobiotics), and are driving changes in the environment faster and in more varied ways than ever experienced by Earth ecosystems. Nowadays the European markets offer around 100,000 chemical substances, and almost a third of them are produced in volumes that exceed 1 ton (1-100 tons) per year (Hengstler et al., 2006). If scientific and regulatory efforts in the 20 th century were centred in establishing which chemicals caused damage to human health (toxicology), a little branch of scientists were worried about which chemicals caused effects or damage to ecosystems (ecotoxicology). Then, the challenge in ecotoxicology for the 21 st century has become to understand the “mechanisms of toxicity” of this milieu of chemicals affecting different wildlife species. Laying on the basis of the way in which wildlife organisms cope with the changing environment, with fluctuations in biotic and abiotic conditions,
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