UNIVERSITÀ DEGLI STUDI DI NAPOLI “FEDERICO II” DOTTORATO IN SCIENZE VETERINARIE XXIX CICLO TESI “Marine Biotechnology: a sea of new resources and solutions for ocean and human health” Candidato Tutor Co-Tutor Dott. Christian Dott.ssa Giovanna Dott.ssa Clementina Galasso Romano Sansone DOTTORATO IN SCIENZE VETERINARIE - Segreteria Dott.ssa Maria Teresa Cagiano Coordinamento - Prof. Giuseppe Cringoli A Carolina, forza, sostegno e futuro della mia vita personale e professionale. A papà e mamma, ai nonni e alla mia famiglia tutta, parte integrante di questo traguardo. 3 4 INDEX List of abbreviations 10 List of figures 14 List of tables 21 Abstract (English version) 22 Abstract (Italian version) 23 GENERAL INTRODUCTION 26 I. Resources from the sea for a sea of resources 27 II. European strategies and Marine Biotechnology 31 Aim of the thesis 31 SECTION 1. 33 The Marine model organism Paracentrotus lividus: an established marine model organism for new biotechnological applications CHAPTER 1. Introduction 34 1.1 The sea urchin model Paracentrotus lividus: an overview 35 1.2 The sea urchin Paracentrotus lividus as model organisms to study the effect of diatom secondary metabolites 45 1.3 PUAs: morphological and molecular effects on Paracentrotus lividus and human cell lines 52 5 CHAPTER 2. Material and Methods 60 2.1 Ethics Statement 61 2.2 Paracentrotus lividus collection, stimulation of spawning and fertilization test 61 2.3 Setting up the in vitro experiments for the treatment of sea urchin embryos with the Oxylipin 62 2.4 Samples collection for gene and protein expression analysis in Paracentrotus lividus 64 2.5 A bioinformatic approach for identifying sea urchin orthologue of human proteins involved in cell death mechanisms 64 2.6 RNA extraction and cDNA synthesis from sea urchin embryos 66 2.7 Primer design and validation 67 2.8 Analysis of variation of gene expression of Paracentrotus lividus samples, by Real Time qPCR 70 2.9 Maintenance, Treatment of A549 and following Cell Viability assay 71 2.10 RNA Extraction and Real-Time qPCR of the cell samples 71 CHAPTER 3. Results 74 3.1 Morphological analysis of the effects of Heptadienal on Paracentrotus lividus embryo development 75 3.2 Gene structures and aminoacid sequences of Paracentrotus lividus identified by the bioinformatic study 77 3.3 Primer validation 92 3.4 Analysis of the variation of gene expression of sea urchin Paracentrotus lividus exposed to heptadienal 95 3.5 Analysis of the variation of gene expression of the A549 cell line exposed to heptadienal 102 6 CHAPTER 4. Discussion 106 Bioinformatic approach and molecular analysis to study programmed cell death in sea urchin Paracentrotus lividus 107 CHAPTER 5. Conclusions and future implications 114 New molecular and environmental applications for the marine model organism Paracentrotus lividus 115 SECTION 2. 117 Drug Discovery from marine microorganisms: Microalgae as source of new natural compounds for applications in pharmacology CHAPTER 6. Introduction 118 6.1 Drug from the sea: current research and market 119 6.2 Biotechnological potential of marine microalgae 121 6.3 The green microalga Tetraselmis suecica 122 6.4 The dinoflagellate Alexandrium andersoni 123 CHAPTER 7. Material and Methods 126 7.1Microalgae biomass production 127 7.2 Chemical extractions and fractionation 127 7.3 HPLC and LC-MS/MS analysis 128 7.4 Scavenging activity against DPPH radical 129 7 7.5 Maintenance, treatment of human cell lines and cell viability 129 7.6 RNA extraction, cDNA synthesis and Real-Time qPCR of the cell samples 130 7.7 Protein extraction and western blotting 131 7.8 Elisa for prostaglandin E2 (PGE2) 132 7.9 Treatment of human epidermis and tissue viability 132 7.10 Cell cycle analysis 132 7.11 Statistical analysis 133 CHAPTER 8. Results 134 Tetraselmis suecica 135 8.1 Chemical characterization 135 8.2 Antioxidant activity assay 138 8.3 Cell viability and recovery experiment 139 8.4 Analysis of the variation in gene expression 141 8.5 Analysis of the variation in protein expression 146 8.6 Assessment of prostaglandin E2 release 147 8.7 Effect on ex vivo tissue 149 Alexandrium andersoni 151 8.8 Viability of tumor cell lines after treatment with Alexandrium andersoni n-butanol extract 151 8.9 Analysis of the variation in gene expression after treatment with Alexandrium andersoni n-butanol extract 153 8.10 Analysis of the variation in protein expression of death receptors after treatment of A549 with Alexandrium andersoni n-butanol extract 156 8.11 Cell cycle analysis in A549 cell line 157 8.12 Viability of tumor and normal cell lines after treatment with Alexandrium andersoni SPE-fractions and Caspase Inhibitor Assay 159 8 8.13 Variation of gene expression analysis after treatment of A549 and HT-29 with Alexandrium andersoni SPE-fractions 161 CHAPTER 9. Discussion 164 9.1 Tetraselmis suecica reduces oxidative damage and induces repairing mechanisms in human cells 165 9.2 Alexandrium andersoni activates caspase-indipendent programmed cell death in tumor cell lines 168 CHAPTER 10. Conclusions and future implications 174 Future perspectives for marine microalgae as high renewable marine resource 175 REFERENCES 177 9 LIST OF ABBREVIATION °C degree Celsius 15-HEPE (±)-15-hydroxy-5Z,8Z,11Z,13E,17Z-eicosapentaenoic acid 5-HEPE (±)-5-hydroxy-6E,8Z,11Z,14Z,17Z-eicosapentaenoic acid A. andersoni Alexandrium andersoni A549 lung adenocarcinoma cell line ACTB actin-beta AFC 7-amino-4- trifluoromethyl-coumarin AIFM1 Apoptosis Inducing Factor, Mitochondria Associated 1 AKR1C2 Aldo-keto reductase family 1, member C2 ANOVA Analysis of variance ATCC American Type Culture Collection ATOX1 ATX1 antioxidant protein 1 homolog ATP6V1G2 V-type proton ATPase subunit G 2 B2M beta-2-microglobulin BAX BCL2 Associated X Protein BCL2 B-Cell CLL/Lymphoma 2 BCL2L11 Bcl-2-like protein 11 BEAS-2B normal lung/brunch epithelial cell line BECN1 Beclin 1 BIRC3 baculoviral IAP repeat containing 3 BLAST Basic Local Alignment Search Tool Blastn Nucleotide-nucleotide BLAST Blastp Protein-protein BLAST Bp base pair BP10 Blastula protease 10 CCL5 Chemokine (C-C motif) ligand 5 CCMP Culture Collection of Marine Phytoplankton cDNA complementary Deoxyribonucleic Acid CDSs coding DNA sequences CH2Cl2 Dichloromethane CHCl3 chloroform Chl a Chlorophyll a Chl b Chlorophyll b COHH Centers for Oceans and Human Health; COLO 205 colorectal adenocarcinoma cell line CRADD CASP2 and RIPK1 domain containing adaptor with death domain Ct cycle threshold CYCS cytochrome c somatic DECADIENAL 2-trans,4-trans-decadienal DECATRIENAL 2-trans-4-trans-7-cis-decatrienal DEPC Diethylpyrocarbonate DEVD Aspartic acid-Glutamic acid-Valine- Aspartic acid DHCR24 24-dehydrocholesterol reductase DHCR24 24-dehydrocholesterol reductase DMEM Dulbecco’s modified Eagle’s medium DNA DeoxyriboNucleic Acid dNTP deoxynucleotide triphosphate 10 DPPH 2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl DPR Decreto del Presidente della Repubblica DPYSL4 dihydropyrimidinase-related protein 4 E efficiency ECVAM European Centre for the Validation of Alternative Models EDTA Ethylenediaminetetraacetic acid EIF5B eukaryotic translation initiation factor 5B ELISA enzyme-linked immunosorbent assay EMBOSS European Molecular Biology Open Software Suite EPI-200 human epidermal tissue model ERA-NET- MBT European Research Area - Network - Marine Biotech; FADD Fas Associated Death Domain FAHs fatty acid hydroperoxides FASLG Fas ligand FBS Fetal Bovine Serum FOXM1 Forkhead box M1 FSW Filtered Sea Water g Relative Centrifugal Force (RCF) or G-Force GADD45 growth arrest and DNA-damage-inducible protein GPX1 Glutathione peroxidase 1 GPX4 Glutathione peroxidase 4 GSTP1 Glutathione S-transferase pi 1 H. sapiens or HS Homo sapiens H2O2 dihydrogen peroxide HAT Human Airway Trypsin-like protease HEPEs Hydroxy-EicosaPentaEnoic acids HepETEs Epoxyalcohols HEPTADIENAL 2-trans,4-trans-heptadienal HIF1A Hypoxia-inducible factor 1-alpha hpf hours post fertilization HPLC High Performance Liquid Chromatography HPRT 1hypoxanthine phosphoribosyltransferase HSP90AA1 Heat shock protein 90kDa alpha A HUBs highly connected nodes IC50 half maximal inhibitory concentration IGF1R insulin-like growth factor 1 receptor KCl Postassium chloride KCNIP1 Kv channel-interacting protein 1 LC-MS/MS liquid chromatography-mass spectrometry LOXs Lipoxygenases MCL1 induced myeloid leukemia cell differentiation protein-Mcl-1 MeOH methanol ml millilitres MTase MethylTransferase enzyme n-BuOH n-butanol NCBI National Center for Biotechnology Information NEAA non-essential amino acids NF-kB Nuclear Factor Kappa B ng nanogram nm nanometres NOAA National Oceanic and Atmospheric Administration 11 OCTADIENAL 2-trans,4-trans-octadienal OCTATRIENAL 2-trans,4-trans,7-octatrienal OHHI Oceans and Human Health Initiative P. lividus or Pl Paracentrotus lividus p38 MAPK p38 mitogen-activated protein kinase p53 Tumor Protein P53 PARP1 Poly ADP-Ribose Polymerase 1 PBS Phosphate-Buffered Saline PCR Polymerase Chain Reaction PDLIM1 PDZ and LIM domain 1 PGE2 prostaglandin E2 PINK1 PTEN Induced Putative Kinase Pl-Z12–1 zinc-finger transcription factor PMSF phenylmethylsulfonyl fluoride PRDX5 Peroxiredoxin 5 PTGR1 Prostaglandin reductase 1 PUAs Polyunsaturated Aldehydes PUFAs Polyunsaturated Fatty Acids qPCR quantitative Polymerase Chain Reaction R expression ratio R2 regression coefficient REST Relative Expression Software Tool RIMAR Research Infrastructures for MArine biological Resources RIP Receptor-Interacting Protein RIPA radioimmunoprecipitation assay RIPK_2 Homeodomain Interacting