The project leading to this application has received funding from the European Union’s Horizon
2020 research and innovation programme under grant agreement No 667387

Training stages

Stage abroad participant: Silion Mihaela

The purpose of travel: participation at Hands-on Course in Ultrafast Sample Treatment for Proteomics”
Place and period travel: Lisbon, Caparica, Portugal, January 22 to 26, 2017
Program: http://www.bioscopegroup.org/index.php/activities/courses
The course has been structured into two modules: theoretical issues concerning the field of "Proteomics” and laboratory practice, particularly on protein handling.
Activities performed / results:

  • Practical aspects of sample collection storage and preservation for the proteome analysis
  • Fundamentals of proteomics
  • Protein fractionation: 1D - GE, 2D-GE, o-gel IEF
  • Sample preparation: In-gel trypsin digestion
  • Protein identification by Mass Spectrometry
  • Sample clean-up
  • Protein separation: 2D gel electrophoresis
  • In-gel trypsin digestion: accelerated method
  • MALDI -TOF/TOF-MS data acquisition
  • Data analysis: Protein identification

The aim of this course was to learn the planning of an experiment in order to separate and identify the proteins from a biological tissue or fluid. The theoretical part of the course started with an overview of the field highlighting the critical points when working with proteins, such as the huge number of proteins in a biological sample or the sample stability. For a successful proteomic experiment, first recommendation was to begin with those techniques that are already successfully established in a laboratory. 2D electrophoresis was performed as a protein separation by both isoelectric point and molecular weight. The protein mixture was first subjected to IEF technique  followed by the electroforetic migration based on molecular weight.
MALDI-TOF and ESI-TOF have been established the most common and effective methods of protein identification, as complementary techniques. Therefore a brief presentation of method principle, along with an exemplification by running the samples (prepared by each participant) using MALDI-TOF instrument. The course was completed by exposing a method for protein identification, based on previously obtained results, using MASCOT software.

  • Stage abroad participant: Uratu Cristina

The purpose of travel: participation at Hands-on Course in Ultrafast Sample Treatment for Proteomics”
Place and period travel: Lisbon, Caparica, Portugal, January 22 to 26, 2017
Program: http://www.bioscopegroup.org/index.php/activities/courses
The course has been structured into two modules: theoretical issues concerning the field of "Proteomics” and laboratory practice, particularly on protein handling.
Activities performed / results:
Schematically, the course program was as follows:
Module I  - Theory

  • Practical aspects of sample collection, storage and preservation for the proteome analysis
  • Description: Description: D:\My Documents\EraChair\Deplasare Lisabona 2017\PICT_1.jpgFundamentals of proteomics
  • Protein  fractionation: 1D - GE, 2D-GE, off-gel   IEF
  • Sample preparation: In-gel trypsin digestion
  • Protein  identification by Mass Spectrometry

Module II  - Hand-on

  • Sample clean-up
  • Sample separation: 2D electrophoresis
  • In gel trypsin digestion: accelerated method
  • MALDI – TOF / TOF – MS data aquisition
  • Data analysis: Protein identification

The theoretical part of the course started with an overview of the field highlighting the critical points when working with proteins, such as the huge number of proteins in a biological sample (e.g. in human about 100,000 proteins can be expected) or the sample stability. For a successful proteomic experiment, first recommendation was to begin with those techniques that are already successfully established in a laboratory. Once good results have been obtained in a reproducible and repeatable way, complementary techniques can be introduced.
The first step in protein detection of a biological specimen is the sample clean-up with methanol and chloroform followed by the determination of protein concentration, using Bradford reagent, based on a calibration curve. 2D electrophoresis was performed as a protein separation by both isoelectric point and molecular weight. The protein mixture was first subjected to IEF technique (an electrophoretic technique for the separation of proteins, based on their isoelectric point) followed by the electroforetic migration based on molecular weight. The resulting spots were visualized using Coomassie Blue R-250. Theoretically, each spot corresponds to a single protein, which should be then identified. After stain removal, gel separated proteins are digested into peptides prior to identification by mass spectrometry. The process involves “in gel” reduction and alkilation, followed by digestion of the proteins with trypsin and the extraction of the peptide from the gel pieces. 
The samples contaning the peptide mixture were subjected to a new clean-up procedure in order to remove salts, detergents, other anions or cations which could obstruct the protein identification.

  •  Stage abroad participants: Dr. Lilia Clima, Dr. Irina Rosca, Gabriela Pricope PhD student

The purpose of travel: research stage at IAB member: Prof. Dr Stéphane VINCENT, Namur Research Institute for Life Sciences (Narilis)/Unit of organic chemistry, Namur University
Place and period travel: Namur Belgium12-19.07.2017
Webpage: https://www.unamur.be/sciences/chimie/cbo/membres/stephane-vincent
Description: Description: D:\Documents\Desktop\20170715_153744.jpgThe stage was organised under the Romania – Belgium bilateral project PN-III-P3-3.1-PM-RO-BE project 99BM /04.04.2017 that Dr. Lilia Clima has applied and wine in cooperation with Prof. Dr Stéphane VINCENT (IAB Member) under the heading of SupraChem Lab Project (see details in D4.2 Second report on SupraChem Lab group integration and networking activities)
Topic of the stage: Molecular biology and Supramolecular complexes
Activities performed / results:
The main objective of the project was establishing a successful scientific bilateral cooperation in the field of adaptive nanoplatforms between the PPIMC and University of Namur for a sustainable collaboration.
During the visit to University of Namur the following objectives have been achieved:

  • Visiting the chemistry department and laboratoires of Prof. Stephane Vincent group
  • Learning about the laboratory infrastructure, meeting team members and establishing complement methodologies, analytical techniques, and testing of nanoplatforms in vitro.
  • Discussing with Prof Vincent the results concerning the antimicrobial activity of the developed library of nanoplatforms based on 1,3,5-benzenetrialdehyde as a trifunctional center, Pegylated squalene derivative for its self-assembly behavior, polyethylene glycol moiety for its biocompatibility properties, aminoguanidine and/or branched polyethylene imine as cationic polymer able to complexate DNA.
  • Dissemination of SupraChem Lab project by giving a presentation with the title: Multifunctional Dynamic Nanoplatforms for Targeted Biomedical Applications (Clima L.)
  • Follow up of the activity of Andrei Diaconu PhD student that works with Prof. Stephane Vincent group under the coordination of SupraChemLab project
  • Stage abroad participant: Andrei Neamtu, Tudor Vasiliu

The purpose of travel: training stage
Place and period travel: Paris, France
Place and period travel: Paris, Institute of physico-chemical biology (IBPC), France, 16 October to 10 November 2017
Trained person: Neamtu Andrei

Description: neamtu-molecula gigantThe purpose of the internship was to familiarize with the techniques, machines and programs used to perform Molecular Dynamics simulations of biological systems, especially natural and synthetic selective channels through phospholipidic membranes.

Activities performed / results:

  • Visit and introduction to the IBPC institute and staff
  • Performing MD simulations of DOTA compound
  • Developing tools to characterize the obtained structures
  • Running HREX simulations of the DOTA/membrane systems
  • Backmapping of the simulated systems from coarse grain to atomistic
  • Using the High-tech 3D visualization system for simulation analysis

Description: 20171027_155041The goal of this internship was to understand and explain the mechanism through which DOTA molecules are able to permeate phospholipidic membranes for water molecules and induce a high water transport rate. To this end, we performed coarse grain molecular dynamics simulation of the auto-assembly of DOTA and POPC molecules in water. In order to confirm that the structured obtained through MD simulation are valid we also melted a DOTA crystal in the membrane and fused a pre-made membrane with a DOTA micelle. To further equilibrate the system, we performed replica exchange simulations to improve the exploration of the conformation space of the systems.
Once the simulations were performed we used the virtualization room in order to visually study the structures that we obtained and to understand the mechanism of channel formation in the POPC membrane.