STARTPAGE

HUMAN RESOURCES AND MOBILITY (HRM)ACTIVITY

MARIE CURIE ACTIONSResearch Training Networks (RTNs)

PART B

“HELP”

Research Training Network on 4-Hydroxynonenal, Lipid Peroxidation and Oxidative Stress

Page 1 of 66

“HELP”

Instructions for preparing proposal Part B “Proposal Description” for

Research Training Networks (RTNs)

In addition to the detailed technical information provided in this Part B, the proposal must also contain a Part A, containing basic information. The forms for Part A are provided elsewhere in this Guide. Incomplete proposals are not eligible and will not be evaluated.

Please note that the information contained in this proposal description will be used by the independent experts to undertake their assessment. We would therefore advise you to address each point fully and consult the HRM Work Programme and Guide for Evaluators for more detailed information concerning the evaluation criteria.

The Proposal Description should be submitted on single-sided A4 pages. Ensure that you print the proposal acronym as a header to each page. All pages should be numbered in a single series on the footer of the page to prevent errors during handling. The numbering format “Page X of Y” should be used.

We would encourage you to write the proposal texts as concisely as possible and advise you not to exceed the lengths defined for each of the sections below.

B1 SCIENTIFIC QUALITY OF THE PROJECT

B1.1. RESEARCH TOPIC (ONE A4 PAGE) B1.2. PROJECT OBJECTIVES (THREE A4 PAGES) B1.3. SCIENTIFIC ORIGINALITY OF THE PROJECT (TWO A4 PAGES) B1.4. RESEARCH METHOD (TWO A4 PAGES) B1.5. WORK PLAN (TWO A4 PAGES, PLUS TABLES AND CHARTS)

B2 TRAINING AND/OR TRANSFER OF KNOWLEDGE ACTIVITIES

B2.1. CONTENT AND QUALITY OF THE TRAINING AND TRANSFER OF KNOWLEDGE PROGRAMME (THREE A4 PAGES)B2.2 IMPACT OF THE TRAINING AND/OR TRANSFER OF KNOWLEDGE PROGRAMME (TWO A4 PAGES) B2.3. PLANNED RECRUITMENT OF EARLY-STAGE AND EXPERIENCED RESEARCHERS (ONE A4 PAGE + TABLE)

B3 QUALITY/CAPACITY OF THE NETWORK PARTNERSHIP

B3.1. COLLECTIVE EXPERTISE OF THE NETWORK TEAMS (ONE A4 PAGE PER NETWORK TEAM) B3.2. INTENSITY AND QUALITY OF NETWORKING (ONE A4 PAGE) B3.3. RELEVANCE OF PARTNERSHIP COMPOSITION (ONE A4 PAGE)

B4. MANAGEMENT AND FEASIBILITY

B4.1. PROPOSED MANAGEMENT AND ORGANISATIONAL STRUCTURE (TWO A4 PAGES) B4.2. MANAGEMENT KNOW-HOW AND EXPERIENCE OF NETWORK CO-ORDINATOR (ONE A4 PAGE) B4.3. MANAGEMENT KNOW-HOW AND EXPERIENCE OF NETWORK TEAMS (½ A4 PAGE PER NETWORK TEAM)

B5. RELEVENCE TO THE OBJECTIVES OF THE ACTIVITY (TWO A4 PAGES)

B6. ADDED VALUE TO THE COMMUNITY (TWO A4 PAGES)

B7. INDICATIVE FINANCIAL INFORMATION (ONE A4 PAGE, PLUS TABLE)

B8. PREVIOUS PROPOSALS AND CONTRACTS

B9. OTHER ISSUES (TWO A4 PAGES)

Page 2 of 66

“HELP”

B1 SCIENTIFIC QUALITY OF THE PROJECT

B1.1. Research topic (one A4 page)

Cardiovascular disease, cancer and neurodegenerative disorders are by far the most prevalent cause of death and morbidity in western and developing nations, and as life expectancy continues to increase, the human and financial cost of these diseases is also set to grow markedly. Cures for these diseases are still far from a reality, and much more research into their prevention and treatment is needed. The cost to the European economy of the primary diseases and subsequent support already impacts greatly on healthcare budgets. For example it is estimated that the cost of coronary heart disease and stroke to the UK economy alone was in excess of £8.7B in 19991. Extrapolate this to the whole of the European Union for cancer and neurodegenerative disease as well as cardiovascular disease, and the cost to the community of these diseases mounts to 100s of billion Euros.

A common factor of these diseases is that they all have an “oxidative aetiology”, which means that their pathology involves oxidative damage to cells and tissues. This arises during the situation of oxidative stress, which results from excess production of free radicals and reactive oxygen species (ROS) that overcomes local and systemic antioxidant defence systems2. During oxidative stress all biological macromolecules (DNA, proteins and lipids) can be oxidized, with serious consequences. However, some lipids (e.g. those containing polyunsaturated fatty acids, or PUFAs) are especially vulnerable to attack, and the resulting process of lipid peroxidation has dire results for the cell, not least because of the release of toxic products. One of these products is an aldehyde called 4-hydroxynonenal (HNE), first identified in 1960´s by the famous Austrian scientist Hermann Esterbauer. In the last ten years, HNE has become a hot topic of investigation, following the increasing realization that it has many specialized and undesirable effects on biological systems3, and 3 years ago the international 4-Hydroxynonenal Club (http://www.kfunigraz.ac.at/hne-club) was established by members of this network to forward research interactions in this field.

Despite considerable advances in this field, many important questions remain about the precise role ROS, lipid oxidation and HNE in disease pathology, in particular the molecular mechanisms which are involved. Further research on the complex nature of oxidative stress and biological effects of HNE will be necessary to improve both the diagnosis and prevention of diseases associated with oxidative stress and lipid oxidation. The primary scientific goal of this project is to integrate the study of the causes of oxidative stress, with the identification of the damage caused and the role of the oxidised products in causing further damage, to give an overall picture of precisely how oxidative stress contributes to the pathology of the diseases named above. In order to achieve this, we will investigate the correlation between the effects of oxidative stress on protein and membrane function, and the cellular response at the genomic and proteomic level. The effects of HNE, and other lipid or protein oxidation products, on cellular processes such as signalling, proliferation, necrosis and apoptosis will be thoroughly investigated, and molecular mechanisms responsible for these effects will be elucidated. Moreover, we will assess whether genetic disorders of lipid metabolism or antioxidative systems might increase the likelihood that oxidative stress and lipid peroxidation can lead to the development these diseases, using modern genomic and proteomic approaches. Synthetic agents with protective activity will also be tested.Thus, improved diagnostic, preventive and therapeutic approaches will be developed and for these purposes we will integrate research activities of scientists performing basic biochemistry, molecular biology, and physiology with clinical researchers and SMEs. A major strength of the network is that it will create the integrated, broad-based, multidisciplinary approach that is required to drive this fundamental area forward.

To achieve the goal of understanding, preventing and treating diseases with oxidative aetiology, a well trained and multidisciplinary research force is essential. We will develop a Research Training Network of senior teachers and researchers from well-know universities and research institutes in 8 European countries, 2 associated states, and Croatia, with the aim of providing the necessary training to a cohort of high quality young researchers. Interdisciplinary collaborations within the network (chemists, physicists, biologists and medical doctors) will attract the best young researchers to join the network, either to undertake supervised research for a doctoral thesis, or for career progression and enhancement.

1 Liu J.L.Y., Maniadakis, Gray A. and Rayner M. 2002 The economic burden of coronary heart disease in the UK. Heart 88: 597-6032 Halliwell B. and Gutteridge J.M.C. 1990 Role of free radicals and catalytic metal ions in human disease: An overview. Methods Enzymol. 186: 1-853 Poli G. and SchaurR.J. 2000 4-Hydroxynonenal in the pathomechanisms of oxidative stress. IUBMB Life 50: 315-321

Page 3 of 66

“HELP”

B1.2. Project objectives (three A4 pages)

The overall, fundamental aim of this Research Training Network is to provide excellent and unique training in the area of oxidative stress to young researchers of outstanding potential, who will develop to be leaders of this field in the future. This will be achieved by appointment of young researchers, either early stage (ESRs) or experienced (ERs), to positions in each participating team. There, in addition to special instructional courses and networking as described in section B2.1 and B2.2, they will conduct original research under the guidance and supervision of the team leaders and senior scientists.

Thus, an excellent research project, with clear and measurable objectives, is central to the training programme envisaged. In this research training network, high quality research projects ongoing in the participating institutions will be brought together through careful planning and extensive interactions, resulting in a synergistic approach to the overall research objectives of the project. The full and ambitious objective of the network is:

“to understand the role lipid peroxidation and 4-hydroxynonenal in fundamental cellular responses to

oxidative stress, and through this, to clarify the role of oxidative stress in the pathology of life-threatening

inflammatory diseases, with the ultimate goal of developing novel diagnostic methods and therapies.”

Within this all-encompassing aim, there are a number of more specific and measurable aims. These are described in some detail below, approximately in the order that such events would occur in pathology.

The Project specifically aims to:

1) Elucidate the mechanisms of free radical production in different conditions . Free radicals and oxidants can be produced in many ways. Some of these are well understood, but for others the mechanism in physiological situations remains little explored and the role of these mechanisms is unclear. Teams from Dublin, Leipzig and Łodz will address the mechanisms by which mitochondria, haem peroxidases, and transition metals contribute to biological oxidative damage.

2) Compare the value of different lipid oxidation products as markers for oxidative stress and methods of assessing them. Lipid peroxidation is an important event as it leads to the formation of many deleterious products. Sensitive and reliable methods for measuring products of this process would be extremely useful in diagnosis of inflammatory conditions, besides providing information about the disease process. Several teams (Glasow, Leipzig, Magdeburg) are using various mass spectrometry methods to detect isoprostanes and oxidized phospholipids, while other teams are using HPLC to detect HNE, other aldehydes, and their metabolites (Zagreb, Dublin, Toulouse-INRA), or monoclonal antibodies to observe HNE-protein adducts (Zagreb). Experimental models will be standardized between these teams to allow a very careful comparison of the strengths and disadvantages of each method of analysis. Ultimately work with clinical samples aims to establish the potential for medical screening.

3) Determine the ability of HNE and other lipid oxidation products to cause cytotoxicity & genotoxicity Mechanisms of cell and DNA damage can result in inflammation and cancer respectively; hence it is vital to establish precisely how this can be triggered in oxidative stress. Apoptosis (programmed cell death) is more complicated, as in some situations this is desirable (e.g. cancer) whereas in others it may not be (e.g. atherosclerosis). The type of response obtained is likely to be concentration dependent. Teams from Salzburg, Torino, Glasgow, and Larissa will investigate these effects in different cell types and situations to provide an comparison of the deleterious effects of these compounds. Bratislava will also participate.

4) Determine the effects of HNE and other lipid oxidation products on signalling pathways, apoptosis & gene expression. Signalling pathways in response to stimuli control many cellular processes, including growth, differentiation and apoptosis. It has been shown that some oxidants can affect these processes. Understanding the effect of oxidative stress on cells requires a dissection of their ability to induce and interact with the multiple signalling pathways in cells. Moreover, the finding of free HNE in various cells

Page 4 of 66

“HELP”

and tissues under physiological circumstances, generates an new open field of research for its possible physiological, as well as pathophysiological, roles. HNE may also induce the activity of other key cellular enzymes. In the relatively short term of the project, we aim only to identify the overall pathways or processes affected (e.g. differentiation/ proliferation/ apoptosis), and gain preliminary insight into some of the mechanisms of action. The teams who will be participating in this area are Toulouse-INSERM, Torino, Glasgow, and Zagreb.

5) Determine the effect upon cells of the formation of HNE- and oxidatively-modified proteins . HNE is known to have a propensity for reacting with proteins to form stable adducts, but many questions remain about their effect on cells and tissues. The project will address the questions by correlating the appearance of protein-HNE adducts, oxidised and glycoxidised proteins with stress responses and other deleterious effects on the cells, in particular determining the harmful and growth modifying activities of modified proteins. This will help to understand the relative importance of these processes in pathology. The value of these modified proteins as diagnostic biomarkers will also be investigated. This research will involve the collaborative effort of teams from Toulouse-INSERM, Lleida, Zagreb and Glasgow.

6) Establish the potential of natural and synthetic antioxidants to limit or prevent oxidative damage . The potential of antioxidant therapy in oxidative diseases is very tempting, although in established diseases the benefits are subject to considerable dispute. It is therefore essential to establish in good experimental models of oxidative stress and pathology, what antioxidants can be beneficial under different circumstances. Moreover, the synthesis of novel pharmacological agents with antioxidant potential would be of considerable value, hence in the project such compounds will be synthesized and tested in a range of oxidative stress conditions. The team from Bratislava will be central to this objective, which will also require collaboration with teams from Torino, Zagreb and Łodz.

7) Analysis of the metabolism of HNE and other aldehydes . As aldehydes cannot be so readily detoxified by antioxidants as ROS, they must with dealt with by other mechanisms. It is now emerging that several enzymatic pathways exist that are capable of metabolizing HNE. However, questions remain about the benefits of these processes and the success of the detoxification. The objective of this part of the project is to understand how the enzymatic processes may be induced, by the presence of aldehydes or other compounds, and whether this is beneficial in terms of the cell’s response to the applied aldehyde, i.e. does it increase or decrease the toxicity? Agents that could stimulate the detoxification of toxic breakdown products of lipid peroxidation without adverse side effects would have important potential for therapy. Teams from Toulouse-INRA and Dublin will collaborate to address this objective.

In addition to these specific research objectives, the project also focuses on particular diseases with known oxidative aetiology and very severe consequences for the health and economy of the European Community. The aims of these disease-specific focuses are given below.

a) Improved understanding of the role of ROS and HNE in CANCER and establishment of novel diagnostics and therapies in oncology. Oxidative stress and consequential lipid peroxidation are, amongst others, causative agents for cancer. Although a variety of studies have been conducted so far demonstrating a major impact of HNE on vital cellular functions, further research on the correlation between and effects of lipid peroxidation on protein and membrane function and the cellular response at the genomic and proteomic level is necessary to improve the diagnosis as well as treatment of cancer. This focus will be supported by the achievements of all the specific aims listed above. The teams who will collaborate within this focus are Salzburg, Larissa and Toulouse-INRA.

b) Improved understanding of the role of HNE in NEURODEGENERATIVE DISEASES for their possible prevention and advanced diagnostics. Oxidative stress and lipid peroxidation is known to play a role the pathology of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and cerebral ischemia. Moreover, many of the neurological deficits associated with the ageing process occur in the absence of neurodegenerative diseases. In fact, these diseases are often superimposed upon an already declining nervous system. These deficits may include decreases in both motor and memory functions which could, in many cases, result in hospitalisation or custodial care. The changes that occur both in Alzheimer's disease and Parkinson's disease, as well as those in ageing, may involve increases in vulnerability to oxidative stress. Although a variety of studies have been conducted so far, further research

Page 5 of 66

“HELP”

on the complex nature of the causes of these neurodegenerative diseases will be necessary for this focus, which again will be supported by the achievements of all the specific aims listed above. The HNE Research Training Network aims at improving the diagnosis and prevention of neurodegenerative diseases by developing novel assays for oxidative stress and lipid peroxidation in perspective of therapeutic approaches by novel antiradical substances. The teams who will collaborate within this focus are Dublin, Blatislava, Lleida and Zagreb.

c) Improved understanding of the mechanisms by which lipid oxidation products contribute to the progression of CARDIOVASCULAR DISEASE and establishment of novel diagnostic markers . It is now well accepted that oxidative stress plays a role in the initiation and progression of atherosclerisis, but many questions remain about the source of free radicals and oxidants in vivo, and about regulation of the molecular events that lead to plaque formation. Understanding of both these aspects will be essential for respectively preventing and treating the disease. As with the other diseases, the research findings of all the specific aims will help to achieve this focus. The teams mainly responsible for work in this area are Glasgow, Toulouse-INSERM, Leipzig and Magdeburg.

d) Identifying ANTIOXIDATIVE METABOLIC DISORDERS that could enhance susceptibility to oxidative diseases. Genetic disorders of lipid metabolism and antioxidative systems might increase the likelihood that oxidative stress and lipid peroxidation can lead to the development of cancer, neurodegenerative diseases and atherosclerosis. Advances in genomics and proteomics might help to identify susceptibility of individuals to develop severe diseases, and hence to improve the diagnosis of these diseases. Identification of enzymes involved in the metabolism lipid peroxidation products (in particular HNE), as well as detoxifying and repairing enzymes might also help to develop preventive measures. This area also overlaps with studies on the age and gender dependence of antioxidant levels and oxidative disease. Teams in Zagreb, Glasgow, Toulouse-INRA and Lleida will be involved in addressing this objective.

This network will enormously advance the knowledge of the field of oxidative stress in pathology. Significant developments are confidently expected for all the aims described above, and together this knowledge will provide an important breakthrough in the overall research area, by providing an integrated overview to many normally disparate research topics.

We wish to reiterate that the major aim of the network is the special training of a cohort of young scientists. Primarily, the network will enhance Europe’s industrial (medical) and scientific competitiveness in this field, through the creation of a network of trained scientists with appropriate multidisciplinary skills. The network will provide young researchers with the opportunity to acquire a unique set of skills through international training, and the opportunity to consolidate these skills in an industrial context. Scientific excellence will be supported in a wider context through the establishment of an educational network. Specific measures to be introduced include the establishment of a series of summer schools and workshops, a seminar series, the introduction of modules into undergraduate and post-graduate training programmes, and subject to demand, the provision of industrial training courses.

Finally, an essential ultimate aim is the dissemination of acquired knowledge throughout the community. This will be achieved by various means, such as the publication of academic papers and presentations to relevant international conferences; the distribution of developed guidelines to relevant medical centres, research and academic organisations in the field; the organisation of specialist workshops affiliated to international conferences; and the maintenance of a dedicated website for publishing scientific reports, demonstration software, material from workshops/summer schools.

Page 6 of 66

“HELP”

B1.3. Scientific originality of the project (two A4 pages)

Although considered for years simply as undesirable by-products of aerobic metabolism, reactive oxygen species (ROS) are now known to play a major role in cell homeostasis, and in oxidative damage of cell components, and are thus potentially responsible for cell injury, dysfunction, mutagenesis and death. The importance of free radical chemistry in biology and medicine has been appreciated only recently by scientists, with the increasing evidence that ROS and bioactive derivatives resulting from lipid peroxidation (in particular 4-hydroxynonenal) are directly involved in ageing and in the pathogenesis of various diseases, including cardiovascular diseases, neurodegenerative disorders, inflammatory and infectious events. Besides their deleterious effects, ROS can also function as second messengers in signal transduction, transcription factor activation and gene induction, and thus play a role in essential cellular functions such as survival, proliferation or apoptosis. The research efforts of many of the teams involved in this network have been responsible for the current understanding of these properties and actions of ROS and especially HNE.

Despite the expanding interest in the field of oxidative stress, many very important questions remain unanswered. A comprehensive knowledge of the role of reactive oxygen species and oxidative stress in the pathophysiology of diseases requires i) to understand the basic chemistry of radical reactions, the cellular oxidative homeostasis and antioxidant defences, ii) to understand the role of lipid peroxidation products involved in the pathophysiological effects of oxidative stress, and iii) to develop and transfer techniques allowing detection of the formation of ROS and lipid oxidation derivatives. The project presented here will provide significant advances at the forefront of the field in all these areas. The outcome will be the best updated overview possible on free radicals and lipid peroxidation products, how they are generated, and how they react, what are their biological effects, how they can be detected using simple or highly sophisticated techniques, and how they can be neutralized or degraded. This information will be essential for future improvements in the diagnosis and therapy of cancer and all inflammatory–based diseases (including atherosclerosis, some neurodegenerative diseases, diabetes, rheumatoid arthritis), and will thus bring a measurable benefit to the European Community.

All the teams in the network are performing novel research in aspects of oxidative stress, and some have outstanding international reputations. The large scientific potential and complementarity of the experienced researchers participating in this network will guarantee the high quality of the training to be offered.

The project will address the outstanding issues at the forefront of most aspects of the actual knowledge in free radicals and lipid oxidation, from their generation in aerobic metabolism, to their consequences in cell homeostasis, and the natural or chemical antioxidant defences. It is divided into several parts, focused first on the basic aspects of free radical generation, the role of transition metals and of the different enzymes and cellular sources (heme peroxidase, phagocytes, mitochondria) involved in ROS production. The role of different reactive oxygen or nitrogen species, including peroxynitrite and hypochlorite will be investigated. The research teams in Toulouse, Leipzig, Łodz, Glasgow, and Dublin are highly recognized in this area).

Particular attention will be given to the different mechanisms leading to protein modification and oxidation, as this field of research is exciting much interest in the international community. The generation of 4-HNE and its subsequent deleterious effects represents a very important event shared by a number of stress situations, elicited by cytokines, bacterial toxins, drug poisoning and others, which all induce oxidative stress, activate transcription factors and trigger inflammation and cellular stress responses. 4-HNE, acrolein, malondialdehyde, are aldehydic by-products of polyunsaturated fatty acids oxidation, which exhibit highly bioactive properties, in particular a rapid reaction (derivatization) of free amino groups and thiol residues of proteins (forming thus ALE (Advanced Lipoxidation End products). This modification impairs progressively the protein properties and leads to uncontrolled cellular responses such as proliferation, cell differenciation or apoptosis, as function of stress intensity and local HNE concentration. Research on this molecular perspective of cellular responses is attracting prizes within the scientific community for its originality and importance, such as the recent Hermann Esterbauer Awards in 2002. Similarly the formation of Advanced Glycation End products (AGE) results from glycoxidation and fragmentation of glucose in diabetic situations (NIDDM or type II diabetes), with same consequences on cellular impairment. The role of AGE and ALE is of particular importance in the mechanism of accelerated atherosclerosis occurring in NIDDM, which represents the major dramatic complication of this disease with an increase in cardiovascular accidents. The respective (and sometimes additive) roles of these agents will be studied and presented as well as an overview of

Page 7 of 66

“HELP”

methodologies used for detecting their presence and expected role in cells and tissues. The teams from Toulouse-INRA, Lleida and Glasgow are making important progress in these areas.

It is important to note that the techniques that will be presented here (paramagnetic spectroscopy (EPR), mass spectrometry, HPLC) are among the most sensitive so far used in this field, allowing a precise and specific identification of free radical species. Another important point is to determine and identify the cellular targets of free radical attack, which can be done by using a wide range of techniques from simple biochemistry to highly sophisticated proteomic analysis or nuclear magnetic resonance which is able to give major information on conformational and structural changes caused by oxidative stress. More simply, the use of antibodies allowing the detection of HNE-, and acrolein-adducts through immunohistochemical and western-blots techniques will be investigaetd and documented, since these techniques are of particular interest in pathologies.

An important aim of the project, in line with international focus, is to understand the role of oxidative stress and lipid peroxidation products such as 4-HNE in pathologies. For instance, the role of oxidation in atherosclerosis is today well established, at least in early lesion formation (fatty streaks), and there is also evidence for later involvement. Low density lipoproteins (LDL) are oxidized in the subendothelial space by free radicals produced by vascular cells. This oxidation transforms LDL into cytotoxins able to trigger inflammatory signalling events, and modify their metabolism by inducing a progressively increasing uptake by the scavenger receptor of macrophages. So far many questions are still without answer, such as the precise mechanism of ROS production involved in LDL oxidation, the biphasic effect of oxLDL on cell signalling towards proliferation (and inflammation) or apoptosis, the nature and role of lipid peroxidation products (4-HNE) involved in biological events potentially involved in plaque erosion and rupture, thrombosis and subsequent cardiovascular diseases. Several teams within the network are conducting highly valued research to address these questions, and continue to make important novel contributions to the subject of atherosclerosis.

On the other hand ROS and HNE have been demonstrated to have a major involvement in age-linked pathologies such as cataract, neurodegenerative diseases, ischemic events, cancers. More than simple lipoperoxidation markers, HNE and related compounds activate various signalling processes and gene expression that result in mitogenic or apoptotic responses (in particular HNE is strongly toxic for neurons). How these molecules are formed, and how they trigger all these events, is still poorly understood and hence forms a critical part of the project which will clearly yield novel and valuable research outcomes.

Lastly, the project will address the role, distribution and properties of natural or dietary antioxidants, and their beneficial and limiting effects in diseases. This is particularly important in atherosclerosis, where the role of antioxidants is still controversial. Moreover antioxidants, although highly efficient at preventing ROS generation, have little ability to neutralize already formed HNE and other aldehydes. In this case, understanding the metabolism of these products, and how it could be manipulated, is a necessity for minimizimg deleterious effects. A new challenge will be represented by the synthesis and use of chemical scavengers able to act synergically with antioxidants, to neutralize and prevent HNE and related compounds generation, and their subsequent deleterious effects. Overall, it will certainly be a great challenge to offer clinicians trustworthy parameters indicating the clinical redox status in patients.

In conclusion, the project presented here represents a transverse approach to ROS and HNE biology, from basic chemical aspects and signalling events at the cellular level, to the pathophysiological consequences, and involvement in diseases. The project is at the forefront of scientific enquiry in all its component directions, and will greatly benefit the European Community owing both to advances in biomedical knowledge and to the unique multidisciplinary training of a cohort of young researchers.

Page 8 of 66

“HELP”

B1.4. Research method (two A4 pages)

The research project “4-HNE, lipid peroxidation and oxidative stress,” forms the basis for the training to be offered to the young researchers, and is critical to their overall development. The research project aims to understand the involvement of oxidative stress in general, and HNE in particular, in the pathology of diseases that are of major importance in the EU, namely cancer, cardiovascular disease, neurodegenerative diseases and liver disease. In order to understand these processes at a molecular level that would allow the development of novel therapies, it is necessary to undertake both fundamental research, and clinical analysis projects. Moreover, for the utmost benefit, it is essential to integrate the outcomes of these two strands of research. It is also very important to break down the barriers between research on individual diseases, to allow advances in the understanding of one inflammatory disease to be extrapolated to improve our knowledge of another disease with comparable underlying aetiology.

The project will be unique in the oxidative stress field, as it will allow integration of many complementary lines of research and technologies. The following research methods will be used to address the aims of the project.

1. DNA Damage Analysis

DNA damage is thought to be a critical event in many situations of oxidative stress, as it can lead to apoptosis, cell death and mutations, and therefore is thought to contribute to the progression of cancer as well as possibly to inflammatory conditions. In this project DNA damage will be measured both by microscopic analysis of chromosomal aberrations and by determination of telomerase activity. These methods are complementary as they bring together the imaging of structural macromolecular changes with a measurement of functionality. The analysis of DNA damage will be used to determine the genotoxic effects of HNE and other lipid oxidation products that result during oxidative stress, and investigate whether there is a specificity of response to particular products. This will then allow analysis of some disease samples to determine whether such damage occurs in pathological situations and give information about the damaging agents likely to be responsible. The teams from Salzburg and Larissa are experts in these techniques.

2. Mass Spectrometric Analysis of Lipid Oxidation

Many lipids are highly susceptible to oxidation, and the resulting lipid oxidation products, which include 4-HNE, are known to have many deleterious biological effects. Hence the detection of oxidized lipids is very important, both in understanding the process of oxidative stress that occurs during pathology, and as potential markers for oxidative stress and inflammation in disease diagnosis. 3 teams of the network are experts in the use of complementary mass spectrometry techniques for the analysis of oxidized lipid products: MALDI- (Leipzig), electrospray ionization- (Glasgow) and GC-MS (Magdeburg). These methods have been shown to detect lipid peroxides, chlorohydrins, lysolipids and isoprostanes, all of which are indicative of oxidative stress. The combination of these techniques, through sharing of samples in the network, will facilitate the comparison of these methods in terms of their clinical diagnostic value, and will provide a comprehensive set of data on the process of lipid damage in vivo and in experimental models. This research approach will also be integrated with the analysis of 4-HNE to yield further information on the balance of pathways in lipid damage.

3. Analysis of 4-HNE and its Metabolism

As indicated above, 4-HNE is a breakdown product of lipid peroxidation with many known deleterious effects. However, it is also emerging that it is present in physiological (as opposed to pathological) situations, suggesting that it may have important signalling functions analogous to those that have been discovered for reactive oxygen and nitrogen species. For this reason it is essential to have sensitive methods for the detection of HNE in a range of biological samples, and to carry out systematic studies to identify and quantitate it in a variety of conditions. Within the network, we have several teams who have specialized in this area. HPLC analysis of HNE was developed extensively in Graz, and passed on to Zagreb and Torino through previous collaborations. Other groups (Dublin, Berlin, Toulouse-INRA) have extensive expertise in studies of the metabolism of HNE, which is vital in cellular protection, and provides further information about the response of the cell to stress and offers alternative markers. These methods are especially valuable in conjunction with the techniques for detecting HNE-modified proteins described below.

Page 9 of 66

“HELP”

4. Analysis of Oxidized and HNE-modified Proteins

4-HNE reacts very readily various protein sidechains, leading to modification of the protein by Schiff base or Michael adducts. In order to study this, specific antibodies to HNE have been developed, and their application has been demonstrated by several network teams (Zagreb, Turin), and this provides a fundamental and sensitive approach to detecting damage by HNE. Immunohistochemical techniques allow the analysis of HNE-modified proteins in a variety of tissues from normal and pathological conditions, and this offers great potential as a clinical diagnostic tool (hence the involvement of the SME, which specializes in this), as well as a research tool in experimental studies of oxidative stress. To support this, other teams are measuring oxidation and glycoxidation of proteins, using NMR (Zagreb), mass spectrometry (Lleida, Glasgow) and biochemical techniques (Łodz).

5. Analysis of Inflammatory and Signalling Properties

4-HNE, and other lipid oxidation products, have been found to have some pro-inflammatory effects such as increasing synthesis of TGF or increasing leukocyte-endothelial adhesion. Further investigation of the bioactive properties of lipid oxidation products are necessary to understand the contribution this could make to disease pathology. Several teams within the network are researching in this area, using a variety of techniques to determine the effects of lipid and protein oxidation products. ELISAs are being used to monitor the formation of inflammatory cytokines (Glasgow and Turin), and molecular techniques are available to study signalling pathways (Toulouse-INSERM, Berlin, Turin and Zagreb). Several teams also have good experience detecting apoptosis and necrosis by flow cytometry, enzymatic assays and microscopy (Salzburg, Glasgow).

6. Proteomics and Metabonomics

Proteomics and metabonomics are modern methods that provide high yield and high quality information about changes in cell behaviour in disease and stress, and they are currently being used by several teams in the network (Glasgow, Toulouse-INRA). Their application to the field of oxidative stress and associated diseases is highly desirable, as they will allow the role of HNE, lipid peroxidation and cellular responses to these to be elucidated in normal and pathological situations. They will provide excellent training for young researchers coming into the field, in addition to potentially identifying new markers of disease states. These approaches really provide complementary information to the other, more narrowly-focused techniques.

7. General Methodology

In addition, to the specialized methods listed above, many routine biochemical and physiological methods will be used in the research. Examples include spectrophotometric, fluorimetric and luminescent assays for enzyme activity and metabolites; histology and microscopic approaches to cell and tissue characterization; FACS analysis; HPLC and FPLC; protein electrophoresis and blotting; and cell and tissue culture methods.

Project Approach

These research methods will be used in combination by teams within the network to address the research tasks that constitute the overall network project. Within the project, the approaches vary from fundamental research on experimental models (cell to animal level) to clinical analysis that will investigate markers for oxidative stress in disease conditions. All the methods described are complementary in the type of information generated.

In all cases, the network will provide the teams and their young researchers with access to techniques and research methods that they would not otherwise have had, and this is a major driving force in the establishment and aims of the network. It will allow comparison of research data output across technologies, thus strengthening the value of the knowledge obtained, both for experimental and clinical investigations. The network will expose the young researchers to an almost unprecedented range of scientific methodology, which will be a tremendous assistance whatever their final career research direction.

Page 10 of 66

“HELP”

B1.5. Work plan (two A4 pages, plus tables and charts)

The aims of the project, described in section B1.2, are to understand the role of oxidative stress and lipid oxidation products and the mechanisms by which pathology occurs in several diseases that are medical priorities in the European community. In addition of the specific research objectives, the project is divided into 4 focus topics, relating to the different diseases that will come under investigation. These, together with the aspect of improved methodology, form the scientific tasks around which the network will be organized.

Task T1. DEVELOPMENT OF IMPROVED METHODOLOGIES FOR ANALYSIS OF OXIDATIVE STRESS AND BIOMARKERS

This is a fundamental area of research that underpins the other tasks and will facilitate their advances. Many of the teams have already been at the forefront of methodology development and application in the field of oxidative stress, and it is essential to continue this effort. The major focuses of this task are: Analysis of protein oxidative damage by nuclear magnetic resonance (NMR), mass spectrometry and

biochemical methods (Zagreb; Toulouse-INSERM; Glasgow; Lleida, Berlin). Analysis of oxidized lipids and lipid peroxidation breakdown products by mass spectrometry and NMR

(Leipzig; Magdeburg; Glasgow). HPLC methods for analysis of HNE and aldehydes in oxidative stress evaluation (Zagreb). Analysis of DNA and chromosome damage (Salzburg; Larissa) Metabonomic and proteomic analysis by high resolution NMR and mass spectrometry (Toulouse-INRA;

Glasgow; SMEs). Development of clinically applicable assays for oxidative stress determination (Toulouse-INRA; SMEs; +

subcontractors i.e. clinics in Zagreb, KB “Dubrava” & KB “Sestre milosrdnice”).

These methodologies will be essential to our interdisciplinary collaborations in the other project tasks (T2 – T5). This task will address specific aim 2 of the project

Task T2. UNDERSTANDING THE ROLE OF ROS AND HNE IN CARCINOGENESIS AND DEVELOPMENT OF IMPROVED DIAGNOSTICS AND THERAPIESParticular attention will be given to experimental and clinical aspects of: Oxidative stress in the process of carcinogenesis (Torino, Salzburg; Toulouse-INRA). Oxidative stress and telomere shortening: mechanisms influencing apoptosis and carcinogenesis (Larissa). The role of granulocytes in carcinogenesis and defense against cancer (Leipzig; Zagreb). HNE in cell growth control (Zagreb). Novel antioxidants in cancer therapies (Bratislava; Lodz; Zagreb).

This task will address specific aims 1, 3, 4 & 6, and focus topic (a).

Task T3. UNDERSTANDING HNE IN NEURODEGENERATIVE DISEASES - EXPERIMENTAL AND CLINICAL APPROACHESThe task will focus on experimental and clinical aspects of: Mechanisms of ROS production in brain mitochondria and in the neurodegenerative processes (Dublin;

Toulouse-INSERM; Zagreb) Role of protein-HNE adducts and their degradation by proteasomes in neurodegenerative disease (Berlin). Acute impairments of central nervous system (CNS) due to oxidative stress (Bratislava; Zagreb; Dublin;

Lleida). Potential of natural and synthetic antioxidants to protect against oxidative stress-related damage in

experimental models of neuropathology (Bratislava; Zagreb, Lodz). Experimental models will be defined jointly and will take advantage of the existing good laboratory

practice of the partner research groups. This task will address specific aims 1, 2, 3, 4 & 6, and focus topic (b).

Task T4. UNDERSTANDING OXIDATIVE STRESS AND LIPID OXIDATION EFFECTS IN CARDIOVASCULAR DISEASE

This task will be investigated in model experimental systems and by analysis of clinical samples. The main focuses of the taks are listed below:

Page 11 of 66

“HELP”

Detection of modified proteins in atherosclerosis, non-insulin dependent diabetes mellitus (NIDDM) and models of oxidative stress (Toulouse-INSERM; Lleida; Zagreb).

Studies of oxidized lipids in atherosclerosis and models of oxidative stress (Glasgow, Leipzig, Magdeburg).

The role of lipid oxidation products in inducing inflammation and signalling processes (Toulouse-INSERM; Glasgow).

Analysis and assessment of the pathophysiological role of glycoxidation (Lleida). Studies of the redox properties of heme peroxidases and pathological significance of hypochlorite and

peroxynitrite (Leipzig; Lodz).This task will address specific aims 1, 2, 3, 4 & 5, and focus topic (c).

Task T5. INVESTIGATING ANTIOXIDATIVE METABOLIC DISORDERS AND THE EFFECT OF AGE AND GENDER ON ANTIOXIDANT STATUS

This complex topic will involve multi-directional collaborations with input of information from several teams. The subtopics comprising this task are as follows: Studies of antioxidants in prevention of pathology, and their sex or age dependence (Zagreb, Bratislava). Metabolism and biological effects of 4-hydroxynonenal (Toulouse-INRA; Dublin). Genomic approaches to antioxidant status (Zagreb). Proteomic approaches to essential protective enzymes (Toulouse-INSERM; Glasgow; Lleida).

This task will address specific aims 1, 6 & 7, and focus topic (d).

In all the tasks, the experimental models will be defined jointly by research partners and based on the interdisciplinary methodologies and research approaches of all members of the network. Test samples that will be analysed in different laboratories will be obtained by simultaneous experiments or by the exchange of the samples between laboratories. The tasks involved in the project are also summarized in Table B1.5.1.

List of major scientific milestones of the network project (see also Table B1.5.2)

1. Comparison of MALDI-MS, ESI-MS and GCMS for the detection of oxidized lipids in experimental models (24 months).

2. Preliminary identification of gene expression and signalling events induced by HNE and lipid oxidation products (24 months)

3. Determination of cytotoxicity, genotoxicity and induction of apoptosis in defined models by HNE and lipid oxidation products (24 months)

4. Synthesis of a number of novel synthetic agents with antioxidant activity (24 months)5. Elucidation of pathways of metabolism of HNE and aldehydes in specific models (30 months)6. Correlation of HNE-protein adduct formation with growth modification and toxicity, and comparison with

oxidised and glycoxidised proteins (36 months).7. Role of different mechanisms of free radical production in generation of oxidative stress in pathology (36

months).8. Determination of the ability of synthetic and natural antioxidants to alleviate oxidative stress in defined

experimental models (42 months).9. Determination of the ability of HNE and lipid oxidation products to induce inflammatory events, and

elucidation of the basic mechanisms involved (42 months).10. Comparison of MS methods with detection of HNE as oxidative stress biomarkers in experimental

systems and clinical samples (48 months).11. Overview of the role of ROS and HNE in diseases of oxidative aetiology generated (48 months).12. Development of several novel clinically applicable assays (48 months).

In addition to the scientific tasks and milestones described above, there are parallel educational and training tasks to be achieved. These are critical to the overall objective of the Research Training Network, and are detailed fully in section B2 (see Table B3.2).

Page 12 of 66

“HELP”

Table B1.5.1. Participation of the research teams in the TASKS of the network

Team No Team Short Name T1 T2 T3 T4 T5

1 Salzburg X X2 Zagreb X X X X X3 Glasgow X X X4 Dublin X X5 Bratislava X X X6 Lleida X X X7 Toulouse-INRA X X X X8 Toulouse-INSERM X X X9 Leipzig X X X10 Magdeburg X X11 Larissa X X X12 Łodz X X X13 EliTec X14 Torino X X X15 Berlin X X X

Table B1.5.2 Progress of Project towards achieving MILESTONESDuration of Project in Months

Milestones 6 12 18 24 30 36 42 48123456789

101112

Page 13 of 66

“HELP”

Table B1.5.3 Contribution of participating teams to the PROJECTS OBJECTIVES.

Team No 1 2 3 4 5 6 7

MechanismsROS

production

LPO markers &

analysis

HNE/ LPO cytotox. & genotox.

HNE/ LPO signalling & gene

expression

protein adducts or oxidation

effects

Natural & synthetic antiox.

HNE metabo-

lism

1 X2 X X X X3 X X X X4 X X X5 X X6 X7 X X8 X X9 X X10 X11 X12 X X13

14 X X X X15 X X X X X

Page 14 of 66

“HELP”

B2 TRAINING AND/OR TRANSFER OF KNOWLEDGE ACTIVITIES

B2.1. Content and quality of the training and transfer of knowledge programme (three A4 pages)

The training objectives are:

1. To establish a network of European researchers who are trained in basic and advanced experimental techniques in the oxidative stress field.

2. To provide interdisciplinary and multidisciplinary research training for both early-stage and experienced researchers in the oxidative stress field.

3. To create a series of courses based on the multidisciplinary research available where young researchers will receive tutoring and lecturing from experts in the oxidative stress network.

4. To train young researchers how to create new diagnostic systems based on oxidative stress markers for use in cancer and neurodegenerative areas.

5. To educate young researchers in how novel experimental findings are commercially exploited and to provide complementary training in management and presentation skills.

The transfer of knowledge objectives are:

1. To create a working environment whereby young researchers will receive mentoring and information on multidisciplinary research from experts in the area of oxidative stress.

2. To allow young researchers to exchange knowledge on oxidative stress systems by spending at least one secondment in another network laboratory.

3. To develop complementary skills in young researchers by exposing them to network organisational activities, seminar activities and scientific publishing activities.

The presence of 15 interdisciplinary research teams in this network will create a critical mass for cutting-edge research in the field of oxidative stress. The network will be organised according to Tasks 1 to 5 and the early-stage researchers contracted to this network will receive instruction in a number of disciplines, hence, an intended outcome will be to produce researchers that have the necessary skills to work in an interdisciplinary setting. Each research partner will provide specialised training in experimental techniques involved in oxidative stress for their respective researcher. For example, some teams will focus on markers of oxidative stress (Task 1) while others will focus on oxidative stress in cancer (Task 2) and neurodegenerative diseases (Task 3). Creating a network of experienced oxidative stress teams and establishing yearly topic-orientated meetings (see below) will benefit early-stage and experienced researchers as they will be educated in new techniques and research systems that would not be possible within most single research organisations. The students will observe first-hand how leading international research groups in the area of oxidative stress function, and the mechanism by which these network scientists combine research efforts to reach an objective. By the inherent nature of these training conferences, researchers will improve their communication skills as they will be required to present their experimental results to their peers and experts in the field of oxidative stress.

As can be seen in the list of network members, a large number of oxidative stress experts of different nationalities are involved. This will provide an opportunity for young researchers to learn about techniques such as mass spectrometry, NMR, ESR, molecular biology, pharmacology, biochemistry, bioenergetics and genomics/proteomics. This will enable students to be expertly trained in important areas of oxidative stress biology and to use this expertise to discover new mechanisms that underlie such biology.

Each network team (except the SME group) will train one ESR for 3 years, because this is an appropriate length of time for the researcher to be registered for a doctoral thesis. The teams will also appoint one ER each for 12 months. These will be critical for the networking and transfer of knowledge aspect of the programme, in addition to assisting with mentoring and support of ESRs. Only the EliTec SME team will not appoint a young researcher, as their environment is not so suitable for students, but instead the ESRs and ERs will be able to visit them to learn about the development of clinical assays.

Page 15 of 66

“HELP”

RESEARCH TRAINING NETWORK EVENTS

During the programme, a variety of different training events will be organised, as detailed below. These training events are also very important in the networking aspect of the programme, and are mentioned in section B3.2.

Instructional Courses

The educational level will involve 2 major instructional courses (year 2 and year 3) to be organised for the young researchers. Holding the courses in the 2 middle years will ensure that all young researchers are able to attend them. In these courses, sessions of 2-6 hours of seminars and workshops addressing specific subtopics will be given by staff in the Research Training Network, helped by a number of invited speakers who are experts in the field. Each partner group will contribute to these meetings. Attending these conferences will bring ECTS credits for participants and will be considered as a part of their Ph.D. education, but ERs will also be encouraged to participate. The precise schedule will be defined when the network is approved, but the following list outlines the main courses that will be offered by the teams.

Team 1 - In vitro test systems for the evaluation of adverse effects of oxidative stress and the protection by antioxidants – Austria. Team 2 - Basic aspects of oxidative stress and lipid peroxidation - Croatia. Team 3 - Glutathione as an antioxidant and marker of oxidative stress – Scotland. Team 4 - Mechanisms of ROS production in brain mitochondria and the neurodegenerative processes; Enzymes and reactive oxygen containing species (ROS)– Ireland. Team 5 - Acute impairments of central nervous system (CNS) and oxidative stress (OS) – Slovakia. Team 6 - Glycoxidation: assessment and pathophysiological role - Spain. Team 7 - Metabolism of 4-hydroxynonenal and development of a lipid peroxidation biomarker – France. Team 8 - Detection of oxidized/modified proteins in diseases – France. Team 9 - Redox properties of heme peroxidases and Polymorphonuclear leukocytes as a source of ROS – Germany. Team 10 - The measurement of protein oxidation –Germany. Team 11 - Oxidative stress and telomere shortening – Greece. Team 12 - Peroxynitrite and hypochlorite: oxidants of pathophysiological significance – Poland. Team 13 - Clinically applicable assays for oxidative stress determination – Austria. Team 14 - Oxysterols in inflammation. Team 15 – Oxidized Protein degradation. External experts will also contribute to the seminars.

Open Week Mobility Scheme

The practical training of ESRs in multiple technologies and research approaches will be achieved by “open weeks”, where each team will invite all the ESRs to visit their laboratory for one week to see ongoing experiments, attempt analysis of their own materials, and obtain some hands-on experience. The proposed schedule of open weeks is shown in Table B3.2. These are aimed mainly at training of ESRs, but ERs will also be welcome to participate.

Scientific Meetings

We plan to organise four major research meetings during a period of four years. These will give young researchers an excellent opportuunity to gain knowledge on the up-to-date research situation in the whole of the oxidative stress field. They will also be very important in the development of transferable skills, as they will be an ideal opportunity for the researchers to present their scientific findings and also to interact with other experts in the oxidative stress field. The scientific meeting provisional plan is as follows:

1. Year 1: Technology (Task 1)

2. Year 2: Cancer and Neurodegenerative Diseases (Tasks 2 and 3)

3. Year 3: Cardiovascular Disease and Antioxidant Disorders (Tasks 4 and 5)

4. Open Conference to publicize results (joint with HNE-Club)

Secondments and Exchanges with other teams

The training of the ERs will be broadened by secondments to collaborating teams for periods of 3-6 months, to enable them to become fully skilled practically in several different types of experimentation. Naturally, this will also forward the transfer of knowledge throughout the network, as well as transfer of

Page 16 of 66

“HELP”

samples for differential analysis. It is felt that the ERs will be sufficiently mature and advanced in their skills to make these exchanges profitable. In a small number of special cases, the management team may also deem that such transfers are appropriate for 3rd year PhD students, but in general this marks a difference between the training or early stage and experienced researchers.

Further Training in Complementary SkillsEarly and experienced researchers will receive instruction in bioethics, gender issues, and

entrepreneurial areas. External experts will be invited to deliver lectures relating to these issues as part of the instructional courses. Here, early-stage researchers discuss in groups a series of experimental and theoretical problems associated with the oxidative stress field. These groups will be split into equal numbers of male and female researchers who will take turns at leading the discussions. In this manner, the early-stage researchers will learn leadership qualities and how scientific problems may be solved in a group.

The proposed network includes a group of SMEs in Austria whose focus is on creating diagnostic assays for oxidative stress. Early-stage and experienced researchers will undergo short training placements in this company where they will learn how to commercialise experimental findings. In addition, all researchers will be required to attend the BioBiz course that specialises on some commercial aspects of scientific research. In this course, researchers will be trained in the use of an electronic template that allows one to write a business proposal for a start-up company. This is an essential component for the commercial exploitation of research findings. Similarly, researchers will receive individual training from their host universities on industrial and commercial enterprises.

There will be 2 Career Development Plan Templates:

1. Early-stage researchers will receive formal training in experimental research in the area of oxidative stress in their own network team. They will also visit other network labs for open weeks, where they will learn other techniques not available to them in their own lab. The instructional courses will instrumental in their scientific education in the area of oxidative stress. In addition, they will develop complementary skills as they will be required to present their scientific results orally at the research meetings; also poster presentations at international conferences will be encouraged. In this way, early stage researchers will receive training in a number of interdisciplinary areas in oxidative stress that will provide a basis for future work as experienced researchers.

2. Experienced researchers’ career development will be focused mainly on using previous experience to make breakthroughs in the oxidative stress research area. These researchers will also learn new techniques from other network teams by secondments while developing their interactive skills. They will be encouraged to present their findings at international conferences and will develop their ability to communicate information to students, as they will be required to give lectures at the yearly scientific conferences. During their fellowships, they will learn the advantages of scientific collaboration and will be encouraged to improve grant writing skills. In this way, the experience should enable experienced researcher to further their scientific careers by starting their own research group in universities or industry.

Both early-stage and experienced researchers will receive intensive and on-going training in their respective laboratories, and continuous mentoring will be provided by the group leaders. Section B2.3 shows that each team possesses a large number of supporting staff who will assist in the training process. All the laboratories participating in the Research Training Network have excellent laboratory facilities, well endowed with modern equipment. All of the proposed network universities have postgraduate support and development programmes, as well as innovation centres that provide training for entrepreneurial enterprises.

In summary, all teams in the Research Training Network are able to provide highly satisfactory training environment for young researchers, and this will be enhanced further by the specialized training programme offered by the network.

Page 17 of 66

“HELP”

B2.2 Impact of the training and/or transfer of knowledge programme (two A4 pages)

Cancer and cardiovascular disease are the major causes of mortality and morbidity throughout Europe, while neurodegenerative diseases also contribute in a major way to poor quality of life in the ageing community. These diseases also have very serious economic consequences, as a large fraction of health care resources are required for chronic treatment, hospitalization and care in the community. Consequently there is an urgent need to understand the mechanisms of pathophysiology, and to forward cutting edge research in this field. This obviously imposes a requirement for highly trained researchers with specialist knowledge.

All these diseases have a common link: they have an “oxidative aetiology”, meaning that damage caused by oxidative stress is involved in the pathology of the disease. This research project focuses on understanding the role of oxidative stress, lipid peroxidation, and HNE in disease, which will allow new, improved approaches to prevention and therapy. It will also identify new markers for the diseases that will facilitate early diagnosis and assessment of the effects of therapy. Thus the project will itself improve the healthcare situation in relation to these diseases. More importantly, however, it will provide a unique type of training to young researchers who are inspired to enter careers in biomedical research.

Currently, many young researchers in the biomedical field specialize in one technology and in one area of research, in part owing to the tendency to remain in one laboratory. While they do become experts in their chosen area, this often has limitations in understanding the potential of other methodologies, the implications of research in other distinct but parallel fields, and in opportunities for lateral thinking in the development of multidisciplinary research. Moreover, biomedical research can be very disease specific, with relatively little crossover of ideas and information between the diseases. Both these problems will be addressed by the research training offered by the Oxidative Stress Network.

In the first place, the network will provide very strong links to theoretical expertise throughout the research project area, which will be available to the young researchers through the Oxidative Stress Portal, where a discussion group will be set up. The researchers will be introduced to a wide range of high quality biomedical research ongoing in Europe, through interaction with the research teams involved in the network, all of whom have international profiles. The “oxidative aetiology” approach to diseases allows the cross-fertilization of ideas in the understanding of mechanisms of pathophysiology, and therefore will provide the young researchers with a much broader knowledge, and a strong basis upon which to build research in any oxidative disease. The specific tasks within the project depend upon the development of underpinning methodologies, many of which are at the cutting edge of science, and through the network the researchers will obtain expertise in a number of different technologies. All of these points are true not only for the young researchers, but also for the senior, established staff. Thus the European human resources in this field will benefit as a result of the training network, not only by high quality multidisciplinary training of young researchers, upon whom our biomedical advances will depend in the future, but also by enhancement of the understanding and expertise of our current research leaders.

The transfer of knowledge is an integral function of the network, in order to ensure a coherent approach to the unravelling of the role of oxidative stress in disease pathophysiology. All the research teams will benefit from the network, and from this sharing of expertise and understanding. Each research team will train one early stage researcher for 3 years (allowing sufficient time for them to obtain a doctorate if desired), who will attend the network organized meetings and courses. Each team will also appoint one experienced researcher (1 year) to facilitate technology and knowledge transfer between the teams, but who will also benefit from training. Thus within the network, the ESRs and ERs are carefully balanced, to ensure that the main aim of training ESRs is achieved while allowing ERs to gain career experience by helping to instruct ESRs.

Page 18 of 66

“HELP”

One way in which transfer of knowledge will be achieved is through the exchange of the young researchers. During their period of appointment, they will be required to undertake secondments to one or two collaborating research teams, for a total length of approximately 25 per cent of their contract. This will allow them to learn several complementary technologies, and expand their understanding of the applications. These secondments will form part of the individual research project being undertaken, and in many cases will involve transfer of materials for analysis by alternative methods, to allow correlation between distinct aspects of oxidative damage.

A second method for transfer of knowledge will be via special “open weeks” hosted by several research teams. This will offer the opportunity for a number of researchers, both junior and senior from elsewhere in the network, to learn about specific technologies and their application, by the observation of ongoing research at the host institute. In some cases, this will also allow exchange of materials for analysis, and some preliminary collaborative tests to be carried out. These open weeks are aimed at facilitating the exchange of information between more disparate areas of the network, rather than between collaborating groups, as the latter will already be achieved by the use of secondments.

There will also be summer schools (2 during the programme) and research meetings (1 per year) throughout the duration of the project. The summer schools will provide theoretical teaching in specific aspects of oxidative stress research and important technologies, and are aimed at training of the young researchers. The courses will be delivered by senior members of the network, with some international guest speakers. The research meetings will be organized by project task, and will involve mainly oral presentation by both senior and junior members of the network. This is an important aspect of training, as it will provide the junior researchers with the opportunity to present their research and receive feedback in a supportive environment, thus preparing them for the experience of an international conference. This is an essential transferable skill for all young scientists.

In addition to these mobility-based methods for transfer of knowledge, use of the network portal will ensure routine and ongoing information exchange between the groups. A junior researcher in each team will be made responsible for regular input from the team, reporting on significant advances within the task and the development of new methodologies.

This network will have a major impact on the oxidative stress field, especially within Europe but also on the international community. In addition to the generation of highly trained young researchers with extensive training in a broad range of technologies, who will be invaluable in the advancement of biomedical science in Europe, the project will achieve the integration of several cutting edge technologies in the study of disease. Moreover, it will enable a communality of approaches to be applied to studies of oxidative pathophysiologies in future research. This should have far-reaching beneficial effects for European science and ultimately for the communities health.

Clearly, throughout Europe there is a high demand for excellent researchers trained to a high level in a variety of techniques. Currently, young researchers tend to have in depth expertise in only one technology, but with the number of multidisciplinary projects ever-increasing, the requirement for researchers with high level experience multiple methodologies is steadily growing. Thus the experience gained by working within this network will offer an invaluable and unique enhancement of their careers, and they are certain to be in great demand for research positions world-wide. In subsequent positions they will be able to contain the transfer of knowledge acquired within the network. The experienced researchers will benefit within their research groups by the import of new research ideas and access to methodologies, which will undoubtedly enhance future research in this area, and consequently is beneficial to their careers.

Page 19 of 66

“HELP”

B2.3. Planned recruitment of early-stage and experienced researchers (one A4 page + table)

In total, 504 person-months of early stage researchers (corresponding to 75%) and 168 person-months of experienced researchers (corresponding to 25%) are requested. In all cases, the ESRs will be appointed for 3 years, to enable them to register for a PhD. The ERs will be appointed for 12 months, and will start their appointment slightly earlier than the ESRs in some teams, so that they are already established and able to mentor the latter when they start. The ERs will start first in the teams that are most central in the networking, or the teams in less favoured countries. All the ERs will undertake secondments (up to 25% of their appointment) to other teams, for the purpose of knowledge transfer and networking. It is also envisaged that ERs may move from one appointment to another in the network, thus also helping transfer of skills.

The emphasis of the network is thus very much on the ESRs, but a number of ERs is essential for the networking and transfer of knowledge activities, which are a fundamental goal of the network. If the ERs were not appointed, the transfer of detailed practical expertise would be much less effective, and the students training would be less outstanding. Having one ER per team will also allow each ER to mentor an ESR, thus gaining valuable supervisory skills.

All the teams involved in the Network are composed of a good number of permanent researchers who are able to supervise young researchers under good conditions. Moreover, as our field is multidisciplinary, it is an excellent opportunity to train young researchers, giving them the possibility of widening their scientific competence, particularly through exchanges between the various teams and via the educational network. Overall, the person-months contributed to the project by research staff who are already part of the teams (2272) greatly exceeds the number of person-months of the appointed young researchers, thus demonstrating that extensive support for research and career advice will be available to the young researchers, and ensuring an excellent training environment.

When the Research Training Network has been approved, all the vacancies will be published in the network Web site, together with two other Web sites which are concerned with HNE (International HNE Club www.kfunigraz.ac.at/HNE-club) or oxidative stress (Society for Free Radical Research, SFRR-Europe www.sfrr-europe.org (employment part)). Vacancies will also be published by the different teams in local Universities and Research Institutes, and advertised nationally. In most of the countries, Email lists of Universities and Doctoral Schools are available and will be used. It is not envisaged that there will be any problem in recruiting young researchers, owing to the number of teams, Universities, and Countries involved in this network. It is anticipated that there will be many instances where doctoral students completing their PhD in one team will be very keen to apply for and ERs position in another team. All the Universities are also generating excellent graduates who are likely to wish to be involved in the Research Training Network.

From all the applications, a shortlist of candidates will be prepared, and this will aim to be evenly balanced in terms of male and female applicants, assuming that this does not compromise the quality of the shortlist significantly. Shortlisted candidates will be invited for interview and will give a short presentation of their research experience or career aims, as appropriate. The management team will be ultimately responsible for all the appointment decisions, although the individual team leaders will be involved at all stages of the process.

The overall criterion for selection of the young researchers will be their intellectual and practical excellence. Their scientific competence and experience, scientific motivation and willingness for communication and exchanges between teams will be considered. The appointments will aim to broaden the research and technical expertise of the appointees, rather than encouraging them to continue in an area where they are already expert. We will also examine carefully the male-female parity, both in terms of the total appointments, and within each team. Dr Spickett, as Assistant Coordinator, will oversee this aspect of recruitment.

Page 20 of 66

“HELP”

Early-stage and experienced researchers to be financed by the contract

Other professional research effort on the network project

Network Team

Early-stage researchers to be financed by the

contract(person-months)

(a)

Experienced researchers to be financed by the

contract(person-months)

(b)

Total (a+b)

(c)

Researchers Likely to contribute(number of individuals)

(d)

Researcherslikely to

contribute(person-months)

(e)1. Salz2.Zagreb3.Glasg4.Dublin5.Bratis6.Lleida7.INRA8.INSER9.Leipzig10.Magd11.Laris12.Lodz13.EliTe14.Torin15. Berli

36363636363636363636363603636

12121212121212121212121201212

48484848484848484848484804848

1911101067889768379

190160220154173891392641921391631444389113

Totals 504 168 672 128 2272

Page 21 of 66

“HELP”

B3 QUALITY/CAPACITY OF THE NETWORK PARTNERSHIP

B3.1. Collective expertise of the network teams (one A4 page per network team)

The participants in the network are:

12 teams from EC countries plus 2 from associated states (Slovakia and Poland) & 1 from other (Croatia)

No Team Short Name Country Team Coordinator Location

1 Salzburg A P. Eckl University of Salzburg

2 Zagreb CR N. Zarkovic Rudjer Boskovic Institute, Zagreb

3 Glasgow UK C.M. Spickett University of Strathclyde, Glasgow

4 Dublin IR G.P. Davey Trinity College Dublin

5 Bratislava SLK S. Stolc Slovak Academy of Sciences,

Bratislava

6 Lleida SP R. Pamplona Medical Faculty, Lleida

7 Toulouse-INRA F F. Guéraud INRA-UMR 1089, Toulouse

8 Toulouse-INSERM F A. Negre-Salvayre Unite INSERM 466, Toulouse

9 Leipzig D J. Arnhold Universität Leipzig

10 Magdeburg D I. Wiswedel Medical Faculty, Magdeburg

11 Larissa GR A. Tsezou University of Thessaly

12 Łodz PO G. Bartosz University of Łodz

13 EliTec/Tatzber A F. Tatzber Tatzber K.E.G. & EliTec GmbH,

Bisamberg, Austria

14 Torino D G. Poli University of Torino

15 Berlin D T. Grune University of Berlin

In the following pages, each teams presents the research staff involved in the Research Training

Network, their expertise and their role in the tasks and objectives of the network.

Page 22 of 66

“HELP”

B3.1.1 Department of Genetics and Developmental Biology, University of Salzburg (Austria)

Address: Institute for Genetics and Developmental Biology, Hellbrunnerstraße 34, A-5020 AustriaPhone: +43.662.8044-2410 Fax: +43.662.8044-144e-mail: peter.eckl@sbg.ac.at URL: http://www.gen.sbg.ac.at/index.html

Staff:

Prof.Dr. Peter Eckl, Vice Rector for Research, University of Salzburg (25%); Dr. Nikolaus Bresgen (40%); Dr. Hans-Christian Bauer, senior researcher at the Austrian Academy of Science and the Institute of Genetics and Developmental Biology (40%); 2 researcher assistants (20%); 3 technicians: Giesshammer Johann, (10%), Glaser Johann (10%), Wimmer Herbert (10%); 2 Secretaries (20%); 8 PhD students: (10% each), plus a Project Manager, to be appointed (100%).

Expertise: The research team of Peter Eckl at the Institute of Gentics and Developmental Biology, University of Salzburg, has been conducting extensive research and development work on HNE, lipid peroxidation, and ROS. In particular this group developed a genotoxicity assay with primary hepatocytes which - due to the metabolic competence of these cells - proved to be a very sensitive and reliable indicator for the cytotoxic and genotoxic potential of suspect compounds. This test system was also applied for the investigation of lipid peroxidation products, especially HNE and the results not only proved the sensitivity of hepatocytes but additionally gave the hint that not HNE itself but one of the metabolites must be responsible for the effect observed. Furthermore,  an indirect involvement in the carcinogenic process has to be assumed. Currently the group studies the action of antioxidants in the primary hepatocyte system.

In addition, oxidative stress appears to play an important role in a variety of pathological events ranging from Alzheimer’s disease to tissue damages after stroke and ischemia. In these cases endothelial cells are thought to be important mediators. In co-operation with Hans Bauer from the Institute of Molecular Biology of the Austrian Academy of Sciences, the institute was able to confirm the sensitivity of brain endothelial cells to HNE and hypoxia / reoxygenation. Currently we are investigating the induction of apoptosis after hypoxia.

Role in the project: In vitro test systems for the evaluation of adverse effects of oxidative stress and the protection by antioxidants.

There are many test systems available to determine in vitro toxicological endpoints of exposure ranging from bacteria (Ames test) to higher eukaryote cell lines and primary cells. Of particular interest are primary cells since they can be considered to more reliably reflect the in vivo situation and thus the potential risk associated with a given exposure. In addition modern culture techniques such as serum-free media formulations allow a precise control of the cellular milieu and reproducible analysis of markers such as metabolites, modified proteins and DNA, induced enzymes, etc. Finally, there are good models of oxidative stress which can be applied to these systems, i.e. for hypoxia / reoxygenation.

Examples of test systems and their advantages and disadvantages related to the endpoints to be determined will be discussed, and the systems will be demonstrated.

Recent publications:

1. Bresgen, N., Karlhuber, G., Krizbai, I., Bauer, H., Bauer, H. C. and Eckl, P.M (2002). Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations, micronuclei and apoptosis. Journal for Neuroscience Research; in press.

2. Slamenová D., Chalupa I., Robichová S., Gábelová A., Farkasová T., Hrusovská L., Bacová G., Sebová L., Eckl P., Bresgen N., Zeitheim P., Schneider P., Wsólová L., Barancoková M., Kazimírová A., Navarová J., Bezek S. (2002) The effect of dietary intake of vitamins A or E on the level of DNA damage, chromosomal aberrations and micronuclei induced in freshly isolated rat hepatocytes by different carcinogens. Nutrition and Cancer, in press.

Page 23 of 66

“HELP”

B3.1.2 Rudjer Boskovic Institute, Division of Molecular Medicine, Zagreb (Croatia)

Address: Rudjer Boskovic Institute, Bijenicka 54, HR-10000 Zagreb, CROATIA.Phone: 00385 1 4560 937 Fax: 00385 1 4561 010 e-mail: zarkovic@rudjer.irb.hrURL: http://www.irb.hr/irb-en.html - Laboratory for Oxidative Stress, former Lab. of Cell & Tissue Differentiation

The team consists of these researchers:

Neven Zarkovic, M.D., Ph.D., senior scientist, group leader, experimental pathology (55%); Kamelija Zarkovic, PhD., clinical and experimental pathologist (40%); Tanja Marotti, Ph.D., biologist, senior scientist, immunology and oxidative stress, in vivo experiments (15%); Marija Poljak Blazi, Ph.D., biologist, senior scientist, iron and oxidative stress, in vivo experiments (15%); Drazen Vikic Topic, Ph.D., physicist, senior scientist, director of the NMR centre, NMR analysis (10%); Karolj Skala, Ph.D., senior research associate, physicist, computer software and internet, web editing (10%); Marina Ilakovac Kveder, Ph.D., physicist, senior research associate, EPR analysis (10%); Suzana Borovic, Ph.D., physicist, pharm. biochemist, senior assistant, HPLC, in vitro experiments (50%); Tihomir Balog, Ph.D., biologist, research associate, NO (RNS), in vitro experiments (25%); Ana Cipak, B.Sc., mol. biologist, junior assistant, immunocytochemistry, in vitro experiments (50%); Tea Vukovic - technical associate (50%); Nevenka Hirsl - technician (25%); 2 Ph.D. Students (100%); 1 Postdoc (100%).

Expertise: Division of Molecular Medicine at Rudjer Boskovic Institute with its affiliated partner laboratories is well equipped and able to provide suitable teaching criteria. Laboratories are fully equipped for in vitro manipulating with nucleic acids and proteins, in vitro cell culturing, protein characterisation, immunological investigations, molecular cloning, gene therapy: equipment for PCR , electrophoresis – horizontal and vertical, ultracentrifuges and other centrifuges, CO2 incubators, sterile units, hybridization oven, ELISA readers, –80°C refrigerators, microinjector, microdissector, Real-time-PCR equipment, microscopes, Fluorescence Activated Cell Sorter (FACS), HPLC systems, chemiluminometer, immunoblotting membrane electrophoresis, lyophilizer, sonicator, scintillation counter, spectrophotometers, amperometer for measuring real-time NO release, electronmicroscopy, EPR, NMR - Bruker AV 600 and AV 300, Varian Gemini 300 11, etc. Recognition as a national/international facility: Animal Facility Unit; Croatian human tumor and DNA bank; Center of excellence in molecular medicine for Croatia and South-East Europe

Role in the project: The Zagreb Team will be involved in basic and clinical research activities (with subcontracting university clinics in Zagreb) of novel clinically applicable assays for oxidative stress and on age and sex dependent studies of the antioxidant enzymes, on growth modulating effects of HNE and oxidative stress as well as on analysis of oxidatively and HNE-modified proteins. Particularly important is involvement of clinical and experimental pathologists (headed by Prof. K. Zarkovic, Head of Zagreb University Division of Neuropathology) that made the Zagreb team well known for their expertise in human and animal immuno-histochemistry and electron-microscopy of HNE-protein adducts. Furthermore, due to the existing project on genetically modified yeast as an advanced in vitro model of cellular response to oxidative stress carried with the group headed by Prof. S. Kohlwein Director of the Institute of Molecular Biology, Biochemistry and Microbiology in Graz, Austria, the Zagreb team will involve also this famous Austrian research group in the network activities. The institute of Biochemistry in Graz became world-wide famous at the time of late Prof. Hermann Esterbauer who discovered HNE. Of additional relevance is also involvement of Prof. R.J. Schaur, one of the closest collaborators of the late Prof. Esterbauer who is an expert in enzymatic aspects of cancer metabolism and oxidative stress and acts now as research partner of the group in Zagreb. Their particular interest is the role of granulocytes in cancer development and therapy.

Two recent articles:1. Fiorella Biasi, Luciana Tessitore, Danialla Zanetti, J.C. Citrin, B. Zingaro, Ellena Chiarpotto, Neven

Žarković, G. Serviddio and Giuseppe Poli (2002) Associated changes of lipid peroxidation and TGF1 levels in human cancer during tumor progression. Gut, 50: 361-367

2. Thomas Semlitsch, Hilmar M. Tillian, Neven Žarković, Suzana Borović, Martin Purtscher, Otmar Hohenwarter and Rudolf J. Schaur (2002) Differential Influence of the Lipid Peroxidation Product 4-Hydroxynonenal on the Growth of Human Lymphatic Leukaemia Cells and Human Peripheral Blood Lymphocytes. Anticancer Res, 22:1689-1697

Page 24 of 66

“HELP”

3. B3.1.3 Department of Bioscience, Royal College Building, Glasgow, (Scotland, UK)

AddressDepartment of Bioscience, University of Strathclyde, Royal College Building, 204 George Street, Glasgow, G1 1XL, UK.Tel. +44 (0)141 5483827 Fax. +44 (0)141 5534124 Email. c.m.spickett@strath.ac.uk

Staff:Dr C.M. Spickett (Lecturer, 60%), Dr A.R. Pitt (Head of Proteomics, 30%), Dr M.H. Grant (Reader, 25%), Dr R. Burchmore (Senior Research Assistant, 20%), Dr S. Kelly (Senior Protein Scientist, 20%), 2 technicians (40%), 2 Graduate students (100%), 1 Graduate student (25%).

ExpertiseThe Glasgow team have an established record in the application of electrospray mass spectrometry to

the analysis of oxidized phospholipids, following our research over the last 6 years. We have shown that intact peroxidized and chlorinated phospholipids can be detected detected in oxidatively stressed cells by this method, and have also applied it to the study of the effect of myeloperoxidase on LDL. Recently we have demonstrated that human phagocytic cells can oxidize phospholipid vesicles to a variety of products, including an epoxyisoprostane derivative of phosphatidylcholine that is known to have bioactive effects. We are now expanding our work to the preparation of specific oxidized phospholipids, such as chlorohydrins, and determination of their inflammatory properties. We also have extensive experience in methods for analysis of glutathione and other cytosolic antioxidants, and are particularly interested in the role of glutathione in protection of cells against oxidative damage and the effect of lipid oxidation products. Dr Grant has been working on aspects of glutathione metabolism during stress and toxicity for more than 10 years, and is developing novel methods for detection of glutathione within cells by laser microscopy. Dr Pitt has many years of experience of mass spectrometry both of lipid and protein modifications, and has recently moved to the Sir Henry Wellcome Functional Genomics Unit, where he now manages the proteomics facility. The senior staff in this team thus have complimentary specializations.

The team has a wide range of modern equipment required for this work, including cell culture facilities, HPLC, equipment for spectrophotometric, fluorimetric, and luminescent assays in microplates or larger samples. We have access to 2-photon laser microscopy at the Biophotonics Centre of Strathclyde University, and mass spectrometry facilities in the Faculty of Science. The Sir Henry Wellcome Facility has extensive equipment for proteomics and mass spectrometry.

Role in NetworkAnalysis of oxidized phospholipids by mass spectrometry. We will continue to develop mass spectrometric methods (mainly electrospray ionization) for the detection of products of lipid oxidation, to improve the sensitivity and application to clinical samples. This will help diagnosis of inflammation and oxidative stress in disease, and provide information on the types of oxidants responsible for pathology. This will require collaboration with Leipzig and Magdeburg for additional mass spectrometric techniques, and Zagreb and Toulouse-INRA for investigation of pathological samples. This research addresses Tasks 1 , 4 & 5.

The inflammatory and cytotoxic effects of oxidized phospholipids. The effects of specific oxidized phospholipids and aldehydes on inflammatory parameters such as cytokine production, leukocyte-endothelial adhesion, and ROS release will be monitored, in addition to the analysis of necrosis and apoptosis. The effects on cellular redox status will also be determined. The mechanisms involved in inducing these changes will be investigated using proteomic methods to observe changes in protein expression in stressed cells. Overall, this will allow us to understand the pathological consequences of different types of lipid oxidation. Collaborations with Toulouse-INSERM, and Lleida are planned, and the research relates to tasks 1 & 4.

Publications relevant to Network1. The detection of lipid oxidation in stressed cells by reversed-phase HPLC coupled with positive-ionization

electrospray MS. Spickett, C.M., Rennie, N., Winter, H., Zambonin, L., Landi, L., Jerlich, A., Schaur, R.J. and Pitt, A.R. (2001) Biochem. J. 355 449-457.

2. Phospholipid chlorohydrins cause ATP depletion and toxicity to human myeloid cells. Dever, G., Stewart, L.J., Pitt, A.R. and Spickett, C.M. (2003) FEBS Letts. 540(1-3) 245-250.

Page 25 of 66

“HELP”

B3.1.4 Department of Biochemistry, Trinity College Dublin, Dublin 2, IRELAND.

AddressDepartment of Biochemistry, Trinity College, Dublin 2, IRELAND.Tel. +353 (0)1 608 1082 Fax. Tel. +353 (0)1 677 2400

PRINCIPAL SCIENTIFIC AND TECHNICAL PERSONNEL, THEIR EXPERIENCE AND RESPONSIBILITY:

Name Qualification Position ExpertiseTime (%)

G.P Davey BA, PhD (M) Lecturer Mitochondria, bioenergetics, inhibitors 50K.F. Tipton BSc PhD (M) Professor Enzymology, kinetics, neurochemistry 30M. Motherway BSc (F) Res. Tech. Assay procedures, Validation 20A.D. McDonald BA, PhD (M) Res. Fellow Statistics, dynamics, modelling, simulation 20J. Healy BSc (M) Res. Student Oxidative enzymes & modification 20J. O'Sullivan BSc (M) Res. Student Enzyme inhibition, ligand binding, receptor

interactions20

C. Chambers MA (F) Res. Assoc. Toxicology assessment 20J. Connolly MA (M) Res. Student Modelling, quantitation 20L.Brady BA (F) Res. Student PTP and ROS assays in mitochondria 201 Post-Doc Bsc/PhD(F) Post-Doc Cell Biology/Biochemistry 100

Expertise: This team has considerable expertise in the areas of nerve transmission and neurodegeneration. Dr Gavin Davey (M) has held a Marie Curie Research Fellowship and is a Senior Research Fellow funded by the Science Foundation, Ireland. His research is centred on energy metabolism and mitochondrial dysfunction in brain and in mathematical modelling of synapse action and in modelling the blood-brain barrier system. He has wide experience in mechanisms of neuroprotection and neuroregeneration, particularly in parkinsonian mouse models. Professor Keith Tipton (M) is Professor of Biochemistry at Trinity College Dublin and is a member of Editorial Boards of major journals of biochemistry and pharmacology and a wide range of international committees. He has research interests in enzymology neurochemistry and neuroprotection and has received support from industrial collaborations and from the EC Framework 5 (EC INTAS, COST D8 and DB). His research group has been active in the areas of enzymology, neurochemistry, bioenergetics and protein chemistry for many years. The laboratory is fully equipped with HPLC systems, spectrophotometers, fluorimeters, bioenergetics apparatus, FACS machine, and molecular biology apparatus.

The University of Dublin, Trinity College, (TCD) has extensive experience in the administration of EU research contracts.

Role in Network: This team will focus on mechanisms of reactive oxygen species production in brain mitochondria and subsequent implications for the neurodegenerative processes. Mitochondrial production of ROS is thought to arise from defects in the electron transport chain and from opening of the permeability transition pore. Research will focus on the nature of such ROS production and any subsequent neuronal damage. In addition to those enzymes that produce hydrogen peroxide and superoxide as potential ROS precursors, some others directly form ROS as a consequence of aberrant reactions or leakage of reactive intermediates. An understanding of the behaviour of these enzymes as well as that of the scavenging enzymes, dealt with elsewhere in the course, is thus essential to an understanding of the oxidative stress load to which the cell may be exposed under different physiological and pathophysiological conditions. Experiments will focus on the kinetic and mechanistic behaviour of the individual enzymes and integrated systems. This work is directly relevant to Tasks 1, 2 and 3 and collaboration will be required with Berlin, Zagreb, Lodz and Lleida to bring the project to completion.

Relevant Publications1. Davey GP, Peuchen S and Clark JB (1998) Energy thresholds in brain mitochondria – potential

involvement in neurodegeneration. J. Biol. Chem 273, 12753-12757.

Page 26 of 66

“HELP”

2. Bianchi L, Della Corte L and Tipton KF. (1999). Simultaneous determination of basal and evoked output levels of aspartate, glutamate, taurine and GABA during microdialysis and from superfused brain slices. J. Chromatogr., B 723, 47-59.

Page 27 of 66

“HELP”

B3.1.5 Institute of Experimental Pharmacology Slovak Academy of Sciences, Bratislava (Slovakia)

Address:Institute of Experimental Pharmacology, Slovak Academy of Sciences, Dubravska 9, 841 04 Bratislava, Slovak Republic. Phone: (+421-2)5941 0657; Fax: (+421-2)5477 5928; e-mail: exfastol@savba.skURL: http://www.uef.sav.sk/homepage.htm

StaffSvorad Stolc, MD, DSc (pharmacology), senior scientist, director (40%); Zdenka Gasparova, PhD (pharmacology), senior scientist (50%); Olga Ondrejickova, PhD (biochemistry), senior scientist (50%); Silvia Skalska, MD (electrophysiology), postgraduate student (100%); Vladmir Snirc (chemistry), postgraduate student (100%); Magdalena Majekova, PhD (drug design) (20%).

ExpertiseThe team has been involved for almost two decades in research of role of reactive oxygen species in damage of nervous tissue induced by ischemia, hypoxia, neurotrauma, as well as by long lasting hyperglycemia. Functional, biochemical, and morphological indicators of central and peripheral nervous system have been assessed. Action of well established antioxidants as well as novel drugs with antiradical properties based on pyridoindol moiety have been analyzed in protection and treatment of these injuries. New molecules were designed with suitable array of pharmacological, pharmacokinetic, and toxicological properties were described. Based on this knowledge new congeners of existing drugs are designed, synthesized and tested in protection of tissues against oxidative impairments induced by controled experimental conditions. The team is focused on electrophysiology (synaptic transmission in brain slice technique in reversible hypxia, condution in peripheral nerve in vitro and in vivo, resistance to ischemic nerve condution failiure, etc.), behavioral assessment after brain ischemia (temporary carotid occlusion in gerbil and rat), sensomotoric stage and mortality after acute head trauma (mice), biochemichal indicators of lipid and protein oxidative impairments (TBARS, protein carbonyls, activity of selected oxidative stress-sensitive enzymes), and morphology (brain bleeding, edema, necrosis, apoptosis).

Role in the project1) Neuroprotection of central nervous system in oxidative stress by conventional and new drugs with enhacned antiradical propertiesThe new antioxidants to be tested further reveal high activity in lipoperoxidation test surpassing that of well established rugs. The putative neuroprotective action has to be proved by more complex techniques. Morover, mechanism of their action has to be elucidated and confirmed on subcellular level. Collaboration with Zarkovic, Croatia and Tipton, Ireland (Task T3)2) Antioxidants in cancer treatmentNew congeners of currently available pyridoindole antioxidants tested so far will be prepared and forwarded to the partner involved in study of oxidative stress in cancer development aimed in improvement of therapy and diagnostics. Collaboration with Zarkovic, Croatia and Bartosz,Poland. (Task T4)

Publications relevant to the Network1. R.Vlkolinsky, S.Stolc: : Effects of stobadine, melatonin, and other antioxidants on

hypoxia/reoxygenation-induced synaptic transmission failure in rat hippocampal slices. Brain Res. 850:118-126 1999.

2. O.Ondrejickova, S.Stolc, A.Ziegelhoffer, L.Horakova, J.Styk, A.Dzurba., R.J.Schaur: Postischemic reperfusioon of the spinal cord: Prolonged reperfusion alleviates the metabolic alterations induced by 25 min ischemia in the cervical and thoracolumbar segments. Gen.Physiol.Biophys. 19:415-426 2000.

Page 28 of 66

“HELP”

B3.1.6 Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina (SPAIN)

Address: Departament de Ciències Mèdiques Bàsiques, Facultat de Medicina, Avda Rovira Roure 44, E-25198 Lleida, SPAIN. Phone: +34-973702408; Fax: +34-973702426

e-mail: manuel.portero@cmb.udl.es ; URL: http://www.udl.es/usuaris/y4371116/eng.htm

The team consists of these researchers:

Dr Manuel Portero-Otin (35%), Dr Reinald Pamplona (35%) , Dr Maria Josep Bellmunt (25%), Dr Joan Prat (5%) , Dr Luis Brieva (10%), Sílvia Garcia (50%), Anna Valls (25%)

Expertise of the team:

The Metabolic Pathophysiology Research Group (MRP), Lleida (Spain) has been working throughout the last 10 years in the role of nonenzymatic protein and aminophospholipid modification in ageing and human pathophysiology. It is known that aldehyde-containing molecules (even glucose) could react with the free amino or sulphydryl groups of proteins or aminophospholipids. Depending on the origin of the aldehyde, the resulting products could be classified as glycoxidative (when the aldehyde arises from glucose or related compounds), or lipoxidative (when the aldehyde comes from lipid peroxidation). This nonenzymatic reaction seems to be involved in the pathogenesis of different degenerative conditions ranging from chronic diabetes complications to neurodegenerative processes. This reaction is also important in the changes associated with ageing. Our group has been applying state-of-the art analytical techniques for the measurement of glyoxidative and lipoxidative products in samples representative of the above-referred processes. Furthermore, we are exploring the cellular response to these aldehydes, in the context of the more general cellular stress response.

The Metabolic Physiopathology Research Laboratory is located at the Department of Basic Medical Sciences (School of Medicine, University of Lleida), and dedicates a substantial portion of time (professionally recognized by National and International Authorities) to facilitate active learning of Medicine to M.D. students.

Role in network

Analysis of lipoxidative and glycoxidative damage in the pathogenesis of neurodegenerative conditions. We will pursue the study of the possible role of glycoxidative and lipoxidative damage, as well as the cellular response over these modifications in the development of a neurodegenerative condition: the amyotrophic lateral sclerosis. The aim is to identify and quantify these and other markers available by the networking in samples representative of the disease, as well as other neurodegeneration models. Furthermore, we would like to characterize the role of these oxidative modification as a trigger of cellular response over unfolded proteins. It is also expected that these analyses could be useful and complementary to the different ones mastered by other network members. We expect to collaborate with Glasgow, Zagreb and Berlin teams. This research is comprised under the Tasks 2 & 3.

Role of fatty acid unsaturation as a determinant of maximum lifespan (longevity). Our previous studies have stressed that -at least for homeoterm species- the maximum lifespan is associated with a lower content of polyunsaturated fatty acid in membranes. This findings has been evaluated using a comparative physiology approach. This association has been confirmed in liver, skeletal muscle, heart and brain lipids, as well as in different subcellular fractions such as mitochondria. The aim is to further identify the molecular pathways responsible for lipid unsaturation maintenance (mainly desaturases) and the relationship with lipoxidative damage. We expect to collaborate with the Graz team. This research is comprised under the Tasks 1& 2

Two recent articles:

1. Portero-Otín M, Pamplona R, Bellmunt MJ, Ruiz MC, Prat J, Salvayre R and Negre-Salvayre A. Advanced glycation endproducts precursors impair epidermal growth factor receptor signaling. Diabetes. 2002; 51:1535-1542.

2. Portero-Otín M, Pamplona R, Ruiz C, Cabiscol E, Prat J and Bellmunt M.J. Diabetes induces an impairment in the proteolytic activity over oxidized proteins and an heterogeneous effect in nonenzymatic protein modifications in the cytosol or rat liver and kidney. Diabetes. 2000; 48: 2215-2220.

Page 29 of 66

“HELP”

B3.1.7 TOULOUSE-Institut National de la Recherche Agronomique-INRA-UMR 1089-Xénobiotiques (France)

Address

INRA-UMR-1089 Xénobiotiques, Toulouse, France.

The team consists of these researchers

Françoise Guéraud CR (70%), Alain Paris DR (30%), Jacques Alary CR (10%), Cécile Canlet IR (NMR) (40%), Estelle Rathahao IE (Mass Spectrometry) (10%), Géraldine Peiro (doc. student) (50%), 2 undergraduate students.

Expertise

In vivo metabolism of HNE has been studied in this group for more than 10 years in the rat with radiolabelled compound. More than ten urinary metabolites were identified and their structure was confirmed by mass-spectrometry techniques. Among the most abundant urinary metabolites, which are much more stable than the parent compound HNE, the dihydroxynonene mercapturic acid derivative (DHN-MA) was evidenced at basal level in the urine of untreated rats but also in human. This compound could be used as a non-invasive biomarker of lipid peroxidation. Mass-spectrometry and ELISA measurements of this compound, together with measurements of classic lipid peroxidation biomarkers (isoprostanes and malondialdehyde) are available in the Laboratory.

Besides, metabonomic approaches are currently used in the Laboratory as High Resolution Nuclear Magnetic Resonance (NMR) and pyrolysis mass-spectrometric methods together with statistical multidimensional techniques are present.

Role in the network

a) Task T2 Relationship between the urinary excretion of DHN-MA and the amount of HNE-protein conjugates detected by immunohistochemistry in a rat model of acute hepatitis and hepatocellular carcinoma (Long Evans Cinnamon (LEC) rats) in order to follow the changes of the excretion of this non-invasive biomarker during the onset of hepatitis and progression of hepatic carcinoma. This rat model would also be a relevant model for the study of the pathological relevance of HNE in these diseases. Collaboration exists with ZAGREB.

b) Task T2 Measurement of the lipid peroxidation process with DHN-MA, isoprostanes and MDA biomarkers in different pathological states (cancer and neurodegenerative diseases). The goal is to determine whether these biomarkers, and particularly DHN-MA, which directly comes from HNE are reliable tools for the diagnosis of the pathological states and could have any predictive value. Collaborations planned with SMEs.

c) Task T2 The efficiency of metabonomic approach in the diagnosis of diseases involving lipid peroxidation process will be assessed to determine if it should provide an early and possibly predictive test in human diseases. Collaborations see b)

d) Task T3 Metabolic disruptions due to lipid peroxidation process on the one hand and to HNE treatment in the rat on the other will be studied and compared using the metabonomic approach on the rat biofluids and organs in order to give a more precise insight of the pathological roles of HNE in the lipid peroxidation pathological consequences. Collaboration with Glasgow.

Publications relevant to Network

1. J. Alary, Y. Fernandez, L. Debrauwer, E. Perdu and F. Guéraud. 2003. Identification of intermedaite pathways of 4-hydroxynonenal metabolism in the rat. Chem Res Tox 16, 320-327

2. M. E. Dumas, C. Canlet, F. André, J. Vercauteren and A. Paris. 2002. Metabonomic assessment of physiological disruptions using 1H-13C HMBC-NMR spectroscopy combined with pattern recognition procedures performed on filtered variables. Anal. Chem. 74, 2261-2273.

Page 30 of 66

“HELP”

B3.1.8 INSERM U466 and Faculty of Medicine Rangueil, Paul Sabatier University, Toulouse, France

Address: INSERM U466, IFR31, CHU Rangueil, 31403 Toulouse, Cedex 4, France.Tel: 33 561 322 059; Fax: 33 561 322 084; Email: anesalv@toulouse.inserm.fr

Staff

Dr Anne Negre-Salvayre PhD, DR CNRS (80%), Prof. Robert Salvayre MD, PhD (30%), Dr Nathalie Auge CR INSERM, PhD (100%), Dr. Meyer Elbaz, MD, PhD (20%), 1 research assistant (40%), 1 research technician (80%), 2 PhD students (100%).

Expertise

Our team has many years experience in the field of biological effects induced by oxidized LDL on cellular vascular cells. We have investigated the cell signaling pathways activated by oxidized LDL and lipid oxidation products (HNE), which result in cell proliferation or apoptosis as function of LDL concentration and cell type. We reported that oxidized LDL exerts a biphasic effect on vascular cells, mitogenic for smooth muscle cells at low concentrations, and apoptotic at higher concentrations on vascular cells. The proliferative effect of oxidized LDL involves diverse signaling pathways such as the sphingomyelin/ceramide/sphingosine 1-phosphate system, the direct activation of tyrosine kinase receptors (RTK), the activation of PI 3 kinase/Akt pathway and of transcription factors AP1 and NFkappaB. Those systems could be activated by intracellular oxidative stress, and by the derivatization of signaling proteins by HNE. Oxidized LDL induce the apoptosis of vascular cells through mechanisms involving both calcium deregulation and caspase activation.This work is now completed by studies on animal models of atherosclerosis (apoE-/- mice), fed with diet containing various antioxidant and carbonyl scavenger molecules in order to screen the in vivo involvement of oxidation in atherosclerosis and to find new molecules able to prevent or slow down plaque formation.

Role in NetworkParticular interest will be given to the mechanism of protein derivatization triggered by lipid oxidation products (HNE and acrolein). This derivatization occurs through the fixation of HNE on free NH2 and SH groups of proteins which impairs progressively their properties. The observed effect is biphasic with activation (proliferation at low concentration) and inhibition followed by protein dysfunction and impairment at higher concentrations. Protein derivatization is a mechanism shared by diverse pathologies in which oxidative stress is involved, in particular atherosclerosis and neurodegenerative disorders. We will study the “when and how” of protein derivatization by HNE occurs in pathologies related to oxidation (atherosclerosis, NIDDM, neurodegenerative diseases) and the consequences on protein dysfunction (for instance gain or loss of function of signaling receptors or transporters in correlation with derivatization). We are experienced in the use of techniques allowing to measure the carbonyl protein content, oxidation markers, western-blots, enzymatic activity of purified proteins with evaluation of loss of function, cell signaling, immunocyto-and immunohistochemistry, molecular biology of signaling proteins, animal models. We will try to develop new molecules able to prevent protein derivatization and to restore their function.

Publications relevant to the Network1. Escargueil-Blanc I, Salvayre R, Vacaresse N, Jurgens G, Darblade B, Arnal JF, Parthasarathy S, Negre-

Salvayre A. Mildly oxidized LDL induces activation of platelet-derived growth factor beta-receptor pathway. Circulation. 2001, 104, 1814-1821.

2. Auge N, Garcia V, Maupas-Schwalm F, Levade T, Salvayre R, Negre-Salvayre A. Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways. Arterioscler Thromb Vasc Biol. 2002, 22, 1990-1995.

3. Andreoletti O, Levavasseur E, Uro-Coste E, Tabouret G, Sarradin P, Delisle MB, Berthon P, Salvayre R, Schelcher F, Negre-Salvayre A. Astrocytes accumulate 4-hydroxynonenal adducts in murine scrapie and human creutzfeldt-jakob disease. Neurobiol Dis. 2002 11, 386-393.

Page 31 of 66

“HELP”

B3.1.9 Institute of Medical Physics and Biophysics, University of Leipzig (Germany)

Address

Institute of Medical Physics and Biophysics, University of Leipzig, Liebigstrasse 27,D-04103 Leipzig, Germany.Phone: +49-341-9715705 Fax: +49-341-9715709

Staff

Dr. Juergen Arnhold (50%), Dr. Juergen Schiller (30%), 1 technician (20%), 6 PhD students (50%).

Expertise

The research team supervised by Juergen Arnhold has been dealing with the investigation of oxidative stress phenomena in biological systems for more than fifteen years. Special interests have been employed to study the production of reactive oxygen species by human polymorphonuclear cells, the biochemistry of tissue destruction by hypochlorous acid, redox properties of myeloperoxidase and other heme peroxidases, pathways of oxidative alterations in membrane phospholipids. Recently, the standard reduction potentials of all redox couples of the peroxidase cycle of human myeloperoxidase were successfully determined. It has been also evidenced as a surprising new result that myeloperoxidase is able to produce lysophospholipids from highly unsaturated phosphatidylcholines via its product hypochlorous acid. Most important methods used in these investigations are different chemiluminescence approaches (luminol-dependent luminescence as well as the highly sensitive Pholasin luminescence), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS), NMR spectroscopy and others.

Role in the network

Analysis of oxidized phospholipids by mass spectrometry. We will continue our work on analysis of lipid alterations in biological systems under the influence of myeloperoxidase and its products by mass spectrometry. A common platform on the application of mass spectrometric approaches in investigation of oxidative stress phenomena of lipids will be formed in this network with the teams of C. Spickett, Glasgow, UK and I. Wiswedel, Magdeburg, Germany. This research addresses Tasks 2 & 4.

Tissue destruction by hypochlorous acid and myeloperoxidase-driven reactions. We will continue to investigate mechanisms of tissue destruction by HOCl and myeloperoxidase in order to clarify their role in the pathogenesis of such common diseases such as arteriosclerosis, rheumatoid arthritis and others. Additionally, cell physiological properties of neutrophils such as ROS production, enzyme release, cytokine production and phagocytosis will be measured. Comparison with effects of NHE and other toxic aldehydes will be also made. Thus, these data will contribute to a better understanding of the role of neutrophils and its products in different pathologies. Collaborations with Graz, Turin and Lodz are planned. This research related to tasks 1 & 4.

Publications relevant to network

1. Panasenko, O.M., Spalteholz, H., Schiller, J., Arnhold, J. Myeloperoxidase-induced formation of chlorohydrins and lysophospholipids from unsaturated phosphatidylcholines. Free Radic. Biol. Med. 34 (2003) 553-562.

2. Arnhold, J., Furtmüller, P.G., Regelsberger, G., Obinger, C. Redox properties of the couple compound I/native enzyme of myeloperoxidase and eosinophil peroxidase. Eur. J. Biochem. 268 (2001) 5142-5148.

Page 32 of 66

“HELP”

B3.1.10 Faculty of Medicine, Magdeburg, Germany

Address

Institut of Clinical Chemistry and Pathological Biochemistry, Department of Pathobiochemistry, Medical Faculty, Otto-von-Guericke-University Magdeburg ,Germany.Tel. Tel. 49-391-6713638 or 42 Fax. 49-391-6713639Email ingrid.wiswedel@medizin.uni-magdeburg.de

Staff: Dr. Ingrid Wiswedel (40%), Prof. Wolfgang Augustin (em.) (25%), Dr Werner Siems collaborating from Julius Herzog Hospital (20%), 2 technicians (50%), 2 MD students (50%).

Expertise:The research team has experiences in investigations of oxidative stress and antioxidants in biological systems, in particular in isolated mitochondria, tissue homogenates, cell cultures and diverse body fluids for more than fifteen years. Special interests of the senior investigator (I.W.) are lipid peroxidation, mitochondrial phospholipids (cardiolipin), enzymatic and non-enzymatic antioxidants (Mn- and CuZn-SOD, glutathione peroxidase, catalase; glutathione, α-tocopherol, β-carotene) and inflammation. Since about 9 years the research team has experiences in gas chromatography-mass spectrometry analyses of oxidized phospholipids. Until now monohydroxyeicosatetraenoic acids (HETEs), F2 - and F4-isoprostanes as nonenzymatic oxidation products of arachidonic and docosahexaenoic acid as well as prostaglandins (PGE2 and PGF2α) and LTB4 as enzymatic arachidonic acid oxidation products were detected and quantified by GC-MS in the negative ion chemical ionisation mode. The UVB-dependent formation of arachidonic acid oxygenation products of the human skin and the influence of non-steroidal antiphlogistica is the subject of a joint research project with the Clinic of Venereology and Dermatology in Magdeburg.

Role in the network:

Analysis of isoprostanes in tissue samples, cultured cells, isolated mitochondria and body fluids (plasma, cerebrospinal fluid, bronchoalveolar lavage fluid, exhaled breath condensate samples, microdialysates, urine) as biomarkers of in vivo lipid peroxidation by gas chromatography/mass spectrometry (negative ion chemical ionisation; SSQ 710, Finnigan MAT). Studies are performed concerning the release of arachidonic acid oxygenation products in cultured keratinocytes and microdialysis samples of the human skin and their role as lipid mediators in UVB induced inflammation.Levels of isoprostanes are measured in different body fluids of patients with chronic obstructive pulmonary disease (Irfan Rahman, Edinburgh), in plasma samples of hemodialysis patients (together with cholesterol oxidation products by GC/FID; Werner Siems, Bad Harzburg), and in plasma samples of rats and guinea pigs treated with different doses of ozone and cigarette smoke (Yoshiki Kawabe, Tokyo, Maria Kadiska, Durham/NC). Furthermore we continue to investigate the role of oxidized phospholipids in mitochondrial functions (interactions between ANT, cytochrome c and cardiolipin).

Publications relevant to network

1. Wiswedel, I., Bohne, M, Hirsch, D., Kühn, H., Augustin, W., Gollnick, H. A sensitive gas-chromatography-mass spectrometry assay reveals increased levels of monohydroxyeicosatetraenoic acid isomers in human plasma after extracorporeal photoimmunotherapy and under in vitro ultraviolet A exposure. J Invest Dermatol 2000; 115: 499-503.

2. Wiswedel, I., Hirsch, D., Nourooz-Zadeh, J., Flechsig, A., Lück-Lambrecht, A., Augustin, W. Analysis of monohydroxyeicosatetraenoic acids and F2 –isoprostanes as markers of lipid peroxidation in rat brain mitochondria. Free Rad Res 2002; 36(1): 1-1.

Page 33 of 66

“HELP”

B3.1.11 Department of Biology and Genetics, University of Thessaly, School of Medicine, Larissa, Greece.

Address

Department of Biology and Genetics, School of Medicine, University of Thessaly, 22 Paoakyriazi Str., 41 222 Larissa, Greece.

Staff

Aspasia Tsezou (As.Prof. 60%), Panagoula Kollia (As. Prof. 30%), Nicholas Vamvakopoulos (Prof. 10%), 2 graduate students (100%), 1 technician (40%).

Expertise

Our team has considerable expertise in the field of carcinogenesis regarding telomerase activation and methods of detecting DNA damage at the level of chromosomes and genome. Our recent interest focuses on the role of telomerase expression and telomere shortening in viral carcinogenesis as useful biomarkers of oxidative stress. Additional expertise exists on the mechanisms of iron metabolism in leukemia as well as on the effect of oxidative stress in heat-shock proteins activation.

The laboratory of Department of Biology and Genetics is a fully equipped molecular biology laboratory and is able to provide suitable training and teaching criteria.

Role in the network

1) Study of telomerase expression and telomere length.

Since telomeres represent sensors for oxidative damage, our team will study telomere length in vivo reflecting the cummulative amount of oxidative damage to the organ/organism. Also, telomerase activation and expression of the catalytic subunit of telomerase will be studied as a marker of genetic instability and malignant transformation in predisposed cells after exposure to oxidative stress metabolites. Collaboration planed with F. Biasi, Italy? This research address Tasks 1 & 5

2) Study of DNA damage

Damaging effects of free radicals through damage to genetic material leading to cancer development and to inefficient repair processes, will be studied at the level of single-stranded damage in telomeric DNA as well as at the level of DNA fragmentation as a pathway leading to apoptosis. Collaboration planed with P. Eckle-Austria ?

This research address Tasks 1 & 5

Publications relevant to network

1. Molecular analysis of transferrin receptor mRNA expression in acute myeloid leukaemia. Kollia P, Stavroyianni N, Stamatopoulos K, Zoi K, Viniou N, Mantzourani M, Noguchi CT, Paterakis G, Abazis D, Pangalos C, Loukopoulos D, Yataganas X. (2001) Br..J. Hem. 115(1) 19-24

2. Expression of the catalytic subunit telomerase in peripheral lymphocytes in patients affected with hepatitis B and C viruses. (Submitted)

Page 34 of 66

“HELP”

B3.1.12 Department of Molecular Biophysics, University of Łódź, Poland

Address:Department of Molecular Biophysics, University of Łódź, Banacha 12/16, PL 90-237 Łódź, POLAND.Tel. +48 42 6354473 Fax. +48 42 6354476 Email. gbartosz@biol.uni.lodz.pl

Staff: Prof. Grzegorz Bartosz, senior scientist, group leader, reactive oxygen species, antioxidants, ESR

(35%) Prof. Krzysztof Gwoździński, senior scientist, reactive oxygen species in pathologies (20%) Dr. Mieczysław Puchała, Ass. Prof., senior scientist, radiation chemistry and physics (20%) Dr. Mirosław Soszyński, Ass. Prof., senior scientist, immunochemistry, peroxynitrite and

hypochlorite, (25%) Dr. Błażej Rychlik, research and teaching associate, membrane transport, fluorimetry, reactive oxygen

species, cell culture (40%) Dr. Łukasz Pułaski, research and teaching associate, membrane transport, cellular signalling,

molecular biology (40%) Agnieszka Grzelak, M. Sc., research and teaching associate, reactive oxygen species, antioxidants

(60%) Aneta Balcerczyk, M. Sc., Ph. D. student, reactive oxygen species, antioxidant, cell culture (60%)

Expertise: Department of Molecular Biophysics, University of Łódź, is involved in studies of detection of reactive oxygen species and footprints of their action, the mechanism of damage of cellular constituents by reactive oxygen species, including peroxynitrite and hypochlorite, mechanisms of action of natural and synthetic antioxidants, and role of oxidative stress in pathologies and in cellular and organismal aging. All members of the team are active university teachers.

Laboratory equipment includes two ESR spectrometers, 2 fluorimeters, 5 spectrophotometers, HPLC system, fluorimetric/luminometric microplate reader, ultracentrifuge, 2 fluorescence microscopes, tissue culture laboratory, X-ray research facility, lyophilizer, -80oC refrigerator, several electrophoresis/immunoblotting units and access to the animal facility of the Faculty.

Institute of Biophysics including the Department has a status of Centre of Excellence of the State Committee for Scientific Research.

Role in the network:

Task T2 Refinement of fluorimetic methods of estimation of production of reactive oxygen species and peroxides, including lipid peroxides

Task T2 Studies on the pathological significance of peroxynitrite and hypochlorite

Task T3 Evaluation of the usefulness of total antioxidant capacity as a parameter reflecting oxidative stress in pathology

Task T3 Role of oxidative stress in cellular and organismal aging (yeast cell model, studies of centenarians)

Task T4 Studies on the antioxidant and prooxidant action of nitroxides and their effects on normal and malignant cells

Two recent articles:

1. Agnieszka Grzelak, Błażej Rychlik, Grzegorz Bartosz (2001) Light-dependent generation of reactive oxygen species in cell culture media. Free Radic. Biol. Med. 30: 1418-1425

2. Grzegorz Bartosz (2003) Total antioxidant capacity. Adv. Clin. Chem. 37: 219-292

Page 35 of 66

“HELP”

B3.1.13 Tatzber K.E.G. & EliTec GmbH, Bisamberg (Austria) represent SMEs

Address:

Leopoldsgraben 38d, Klosterneuburg, A-3400, Austria.Tel. +43 650 8289273 Email. tatzi@ping.at

Staff Involvement in Network:Dr Franz Tatzber (20%), Dr Willibald Wonisch (40%), Prof Sinzinger (10%), 1 technician (20%)

Expertise:

Both companies are SMEs located in Austria and focused on detection and measurement of several aspects of oxidative stress (OS). EliTec Laborreagenzien GesmbH has developed an ELISA system for detection of antibodies against oxidatively modified LDL (oLAb), which is distributed world wide by several license partners. The other SME, Dr. Franz Tatzber KEG, put its focus on simple enzymatic and fluorescence methods for the measurement of direct effects of oxidative stress (OS). including peroxides, antioxidants and endogenous peroxidases. Recently, the company has developed OS-control sera for quality management systems concerning OS. Another milestone in research and development on methods for detection of OS was the production of several different antisera raised against various oxidatively modified (lipo)proteins, which were used in numerous immunohistochemical studies concerning connections between OS and degenerative diseases like atherosclerosis. Beside these achievements, both companies have cooperated with major representatives of the Marie Curie HNE-network for several years with the aim to develop and produce additional methods for measurement of direct and indirect consequences of OS in biological systems. Furthermore, both companies received export and innovation awards of the Lower Austrian Chamber of Commerce for the development of methods for OS measurement. In Conclusion, both SMEs possess know how in development, production and distribution of biochemical methods for detection of direct and indirect effects of OS in humans and animals. Further achievements include designs and scientific publications of epidemiological and clinical studies focussed on general, physiological, pathological and nutritional consequences of OS.

Role in Network:Dr Tatzber has been collaborating for many years with some teams of the network on research that has

helped in the development of commercial products. He has been collaborating with the team in Zagreb in the clinical testing of the diagnostic kits. Prof Sinzinger is based at the Department of Nuclear Medicine in Vienna, and acts as advisor to Dr Tatzber, he also has links with teams in the network.

This team will collaborate with the other participants in the network with the aim of testing the diagnostic assays in a variety of experimental models and clinical samples. It will also be advise the other teams on the possible application and development of new biomarkers. Members of this team will visit the other teams who are working on improved methods of diagnosis (Task 1). Young researchers will also make short stays at EliTec to find out more about the commercialisation process.

Publications:

1. Resch U, Helsel G, Tatzber F, Sinzinger H. (2002) Antioxidant status in thyroid dysfunction. Clin Chem Lab Med, 40(11):1132-4

2. Zarkovic N, Hayn M, Tatzber F, Reichel H, Kor ic M, Zarkovic K, Plav ic V, Breskovac LJ, Klingmuller M, Paladino J. (2000) Growth promoting effect of human plasma ultrafiltrate bioactive fraction (TBP) for human non-functioning pituitary adenoma cells in vitro. J Endocrinol Invest, 23(11):737-43

Page 36 of 66

“HELP”

B3.1.14 Department of Clinical and Biological SciencesUniversity of Torino, Italy.

Address:

Department of Clinical and Biological Sciences, University of Torino, Via Verdi 8, 10124 Torino, Italy

Tel. +39 011 6708101 Fax. +39 011 6708113 Email. giuseppe.poli@unito.it

URL. http://www.medicina.unito.it/sluigi/index2.html

Staff Involvement in Network:Prof. G. Poli (20%), Dr Fiorella Biasi (30%), Dr G. Leonarduzzi (30%), 1 research assistant (25%), 1 technician (20%), 2 PhD students (30%).

Expertise:

Prof Poli’s team has many years experience in the field of oxidative stress, and biological effects of oxysterols and other lipid oxidation products. They have many important publications reporting the effects of these compounds on TGF-beta, signalling processes and gene expression. There is a strong interest in cancer, and a strong collaboration with the CNR Centre of Immunogenetics and Experimental Oncology. This team collaborated very strongly with Prof Esterbauer on the effects of HNE, and now has collaborations with Dr Zarkovic in the Zagreb team in this area. Prof Poli also carried out extensive research relating to the role of lipid oxidation in atherosclerosis, as well as many other aspects of oxidative stress and antioxidant defence.

Role in Network:

The Torino team will participate in tasks 2 (cancer), 4 (cardiovascular disease) and 5 (antioxidant disorders), contributing mainly to the objectives on cytotoxicity and signalling. Some research on the potential of antioxidants to prevent damage will also be undertaken.

Collaborations will continue with Zagreb and Berlin, and new interactions will be set up with ToulouseINSERM, Glasgow and Bratislava.

Publications:

1. Leonarduzzi G, Sevanian A, Sottero B, Arkan MC, Biasi F, Chiarpotto E, Basaga H, Poli G (2001) Up-regulation of the fibrogenic cytokine TGF-beta1 by oxysterols: a mechanistic link between cholesterol and atherosclerosis. FASEB J, 15(9):1619-21.

2. Biasi F, Tessitore L, Zanetti D, Cutrin JC, Zingaro B, Chiarpotto E, Zarkovic N, Serviddio G, Poli G. (2002) Associated changes of lipid peroxidation and transforming growth factor beta1 levels in human colon cancer during tumour progression. Gut, 50(3):361-7.

Page 37 of 66

“HELP”

B3.1.15 Neuroscience Research Center, Medical Faculty (Charité), Humboldt University Berlin

Address: Free Radical Research Laboratory, Neuroscience Research Center, Medical Faculty (Charité), Schumannstr. 20/21, 10117 Berlin, Germany

E-mail: tilman.grune@charite.de; URL: http://www.charite.de/nwfz

The team consists of these researchers:

Dr Tilman Grune (35%), Dr Grit Sandig (35%) , Alexandra Stolzing (25%), Diana Poppek (5%) , Tobias Jung (25%), Rebecca Widmer (15%), Katrin Merker (50%), Manuela Jakstadt (30%), Deborah Horn (15%)

Expertise of the team:

My laboratory has been working on the oxidative stress mediated damage of cells and organs. The cascade of biochemical alterations in cells after a hypoxic damage was investigated with special emphasis on the nucleotide metabolism, changes in the redox equilibrium, lipid peroxidation and glutathione system. Special attention was given to formation and to the degradation of the lipid peroxidation product 4-hydroxynonenal. Since this aldehyde is able to modify proteins we followed the fate of 4-hydroxynonenal modified and oxidised proteins. We found an increased degradation of proteins in cells treated with 4-hydroxynonenal. In further investigations using various oxidants we demonstrated, that mammalian cells are able to degrade oxidised proteins preferentially.Today we focus our research on the turnover of oxidized proteins the degradation of oxidised proteins in human primary cells and the role of the proteasome in this process. Further studies are focused on the accumulation of oxidized proteins and changes of the proteasomal system during aging and neurodegeneration. Projects investigating the regulation and activity of the proteasome during aging are also in progress. Therefore, we most of the state of the art biochemical, molecular biologcal and analytical methods of free radical research are used in our laboratory. Especially those related to protein oxidation, protein modificcation and protein degradation.Our free radical research laboratory is located within the neuroscience research centre building at the faculty campus. The neuroscience research centre of our faculty is a research facility combined with the training facility for post-docs and researchers early in their carrier.

Role in network

Proteolysis of lipid peroxidation product modified proteins. Our research will be aimed at the recognition and degradation of proteins modified by lipid peroxidation products, especially 4-hydroxynonenal. Special attention will be given to the possible cross-linking effects of these aldehydes and their role in the accumulation of protein aggregates. This will be investigated in neuronal cells due to the important role of these processes in neurodegeneration. The aim is to find the condition at which the proteins are degraded and under what influences this degradation is not sufficient. Furthermore, the effects of these aldehydic lipid peroxidation products on the regulation of intracellular proteolytic systems will be investigated. Since today's knowledge is limited to some of the in vitro effects of these aldehydes, special emphasis will be given to the effect of aldehydic lipid peroxidation products in living cells. We plan to work together with the Lleida Glasgow, Zagreb, Bratislava and Lodz teams. This research is comprised under the Tasks 2, 3 & 4.

Hydroxynonenal and protein cross-linking in aging. Our previous indicated that oxidatively modified protein aggregates play a key role in the age-related decline in the proteasomal activity. Therefore, it is important to investigate whether the protein cross-linking agents during the senescence process are lipid-borne and which role the lipid peroxidation process plays during the aging of a cellular system. For theses investigations we will explore a human fibroblast senescence model and study the effects of endogenous and exogenous lipid peroxidation products on the protein aggregate formation. We expect to collaborate with the Lleida and the Bratislava team. This research is comprised under the Tasks 1& 5

Two recent articles:

1. Sitte, N., K. Merker, T. von Zglinicki, K.J.A. Davies, T. Grune: Protein oxidation and degradation during cellular senescence of human BJ-fibroblasts. FASEB J. 14 (2000) 2503-2510.

2. Grune, T., T. Reinheckel, J.A. North, R. Li, P.B. Bescos, R. Shringarpure, K.J.A. Davies: Ezrin turnover and cell shape changes catalyzed by proteasome in oxidatively stressed cells. FASEB J. 16 (2002) 1602-1610.

Page 38 of 66

“HELP”

1.1 B3.2. Intensity and quality of networking (one A4 page)

The network consists of 12 research groups plus a group of SMEs from 7 European countries, 2 associated states and 1 other country. Several of these teams have already been collaborating in research over the past few years, but the network will enormously enhance the possibility for interactions between teams.

Every team has its own individual research lines and areas of specialization, but between many of the teams there is some overlap in either methodology, research models or research aims, and the research of all the teams contributes to the overall aims and tasks of the project. While each team pursues its own research line, it will also be interacting closely with at least 2 and as many as 5 other teams to ensure transfer of technology and research knowledge. The methods utilized to achieve this are explained below. The network will moreover generate many new international collaborations between teams which have hitherto not interacted, through increasing awareness of the ongoing research areas and available technology. Thus the network is of huge importance in obtaining maximum value from oxidative stress research within Europe.

The network management will ensure that teams containing younger and less experienced team leaders, and also the teams from Slovakia, Poland and Greece, are given additional assistance in terms of mobility. This will facilitate their full integration into the network and the acquisition of research experience and leadership skills from more senior staff. It will also ensure that the appointed young researchers receive the best possible training and fullest interaction with other colleagues, both junior and senior.

The collaborations from one team to another have been summarized by charts presented in B1.5, and will take place through the events and exchange programs described below:

NETWORK-SCIENTIFIC MEETINGS: 4 major research meetings (1 per year), each relating to one of the project tasks, will be organized in turn by the group of teams most closely associated with the topic. These aim to facilitate broad interaction between all the teams, and allow young researchers a platform for presenting their research in a supportive environment (part of the communication skills training). Young researchers will improve their knowledge of the field of oxidative stress by hearing presentations from senior scientists.

NETWORK INSTRUCTIONAL COURSES: 2 instructional courses for early stage researchers will be organized over the duration of the project. These will consist of lectures, seminars or workshops delivered by senior staff of the network or invited eminent scientists. ECTS credits towards the doctoral degrees will be awarded for ESR attendance, but the courses will of course be open to ERs who wish to attend.

MOBILITY AND SECONDMENTS: a major part of the networking will be achieved by mobility of experienced and senior researchers between teams, for periods of 1-6 months. The aim of this is to enable transfer of technology and research expertise within the teams. Every ER appointed in the network will undertake at least one secondment to another laboratory (total length 6 months), while senior researchers will make shorter exchange visits.

OPEN WEEKS: a special feature of the network will be the organization of an open week in each team’s laboratory, to which all ESRs of the network will be invited with the aim of learning new research techniques. 3-4 open weeks will be organized each year. ERs may also participate, but this will be the main form of inter-laboratory mobility for the early stage researchers.

NETWORK PORTAL: networking will also be achieved through the HELP network portal, and one ESR from each team will be responsible for monthly reporting and participation in discussion groups.

Page 39 of 66

“HELP”

Table B3.2 Provisional Schedule for Networking and Educational Events

ProjectYear

Network Scientific

Meetings

(summer)

Network Instructional

Courses

(summer)

Open Weeks

(months from start of project)

1

Germany

Task 1

(Technology) Bratislava (9 months),

Zagreb (11 months)

2

Dublin

Tasks 2 & 3 (Cancer and

Neurodegenerative Diseases)

Bratislava

Sources of ROS, Analysis and

Biomolecular Damage

Toulouse-INSERM (15 mths),

Glasgow (18 months),

Berlin (20 months),

Salzburg (23 months)

3

Toulouse

Tasks 4 & 5 (CVD and

Antioxidative Disorders)

Lleida

Antioxidants, Signalling and

ROS in Pathologies

Łodz (27 months), Leipzig + Madgeburg (30 months),Larissa (33 months)Dublin (36 months)

4

Zagreb

Final Open Meeting joint with

HNE-Club

Toulouse-INRA (38 months),

Lleida (41 months),

Torino (44 months),

Page 40 of 66

“HELP”

B3.3. Relevance of Partnership Composition (one A4 page)

There has already been both preliminary and advanced co-operation between several of the network partners in the field of HNE and lipid peroxidation research (e.g. Zagreb-SME, Zagreb-INRA, Zagreb-Torino, Zagreb-Graz, Zagreb-Salzburg, Zagreb-Bratislava, Zagreb-Dublin). Some of these arose as a result of interactions at the Society for Free Radical Research International and European Conferences, and the setting up of the HNE-Club in 2000 furthered the extent of co-operation, mainly but not exclusively in the area of HNE research. Some are based on joint research activities of the scientists who were working together with the late Prof. Hermann Esterbauer and extended collaborations between their partner institutions in various frameworks (personal or collaboration agreements, joint ventures, bilateral agreements, etc.). The HNE-Club is the most prominent forum worldwide in this area and comprises the most important players in this field of research. In July 2002, a Conference on “4-Hydroxynonenal and Related Lipid Peroxidation Products” was held in Salzburg, and attracted leading researchers form all over the world, including the United States, Japan and Europe. Accordingly, our network will by affiliated to all relevant international laboratories worldwide and will allow the leading position in the fields of oxidative stress research and HNE for the European network partners (our teams published in the period of 5 years more than 300 articles in Current Contents, i.e. more than one per week).

The Salzburg meeting succeeded in introducing many previously unacquainted researchers, and as a result it became apparent that the field of research would benefit from a much more extensive co-operation between members. This would allow complementary approaches to important research questions, comparison of technological developments and oxidative involvement in distinct disease pathologies, and a much enhanced transfer of knowledge between groups with common interests.

This Research Training Network is composed of 15 teams who all share a common drive to understand the mechanisms of oxidative damage and HNE involvement in disease pathologies. The teams all have highly complementary research focuses and expertise. The composition of the network is very well balanced in terms of the research area: we have teams working on HNE, on lipid oxidation, and on other aspects of oxidative stress. Together, the teams address all aspects of oxidative damage to cells, namely lipid oxidation, protein oxidation and modification, and DNA damage. Some teams have experience in working on very fundamental events in oxidative stress, such as the generation of free radicals and ROS, while others have physiological experimental models, and the remainder are at the clinical interface. One of the teams consists of 2 SMEs, who will ensure commercial relevance of the project and collaborate with other teams in developing diagnostic assays.

The network will be invaluable, as it will allow new collaborations to be established between many of the teams in apparently disparate areas that nevertheless contribute synergistically to the whole project. For example, new collaborations of Zagreb with Glasgow, Dublin and Lleida will enable these teams to adopt the use of antibodies to HNE-proteins adducts to relate this to other types of damage observed, and streamline efforts in proteomics. New interactions between Toulouse-INSERM, Dublin, Bratislava and Larissa will allow an understanding of how pathology between different diseases varies, and may be helped by synthetic antioxidants. Overall, each teams will initiate between 2 and 4 new major collaborations, and will develop interactions with all the other teams in the network. The existing collaborations between groups will be considerably strengthened by the structured interactions that are planned, and the clear united aims and objectives of the Research Training Network.

Page 41 of 66

“HELP”

JUSTIFICATION FOR ZAGREB’S INVOLVEMENT

The involvement of Rudjer Boskovic Institute (RBI), Zagreb is absolutely crucial and central to this network. The team at Zagreb have extensive expertise in areas of HNE analysis, HNE-protein adduct detection by several methods, and the study of HNE and lipid peroxidation in neurodegenerative diseases, cancer and in particular in the cell growth control that otherwise would not be available to the other teams in the network. Zagreb can also offer one of the very few neuropathologists with both clinical and experimental disease in the whole of Europe. Moreover, Zagreb is the team with the largest current number of collaborations, and thus acts as a HUB to bring the other teams together. It also has an extraordinarily well funded and equipped facilities of electron-microscopy, NMR, EPR and several molecular biomedicine laboratories that offers excellent training facilities to all the young researchers in the network.

RBI is the biggest Croatian research institution (752 scientists, 314 with Ph.D.) that publish two scientific papers daily on the average, while members of the RBI team participating in this network are among the best experts in the field. The RBI team includes also a clinical pathologist (Prof. Kamelija Zarkovic, Head of University Division of Neuropathology) who has great experience in animal experiments and in vitro studies. This team also performs several clinical studies on the applicability of the novel diagnostic assays (jointly with the Austrian partners).

Due to the existing project on yeast as an advanced in vitro model of cellular response to oxidative stress carried with the group headed by Prof. Sepp Kohlwein at the Institute of Molecular Biology, Biochemistry and Microbiology in Graz, Austria, the RBI team will involve also this famous Austrian research group in the network activities. The institute of Biochemistry in Graz became world-wide famous at the time of late Prof. Hermann Esterbauer who discovered HNE. Of additional relevance is also involvement of Prof. R.J. Schaur, one of the closest collaborators of the late Prof. Esterbauer who is an expert in enzymatic aspects of cancer metabolism and oxidative stress and acts now as research partner of the group in Zagreb. Their particular interest is the role of granulocytes in cancer development and therapy. All members of the team are university teachers and already provide training for Ph.D. students and postdoc fellows from Austria and France. Without Zagreb, the HNE focus of the network would be significantly weakened; this is very undesirable as HNE is perceived as an exciting aspect of oxidative stress and related pathology, and one that has great potential for scientific innovation and diagnostic developments in biomedical science.

Finally, Prof. Neven Zarkovic, leader of the Zagreb Team is initiator of our network.

Page 42 of 66

“HELP”

B4. MANAGEMENT AND FEASIBILITY

B4.1. Proposed management and organisational structure (two A4 pages)

Network Management Committee

The management structure of the network in shown in Table B4.1. The overall responsibility for the management of the network rests with the Network Co-ordinator, Prof.Dr. Peter Eckl, University of Salzburg, Institute for Genomics and General Biology. He will be assisted by 4 Assistant Co-ordinators, Neven Zarkovic from the Rudjer Boskovic Institute, Juergen Arnhold from the University of Leipzig, Corinne Spickett from University of Strathclyde, and Anne Negre-Salvayre from INSERM at Toulouse. This will constitute the Network Management Committee, chaired by Prof. Eckl, which has executive power over all aspects of the management of the network. This team will be advised by 3 consultants: Tilman Grune (Berlin) and Giuseppe Poli (Turin), who are leading European scientists in the field of HNE and lipid peroxidation (members of the committee of the SFRR-Europe), and science-manager consultant Dr. Raoul Kneucker, former general director for science and international affairs at the Austrian Ministry of Science, Culture and Education.

Co-ordinating Group

Reporting to the small Management Committee will be the larger Co-ordinating Group, consisting of 1 representative from each institute (the assistant co-ordinators will represent their teams). This group will be chaired by the Director, Prof Eckl, and will oversee the general operation of all aspects of the network. It will delegate responsibility in specific areas to 5 subcommittees. University of Salzburg will appoint a secretary to assist with the administrative chores and day to day running associated with the group.

Subcommittees

Each of the subcommittees will be chaired by one of the assistant coordinators or the Director, and will report directly to the Co-ordinating Group. The responsibilities of the sub committees are detailed below.

Finance Subcommittee

Chaired by Prof Eckl, this will propose the budget and manage the distribution of funds to the participating teams. It will receive the financial reports of all the teams, and will monitor the teams expenditure. A financial administrator will be appointed, and financial officers from some of the other institutes will assist. The budget will be cross-checked by the Network Management Committee. Visits from a member of the Finance Committee, plus advisor(s), to each team on twice yearly basis will ensure that expenditure is properly controlled and in accordance with the official auditing requirements. This is especially important in view of the relatively large size of the network. The University of Salzburg will accept receipt of the Community Contribution, and distribute it according to the budget and when instructed by the Finance subcom.

Appointments Subcommittee

Chaired by Dr Spickett, this will monitor all aspects of the appointment procedures for the young researchers, from advertisment, interviewing, selection and issues of gender parity and mobility. This subcom. will be especially important in the early stages of the network, but will subsequently monitor career progression and will advise on transfer of ERs to appointments at a second and different team. It will also overview the pastoral arrangements for proper accommodation, language training, where appropriate, and mentoring of the appointed, as well as the exchanged, research workers. Research Subcommittee

Chaired by Dr Zarkovic, this will be responsible for overseeing research direction, receiving regular reports from the participating teams, and drafting the annual scientific reports, to the Co-ordinating Group. They will also track external research developments, and advise on actions which might need to be taken in the light of changing circumstances. Tilman Grune and Giuseppe Poli will act as referees and moderators.

Page 43 of 66

“HELP”

This subcom. will also ensure that ethical issues are properly considered. The day-to-day organisation of the work of the researchers employed under the contracts will be the responsibility of theeir team leader at each institution.

Training Subcommittee

Chaired by Dr Arnhold, this will be responsible for overseeing training programme of the network, including instructional courses and open weeks, for which a provisional programme has already been prepared. The subcom. will ensure the operation of an appropriate schedule for the open weeks, and will support and advise the teams hosting the courses, if necessary co-opting additional staff to assist. The subcom. will be responsible for preparing reports on the courses, and collecting short repports from each team hosting an open week.

Conference Subcommittee

Chaired by Dr Negre-Salvayre, this will be responsible for organisation of the scientific meetings, and will liaise closely with the teams hosting these events to ensure that all goes to plan. They will determine the overall format of the meetings, and advise on the programme. If necessary, further members with be coopted to help, in particular with the final meeting, which will be joint with the HNE-Club.

Much of the co-ordination will be carried out either by email, or via the Network Portal (see below). The Co-ordinating Group and the Network Management Committee will normally meet twice yearly, coinciding with networking events were possible. The subcommittees will convene more regularly, but this may be achieved by conference calls rather than mobility for some meetings. will also meet regularly.

Management Summary

This structured organization of the network is essential for its smooth operation. The arrangements described above will optimize use of the established management abilities and strengths of each of the participating teams. All of the institutes involved a full infrastructure for personnel and financial management, but nevertheless monitoring by the Network Management Committee is critical for quality control. Any disputes arising will be resolved by this committee.

Website and Portal

The continuous exchange of information among the groups will be achieved using the website PORTAL (http://ptp.irb.hr/en/olprojekti.php?id_projekta=53) currently developed for the network by the RBI-Team. This will be a showcase presenting the activities of the network. Karolj Skala (Zagreb) will be responsible for mainteance of the website. Research findings will be disseminated via the web, as well as through conferences, and publication of special guidelines and information for clinic or healthcare bodies. We will also use the web site of the international HNE-club to promote our activities http://www.kfunigraz.ac.at/hne-club.

The network website will include:

(1) A general presentation and motivations of the network, information on the various groups (with links to existing Web pages), list of publications and preprints with available files to be downloaded, workshop announcements and programs, and position vacancies.

(2) An area devoted to the exchange of information among young scientists, a technical e-magazine where young scientists will be encouraged to publish articles describing technical innovation;

(3) A unique e-mail address: for news transmitted by groups, which will be automatically transmitted to all other groups, and recorded on the Web page.

Page 44 of 66

“HELP”

Peter Eckl (Ph.D) is Vice Rector for Research at the University of Salzburg and Assistant Professor at the Institute of Genetics and Developmental Biology. Since 1989, he is head of the research group Genetic and Environmental Toxicology at the institute. Councilor for Austria of the European Environmental Mutagen Society. He has organised the 1998 Annual Meeting of the European Environmental Mutagen Society in Salzburg, the 1st International Meeting of the HNE-Club, Salzburg, 2002, and is doing now the organisation of the 200th Anniversary of Christian Doppler event. Peter Eckl is member of a variety of national and international scientific societies, such as New Yoork Academy of Science, the Society of Environmental Toxicology and Chemistry, the American Association fir Chemistry Research, or the European Society for Free Radicals.

Neven Zarkovic (M.D., Ph.D.) is senior scientist, associate director for science at Rudjer Boskovic Institute, leading scientific institution in Croatia. He got his M.D. degree in 1984, and Ph.D. in 1989. Since 1994 he is associate scientist at the Institute of Biochemistry of Karl Franzen’s University in Graz, Austria. Leading three Ph.D. courses on oxidative stress N. Zarkovic is teacher at Zagreb University, Medical Faculty from 1998 (http://bio.mef.hr/druga.html) and Faculty of Science and Mathematics from 2000 (http://www.biol.pmf.hr/~postdip/planiprogram/planfiz.html ). Since year 2000 he is head of Laboratory for Oxidative Stress and a member of the Steering Committee of the International HNE-Club (a group of interest of the International Society for Free Radicals Research, SFFR http://www.kfunigraz.ac.at/hne-club ).

Corinne Spickett (M.A., D.Phil. Oxon) is a lecturer at the Department of Bioscience, University of Strathclyde in Glasgow. She obtained her D.Phil in 1990, and after several years of postdoctoral work in multidisciplinary areas, took up post as a Glaxo-Jack Research Lecturer in the Dept. of Immunology at Strathclyde in 1994. Dr Spickett moved to the Dept. of Bioscience in 2001, where her teaching duties expanded considerably; she is 2nd Year Coordinator and teaches many undergraduate courses in biochemistry, both theoretical and practical, as well as running courses in oxidative stress. She was Socrates exchange coordinator in the Dept. of Immunology, and continues to be involved in annual teaching and research exchange with this programme. From 1995-2000 she was a member of the Celsus Research Group on Inflammation, and was responsible for organizing many of its activities. Dr Spickett is a member of the HNE-Club, and has an active research group in oxidative stress, which is involving in training a steady progression of young researchers.

Page 45 of 66

“HELP”

Table 4.1 showing the Management Structure of the Network

Prof. Peter Eckl, Network Co-ordinator and Financial Director, Responsibility for Task 2

NETWORK MANAGEMENT COMMITTEE

with executive power

Assistant Co-ordinator

Dr. Neven Zarkovic

Responsibility for Task 3 & Research Direction

Assistant Co-ordinator

Dr. Corinne Spickett

Responsibility for Task 5 & Appointments

Assistant Co-ordinator

Dr. Jürgen Arnhold

Responsibility for Task 1 & Educational Courses

Assistant Co-ordinator

Dr. Anne Negre-Salvayre

Responsibility for Task 4 & Scientific Meetings

Consultants to the Network Management Committee: Prof. G. Poli, Dr T. Grune, Dr. Raoul Kneucker

Co-ordinating Team oversees Network Research and Education Progress

and delegates specific actions and responsibility to subcommittees (below)

One Representative from each Participating Team (including assistant co-ordinators)

Network Secretary (to be appointed)

Finance Subcommittee

Prof Eckl

+ 3 members to be chosen

Appointment Subcommittee

Dr Spickett

+ 3 members to be chosen

Training Subcommittee

Dr Arnhold

+ 3 members to be chosen

Research Subcommittee

Dr Zarkovic

+ 3 members to be chosen

Conference Subcommittee

Dr Negre-Salvayre

+ 3 members to be chosen

Dr Karolj Skala, Website Manager

15 Participating Teams under Team Leaders

Page 46 of 66

“HELP”

B4.2. Management know-how and experience of network co-ordinator (one A4 page)

Professor Dr. Peter Eckl has extensive experience in project management, training activities, as well as the organisation of conferences and seminars – all tasks which are of importance for Marie Curie Research Training Networks.

Research projects that Mr. Eckl is currently managing are the international projects “In vitro and in vivo studies on the antimutagenic properties of glucans” as well as “Elimination of Genotoxity by Natural and Synthetic Antoxidants” (a project including partners from Austria, the Slovak Republic, and Hungary). Furthermore, he is leading the INTAS project “Assessment of Children’s Health and Psychological Status of Families in Ecological Stress in the Aral Sea Basin”. Within the university, Mr. Eckl has been specialising in HNE, lipid peroxidation and oxidative stress and conducting numerous internal research projects at the Institute of Genetics and General Biology.

In 2002, Prof. Eckl has been organising the conference on “4-Hydroxynonenal and related Lipid Peroxidation Products” from July 13-15 in Salzburg. The conference included lectures, oral presentations, posters, as well as workshops. The high-level participants at the international conference came from Europe, Asia, and the United States. Previously he has organised the 1998 Annual Meeting of the European Environmental Mutagen Society in Salzburg. Currently, he is organising the 200th Anniversary of Christian Doppler Symposium (http://www.sbg.ac.at/doppler).

Since 1998, Peter Eckl is also Vice Rector for Research for the University of Salzburg. In this position he has been co-ordinating national and international research and development projects for all institutes of the University of Salzburg. The university of Salzburg has four university divisions, natural and life sciences, social sciences, theology, as well as law. Within these there are several institutes. Furthermore, Mr. Eckl is also responsible for co-ordinating research collaboration of the university with regional, national, and international industry partners, universities, and non-university research institutes.

Prof. Eckl has also been a reviewer for EU research and development projects in the Fifth Framework Programme.

The Institute of Genetics and General Biology, University of Salzburg has extensive experience in both national as well as international research projects and training programmes. A recent project the Institute is participating as partner in “CREATE – Development of Certified Reference Materials for Allergenic Products and Validation of Methods for their Quantification”, which has started late 2001. Other projects include the participation in the EU project EUROFAN where 750 unknown yeast genes were identified during the yeast genome sequencing project.

Page 47 of 66

“HELP”

B4.3. Management know-how and experience of network teams (½ A4 page per network team)

B4.3.1 Salzburg Team: Prof P.Eckl

See section B4.2 above

B4.3.2 Zagreb Team: Dr N. Zarkovic

The Zagreb team currently (since summer 2002) works on following project supported by the Ministry

of Science and Technology: “Oxidative stress and malignant diseases" (0098101), “"NMR of bio-organic

molecules" (0098059), “Biophysics of interactions between lipoproteins and bioactive substances" (0098037),

"Status of oxidants/antioxidants after opioid and opiate applications" (0098096), “Primary percutaneous

coronary angioplasty and oxidative stress” (0108337), “Surgical and oxidative stress in colorectal

malignancies” (0198016), “Pathomorphological features of oxidative stress in liver regeneration” (0108254),

“Infectious diseases and oxidative stress of the CNS” (0108298) and several other technological projects or

contract research activities with industry. The Team leader, Neven Zarkovic, is Science Director of the Rudjer

Boskovic Institute and Deputy Manager of Institute for the World Bank Project of R&D Restructuring in

Croatia. So far he was a principal researcher or co-ordinator for 16 Croatian and international projects (FWF-

Austria, AO/ASIF Switzerland). He is about to take duty of the Joint Study Director for doctoral (Ph.D.) study

program prepared now by the University in Zagreb (Medical Faculty) and Rudjer Boskvoic Institute.      

B4.3.3 Glasgow Team: Dr C.M.Spickett, Dr M.H. Grant, Dr A.R. Pitt

Overall, we have considerable experience in the management and operation of collaborative research projects,

both with industry and within the academic sector. Successful collaborative projects have been undertaken

previously with Karl Franzens University, Graz, and the University of Bologna. Dr Spickett was for several

years on the Steering Committee of the Celsus Research Group, which involved organizing interdisciplinary

meetings and networking. Dr Pitt is the Head of the Proteomics Unit of the Sir Henry Wellcome Functional

Genomics Unit, and thus has an abundance of management and collaborative experience. The University of

Strathclyde has participated in many EC funded networks and other mobility programmes, and is experienced

in the necessary financial arrangements. Dr Spickett will be an Assistant Co-ordinator for the project,

responsible for Task , while Dr Pitt will also be a member of the management team.

We will support 2 young researchers based mainly at Strathclyde for the period of the contract, and they will

be jointly supervised by the senior members of the team. We also expect to host 3-4 students visiting from

other laboratories over the course of the project. Strathclyde has a well organized Postgraduate and

International Office, so they will be well looked after at the university level as well as within the team.

Page 48 of 66

“HELP”

B4.3.4 Dublin Team: Dr. G. Davey, Prof. K. Tipton

The team of Dr. Davey and Prof. Tipton has a long-standing expertise to assist with the organisation and

management of the network as it has participated in several earlier Framework projects (Contract Nos. B104-

Ct97-2099, - BIO4-CT98-0226, BIO4-CT96-4838 and BMH1-CT94-1402) and acted as coordinator of the

Human Capital and Mobility project EU-SSAO Network” (CHRXCT930256). Additional expertise in

assisting network management has come from involvement in the COST chemistry action D8 (Chemistry of

Metals in Medicine), within the D8/0005/97, D13 (New Molecules towards Human Health Care), within

projects WGs D13 0011 and D13 WG 0018, as well as being a member of the Management Committee of

COST B10 (Brain damage and repair).

B4.3.5 Bratislava Team:Dr. S.Stolc

Dr. Stolc (Director and Head of Neuropaharmacology Dpt.) has been involved in numerous research projects

on national and international level headed mostly by him. Current projects have been supported by the Slovak

Grant Agency for Science (VEGA 2/2054/22), the Agency for Science and Technology (APVT-20-020802).

The projects involve several research teams both from his Institute as well as from related clinical institutes.

He has also experience in management of foreign 5-year research grant awarded by a US pharmaceutical

partner as well as the teaching grant awarded by NIH-Fogarty International Center (MIRT 1T37

TW0008005). He is currently participating in COST B10 Action (Brain Damage Repair) as one of two

national Management Committee members.

B4.3.6 Lleida Team: Dr M. Portero-Otín, Dr R. Pamplona, Dr MJ Bellmunt, Dr J Prat, L Brieva

Our team has been involved actively in the research of lipid peroxidation and other oxidative damage-

derived reactions in the development of several diseases as well as in the study of the ageing. This has been

achieved by applying mass-spectrometry techniques and proteomic approaches. We have an extensive

experience of collaborations with different leaders on the field which has allowed the publication of several

articles. The quality of this research has been supported by the funding of different project, both at a national

and at international level (ALS Association, USA). We have considerable experience in the management and

operation of multidisplinary research tasks. This is evidenced by the participation in the Catalonian Network

of Oxidative Stress research, where more that 12 groups (comprising a rough 100 different research

professionals) are coordinated at a national level for increasing the outcomes of research on this important

issue.

We currently maintain an active international research schedule collaborating with more that five

different groups in the measurement of lipoxidative, glycoxidative and oxidative damage in proteins of

different experimental models, ranging from S. cerevisiae to human beings. In this context we hope to host

several different students from different laboratories over the course of the contract, in order to increase the

Page 49 of 66

“HELP”

exchange of techniques and the advancement of research. We expect to support, through this contract, a

doctoral student for the four years, as well as to undertake different visits to other teams in the network. We

also hope to participate in the different topic-oriented meetings. Our group has been involved actively in the

PhD studies of several students, coming from many different disciplines. Furthermore, we are participating in

the development of the PhD curricula from our department, which is now under evaluation for achieving the

qualification of High-Quality Doctorate Program.

B4.3.7 Toulouse-INRA Team: Dr F. Guéraud, Dr J. Alary, Dr E. Rathahao, Dr A. Paris, Toulouse, F.

Our team has been working in the field of metabolism of xenobiotics, and using the same analytical

procedures on the metabolism of endogenous compounds, and more particularly on 4-hydrononenal in rodents

for nearly ten years. Related methods based on mass spectrometry have been elaborated to assay the main

metabolite in urine as a putative biomarker of inflammatory processes. A useful model of copper-induced

hepatocarcinoma using Long-Evans Cinnamon rats is currently evaluated, in particular for some progressive

metabolic disorders which are appearing in parallel to the increase in lipoperoxidative events, and also for

protective strategies using drugs or nutrients.

Our team is integrated in the Food & Nutrition Research Department of the National Institute for Agronomic

Research, and is involved in some national and European collaborative projects, but has also full research

projects on xenobiotics metabolism with industry. Multidisciplinary formation of numerous doctoral students

has been done in our laboratory on metabolism, analytical sciences, physiology and analysis of disruption

processes, and statistics in connection with Universities of Toulouse, Paris IV and VI, and Marseille.

Our multidisciplinary approach will be helpful to support one or two young researchers for the period of the

project. Some doctoral students coming from other laboratories of the network will be invited to perform

specific experiments in connection with their thesis, which will be an opportunity to prepare some fruitful

transitions for a postdoctoral period.

B4.3.8 Toulouse-INSERM Team: Dr. A. Negre-Salvayre, Pr. R. Salvayre, Dr. N. Auge, Dr. M. Elbaz

Our team (Team 2 INSERM U466, Toulouse), has been working in the field of oxidized LDL and

atherosclerosis, more precisely on cell signaling triggered by oxidized lipids and mechanisms of LDL

oxidation by vascular cells. Related methods have been developped to establish links between the extent of

protein derivatization content (by HNE), cellular dysfunctions and pathologies (atherosclerosis, NIDDM). In

addition we start experiments on animal models of atherosclerosis and NIDDM, in order to better understand

the role of oxidation in those pathologies, and to develop new therapeutical strategies associating antioxidants

and carbonyl scavengers.

Our INSERM Unit is located on the site of the CHU Rangueil Hospital, and constitutes an active part of IFR

31 Louis Bugnard Institute, an important Research Institute which integrate 5 INSERM Research Units, 4

University teams and 1 CNRS Research Unit, thematically orientated towards cardiovascular, metabolic and

cancer research. This Institute offers all facilities to student for theoretical and practical formation with highly

Page 50 of 66

“HELP”

sophisticated equipment, lectures, national and international collaborations. Our team has been engaged in

collaborative european networks BIOMED-1 (1993-1996) and BIOMED-2 (1998-2001).

Our group should be helpful to support one or two young PhD (or PostDoc) researchers for the period of the

project, and to exchange doctoral or postdoctoral students to perform some specific work during their thesis, in

relation with the topic of their search.

B4.3.9 Leipzig Team: Juergen Arnhold, Juergen Schiller

Scientific and methodological collaborations exist and have existed with many others groups. These

collaborations include the exchange of junior and senior researchers, common scientific projects, exchanges of

tools and the common use of special equipment in other laboratories. Thus, numerous common publications in

peer-reviewed journals and presentations on international conferences are the result of these collaborations.

Important collaborations exist with other teams of this network including the groups of Jörg Schaur (Graz,

Austria), Corinne Spickett (Glasgow, UK) and Ingrid Wiswedel (Magdeburg, Germany). Especially with the

both last groups, a common platform on the application of mass spectrometric approaches in investigation of

oxidative stress phenomena of lipids will be formed in this network. This team has received much research

funding and thus is expert in organizing research projects, also in administration of financial matters.

Dr Arnold will be the Leipzig representative on the Network management team, and will be an active

participant in the overall co-ordination of the Network.

B4.3.10 Magdeburg Team: Dr I. Wiswedel, Prof Augustin, Dr W. Siems

Ours team has experience of managing many research projects, including international collaborations.

We collaborate with several clinical groups, and have close links with other teams of the network (e.g.

Leipzig). The Faculty of Medicine at Magdeburg has much previous experience of adminstering international

projects and consortiums. Some examples of current collaborative projects are:

“Highly sensitive GC-MS quantification of hydroxy fatty acids and isoprostanes as biomarkers of oxidative

stress in vivo. (Research fund of Saxony Anhalt)”

“Role of oxidized eicosanoids in UV-induced erythema of human skin (microdialysis samples and cultured

keratinocytes) and influence of non-steroidal antirheumatica (BMBF)”

“Biomarkers of oxidative stress and inflammation in chronic obstructive pulmonary disease (COPD)

(NIH/NHLBI)”.

B4.3.11 Larissa Team : Dr. A. Tsezou, Dr. P. Kollia, Dr. N. Vamvakopoulos.

Our team has been working in the field of carcinogenesis especially involving telomerase activation after

hepatitis virus infection as well as human papilloma virus induced cervical abnormalities. Advanced molecular

biology methods will be used for the expression of telomerase and the measurement of telomere length which

Page 51 of 66

“HELP”

constitute useful biomarkers of oxidative stress. Previous work of our group has been concentrated in the

activation of heat-shock proteins as a result of oxidative stress. Additional expertise exists in the field of the

mechanisms of iron metabolism in leukemia and the association of iron-induced oxidative stress with lipid

peroxidation and oxidative DNA and protein damage.

Our team has previous experience in management and operation of collaborative research projects within the

Academic Sector. Dr. Tsezou, Dr. Kollia and Dr. Vamvakopoulos have all previously supervised and are

currently supervising doctoral students. The management of this group and responsible for interactions with

the network will be Dr Tsezou.

B4.3.12 Łódź Team: DR Grzegorz Bartosz, Agnieszka Grzelak, Aneta Balcerczyk, Blazej Rychlik,

Miroslaw Soszynski, Lukasz Pulaski.

The team has a long experience in studies of reactions of reactive oxygen species with biomolecules

(including radiation chemistry approach), detection of reactive oxygen species (including spin trapping and

fluorogenic probes) and studies of antioxidants, especially glutathione. ESR spectrometer and research X-ray

facility are available; the team has also an access to a linear beta accelerator at the Technical University of

Łódź. The laboratory has used erythrocyte as a simple model cell for years; other cellular models employed

include the yeast Saccharomyces cerevisiae and mammalian cells in vitro. The team has numerous

collaborations with research centers within Poland and abroad, including Tomasz Biliński (Rzeszów, Poland;

yeast physiology and aging), Ilya Zavodnik (Grodno, Belarus; reactions of hypochlorite) and Jan Gebicki

(Sydney, Australia; oxidative damage to proteins).

Institute of Biophysics including the Department has a status of Centre of Excellence of the State Committee

for Scientific Research. Since ten years the Institute is the centre of Polish Section of the Society for Free

Radical research, organizing symposia on Free Radicals in Biology and Medicine, usually every other year,

and is preparing Summer Meeting of SFRR-Europe in 2004.

B4.3.13 Elitec/Tatzber Team: staff involved Dr F. Tatzber, Dr Willi Wonisch, Prof H. Sinzinger.

This team consists of 2 SMEs located in Austria and focused on the detection and measurement of

several aspects of oxidative stress (OS). Both Dr Tatzber and Prof Sinzinger also hold academic posts in

Austrian universities, and have been highly effective at the exploitation of research knowledge. Their

involvement with the small enterprises clearly demonstrates their management and administrative abilities.

Both SMEs have cooperated with major representatives of the Marie Curie HNE-network for several years

with the aim to develop and produce additional methods for measurement of direct and indirect consequences

of OS in biological systems. Furthermore, both companies received export and innovation awards of the

Page 52 of 66

“HELP”

Lower Austrian Chamber of Commerce for the development of methods for OS measurement. Thus this team

is well experienced in project and financial management as well as conducting international collaborations.

B4.3.14 Torino Team: Prof. Guiseppe Poli

Prof. Poli has managed a large number of multicentre collaborative projects, both in Europe

and with Prof Sevanian in the USA. The large number of publications testifies to the success of these

interactions. He has been highly involved in the organization of the European Committee of the Society

for Free Radical Research (SFRR), requiring many intense management skills.

B4.3.15 Berlin Team: Dr. Tilman Grune

Dr. Grune (Head of Free Radical Research Laboratory and Director of the Neuroscience Research Center)

is the principal investigator of numerous national research projects. Currently 4 projects are sponsored by the

German Research Council (DFG). He has also experience in managing of long term projects. Dr. Grune is

currently member of the European Committee of the Society for Free Radical Research (SFRR). He has

experience in organising international conferences and is the organiser of the 'International Conference on

HNE and Lipid Peroxidation Products' scheduled in 2004. Furthermore he was the participant and is the

organiser of the next 'Free Radical Research School' supported by SFRR-International, SFRR (Europe) and

several other European and international organisations.

Page 53 of 66

“HELP”

B5. RELEVENCE TO THE OBJECTIVES OF THE ACTIVITY (two A4 pages)

This project joins 15 teams working in different areas of the biomedical, biological, biophysical and

biochemical sciences, each of which has unique experience and specialized technologies. The project also

targets certain diseases, all of which are linked through oxidative stress, but that have traditionally been

researched rather separately. The pooling of these resources into an integrated, multidisciplinary research and

training network will allow members of the individual teams to gain insights and practical experience of

approaches that would not otherwise be available to them. The regular topic-oriented meetings and educational

events will allow the new young scientists to obtain knowledge and research experience necessary for their

future career development as experts in the field of oxidative stress.

The requirement for expertise in these disparate fields, each of which shares interests and

involvements in the central theme of the proposal, can only be fulfilled at the trans-european level. Each of the

teams involved has extensive experience of teaching and training at both the undergraduate and post-graduate

levels. This project will open up new avenues of training that are not available in the individual institutions.

This will be achieved through:

Instructional Courses (similar to Summer Schools) involving seminars and workshops. In addition to

facilitating the exchange of ideas and training amongst members of the network, these will also allow

dissemination to other scientists.

An Exchange Programme for the young research workers, who will be hired within the framework of

the network. It is envisaged that each experienced researcher hired by one team will spend 25 % of

their time working and learning in the laboratories of at least one other participating team. Shorter

visits will also be possible for early stage researchers.

Open weeks organized by each of the participating teams will provide unique, hands-on training in a

large number of technologies and research approaches.

Regular visits of established individuals between groups will be an essential feature to ensuring the

continued success and cohesion of the network.

Electronic communication in addition to regular communication through e-mail and conference calls,

communication within the network and wider dissemination will be effected through a specific group

web-site. Effective visibility will be ensured by links from existing sites, such as the Society for free

radical research (http://www.sfrr.org/ and http://www.sfrr-europe.org) and the HNE-Club

(http://www.kfunigraz.ac.at/hne-club/); a site with which some members of the group have detailed

experience and involvement. Zagreb Team established already an internet portal that was used to

prepare the network application (http://ptp.irb.hr/en/olprojekti.php?id_projekta=53). Networking will

also be achieved through the HELP network portal.

Page 54 of 66

“HELP”

These activities will ensure a degree of experience and technical competence that will make those trained

within the network uniquely qualified to for valuable research contributions in the future. In addition to

providing an educational framework, the activities described above will ensure the continuing coherence of the

network

In summary, the Research Training Network, will strengthen and extend existing research contacts

between the teams involved and the development of continuing collaborations, exchanges and the pooling of

expertise. The size of the network is such as to ensure a very extensive and special training is available, yet

the excellent level of interactiveness between teams and the clear management structure allows us to be

confident that it will operate successfully. The Research Training Network will foster many new

collaborations, which will persist well beyond the end of the programme. The sharing of technology will also

encourage durable collaborations and interdependence. Moreover, as the young researchers pursue their

career path and establish their own research groups in Europe, they will already have acquired an excellent

network of acquaintances who will support them. This will make a very material difference to the

effectiveness of their early research efforts, and hence to their ultimate success. It can be seen that the network

will have ramifying and far-reaching effects on the quality of collaborative research in this field within

Europe.

Page 55 of 66

“HELP”

B6. ADDED VALUE TO THE COMMUNITY (two A4 pages)

Page 56 of 66

“HELP”

This Research Training Network will contribute to several objectives from the European

Research Area as drawn from the thematic priority 1.1.1 “Genomics and biotechnology for health”.

The scientific objectives of the present network relate to the actions of 1.1.i.a “Fundamental

Knowledge and basic tools for functional genomics in all organisms”, 1.1.ii.a “ Application oriented

genomic approaches to medical knowledge and technologies”, specially in the “Combating

cardiovascular diseases, diabetes and rare diseases”, “Studying the brain and combating diseases of

the nervous system”, and “Studying human development and the ageing process”.

This proposal entirely supports one of the goals of the European Research Area, “the

networking of existing centres of excellence in Europe and the creation of virtual centres through the

use of new interactive communication tools”. This is supported by the extensive experience of all the

participants in this proposal in the field of lipid peroxidation. The interaction between these different

groups will help to develop the communication tools described in the objectives. By its nature, the

proposal of this network fits perfectly in the enhancement of mobility and abundance of human

resources for researchers. This is justified by the multidisciplinary objectives of the network: both

research and training (achieved through increasing mobility of researchers) are addressed in this

action. One of the goals of the network, to recruit young scientists for its scientific career

development is clearly in line with the stimulation of “young people’s taste for research” described

by the goals of the European Research Area.. Furthermore, the high proportion of women in charge

of the different groups or participating directly in the achievement of aims will improve the gender

equality and balance of this area of research. The management of the network will monitor the

appointment of female and male researchers very carefully to ensure that gender parity is maintained

in the teams and the whole network.

Page 57 of 66

“HELP”

This Research Training Network, by its intrinsic properties, will facilitate and help to increase

the mutual interaction between different groups on the same background. Thus, this relationship will

contribute to the development of the European Research Area in several synergistic modes: i) the

sharing of experience and dissemination of mastered methodologies will increase the efficiency of

the funds dedicated for research; ii) the same principle could be applied in the training of scientists,

that will provide a first-hand communication from different, field-leading research teams. This

characteristic will increase the attractiveness of Europe for researchers and its competitiveness, since

this network counts with the major experts of lipid peroxidation in Europe; iii) the sharing of know-

how between different teams, and the actual intercommunication that this training network will

require would help to establish the basis for future research enterprises, such as the elaboration of

proposals for integrated projects or network of expertise. This is evidenced by the present existing

and fruitful collaborations between the different teams, even allowing to the development of a “HNE

Club”; iv) the high degree of specialization and quality of the different teams, supported by their

extensive list of high quality references will help to increase the role of European science in this field

of research.

This network has addressed as a part of its process of constitution the requirement for

inclusion of teams from Less-Favoured regions, and Candidate Countries. Specifically, teams from

Magdeburg, Poland, Slovakia and Croatia are actively participants in this network. Their role will be

been extremely important, not only under a point of view of coordination tasks, but also for its

necessary participation in the development of the different aims of the network.

This network, through the recruitment of doctoral and postdoctoral students will help to

increase the quality of regional and national higher education courses. The mobility of postdoctoral

and experienced researchers in this network will support the development of a PhD program, where

the doctoral students will be enrolled, specialized in lipid peroxidation and oxidative stress in health

and disease. It is envisaged that the instructional courses of seminars and the research conference

conferences will be coordinated with the existing national programs of research and higher education

within each country. This program, by its uniqueness and quality, should be a benchmark in the

field, and is expected to attract many high quality young researchers who might otherwise be tempted

by the USA. Moreover, the network will have long-lasting effects, as it is almost certain that the new

collaborations formed will continue after the conclusion of the project, and the instructional courses

are likely to be very successful in the training framework, and will also continue to be desirable.

Page 58 of 66

“HELP”

Page 59 of 66

“HELP”

B7. INDICATIVE FINANCIAL INFORMATION (one A4 page, plus table)

This research training network will appoint 14 early stage researchers for 36 months each, and 14 experienced researchers for 12 months each. The appointment costs were all calculated based on salaries, with a married person’s mobility allowance for all the ERs and 9 out of 14 ESRs. The travel allowance was calculated at 750 Euros for France and Germany, in view of their central location, and 1000 Euros for all other countries. The total appointment costs are as follows:

ESRs: 1864458 Euros

ERs: 868589 Euros

Total: 2733047 = 65%

Hence this corresponds to a total budget of 4204688 Euros

After subtraction of the overhead proportion, the amount remaining to be divided in B7 was 1089396 Euros.

Within B7, the finances have been distributed according to:

A) the overall staff input from the nonappointed researchers in each team together with the involvement of the team in organizational activities;

B) the number of researchers to be appointed per team (the same in all cases except 1). This is based on an amount of 9000 Euros per person year of reseacher, which is a very reasonable sum considering modern biomedical research costs;

C) the involvement of the team in organizational or management activities;

D) no equipment was required as all teams have well furnished laboratories, but special support for dissemination expenses are included for Salzburg as detailed below.

Central Austrian Technology Transfer and Training (CATT-Salzburg), AustriaThe network will employ the services of CATT Salzburg, a regional contact and support point for

European research and development as well as innovation projects. CATT Salzburg will support the dissemination and transfer of knowledge of scientific research and training results to a variety of target groups, such as industry (companies which might be interested in applying research results or are searching for respective employees), the public sector (for example hospitals or health organizations to distribute new findings), as well as academia in Europe. In this context, CATT Salzburg will support the overall project aims and contribute to make sure that results will be made available for a wide audience throughout Europe. CATT Salzburg will also support the promotion of open weeks and scienctific events for the project. Mag. Andreas Strasser will be employed in this regard.

Support costs for the CATT-Salzburg, involvement have been calculated as 11200 Euros, and have been included in [D].

Page 60 of 66

“HELP”

Indicative financial information on the network project (excluding expenses related to the recruitment of early-stage and experienced researchers)

Network TeamNo.

Contribution to the research/ training / transfer of knowledge expenses

(Euro)

Management activities (including audit certification)

(Euro)

Other types of expenses / specific

conditions

(Euro)

(A) (B) (C) (D)

1.2.3.4.5.6.7.8.9.10.11.12.13.14.15.

24650207502025024250184502195022250222502155020500165251650041001852019500

360003600036000360003600036000360003600036000360003600036000

03600036000

22500204001895021950197002170021500219502070020700164501645030001840018250

11200

Totals 291995 504000 282300 11200

The essential nature of Zagreb’s involvement is fully detailed in section B3.3. Zagreb have been largely instrumental in setting up the network , and have collaborations with many of the teams already. They have essential expertise to several areas, and will form a hub about which the network will revolve.

Page 61 of 66

“HELP”

B8. PREVIOUS PROPOSALS AND CONTRACTS

Department of Biochemistry, University Trinity College

“EU-SSAO Network” - CHRXCT930256 (Coordinator) Duration: 1993-1996

"Eurotaurine” BMH1-CT94-1402 (partner). Duration: 1994-1997

“Pilot study to establish a system for rapid integration of biochemical functional data into the databases enzyme and SWISS-PROT + TREMBL” FP3 - BIO4-CT96-4838 (partner). Duration: 1996-1997

“Enzyme Function Data Base” FP4 - BIO4-CT98-0226 (partner). Duration: 1998-2000

“BIOBABEL” FP5 - B104-Ct97-2099: 2002-2004 (partner) Started: 2002

Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovak Republic

The suggested MC-RTN Project is fitting well to the profile of contemporary research activities at the Institute. It will be supported from the “institutional” budget of the Institute. Other resources are:

“VEGA 2/2054/P22” (Coordinator) Duration: 2002-2004

“APVT-20-020802” (Partner) Duration: 2002-2005

Page 62 of 66

“HELP”

B9. OTHER ISSUES (two A4 pages)

All teams participating in the research are fully aware of the importance of ethical issues. Most institutes have their own ethical committee, or access to a local Clinical Board. All currently ongoing research is approved by the relevant local or national Ethics Board, and appropriate applications will be made by the individual teams as required by changes in research direction, as will almost certainly occur on starting the network.

Nevertheless the network will regularly communicate with relevant ethical boards in Europe, e.g. the European Group on Ethics in science and new technologies (EGE) or the European Science Foundation. The network will also consider principles of relating official documents, such as the Declaration of Helsinki by the World Medical Association, declarations of the UNESCO such as the Cartagena Protocol on Biosafety. Central in our consideration will also be the Charter of Fundamental Rights of the European Union as well as the respective Directives of the European Union on ethical aspects.

In May 2001, the European Science Foundation (ESF), strongly encouraged by its Member Organisations, in particular its European Medical Research Councils Standing Committee (EMRC), issued a wide ranging Policy Briefing on Controlled Clinical Trials. A key element of the Briefing concerned the registration of planned, recruiting and unreported controlled trials, which, it was noted, is ‘essential for good financial management, good scientific practice and good ethical practice’. Research funding agencies, as well as pharmaceutical companies, need to take their decisions in the light of reliable information about all studies, to avoid duplication of effort, to promote appropriate replication and to promote collaboration. In addition, patients, health professionals, clinical researchers, members of ethics committees and other decision-makers need to be informed about trials in which they can participate, or to which they can contribute in other ways.

The consortium will also direct its work according to the national guidelines of the participating countries, such as the Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine, which was adopted in November 1996 by the Committee of Ministers of the European Council.

Information required from proposers on the ethical aspects of the research presented

A. Proposers are requested to fill in the following table

Does the research presented in this proposal raise sensitive ethical questions related to:

YES NO

Human beings X

Human biological samples X

Personal data (whether identified by name or not) X

Genetic information X

Animals X

Page 63 of 66

“HELP”

B. Proposers are requested to confirm that the research presented in this proposal does not involve:

Research activity aimed at human cloning for reproductive purposes,

Research activity intended to modify the genetic heritage of human beings which could make such changes heritable1;

Research activity intended to create human embryos solely for the purpose of research or for the purpose of stem cell procurement, including by means of somatic cell nuclear transfer;

Research involving the use of human embryos or embryonic stem cells with the exception of banked or isolated human embryonic stem cells in culture2.

Further information on ethics requirements and rules are given at the science and ethics website at http://europa.eu.int/comm/research/science-society/ethics/ethics_en.html.

The research presented will not involve any of the research activities stated above.On behalf of proposers:

Prof. Dr. Peter EcklCo-ordinatorVice-rector for ResearchUniversity of SalzburgAustria

1 Research relating to cancer treatment of the gonads can be financed2 Proposers should note that the Council and the Commission have agreed that detailed implementing provisions concerning research activities involving the use of human embryos and human embryonic stem cells which may be funded under the 6th Framework Programme shall be established by 31 December 2003. The Commission has stated that, during that period and pending establishment of the detailed implementing provisions, it will not propose to fund such research, with the exception of the study of banked or isolated human embryonic stem cells in culture.

Page 64 of 66

“HELP”

ENDPAGE

HUMAN RESOURCES AND MOBILITY (HRM)ACTIVITIES

MARIE CURIE ACTIONSResearch Training Networks (RTNs)

PART B

“HELP”

Page 65 of 66

“HELP”

66

HRM Activity Guide for Proposers for Marie Curie Research Training NetworksCall Identifier FP6-2002-Mobility-1, February 2003