Investigating Integrating Physiological Systems  in a Changing Environment

Göteborg university Research platform on 

Integrative Physiology

GRI

Investigating Integrating Ph

ysiological Systems  in a

Cha

nging En

vironm

ent

Göteborg university Research platform on 

Integrative Physiology

MAIN APPLICANTProf. Susanne HolmgrenDepartment of Zoology/ZoophysiologyGöteborg UniversityBox 463,405 30 Göteborg, Sweden

GRI

APPENDICESA. DeliverablesB. List of participants and letters of supportC. CVs  from  Susanne  Holmgren,  Michael  Thorndyke  and

Thrandur BjörnssonD. 20 selected publications E. Strategy for advancing female participation and leadershipF. Description of the link to educationG. BudgetH. Communication strategyI. Suggestions for five external referees

PARTICIPANTSMichael  Thorndyke,  Thrandur Björnsson,  Michael  Axelsson, Magnus Andersson, Susanne Baden, Paul Bland, Stig Boström, Malin Celander,  Ingibjörg Einarsdottir, Susanne Eriksson, Lars Förlin, Bodil Hernroth, Sten Holm, Elisabeth Hörnquist, JörgenJohnsson,  Elisabeth  Jönsson Bergman,  Joakim Larsson,  Lena Mårtensson, Catharina Olsson, Bengt Silverin, Kristina Sundell, Helen Sköld, Lena Sundin Rådström, Kerstin Wiklander

RESEARCH PROGRAMME

Göteborg University Research Platform on

INTEGRATIVE PHYSIOLOGY

Investigating Integrating Physiological Systems in a Changing Environment

GRIGRI

Physiology is ‘the science of the normal processes and functions of all or part of an organism’. Integrative physiology is the study of ‘how gene products integrate into the function of cells, whole tissues and intact organisms’ (Dow & Davies 2003). The uniqueness of the proposed platform lies in the comparative and ecophysiological approach and in the combination of organizational levels from molecule to the whole organism in its environment.

VISION To establish a critical research mass in the field of integrative physiology within Göteborg University, by bringing together unique competences in the fields of invertebrate and vertebrate physiology, and related disciplines, and to apply this multidisciplinary competence to multifaceted platform objectives. OBJECTIVES To obtain a holistic understanding of how environmental changes affect life processes in the whole animal Göteborg university Research platform on Integrative Physiology (GRIP) will have the potential to generate a holistic view on the impact of environmental changes on complex animal life processes, thereby placing the Faculty of Sciences at Göteborg University at the international forefront of the rapidly expanding fields of systems biology and environmental physiology. GRIP will combine the use of powerful molecular tools and advanced physiological measurements to elucidate function at the organism level in an ecological context. To increase scientific and educational quality The overall goal is to form an interdisciplinary network with a focus on the integrative physiology of animals, and thus to promote innovative research across traditional boundaries. Such a structure will not only create scientific momentum, but will also lead to major innovations in the education of biology students. Our ambition is to form a dynamic research platform that stimulates communication at all levels, by sharing ideas and methodologies and creating new training and collaboration opportunities, including that with industry. In a similar manner, an open and active communication strategy targeting the scientific

community, schools, and the general public is envisaged. An important principle of GRIP will be to offer PhD students challenging, interdisciplinary research projects, where the potential of the platform is fully utilized in regard to research and supervision. To promote renewal and secure sustained platform activities, stressing the importance of female participation GRIP will create the basis for future joint multidisciplinary research applications. Active participation will be sought in research projects, networks of excellence, and other research instruments offered to support mobility and transfer of knowledge and techniques. GRIP intends to support the recruitment of young researchers and lecturers, and use available means to promote female participation in research and leadership. RESEARCH FOCUS Major scientific advances to better understand physiology at the whole organism level can be made by increasing communication between classical physiologists, ecologists and researchers trained in molecular biology. This includes linking physiological functions to behaviour and ecology, bridging classical laboratory and field work, finding scientific and methodological synergies between vertebrate and invertebrate physiology, as well as placing data from genomic/proteomics into a functional context. The general research aim is to advance the knowledge of how animals respond and adapt to their environment, by addressing fundamental questions about physiological mechanisms, and how these have evolved. This will be achieved by focusing on three major research areas which are central for the optimal performance of organisms

- Göteborg university Research platform on Integrative Physiology - (Figure 1): Homeostatic mechanisms, growth and development, and immune systems. GRIP will focus its research efforts on two integrated approaches (Figure 1): 1. The comparative approach: Integrating comparative data from phylogenetic and ontogenetic studies of responses to environmental change, models will be constructed to elucidate the evolution of physiological processes and for prediction of stress responses. 2. The ecophysiological approach: We will extend the use of advanced technologies from the laboratory into the field for cross-validation, and use the generated knowledge to further improve laboratory experimental design. This will advance our understanding of physiological limits/capacities in complex and changing environments. UNIQUE POSITION AND FUTURE POTENTIAL The collected research competence represented by the platform scientists who are equipped to examine a breadth of physiological functions in a wide range of animals, encompassing vertebrates and invertebrates, together with the multidisciplinary research approach and methodologies which can be applied is unique in Sweden. Adding the strong scientific competence of the researchers, the platform should be highly competitive on the international scale. Interdisciplinary collaboration will further strengthen our scientific profile.

Integrative physiology is the central discipline for understanding complex life processes in the whole animal. During past decades, molecular biology has led to major progress in biology. Now, the challenge is to take this information to a new level, ultimately explaining organ and organism physiology. Conversely, studies of whole organisms or living systems can shed light in molecular and cellular biology. A full understanding of how organisms respond to their environment, and to disease, effects of medicines, treatments and vaccines requires exploration at the level of the whole animal. The combination of laboratory-based studies and field studies where the natural behaviour of the animal (on the individual and social level) and physiology can be verified against the results obtained in the laboratory will lead to a better understanding of the limit or potential for the animal to cope with environmental changes. This can give synergistic effects and lead to a substantial leap forward in

science, as well as adding valuable knowledge when e.g. predicting ecosystem resilience. There is a well-equipped technological infrastructure at the involved departments, as well as at resource centres linked to Göteborg University, to support such developments. Thus, both basic and applied research in integrative physiology will be of significant importance, and there will be a continuous need for trained physiologists in the academy, environmental management-, pharmaceutical- and bio-technology industry as well as in animal farming.

The research focus of GRIP lies well within the strategic aims of the Faculty of Sciences (the new biology and partly within the marine profile), the major Swedish research councils VR and FORMAS (climate change, sustainable development and marine environment), as well as the program for the 7th framework of the EU (environment, including climate change; food, agriculture and biotechnology; health). GRIP will thus have extensive opportunities to apply for external funding while maintaining the focus of the Faculty. Our intention is to generate a number of both smaller and larger applications within GRIP. Applications to the Marie Curie Program

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- Göteborg university Research platform on Integrative Physiology - will aim to support the training and promotion of young scientists.

The large number and broad spectrum of undergraduate courses given by GRIP participants (see appendix F) are linked to integrative physiology and attracts students from all over the country. The students are given a thorough competence in state-of-the-art research-related techniques of integrative physiology. To further extend this unique competence, GRIP will start a new Master’s program (see appendix F) with speciality Integrative physiology – this will include a new mandatory course (10 weeks) given by GRIP researchers. PLATFORM ORGANIZATION GRIP will be governed by a board of scientists representing the major participating departments and/or subdisciplines, and led by a GRIP coordinator. The coordinator will hold an 80% project position at Associated Professor level (and given an opportunity to combine this with 20% teaching within the Faculty). GRIP coordination will be combined with research (for details see appendix G). In addition, the platform will employ one full time Assistant Professor, for four years. This position will mainly be focused on research within GRIP, but with 20% of the time allocated to assist the main coordinator with platform issues. The PhD-students financed by GRIP will play a central role implementing the inter-disciplinary projects. The core participants of GRIP will be scientists, and project and graduate students from several departments/institutes at Göteborg University, with broad competence in integrative and comparative physiology, human physiology, molecular biology, ecotoxicology, ecophysiology, behavioural ecology and mathematical statistics. In addition, industrial partners from AstraZeneca and Flextronics will provide methodological resources and training opportunities. Furthermore, members of GRIP have extensive national and international networks, including involvement in high-quality EU projects, all of which can be linked to GRIP. A horizontal rather than hierarchical leadership style will be applied. A particularly important principle will be to delegate leadership roles to younger female scientists within the platform. Full platform meetings will be scheduled on a regular basis to ensure open communication between

platform disciplines, as well as between scientists, post-docs and students. RESEARCH PLAN Background The physiology of the organism is closely linked to its physical and biological surroundings. All environmental fluctuations that are sensed by an animal will lead to physiological responses (Willmer et al. 2005). In the worst case, the responses may ultimately compromise fitness, i.e. the ability to reproduce and survive, which in turn, may affect populations and ecosystems. Interrelationships between physiological and ecological adaptations to environmental changes are still largely unexplored, and it is essential to gain knowledge in this area to allow predictions of how environmental changes, natural as well as anthropogenic, may affect animal populations. This requires the bridging of diverse scientific disciplines from molecular and cellular biology, to integrative physiology and ecology. Climate change is a widely recognized potential threat to biodiversity and ecosystems. Increasing greenhouse gas concentrations, trapping infrared radiation near Earth’s surface, have led to thermal expansion of the ocean which in turn may increase stratification and thereby the oxygen supply (see Hegerl & Bindoff 2005). Consequently, organisms on e.g. the continental shelves are at great risk. Many fish species in the North Sea such as cod, whiting, and anglerfish show a strong northward shift (from 50 to 800 km) during the last 25 years. If this trend continues, several commercially important species may have withdrawn completely from the North Sea by 2050 (Perry et al. 2005). In light of this scenario, it is vital to understand adaptive and non-adaptive strategies, ranging from genetical to physiological to behavioural adjustments, that organisms use to cope with environmental changes. Equally important is to analyze those strategies through the comparative approach. For instance, the use of a range of species inhabiting different environments greatly increases the potential to understand how and why different mechanisms to solve physiological problems have evolved. Studies of invertebrates as well as vertebrates are important to understand evolutionary relationships of physiological mechanisms (Willmer et al. 2005). Furthermore, the majority of physiological research has been conducted in the laboratory. Corresponding experiments in the field are necessary for a more holistic understanding of how animals function in

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- Göteborg university Research platform on Integrative Physiology - their natural environment (Costa & Sinervo 2004). Again, these are all examples of where molecular biologists, physiologists, ecologists and evolutionary biologists can meet and carry out common, interdisciplinary research projects. This research programme will increase the knowledge of how environmental factor(s) (oxygen, temperature, salinity, food availability) alone or combined affect homeostasis, growth, development and immune system of animals. The ambition is to create a high level of interaction and integration between these research areas in the suggested projects. General Research Approach The Comparative Approach From an evolutionary perspective, genetic adaptation is achieved by the process of natural selection and takes place over a period of time much longer than the lifetime of individuals. An informative means of analyzing adaptations is thus by comparing different species, but also the same species across a geographical range (Cossins & Crawford 2005). Environmental changes can, for example, give adaptive responses in one animal group but a non-adaptive in another. GRIP comprises a complementary expertise for a range of (marine) invertebrate systems (e.g. Bivalves, Crustaceans, Echinoderms, Tunicates) as well as vertebrates, in particular non-mammalian vertebrates. Comparative studies have been an integral part of earlier work for many of the GRIP members, but through platform collaboration a more holistic view using a broader comparative approach is made possible. The studies will include several “model organisms” for which the genome mapping is (or soon will be) complete. Within GRIP we also have excellent resources and increasing expertise in a range of modern methods including qPCR, proteomics, bioimaging, cell culture, in situ hybridization, microarrays, masspectrometry and NMR. The Ecophysiological Approach Field physiology provides insight into the actual mechanisms an organism employs to maintain homeostasis in its natural environment rather than in the lab. This requires an understanding of an organism's natural history and is a prerequisite to developing hypotheses about physiological mechanisms. State-of-the art facilities and technical knowledge are available and will be used to cross-validate measurements found in the field environment and controlled laboratory settings. This includes the full-scale trial of a recently

developed telemetry technique (by participants in GRIP) with a novel system for long-range monitoring of blood flow in combination with blood pressure. Non-invasive methods for assessment of foraging activity and food-intake in the natural environment will be developed. This will be combined with modern methods as mentioned above. Experimental facilities Easy access to the environment and thus in situ experiment and hypothesis testing is an important cornerstone of the GRIP infrastructure. A broad range of fresh water and marine organisms, experimental climate-controlled rooms with flow-through water systems and laboratories that can be used within minutes after obtaining the animals are provided at Kristineberg Marine Station. Temperature-controlled fresh- and saltwater aquaria, and amphibian, reptile are available at the Department of Zoology. Cell culturing facilities for invertebrates, fish and mammals are available at Kristineberg Research Station, Department of Zoology, and Department of Immunology. The access to wild stream populations of anadromous brown trout (Salmo trutta) on the west coast of Sweden is especially valuable since long-term background data are available on their general growth patterns and ecology, and since the high population densities allow efficient field experiments, and reasonable recapture rates. Furthermore, one of Europe’s most modern research facilities at Matre Aquaculture Station in Norway is available for GRIP. This allows large-scale fish trials with complete control of environmental factors, and the access to a large-scale sea-cage laboratory for performing trials in the sea with continuous monitoring of naturally fluctuating environmental factors. Similarly, existing international collaboration with Australia gives access to both wild and cultured populations of reptiles. The BioXmed campus at GU provides an excellent large-scale infrastructure and competence for high-tech applications in life-sciences, including facilities for genomics, proteomics, cellular- and bioimaging, transgenics, protein expression, bioinformatics, phenotyping, NMR-spectroscopy and more. Statistics Sophisticated statistical models will be adopted in order to treat complex situations. As multifactor studies will be performed, emphasis will be placed on experimental planning, design and modelling. This is particularly important in field studies where many factors can not be controlled for and

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- Göteborg university Research platform on Integrative Physiology - dependencies can be difficult to avoid. Investigations on threshold values, when environmental factors become stressors, are also important. By studying them one by one, there is a considerable risk to estimate those values incorrectly. Designs and models will therefore be developed to take the interaction of factors into account. Description of research projects 1. Effects of environmental changes on homeostatic mechanisms Homeostasis is the maintenance of a stable internal environment. It describes the physical and chemical parameters that an organism must sustain to keep the internal environment within tolerable limits to allow proper performance. Endocrine and nervous systems exert the major homeostatic control, often acting in complementary ways (Willmer et al. 2005). Sensory receptors on the body wall and in the body respond to different stimuli, and trigger intrinsic mechanisms to maintain homeostasis. Thus, a deviation of the animals’ physiology will be rapidly corrected. Fundamental questions to be addressed by GRIP are: What key regulatory mechanisms are present among the phyla, and how have these evolved? In a next step, GRIP will investigate the important issue of what consequences environmental changes have on the homeostatic capacities/limits of e.g. aquatic animals. Some species have physiological properties that allow them to live in extreme environmental conditions, such as at subzero temperatures, anoxia, pH down to 3.5, and water as dilute as distilled water (Franklin et al. 2004, Val et al. 2005). What physiological adaptations enable some species to live in such conditions? How do some species cope with environmental fluctuations in temperature, oxygen levels and ion concentrations better than others? How sensitive are species that live at their tolerance limit for one environmental variable to fluctuations in other environmental variables? Temperature strategies With the exception of birds and mammals, most animals are exothermic, and their body temperature dependent on the environment. These animals can only regulate their temperature by behavioural means, by migrating to areas with a species-specific optimal temperature range. Continuous increases in temperatures will impose serious problems particularly for species living in enclosed environments (Pörtner 2002). By

necessity, thermo-sensitivity must be present to allow for proper physiological and behavioural strategies to survive seasonal temperature changes. GRIP will survey behavioural responses that help aquatic animals to maintain homeostasis. At the molecular level, thermoreceptor proteins have recently been identified (McKemy et al. 2002; Zuker 2002). Different subsets of ion channels respond to different, specific temperature intervals. GRIP will investigate the composition and up- or down-regulation of thermoreceptor proteins between and within species adapting to different environmental temperatures. Oxygen level changes Due to the low O2 capacitance and unreliable oxygen levels in aquatic systems, a variety of control strategies to optimize the convective and diffusive blood/gas transport components of the gas exchange systems are expressed in water breathing animals. In addition, the development of acute oxygen sensing mechanisms and hypoxia tolerance are traits vital for their survival. Strategies to optimize oxygen uptake and reduce oxygen consumption consequently span from cellular mechanisms to behaviour and recent evidence show that fishes have oxygen-dependent gene expression (Nikinmaa & Rees 2004) Questions to be addressed by GRIP are: How sensitive are the sensing mechanisms and where are the oxygen sensing receptors located? What types of transmitters and/or hormones are involved in the regulatory response pathways? What are the physiological adaptations to low and high environmental oxygen levels? Ion concentration changes Osmoregulation and conformation are crucial for all living organisms. Two strategies have evolved depending on the environment: to absorb or to excrete ions and water. Animals from different phyla inhabit both the freshwater- and the marine environment , and may have evolved osmoregulatory mechanisms independent of each other. There are also animals that can migrate between the two environments. Cloning and sequencing of genes for ion-transporting proteins from gills and immunocytochemical localization of key transport proteins provide new tools for structure/function and comparative studies (Cutler & Cramb 2001). Equally important are electrophysiological studies, where GRIP scientists have extensive and complementary competence in epithelial transport and electrical processing of information from sensory receptors. Important aspects that GRIP will investigate are:

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- Göteborg university Research platform on Integrative Physiology - Changes in osmoregulatory mechanisms during migration and the endocrine regulation of these events. Synergistic effects increasing temperature and decreasing oxygen availability have on the osmoregulatory capacity of aquatic animals. Osmoregulatry strategies in different environ-ments. Environmental changes and ecotoxicology Natural and anthropogenic changes in the environment may directly or indirectly affect organismal exposure to toxic compounds. Directly, through natural chemical processes and anthropogenic discard. Indirectly, by increasing the bioavailability, as when low pH and salinity increase metal toxicity in aquatic environments (Kautsky 1998). Toxic compounds add yet another dimension to the multivariate level of stress an organism face. GRIP includes ecotoxicologists with expertise in effects and metabolism of inorganic and organic compounds (e.g. metal, sulphide, steroids, pharmaceuticals and pesticides) in both vertebrates and invertebrates. Initially we will use molecular biology and biochemical approaches to better understand involvement of the cytochrome P450 system in drug-metabolism and interactions with endocrine systems, combined with general pharmacological and physiological expertise within GRIP for effects on organs and the whole animal. This project together with our common long-standing experience of working with marine organisms, as well as integrating genomics, proteomics and metabonomics connects GRIP with the platform “Ecotoxicology – from Gene to Ocean”. 2. Effects of environmental changes on growth and development Winter growth Growth affects processes such as developmental rate, sexual maturation and survival. It is highly dependent on energy status and is the result of interrelated mechanisms ranging from food availability, ingestion and digestion, to cellular and tissue growth, via neuroendocrine and endocrine control, and foraging behaviour (Mommsen 2001, Stearns 1992). Winter growth is an interaction between animals and environment which is unique to temperate zones. Few data exist and further research into underlying physiological mechanisms to seasonal changes in growth, and physiological and behavioural strategies for winter survival will help understand how climate change may affect animal physiology. An important part of this is to link growth and energy status to

density-related population processes. The research will comprise GRIP expertise in physiology, endocrinology, behavioural ecology, genomics, metabonomics and statistical modeling. Novel markers for instantaneous growth rate and energy status will be developed, using identified candidates, transcriptome analyses (microarrays), and NMR-metabonomics, recently developed for fish at GU (www.physiology.gu.se/endo). The microarray work includes an advanced benchtop printing and microarray-facility where species for which there are no pre-printed chips can be studied, requiring only sequence information (geniom®one)*. Specificity, sensitivity and robustness of growth/energy markers, which can include hormones, binding proteins, enzymes and/or gene expression clusters involved in the growth axis, will be evaluated in the lab and used in the field. Here, density manipulations will be combined with novel telemetry methods for scoring physical activity, growth, and food intake. Adaptive and non-adaptive responses during development Many animals go through dramatic physiological changes during their life cycle, and the development of regulatory mechanisms follow an ontogenetic blueprint that may be altered by environmental variables, so called phenotypic plasticity (Schlichting & Pigliucci 1998). Plasticity can be adaptive, when it allows an individual to adjust physiologically to increase its fitness in its particular environment (Hodin & Riddiford 2000) or non-adaptive, when it is a simple expression of how environmental inputs modify development because of physiological constraints (Reznick 1990). Life-history plasticity is taxonomically widespread, but where environmental changes give adaptive responses in one animal group the response may be non-adaptive in another. It thus becomes challenging to examine these relationships in greater detail both from an evolutionary as well as an ecophysiological point of view. Environmental changes and ontogeny Equally important is to compare physiological responses depending on the character of the environmental change itself such as: Level (amplitude), duration (length) and repetitiveness (frequency), and the moment in time of the insult during development, as these four parameters will work alone or in concert to shape the physiological systems of the adult animal. GRIP is well equipped to answer ontogenetically-oriented

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- Göteborg university Research platform on Integrative Physiology - questions involving both invertebrates (e.g. Ciona intestinalis for which the genome is available) and vertebrates (e.g. model animals like zebrafish and the African clawfrog). Central operative techniques in GRIP are in vivo imaging and analysis of the developing gastro-intestinal and cardio-respiratory systems. Critical questions that will be addressed are: What impact does unfavourable environmental conditions (and their appearance) in juveniles have on the adult phenotype? When, and in what order, do regulatory systems develop in invertebrates and non-mammalian vertebrates? How does evolution of these mechanisms correlate with the ontogeny of a physiological function in mammals? Stem cells and cell proliferation in physiological plasticity During embryonic development, and to some level in the adult, there is a constant renewal of tissues from stem cells. To understand the role of stem cells during the life cycle of animals, analysis of if/how environmental stimuli change cell proliferation in certain areas of the body is necessary (Eriksson et al. 1998). Such an approach would be new in the area of stem cell research, where most focus has been on internal stimuli (Abrous et al. 2005). Higher vertebrates have a limited regeneration capacity, whereas invertebrates and lower vertebrates have a high capacity to regenerate whole tissues, and even the whole animal, from adult stem cells. The strong GRIP expertise in comparative physiology of the nervous system, the immune system, endocrinology, stem cell research and behavioural ecology gives excellent opportunities to clarify the role of stem cells, particularly neuronal and hematopoetic, for the phenotypic plasticity an individual must have to live in a changing environment at all life stages. A better knowledge of stem cells in lower animals may also have medical potential for humans. GRIP will investigate: Cell proliferation during different seasons in animals with seasonal adaptations in behaviour and/or morphology. Cell proliferation as a marker for phenotypic plasticity, together with physiological parameters at different ages. Effects of inhibiting cell proliferation on stem cells and the animal. 3. Effects of environmental changes on immune systems Immune defences are essential for the well-being of all organisms. It is becoming increasingly recognised that the immune “system” interacts with all other body systems and can only be

effectively understood by application of integrated physiology. Inflammation The inflammatory process encompasses immune responses to environmental challenge at several levels: the immediate response of the “innate” system, which is responsible for rapid clearance of pathogens; the adaptive response, which provides memory of the environmental challenge; and the extracellular matrix changes which are responsible for wound healing. GRIP has model systems for integrating these responses at three phylogenetic levels: mammals, teleost fish and invertebrates. Mammals and teleost fish have innate and adaptive immune responses. Invertebrates, on the other hand, lack adaptive immunity but can provide insight into the origin of vertebrate immune networks, the identification of novel factors, and biomarkers of environmental stressors (Fearon 1997). Within GRIP, workers are already linking microbial changes to immune function and as the GRIP progresses, these studies will integrate with other stress parameters and link with inflammatory function studies in representative species. Critical questions to be addressed are: Can we define the effects of stress and thereby use the invertebrate inflammation-regeneration models as biomarkers for environmental changes? Can we predict, through the use of immunological markers, the effect of environmental stressors on disease susceptibility and productivity and, as a result, define limiting parameters within the environment? Can we through comparative studies develop new strategies for regulating inflammation? Interactions of the immune, the endocrine and the nervous systems The immune and endocrine/nervous systems of vertebrates are intimately linked; hormones and neuropeptides control immune cell function and immune cytokines exert biological actions on endocrine and nerve tissues (Harris & Bird 2000, Salzet 2001). Invertebrates, possessing only the innate immune response, reveal similarities with the vertebrate neuro-endocrine-immune systems - e.g., in producing neuropeptide-derived antibacterial peptides (Salzet 2001). The three systems share receptors and signal molecules that support bi-directional communication, essential for the maintenance of homeostasis. For cytokine, neuropeptide and endocrine growth factor components, it may even be difficult to clearly assign them to either system, as many of them function in more than one. Endocrine systems are

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- Göteborg university Research platform on Integrative Physiology - to a large extent under the influence of environmental factors and the nervous system mediates external changes perceived by sensory receptors. Thus, the immune system may indirectly be affected by environmental changes, via neurons and endocrine factors. In search for bi-directional communication between the systems integration between immunology, neurology, endocrinology and ecology is crucial. High throughput molecular techniques (microarrays, e.g. using geniom®one*), in vivo and in vitro endocrine treatments and analyses, will be developed, validated and used within GRIP for this task. Critical questions that will be addressed are: How do the systems interact at the molecular level, with special focus on the corticosteroids and the GH-IGF-I system? How are environmental changes like temperature and salinity reflected in changes in the immune systems and further reflected in the health and limits of survival of the organism?

REFERENCES Abrous DN, Koehl M & Le Moal M (2005). Physiol Rev 85:523-69 Cossins AR & Crawford, DL (2005). Nature. 6:324-333. Costa P & Sinervo B (2004). Ann Rev Physiol 66:209-38 Cutler CP & Cramb G (2001). Comp Biochem Physiol 130:551-564 Dow JT & Davies SA (2003) Physiol Rev 83:687-729 Eriksson PS, Perfilieva E, Bjork-Eriksson T et al (1998). Nat Med. 4:1313-7 Fearon DT (1997) Nature 388:323-324 Franklin CE, Axelsson A, Sundin L, Davisson W (2004). Antarctic challenges 233-244 Harris J & Bird DJ (2000). Vet Immuno Immunopath 77: 163-176 Hegerl GC & Bindoff NL (2005). Science 309 (5732): 254-255 Hodin J & Riddiford LM . (2000). Evolution 54: 1638-1653 Kautsky L (1998). Pure Appl Chem 70: 2313-2318 McKemy DD, Neuhausser WM et al. (2002). Nature 416: 52-8 Mommsen TP (2001). Comp Biochem Physiol B 129:207-219 Nikinmaa M & Rees BB (2005). Am J Physiol Regul Integr Comp Physiol 288: R1079-R1090 Perry AL, Low PJ, Ellis JR & Reynolds JD et al. (2005). Science 308: 1912-1915 Pörtner HO (2002). Comp Biochem Physiol A Mol Integr Physiol 132: 739-61 Reznick DN (1990). J Evol Biol 3:185-203 Salzet M (2001). TRENDS in Immunology 22:285-288

GRIGRISchlichting CD & Pigliucci M. (1998). Phenotypic Evolution: A reaction norm perspective. Sinauer, Sunderland. MA, USA Stearns SC (1992). The Evolution of Life-histories. Oxford University Press, UK Val AL, Almeida-Val VM, Randall DJ (2005). In The Physiology of Tropical Fishes.Vol 21, pp 1-45. Academic Press, Elsevier, USA Willmer P, Stone G & Johnston I (2005) Environmental Physiology of Animals. Blackwell Publishing, UK Zuker CS (2002). Nature 416: 27-8 *http://www.febit.de/geniom/go_start.htm

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- Göteborg university Research platform on Integrative Physiology - Appendix A

Deliverables GRIP activities will be documented at the annual platform meetings, where the conducted research and education will be evaluated. Scientific progress will be continuously disseminated through scientific publications, workshops, seminars and national and international meetings. External funding obtained by the platform will be monitored as a measure of success. Research

• Interdisciplinary scientific publications • Interdisciplinary master’s and PhD-theses • Interdisciplinary national and international research applications • Interdisciplinary workshops and seminars • Extended platform workshops with invited international collaborators • Transfer of techniques and methodologies within the platform • Novel applications for established techniques and methods • Development of novel techniques and methods for field measurements • Collaboration between academic departments • Collaboration with industry in western Sweden

Female leadership (see appendix E)

• Improved leadership competence through mentor programs, seminars and research networks

• Improved opportunities for women to accept leadership roles, through a leadership fund supporting scientific assistance

• Literature study groups and seminars on women leadership and gender issues, open for both women and men

• Increased women participation in postdoctoral research through platform information and support

Education (see appendix F)

• Master programme in biology with focus on integrative physiology • Development of master and PhD courses • Development of experimental practicals suitable for undergraduate training • Interaction with public schools, e.g through mini-projects for selected students

Communication to the general public (see appendix H) • Annual participation in Vetenskapsfestivalen, Göteborg university • Web-site with open access communicating scientific data to the general public • Regular, scheduled meetings with the information office at the Faculty of Sciences

at Göteborg University • Newspaper and magazine articles; scientific radio and TV-programs when possible • Coordinator participation in communication events

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- Göteborg university Research platform on Integrative Physiology - Appendix B GRIP list of participants and letters of support Participant Title Affiliation Major Field of Competence Magnus Andersson Consultant Flextronics Computer aided measurements Michael Axelsson Professor Zoophysiology

Faculty of Sciences Integrative comparative cardiovascular physiology

Susanne Baden Professor Marine Ecology, Faculty of Sciences, Kristineberg Marine Research Station

Invertebrate ecophysiology

Thrandur Björnsson Professor Zoophysiology Faculty of Sciences

Fish endocrinology, osmoregulation, growth, aquaculture

Paul Bland Professor Dept. Clin. Immunology Sahlgrenska Academy

Immunology, inflammation

Stig Boström Professor AstraZeneca Drug development, cardiovascular biology

Malin Celander Associate Professor

Zoophysiology Faculty of Sciences

Metabolism of bioactive organic substances in vertebrates

Ingibjörg Einarsdottir Researcher Zoophysiology Faculty of Sciences

Fish endocrinology, development, stress

Susanne Eriksson Assistant Professor

Marine Ecology, Faculty of Sciences, Kristineberg Marine Research Station

Invertebrates, development, bio-geochemistry, proteomics

Lars Förlin Professor Zoophysiology Faculty of Sciences

Fish toxicology, biomarker development, proteomics

Bodil Hernroth Researcher Royal Swedish Academy, Kristineberg Marine Research Station

Invertebrate immunology, immunotoxicology, environmental microbiology

Sten Holm Professor Dept. Orthopaedics Sahlgrenska Academy

Nutrition of calcified tissues. neuromuscular reflex systems

Susanne Holmgren Professor Zoophysiology Faculty of Sciences

Gut physiology, pharmacology, development

Elisabeth Hultgren Hörnquist Associate Professor

Dept. Clin. Immunology Sahlgrenska Academy

Mucosal immunology esp adaptive immunity, animal models

Jörgen Johnsson Associate Professor

Zooecology Faculty of Sciences

Behaviour and ecology, fish biology

Elisabeth Jönsson Bergman Assistant Professor

Zoophysiology Faculty of Sciences

Fish endocrinology, appetite, energy balance

Joakim Larsson Assistant Professor

Dept. Physiol. and Pharma-cology, Sahlgrenska Academy

Transcriptomics, proteomics, metabonomics, reproductive endocrinology, ecotoxicology

Lena Mårtensson Researcher Zoophysiology Faculty of Sciences

Pharmacology, invertebrate physio-logy, neurobiology

Catharina Olsson Researcher Zoophysiology Faculty of Sciences

Gut physiology, neurobiology, bioimaging

Bengt Silverin Professor Zoomorphology Faculty of Sciences

Field and stress endocrinology in birds

Helen Sköld Post doc Royal Swedish Academy, Kristineberg Marine Research Station

Intracellular transport, cell division and stem cell biology

Kristina Sundell Professor Zoophysiology Faculty of Sciences

Fish endocrinology gut physiology, osmoregulation

Lena Sundin Rådström Associate Professor

Zoophysiology Faculty of Sciences

Integrative cardio-respiratory physiology, neurobiology

Michael Thorndyke Professor Royal Swedish Academy, Kristineberg Marine Research Station

Cell and molecular biology marine invertebrates

Kerstin Wiklander University lecturer

Mathematical sciences Faculty of Sciences

Statistical methods and experimental design

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- Göteborg university Research platform on Integrative Physiology - Appendix D

Selected publications Listed is a selection of 20 publications representing the participants in GRIP. Participant names are in bold.

Axelsson M, Altimiras J and Claireaux G (2002). Post-prandial blood flow to the gastrointestinal tract is not compromised during hypoxia in the sea bass Dicentrarchus labrax J Exp Biol 205: 2891–2896

Einarsdóttir IE, Silva N, Power D M, Smáradóttir H and Björnsson BTh (2005). Thyroid and pituitary gland development from hatching through metamorphosis of a teleost flatfish, the Atlantic halibut. Anat and Embryol. In press

Eriksson SP (2000). Temporal variations of manganese in the hemolymph and tissues of the Norway lobster, Nephrops norvegicus (L.) Aquat Toxicol 48: 297-307

Hernroth B, Baden SP, Holm K, André T and Söderhäll I (2004). Manganese induced immune suppression of the lobster, Nephrops norvegicus Aquat Toxicol 70: 223–231

Holm S, Indahl A and Solomonow M. Sensorimotor control of the spine (2002). J Electromyogr Kines 12: 219-234

Holmberg A, Schwerte T, Pelster B and Holmgren S (2004). Ontogeny of the gut motility control system in zebrafish, Danio rerio, embryo and larvae. J Exp Biol 207:4085-4094

Johnsson JI, Höjesjö J and Fleming IA (2001). Behavioural and heart rate responses to predation risk in wild and domesticated Atlantic salmon. Can J Fish Aquat Sci 58: 788-794

Jönsson E, Johansson V, Björnsson BTh and Winberg S (2003). Central nervous system actions of growth hormone on brain monoamine levels and behaviour of juvenile rainbow trout. Horm Behav 43: 367:374

Larsson D, Nemere I Aksnes L and Sundell K (2003). Environmental salinity regulates receptor presentation, cellular effects and circulating levels of two antagonizing hormones 1,25-dihydroxyvitamin D3and 24,25-dihydroxyvitamin D3, in rainbow trout. Endocrinol 144: 559-566

Larsson DGJ, Hällman H and Förlin L (2000). More male fish embryos near a pulp mill. Environ Toxicol Chem 19: 2911–2917

McArthur AG, Hegelund T, Cox RL, Stegeman JJ, Liljenberg M, Olsson U, Sundberg P and Celander MC (2003). Phylogenetic analysis of the CYP3 gene family. J Mol Evol 57: 200-211

Mårtensson LGE, Wärmländer S and Hildebrand C (1999). Noradrenaline-induced pigment aggregating response of melanophores in normal, denervated and reinnervated cichlid skin. Neursci Lett 275: 113-116

Nilsson Sköld H, Komma DJ and Endow SA (2005). Assembly pathway of the anastral Drosophila oocyte meiosis 1 spindle. J Cell Sci 118:1745-1755

Olsson C (2002). Distribution and effect of PACAP, VIP, nitric oxide and GABA in the gastrointestinal tract of the South African clawed frog, Xenopus laevis. J Exp Biol 205:1123-1134

Patruno M, McGonnell I, Graham A, Beesley P, Candia Carnevali MD and Thorndyke M (2003). AnBMP2/4 is a new member of the transforming growth factor-b superfamily

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- Göteborg university Research platform on Integrative Physiology - Appendix D

isolated from a crinoid and involved in regeneration. Proc R Soc Lond B 270: 1341–1347

Silverin B, Baillien M and Balthazart J (2004). Territorial aggression, circulating levels of testosterone and brain aromatase activity in free-living pied flycatchers. Horm Behav 45: 225-234

Sundin L, Turesson J and Taylor EW (2003). Evidence for glutamatergic mechanisms in the vagal sensory pathway initiating cardiorespiratory reflexes in the shorthorn sculpin Myoxocephalus scorpius. J Exp Biol 206: 867-876

Tarlton JF, Whiting CV, Tunmore D, Bregenholt S, Reimann J, Claesson MH and Bland PW (2000). The role of up-regulated serine proteases and matrix metalloproteinases in the pathogenesis of a murine model of colitis. Am J Pathol 157: 1927-1935

Wiklander K and Holm S (2003). Dispersion effects in unreplicated factorial designs. Appl Stoch Models Bus Ind. 19: 13-30.

Öhman L, Franzén L, Rudolph U, Birnbaumer L and Hultgren Hörnquist E (2002). Regression of Peyer's patches in Gai2 deficient mice prior to colitis is associated with reduced expression of Bcl-2 and increased apoptosis. Gut 51: 392-397

GRIGRI

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- Göteborg university Research platform on Integrative Physiology - Appendix E

Strategy for advancing female participation and leadership We propose to promote female leadership by providing support, information and opportunities to female researchers in GRIP through four actions: A. Affirmative action B. Leadership fund C. Mentorship program D. Targeted information network Of the 23 current academic partners of the platform, there are 9 males and 14 females. However, this equality is only apparent, as the majority of the females (64%) are supported by soft money, while most of the males (89%) have permanent faculty positions. This situation reflects quite accurately the major current gender equality problem in Swedish academia, where the number of females in natural sciences equals or is larger than the number of males at the undergraduate and PhD levels, but where women hold only a small percentage (18% among the professors and 21% among the lecturers at the Faculty of Sciences, GU) of permanent faculty positions. A. Affirmative action Gender mainstreaming is one of the most important tasks in today’s society. A major current problem at Swedish universities is that significantly fewer women than men hold permanent positions as lecturers and professors. When advertising and appointing research positions within the platform, affirmative action will be practiced (on equal merits, the underrepresented gender will be favoured), and females will be actively encouraged to apply. This is in line with the policy of the Faculty of Sciences, GU. B. Leadership fund A leadership fund will provide funding for technical assistance to enable young scientists to allocate time for leadership appointments, without having to downgrade their research activities. While this problem may seem gender neutral, it is not. A current dilemma within Swedish academia is that although only around 20% of the senior scientists are females, all academic committees aim at 40% female participation. As a result, Swedish female scientists in general have to devote more time to administrative appointments than their male colleagues, frequently to detriment of their scientific careers. The funding will pay for scientific assistance (eg. analytical or statistical) in order to compensate for this. A short application addressed to the platform coordinator and granted or dismissed by the platform board is required. The application should state the leadership appointment, the estimated time consumption, the amount of assistance required, and relevance to the platform. The support would normally be in the range of 1-2 month salary for a laboratory technician or equivalent. C. Mentorship programme for women leadership The mentorship programme will be open for both graduate students and PhDs, and the duration will be one year. Seminars and literature studies on gender issues will be open both for men and women. The mentorship programme will be administered by the platform coordinator and consist of two parts:

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- Göteborg university Research platform on Integrative Physiology - Appendix E

1. Younger female scientists will interact with women in leading positions, acting as role models, to create a forum for questions, the sharing of experiences and knowledge. The mentors may come from the academia, industry or government. Important is that the adept is not otherwise dependent of the mentor. The adept and mentor will have scheduled meetings at least four times, and will also have the opportunity to meet at seminars. 2. Seminars and other activities such as group discussions, literature studies and role-plays to prepare and inspire for leadership. The participants will have the opportunity to have input on the seminar contents. These should highlight different issues to give insights and tools for academic leadership, female academic networks, organization skills, and communication techniques, as well as how to handle conflicts, rhetoric, and gender issues. Female role models will be invited. D. Targeted information network The platform coordinator will initiate and stimulate an information network for all junior researchers linked to GRIP. This network will be of value for every student, but in particular for those who do not participate in the mentorship programme. Within the network, work will be organized to keep the participants fully updated on events which may further their scientific career, such as nationally and internationally available funding opportunities, research positions, seminars and courses. Special emphasis will be placed on women participation and monitoring of specific career opportunities available for women. This programme has been approved from gender equality and discrimination aspects by the Göteborg University equality secretary

GRIGRI

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- Göteborg university Research platform on Integrative Physiology - Appendix F

Description of the link to education The role of GRIP in education The platform contains from the start, a nationally distinct list of undergraduate courses at the C and D-levels in integrative physiology, both in terms of number and contents of courses, and the number of qualified, active researchers participating as lecturers. Thus, not surprisingly, these courses already attract students from all over the country. One prominent aspect of these courses is the great emphasis on advanced laboratory exercises at the tissue as well as at the whole animal level. This allows the students to obtain unique competence in state-of-the-art research techniques in integrative physiology. As a consequence, these students are not only well equipped to embark upon an academic research career, but are also much sought-after by major pharmaceutical companies which often have difficulties recruiting researchers with expert training in animal experimentation. The platform intends to build further on this foundation, by creating a national training center for integrative physiology. This will be done by establishing a new Master’s (MSc) programme in integrative physiology, which will include some existing courses, as well as major new courses in integrative physiology, especially designed for the programme. Basic level (BSc) The education within the Biology Section of the Faculty of Sciences, GU, is already well organized, and the participating departments all have a successful and internationally recognized research. This gives the students a natural and close contact with ongoing research. Thus, our assessment is that education at the basic level is already well linked with the platform, and immediately operational. Advanced level (MSc, PhD) Master (MSc) programme GRIP will give a MSc-programme in biology with focus on integrative physiology. Founded on the basic level, the objective of the programme is to give students a broad, multidisciplinary view of animal physiology, combined with scientific depth of knowledge and methodologies within integrative physiology. This will provide a solid basis for further studies (PhD) or industry-related research careers. Several of the courses suggested to be elective in the MSc-programme (‘Special courses’ in Table 2 below) may be included in a future PhD according to the proposed new Swedish system. All of the elective courses within the programme have certain prerequirements for the participating students. The programme is designed to allow mobility of students among Swedish as well as foreign universities. Given the expected heterogeneity among applicants to the programme, each student will follow a custom-tailored course plan, which takes into account strengths and weaknesses in theoretical as well as practical skills of that particular student. The students will be encouraged to maximize the time spent on experimental thesis work. However, it should be noted that certain courses are mandatory, indicated with different color codes in the programme plan (see next page).

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- Göteborg university Research platform on Integrative Physiology - Appendix F

Table 1. Master’s programme: Biology with focus on Integrative Physiology The table shows the Master’s programme outline for a) students beginning in the fall (left column) and b) students beginning with the spring term (right column). Mandatory courses are colour coded. Courses marked with green and underlined are mandatory for GRIP. Courses marked with brown and italics are mandatory for the Master´s programme in biology. Courses marked with red and bold are mandatory for the Master´s programme within the faculty. Courses marked with blue/bold and underlined are mandatory for handling experimental animals (vertebrates) and isotopes. * The course Comparative and marine zoophysiology 10 p (BIN251) will be mandatory for students without this course in their BSc. Other students will pick elective courses (Table 2) instead.

Science theory/Philosophy and scientific communication 5 p, (NGT100) Elective special courses (see Table 2) and parts taken in the seminar block 5p

Term 1 1-20 p

Integrative Physiology 10p

Statistical analyses and experimental design 5 p, (MSN460) Elective special courses (see Table 2) and parts given in the seminar block 5p

Statistical analyses and experimental design 5 p, (MSN460) Elective special courses (see Table 2) and parts given in the seminar block 5 p

Term 2 21-40 p

*Comparative and marine zoophysiology 10 p, (BIN251) Start 40 p thesis work

Term 1 1-20 p

*Comparative and marine zoophysiology 10 p, (BIN251)

Science theory/Philosophy and scientific communication 5 p, (NGT100) Elective special courses (see Table 2) and parts given in the seminar series 5 p Start 40 p thesis work

Term 3 41-60 p

Continue 40 p thesis work Start 20/30/40 p thesis work Elective advanced courses (see Table 2)

Term 2 21-40 p

Integrative Physiology 10p

Term 4 61-80 p

Continue 30/40 p thesis work 20 p thesis work Elective advanced courses (see Table 2)

Term 3 41-60 p

Continue 40 p thesis work Start 20/30/40 p thesis work Elective advanced courses (see Table 2)

Seminars 2 p The series will include research seminars as well as seminars on research ethics, leadership, equality and gender aspects Own seminars and conference contributions 2p Literature 2-3p Mandatory courses given as seminars Experimental animal science 2p (FKP850) Radiation safety 1 p (internal course)

Term 4 61-80 p

Continue 30/40 p thesis work 20 p thesis work Elective advanced courses (see Table 2)

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- Göteborg university Research platform on Integrative Physiology - Appendix F

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Table 2. Summary of elective courses available for students entering into the Master’s programme. Courses with a course code are given regularly in the current biology programmes. Remaining courses are either novel (planned for the programme and described below) or intermittently given as courses at PhD levels. p = point, current Swedish credit for 1 week studies. Special courses MSc/ PhD level

Elective advanced level courses

Autonomic nervous system, 2-3 p

Bioinformation and functional genomics, 10 p (BIN871)

Basic comparative experimental surgery, 2-3 p

Ecological zoology, 10 p (BIN810)

Central nervous system, 1-2 p

Ecophysiology, 10 p

Embryology and development, 2-3 p

Experimental design and statistics for natural sciences, 10 p (BIN920)

Endocrinology, 3 p

Fisheries ecology and management, 10 p (BIN700)

Immunological techniques, 2 p (course, No 5)

Fish physiology, 10 p (BIN 71)

Inflammation, 1-2 p

Human physiology, 10 p (BIN630)

Molecular biology methods, 1 p

Immunology, 10 p (BIN360)

Pedagogic course, 2 p

Marine Organisms and their habitat, 10 p (BIN180)

Problem-based learning, 1 p

Marine population ecology, 10 p (BIN380)

Proteomics and bioinformation, 4 p

Marine systems ecology, 10 p (BIN390)

Statistical modeling and methods, 5 p

Neurobiology, 10 p (BIN730)

Pelagic biogeochemistry, 10 p (BIN930)

Zool. Cell physiology, 10 p (BIN760)

Short description of planned courses: -Integrative physiology: A 10 week course, which will give students hands-on experience with whole animal methodologies crucial for the subject, as well as bioimaging and microscopy. Focus of lectures is placed on how physiological systems interact with each other in the living animal -Ecophysiology: This 10 week course will focus on how environmental factors are integrated with physiological mechanisms and behaviour of animals, to help animals cope with predictable and unpredictable changes in biotic and abiotic environmental variables. -Basic comparative experimental surgery: A two week course to learn basic surgical techniques necessary for placing devices onto different animals for physiological measurements. -Statistical modeling and methods: This course is a continuation of questions raised in the Master´s programme course ‘Statistical analysis and experimental design’. It will deal with more complex situations, e.g. models incorporating both controllable and nuisance factors. Several statistical methods suitable for experiments as well as field studies are treated.

- Göteborg university Research platform on Integrative Physiology - Appendix F

PhD programme The coordinator attached to GRIP will, apart from the Master’s programme, also be responsible for developing new, interdisciplinary PhD courses, reflecting the broad competence in this platform. A budget will be set aside for such course development. Focus will be on methodologies, which are crucial for mastering interdisciplinary research questions. As this platform highlights the need for novel research on complex environmental interaction with various aspects of animal physiology, there is a need for developing a course in statistical mathematical modeling for multifactorial analysis. Furthermore, as the international research profile of the platform is strong, efforts will also be made to open up the platform courses to international students by applying for external funding.

GRIGRI

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- Göteborg university Research platform on Integrative Physiology - Appendix H

Communication strategy GRIP will be organized and operated to stimulate informal, spontaneous, as well as formal communication, aiming at a high level of interaction within the platform. Already today there is a strong tradition of scientific communication training for PhD-students within the platform. It is for example mandatory for PhD-students to participate in international conferences and to give regular seminars at their departments. PhD-students and researchers are strongly encouraged to participate in courses in pedagogic and presentation techniques to improve communication skills. GRIP will further emphasize the importance of outreach and dissemination of research results to the public and the press. The platform coordinator will be responsible for the implementation of the communication strategy. Internal platform communication

• Regular information letters about platform-related activities through e-mail and an intranet

• Annual platform meetings • Workshops and thematic meetings where PhDs, supervisor and other invited

scientists meet for scientific discussions • Seminar series with invited speakers and platform members

Outgoing platform communication Scientific

• Dissemination of scientific data by publishing scientific papers in international journals and at international conferences

• An extended platform network meeting during the second year, with invited selected, established international collaborators for seminars and workshops

• This platform has a natural link with a number of other putative platforms at the Faculty of Sciences, and some members are also members of other platforms (Ecotoxicology-from Gene to Ocean and Forcing factors). Communication will be achieved through common workshops, courses, seminar series and mentorship programme.

General public and schools

• A platform web-site with open access will be constructed, targeted to the general public and schools to communicate scientific results

• Regular meetings with the information office at the Faculty of Sciences at Göteborg University to present interesting results and activities.

• Media briefings (newspapers, scientific radio and TV-programs when possible) to communicate platform activities and results

• Participation in the annual “Vetenskapsfestivalen” in Göteborg, and similar events, such as the international conference “Communicating European Research” in Brussels.

• Communication through the website “Bioscience explained”

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