Multibiomarker approaches as an indicator of the health of organisms

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Table of contents

CHAPTER I STATE OF THE ART
1. PROTEIN BIOMARKERS IN ECOTOXICOLOGY AND MULTI-BIOMARKER APPROACHES
1.1. Biomarkers: definition, classification, examples and limitations
1.1.1. Examples of protein biomarkers used in animal Ecotoxicology
1.1.2. Limitations and perspectives
1.2. Multibiomarker approaches as an indicator of the health of organisms
2. OMICS TOOLS AND NEW STRATEGIES FOR MOLECULAR BIOMARKER DEFINITION
2.1. Transcriptomics
2.2. Proteomics
2.2.1. Protein identification by mass spectrometry
2.2.2. Proteogenomics for protein discovery in non-model species
2.2.3. Mass-spectrometry-based protein quantitation
2.3. Proteomics applications in Aquatic Ecotoxicology
2.4. Proposal of a proteomics biomarker pipeline for environmental applications ]
3. GAMMARIDS AS SENTINEL SPECIES FOR ENVIRONMENTAL MONITORING OF FRESHWATERS
3.1. Classification, morphology and ecology of Gammarus fossarum
3.2. Crustacean endocrine systems
3.3. Gammarus in Ecotoxicology and currently used biomarkers
3.4. Current status in “omics” approaches using Gammarus
4. OBJECTIVES OF THE THESIS
Development and application of a multiplexed protein biomarker measurement by mass spectrometry
Endocrine disruption biomarkers
CHAPTER II MATERIALS AND METHODS
1. BIOTESTS IN GAMMARUS FOSSARUM
1.1. Sampling and maintenance of organisms
1.2. Biological procedures
1.2.1. Reproductive toxicity test
1.2.2. Gonad dissection
1.2.3. Sampling of organisms at different physiological conditions
1.3. Protocols of contamination exposures
1.3.1. Laboratory exposures to model contaminants
1.3.2. In situ exposures
2. PROTEOMIC ANALYSES
2.1. Targeted proteomic analysis by Selected Reaction Monitoring
2.1.1. Candidate selection based on proteogenomics-derived protein sequences
2.1.2. Analytical validation of the methodology
2.1.3. Protein extraction and digestion
2.1.4. LC-MS/MS analysis
2.1.5. Data exploitation and absolute quantification
2.2. Shotgun proteomics of male gonads
2.2.1. Protein extraction and digestion
2.2.2. LC-MS/MS analysis
2.2.3. Protein identification and relative quantification by spectral counting
3. GENETIC ANALYSES
3.1. Sequence alignments and phylogenetic analyses
3.2. Protocols for gene expression
3.2.1. Total RNA extraction and cDNA synthesis
3.2.2. PCR amplification, cloning and sequencing
3.2.3. Quantitative real-time PCR
3.2.4. Data exploitation and relative quantification of gene expression
CHAPTER III DEVELOPMENT AND APPLICATION OF A MULTIPLEXED PROTEIN BIOMARKER MEASUREMENT BY MASS SPECTROMETRY IN GAMMARUS FOSSARUM
PUBLICATION Nº1
PUBLICATION Nº2
PUBLICATION Nº3
CHAPTER IV DEVELOPMENT OF SPECIFIC BIOMARKERS FOR ENDOCRINE DISRUPTION ASSESSMENT IN GAMMARUS FOSSARUM
PUBLICATION Nº 4
NOTE 1: IDENTIFICATION OF CANDIDATE GENES VIA LITERATURE SEARCH
NOTE 2: REPROTOXICITY ASSESSMENT OF MODEL MOLECULES IN FEMALE GAMMARIDS
PUBLICATION Nº5
CHAPTER V SYNTHESIS AND GENERAL DISCUSSION
1. DEVELOPMENT OF A NEW MS-BASED MULTIBIOMARKER ABSOLUTE QUANTIFICATION IN GAMMARUS
FOSSARUM 205
2. ENDOCRINE DISRUPTION BIOMARKERS
2.1. Comparative shotgun proteomics for ED biomarker discovery
2.2. Candidate gene approach
CONCLUSIONS & PERSPECTIVES
REFERENCES