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Reporting period: 2018-01-01 to 2019-12-31

The project proposes to organize the exchange of staff of three Academic institutions from Serbia (coordinator), Turkey and Finland, two SMEs from France and Turkey and three TC institutions (two from USA and one from Costa Rica) towards the production of an innovative automated multifunctional device for fluorescence-based diagnostics of neurodegenerative diseases.
The objectives addressed will be:
• Development of experimental cellular models and procedures with immunoglobulins (IgGs) from patient sera as diagnostic and prognostic technologies related to neurodegenerative diseases (particularly based on studies of amyotrophic lateral sclerosis - ALS).
• Defining mark-up characteristics of the standardized in vitro approach for personalized diagnostic protocols.
• Design of a small-scale platform based on automated fluorescence microscopy.

These objectives are based on previous studies on ALS of the coordinators group, however the project also proposes to study the applications on other neuroinflammations and neurodegenerative conditions. This addresses a relevant R & I as well as a socioeconomic medical issue. This research will be chalanged towards application by means of networking that will deal with interesectorial and international exchange of expertise. In addition, three workshops and two training schools will be organized followed by an elaborate dissemination programme. Career plans will be designed for the seconded staff in order to maintain the sustainability of the Action.
WP1. Standardization and benchmarking – in progress
Rodent astrocytes as the main cell type were studied for Ca2+ transients (dynamic fluorescent microscopy) induced by ALS IgGs.
Main reults and tasks were:
- A protocol for automated data acquisition has been designed.
- Multiple recordings of Ca2+ transients from neurons in culture induced by ALS IgG were charachterized.
- A software pilot to analyse multiple recordings of Ca2+ transients from neurons in culture is designed.
- An automated unitary microfluidic device with flow control and measurment protocols is designed.
- A study of microglia inflammation was initiated on the ALS G93A mouse model.
- Image segmentation analysis for automated data acquisitions from cells in culture has been accomplished.
- Preparation of samples of ALS IgG-treated cells for advanced microscopy (TEM, SEM, AFM) was acomplished.

Main training and transfer of knowledge activities thus related to Ca2+ imaging and signal analysis, and machine learning for automated fluorescent signal analysis.

WP2. Pilot platform design – in progress
A design and fabrication of a microfluidic chip was performed.
Main reults and tasks were:
- Pilot microfluidic chips were positively tested for flow and solution exchange. The next step – testing with cells is awaited for the next period.
- Testing procedures for automated data acquisitions using data samples from Belgrade.
- A study of microglia in culture response to ALS IgGs – electrophysiology and fluorescent indicators of ROS is in progress.

Main training and transfer of knowledge was in microfluidic biochip fabrication and functional characterization.

WP3. Dissemination, communication and net working – in progress
• A conference with representatives of 3 other RISE projects was organized at ELVESYS.
• A Training School was organized at UEF
• Two Workshop was organized at YEDITEPE
• Two press releases (one in a public newspaper in Slovenia and one in a
student journal at the University of Belgrade)
• Beginning of year 2018 one appearance at the Television Belgrade to talk about the granted project.
• A project website was setup at FBUB and a page for the project was designed at the ELVESYS site.
• There were participations of FBUB personnel at 8 conferences and 4 workshops. At one of the conferences - Regional Biophysis conference FBUB personnel took part at pitch event with a talk about the project
• In Sept 2018 and 2019 FBUB personnel with seconded staff from ARGENIT and LANOTEC, resp. took part at Researchers Night in Belgrade presenting the project to the general public.
• Two videos were made and posted as YouTube on the project site and FBUB site.
• One Open access publication related and supported by the project has been uploaded on the Participant Portal

WP4. Project management and sustainability – in progress
- At the Kick-off meeting Consortium agreement was signed as well as Partner agreements with LANOTEC (Costa Rica) and UCHC (USA).
- During the Kick-off meeting relationship of participants was established by the formation of several management bodies: Supervisory board, Secondments Committee, and R & I Council
- Ethics advisor was subsequently nominated and approved.
- At the end of the 1st year the Annual meeting was organized at FBUB in Belgrade with participation of representatives of beneficiaries from YEDITEPE, ARGENIT and a participant from UCHC (USA). Minutes have been recorded and posted online.
- In April 15-16 a Mid Term meeting was organized in Jarnac (France) with participation of representatives of beneficiaries FBUB, ARGENIT, ELVESYS, YEDITEPE, UEF, and partner LANOTEC and in the presence of PO. Minutes have been recorded and posted online.
- By the end of 2nd year the Annual meeting was organized at FBUB in Belgrade with participation of representatives of beneficiaries from YEDITEPE, ARGENIT, UEF and from the partner UCHC (USA).Minutes have been recorded and posted online.
An Amendment has been introduced. The reason was the human resources change of a USA partner (UCHIC) that was not be able to effectuate completely the planned tasks. A new partner at Northwestern University, NWU (also in Chicago USA) was found that could cover the lacking seconded months and tasks.
Ca2+ transients induced by ALS IgGs in astrocytes and neurons were defined and characterized as neuroinflammatory markers, thus openning the field for novel diagnostic procedures. One PhD is going to be founded on these results. In addition, networking signaling parameters in ALS IgGs of plated neurons were also analysed and characterized as a potential innovative diagnostic marker.
Machine learning pilot protocols were designed in order to understand the diagnostic signalling patterns. One PhD will be based on these studies.
An automated unitary microfluidic device with flow control and measurement protocols is designed as a pilot device. It is meant to be the basic device for the final multicomponent micro-chip system with integral micro-optics. The research of marketing opportunities has been already started.

SMEs in the project and potential impact
ELVESYS - microfluidics design of the pilot device and further training of seconded personnel in microfluidic biochip fabrication was of potential impact for the progress of the project.
ARGENIT - automated signal analysis using machine learning was instrumental as the impact on basic studies of cellular dynamic responses to ALS IgGs and automated recording procedure of the pilot device.