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LP - UNITS - Università degli Studi di Trieste

PROJECT COORDINATION AND MANAGEMENT

Project coordination: monitoring internal scientific activities; promotion of knowledge and technology transfer; contact with external bodies; sustainability and development of professional profile of young researchers; dissemination).
Project management: ensuring the achievement of the project goals, by monitoring activities, budget and by identifying opportunities of performance improvement. Supported by T&B Associati Srl.
 

FIELDS OF INTEREST
Biochemistry, cell and molecular biology, physiology, molecular pathology, pharmacology, environmental sciences (peptide, science membrane proteins, drug transport). Study of antimicrobial peptides: structure, mode of action, gene and protein expression.

ACTIVITIES
Bilitranslocase group

Investigation of  bilitranslocase, a plasma membrane bilirubin and flavonoid transporter, in fish tissues. This membrane protein is found in various tissues of mammals, where it transports endogenous (e.g. bilirubin) and dietary antioxidants (e.g. flavonoids) into cells. A functional homologue has been discovered in sea-bass hepatopancreas. It binds both bilirubin and biliverdin with high affinity. This homologue is inactivated by heavy metals and thiol oxidants. It is regarded as a potential biomarker of fish health. Therefore, its primarys structure and gene is under investigation.

Antimicrobial peptides group

The field of interest of this group is antimicrobial peptides, studing their mode of action and the possibility of using them for the drug delivery and/or as antibiotics. In particular in the Innovaqua project the labratory is involved in the sperimental use of peptides as vaccination adjuvants, in order to improve an existing vaccine. Another goal is the analysis of expression of cathelicidins/immunity genes and proteins to evaluate their differential expression in response to bacterial and viral infections with the aim of using the differential expressed genes, for diagnostic purposes, developing a diagnostic kit for the identification of bacterial and viral stress conditions in fish.

Bilitranslocase group

Long experience in: 

Functional assays in membrane vesicles, isolated cells and organ fragments. Two anti-sequence monoclonal antibodies are available, with the property of inhibiting transport function and allowing for immune-detection of fixed samples.  A QSAR model with strong predictive capacity has been built by the Laboratory of Chemometrics of National Institute of Chemistry, Ljubljana, Slovenia (Prof. dr. Marjana Novic), coupled with established biological assays, allows screening new bilitranslocase ligands and assessing them for transport and bioactivity.

Developed Methods:

Primer design for PCR and RACE-PCR of mRNA extracted from rat liver to characterise mRNA transcribing for mature bilitranslocase. These methods will be translated to sea-bass hepatopancreas. The expression of bilitranslocase in fish farmed under various conditions will enable to assess its sensitivity to the environmental and feeding conditions.
Detection of bilitranslocase protein in tissue extracts by Wester blot analysis, with purified anti-sequence antibodies, enables to follow the protein expression and integrate the data obtained by PCR.

Antimicrobial peptides group

 

Long experience in:

- isolation, chemical synthesis, characterization and modification of antimicrobial peptides (AMPs) and proven experience in the design of novel AMPs. It has been created a large collection of structurally diverse natural (defensins, cathelicidins) and artificial peptides;

- in vitro and in vivo assays to evaluate the antibiotic properties of AMPs;
 
- production of antibodies active against different AMPs and bacterial targets;
 
- patenting.
 

Developed Methods:

 
- validated antimicrobial assays:
 
              1. in vitro: susceptibility testing as microdilution susceptibility testing (MIC and MBC),
                                growth kinetic and radial diffusion assay;
                                bacterial killing assays as killing kinetics, inhibitory effects of blood,
                                body fluids, salts etc.
 
              2. in vivo: acute toxicity tests (Lethal dose, toxic effects etc);
                                acute peritonitis mouse models;
                                systemic candidiasis model in mouse;
 
- validated methods for the determination of the mode of action of antimicrobial and cytotoxic peptides:
 
              1. seach for molecular targets:    affinity chromatography;
                                                                      selection of resistant-mutants and genetic analysis
                                                                      of the mutants to identify gene(s) involved in the
                                                                      action of the AMPs;
                                                                    
               2. Electron microscopy techniques:     cell morphology (SEM);
                                                                               peptide internalization and drug delivery features
                                                                               (immunogold-TEM);
                                                                               confocal microscopy;
 
              3. cell-permeabilization and membrane depolarization studies by flow cytometry
                  (propidium iodide uptake etc.);
 
              4. hemolytic activity assay on peripheral human blood to evaluate the toxicity of
                  antimicrobial peptides against eukaryotic cells;
 
              5. spectrophotometric and fluorimetric methods;
 
- production of recombinant proteins from bacteria;
 
- conjugation strategies that may reduce toxicity and improve their systemic delivery (pegylation);
  
- Real Time PCR and Western blot analyses to evaluate the gene and protein expression of AMPs and other immunity markers, on fish stressed by microbial infections.