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  1. 1. 1 Questionnaire Summary of the main activities of a scientific Organisation of the Slovak Academy of Sciences Period: January 1, 2003 - December 31, 2006 I. Formal information on the assessed Organisation: 1. Legal name and address Institute of Experimental Endocrinology, SAS Vlárska 3 833 06 Bratislava 2. Executive body of the Organisation and its composition Head of the Scientific Board Prof. MUDr. Iwar Klimeš, DrSc. 3. Basic information about the research personnel i. Number of employees with a university degree (PhD students excluded) engaged in research and development and their full time equivalent work capacity (FTE) and average number during the assessment period in 2003 = 39 (36,75), 2004 = 39 (37,28), 2005 = 40 (37,63), 2006 = 39 (36,89) ii. Organisation units/departments and their FTE employees with the university degree engaged in research and development
  2. 2. 2 4. Basic information on the funding i. Total salary budget1 of the Organisation allocated from the institutional resources of the Slovak Academy of Sciences (SAS) in 2003, 2004, 2005, 2006, and average amount for the assessment period 5. URL of the Organisation’s web site www.endo.sav.ak II. General information on the research and development activity of the Organisation: 1. Mission Statement of the Organisation as presented in its Foundation Charter The Institute of Experimental Endocrinology of the Slovak Academy of Sciences is focused on the basic research of neuroendocrine regulation systems with the aim to clarify their roles in regulation of different physiological functions of an organism using model situations including development, loading and in relation to etiopathogenesis of various diseases. Institute performs studies of mechanisms of hormone effects and their role in metabolism, aging, nutrition, tissue regeneration and development of major diseases. Institute elaborates new investigative methods of endocrine system for helping to solve problems in detection of endocrine system disorders. Institute provides consulting and expertise services in accordance with its main activities. Institute is pursuing scientific education in accordance with valid legislation. Results of scientific activities are published in relevant periodic and non periodic press. 2. Summary of R&D activity pursued by the Organisation during the assessed period, from both national and international aspects and its incorporation in the European Research Area (max. 10 pages) 1 Sum of the brutto salaries without the fund contributions.
  3. 3. 3 Introduction: The scientific research activities of the Institute over the last four years have continually followed up the existing main research focuses in the area of endocrinology and metabolism. They have been performed in harmony with the most progressive trends and accent at investigations of molecular mechanisms of the genesis, development and innovative diagnostics of non-infectious civilization diseases. In agreement with Mission Statement presented in the Foundation Charter of the Institute a special attention has been paid to two directions, in particular to a) The neuroendocrine regulation of body functions with special emphasis on the elucidation of processes operating in stress situations and their significance for the cardiovascular and immune systems, or physiology and/or pathology of brain functions and mental states b) Studies of biological action of hormones, cytokines and other bioactive molecules of endo- or exogenous origin at the DNA, RNA and protein level during their influencing of metabolic processes in the cells, both the in vitro situation and/or under conditions of integrated organism with the aim to identify the principle diseases mechanisms or to improve and innovate diagnostics and treatment of selected diseases A significant part of the research capacity of the Institute has been concentrated on the role of the endocrine system in the development and progression of socially important diseases as obesity and the metabolic syndrome, type 2 diabetes, cardiovascular and rheumatoid diseases, or mental impairments. Five research projects of the 5th Framework Program of the European Commission were successfully concluded within the evaluated period. This concerns particularly the investigations on the role of retinoic acid in regulation of gene expression in relation to the processes of biological aging /acronym VITAGE; 2000-03/ and the project on evaluating the effect of environmental pollution with endocrine disruptors on the health status of the populations of selected areas of East Slovakia /acronym PCBRISK; 2001-04/. The latter project won also the Slovak Academy of Sciences Award for “Excellent research data achieved in collaboration with the Safarik University and the Slovak Health University”. A special project of the 5th FP, STRESSNUTS /2000-04/ had also concluded within the evaluated period. The project dealt with reduction of negative impact of environmental factors on human health and was the base for granting our Institute with the status of a „Centre of Excellence“supported financially by the European Commission. The main achievements of the project, which was a collective endeavour of the core Laboratories of the Institute and their international collaborations, included generation of important scientific data with relevance to clinical research. In particular, evidence has been obtained to support further research on several molecules known to modulate hormone release and potentially interesting for treatment of the negative impact of stress, anxiety and depression (e.g. felbamate, phenytoin, lamotrigin, citalopram, tianeptine, Ginco biloba extracts or pentoxifylline). Also, new mechanisms involved in the stress response have been revealed; e.g. the findings on gene expression of adrenaline synthesizing enzyme in cardiac tissue in normal and corticoliberin gene deficient mice. The results contributed to understanding of regulation of the heart function by glucocorticoids, or the proof for a direct biosynthesis of adrenaline in the sympathetic ganglia which might clarify the mechanism of signal transmission at the ganglionic level. The original data on evaluation of the non-verbal behaviour during stress exposure and its modulation by drug treatment etc. were obtained. Important data were gathered in the field of nutritional manipulations and metabolic research; e.g. whole body insulin sensitivity and glucose utilization depends on fatty acid composition of the diet, where dietary fat rich in long chain saturated fatty acids led to insulin resistance, meanwhile feeding the medium chain triglyceride prevented the high fat diet-induced decrease of insulin action. Studies on mechanism of leptin action have underlined its important role in the regulation of fuel sensing mechanism in health and obesity. It was also shown that dietary supplementation of specific amino acids can be recommended for prevention of stress-related anxiety disorders. Funding of the STRESSNUTS project enabled further increase in the quality of the scientific research at our Institute, to become an international center for training of doctoral students and post-doctoral fellows from EU countries, and also to organize three important international scientific symposia attended by the top researchers from the field. Further two research grants from the 5th FP of the EC dealt also with very interesting themes. The goal of ENDOMET project (2003-2006) was to determine whether plasticisers, which are widespread environmental
  4. 4. 4 contaminants, could affect only the human reproductive system or also human neuronal and thyroid development and function and which mechanisms might be involved. Within the basic research, a successful application into the 5th FP encompassed a project devoted to a construction of novel collection of chromosomal applications in drosophila melanogaster /acronym DROSDEL; 2003-04/. The latter studies, partly originated also from data gained within a NATO supported project, which had been devoted to genes controlling the apoptosis during the metamorphosis of drosophila. Also within the 6th FP of the EC, the Institute had two successful grant applications, i.e. one on the role of chemical contaminants in the food chain /acronym CASCADE; 2004-09 / and the Danubian Biobank Initiative: Towards Information Based Medicine (acronym DANUBIOBANK; 2006-07). The above mentioned projects of the European Commission in Brussels, and particularly several grants from the Science and Technology Assistance Agency /APVT/ or from the Agency for Support of Science and Research / APVV/ as well as the state program Cardiogenomics, the grants from the Scientific Grant Agency /VEGA/, the Slovak Ministry of Health, the Slovak Ministry of Education, the European Social Fund/ enabled in the accredited period a significant innovation of the methodological approaches used at our Institute, even establishment and progressive development of new workplaces within our Institute. In 2003, the Diabetes and Nutrition Laboratory of the Institute established in collaboration with the National Institute of Endocrinology and Diabetes in Lubochna, the DIABGENE laboratory, a joined workplace residing at our Institute in Bratislava. Its mission has been the screening and DNA diagnostics of monogenic forms of diabetes in the Slovak Republic. Since 2004, the DIABGENE has been participating in endeavors of an international team which has led in 2006 to discovery of a genuinely new, pharmacogenomics-based treatment of permanent neonatal diabetes. In 2005, a second joined laboratory – the MicroPET Laboratory - was established at this Institute due to cooperation between the BIONT Company and our Institute. The aim of this laboratory is to carry out the positron emission tomography in small laboratory animals. In terms of analytical equipment availability at the Institute, the aforementioned domestic and foreign research grants have allowed a significant progress when compared to the previous accredited period. Regarding larger investments, beside of the miniPET apparatus, the DIABGENE Laboratory succeeded to acquire a second capillary automatic sequencer, which in combination with a recently purchased dHPLC apparatus enabled creation of a high throughput sequencing center. In agreement with the aims of the research strategy presented at the previous evaluation period, intensive efforts have been launched with the aim to strengthen the international collaboration on base of joined projects, for which financing from the research grant agencies had been obtained, particularly from the European Commission. The international collaboration is a considerable factor, traditionally contributing to an adequate publication and citation activity of our Institute. Moreover, the latter is keeping a moderately increasing trend also in this evaluation period. 2. A. THE RESEARCH STATUS AND A BRIEF CHARACTERISTICS OF RESULTS ACHIEVED WITHIN THE EVALUATED PERIOD 2. A.1. The most remarkable results obtained - Mutations in the KCNJ11 and ABCC8 genes encoding the Kir6.2 and SUR1 subunits of the pancreatic B cell KATP channel have recently been shown to be the most common cause of permanent neonatal diabetes (PNDM). Information regarding the frequency of PNDM has been based mainly on non-population or short- term collections only. Therefore, using the unique Slovak Children Diabetes Registry eight patients with diabetes fitting the PNDM definition (i.e. diabetes onset before the 6 th month of life without remission) were identified and for the first time a true, diabetes registry based PNDM incidence was calculated. In Slovakia, it is much higher /1: 215,417 live births/ than international estimates /1: 800,000/ shown elsewhere. In four patients, 3 different Kir6.2 mutations were found (two cases with R201H, and two novel mutations H46Y and L164P). Three patients with the Kir6.2 mutations R201H and H46Y were transferred from insulin to sulphonylurea, decreasing their HbA1c from 9.3-11.0% on insulin to 5.7-6.6% on sulphonylurea (SU) treatment. One patient has a novel V86A mutation in the SUR1 gene, and was also successfully transferred to SU. The results obtained are an excellent example of the translational approach in medicine, and the very first classical example of pharmacogenomics in diabetology, since the genetic cause of the insulin deficiency determines the response to treatment. The data has been published also in the prestigious NEJM with the top third impact factor in biomedicine at all (I. Klimeš)
  5. 5. 5 - It was shown that cell swelling (e.g. induced by hypotonicity) induces insulin secretion exploiting novel signaling pathway different of that involved in the glucose-induced insulin release. Cell swelling-induced exocytosis possesses specific features. This mechanism is relatively unspecific, thus advantage can be taken when studying control of peptides secretion in localization where their role is unknown (e.g. TRH in heart). It is supposed that signaling steps of swelling-induced secretion may soon become a drug target for molecules affecting secretion of many proteins and peptides. This might be of special interest for regulation of insulin secretion both for research and/or therapeutic purposes (V. Štrbák) - Using a model of psychosocial stress, it was shown /in contrast to traditional believes/ that secretion of stress hormones in persons with high trait anxiety may be rather low, and that increased levels of hormones during stress situations contribute to better coping with stress. Consistent with the traditional view, the rise in heart rate during stress was higher in anxious subjects than in non-anxious ones. However, plasma levels of cortisol, adrenaline and noradrenalin during stress were lower in anxious subjects. It was also shown that subjects with better cognitive performance in another model of mental stress had significantly higher cortisol levels compared to persons with worse performance. Correlation analysis in anxious subjects showed that lower ACTH and cortisol stress responses were associated with exaggerated perception of stress and depression feelings. The data obtained suggest that an adequate concentration of cortisol is needed for optimal emotional and cognitive coping with stress (D. Ježová) - It seems that imbalance between immuno-stimulating and immuno-suppressive hormones may predispose to development of rheumatic disorders. Therefore, hormone levels were analyzed in the synovial fluid of patients with rheumatoid arthritis (RA) and in patients with osteoarthritis (OA). Testosterone and estradiol levels were lower, and aldosterone, insulin and C-peptide levels were higher in the synovial fluid than in plasma. Overall levels of proinflammatory hormones were higher in synovial fluid of the RA patients. Low levels of dehydroepiandrosterone, the precursor of active androgens and estrogens, were confirmed in females with premenopausal onset of RA. Elevated plasma PRL levels were observed in about one third of patients with RA and Systemic Lupus erythematosus (SLE). Increased frequency of glucocorticoid treatment in hyperprolactinemic RA patients compared to normoprolactinemic suggested that the imbalance between glucocorticoids and PRL might play an aggravating role in RA. Taken together, the data supports the introduction of dehydroepiandrosterone supplementation in therapy of RA (L. Macho) - Obesity due to high caloric intake or due to growth hormone deficiency (GHD) may result in subclinical proinflammatory situation. Thus, molecular mechanisms of both situations were studied. The mRNA levels for CD68 and CD14 (markers of macrophage activity) and proinflammatory adipokines (TNFalpha, IL-6, PAI-1) were higher in fasting state or during euglycemic hyperinsulinemia. Gene expression for proinflammatory parameters showed a continual increase over a range of the s.c. adipose tissue (AT) mass, both in obesity and GHD. Inflammation in AT of obese subjects was analyzed using a high-throughput protein cytokine assay showing 27 proteins presented at higher levels. Among them were leptin, growth factors and their receptors, number of chemokines, cytokines and their receptors, and other receptors and enzymes. Selected protein array results were verified using the real-time PCR. This is the first evidence on „molecular disease” mechanisms of chronic inflammation in AT of clinically yet healthy obese young men. Chronic inflammation is present also in GHD, where accumulation of central AT together with an insufficient insulin secretion during oGTT seem to contribute to impaired glucose tolerance seen in the GHD subjects (D. Gašperíková) - Elaboration of experimental models of disease states resulted into new information on the mechanisms involved in development of depression and addictive behaviour. Gender dependent differences of gene expression in selected brain regions of a rat model of experimental depression seem to support the gender differences in incidence of depressive disorders in man. Anhedonic animals also exhibited depression-like symptoms, e. g. increased basal and decreased stress-induced hormone levels, increased gene expression for corticoliberin (CRH) in the hypothalamus and selective changes in glutamate receptor subunit gene expression in regions of brain reward system. The aforementioned indicates a role of CRH and glutamate in pathogenesis of depression. 3 weeks of voluntary wheel running by the Lewis rats resulted in development of compulsive running for 4-6 km/day. In this model of non-substance addiction, the most significant changes were observed in stress hormones and glutamate receptor gene expression in the brain. Changes observed might be important for development of the addictive behaviour as pharmacological blockade of glutamate release by phenytoin prevented development of compulsive running (D. Ježová) - High prevalence of disorders of the reproductive system in Europe seems to be in part result of environmental contamination by endocrine disruptors. This is a very diverse group of chemicals intensively used as a plasticizers, cosmetic ingredients, pharmaceuticals and pesticides. Effects of selected environmental plasticizers were studied in primary cell culture system of granulosa cells isolated from porcine ovarian pre- ovulatory follicles. The results indicated that the tested phenol and phthalate derivatives could induce changes in basal as well gonadotropin-stimulated progesterone and estradiol production by granulosa cells. Marked changes
  6. 6. 6 were observed by the action of bisphenol A, chlormethyl phenol, benzylbutyl phthalate and di (2-ethylhexyl) phthalate. We suppose that environmental plasticizers may act at the level of enzymes involved in steroid genesis in follicular cells as well as interfere with signal pathways of gonadotropin action in steroid hormone production. The inhibitory action of phenols on oocyte nuclear maturation and hyaluronic acid retention in the oocyte-cumulus complex matrix suggests ability of these compounds to influence the processes required for successful ovulation and fertilization (M. Ficková) - The age related alterations in the gene regulating abilities of retinoic acid, the biologically active form of vitamin A were investigated. Its counterparts, nuclear retinoic acid receptors belong to a superfamily of the retinoid-inducible transcription factors. Our data has shown for the first time that human lymphocytes express - besides the known two subtypes of all-trans retinoic receptor RAR alpha, RAR gamma, and the known one subtype of 9-cis retinoic acid receptor RXR alpha  - also the second 9-cis retinoic acid receptor, RXR beta. The data has shown that RXR beta receptor expression is significantly decreased in lymphocytes in a group of old men when compared to that of young men. We have shown also for the first time the rate of distribution of retinoic acid receptor subtype expression in human lymphocytes from young men who are in the following order: RXR beta (39.8 %), › RAR gamma (32.4 %), and › RXR alpha (17.8 %), › RAR alpha (10.3 %). Supplementation with 13-cis retinoic acid changes the distribution pattern predominantly of retinoid X beta receptor expression in human lymphocytes: RXR beta (34.2), › RAR gamma (35.9 %), and › RXR alpha (20.1 %), › RAR alpha (9.8). Decreased RXR beta expression may indicate toward higher risk of altered immune function in healthy elderly subjects. Consequently, retinoic acid supplementation might be beneficial in prevention of the immune dysfunction (J. Brtko) - By studying hormone-triggered programmed cell death (PCD) in larval salivary glands during metamorphosis of Drosophila we have identified a series of genes (P58IPK, Hsc70.3, Ca-P60A, Crc, Cnx99A, Cct5 and Hop) responsible for immediate activation of execution phase of the apoptosis which takes place by disintegrating endoplasmic reticulum (ER) into vesiculated lamellae. Within rapidly forming ER vesicles we have found increased abundance of myelin-like structures that are typical for accumulation of incorrectly folded proteins indicating lowered function of ER chaperones. This suggests that Drosophila salivary gland PCD reflects accumulated ER stress as a result of the unfolded protein response (UPR). The inappropriate UPR signaling is often associated with several degenerative diseases including Parkinson’s, Huntington’s, Alzheimer, Lou-Gehrig disease or amyotrophic lateral sclerosis (ALS) the common feature of which is formation of inclusions inside of ER lumen. Identification of novel components of signaling pathways that take part in the control of cell transformation, hormonal response and apoptosis can provide new therapeutic or diagnostic vistas at much higher efficiency if we use well-defined model system such as Drosophila (R. Farkaš) 2. B. SPECIFIC RESULTS IN INDIVIDUAL RESEARCH AREAS 2. B.1. In elucidation of the role of neuroendocrine regulation of body functions with special emphasis upon the knowledge on stress situations - Measurements of gene expression for enzymes responsible for synthesis of adrenaline and noradrenalin in the heart of rat have shown a gene expression for PNMT which is responsible for the adrenaline synthesis; meanwhile gene expression for the tyrosine hydroxylase was not detected. The data has shown that PNMT mRNA is not localized in the neural cells of the heart as has been anticipated; instead it is present within the cardiomyocytes where it responds by an increase of PNMT gene expression to stress. Gene expression for PNMT was detected also in human heart and we hypothesize that changes in the gene expression may play an important role for example in regulatory processes following heart transplantation (R. Kvetňanský). - During studies aimed on regulation of gene expression for enzymes of the catecholaminergic system and their modulation by transcription factors in mammals during stress, it was found that both the tyrosine hydroxylase (TH) activity and the gene expression for TH of cold adapted rats did not differ from the control animals. However, in the repeatedly immobilized rats remained those parameters highly increased. The substance for the differences found is the probably the impaired activation of the transcription factors Fra-2, phosphorylated CREB and Egr-1. The results indicate that absence of those transcription factors may cause the adaptation of the TH expression in response to coldness (R. Kvetňanský). - Investigations after short- and long-term stress have shown in the adrenal gland cortex of CRH „knockout“ mice (which are not capable to synthesize a functional neuropeptide corticoliberin axis) a substantial reduction of gene expression for and protein levels of the fenylethanolamine-N-methyltransferase (PNMT) when compared to the „wild-type“ animals. Our findings confirm the crucial role of glucocorticoids for a normal production and function of the PNMT in stress (R. Kvetňanský). - Effect of acute peripheral administration of Zolpidem (a GABAA receptor agonist with selectivity for α1 subunits) was studied on functional activation of oxytocinergic (OXY) cells in the hypothalamic paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei using dual Fos/OXY immunohistochemistry. The data of this study provide insight into the topographic patterns of brain activity within the clusters of magnocellular OXY cells in the hypothalamus. They indicate that stimulation of GABAA benzodiazepine receptors activates not
  7. 7. 7 only the stress responsive CRH neurons, but also the OXY neurons located in PVN and ACC, and not in SON. We showed that the regulatory role of this GABAA receptor is wide-ranging and different even within the functionally very similar magnocellular population of OXY neurons in the hypothalamus (A. Kiss). - The role of central alpha2-adrenoceptors in the regulation of hypothalamic magnocellular cells was studied under treatment with alpha2-adrenoceptor agonist, xylazine (XYL) and compared with effects of hyperosmotic challenges induced by hypertonic saline (HS). It was found that the peripheral administration of XYL is a potent stimulus for oxytocinergic (OXY), but not vasopressinergic (AVP) population of magnocellular cells eliciting a marked activation (Fos expression) in discrete population of OXY neurons in both the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. It was also found that higher osmotic challenge entirely overlaps the effect of XYL. Moreover, XYL pretreatment considerably reduces the activity of the CRH- producing parvocellular population of PVN cells activated by higher concentrations of HS. These data indicate for the first time that alpha2-adrenoceptors have broader impact in the regulation of hypothalamic neurons as assumed so far (A. Kiss). - Studies aimed at traditional and unconventional mechanisms of regulation of peptide secretion have proven gene expression for TRH in the rat heart. The gene expression is raised two fold in the left auricle when compared top the left ventricle. TRH secretion by slices of both the auricle and ventricle in vitro displays attributes of regulated secretion: it can be stimulated by hypoosmosis (i.e. by cell swelling) as well as by a depolarization of the plasma membrane using KCl. TRH secretion can be inhibited by angiotensin II, where this action is mediated via the AT1 receptors. TRH seems to be a part of a system determining some functions of the cardiovascular apparatus. Moreover, also the expression of the prepro-TRH in heart was proven, which significantly increases in transgenic rats with an extra gene for renin. TRH is more intensely produced and expressed in the atrium than in the ventricle. The cell swelling-induced secretion can be unmasked also in specifically (i.e. reversely) responding cells which are engaged in regulation of the water and electrolyte metabolism. This can take place by inhibiting a specific response – via mechanosensitive receptors activated channels with aid of the GdCl3 (V. Štrbák). - Within the project aimed at evaluation of neuroendocrine mechanisms in relation to mental state and the effects of psychotropic drugs, using the model of long-term voluntary wheel running of rats (which has a compulsory character), the possible role of the stress hormone oxytocin, which has been anticipated to participate in regulation of some cardiovascular functions, was studied. It was found in the running rats that the oxytocin concentration decreases in neurohypophysis without a parallel change of its plasma levels. Voluntary running led however to a decrease of oxytocin content in the heart auricle, which indicates to a possible role of oxytocin during a long term physical activity coupled with compulsive behavior (D. Ježová). - Proofs about a modulatory effect of the antiepileptic drug lamotrigine on the hormonal and cardiovascular response to mental stress were obtained in healthy volunteers. Lamotrigine has an inhibitory effect on the glutamatergic neurotransmission and the data obtained represent the first, although an indirect proof of the role of endogenous excitatory amino acids during stress in man. The results brought also the first information about changes of the non-verbal behaviour during psychosocial stress in response to endocrine changes. The effect of lamotrigine on hormone secretion plays probably an important role during its thymoprophylactic action in treatment of the bipolar disorder (D. Ježová). - During assessment of the importance of changes of neuroendocrine mechanisms at the origin of hypertension and insulin resistance in young, non-obese males with newly-diagnosed and yet untreated hypertension of the 1. grade and/or with high normal blood pressure, no changes in plasma adrenaline, noradrenalin, cortisol, PRA and aldosterone were found in response to a metabolic stress stimulus (i.e. to insulin-induced hypoglycemia). The hypertensive subjects had, however, a lower response of the somatotrophic and lactotrophic axis to hypoglycemia. Interestingly, the hypoglycemia nadir was in spite of the insulin resistance in the hypertensive subjects in both groups identical. The cause for the aforementioned could be located at the level of central mechanisms of the contraregulatory response. In particular, based upon the decreased response to the alpha2- adrenergic stimulation with clonidine, it was hypothesized that the decrease of growth hormone and prolactin response to hypoglycemia is given by a defect at the level of the alpha-adrenergic receptors at the hypophyseotrophic neurons (M. Vigaš). 2. B.2. In the area of studies on the biological action of hormone, cytokine and other bioactive molecules of endo-or exogenous origin at the DNA, RNA and protein level for 2. B.2.1. Influencing of metabolic processes - Studies on elucidation of the role of the local renin-angiotensin systems in adipose tissue, spleen, lymphocytes and carcinoma of the mammary gland have shown that losartan administration, a blocker of the AT1 receptors, to rats led to the known effect of increasing the gene expression for renin in the kidney cortex
  8. 8. 8 with a subsequent enormous increase of plasma rennin activity /PRA/. Furthermore, raised renin kidney activity (KRA) after losartan administration was found both in the kidney cortex and the medulla, respectively. It is proposed that the increase of KRA and PRA arises due to the raised renin expression in the kidney cortex as a consequence of lower levels of the cytoplasmic calcium as induced by inhibition of the angiotensin II. action. Renin expression was noted also in the kidney medulla although it did not increase after losartan administration. This is the first report on this type of losartan action (Š. Zórad). - During studies on monitoring the effect of 13-cis retinoic acid (a differentiation-inducing agent) administration on adipose tissue parameters, gene expression for the components for the renin-angiotensin system /RAS/ and for the adipokines, a significant increase of the gene expression of the aP2 (fatty acid binding protein) marker was noted. This confirmed the raised adipocyte differentiation after administration of the retinoic acid. The increased transformation of preadipocytes into adipocytes became manifested also via the increased gene expression for the angiotensin AT1 receptors, and by the increased content of leptin and adiponectin in the adipose tissue. Due to the production of new small fat cells significantly increased also the gene expression for markers of insulin sensitivity /GLUT 4 and PPARγ/. It is suggested that 13-cis retinoic acid induces the conversion of preadipocytes into adipocytes by creating heterodimers of the retinoic nuclear receptors with PPARγ (Š. Zorad). - Data on the importance of the transmembraneous /TM/ segment of membrane receptors in the mechanism of their activation have shown in A431 cell line that inhibition could be induced by: a) incorporation of TM into plasma membranes and b) by rigidisation of plasma membranes as induced by modification of the lipid composition. Decreased autophosphorylation of the EGF receptor was followed by inhibited phosphorylation of intracellular signaling proteins, what may significantly influence the terminal effect at the level of DNA replication, cellular proliferation and differentiation. The importance of these results relate to malignant cells over expressing EGF receptors (M. Ficková). - In studies focused at development of experimental arthritis in rats under various nutritional conditions, levels of leptin, ACTH, corticosterone, IL-1beta and production of NO on the 10th and the 22nd day of adjuvant arthritis of male rats were measured in order to assess the regulatory relationship of leptin and its modulators during the early and late phase of experimental inflammation. Circulating leptin levels were significantly decreased already on the 10th day of the diseases similarly as its mRNA in the epididymal fat. The IL-1beta concentration, which is an activator of leptin production, was increased in the spleen already on the 10th day. A significant activation of the HPA axis took place in the chronic disease phase. NO production was significantly raised in both intervals monitored. These results point to a dysregulation of leptin during adjuvant arthritis (J. Jurčovičová). - During investigations of molecular mechanisms, which participate in raised fatty acid oxidation v liver and skeletal muscle of rat as induced by leptin administration, it was found that a single application leads to higher levels of triglycerides, fatty acids and glycerol in circulation with a parallel decrease in triglyceride concentrations in both tissues. The aforementioned effects associate with a higher fatty acid beta-oxidation in both tissues, though with different kinetics for the liver and muscle. Measurements of activities and gene expressions for key enzymes of mitochondrial and peroxisomal oxidation of fatty acids have shown that leptin stimulates oxidation particularly in the peroxisomes (E. Šeböková). - Monitoring the effect of transfection of DNA vaccines in vivo in relation to the biological action of retinoic acids in normal and neoplastic tissue of the mammary gland, the effect of the 13-cis retinoic acid and the effect of CpG sequence containing oligodeoxynucleotides on the gene expression profile in spleen and in malign tumours of the mammary gland of the Sprague-Dawley rats, were studied using the cDNA macro array. The effect of the aforementioned components, whether independently or in combination, has induced both in the spleen and in the tumours of the mammary gland important changes in expression of more than 200 genes, which have not been described as yet (J. Brtko). - Molecular-genetic studies of function and expression of the hormone responsive dehydrogenase in Drosophila melanogaster have disclosed with aid of the analysis of the transcription units for the individual malate dehydrogenase (MDH) genes, that the transcription unit MDH53C1 codes for the synthesis of 4 and the MDH53C2 gene of 2 mRNA, whereby the two longest MDH53C1 transcripts reach by their second half of their 3‘-terminus into the transcription unit of the MDH53C2 gene. Both the 4 MDH53C1 mRNA and the 2 MDH53C2 mRNA originate via alternative splicing of different exons assuring identical N-terminal and middle areas, and different C-terminal ends of the MDH53C proteins. In contrast, the gene MDH87D1 for the cytosolic malate dehydrogenase codes production of only 2 mRNA using 2 independent promoters securing this was the origin of two isoforms of the MDH87D1 proteins, which differ in the N-terminal area and share identical C- terminal domains (R. Farkaš).
  9. 9. 9 - The Juvenile hormone, similar to retinoids and thyroid hormones, as the last orphan ligand in animal kingdom plays a crucial role in regulation of cell differentiation and morphogenesis of Drosophila. Therefore, we have cloned and fully sequenced whole genomic region of the Met gene, residing on X chromosome and encoding bHLH-PAS protein homologous to dioxin receptors. Analysis of 13 Met alleles generated by radiation and P-element mutagenesis revealed unexpected complexity of MET protein action and provided groundwork for understanding Met- phenotypes including animal's resistance to JH. By combining pharmacological and genetic approaches we were able to disclose major pathway of JH cellular mode of action which involves Broad- Complex (BR-C) BTB/POZ transcription factor signaling by influencing timing of its action in context with ecdysone cascade. These observations have been confirmed by genetic interaction using double heterozygote crosses as well as by protein-protein interaction studies in yeast two-hybrid system (R. Farkaš). 2. B.2.2 innovative diagnostics and treatment of selected diseases - With help of the National Diabetes Program (NDP), in 2003 the activities of the joined DIABGENE laboratory, which has been established under the patronage of the IEE SAS and of the National Institute for Endocrinology and Diabetes in Lubochna, were launched. The DIABGENE Laboratory as such resides at the IEE SAS in Bratislava. The Laboratory became over the three years step by step equipped with relevant hardware comparable with international standards (2 automatic sequencers, 1 dHPLC, 2 gradient PCR thermocyclers etc.), the DNA repository for monogenic diabetes was established and techniques for routine sequencing of the most common forms of the MODY diabetes and the real-time PCR diagnostics of the mitochondrial diabetes were set up. Moreover, mutation pre-screening using the dHPLC apparatus has been initiated. Based upon the DNA diagnostics of MODY diabetes subtypes clinical diabetologists in Slovakia are more and more utilizing the DNA results for introduction of „to the person tailored treatment“ plans /e.g. return from insulin to sulfonylurea in suitable cases of MODY 3 or the use of the diet treatment in the MODY2/. In collaboration with the Exeter University in UK, the diagnostic spectrum covers also the DNA diagnostics of various forms of the neonatal diabetes mellitus. Beside routine diagnostics of MODY subtypes within the territory of Slovakia, a couple of so far unknown mutations of permanent or transient neonatal diabetes have been discovered. These are now being investigated in terms of structure-function studies at the University of Oxford, UK. In addition, very important original results were achieved in respect to treatment of permanent neonatal diabetes producing the first pharmacogenomics data for practical use in clinical diabetology (I. Klimeš). - Evaluations of the health risk due to a long-term exposition to low dose of polychlorinated biphenyls (PCB) have discovered that a significant contamination of the environment as documented with 4-times higher PCB serum levels of 1010 adults form the contaminated adults in comparison to 1040 subjects from non- contaminated counties was associated with increased volume of the thyroid gland, higher levels of thyroidal hormones (free thyroxin and total triiodothyronine) and with raised appearance of low levels of TSH in the zone of subclinical and manifest hyperthyroidism. This points to the role of PCB in the origin of an increased thyroid gland function as based upon displacement of thyroxin from its binding to protein carriers, and to a consequent increase of its turnover in the periphery (P. Langer) - Moreover, significant correlations between the environmental pollution and the appearance of impaired glucose metabolism were found in 2047 subjects from a population chronically exposed to increased concentration of several organochlorine compounds. Their concentrations in circulation of the exposed persons correlated highly significantly with each other, which indicate to presence of a polycontamination. Associations between them depend upon the age and BMI, which are though simultaneously risk factors for diabetes and dysglycemia. With help of mathematical models for estimation of indices of insulin sensitivity (R HOMA, ISI McAuley, ISI Matsuda, ISI Cederholm) a higher frequency of insulin resistance in inhabitants of the contaminated areas was found what is in close harmony with a clearly higher appearance of diabetes and prediabetes in individuals with proven contamination by organochlorines (I. Klimeš) - During studies of interaction of central and local hormonal and immunologic factors in pathogenesis and course of rheumatoid arthritis it was proven that the relatively modest hypocortisolemia in these patients was not caused by a decreased hypothalamic-hypophyseal regulation, but rather by a change in biosynthesis of corticoids in the adrenal cortex. Moreover, an isolated decrease in basal production of androgens (dihydroepiandrostendione sulphate, DHEAS), which have an important immunomodulatory action, is also taking place. An interesting finding was also a substantial lowering of the sympathetic adrenomedullary response to hypoglycemia, which may show up also during common stress situations in patients and alter regulation of immunity. The results point to an increase of the sulphotransferase activity in biosynthesis of corticoids and its contribution to a normal cortisol production (R. Imrich). - Within the retinoic acid research project, further interesting data were achieved. In particular, using the Real-Time PCR original results on the expression of all subtypes of retinoic receptors (RARalpha, RARbeta RARgama) and rexinoid receptors (RXRalpha, RXRbeta a RXRgama) in an extremely malignant and rare tumor (Triton tumor) of child patient were obtained. Moreover, also a standard protocol was prepared for the
  10. 10. 10 diagnostics of retinoid receptors in malignant tumours of the human thyroid gland and mammary gland, which should enable to make an estimate of the success rate of treatment with retinoic acid derivatives in clinical cases (J. Brtko). - Evaluations of the relation structure-function of insulin analogues as prepared by modification of the C- terminus from the B-chain of insulin included also testing of a series of insulin analogues derived from the des- tetrapeptide (B27 – B30) of insulin. These were modified via the N-methylation of the peptide binding in the B23 – B24 position and also by exchanging of aminoacids in positions B25 or B26. Measuring the ability of the analogues to stimulate glucose transport into the rat adipocytes and binding to the receptor of the adipocyte membranes, it was found that the substitution TyrB26 with histidine has a positive influence on the biological activity of the peptide. N-methylation of the peptide bond B23 – B24 and B24 – B25 has a negative impact on the analogue potency. On the contrary, combination of HisB26 with methylation in the B25 – B26 position leads to a “super active“ analogue [NMeHisB26]-des-tetrapeptide (B27-B30) insulin. A further similar analogue [NMeAlaB26]-des-tetrapeptide (B27-B30) has also a high biological activity. Currently are both active analogues being tested in two top laboratories in the US and in Denmark (Š. Zórad). - The MicroPET equipment enables to study distribution and effects of drugs, tumor development as well as the effect of treatment in living small laboratory animals. It is also a valuable tool especially for testing the effects and distribution of newly developed drugs (V. Štrbák) C. THE RESEARCH STRATEGY AND FOCUSING OF THE RESEARCH ACTIVITIES OF THE INSTITUTE OVER THE NEXT 4 YEARS C.3.i Present state of knowledge and status of ongoing research related to subject of the Concept, from both international and national perspective The global trends in experimental and clinical endocrinology are concentrating at elucidation of the neuroendocrine regulatory processes with special emphasis on assessments of their significance for impairments of pathogenesis of important non-infectious diseases, on understanding of mutual relations of the neural, endocrine and immune systems. In the field of neuroendocrinology, increasing evidence has accumulated supporting the fact that neuroendocrine factors are involved in the pathogenesis and course of several mental disorders. In particular, exposure to intensive or chronic stress stimuli and dysregulation of stress hormone release play an important role in currently highly prevalent depressive disorders. The main research focus turned from classical monoaminergic neurotransmitters to the factors participating in neural plasticity and neurodegeneration. The influence of stress exposure on the susceptibility to the development of depression is thought to be based on both genetic and environmental factors. The environmental factors include prenatal and perinatal exposure to demanding events and the present knowledge allows to suggest an important role of neuroendocrine alterations in long-term consequences. Measurements and correlations of genetic variations, neuroendocrine changes and psychological characteristic are likely to reveal new aspects for the treatment and prevention of mental disorders. Intensive attention is being paid to investigations of the endocrine functions of the various adipose tissue depots, to the role of the many molecules being either produced inside (e.g. adipokines) or being brought into the adipose tissue via blood stream (e.g. cytokines, circulating blood elements) for the origin and progression of the low-grade systemic inflammation and related disease processes. The research further focuses at deeper elucidation of the mechanism of hormone action with a special accent on regulation of gene expression for enzymes, receptors, transcription factors which could be of great importance for development of socially important diseases like obesity, type 2 diabetes, atherosclerosis, cardiovascular disease, hypertension etc. More recently several gene defects have been identified which modulate the hormone receptor cell signaling pathways causing obesity, insulin resistance and atherosclerosis. Analysis of gene polymorphism or mutation in human subjects is used for detection of risk factors for development of obesity, the metabolic syndrome, monogenic forms of diabetes, type 2 diabetes and cardiovascular diseases. Pharmacogenomics data (e.g. in diabetes, lipids, cardiovascular medicine, in psychiatry) are also emerging indicating to new possibilities for personalization of the diagnostics and treatment to the individual patient. The hormone and cytokine receptor transgenic and specific gene knockout animals are used in studies aimed at identification of the ethiopathogenesis of diseases states like the metabolic syndrome, diabetes and its complications or psychiatric disorders. The cell membrane initiated action of hormone ligand for nuclear receptors (e.g. steroid or thyroid hormones) and their effects on transcription processes are studied. The idea of a synergistic coupling between membrane initiated and genomic action of hormone revises fundamentally the cell
  11. 11. 11 signaling pathways in endocrinology. Alternative pathways for regulation of biosynthesis and secretion of hormones and other active molecules are being intensively sought as new drug targets. Due to a striking methodological progress over the last decades in all areas of medicine and molecular biology, there are much greater possibilities and much less ethical concerns than in the past to use for research purposes samples of human origin. The high throughput techniques at the DNA, RNA and/or protein level allow running of hundreds of parameters in very small amounts of human material, which is certainly more informative for the scientific progress, more ethical to the patients and/or volunteers. Beside these analyses, further continues the use of selected animal models with various genetic modifications, tissue cultures of selected cell lines of both human and animal origin for solution of mechanistic questions arising from studies of the endocrine system- related human diseases. In harmony with the aforementioned, all around Europe large genotype and phenotype repositories are being established on the general population and/or selected diseases states. They allow rapid responses of the research community to newly emerging challenges in their area of interest, by simply employing the research tools of molecular biology and genetics on the samples readily available due to the existing PC supported databases. 3.ii. Organisation´s role of significance in overall research effort within the field of the Concept on both national and international scales The Institute is the only scientific research place in the Slovak republic which deals with the basic explorations in the area of endocrinology combined with research outputs specifically aimed for the use in clinical practice. The Institute is an internationally accepted and renowned research institution, which has been over several decades engaged into research of the function and regulation of the thyroid gland in various disease states. Furthermore, the Institute has won an outstanding position in the research of neuroendocrine response to stress situations; in particular in the area of function of the sympathoadrenal system after application of various stress stimuli including the space flight of both, the rats and the astronauts. International recognition has been also received with respect to new insights into the role of neuroendocrine factors in depression and anxiety. Studies on the role of cell volume changes for regulation of peptide hormone secretion have also a pioneering character. A very intensive attention has been paid at the Institute in the last couple of years to identification of various disorders of lipid metabolism, and production of various adipokines and cytokines in adipose tissue in relation to the low grade systematic inflammation and origin of the metabolic syndrome. The Institute has become also the leading center in the screening and DNA diagnostics of the various forms of monogenic diabetes in Slovakia and has established the first DNA repository in diabetology in this country. Of utmost importance for the Slovak endocrinology has been also the publication of the postgraduate textbook on basic and clinical endocrinology which is being used as the main textbook for taking the specialization boards in clinical endocrinology in this country. Two leading researchers – employees of this Institute served as editors. Moreover, researchers from this Institute contributed in total to completion of 20 chapters of this book. The textbook received the Award of the Slovak Literature Foundation for the Best Book in section Medicine and Molecular Biology as published in 2004. A national reward of the current achievements of the Institute has been also the awarding of our Institute by the status of an Centre of Excellence presented to us by the Slovak Academy of Sciences SAV based upon the project entitled “Center of Excellence for research of neuroendocrine mechanisms in the pathogenesis of weighty diseases /acronym CENDO/”. The project anticipates an active collaboration with the Slovak Health University, with the faculty of Natural Sciences as well as continuation of collaboration with other research and health care organizations. An important international recognition of the Institute’s research has been the awarding of our Institute with the statute of a Centre of Excellence by the European Commisssion in Brussels, Belgium. This was received after a successful evaluation of our project entitled „Reduction of negative impact of environmental factors on human health“. With participation of important foreign researchers several research projects were carried out, e.g. in the area of regulation of vasoactive hormone secretion in stress (H. Hinghofer-Szalkay), investigations of neuroendocrine regulations of behaviour and brain plasticity (B. Johansson), hormone secretion in relation to mental stress (I. Loder), or mechanisms of insulin secretion regulation in relation to changes of cell volume (M. Ritter). Within the CE project, several long-term study stays of Post-Docs from abroad took place, who worked on, e.g. regulation of fatty acid oxidation in liver and skeletal muscle by leptin in relation to insulin resistance development (S.Wein, FRG), on elucidation of central regulatory mechanisms of stress-induced prolactin secretion (I. Bodnár, Hungary). Moreover, two Post-Docs (L. Rondahl, Sweden and S. Mostböck, Austria) studied the effect of heavy metals appearing in pollutants on nuclear receptor pathway. A PhD student from Greece (A. Makatsori) had studied pharmacological possibilities for alteration of stress in man and animals.
  12. 12. 12 The important international position of our Institute is underlined by the participation of our research teams in 6 international projects within the EU Framework Programs. The aforementioned encompasses within the 5th FP the following projects, i.e. the PCBRISK (Evaluating human health risk from low-dose and long-term PCB exposure), VITAGE (Vitamin A, vitamin E and carotenoid status and metabolism during ageing: Functional and nutritional consequences), ENDOMET (Dysregulation of endogenous steroid metabolism alters neuronal and reproductive system development: effects of environmental plasticisers) and the DROSDEL (Construction of a new Drosophila duplication collection). Within the 6th FP, the research teams of this Institute participate in the following projects: CASCADE (Chemical contaminants in the food chain) and in the DANUBIOBANK (The Danubian Biobank Initiative: Towards Information-Based Medicine). Several research groups of our Institute had been active in research projects supported by the biomedical programs of the EC like the COST B17 (Insulin resistance, obesity and diabetes mellitus in the elderly: Fatty acid oxidation and insulin resistance) or other international organizations, e.g. NATO (Genes in Control of Apoptosis during Drosophila metamorphosis). Three research projects have been supported within the scheme of the Slovak-American scientific and technical collaboration scheme, e.g. the project on „Ethanol and cell swelling induced peptide secretion: intracellular signalling and regulation“; „Function and molecular interactions of the LAMMER kinase“ and the „Stressor- specific regulation of catecholamine biosynthetic enzyme gene expression“. Successful scientific collaboration has been pursued also at further important international projects, i.e. on „Environmental influences on hormone release during stress in humans: continuation of previous common studies“, which has been executed within an Austrian-Slovak collaboration, and a project with a Greek partner dealt with „Environmental stimuli and neuronal plasticity“. Within a bilateral collaboration with a US partner successfully continued the work at the project „Slovakia PCB Exposures and Early Childhood Development“. The very important and highly innovative results on „Pharmacogenomics of Permanent Neonatal Diabetes“ have been achieved within a long-term bilateral collaboration with the Biomedical Research Institute of the Exeter University in the UK. Several leading scientists of the Institute have been members of the editorial/international boards of various international professional journals what resembles their high professional credit in their professional community abroad. Out of the 9 colleagues who are active in the editorial boards, we like to underline particularly the positions of the Executive Editor for Europe in the journal Cellular and Molecular Neurobiology (USA), the Associate Editor in the official journal of the European Association for the Study of Diabetes, i.e. Diabetologia, members on boards of Stress (USA), Neuroendocrinology (USA), Neuroscience News (USA), and Archives Physiol. Biochemistry (Netherlands). 3.iii. Objectives of the Concept The main lines of the thematic orientation for scientific research activities of the Institute for the next 4 years shall involve: A/ research of mechanisms of the neuroendocrine regulation of body functions with special emphasis upon identification of the effects of genetic background, gender, physiological, pharmacological or environmental factors or interventions on the responses to various stress situations, and elucidation of their changes in pathogenesis of various diseases, including those of the cardiovascular and immune system, rheumatic disorders, and the physiology and pathology of behaviour. B/ investigations on the action of hormones, cytokines and other biologically active molecules of endogenous and/or exogenous origin influencing metabolic processes both in cells /in vitro/ conditions and in integrated organism /in vivo/. Furthermore, molecular biology tools will be employed to disclose the role of gene polymorphisms and mutations for regulation of metabolic pathways in health and disease at the DNA, RNA and protein level. The aforementioned shall be carried out with the aim to unmask the presence of disease risk factors, to elucidate detail mechanisms of disease origin and development, and to innovate diagnostics and treatment of selected diseases /e.g. obesity, diabetes, specific oncology diseases etc.). In the area of neuroendocrine regulations, the research shall focus at: 1. physiology and pharmacology of the catecholaminergic system in response to stress, in particular at a) elucidation of the genetic nature of the adaptive and maladaptive processes during adaptation to a long-term acting of psychological or physical stress stimuli
  13. 13. 13 b) evaluations of the molecular genetic nature of the hyper-reactivity of organism adapted to one type of stress stimulus after their exposure to a different stressor c) studies of the adaptation processes to repeated exposure to different stressors in a new line of genetically modified animals lacking the key enzyme of catecholamine synthesis, i.e. the PNMT d) broadening of knowledge on the genetic apparatus of catecholamine biosynthesis in the adipose tissue which may be critically involved into the stress response and into deterioration of the insulin action as well e) mapping of changes of the sympatho-adrenal and hypothalamic-hypophyseal-adrenocortical system in a rat model of the Alzheimer disease 2. mental disorders, in particular at a) investigations of principles of the neuroendocrine regulation of the organism in conditions of stress and their relation to the induction and development of mental impairment. Attention will be also given to identification of gender differences in the regulatory processes of the body as the appearance of individual mood disorders seems to be deeply sex dependent, b) elucidation of the degree to which the neuroendocrine changes in stress can be determined by the individual psychologic and behavioural characteristics with special emphasis on anxiosity and depressive situations and c) studies of prenatal stress effects with the aim to test possible long-term consequences for coping with stress depending on the genetic background and epigenetics The aforementioned goals shall be executed in both, the animal and human models, as well as in specific patients selected in collaboration with the clinical psychiatry department in Bratislava 3. rheumatic diseases, in particular at a) elucidation of the mechanism for the reduced adrenocortical and adrenomedullary function in rheumatic patients b) following up the consequences of the above endocrine changes on the regulation of immune functions looking at the effect of cortisol, dehydroepiandrosterone and catecholamines on cytokine production, expression of receptors mediating the antigen presentation, apoptosis and other functions c) contribution of the genetic predisposition to these endocrine changes in the first degree relatives d) studying the role of the chronic inflammation on lipid and carbohydrate metabolism with parallel measurements of production of proinflammatory cytokines in subcutaneous adipose tissue e) investigations of the subsystems of the autonomic nervous system in rheumatic patients The goals of this project shall be achieved in the ongoing long-lasting collaboration with the Institute of Rheumatic Diseases in the town Piestany 4. hypertension and diabetes, in particular at a) investigations of the subsystems of the autonomic nervous system in diabetic patients b) elucidation of mechanisms of insulin resistance and insulin hypersecretion in subjects with juvenile hypertension at the level of tissues The goals of this project shall be achieved in the ongoing long-lasting collaboration with the Institute of Rheumatic Diseases in the town Piestany. 5. tumorigenesis, in particular at a) studies aimed at ascertaining of the n. vagus participation in monitoring and modulation of function of the immune system components with a special emphasis on the role of n. vagus in tumorigenesis b) definition of humoral and neural pathways participating in transmission of information about tumorigenesis to the central nervous system and c) identification of those brain areas which are receiving the above information, digest them and reciprocally intervene with the tumorigenesis processes d) describing the role of the vagus nerve in regulation of inflammatory processes with emphasis on the role of vagus stimulation for induction of the ischemia-reperfusion liver damage and the role of spleen in these processes. The goals of this project shall be achieved in close cooperation with the Institute of Pathophysiology of the Comenius University School of Medicine in Bratislava. In the area of hormone secretion and action and their role in pathogenesis of diseases, the research shall focus at the: 6. role of adipose tissue and low-grade systemic inflammation for the metabolic syndrome in man, in particular at a) analyses of the gene expression for transcription factors involved in the origin and maintenance of the inflammatory processes in simple obesity, glucose intolerance and growth hormone deficiency b) measurements of the oxidative capacity of the mitochondria and their gene expression for relevant enzymes in the above situations c) pathogenesis of the subclinical inflammation processes in circulation, adipose and muscle tissue in patients with morbid obesity before and after surgical intervention /gastric banding/
  14. 14. 14 d) testing the hypothesis that the decreased amount of adipose tissue in patients with the chronic obstructive disease of the lung produces higher amounts of adipokines and leads thus to metabolic abnormalities typical for low-grade systemic inflammation 7. role of the renin-angiotensin system in the adipose tissue of rats, in particular at a) measurements of the components of the renin-angiotensin system in the adipose tissue in response to high fat feeding b) up-regulation of the angiotensin AT1 receptors in the fat tissue during diet- or genetically-induced obesity of rats c) investigation of changes in the cellular organization of the adipose tissue and in the adipokine production after blockade of the AT1 receptors d) comparison of the action of the ACE inhibitors and blockers of AT1 receptors on the cellularity of the adipose tissue and adipokine production 8. DNA diagnostics and screening of monogenic forms of diabetes in Slovakia, in particular at a) continuation of the ongoing activities in DNA diagnostics of monogenic diabetes across the whole country b) active screening for mutation carriers yet without the manifest disease in all blood relatives of the positive probands having clinically manifested diabetes c) active counselling on the DNA based recommendations for change of treatment /insulin vs. sulphonylurea or diet/ and long-term clinical physiology monitoring of those patients d) identification of new mutations and collection of extensive phenotypes in order to enable proper structure- function studies e) setting-up the high throughput analytical performance profile /i.e. dHPLC mutation pre-screening plus mutation identification via ABI direct sequencing/ for all types of monogenic diabetes /currently about 15 diagnoses/ f) maintenance of the DNA repository, opening of a sub-registry on monogenic diabetes within the National Children Diabetes Registry and loading it up with the gene- and phenotypic data obtained 9. alternative mechanisms of hormone secretion and action, in particular at a) identification of a novel mechanism of secretion and exocytosis of peptide hormones with accent at possibilities to influence the insulin secretion b) characterization of the signaling pathway leading from cell swelling to exocytosis of insulin and its comparison with the glucose-induced insulin secretion pathway c) classification of the function and regulation of peptides in heart with emphasis on the appearance of TRH in the heart, including its regulation and interaction with the renin-angiotensin system 10.insulin analogues and their mechanisms of action, in particular at a) identification of the effects of the super potent insulin analogues on insulin aggregation and b) the interaction of insulin with specific receptors 11. the retinoids and rexinoids, in particular at a) elucidation of the mechanism of action of both, the retinoids and rexinoids as well as of their structural derivatives b) investigation of mechanism of action of the steroid hormone dihydroxyvitamine D3 and its structural derivatives with their potentially anti-cancer effects c) the use of the relevant analytical methodology in human endocrinology, especially for measurements of expression of subtypes of the nuclear receptors of the all-trans retinoic and 9-cis retinoic acids in the tumor tissue of the thyroid gland, mammary gland and in the prostate with the possibility to use retinoic acid and its derivatives in treatment of oncological diseases d) studies on the negative effects of chemical compounds on the regulatory mechanisms mediated via the nuclear receptors of retinoic acids, or hormones of the thyroid gland and estrogens 12.on further explorations of endocrine disruptors, in particular at a) studies on the mechanisms of action of the individual phthalate/phenols derivatives and their combinations on the cell growth via elucidation of the intracellular and nuclear mechanisms of action of the estrogen receptor-α b) identification of the relation between the length of exposition and dosage of the individual compounds and their mixtures tested in relation to cell proliferation with the aim to define safe exposition dosages, which do not induce cell proliferation 13.characterisation of molecular and cellular mechanisms of action of the Juvenile hormone /JH/ elucidation of the programmed cell death, in particular at a) learning the properties of a so far not known JH receptor b) deepening of knowledge of its signaling pathway used for its effect on morphogenesis c) explaining the evolutionary consequences of this development d) investigations of the hormone-induced apoptosis of the Drosophila salivary gland
  15. 15. 15 e) learning the molecular and genetic interactions between the individual components of this signaling pathway in order to understand the origin of the Parkinson, Huntington, Alzheimer or Lou-Gehrig diseases 3.iv. Proposed strategies and methods to be applied, and time schedule The research strategy as outlined above can be pursued proposed that the financial requirements of the individual projects shall be met in full over the years to come. The current situation with an overview of the type and duration of the research grant support is displayed in the table below: * Time Types of Research Grants Number of projects in each category schedule Objective VEGA APVV EC ESF NATO COST Other From-To number 1 X X 2007-09 2 X X X X 2007-09 3 X 2007-08 4 X X X 2007-09 5 X, X X X 2007-09 6 X, X X X 2007-09 7 X 2007 8 X X X X 2007-09 9 X X X X 2007-09 10 X 2007 11 X X X 2007-09 12 X, X X 2007-09 13 X X 2007-09 * Types of Research Grants: VEGA = Scientific Grant Agency /small grants/, APVV = Agency for Support of Science and Development /the major research grant agency in Slovakia/, EC = European Commission /6th and 7th FP/, ESF = European Social Fund, COST = Cooperation in Science and Technology Beside external fund raising, which is essential for successful accomplishment of any research project per se, it will be necessary to continue in the broad international collaboration /for more details see Appendix No. 1/. In this respect, our Institute has a long and proven track which is resembled at best in the Institute’s List of Publications and their citations, majority of which comes from impacted foreign journals. The scientific goals of the Institute as outlined for the four years to come, shall require a significant move forward in the overall methodological development. The research strategy shall require beside a series of currently used methods of molecular biology, genetics, biochemistry, histochemistry and clinical physiology development of a series of other techniques, in particular /hardware already available/: - dHPLC pre-screening and identification of new mutations by direct sequencing for all types of monogenic diabetes /all hardware already available and working/ - Real time PCR assays of nuclear receptors and reversed transcription of them - Electrophoretic mobility shift assay /hardware to be obtained or to be used at collaborating centers/: - DNA and protein arrays - Fluorescence and laser confocal microscopy - Transmission microscopy and reverse electron microscopy - In situ hybridization with non-radioactive markers - Laser micro dissection of specific areas in the brain - Gene transfection and gene silencing Potential risks for fulfillment of the Concept encompass mainly low institutional financing from the Academy resources coming in from the state budget, constant increase in the energy prices, persisting administrative and financial obstacles for joining and utilization of the EU structural funds, very low increase of wages and insufficient inflow of new permanent positions for middle-age investigators. In fact, currently at the Institute we experience a large gap between the researchers in their fifties and the new generation in late thirties, of whom we
  16. 16. 16 have currently employed just three investigators, out of them two are working abroad for more than three years so far and their return chances are rather low. Lastly, additional finances shall be needed to keep up with the proposed methodological advancement of the Institute, in particular for purchase of larger pieces of hardware, requiring capital outlays. III. Partial indicators of the main activities: 1. Research output i. List of the selected publications documenting the most important results of basic research. Total number of publications in the whole assessed period should not exceed the average number of the research employees 2003 (IF 2002) 1. KLIMES I, WESTON K, KOVACS P, GASPERIKOVA D, JEZOVA D, KVETNANSKY R, THOMPSON JR, SEBOKOVA E, SAMANI NJ: Mapping of genetic loci predisposing to hypertriglyceridaemia in the hereditary hypertriglyceridaemic rat: analysis of genetic association with related traits of the insulin resistance syndrome. Diabetologia, 46: 352-358, 2003. IF= 5,136 2. BRTKO J, THALHAMER J: Renaissance of the biologically active vitamin A derivatives: Established and novel directed therapies for cancer and chemoprevention. Current Pharmaceutical Design, 9: 2067-2077, 2003. IF= 4,692 3. ROVENSKY J, IMRICH R, KOSKA J, KOVALANCIK M, KILLINGER Z, PAYER J, VIGAS M, JEZOVA D: Cortisol elimination from plasma in premenopausal women with rheumatoid arthritis. Ann Rheum Dis, 62: 674-6, 2003. IF=3,593 4. MONCEK F, AGUILERA G, JEZOVA D: Insufficient activation of adrenocortical but not adrenomedullary hormones during stress in rats subjected to repeated immune challenge. J Neuroimmunol, 142: 86-92, 2003. IF= 3,577 5. NAJVIRTOVA M, GREER SE, GREER MA, BAQI L, BENICKY J, STRBAK V: Cell volume induced hormone secretion: Studies on signal transduction and specificity. Cell Physiol Biochem, 13: 113-122, 2003. IF= 3,059 6. MICUTKOVA L, KVETNANSKY R, KRIZANOVA O: Repeated immobilization stress reduces the gene expression of type 1 and 2 IP3 receptors in stellate ganglia. Neurochem Int, 43(6): 557-561, 2003. IF= 3,040 7. MICUTKOVA L, RYCHKOVA N, SABBAN EL, KRIZANOVA O, KVETNANSKY R: Quantitation of changes in gene expression of norepinephrine biosynthetic enzymes in rat stellate ganglia induced by stress. Neurochem Int, 43(3): 235-242, 2003. IF= 3,040 8. MAKATSORI A, DUNCKO R, SCHWENDT M, MONCEK F, JOHANSSON BB, JEZOVA D: Voluntary wheel running modulates glutamate receptor subunit gene expression and stress hormone release in Lewis rats. Psychoneuroendocrinol, 28: 702–714, 2003. IF= 2,982 9. PIRNIK Z, MIKKELSEN JD, KISS A: C-Fos induction in the rat deep cerebellar and vestibular nuclei after central administration of colchicine: a qualitative and quantitative time-course study. Brain Res Bull, 61(1): 63-72, 2003. IF= 2,283 10. UKROPEC J, RESELAND JE, GASPERIKOVA D, DEMCAKOVA E, MADSENL, BERGE RK, RUSTAN AC, KLIMES I, DREVON CA, SEBOKOVA E: The hypertriglyceridemic effect of dietary n-3 fatty acids is associated with increased β-oxidation and reduced leptin expression. Lipids, 38 (10): 1-7, 2003. IF= 2,044 2004 (IF 2003) 1. BENNASROUNE A, FICKOVA M, GARDIN A, DIRRIG-GROSCH S, AUNIS D, CREMEL G, HUBERT P: Transmembrane peptides as inhibitors of ErbB receptor signaling. MOL BIOL CELL 15(7):3464-74, 2004. IF = 7.454 2. ZAKOVA L, BARTH T, JIRACEK J, BARTHOVA J, ZORAD S: Shortened insulin analogues: marked changes in biological activity resulting from replacement of TyrB26 and N-methylation of peptide bonds in the C-terminus of the B-chain. BIOCHEM 43 :2323-2331, 2004. IF = 3.922
  17. 17. 17 3. IMRICH R, ROVENSKY J, ZLNAY M, RADIKOVA Z, MACHO L, VIGAS M, KOSKA J: Hypothalamic-pituitary-adrenal axis function in ankylosing spondylitis. ANN RHEUM DIS 63: 671-674, 2004. IF = 3,827 4. KUBOVCAKOVA L, KRIZANOVA O, KVETNANSKY R: Identification of the aromatic L- amino acid decarboxylase gene expression in various mice tissues and its modulation by immobilization stress in stellate ganglia. NEUROSCIENCE 126: 375-380, 2004. IF = 3,601 5. MONCEK F, DUNCKO R, JOHANSSON BB, JEZOVA D: Effect of environmental enrichment on stress related systems in rats. J NEUROENDOCRINOL 16: 423-431, 2004. IF = 3,418 6. BODNAR I, MRAVEC B, KUBOVCAKOVA L, TOTH EB, FULOP F, FEKETE MIK, KVETNANSKY R, NAGY GM: Stress- as well as suckling-induced prolactin release is blocked by a structural analogue of the putative hypophysiotrophic prolactin-releasing factor, salsolinol. J NEUROENDOCRINOL 16: 208-213, 2004. IF =3,418 7. PIRNIK Z, MRAVEC B, KISS A: Fos protein expression in mouse hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei upon osmotic stimulus: colocalization with vasopressin, oxytocin, and tyrosine hydroxylase. NEUROCHEM INT 45(5): 597-607, 2004. IF = 3,261 8. MAKATSORI A, DUNCKO R, MONCEK F, LODER I, KATINA S, JEZOVA D: Modulation of neuroendocrine response and non-verbal behavior during psychosocial stress in healthy volunteers by the glutamate release inhibiting drug lamotrigine. NEUROENDOCRINOL 79:34-42, 2004. IF= 2,844 9. POHORECKY LA, BLAKLEY GG, KUBOVCAKOVA L, KRIZANOVA O, PATTERSON- BUCKENDAHL P, KVETNANSKY R: Social hierarchy affects gene expression for catecholamine biosynthetic enzymes in rat adrenal glands. NEUROENDOCRINOLOGY 80: 42-51, 2004. IF =2,844 10. JEZOVA D, MLYNARIK M, ZELENA D, MAKARA GB: Behavioral sensitization to intermittent morphine in mice is accompanied by reduced adrenocorticotropine but not corticosterone responses. BRAIN RES 1021(1):63-8, 2004. IF = 2,474 2005 (IF 2004) 1. BROUWERS FM, PETRICOIN EF, KSINANTOVA L, BREZA J, RAJAPAKSE V, ROSS S, JOHANN D, MANNELLI M, SHULKIN BL, KVETNANSKY R, EISENHOFER G, WALTHER MM, HITT BA, CONRADS TP, VEENSTRA D, MANNION DP, WALL MR, WOLFE GM, FUSARO VA, LIOTTA LA, PACAK K: Low molecular weight proteomic 2. information distinguishes metastatic from benign pheochromocytoma. ENDOCR RELAT CANCER 12 (2): 263-272, 2005. IF =4,597 3. IMRICH R, ROVENSKY J, MALIS F, ZLNAY D, KILLINGER Z, KVETNANSKY R, HUCKOVA M, VIGAS M, MACHO L, KOSKA J: Low levels of dehydroepiandrosterone sulphate in plasma, and reduced symphatoadrenal response to hypoglycaemia in premenopausal women with rheumatoid arthritis. ANN RHEUM DIS 64 (2): 202-206, 2005. IF = 3,916 4. IMRICH R, ROVENSKY J, VIGAS M: Different threshold for prolactin response to hypoglycaemia in patients with rheumatoid arthritis? ANN RHEUM DIS 64 (2): 515-516, 2005. IF = 3,916 5. KLIMES I, WESTON K, GASPERIKOVA D, KOVACS P, KVETNANSKY R, JEZOVA D, DIXON R, THOMPSON JR, SEBOKOVA E, SAMANI J: Mapping of genetic determinants of the sympathoneural response to stress. PHYSIOL GENOM 20: 183-187, 2005. IF = 3,855 6. PISTOVCAKOVA J, MAKATSORI A, SULCOVA A, JEZOVA D: Felbamate reduces hormone release and locomotor hypoactivity induced by repeated stress of social defeat in mice. EUR NEUROPSYCHOPHARMACOL 15: 153-158, 2005 IF = 3,545 7. STEFANIK P, MACEJOVA D, MRAVEC B, BRTKO J, KRIZANOVA O: Distinct modulation of a gene expression of the type 1 and 2 IP3 receptors by retinoic acid in brain areas. NEUROCHEM INT 46 (7): 559-564, 2005. IF = 3,211 8. PIRNIK Z, JEZOVA D, MIKKELSEN JD, KISS A: Xylazine activates oxytocinergic but not vasopressinergic hypothalamic neurons under normal and hyperosmotic conditions in rats. NEUROCHEM INT 47(7): 458-465, 2005. IF = 3,211 9. BACOVA Z, BENICKY J, LUKYANETZ EE, LUKYANETS IA, STRBAK V: Different Signaling Pathways Involved in Glucose and Cell Swelling-Induced Insulin Secretion by Rat Pancreatic Islets in Vitro. CELL PHYSIOL BIOCHEM 16: 59-68, 2005. IF = 3,093
  18. 18. 18 10. MRAVEC B, BODNAR I, UHERECZKY G, NAGY GM, KVETNANSKY R, PALKOVITS M: Formalin attenuates the stress-induced increase in plasma epinephrine levels. J NEUROENDOCRINOL 17 (11): 727-732, 2005. IF = 2,920 2006 (IF 2005) 1. PEARSON ER, FLECHTNER I, NJOLSTAD PR, MALECKI MT, FLANAGAN SE, LARKIN B, ASHCROFT FM, KLIMES I, CODNER E, IOTOVA V, SLIGERLAND AS, SHIELD J, ROBERT JJ, HOLST JJ, CLARK PM, ELLARD S, SOVIK O, POLAK M, HATTERSLEY AT: for the Neonatal Diabetes International Collaborative Group (including GASPERIKOVA D, STANIK J): Switching from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 mutations. N ENGL J MED 355 (5): 467-77, 2006. (IF=44,016) 2. KUBOVČÁKOVÁ L, MIČUTKOVÁ L, BARTOŠOVÁ Z, SABBAN EL, KRIŽANOVÁ O, KVETŇANSKÝ R: Identification of phenylethanolamine N-methyltransferase gene expression in stellate ganglia and its modulation by stress. J NEUROCHEM 97:1419-30, 2006. (IF=4.604) 3. IMRICH R, LUKAC J, ROVENSKY J, RADIKOVA Z, PENESOVA A, KVETNANSKY R, HUCKOVA M, VIGAS M, MACHO L, KOSKA J: Lower adrenocortical and adrenomedullary responses to hypoglycemia in premenopausal women with systemic sclerosis. J RHEUMATOL 33(11): 2235-2241, 2006 (IF=3,010) 4. TILLINGER A, NOVAKOVA M, PAVLOVICOVA M, LACINOVA L, ZATOVICOVA M, PASTOREKOVA S, KRIZANOVA O, KVETNANSKY R: Modulation of gene expression of the PNMT in the heart during immobilization stress by 6–hydroxydopamine. STRESS 9: 207–213, 2006 (IF=2.967) 5. BAČOVÁ Z, BAQI L, BEŇAČKA O, PAYER J, KRIŽANOVÁ 0, ZEMAN M, SMREKOVÁ L, ZORAD Š, ŠTRBÁK V: Thyrotropin-releasing hormone in rat heart: effect of swelling, angiotensin II and renin gene. ACTA PHYSIOLOGICA 187:313-319, 2006 (IF=2,865) 6. DUNCKO R, MAKATSORI A, FICKOVA E, SELKO D, JEZOVA D: Altered coordination of the neuroendocrine response during psychosocial stress in subjects with high trait anxiety. PROG. NEUROPSYCHOPHARMACOL. BIOL PSYCHIATRY 30: 1 058-1066, 2006 (IF=2,769) 7. WILSON TG, WANG S, BEŇO M, FARKAŠ R: Wide mutational spectrum of a gene involved inhormone action and insecticide resistance in Drosophila melanogaster. MOL GENET GENOMICS 276 (3): 294-303, 2006 (IF=2,632) 8. KISS A, SØDERMANA, BUNDZIKOVA J, PIRNIK Z, MIKKELSEN JD: Zolpidem, a selective GABAA receptor α1 subunit agonist, induces Fos expression in oxytocinergic neurons of the hypothalamic paraventricular and accessory but not supraoptic nuclei in the rat. BRAIN RES BULL 71(1-3), 200-207, 2006. (IF=2.481) 9. ZORAD S, DOU J, BENICKY J, HUTANU D, TYBITANCLOVA K, ZHOU J, SAAVEDRA J.M.: Long-term angiotensin II AT1 receptor inhibition produces adipose tissue hypotrophy and increases markers of insulin sensitivity adiponectin and PPARγ. EUR J PHARMACOL 552: 112 = 122, 2006 (IF=2,477) 10. RADIKOVA Z, PENESOVA A, CIZMAROVA E, HUCKOVA M, KVETNANSKY R, VIGAS M, KOSKA J: Decreased pituitary response to insulin induced hypoglycemia in young lean male patients with essential hypertension. J HUM HYPERTENS 20(7): 510-516, 2006 (IF=2,405) ii. List of monographs/books published abroad PACAK K, AGUILERA G, SABBAN EL, KVETNANSKY R (Eds.): Stress – Current Neuroendocrine and Genetic Approaches. NY Acad Sci, New York, p. 1-590, 2004 List of chapters published in monographs abroad
  19. 19. 19 1. STRBAK V, BENICKY J, GREER SE, BACOVA Z, NAJVIRTOVA M, GREER MA: Cell swelling-induced peptide hormone secretion. In: Lauf PK, Adragna NC (eds.) Cell Volume and Signaling, Advances in Experimental Medicine and Biology, New York, Springer Science and Business Media, vol 559, 2004, p.325-330. 2. BAČOVÁ Z, BENICKÝ J, GREER MA, RITTAR M, GREER SE, ŠTRBÁK V: Cell volume induced hormone secretion: studies on signal transduction and specificity. In: Receptors Channels Messengers, Platon Kostyuk, Elena Lukyanetz (eds), DUS, Kiev 2005, p. 212-219. 3. BENICKÝ J, KRIŽANOVÁ O, ŠTRBÁK V: Effect of thyrotropin releasing hormone (TRH) on insulin secretion. In: P. Joseph-Bravo(ed) Molecular Endocrinology, Research Signpost, Trivandrum, Kerala, 2006, pp 109-125. 4. JEZOVA D, SCHWENDT M: Adrenal glutamate receptors: a role in stress and drug addiction? In: Glutamate Receptors in Peripheral Tissue: Excitatory Transmission Outside the CNS. Gill, S. and Pulido O. (eds.). Kluwer Academic/Plenum Publishers: New York, 2005, pp. 169-178. 5. ŠTRBÁK V: Cell Volume and Peptide Hormone Secretion. In: Lang F. (ed): Mechanisms and Significance of Cell Volume Regulation. Contrib. Nephrol. Basel, Karger, 2006, vol. 152, pp. 210-220. iii. List of monographs/books published in Slovakia 1. Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), AEP, Bratislava, 2004, 910 pages. iv. List of other scientific outputs specifically important for the Organisation 1. Všeobecná a klinická endokrinológia (Basic and clinical endocrinology).(A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), AEP, Bratislava, 2004, pp. 1-910. List of chapters published in monographs in Slovakia 1. JEŽOVÁ D, KRISTOVÁ V, KÁKOŠOVÁ V. Interakcie hormonálnych liečiv. (Interactions of hormonal medicaments) In: Interakcie liečiv v klinickej praxi, L. Magulová, L. Božeková, M. Kriška (eds.), Slovak Academic Press, Bratislava, 135-148, 2003. 2. Langer, P.: Ciele a spôsoby vedeckého bádania (Aims and methods of scientific research). In: Hulín, I.: Úvod do vedeckého bádania I., Bratislava, Slovak Academic Press, s. 151-172, ISBN80-89104-29-0, 2003. 3. HUDÁK J, VLČEK D, FARKAŠ R a kol.: Encyklopédia biológie, zv. 1, Genetika, molekulárna biológia a evolúcia. (Encyclopedy of biology, Vol. 1, Genetics, molecular biology and evolution). Slovenské pedagogické nakladateľstvo, Bratislava, 2005. 4. BRTKO J: Hormone action mediated via nuclear receptors /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 23-29, AEP, Bratislava, 2004. 5. BRTKO J, THALHAMER J: Gene expression as a response to the hormone action /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 101-106, AEP, Bratislava, 2004.
  20. 20. 20 6. GASPERIKOVA D, SEBOKOVA E, KLIMES I: Mitochondrial DNA and diabetes /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 620-623, AEP, Bratislava 2004. 7. HUCKOVA M: Laboratory methods for hormone analyses /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 62-70, AEP, Bratislava 2004. 8. JEZOVA D: Suprahypothalamic regulation of endocrine functions /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 107-116, AEP, Bratislava, 2004. 9. JEZOVA D: Pathophysiology of the sympatho-adrenal-medullary system /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 403-411, AEP, Bratislava, 2004. 10. KLIMES I, SEBOKOVA E, TKAC I: Metabolic syndrome /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 610-613, AEP, Bratislava 2004. 11. LANGER P: Endocrine disruptors /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 51-58, AEP, Bratislava 2004. 12. LANGER P: Pathophysiology of the thyroid gland /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 189-201, AEP, Bratislava 2004. 13. LANGER P: General ethiology and pathogenesis of thyreopathies /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 202-211, AEP, Bratislava 2004. 14. LANGER P: Laboratory diagnostics of the thyroid gland diseases /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 212-218, AEP, Bratislava 2004. 15. NEMEC J, ZAMRAZIL V, RACEK P, CHYTRY P, LANGER P: Tumors of the thyroid gland /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 275-281, AEP, Bratislava 2004. 16. MACHO L: Biosynthesis, secretion, transport and metabolism of hormones /in Slovak/. In.: Všeobecná a klinická endokrinologia (A. Kreze, P. Langer, I. Klimeš, L. Starka, J. Payer, J. Michálek eds.), s. 7-15, AEP, Bratislava, 2004. 17. MACHO L, LANGER P, KREZE A, STARKA S: Pathophysiology of the adrenal cortex. In: Všeobecná a klinická endokrinológia, (A.Kreze, P.Langer, I. Klimeš, L. Starka, J. Payer, J. Michálek eds.), s. 351-359, AEP, Bratislava, 2004. 18. MACHO L: History of endocrinology. The Institute of Experimental Endocrinology /in Slovak/. In Všeobecná a klinická endokrinologia (A. Kreze, P. Langer, I. Klimeš, L. Starka, J. Payer, J. Michálek eds.), s. XVIII-XIX, AEP, Bratislava, 2004. 19. SEBOKOVA E, KLIMES I: Endocrinology of the adipose tissue /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 515-521, AEP, Bratislava 2004. 20. SEBOKOVA E, GASPERIKOVA D, KLIMES I: Membrane receptor-mediated hormone actions /in Slovak/. In: Všeobecná a klinická endokrinológia (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 16-22, AEP, Bratislava 2004. 21. VIGAS M: Endocrine system and stress. In: Všeobecná a klinická endokrinológia. (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 30-35, AEP, Bratislava, 2004. 22. VIGAS M, KOSKA J: Pathophysiology of the hypothalamus-hypophysis system /in Slovak/. In: Všeobecná a klinická endokrinológia. (A. Kreze, P. Langer, I. Klimeš, L. Stárka, J. Payer, J. Michálek, eds.), s. 117-131, AEP, Bratislava, 2004.
  21. 21. 21 1. Table of research outputs Table Research outputs shows research outputs in number of specified entries; these entries are then divided by FTE employees with a university degree (from Tab. Research staff) for all Organisation at the respective year; finally these entries are divided by the total salary budget (from Tab. Salary budget). 2. Renormalized publications2 Renormalized publications = number of CC publications in the given year times authorship’s portion of the Organisation times the journal impact factor in 2005 divided by the median impact factor in the research field 3. Standard manuscript page count3 4. List of patents and patent applications 2003: Patents: None Patent applications: None 2004: Patents: None Patent applications: None 2005: Patents: None Patent applications: None 2006: Patents: None Patent applications: None 5. Supplementary information and/or comments on the scientific output of the Organisation 2 This information is required only from the Organisations of the Section 2 of the Slovak Academy of Sciences. 3 This information is required only from the Organisations of the Section 3 of the Slovak Academy of Sciences.
  22. 22. 22 Generally continuing positive trend in quantity and particularly in quality (illustrated by IF) of CC publications has been observed. Exceptionally high number of publications in 2004 was due to publishing papers from Symposium contributions. 2006 2005 2004 2003 2002 CC publications 39 37 46 37 30 Average IF 3,65 2,16 2,18 1,87 1,96 Top 10 IF 7,0 3,67 3,73 3,47 2,8 Several breakthrough papers were produced from our Institute two of them concern improvement of healthcare – diagnostic and therapy as a part of top international collaboration: Brouwers FM, Petricoin EF, Ksinantova L, Breza J, Rajapakse V, Ross S, Johann D, Mannelli M, Shulkin BL, Kvetnansky R, Eisenhofer G, Walther MM, Hitt BA, Conrads TP, Veenstra D, Mannion DP, Wall MR, Wolfe GM, Fusaro VA, Liotta LA, Pacak K: Low molecular weight proteomic information distinguishes metastatic from benign pheochromocytoma. ENDOCRINE-RELATED CANCER 12: 263-272, 2005, IF = 4,597 (2004). Paper specifies patients endangered by metastasic pheochromocytoma. This improvement of diagnostics was discussed in the editorial article of the NATURE CLINICAL PRACTICE ONCOLOGY 9 (2): 439-440, 2005 (Rebeca Orreland: Novel method for distinguishing metastatic and benign pheochromocytoma). Another paper: Pearson ER, Flechtner I, Njolstad PR, Malecki MT, Flanagan SE, Larkin B, Ashcroft FM, Klimes I, Codner E, Iotova V, Sligerland AS, Shield J, Robert JJ, Holst JJ, Clark PM, Ellard S, Sovik O, Polak M and Hattersley AT (for the Neonatal Diabetes International Collaborative Group including Gasperikova D and Stanik J): Switching from insulin to oral sulfonylureas in patients with diabetes due to Kir6.2 mutations. N ENGL J MED 355 (5): 467-77, 2006, IF = 44,016 (2005). Paper describes application of the top diagnostic approach resulting in diabetes therapy improvement in neonatal patients. Importance of both papers has been underlined also by editorials published in parallel in Nat Clin Pract Oncol and N Engl J Med, respectivelly. Dr. Kvetňanský was invited to write a review on Stress and catecholamines for PHYSIOLOGICAL REVIEWS (IF = 28,7). Paper was submitted and is currently under evaluation. 2. Responses to the scientific output Table Citations shows specified responses to the scientific outputs; these entries are then divided by the FTE employees with a university degree (from Tab. Research staff) for all Organisation at the respective year; finally these entries are divided by the total salary budget (from Tab. Salary budget). i. List of 10 top-cited publications and number of their citations in the assessment period 1. SEDLAK J, LINDSAY RH: Estimation of total protein-bound and nonprotein sulfhydryl groups in tissue with ellmans reagent. ANALYT BIOCHEM 25: 192-205, 1968
  23. 23. 23 Cited times: 351 2. SMITH MA, MAKINO S, KVETNANSKY R, POST RM: Stress and Glucocorticoids Affect the Expression of Brain-Derived Neurotrophic Factor and Neurotrophin-3 Messenger-RNAs in the Hippocampus. J NEUROSCI 15 (3): 1768-1777 Part 1, 1995 Cited times: 162 3. BARTANUSZ V, AUBRY JM, PAGLIUSI S, JEZOVA D, BAFFI J, KISS JZ: Stress-induced changes in messenger-RNA levels of n-methyl-d-aspartate and ampa receptor subunits in selected regions of the rat hippocampus and hypothalamus. NEUROSCI 66 (2): 247-252, 1995 Cited times: 41 4. SABBAN EL, KVETNANSKY R: Stress-triggered activation of gene expression in catecholaminergic systems: dynamics of transcriptional events. TRENDS IN NEUROSCI 24 (2): 91-98, 2001 Cited times: 36 5. BARTANUSZ V, JEZOVA D, BERTINI LT, TILDERS FJH, AUBRY JM, KISS JZ: Stress- Induced Increase In Vasopressin And Corticotropin-Releasing Factor Expression In Hypophysiotrophic Paraventricular Neurons. ENDOCRINOL 132 (2): 895-902, 1993 Cited times: 35 6. KVETNANSKY R, FUKUHARA K, PACAK K, CIZZA G, GOLDSTEIN DS, KOPIN IJ: Endogenous Glucocorticoids Restrain Catecholamine Synthesis And Release At Rest And During Immobilization Stress In Rats. ENDOCRINOLOGY 133 (3): 1411-1419, 1993 Cited times: 34 7. SMITH MA, MAKINO S, KIM SY, KVETNANSKY R: Stress Increases Brain-Derived Neurotropic Factor Messenger-Ribonucleic-Acid in the Hypothalamus and Pituitary. ENDOCRINOL 136 (9): 3743-3750, 1995 Cited times: 32 8. DUNCKO R, KISS A, SKULTETYOVA I, RUSNAK M, JEZOVA D: Corticotropin- releasing hormone mRNA levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mRNA levels decrease in both sexes. PSYCHONEUROENDOCRINOL 26 (1): 77-89, 2001 Cited times: 30 9. KISS A, JEZOVA D, AGUILERA G: Activity of the hypothalamic-pituitary-adrenal axis and sympathoadrenal system during food and water-deprivation in the rat. BRAIN RES 663 (1): 84-92, 1994 Cited times: 30 10. DE GOEIJ DCE, KVETNANSKY R, WITHNALL MH, JEZOVA D, BERKENBOSCH F, TILDERS FLH: Repeated stress-induced activation of corticotropin releasing factor neurons enhanced vasopressin stores and colocalization with corticotropin releasing factor in the median eminence of rats. NEUROENDOCRINOL 53: 150, 1991 Cited times: 27 ii. List of top-cited authors from the Organisation (at most 10 % of the research employees) and their number of citations in the assessment period Author of top cited paper (over 2 200) from the Institute Jozef Sedlák passed away several years ago. Here is the list of top cited authors working at the Institute. Number citations of papers with the Institute displayed on first page (only WOS + Scopus) without self citations are shown. 1. RNDr. R. Kvetňanský, DrSc. 786 2. Prof. PharmDr. D. Ježová, DrSc. 703

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