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Upgrading the research performance in molecular medicine at the Faculty of Veterinary Medicine University of Zagreb

Final Report Summary - VETMEDZG (Upgrading the research performance in molecular medicine at the Faculty of Veterinary Medicine University of Zagreb)

Executive Summary:
Faculty of Veterinary Medicine University of Zagreb (FVM) is well established research institution in Croatia with more than 178 staff members who have excellent education and scientific experience. In order to conduct structural changes in veterinary medicine, scientists at FVM have accepted ERA Chair initiative, aiming at fostering structural changes, synergies between cohesion and research funding, and to raise research quality in the application of molecular technologies to international levels of excellence. The project proposal aims to reinforce research performance of FVM through enhancing its capacities in molecular veterinary medicine, by attracting outstanding experienced researchers, transfer of knowledge and research potential, especially in the fields of proteomics and metabolomics. The exchange of know-how and experience with esteemed outstanding experienced researchers will be accomplished through coherent set of measures: training, workshops and trans-national two-way secondments of research staff. The workshops on different aspects in molecular medicine will be organised in Zagreb and will mostly cover three chosen fields: comparative tumour analysis, post-genomic technology and wildlife research at the FVM departments. By the upgrading of its research performance and excellence FVM will improve performance in competitive research funding, prevent brain drain, enhance its competitiveness and visibility in broader region and become internationally recognized partner. The proposed activities should increase research performance in specific field of molecular veterinary medicine and FVM will be able to increase its involvement in international research projects with EU partners and to be better integrated into the ERA. The ERA Chair Initiative will be unique in croatian veterinary science with potential benefits for the broader research community and neighbouring countries.
Project Context and Objectives:
Faculty of Veterinary Medicine University of Zagreb (FVM) is a well-established research institution in Croatia with highly educated and experienced scientific staff. The members of the Faculty are aware that they must strengthen its scientific potential and researchers to successfully participate in research activities at the European level, in order to be best prepared for Europe 2020. In order to reach the full strategic, research potential, FVM must attract outstanding researchers at FVM as a part of a strategy to upgrade its research performance effectively widening participation in competitive European funded research, and simultaneously enhance innovation, personnel skills and research equipment.
Unfortunately, the FVM does not by far have sufficient funds to build research infrastructure, as well as to develop new new high-tech laboratories, such as proteomics laboratory, or to acquire new capital equipment. Simultaneously, FVM has not enough experience with application to Structural Funds in the capacity building of the institution.
In order to conduct structural changes in veterinary medicine, scientists at FVM have accepted the challenge of the ERA Chair initiative, aiming at creating structural changes and synergies between cohesion and research funding, and raising research quality in the application of molecular technologies to international levels of excellence.
The project VetMedZg has been continuously working on the reinforcement of research performance of FVM through enhancing its capacities in molecular veterinary medicine, by attracting outstanding experienced researchers, and with transfer of knowledge and research potential, mostly in the fields of proteomics and metabolomics. The exchange of know-how and experience with esteemed outstanding experienced researchers has been provided through coherent set of measures: trainings, workshops and scientific trainings of FVM research staff. The workshops on different aspects in molecular medicine have been organized in Zagreb to cover three selected fields: comparative tumour analysis, post-genomic technology and wildlife research at FVM departments. By upgrading its research performance and excellence, FVM has improved performance in competitive research funding, it has enhanced its competitiveness and visibility in broader region and has become more recognized in international partnership projects. The proposed and conducted activities have shown to increase research performance in specific field of molecular veterinary medicine and FVM is able to increase its involvement in international research projects with EU partners and is becoming more integrated into ERA.
The main objective of VetMedZg project was to upgrade, encourage, develop, and enlarge the capacities for research and improve research performance in molecular veterinary medicine at FVM, to be more successful in obtaining competitive funding, and to enable better integration into ERA, all under the direction of an outstanding researcher; ERA Chair holder.
The major objectives of the project implied to recruit, develop and retain the best researchers at FVM; to increase the research potential and skills of researchers in specific field of molecular veterinary medicine; to increase the visibility of the researchers and FVM at the international citation database (WoS); to increase the number of articles that FVM's researchers have published in international citation databases; to increase participation of researchers from FVM as coordinators on at least two Horizon 2020 projects; to establish core facility in proteomics at FVM; to upgrade and enlarge the laboratory equipment and research capacities at FVM, and to enable the highest investigations in molecular veterinary medicine; to improve know-how and experience, and transfer the highest technology and knowledge onto the researchers of FVM with the help of partner research institutions; to increase the visibility of FVM and increase the participation of researchers from FVM on European projects and to facilitate the inclusion of molecular veterinary science into ERA; and finally to increase the knowledge on possibilities for the protection of intellectual properties. VetMedZg project has brought a positive effect on all of the proposed objectives and has led to some significant structural changes at the FVM.

Project Results:
Background
The Lisbon Strategy from the year 2000, together with other European Union documents has its goal
to make Europe the most competitive and dynamic knowledge-based economy in the world, capable
of sustainable economic growth with more greater job positions and better social cohesion.
One of the prerequisites required to accomplish this goal is to decrease the scientific and technological gap
between the developed and less developed regions of Europe. It was assumed that the Europe
convergence regions, based on knowledge-based information society, promotion of R&D, new
technologies and easier mobility, would quickly modernize older technology with state-of-the-art
equipment, which would in turn lead to improvement in the technology capacity.
The fundamental premise of the European Union policy was the integration of new members into the
European Research Area (ERA). To accomplish this and enable further European growth, the
continent has to have appropriately developed regions. However, European regions vary considerably
in their capacity to absorb and develop knowledge and technology. Many regions and organisations
could not play a significant role in the ERA, because of the continuous brain drain, problems with
infrastructure, economic and institutional organisation or lack of appropriate access to finance.

Republic of Croatia (RH) has entered the ERA in 1998 as part of the Fifth Framework Program and
has continuously participated in FP6 and FP7 Programs. However, the success in participation of Croatian scientists and scientific institutions in the ERA has not been corresponding.. In year 2013 Croatia was expected to join EU and scientific institutions were preparing to become a part of broader scientific community.
Faculty of Veterinary Medicine, University of Zagreb (FVM), is the main and only research and educational institution in the field of veterinary medicine in Croatia (HR), and at the same time, the leading institution in the area of veterinary science in the Western Balkan region.
Faculty of Veterinary Medicine was founded in 1919 as a Veterinary College, and was in 1924 transformed into the Faculty of Veterinary Medicine to make an integral part of the University of Zagreb. Faculty of Veterinary Medicine has a long history of promoting high quality education through research and exchange of information between international institutions. It employs more than 178 academic and research staff who work on the development and shape of the future careers of about 650 students. International activity of the FVM is especially bound to the work of the European Association of Establishments for Veterinary Education (EAEVE). EAEVE is an association of veterinary higher education establishments, and FVM is an active member of it since 2002, the year when it was accepted as a positively evaluated establishment.
Even before Croatia entered the European Union, with wishes to facilitate structural changes and provide a strategy to develop research excellence, to become more open, attractive and competitive, the continuation of scientific progress and revaluation by the EAEVE, FVM has decided to revise the progress since last evaluation by the EAEVE and to perform a SWOT analysis. SWOT analysis is the result of analysis of various data significant to the FVM, as well as formal and informal discussions
with collaborative, research and education staff. The results of this analysis are organised into six
areas: (1) science, (2) human resources, (3) infrastructure and equipment, (4) finances, (5) education,
and last, but not least, (6) international cooperation.

Scientific and technological capacity of the University of Zagreb, Faculty of Veterinary Medicine – SWOT analysis
The main R&D objectives of the Faculty of Veterinary Medicine (FVM) is fostering structural change to raise research quality to international levels of excellence, to upgrade, develop, and enlarge the capacities for research in molecular veterinary medicine as well as significantly improve their performance in competitive research funding, and at the end, to enable better integration into ERA.
Faculty of Veterinary Medicine is the highest scientific and educational veterinary institution in Croatia.
The research is organized through 25 departments and clinics, which constitute Faculty of Veterinary Medicine and Institute for veterinary medicine.
The strength is a great number of motivated human resources and experienced researchers open for structural change, a
significant number of national scientific projects, as well tendency of increase in the
number of scientific papers at the Faculty. In addition to the above, faculty has significant predispositions for development of the Faculty training ground with final aim to improve clinical teaching in the studies, as well as research investigations. Namely, Faculty owns about 99 hectares of land near Zagreb. Also, the facilities of the faculty Clinics (Internal medicine, Reproduction, and Surgery) have been modernised. A computer program for universal management of veterinary data has been introduced. New premises have been built to house the Virology Laboratory, and new equipment has been bought for the Laboratory of the Internal Medicine Clinic, with new haematology and biochemistry analyzers, as well as coagulation and acid-base and electrolyte analyzer. Also, a new centrifuge with cooling unit, a refrigerating unit for sample storage (up to -80°C), flow-cytometry analyzer, and a high-pressure liquid chromatography (HPLC) machine were bought. Four accredited laboratories have the status of a national reference laboratory (Laboratory for IAK-IAKLab, Laboratory for Leptospirosis-LEPTOLab and Laboratory for equine viral arthritis-ARTERLab) The laboratory for trichinella-LabT. In addition, Faculty has improved a unitary 3-year Doctoral Study in Veterinary Sciences (DSVS), which is organized in six semesters (180 ECTS).
At the same time, the awareness that international cooperation is imperative for FVM development has led to signing of bilateral agreements with various veterinary faculties. In addition, international cooperation has led to partnership in various international projects. International cooperation is also bound to cooperation with EAEVE and VetNEST (Veterinary Network of European Student and Staff Transfer).
Unfortunately, the FVM does not, by far, have sufficient funds to build research infrastructure, as well as to develop new high-tech laboratories, such as proteomics laboratory, or to acquire new capital equipment. Simultaneously, FVM does not hold enough experience with application to Structural Funds in the capacity building of the institution. Potential funding outside the FVM and Republic of Croatia could result young scientists, to gain knowledge and skills and additional to apply a molecular technologies in research. This should facilitate structural change in the FVM, and facilitate necessary additional financing through Cohesion funding, so that synergies between cohesion and research funding are strengthened. This way, new research possibilities and excellence in various fields of veterinary molecular medicine will present itself, and generally encourage scientists to new ideas and research.
Considering all the above facts, to develop research excellence and improved performance, FVM based on SWOT analysis, had to take the following activities and objectives:
1) Enable researchers at the FVM that under the direction of an outstanding researchers and
research managers in the given field of research, significantly improve its research performance, and to be much more successful in getting international projects.
2) To upgrade present facilities with sophisticated equipment for application of molecular
technology and enable young and motivated scientists at the FVM under the direction of an
outstanding researchers to significantly improve its research performance and gain domestic and
international research projects. At the same time, the evolution of science based on highly
sophisticated, postgenomic technology will enable just graduated individuals to form their career
in Croatia. Also, new cooperation possibilities will present itself, especially in the view of
cooperation with leading European researchers, which will result in transfer of the scientific
knowledge and experience, and which will facilitate the inclusion of veterinary science in ERA.
3) International cooperation of FVM with outstanding researchers should facilitate the transfer
of know-how, and increase the capacities for research in molecular veterinary medicine. This
would lead to modernisation of scientific capacities of FVM, more success in getting
international projects and better integration into ERA.
4) Prominent scientists from the FVM have concluded that the faculty should be equipped with high-tech equipment which would facilitate research in the molecular medicine, especially postgenomic research in the fields of proteomics and metabolomics.
5) All of the aforementioned structural changes at the FVM should result in the increased
development of competitivity and innovations, significantly improve research quality to
international levels of excellence and to be more successful in obtaining competitive funding. The FVM will also contribute to Western Balkan region through reinforcing the dissemination of the scientific information, and by facilitating the cooperation on the regional level.
After all, the Faculty of Veterinary Medicine SWOT Conclusions are:
Major strength
One of the major strengths of the FVM is respective number of researchers, open to structural changes, open to upgrade their knowledge, skills and research performance, as well as in competitive research funding. Moreover, there is increasing number of assistants interested in application of molecular methodology in veterinary medicine.
Major weaknesses
The lack of experience in managing large projects, and the lack of financial resources for acquisition of capital, state-of-the-art equipment for molecular veterinary medicine and financial resources for upgrading the knowledge and skills for application of proteomics and genomics in veterinary medicine is a major weakness.
Major opportunity
By upgrading of infrastructure for research, application of proteomics and genomics and by
upgrading research human potentials faculty of Veterinary medicine will be the leading institution
for research in molecular veterinary not only in Croatia, but in region and Europe as well.
By upgrading the knowledge in proteomics, application of proteomics and genomics, and upgrading, the equipment for research and research human potentials Veterinary faculty will be the leading institution for research in molecular veterinary not only in Croatia, but in region and Europe as well.
Major threats
Inadequate conditions for advance of young researchers and inability to use modern technology results in brain drain of perspective scientists in wealthier EU states, in which science and research is recognised as being a basis for better future.
Based on SWOT analysis the following recommendations were formed:
1. Enable researchers at the FVM that under the direction of an outstanding researchers and
research managers in the given field of research, significantly improve its research
performance, and to be much more successful in getting international projects.
2. The upgrade present facilities with sophisticated equipment for application of molecular
technology will enable young and motivated scientists at the FVM under the direction of an
outstanding researchers to significantly improve its research performance and gain domestic
and international research projects. At the same time, the evolution of science based on highly
sophisticated, postgenomic technology will enable just graduated individuals to form their
career in Croatia. Also, new cooperation possibilities will present itself, especially in the view
of cooperation with leading European researchers, which will result in transfer of the scientific
knowledge and experience, and which will facilitate the inclusion of veterinary science in
ERA.
3. International cooperation of FVM with an outstanding researcher should facilitate the
transfer of know-how, and increase the capacities for research in molecular veterinary
medicine. This would lead to modernisation of scientific capacities of FVM, much more
successful in getting international projects and better integration into ERA.
4. Prominent scientists from the FVM have concluded that the faculty should be equipped
with high-tech equipment, which would facilitate research in the molecular medicine,
especially postgenomic research in the fields of proteomics and metabolomics.
5. All of the aforementioned structural changes at the FVM should result in the increased
development of competitivity and innovations, significantly improve research quality to
international levels of excellence, and to be more successful in obtaining competitive
funding. The FVM will also contribute to Western Balkan region through
reinforcement the dissemination of the scientific information, and through facilitation the
cooperation on the regional level.

Scientific and technological objectives
The main objectives of the FP7-ERAChairs project VetMedZg are, under the direction of an outstanding researcher, to upgrade, encourage, develop, and enlarge the capacities for research and improve research performance in molecular veterinary medicine at the Faculty of Veterinary Medicine University of Zagreb, to be more successful in obtaining competitive funding, and to enable better integration into ERA.
The major objectives of the project are:
- To recruit, develop and retain the best researchers at the FVM
- To increase the research potential and skills of researchers in specific field of molecular veterinary
medicine under the direction of an outstanding researchers
- To increase the visibility of the researchers at international citation database (WoS) and FVM under the direction of an outstanding researchers
- To increase the number of articles that will FVM researchers publish in international citation databases under the direction of an outstanding researcher
- To increase participation researchers from FVM as a coordinators at least two Horizon 2020 project
- To establish core facility in proteomics are the FVM
- To upgrade and enlarge the laboratory equipment and research capacities at the FVM, and to enable the highest investigations in molecular veterinary medicine
- To improve know-how and experience, and transfer the highest technology and knowledge on researchers of the FVM under the direction of outstanding researchers and partner research institutions
- To increase the visibility of the FVM
- To increase the participation of researchers from FVM on European projects and to facilitate the inclusion of veterinary molecular veterinary science into ERA
- To increase the knowledge on possibilities for the protection of intellectual properties

To accomplish all these scientific and technological objectives the ERA Chairs project VetMedZg has achieved:
- employed outstanding, leading, experienced researcher (ERA Chair holder)
- employed four distinguished experienced researchers
- gained the Horizon 2020 project, as well as several bilateral international and national project
- established core facility in proteomics
- updated laboratories in molecular veterinary medicine
- organised nine workshops and many trainings for young and promising researchers
- invited eminent visiting scientist from the partnering organisations for transfer of
know-how and skills
- promoted the capabilities of the FVM by showing its scientific excellence
All these scientific and technological objectives of the ERA Chairs project VetMedZg have been realized through several work packages.

Inside WP1 - Brain gain - recruitment of ERA Chair holder and experienced researchers -at the FVM was employed one outstanding researcher directly involved in the research activities at FVM and activities at VetMedZg project. ERA Chair holder, an outstanding researcher and research manager, applied as lead scientist/principal investigator for several international/national projects at the FVM. Simultaneously, all the recruited and experienced researchers participated together with ERA Chair holder in all national and international research grants within the duration of the VetMedZg project. In addition, each of them introduced all the technology and training activities proposed for suggested WPs, as well as participated on the workshops within the duration of the VetMedZg project.

The ERA Chair holder and his team, helped the researchers from FVM through different activities such as: transfer of know-how for research in molecular veterinary medicine through training activities, lectures, organisation of workshops, meetings, writing articles in peer-reviewed research journals, as well as transfer of knowledge in organization, management and implementation of large joint international projects (Horizon 2020), as well as bilateral and national projects.
ERA Chair holder and experienced researchers (ERA Chair team) helped researchers at the Faculty to translate state of the art knowledge, skills and experience into the respective areas of molecular veterinary medicine, especially, post-genomic technology in companion and large animals, comparative tumour analysis and wildlife research, not only at the FVM but also in Croatia. ERA Chair team enabled the development of research excellence at the FVM through increased number of papers published in the database CC, SCI, and SCI expanded, which increased the average impact factor of papers published, increased visibility of the researchers at international citation database, as well as better success in obtaining competitive research funding within the duration of the VetMedZg project.

ERA Chair holder and his team stimulated scientific excellence in the field of molecular
veterinary pathology with particular emphasis on neoplastic diseases. Namely, very often molecular
genetic analysis is prerequisite for the correct diagnosis, prognosis and especially therapy of the
tumour. Translational medicine and individualisation of the therapy protocols are very recent goals not
only in oncology but also in the medicine as a whole. Experienced researchers upgraded the
knowledge of professionals in the field of laboratory diagnostics, diagnosis and therapy of neoplastic
diseases. They also increased the research excellence of FVM in the research using various ‘’-omics’’ technologies such as proteomics and metabolomics in companion and large animals, and in the same time, they increased the participation of FVM in proteomics research.

ERA Chair holder and his team enabled entrance into clinical proteomics, with focusing on biomarkers in small animals but also in farm animals. The special emphasis was on low-molecular weight region of the blood proteome that can be used as biomarkers of health and disease status in companion animals but also in the production animals and for monitoring quality traits in meat and food products. ERA Chair team also increased the competitiveness in wildlife research, especially in the field of wildlife epidemiology, with emphasis on fascioloidosis, treatment effect, levels of active components in the liver tissue and oxidative stress biomarkers in the blood determined by HPLC and compared with necropsy findings.

Cooperation of ERA Chair team with researchers at the Faculty made possible the transfer of advanced know-how and skills in modern, innovative technology, and insured the training of PhD researchers on highest international standards. New methods and technologies in investigation and study with the top scientists have provided knowledge about the higher scientific research, new knowledge and experience about the competitive funding of science. New technology and knowledge, as well as new projects provided new sources of funding, and thus a strategy towards raising research excellence in the chosen research area on a sustainable basis. All this contributed to increasing the innovation potential and economic growth.
All four established researchers were involved in three different teams to lead the research activities on different departments and clinics. One established researcher was primarily a chemist, and assigned to work with mass spectrometer. In parallel, he established a core facility for proteomic investigations, conducted training activities related to the mass spectrometer, and conducted workshops in proteomics. He participated in lectures and workshops. Also the expert was included in the process of publication of the scientific data and preparation of the project proposals. He was in everyday contact with one established researcher at the Clinic for Internal Diseases, who lead the research team in clinical proteomics and metabolomics. Their close cooperation was very important because of their association in clinical proteomic studies. Expert in proteomics and metabolomics, particularly proteomics, is extremely necessary for improvement of the overall knowledge in the field of proteomics investigations at the FVM, with the possible application in clinical research. He conducted training activities related to the proteomics, as well as workshops. He participated in lectures and workshops, as well as the process of publication of the scientific data, and future application to EU project. Both established researchers were in daily contact with the ERA Chair holder who was in direct control of the research activities. Third established researcher was employed at the Department of Pathology, and lead the group of researchers. He lead research in the field of comparative human and animal tumour pathology, work particularly based on the PCR method. He participated in workshops, and lectures. Also, the expert had to be included in the process of publication of the scientific data and preparation of the project proposals. He was also in daily contact with ERA Chair holder. Fourth established researcher was at the Department for Game Biology, Pathology and Breeding of FVM. He lead research group whose aim was to raise competitiveness in wildlife research, particularly in the field of wildlife epidemiology, with emphasis on fascioloidosis, treatment effect, levels of active component in the liver tissue and oxidative stress biomarkers in the blood. He also participated in workshops and lectures. He lead research team in applications to EU projects. ERA Chair holder and his team had daily consultations with researchers who were engaged in proteomics.
All the established researchers were under the daily supervision of ERA Chair holder. ERA Chair holder directed and controlled the ideas and research activities, giving assignments, ERA Chair holder gave ideas, elaborating them with established researchers, and gave assignments
to researchers at the FVM, applying and leading all Horizon 2020 projects at the Faculty of Veterinary
Medicine University of Zagreb.

ERA Chair team increased the different aspects of molecular medicine at the FVM, enhancing research performance and excellence, as well as innovation systems in order for FVM to become successful in a number of EU programs. In addition to these objectives, ERA Chair Holder, during the third year of the project, created own research group, and opened new research areas in order to build scientific excellence at the FVM. In regard to non-research personnel, highly educated and trained person, was recruited for VetMedZg project administration. She worked with administrative and financial issues, organisation of workshops and conferences, and engaged on the dissemination activities to increase the visibility of FVM. Her work assessed in the quality of periodic reports and questionnaires at the end of workshops and conferences.

In WP2 - Establishment the research potential to conducting proteomic and metabolomic analysis at FVM, we bought new generation of mass spectrometer that made possible for FVM to enter in-omics technology, and in the same time to re-establish core facilities at the FVM. In this sense, the major bonus was that the price of such equipment was continuously decreasing. In addition to this equipment, we purchased several instruments, which are essential to restitute, or upgrade the full capacity of the Faculty. The direct goal of the proposed activities was to coordinate the training of investigators with the efforts to upgrade and establish research potential to be served as a core facility for the whole faculty. Through different WPs VetMedZg project enabled specific training acitivities and specific instruments which improved their capacities and competence in molecular veterinary science. Inside this WP, under the direction of the outstanding researchers and our international partners from VetOMICS, University of Glasgow, Glasgow, UK, Veterinary Medicine Vienna, Austria and
MS - Mass Spectrometry Laboratory of the Chemical and Biological Technologies Institute, Portugal,
our ERA Chair project VetMedZg organized one workshop about mass spectrometry and technical aspects of animal proteomics.
Today, researchers at the FVM can participate in the proteomic research. Namely, several factors contributed to the current success of proteomic research in the world. Sample size nowadays increased to include thousands of cases. While procedures such as Western blot detect one protein per
experiment, proteomics can analyze thousands of proteins in a single experiment. In the same time
proteomics is giving us potentially unexpected changes in protein expression, interaction or
modification as a result of a treatment and in that way would facilitate the understanding of the molecular mechanism critical for cellular functions. Proteomic technologies have been applied to the identification of new biomarkers in animal disease states. To the satisfaction of the researchers at the FVM, they can use all these achievements of proteomic research in the world.
The researchers at the FVM can use mass spectrometer-based proteomics to investigate aetiology of diseases, develop predictive biomarkers in therapeutic interventions and potential strategies for treatment of complex and toxicant mediated diseases. Proteomics can be used for developing predictive biomarkers in animal health and disease. These approaches can be used also to identify a protein targets for new diagnostic tests and therapies. Currently, a proteomic approach requires a combination of efficient and stringent separation technologies and high-resolution mass spectrometry. The most common form of peptide separation in proteomic research, liquid chromatography, directly linked to mass spectrometers (LC - MS) can detect and identify all proteins down to low femtomolar concentrations. New instruments, such as Orbitraps, allow the sensitivity, accuracy, and resolution necessary to sequence peptides. So far, researchers at the Faculty of Veterinary Medicine have used old and inefficient methods, such as 1-dimensional or 2-dimensional gel electrophoresis (1 - DGE or 2 - DGE). Nowadays, researchers can use state of the art methods in the proteomic research. Except complete new Laboratory for proteomics with main instrument in the laboratory (mass spectrometer), FVM is equipped with several instruments: chromatography manager software for HPLC, laboratory freezer, low speed saw, and PCR equipment. The majority of the project members’ experiences were gained on postdoctoral fellows at the Croatia or at scientific institutions in Europe. Moreover they gained experience in designing and conducting projects in collaboration with colleagues from EU through FP7 projects (COST) or through collaboration on domestic projects.

Now, state-of-the-art research equipment enables a huge step in research investigations, at the FVM and also in veterinary science in Croatia. State-of-the-art research equipment is the base for strengthening the competitiveness and innovative capacity of FVM researchers, the base for new knowledge, excellent research, and new ideas in research. Simultaneously, outstanding researchers with their experience made it possible that researchers at the Faculty can use this research equipment in their investigation. ERA Chair holder and his team have significantly improved research performance, and allowed for much more success in the competitive funding, and at the end, an easier inclusion in the European Research Area (ERA). Positive impact of purchased apparatuses and respective technologies on the success of VetMedZg project in competition for research grants is the main and major accomplishment.

However, ERA Chair project and ERA Chair holder and his team have given a new dimension to development of post genomic technology in Croatia. Together with researchers from the Faculty of Veterinary Medicine University of Zagreb they gained structural funds project ‘’Omica approach in veterinary molecular medicine’’ (KK.01.1.1.06 Contract KK. 01.1.1.06.0004) inside call Developing and Enhancing Synergies with Horizon 2020 Horizontal Activities: Twinning and ERA Chairs. Thanks to this project, Faculty of Veterinary Medicine University of Zagreb purchased, respectively upgraded, the main instrument (LC-MS/MS) in the Laboratory for Proteomics. Nowadays, the Laboratory for proteomics can perform metabolomics research. Two researchers spent almost two months in the partner university (Glasgow Polyomics, University of Glasgow) on training in metabolomics.

Faculty of Veterinary Medicine University of Zagreb is today the only university at the Western Balcans with such equipment and possibility to perform proteomics and metabolomics research.

First conference abstract about metabolomics research in the Babesia canis infection is prepared (Rubić et al., ‘’Study of serum metabolom in canine babesiosis by mass spectrometry’’; ''Central and Eastern European Proteomic Conference'' (CEEPC), Ustron, Poland, September 23-25, 2019).
Significant success has been achieved in WP 3 - Strengthening of the research capacity in comparative tumour analysis. Namely, although the field of scientific oncology is probably the most explored area in biomedicine, comparative analysis of tumours in different domestic animals and humans is still not an established research focus. In veterinary oncology the main determinants of the biological behaviour of tumours include sex, species and breed of the animal, which also impact on prevalence and histological tumour subtype (mammary gland tumours, prostatic tumours, lung tumours, testicle tumours, mast cell sarcoma, hemangiosarcoma, eg.). These might be directly connected to diagnostics, prognosis, and therapy of malignant diseases in human and veterinary medicine as well. However, this comprehensive approach is impossible to conduct without state of the art scientific methodology which will be conducted by highly trained researchers in the field of molecular pathology. Over the last 15 years the scientists from the Department of Veterinary Pathology at the Veterinary Faculty, University of Zagreb have been trying to join the scientific efforts from all over the world to understand the reasons for such variety and to determine the frequencies and types of tumours in Croatia. All research has improved significantly under the direction ERA Chair holder and his team.
Collaboration with academic institutions in Europe particularly with the Institute of Veterinary
Pathology of the University of Zurich has been significantly improved. Several young researchers (researchers Ivan Conrado Šoštarić Zuckermann, and Marko Hohšteter) from department of Veterinary Pathology visited at different occasions this institution due to training and to join on different research investigations. Researchers Andrea Gudan Kurilj and Ana Beck spent several weeks in training in the laboratory in Zurich, but also in the Laboratory of the University of Veterinary Medicine, Kosice, Slovakia.
Researcher Šoštarić Zuckermann has during his visit to the laboratory gained new knowledge in the molecular cloning, immunoblotting.This knowledge and experience has resulted with the paper published in the journal (Šoštarić-Zuckermann, Ivan-Conrado et al., European journal of histochemistry. 60 (2016), 2; 91-98). Researcher Hohšteter applied the new knowledge and experience gained in the laboratory in Zurich, which resulted with new papers (Hohšteter, Marko et al., Veterinarski arhiv. 88 (2018) , 4; 569-579.; Hohšteter, Marko et al., Ljetopis Akademije medicinskih znanosti Hrvatske. 1 (2017) ; 10-27).
Researcher Ana Beck applied new knowledge from training in the recent paper (Beck, Ana et al., Veterinarski arhiv. 86 (2016) , 6; 857-865). During training in Zurich researcher Hohšteter gained the new knowledge about the cloning in plasmid vectors, particularly connected with Escherichia coli bacterial colonies, which will be used in the new research investigations. Resarcher Gudan Kurilj during her staying in the laboratory in Zurich gained the knowledge about molecular pathology techniques.
At the same time, focus was on the improvement of application of advanced methodology in the field of oncology, which are essential for the scientific work in oncology. ERA Chair holder and his team upgraded the knowledge of professionals in the field of laboratory diagnostics, diagnosis and therapy of neoplastic diseases. Particularly researcher Nicolas Guillemin from ERA Chair team, translated the cutting edge knowledge about how to use PCR equipment in the molecular pathology laboratory. He explained and showed the application of RT-PCR to routine laboratory work at the Department, especially in the frame of diagnosis and therapy of neoplastic diseases in veterinary medicine. On this way Laboratory for molecular pathology at the Department of Pathology Faculty of Veterinary Medicine University of Zagreb got the possibility to include in the new research. In addition to that, Department for Pathology Faculty of Veterinary Medicine University of Zagreb exchanged know-how and experience with the Institute of Veterinary Pathology The Vetsuisse Faculty, University of Zurich on the use of RT-PCR array technology and analysis as well as interpretation of obtained results. After all, the Department of Pathology has become a central place for the application of the latest methods in molecular pathology related to the diagnosis and therapy of neoplastic diseases in veterinary medicine in Croatia and beyond. All these activities resulted in the application of two grants in collaboration with ERA Chair team and researchers from abroad, but unfortunately without success in funding.
Recent efforts have been directed to the application of proteomics research in the diagnosis and therapy of neoplastic diseases using mass spectrometry in the Laboratory for Proteomics. In doing so, researchers from the Department of Pathology became involved in the latest research in veterinary medicine in the world.
WP 4 - Strengthening of the research capacity in post genomic technology at FVM is probably the main work package at the project. Namely, WP 4 was focused on establishment post-genomic technologies into veterinary science in Croatia. The ultimate goal of this WP in collaboration with outstanding foreign researchers (ERA Chair team) is to foster excellent research, particularly to develop proteomics and metabolomics at pre-clinical and clinical science.
Namely, the rapid advancement of the post-genomic technologies in transcriptomics, proteomics and metabolomics has led to the development of strategies aimed at identifying biomarkers from the thousands of molecules present in a tissue or biological fluid. The resulting pathophysiology was reflected in an altered expression of a broad range of proteins. Consequently, proteomics was increasingly being used to identify diagnostic biomarkers, monitor novel therapeutic strategies and explore the pathophysiology of prevalent and important diseases in medicine. Such as in the veterinary medicine, babesiosis is one of the disease, which is the subject of interest in many investigations. ERA Chair Holder and his team together with project coordinator have increased the research about babesiosis especially with proteomic approach.

Babesiosis, which is caused by intraerythrocytic parasites of the protozoan genus Babesia (phylum Apicomplexa), is an important parasitic disease of free-living animals worldwide. Canine babesiosis is a common and clinically significant tick-borne disease in Croatia. The disease can be divided clinically into uncomplicated babesiosis, where clinical signs are attributable to the anaemic state alone, and complicated babesiosis with well-recognised complications of B. canis infection. Complicated canine babesiosis shares a common pathomechanism with other inflammatory diseases such as malaria. Over the last 10 years, the scientists from the Clinic for Internal Diseases Faculty of Veterinary Medicine have been trying to come along the scientific effort to understand the pathogenesis of the babesiosis, especially at the molecular level. Their scientific results were published in respected international veterinary journals. The latest investigations were focused on genomic and proteomic research, like International Journal for Parasitology and the detection of two equine piroplasms, Theileria equi and Babesia caballi between 848 randomly selected, asymptomatic dogs and 81 symptomatic dogs. Namely, B. cabali has never been detected in dogs.

Recently, the focus of the babesiosis group has shifted toward the application and the role of post-genomic technologies in the biomarker discovery for animal health and disease. Early detection can lead to significant benefits in terms of efficient and timely treatment. The blood contains a multitude of unstudied and unknown proteins (biomarkers) that could reflect the ongoing physiologic state of tissues and organs. The low-molecular weight region of the blood proteome can be used also as biomarkers of health and disease status in production animals and to monitor quality traits in meat and food products. The wide range of low molecular weight proteins and metabolites in complex biological systems demands a variety of analytical platforms for detection, identification and quantification. However, the group could not continue the research without new instrument for proteomics investigation, respectively new laboratory with a lot of small equipment which were missing. ERA Chair project VetMedZg together with ERA Chair holder and his team opened a new page in the proteomic investigation of babesiosis with mass spectrometry in Croatia. ERA Chair team has helped FVM to significantly improve its research performance and to be more successful in obtaining competitive funding. The activity in WP 4, like other WPs, have resulted in increased international research collaboration, as well as increased scientific mobility at the FVM. Application of new technology under the guidance of ERA Chair team enabled cutting-edge research, which resulted in papers in high-ranking journals with high impact factor, and thereby better visibility of scientists in international citation databases. All this hasallowed better recognition of the FVM in the ERA. New methods and technologies in investigation and study with the top scientists will provide knowledge about the higher scientific research, new knowledge and experience about the competitive funding of science. New technology and knowledge, as well as new projects will provide new sources of funding, and thus a strategy towards raising research excellence in the chosen research area on a sustainable basis. Such equipment and methodology was an enormous enhancement in research potential and competitiveness in veterinary medicine at the FVM overall, and particularly in the field of babesiosis research.

Within the given field, researcher from ERA Chair team, Josipa Kuleš published a new paper (Kuleš et al., BMC Veterinary Research. 10 (2014); 1-11) about identification serum biomarkers in dogs with B. canis infection by proteomic approach. In that research a number of differentially expressed serum proteins which are involved in inflammation mediated acute phase response, complement and coagulation cascades, apolipoproteins and vitamin D metabolism pathway were identified in dogs with babesiosis. Their findings confirmed two dominant pathogenic mechanisms of babesiosis, haemolysis and acute phase response. These results may provide possible serum biomarker candidates for clinical monitoring of babesiosis and this study could serve as the basis for further proteomic investigations in canine babesiosis.
It is well known, that in babesiosis can developed systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndrome (MODS), which are of major importance in clinical practice for providing information about severity and outcomes of the disease and therapy. Researcher Josipa Kuleš published a new paper together with the members of Clinic for Internal Diseases (Kuleš et al., Research in Veterinary Science 105 (2016); 222-228) about biomarkers of SIRS and MODS in babesiosis of dogs. Scientists have found that SIRS in babesiosis was characterised by increases in paraoxonase 1 and apoA-I, whereas MODS with decrease of complement inhibitors leading to prolonged complement activation and decrease of vitamin D binding protein due to haemolysis and activation of the coagulation cascade.
Project coordinator and ERA Chair holder together with Professor Fabrizio Ceciliani were guest editors of the journal Molecular BioSystems with the themed collection: ''Omics in Animal Sciences'' (http://pubs.rsc.org/en/journals/articlecollectionlanding?sercode=mb&themeid=826968be-c72a-4db4-a563-d51cf7dc30b7). Bringing together researchers taking advantage of the omics approach in the animal sciences and allied subjects this themed collection aims to integrate various omics expertise to help provide the “big picture” necessary to move towards unravelling the complexity of biological phenomena in the animal sciences. In this collection were participated all ERA Chair team with overview papers (Kuleš et al., Molecular biosystems. 12 (2016), 9; 2680-2694; Horvatić et al., Molecular biosystems. 12 (2016); 2373-2384; Guillemin et al., Molecular biosystems. 12 (2016); 2036-2046; Galan et al., Molecular biosystems. 12 (2016), 8; 2342-2358). In these papers, ERA Chair team presented cutting edge knowledge about different themes with omics approach in the animal sciences. Such as, researcher Josipa Kuleš presented a new approaches and omics tools for mining of vaccine candidates against vector-borne diseases. Researcher Anita Horvatić in her review addressed high-throughput mass spectrometry-based technology for monitoring protein-protein interactions in vitro and in vivo and discussed about pathogens cultivation, model host cells and available bioinformatic tools employed in vaccine development. The member of ERA Chair team, researcher Asier Galan in his paper presented the latest knowledge about library-based display technologies. Researcher from ERA Chair team Nicolas Guillemin also published in the same collection very interesting review paper about omics approaches in the probe markers of disease resistance in animal sciences. All these papers presented ERA Chair team of project VetMedZg on the best way in the in the scientific community.
Project coordinator together with the members of ERA Chair team published a paper (Mrljak et al., Vector-borne and zoonotic diseases 17 (2017) 6; 398-408) about prevalence and geographic distribution of vector-borne pathogens in healthy dogs in Croatia. Namely, vector-borne pathogens are a group of globally extended and quickly spreading pathogens that are transmitted by various arthropod vectors. As the climate changes, they are a growing problem in the world and in Croatia. The main aim of the study was to investigate the seroprevalence against Babesia canis, Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato, Leishmania infantum, Dirofilaria immitis, and Ehrlichia canis in dogs in Croatia. The results of the researchers confirmed that all studied pathogens are existing in Croatia, and that Babesia canis is the most prevalent pathogens in dogs in Croatia. The research also confirmed that in dogs there is a possible coinfection with two or more pathogens in the same animal. This study is the first comprehensive survey of vector borne pathogens seropositivity conducted in Croatia.
Researcher Josipa Kuleš together with ERA Chair team and the members of the Internal Clinic published papers about diagnosis of vector-borne diseases (Kuleš et al., Vector-borne and zoonotic diseases. 17 (2017) 5; 285-296). The paper emphasizes that vector-borne diseases have been emerging or re- emerging in many geographical areas, alarming new disease threats and economic losses. It also stands out that precise diagnosis of many of these diseases remains a major challenge because of the lack of comprehensive data available on accurate and reliable diagnostic methods. In the paper is given a systematic and in-depth review of the former, current, and upcoming techniques employed for the diagnosis of vector-borne diseases.
In babesiosis there is a lot of pathological changes, with result in different clinical symptoms. Josipa Kuleš together with the members of babesiosis group published two papers about the alterations in the coagulation system during B. canis infection (Kuleš et al., Comparative immunology, microbiology and infectious diseases 53 (2017); 1-6; Kuleš et al., BMC Veterinary Research. 13 (2017); 82-1). In the first study researcher described alteration of haemostatic parameters in uncomplicated canine babesiosis. Researchers concluded that haemostatic alterations in uncomplicated babesiosis represent a procoagulant state that is mostly reversed during treatment. In the same time, antithrombin III activity was decreased as a result of a negative acute phase response, degradation by elastase, reduced availability of glycosaminoglycans, and, most importantly, consumption as a consequence of thrombin formation. In the second paper researchers confirmed that the concentration of soluble thrombomodulin, HMGB-1, VCAM-1 and suPAR were increased in dogs with babesiosis at admission compared to healthy dogs. After treatment, concentrations of TM were lower in infected dogs compared to healthy dogs. Dogs with babesiosis also had increased concentrations of TM, ICAM-1 and HMGB-1 and decreased plasminogen and PAI-1 at presentation compared to day 6 after treatment. Dogs with complicated babesiosis had higher concentrations of TM, HMGB1 and TAFI at admission compared to the 6th day. Finally, researchers concluded that biomarkers of endothelial activation and fibrinolysis were altered in dogs with babesiosis.
In one of papers, the researchers from the babesiosis group observed if the oxidative stress could be related to the severity and outcome of canine babesiosis (Crnogaj et al., BMC Veterinary Research 13 (2017), 114; 114, 9). The results of study showed significantly decreased SOD, catalase and TAS values in diseased dogs compared to controls, while there were no significant differences in GPx between these groups. Dogs that developed MODS showed lower activities of SOD and GPx and higher MDA values compared to dogs with uncomplicated babesiosis as well as with dogs that developed one complication. Superoxide dismutase, catalase and GPx were negatively correlated whereas MDA was positively correlated with the lethal outcome of the disease. Furthermore, this study detected more pronounced decrease in antioxidant biomarkers (SOD, GPx and catalase) in dogs with moderate anaemia compared to those with mild anaemia. Based on all data, researchers concluded that the results of this study showed changes in biomarkers related to the antioxidant status of dogs naturally infected with B. canis canis. These biomarkers could be used as indicators of disease severity and outcome in dogs suffering from babesiosis.
Researchers from ERA Chair team and members of the FVM participated with Spanish researcher's in the investigation of canine leishmaniasis (Martinez-Subiela et al., Veterinary immunology and immunopathology, 191 (2017), 60-67). They identified novel biomarkers for treatment monitoring in canine leishmaniosis by high-resolution quantitative proteomic analysis. With gel-free, label-based quantitative proteomic approach enabled identification of 117 canine proteins. Among these, 23 showed significant difference (p < 0.05) in expression (two downregulated and 21 upregulated ranging from 1.25 to 2.5 fold change). Comparison of gel-free TMT-based quantification and a gel-based approach previously applied to the same samples resulted in the identification of some common markers (Apo-A1, vitamin D binding protein and RBP4). However, 20 additional differentially represented proteins were highlighted by the gel-free approach, 13 of which have not been previously reported in canine leishmaniosis. In conclusion, the TMT-based proteomic approach allowed identification of new serum proteins that significantly change in concentration after canine leishmaniosis treatment. These proteins are involved in various physiopathological processes such as inflammatory, coagulation or defense mechanisms, and could potentially be suitable biomarkers for treatment monitoring of this parasitic disease.
Obesity in dogs is also one of the scientific subjects of the researchers at the Faculty of Veterinary Medicine University of Zagreb. Professor Barić Rafaj together with the member of ERA Chair team and researchers from partner institution in Spain has investigated plasma markers of inflammation and hemostatic and endothelial activity in naturally overweight and obese dogs (Barić Rafaj et al., BMC Veterinary Research, 13 (2017), 13-1). Obesity is a huge problem in humane population. Based on this facts, this research investigation is gaining importance. This study was designed to evaluate the effect of naturally gained overweight and obesity in inflammatory, hemostatic, fibrinolytic and endothelial biomarkers of dogs. Researchers concluded that weight excess in dogs reflects a chronic low degree of inflammation, shifts the hemostatic balance and features a hypercoagulable state, where primary and secondary hemostasis are both affected. Overweight and obesity are not associated with impairment of endothelial function in dogs.
A group of authors including the project coordinator and member of Clinic for Internal Diseases Faculty of Veterinary Medicine published a paper about human and canine obesity (Munoz-Prieto et al., Scientific Reports. 8 (2018), 1; 1-10). Authors analized owner perception of obesity and factors associated with human and canine obesity. Obesity is a common nutrition-related disorder leading to reduced life expectancy in both humans and dogs. The researchers concluded that common factors associated with obesity in owners and their dogs were age, gender and owners’ attitudes to diet and physical activity. Dog owners who did not consider obesity to be a disease were more likely to have obese dogs.
The member of ERA Chair team Josipa Kuleš together with project coordinator and members of Clinic for Internal Diseases published papers about kidney damage markers in canine babesiosis caused by Babesia canis (Kuleš et al., Ticks and Tick-Borne Diseases 9, (2018), 6; 1508-1517). Authors evaluated glomerular and tubular kidney damage markers in canine babesiosis caused by Babesia canis. All tested markers, with exception of uromodulin, showed significant differences between dogs with babesiosis and healthy dogs, and also showed strong or very strong positive correlation with UPC. Increases of urinary albumin and IgG suggested glomerular damage, and increases of KIM-1, RBP and NAG proximal tubular damage in dogs with babesiosis. They demonstrated clear advantages compared to conventional parameters by showing earlier changes in detecting renal damage.

Researcher Lorena Franco Martinez together with the members of ERA Chair team and project coordinator published papers about changes in salivary analytes in canine parvovirus and a high-resolution quantitative proteomic study. Bioinformatics’ analysis revealed that saliva reflects the involvement of different pathways in parvoviral enteritis (PVE) such as catalytic activity and binding, and indicates that antimicrobial humoral response as a pathway with a major role in the development of the disease. These results indicate that saliva proteins reflect physiopathological changes that occur in PVE and could be a potential source of biomarkers for this disease.
Bilić Petra, PhD student at the Faculty of Veterinary Medicine published paper together with member of ERA Chair team and project coordinator about babesiosis. Researchers presented cutting edge knowledge about canine babesiosis (Bilić et al., Acta veterinaria (Beograd). 68 (2018), 2; 127-160). In another paper researcher Bilić together with ERA Chair holder and his team published paper about proteomics in veterinary medicine and animal science (Proteomics, 18 (2018), 1800047, 7 doi:10.1002/pmic.201800047). The researchers provided an overview of the latest insights into proteomics in veterinary medicine and animal science.
Bilić Petra, PhD student and ERA Chair team together with researchers from Clinic for Internal Diseases University of Zagreb and Glasgow Polyomics University of Glasgow published paper about serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach (Bilić et al., J. Proteomics 179, (2018) 110-121). Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, researchers used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril- associated glycoprotein 4 and apolipoprotein A- IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain.
In one of the last papers ERA Chair team has had cooperation with different researchers from abroad, but cooperation with researchers from Spain was very common. Namely, researchers analysed changes in serum analytes and possible metabolic pathways associated with canine obesity-related metabolic dysfunction (Tvarijonaviciute et al., Veterinary journal 244 (2019); 51-59). Proteomic analysis revealed that 23 proteins related to lipid metabolism, the complement factor system, cellular adhesion and functionality, inflammation, and coagulation were altered in dogs with obesity-related metabolic dysfunction (ORMD). Also, these data suggested that canine ORMD is associated with changes in analytes that reflect altered lipid metabolism, and liver and immune function impairment and suggests the potential for a prothrombotic state and lung function alterations.
Investigating saliva from dogs with and without obesity-related metabolic dysfunction by proteomic approach (Lucena et al., Journal of Proteomics 201 (2019); 65-72), project coordinator in cooperation with researchers from Spain concluded that salivary proteome of obese dogs with ORMD was different in comparison with obese dogs without ORMD. Researchers also concluded that approximately 20% of dogs with naturally occurring obesity were described to suffer ORMD associated with insulin resistance and hypoadiponectinemia, the fact that indicates possible links between ORMD and associated diseases.
The ERA Chair team was also active in the study of cytokines in canine babesiosis. Researcher Asier Galan together with researchers from Clinic for Internal Diseases Faculty of Veterinary Medicine University of Zagreb and researcher from University of Murcia, Spain concluded that cytokines MCP-1, KC-like and IL-8 as critical mediators of pathogenesis caused by Babesia canis (Galan et al., PLoS One 13 (2018), 1; 1-21). Also, researchers have come to the conclusion that infection by B. canis elicits a cytokine pattern that is distinct from that observed with B. rossi and that some of the inflammatory mediators can be useful to predict complications.
In another paper by a researcher from the ERA Chair team together with researchers from Clinic for Internal Diseases Faculty of Veterinary Medicine in Zagreb was analysed serum phosphoproteome in dogs with babesiosis caused by B. canis (PLoS One (1932-6203) 13 (2018), 11; E0207245, 24). Namely, phosphorylation is the most commonly studied protein post-translational modification (PTM) in biological systems due to its importance in controlling cell division, survival, growth, etc. In this reasearch was compared serum from 10 healthy dogs and 10 dogs affected by B. canis- caused babesiosis with no organ dysfunctions by employing gel-free LC-MS/MS analysis of individual samples and tandem mass tag (TMT) label-based quantitative analyses of pools, both supported by phosphopeptide enrichment. Results showed a moderate number of phosphorylated proteins (50–55), with 89 phosphorylation sites not previously published for dogs although a number of them matched phosphorylation sites found in mammalian orthologs. Three phosphopeptides showed significant variation in babesiosis-affected dog sera compared to controls: Serum amyloid A (SAA) phosphorylated at serine 101 (up-regulation), kininogen 1 phosphorylated at threonine 326, and fibrinogen α phosphorylated at both threonine 20 and serine 22 (down-regulation). This study represents the first attempt to characterize canine serum phosphoproteome.
ERA Chair holder and his team gave support in the investigation of farm animals. ERA Chair holder David Eckersall published two papers in the themed collection: Omics in Animal Sciences (Thomas et al., Mol. BioSyst., 2016,12, 2762-2769; Mudaliar et al., Mol. BioSyst., 2016,12, 2748-2761).
ERA Chair holder and his team were also involved in investigating poultry diseases. In one paper, they applied quantitative proteomics using tandem mass tags in relation to the acute phase protein response in chicken challenged with Escherichia coli lipopolysaccharide endotoxin (Horvatić et al., Journal of Proteomics 192 (2019); 64-77).
Researchers from ERA Chair team gave their support with one chapter in the book (Horvatić et al. Methods in Molecular Biology: Host-pathogen interactions, Methods and protocols / Medina, Carlos; López-Baena, Francisco Javier (ed.). New York : Springer, Humana Press, 2018.; 83-96). In the chapter researchers described the methodology for the enrichment and identification of pathogen surface proteome using cell surface protein biotinylation followed by LC-MS/MS and bioinformatic analyses of such data. Researchers concluded that this strategy could be employed for the determination of protein subcellular localization and prediction of potential pathogen interacting proteins.

The WP5 Increasing the competitiveness in wildlife research is focused on the improvement of wildlife research with emphasis on understanding of problems related to more efficient treatment of fascioloidosis. Its main goal was to establish excellence in wildlife research and to acquire practical knowledge and experience in advanced HPLC, age determination and epidemiology of wildlife diseases through collaboration with ERA Chair team. The main aim of the WP5 program was to strengthen the capacity for application of wildlife epidemiology and toxicology in veterinary pre-clinical and clinical science. Increasing human population has led to increased contact between domestic animals and humans with wildlife, enabling easier spread of mutual diseases but also ensuring outbreaks of zoonotic diseases that were until recently of minor importance. Therefore, means for establishment of safer environment and conservation of endangered species (and ecosystems) play increasingly important role, truly defining the concept ‘’one world one health’’.
During the first part of the VetMedZg project implementation obtained and improved knowledge on molecular diagnostic techniques, wildlife diseases, wildlife epidemiology and age evaluation of wildlife were used within the project of the Croatian Science Foundation (CSF) UIP 3421 ''Molecular epidemiology of selected parasitic diseases of wildlife'', which financed the research part. From that integration following papers were published:

Sindičić, M., F. Martinković, T. Strišković, M. Špehar, I. Štimac, M. Bujanić, D. Konjević (2017): Molecular identification of the rumen flukes Paramphistomum leydeni and Paramphistomum cervi in a concurrent infection of the red deer Cervus elaphus. Journal of Helminthology 91, 637-641 - in this research WP5 researchers have applied knowledge obtained on molecular diagnostic tools, to identify for the first time the ruminal fluke Paramphistomum leydeni in wild cervids in Croatia. Amplification of DNA extracted from 61 ruminal flukes led to a 320-bp product. In 59 samples, the sequence was identical to a P. leydeni, while the remaining two corresponded to P. cervi. Researchers also compared morphological and molecular diagnosis with a conclusion that previous reports of paramphistomosis should be interpreted with caution, since many authors did not report attempts to differentiate these two species. Research was supported by the CSF (Croatian Science Foundation) grant.

Konjević, D., M. Bujanić, V. Erman, A. Gudan Kurilj, T. Živičnjak, K. Severin, S. Tomić, F. Martinković (2017): New data on wild boar (Sus scrofa L.) a dead-end host for large American liver fluke (Fascioloides magna). Helminthologia 54, 77-80 - one of the goals of the WP 5 was to improve research on fascioloidosis in Croatia. Previously established research group benefited from experienced researchers recruited on the ERAChair project and also from exchange of knowledge with Professor Peter Lazar. This was applied in this research financed by CSF. The impact of Fascioloides magna on wild boar as dead-end host was rarely described in scientific literature. The common conclusion of those papers is that wild boar forms thick-walled pseudocyst leading to the death of the flukes before reaching sexual maturity. In our paper we have described results of one year monitoring conducted on the area of Posavina. Along with the previously reported thick-walled pseudocysts we have also described a thin-walled pseudocyst (characteristic for typical hosts) with sexually matured (7 cm long flukes) with numerous eggs. With this finding we have described for the first time potential change in host-parasite interaction between wild boar and F. magna. Research was supported by the CSF grant.

Škvorc, N., M. Bujanić, J. Grbavac, D. Konjević (2018): Application of dental incremental lines in age estimation of roe deer (Capreolus capreolus L.). Hrvatski veterinarski vjesnik - Hrvatska veterinarska komora 26 (5-6), 40-44 - proper age estimation is one of crucial factors for better management of wildlife populations. In this paper using new Isomet diamond saw we cut teeth of 36 roe bucks collected at the area of Zagreb County. On tooth sections cementum annuli were counted. Majority of animals were aged at 3 years (n=24, 66%), followed by 2 years (n=9, 25%) and 4 years old (n=3, 8%). Mean value is 2.83 years. If potential error is included, majority of shot animals are between 3 to 5 years of life. This pattern shows bias from hunting plan, but also points out on a certain dynamic of body and trophy development in different habitats.

Konjević, D., Z. Janicki, P. Calmels, D. Stojčević Jan, A. Marinculić, M. Šimunović, M. Pavlak, K. Krapinec, Z. Poljak (2018): Evaluation of factors affecting the efficacy of treatment against Fascioloides magna in wild red deer population. Veterinaria Italiana 54, 33-39 - the disease (fascioloidosis) control program (DCP) was launched in 2006 in the hunting grounds managed by Croatian Forests. Since then it was regularly conducted with several short-term interruptions. The prevalence of positive animals was 36.42 % (excluding fawns). The epidemiological analysis was applied to analyse relationship of demographic and environmental factors that contributed to maintaining of relatively high prevalence despite the DCP. Each demographic variable was tested at the seasonal, individual and location level. Model for pathological lesions suggested that the likelihood of lesions was dependent on age (p=0.003). We did not find any locality or sex related significant differences. Finally, environmental characteristics and migratory patterns were analysed using Geographic Information System (GIS) and showed that Spačva region represents an epidemiological unit for red deer fascioloidosis. Research was supported by the CSF grant.

Sindičić, M., M. Bujanić, I. Štimac, F. Martinković, N. Tuškan, M. Špehar, D. Konjević (2018): First identification of Echinococcus multilocularis in golden jackals in Croatia. Acta Parasitologica 63, 654-656 - tapeworm Echinococcus multilocularis is a causative agent of alveolar echinococcosis one of the world’s most dangerous zoonosis and an emerging disease with growing incidence in humans. For a long time there was no confirmation of E. multilocularis in Croatia. In 2016 it was confirmed in red foxes. In this paper we have analysed 29 carcasses of golden jackals and confirmed presence of E. multilocularis in one animal using molecular techniques. These results suggest that alveolar echinococcosis should be treated as an emerging disease in Croatia. Research was supported by the CSF grant.

Čurlík, J., D. Konjević, M. Bujanić, Ž. Sabol, F. Martinković, M. Sindičić (2019): First description of Setaria tundra (Issaitshikoff & Rajewskaya, 1928) in roe deer from Croatia. Helminthologia 56, 252-255 - Genus Setaria, Viborg 1795, comprises 46 species that parasitize in the peritoneal cavity of Artiodactyla, Perissodactyla and Hyracoidea. The majority of these infections pass unnoticed, but occasionally they can induce severe peritonitis or neurological signs in aberrant hosts and, rarely, even in humans. In Croatia, Richter (1958) conducted one study of roe deer parasites and confirmed Setarial nematodes in 34% of animals. He used morphological keys for identification and concluded that observed parasites were Setaria labiatopapillosa. In this paper we have analysed 45 complete digestive systems of roe deer and found Setaria spp. in 24.44 % of samples. Following the DNA isolation a 514 bp subunit of cox1 gene was amplified. Comparison with archived data in GeneBank revealed that analysed nematodes belong to the species Setaria tundra. This was the first scientific proof of existence of S. tundra in roe deer in Croatia. However, the impression in our case is that these nematodes were present in the parasitic fauna of roe deer long before this description but where misdiagnosed as other Setaria species due to the high morphological similarity and low quality of diagnostic equipment at that time. Research was supported by the CSF grant.

Arbanasić, H., D. Konjević, L. Vranković, M. Bujanić, S. Stipoljev, M. Balažin, N. Šprem, D. Škorić, A. Galov (2019): Evolution of MHC class II SLA-DRB1 locus in the Croatian wild boar (Sus scrofa) implies duplication and weak signals of positive selection. Animal Genetics 50, 33-41 - the wild boar is autochthonous wild species in Croatia with expanding range. It is also an ancestor of the domestic pig. Although a large amount of data are available on major histocompatibility complex (MHC) variability in domestic pigs, only a few studies have been performed on wild boars. Due to their crucial role in appropriate immune responses and extreme polymorphism, MHC genes represent some of the best candidates for studying the processes of adaptive evolution. In this research we have presented the results on the variability and evolution of the entire MHC class II SLA- DRB1 locus exon 2 in 133 wild boar from Croatia. Using direct sequencing and cloning methods, we identified 20 SLA-DRB1 alleles, including eight new variants, with notable divergence. In some individuals, we documented functional locus duplication, and SLA- DRB1*04:10 was identified as the allele involved in the duplication. The expression of a duplicated locus was confirmed by cloning and sequencing cDNA-derived amplicons. Based on individual genotypes, we were able to assume that alleles SLA-DRB1*04:10 and SLA- DRB1*06:07 are linked as an allelic combination that co-evolves as a two-locus haplotype. Our study of evolutionary processes at the SLA-DRB1 locus confirmed the role of intralocus recombination in generating allelic variability, whereas tests of positive selection based on the dN/dS (nonsynonymous/synonymous substitution rate ratio) test revealed atypically weak and ambiguous signals. This research beside the knowledge on variability of MHC genes in wild boar also makes basis for studies in molecular epidemiology, i.e. adaptation of the organism on different pathogens. Research was supported by the CSF grant.
Project application
Based on the previous CSF project (UIP 3421), obtained knowledge on molecular and postmolecular techniques, established lab and research group, WP5 researchers have applied for new CSF project, IP 8936 ''Host-parasite interactions: differences in relation between three types of hosts to Fascioloides magna infection’’. Project proposal was evaluated and granted for the following four years.
Short abstract of the project:
Parasitic infections represent almost 70% of all wildlife diseases. While parasites and their hosts have evolved together over a thousands of years, non-native parasites can represent serious threat to naïve hosts. Fascioloidosis is a parasitic disease caused by non-native digenean trematode Fascioloides magna, also known as a large American liver fluke. Originally a parasite of the North American deer species, F. magna was introduced to Europe at least twice, and consequently had spread over a vast area along the Danube River. In Croatia it was described for the first time in 2002 in red deer from Baranja region. According to its relation to hosts, three types of final wild hosts can be recognized in Europe: definitive hosts (red and fallow deer), dead-end hosts (wild boar) and aberrant hosts (roe deer, mouflon and chamois). As expected, this parasite attracted large scientific interest, resulting in numerous papers describing its first appearance, gross and microscopic lesions of livers, intermediate hosts, treatment possibilities and lately genetic characteristics of the parasite. However, host-parasite interactions responsible for the outcome of the disease (type of host) still remains unclear. In its final host’s parasite migrate through the liver, stops at one place and pseudocyst is formed around it. In dead-end hosts pseudocyst formation is excessive, forming a thick-walled pseudocyst that eventually lead to the death of the parasite. In aberrant hosts parasite continues to migrate causing excessive damage of the liver parenchyma with haemorrhages. Such course of the events finally results in host’s death. Only final hosts shed eggs into the environment. Such type of disease in roe deer is one of the factors that led to a decline of roe deer population in areas infested with F. magna. The aim of this project is to understand the mechanisms of host-parasite interactions in different types of hosts. It is based on two hypothesis: i) the continuing migration of a parasite is a result of inadequate host immune response, and ii) the continuing migration of a parasite is a result of its inability to reach sexual maturation. Consequently project goals are: a) to analyse gross and microscopic liver lesions in different types of hosts, b) to understand immune responses to F. magna in different types of host, c) to compare variability/presence of the specific alleles of the MHC genes in different types of hosts, d) to analyse different stages of the flukes in different types of hosts, with emphasis on sexual maturity, e) to assess chemical composition of the livers in different host types. To achieve these goals a case-control study and sampling based on the disease status will be applied. Samples of blood, whole livers and flukes will be collected following regular hunting operations. Thorough examination of each liver will be used to classify animals as negative, infected (livers containing cysts) and reinfected (livers containing cysts and migratory stages of the fluke). Animals from disease free areas (parts of the Zagreb County, Moslavina and Gorski kotar) will be used as full negative controls. Methods applied to achieve project goals include parasitological analysis of livers and faeces; whole proteome analysis approach will be used to pinpoint the essential parasite proteins relating to virulence, to investigate important parasite protein interactions (proteins that are correlated in expression or additive in their effect) and to identify mechanisms to prolong infection; exon 2 of MHC class II loci will be analysed using classical approach and Next generation sequencing; collected different stages of parasites will be sectioned and analysed for maturity; chemical composition of liver samples will be assessed. The analysis will be performed mainly by the team formed during the installation grant (UIP 3421), strengthened by ERA Chairs (VetMedZg) member for proteomic analysis and domestic and foreign expert in fascioloidosis research. The deliverables of the proposed project should provide answers for important host-pathogen interactions in fascioloidosis.

Potential Impact:
Strategic impact of the ERA Chairs project VetMedZg
Considering potentials and limitations of current development of biomedical research at the Faculty of Veterinary Medicine University of Zagreb (FVM), the project has a strategic impact on the development of the research capacity and capability of the veterinary science at the FVM at several levels. The VetMedZg project has also created an impact on complete veterinary science in Croatia, as well as positive influence on the region of Western Balkan. All of strategical measures proposed in this project have hopefully reinforced the existing research teams through attracting to FVM the ERA Chair holder and experienced researchers who gained their expertise in the leading research institutions of the world. All top level scientists employed for the project have been participating in research activities at the FVM, thus, their input was stimulating the research performance and excellence at the entire Faculty. ERA Chair team has helped FVM to ensure multiple interactions with high profile researchers from collaborating institutions, thus improving the levels of integration of FVM in the ERA.
In addition, strategical measures proposed in this project were aimed at upgrading the RTD capacity and capability of the FVM through the upgrade of present laboratories with modern equipment and implementation of new research methods, especially in the fields of proteomics and metabolomics.
New equipment and novel research technologies have upgraded the research potential so that FVM has moved forward to becoming a leading veterinary scientific institution in the broader region. Improving the RTD capacity and capability has led to better utilization of current human potentials through recruitment of new researchers and increased mobility of existing scientific staff. The founding of the proteomics laboratory has made it possible to apply new research and innovative technologies in veterinary medicine.
The activities planned in the project through workshops, trainings and visiting professorship programmes have enabled for the FVM to increase the research performance and excellence, to become more successful in obtaining competitive funding, as well as to increase its visibility and widen its partnership with leading European R&D organizations. The project workshops have additionally enabled FVM to become a mediator in dissemination of new technologies in the broader area, providing research platform in veterinary medicine for Western Balkan region and closer cooperation with regional centres.

The project has also created various positive impacts at the national and EU level through better integration of FVM into ERA, and strengthening of its existing and new collaborations. The expected results of the VetMedZg project have increased the opportunities for the involvement of FVM in Horizon 2020 projects, and participation in other grants as well. By realization of the project, FVM has become one of the leading institutions for research in molecular veterinary medicine not only in Croatia, but in region as well.
Faculty of Veterinary Medicine University of Zagreb is the highest scientific and educational veterinary institution in Croatia, with a relatively young educational staff, open to changes at the beginning of the 21st century. The SWOT analysis presented in the VetMedZg project had identified several weaknesses, which have been managed to change with the implementation of ERA Chair programme. Faculty of Veterinary Medicine became one of the leaders in the molecular medicine with the realization of the ERA initiative. The existing teams on the Faculty have been reinforced by attracting to FVM a number of new, experienced researchers who gained their expertise in the leading research institutions of the world. All top-level scientist employed for the project have participated in research activities at the FVM, thus, their input stimulated the development of scientific environment at the entire institution. With various activities, the development of the post-genomic technology in veterinary medicine in Croatia has been upgraded, and at the same time, the capacities in molecular pathology on several different departments of the FVM have been increased. All of the proposed has resulted in better scientific and research cooperation between the FVM departments and clinics, and cooperation with partners in neighbouring countries of the Western Balkans. All the activities in the framework of WP1 and WP2 have enabled FVM to increase its potentials in the fields of molecular medicine, and application of post-genomic technology, which resulted in the opening of new research areas in veterinary medicine. The acquisition of the planned equipment has led to establishment of the core facility at the FVM, and at the same time, has led to the application of proteomics and metabolomics in veterinary medicine in Croatia. The newly acquisitioned equipment under the direction of ERA Chair holder and recruited experienced researches, has led to a new research spurt, design of new studies and more successful obtaining of competitive funding in the ERA. With the VetMedZg project cooperation between the Laboratory of proteomics and newly equipped laboratories of the departments and clinics of FVM were increased, and the result was a formation of new research teams at the FVM. Now, such interdisciplinary cooperation, based on the application of molecular research, especially post-genomic technology, proteomics and metabolomics, has been made possible.
WP3, WP4 and WP5 had been designed to support transfer of knowledge and experience from ERA Chair holder and experienced researches and outside partners to the members of VetMedZg project, and all researchers at the FVM, through upgrading the capacities for research in molecular medicine. By increasing the capacity for research in molecular medicine through VetMedZg ERA Chairs project, human and material resources at the FVM (training members of VetMedZg project in molecular medicine, procurement of equipment, equipment upgrade, and recruitment of experienced researchers as well as know-how transfer from EU) have been strengthened, making the FVM to become suitable for integration into ERA.

The ERA Chairs project has led to strengthening of the research capacity in comparative tumour
analysis through obtaining specific know-how and skills from the experienced researchers and partner institutions for the methodology of development in the molecular pathology field. The VetMedZg ERA Chairs project improved existing technologies in molecular pathology through upgrading infrastructural and intellectual support for development of molecular pathology in the diagnosis and therapy of tumour diseases of animals aimed within the WP3.
The first impact of the VetMedZg ERA Chairs project was setting up of the Laboratory of Proteomics at the FVM, respectively, better put, the development of post-genomic technology in veterinary research in Croatia. Core facility will continue to serve as a central support unit to most research groups involved within VetMedZg and also for the rest of the researchers at the FVM. The project has resulted in the application of proteomics in veterinary science, especially in clinical science. One of the great impacts of the VetMedZg ERA Chairs project is application of proteomics and metabolomics in biomarkers discovery, especially discovery of proteins in connection with the development of complications of canine babesiosis. At the same time, the VetMedZg project has contributed to the strengthening of the application of proteomics in companion and large animals, aimed within the WP4.
The VetMedZg ERA Chairs project has resulted in improvement of the understanding of treatment of fascioloidosis in wildlife animals, and better understanding of pharmacokinetics of triclabendazole metabolites and their withdrawal time from the liver tissue. In addition, another impact of the project within the WP5 is the establishing of a laboratory for independent age determination in cooperation with Department of Pharmacology and Toxicology.

a) Impact on development of research capacity and research excellence in combining different funding streams
In order to fostering and conduct of structural changes in veterinary medicine at the FVM, ERA Chairs initiative has been accepted, with final aim to raise research quality at the Faculty to international levels of excellence. Simultaneously, the ERA initiative has made it easier to propose new structural projects and on such way has been increased the capacity building of the institution. Based on the ERA Chairs initiative FVM had the possibility that young scientists at the FVM gain knowledge and skills and additionally apply molecular technologies in their research. That has made easier structural changes at the FVM, and has facilitated necessary additional financing through Cohesion funding, so that synergies between cohesion and research funding are strengthened. This way, new research possibilities and excellence in various fields of veterinary molecular medicine have opened for FVM. That was encouraging for scientists to start new ideas and research during the lifeline of the project. The ERA Chairs project opened a possibility to set up new spin-off companies (especially in the area of proteomics- ‘’Center for Proteomics’’) which would increase incomes for the FVM in the future.

b) Impact on the increased participation of the FVM in Horizon 2020 and other important European programmes; effectively widening participation
All measures under the ERA Chairs initiative enabled the FVM to increase its applications for EU grants. The proposed activities under the direction of ERA Chair holder and his team have made it possible for the FVM to rise up on the quality scale, and become a real scientific and research leader in the region. At the same time, the VetMedZg ERA Chairs project made possible, through its comprehensive actions, the full competitiveness of FVM in the European Research Area. The newest trends are already visible, since several scientists at the FVM are partners in the Horizon 2020 research projects. The ERA Chairs project enabled that FVM will try to initiate and coordinate more new framework programmes of EU and other important European programmes on national and international levels.

c) Impact on compliance with the ERA priorities
The FVM has accepted all ERA priorities. Inside ERA Chairs project VetMedZg were included all ERA priorities. Based on the activities in the project, FVM raised up on the quality scale, and has positioned itself better at the national level and in a new common European Research Area. At the same time better implementation of all the proposed measures has enabled FVM greater investment in science. This will result in sustainable science. The ERA Chairs project has enabled transnational cooperation at regional level (in cooperation with the countries of the Western Balkans) and with the developed countries of Europe. Increased quality of scientific research in veterinary science of Croatia has allowed greater competition in the European space. VetMedZg project has enabled the construction of their own scientific system based on own strength, in line with smart specialisation strategy. This generates both more competition and more cooperation. The project enabled the conditions in which researchers, scientific knowledge and technology circulate freely, and that Croatia strengthens its scientific and technological bases, and finally its competitiveness. In parallel, the greater possibility of training in various scientific laboratories enables greater mobility, and on that way the basis for attractive research careers. Based on the experience of the ERA project, FVM completely accepted gender equality through integration of a gender dimension and using incentives in all future projects. All ERA priorities have become the standard at the FVM including open recruitment, gender balance, peer review and doctoral training.

d) Impact on stronger interaction with national and regional authorities designing and implementing research and innovation strategies for smart specialisation
With acceptance and conduction of the all measures inside the ERA Chairs initiative, researchers at the FVM became active players in ERA. FVM has strengthened research potential by attracting outstanding researchers to research institution. In addition, FVM reinforced collaboration with the partnering organisations from different EU countries (Spain, Austria, Switzerland, Portugal, Great Britain, Italy, and Greece). At the same time, FVM has been cooperating with several national authorities (Ministry of regional development and EU funds, Ministry of Science, Education, Croatian Veterinary Institute, School of Medicine Rijeka, and Institute Ruđer Bošković). That way, ERA Chair initiative is an opportunity for FVM to take responsibility for reforming their research system, driving a process of smart specialization and helping to close the innovation divide. ERA Chairs project VetMedZg enabled competitive advantages FVM in Croatia and the Western Balkan region. In addition, ERA initiative enabled the strengthening of the regional innovation systems, to maximize the flow of knowledge and spread the benefits of innovation by region. Researchers at the FVM identified areas that have the best potential for innovation through the upgrading of research potential in the field of molecular medicine, especially in proteomics and metabolomics. Namely, Ministry of Economy is determined within benchmarks of SMART specialization strategies for Croatia as one of the priority sectors, the advanced technology, nano-technology, and bio-technology. Faculty of Veterinary Medicine, after appliance of all research results within VetMedZg project, is able to join the food and the health cluster of Croatia, and thus become regionally competitive. All of that has increased the innovation potential of the Faculty and veterinary science in Croatia and contributed to economic growth. All of these conducted activities on the VetMedZg project have unlocked and developed research excellence. The project increased the visibility of excellence of the FVM and ensured the setting up of long lasting strategic partnership in ERA. Proteomics and metabolomics are powerful instrument to tackle social and environmental challenges. In addition, proteomics and metabolomics methods ensure that research and innovation resources reach critical mass, and become self-sustaining or critical potential to boost human resources and knowledge infrastructure. All these activities have contributed the sustainability of FVM like regions' RTD competitors.
e) Impact on research activities to contribute to regional/national development plans on research infrastructure and innovation
ERA Chair project VetMedZg has enabled development of regional/national plans on research infrastructure and innovation through establishing the novel technology and introducing it into the veterinary science in Croatia. The application of the state-of-the-art equipment in the field of proteomics with simultaneous transfer of knowledge by the leading post-genomic scientists has led to increased research excellence in Croatia, and even wider, in the region of Western Balkans. The application of proteomics and metabolomics in the discovery of new biomarkers of disease and health, and its direct control in clinical practice, i.e. in research of protozoal diseases treatment (especially in babesiosis and leishmaniosis, problems with obesity in dogs), in research of farm animal production, were some of the fields in which the highest impacts were achieved. Based on the results of project activities, innovativeness is manifested through a new approach to therapy of wildlife diseases and its application in the area of the natural reserve in Baranja, eastern Croatia, respectively through the facts that transfer of know-how and skills gave answers to questions regarding effectiveness of therapy of wildlife diseases. The results of the project also contributed to the wellbeing of Croatian science through a new approach and application of proteomics and metabolomics in discovering new biomarkers, and new approach to therapy and control of wildlife diseases, as well as diagnosis of leishmaniosis.

f) Impact on the research capacity for an effective contribution to regional economic and social development
Application of new methods in the science at the FVM and Croatia research significantly increased research potential, and contributed to regional economic and social development. Namely, global approaches that screen large numbers of molecular targets simultaneously are playing increasingly important roles as discovery tools in the basic biological and clinical sciences. Especially, the rapid progress in the post-genomic technologies of proteomics and metabolomics has led to the development of strategies aimed at identifying biomarkers from the thousands of molecules present in a tissue or biological fluid. VetMedZg project and new methods of research have served as a motivator on the local level and on other institutions of the region. That way, the project had an impact on the development of veterinary research potential to organize training and networking activities in the post-genomic technologies. One of the important aspects of the ERA Chairs project is building of regional capacity, mainly through reduction in regional disparities in the region of the Western Balkans. The formation of the collaborative milieu between VetMedZg and different scientific groups within the region has contributed to a more geographically balanced scientific and economic development. Based on the results of the project, FVM became a positive example, and at the same time, a supporting institution in the process of development of similar institutions with capacities comparable to those found in the EU. The new generation of veterinary scientists trained at FVM can induce the founding of biotech companies and high-tech enterprises in Croatia. It is widely known that the quality of veterinary research personnel, or better said scientists at a university level is mainly dependant on the quality of veterinary education. Because of that, the implementation of all aims of the VetMedZg project has resulted in extremely positive effect on veterinary education in Croatia, and on veterinary care quality. The VetMedZg project opened a new chapter in the veterinary science in Croatia, by bringing the post-genomic technology into various disciplines head-on. New initiatives in the Croatian research contributed to the challenges on how to improve economic and financial situation in Croatia and in the region of Western Balkan. VetMedZg project contributed to the improvement of the economic situation in the region by organizing numerous international workshops with experienced researchers from EU as well as young researchers from the region thus facilitating communication between researchers with similar scientific interests. This improved the response to socio-economic needs of the region.

g) Impact on the level of interaction of the ERA Chair with additional stakeholders, such as clusters or connections to industry/SMEs
The ERA Chair initiatives has had significant impact on the development of biomedical research at the Faculty of Veterinary Medicine University of Zagreb (FVM). The enhancement of the research potential of the FVM has been realised by attracting outstanding researchers. ERA Chair significantly improved research performance of FVM through activities designed to introduce new knowledge and novel, innovative technologies in the research with the aims to stimulate economic growth. In the same time, ERA Chair encouraged innovation in research, and their beneficiaries may be meat and dairy industry. Namely, there is an increasing awareness of the potential of proteomic technologies to study production animals. Proteomics has also been used to characterize disease states in production animals, but also proteomic strategies have been employed to measure the dynamics of muscle growth in poultry and advance farm animal reproduction. To make research results in the FVM, specifically the results of VetMedZg project, closer to stakeholders and industry, we presented the results of the project to meat and dairy industry, respectively to the different farms of milk cows. In the future, the result of the ERA Chair initiative could be collaboration with the pharmaceutical industry and food industry, start-ups, two-way exchange of staff. All together the results of the project have influenced on a possible economic growth, but also on human health and animal welfare. ERA Chair influenced on closer cooperation with regional centres (Veterinary Faculty University of Belgrade, Veterinary Faculty University of Sarajevo). Namely, the formation of the collaborative milieu between VetMedZg and different scientific groups within the region contributed to a more geographically balanced scientific and economic development.

Dissemination is an important and critical component of this project, and therefore has been assigned to a separate work package, WP6. The dissemination activities were conducted through the whole period of the VetMedZg ERA Chairs project implementation. The main objectives were: a) to increase the visibility of the Faculty of Veterinary Medicine University of Zagreb on national, regional and EU level; b) to increase the visibility of the researchers from the Faculty of Veterinary Medicine University of Zagreb in the international citation databases; c) to enlarge the presence of researchers in the community through dissemination activities, and in this way reduce the gap between researchers and the public; d) to promote the capacity of the FVM by showing its scientific excellence and to attract new possibilities of collaboration with industry and additional stakeholders; e) to increase the capacity of FVM to compete for structural and cohesion funds.

One of important dissemination activities are published papers. Through the papers, ERA Chair project presented the main research fields and results conducted during the project, and in the same time increased the visibility of the researchers from the FVM in the international citation databases. This way the visibility of the Faculty of Veterinary Medicine University of Zagreb was increased on national, regional and EU level. In addition to that, ERA Chairs project has disseminated the activities and the results of the project not only to the scientific and research community, but to the general public as well, by placing the information to local authorities and local media in the form of short articles and interviews. Croatian television has filmed and prepared videos for two TV shows about the project. Also, Croatian radio made an interview with Coordinator of the project. In order to maximize visibility, the ERA Chair project has set up a project website (www.vetmedzg.eu) as well as a project logo. On the project website, all project activities and results are registered. In order to maximize the visibility of the project, logo of the project is set to all propaganda materials.
In the research, very important dissemination activities are international and national projects. The members of the ERA Chairs team and researchers of the FVM prepared more than 16 different projects, and on such way improved research activities and results on the FVM. The members of the ERA Chairs project prepared two leaflets during the project, but also several VetMedZg posters and bookmark of the project.

In the aim of better dissemination of the ERA Chairs project and its results, FVM established better connection with stakeholders and industry. Our liaison person with stakeholders and ERA Chairs holder and Project coordinator informed different authorities about the project results and possibility of using state of the art methods and equipment in the molecular medicine.

ERA Chairs team enhanced the innovation and research infrastructure and capacity through structural and cohesion funds. It is important to underline that VetMedZg project made it possible the research equipment for further investigation was obtained through structural and cohesion funds. Different workshops and their hands-on part were one of the main dissemination activities. Eight workshops were held during the project, with the latest knowledge about the different parts of molecular veterinary medicine, in particular proteomics and metabolomics, were transmitted to the wider scientific community. During the project were organized the following workshops: 1) Post-genomic era of molecular biology-proteomics era I; 2) Post-genomic era of molecular biology-proteomics era II; 3) Comparative molecular analysis of the hepatocellular carcinoma in humans and dogs; 4) Clinical and applied Proteomics; 5) Intellectual property management in support of innovation; 6) Biomarkers in veterinary medicine: ; 7) Wildlife medicine - parasitic diseases; 8) Wildlife medicine - epidemiology.
More than 50 invited speakers during the workshops visited FVM to translate the cutting edge knowledge and skills in the molecular veterinary medicine to researchers at the FVM and wider research community. During workshops researchers at the FVM gained specific new knowledge in three chosen fields: a) in the methodology development of the molecular pathology field, in the field of molecular pathology, respectively the comparative tumour analysis; b) in the post genomic technology, respectively in the proteomics and metabolomics and their application in the veterinary research in Croatia, specifically in the discovering of new biomarkers in companion animals, farm animals and horses; c) in the wildlife epidemiology, toxicology, health monitoring, and HPLC instrumentation.

During the duration of the project were organized more than 25 different trainings in the partner institutions abroad for FVM research staff. During the training, researchers from the FVM received the latest, innovative knowledge and skills in the field of veterinary molecular pathology.

The FVM was visited by more than 15 invited experts from different countries with main aim to translate the knowledge and skills to the researchers of the FVM.

The main exploitation of results of the ERA Chairs project VetMedZg are:
- the results of the investigations in molecular veterinary medicine conducted on the VetMedZg project
- specific new knowledge in the methodology development of the molecular pathology field, in the field of molecular pathology, respectively the comparative tumour analysis
- specific new knowledge in the post genomic technology, respectively in the proteomics and metabolomics and their application in the veterinary research in Croatia, specifically in the discovering of new biomarkers in companion animals, farm animals and horses
- specific new knowledge in the wildlife epidemiology, toxicology, health monitoring, and HPLC instrumentation
- newly established cooperation with universities in different parts of the world, as well as newly established cooperation with research groups in the world
- newly gained international research projects
- newly gained structural funds project
- better visibility of FVM in this part of Europe, respectively researchers in the international citation databases
- new scientific excellence at the FVM, and new possibility of collaboration with industry and additional stakeholders
- new initiatives of young researchers to apply new methods and technologies at the FVM
- acceptance of creation of ERA Chair culture and standards at the FVM
- and finally new state of the art proteomics and metabolomics laboratory at the Faculty of Veterinary Medicine University of Zagreb.
List of Websites:
www.vetmedzg.eu

Project coordinator Professor Vladimir Mrljak
Faculty of Veterinary Medicine University in Zagreb
Clinic for Internal Diseases
Heinzelova 55
10000 Zagreb
Croatia
e-mail: vmrljak@vef.hr
phone: +385 1 2390 450
+385 1 2390 346