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Uloga neuropeptida Y (NPY) u modulaciji boli nije potpuno objašnjena. Cilj je bio istražiti utjecaj izravnog unosa NPY-a i antagonista njegovih Y1 i Y2 receptora u spinalni ganglij na bolno ponašanje štakora koristeći dva eksperimentalna životinjska modela boli; model neuropatske boli izazvan podvezivanjem spinalnog živca (SNL) i model upalne boli uzrokovan injiciranjem karagenana u šapu štakora. Odmah nakon injekcije karagenana ili nakon SNL-a, NPY ili njegovi antagonisti injicirani su izravno u spinalni ganglij. Testiranje ponašanja provedeno je dan prije injiciranja karagenana ili SNL-a, i četiri puta nakon injekcije tj. tri puta nakon SNL-a. Imunohistokemijska analiza pomoću biljega aktivacije satelitskih stanica u spinalnom gangliju i astrocita u kralježničnoj moždini (glijalni fibrilarni kiseli protein – GFAP) provedena je osam dana nakon operacije u upalnom modelu i 16 dana nakon operacije u SNL modelu. U modelu neuropatske boli, ustanovljeno je da injiciranje NPY-a pogoršava bolno ponašanje potaknuto SNL-om. Nakon injiciranja Y1 i Y2 antagonista u spinalni ganglij utvrđeno je da antagonist Y2 receptora poništava djelovanje NPY-a. Pogoršanje bolnog ponašanja nakon NPY injekcije bilo je popraćeno aktivacijom astrocita i satelitskim stanicama ipsilateralno od ozljede. Ova aktivacija smanjena je nakon primjene antagonista Y2 receptora. U modelu upalne boli NPY je pogoršao proalgezijski učinak koji je inducirao karagenan samo u testovima s podražajem hladnoćom. NPY u tom modelu nije dodatno pogoršao aktivaciju satelitskih stanica i astrocita uzrokovanu intraplantarnom injekcijom karagenana. Injiciranje antagonista Y1 i Y2 receptora izravno u spinalni ganglij poništilo je proalgezijski učinak injekcije karagenana i smanjilo gliozu u spinalnom gangliju i kralježničnoj moždini. Zaključno, ovi rezultati ukazuju na važnu vezu između bolnog ponašanja i neuroimunološkog djelovanja NPY Y1 i Y2 receptora.

Different types of interneurons in the striatum are still relatively poorly understood, especially compared to other parts of the brain such as the cortex or hippocampus. One of the lesser-known types of striatal interneurons that has received increasing attention recently, are neuropeptide Y-expressing interneurons (NPY+ interneurons). Available work indicates that these interneurons are controlled by striatal cholinergic interneurons and thus they may serve as mediators of cholinergic modulation in the striatum. The first aim of this thesis is to determine the absolute number of NPY+ interneurons in the mouse dorsal striatum by stereology, using a transgenic mouse model that shows GFP expression in NPY+ neurons. The second aim of this thesis is to use stereology to determine the volume of the dorsal striatum that we are able to target with viral expression when using a standard stereotactic injection protocol commonly used in our laboratory. The third aim of this study is to test the behavioural effect of knock down of the beta2 subunit of the nicotinic acetylcholine receptor expressed by NPY+ interneurons in the dorsal striatum. The work should serve as a first step in a study that will ultimately allow us to develop a quantitative idea of the absolute and relative number of NPY+ interneurons in...

Objectives: The main aim of this study was to compare serum concentrations of NPY between patients presenting with ST elevation myocardial infarction (STEMI) and patients presenting with non ST elevation myocardial infarction (NSTEMI). In addition, we sought to compare NPY concentrations with various anthropometric, clinical and laboratory data. Subjects and methods: In this cross-sectional study we enrolled a total of 60 patients with acute myocardial infarction (MI), 30 of which were diagnosed with STEMI, and 30 with NSTEMI. Patients were submitted to thorough clinical examination where anthropometric data was obtained. Subsequently, peripheral blood sample was drawn, and serum Neuropeptide Y (NPY) levels were determined. Results: NPY levels were significantly higher in patients with NSTEMI compared to patients with STEMI (0.590 (0.550 – 0.650) vs. 0.735 (0.630 – 0.820) ng/mL, P < 0.001). Furthermore, patients with DM had significantly higher NPY serum concentrations (0.640 (0.550 – 0.650) vs. 0.740 (0.720 – 0.754) ng/mL, P=0.046) than patients without DM. The Spearman’s correlation showed that there was no correlation between NPY serum concentrations and neither LVEF (r = 0.073, P=0.609) nor diastolic blood pressure (r = -0.089, P=0.499), respectively, however NPY serum concentrations positively correlated with GRACE score (r = 0.293; P=0.023). Finally, NPY serum concentrations positively correlated with urea (r = 0.318, P=0.013) and creatinine (r = 0.298, P=0.020) levels, and negatively with estimated glomerular filtration rate (r = -0.374, P=0.003). Conclusions: We concluded that NPY concentrations are significantly higher in patients with NSTEMI in comparison to patients with STEMI, and that NPY concentrations in patients with acute MI are significantly higher among patients with diabetes but with no difference between hypertensive and non-hypertensive patients. In addition, NPY concentrations correlate with the risk of mortality and renal function parameters in patients with MI., Cilj: Glavni cilj ovog istraživanja bio je usporediti serumske koncentracije neuropeptida Y (NPY) među bolesnicima s infarktom miokarda sa ST elevacijom (engl. ST elevation myocardial infarction, STEMI) i infarkta miokarda bez ST elevacije (engl. non ST elevation myocardial infarction, NSTEMI). Također, usporedili smo razine NPY s raznim kliničkim i laboratorijskim parametrima. Materijali i metode:. U ovu presječnu studiju uključili smo 30 bolesnika sa STEMI-jem i 30 s NSTEMI-jem. Bolesnici su podvrgnuti detaljnom kliničkom pregledu gdje su uzeti i antropometrijski podaci. Konačno iz uzoraka krvi smo analizirali razne laboratorijske parametre uključujući NPY. Rezultati: Serumske razine NPY-a su bile značajno veće u bolesnika s NSTEMI-jem u usporedbi s bolesnicima s STEMI-jem (0,590 (0,550 – 0,650) vs. 0,735 (0,630 – 0,820) ng/mL, P < 0,001). Nadalje, bolesnici koji boluju od šećerne bolesti imali značajno veće koncentracije NPY-a od ispitanika koji ne boluju od iste (0,640 (0,550 – 0,650) vs. 0,740 (0,720 – 0,754) ng/mL, P=0,046). Spearmanova korelacijska analiza pokazala je da serumske koncentracije NPY-a ne koreliraju s ejekcijskom frakcijom (r = 0,073, P=0,609) ni s vrijednostima dijastoličkog tlaka (r = -0,089, P=0,499), ali koreliraju s GRACE zbirom (r = 0,293; P=0,023). Konačno, serumske koncentracije NPY-a pozitivno su korelirale s urejom (r = 0,318, P=0,013) i kreatininom (r = 0,298, P=0,020), a negativno s procijenjenom glomerularnom filtracijom (r = -0,374, P=0,003). Zaključci: Utvrdili smo značajno veće koncentracije NPY u bolesnika s NSTEMI-jem u odnosu na bolesnike sa STEMI-jem. Uz to, demonstrirali smo da su koncentracije značajno veće u dijabetičara u odnosu na ne-dijabetičare, te da nema značajne razlike između hipertoničara i ispitanika koji nisu bolovali od hipertenzije. Konačno, pronašli smo značajnu korelaciju razina NPY-a s GRACE zbirom i markerima bubrežne funkcije.

Neuropeptid Y (NPY) je peptid koji se sastoji od 36 aminokiselina. U posljednjih nekoliko godina ustanovljeno je da je NPY važan ko-transmiter u perifernom živčanom sustavu. U ovom radu dan je pregled literaturnih podataka o kardiovaskularnim bolestima, lijekovima koji se koriste u njihovom liječenju i ulozi estrogena. Detaljnije su opisane ishemijska bolest srca i hipertenzija jer se smatra da će NPY antagonisti biti korisni u njihovom liječenju, budući da otpušteni NPY uzrokuje konstrikciju krvnih žila i smanjenje srčanog tonusa. U svrhu daljnjeg istraživanja potencijalnog učinka na kardiovaskularne bolesti sintetiziran je hidroklorid amida (R)-N 2 -(difenilacetil)-(R)-N-[1-(4- metoksifenil)etil]arginin (7), a njegova struktura je dokazana spektroskopskim metodama (1H i 13C NMR). U daljnjim istraživanjima, koja prelaze okvire ovoga diplomskog rada, bit će ispitano njegovo djelovanje na kardiovaskularni sustav Neuropeptide Y (NPY) is a peptide consisting of 36 aminoacids. Recently, it has been established that NPY is an important co-transmitter in peripheral nervous system. This thesis gives an overview of the current literature regarding cardiovascular diseases, drugs used in cardiovascular medicine and the role of estrogen. Myocardial ishemic heart disease and hypertension are described in more detail since NPY antagonists might be useful in the treatment of these cardiovascular diseases because NPY causes constriction of blood vessels in both the heart and it reduces the cardiac vagal tone. In this work we have prepared (R)-N 2 -(diphenylacetyl)-(R)-N-[1-(4- hydroxyphenyl)ethyl]arginine amide hydrochloride (7), NPY antagonist, which was characterized by standard spectroscopic methods (1H and 13C NMR). Future investigations will focus on the evaluation of its role in cardiovascular diseases.

Die Huntington-Krankheit (HD) ist eine autosomal dominante vererbbare neurodegenerative Polyglutamin-vermittelte Krankheit für die zurzeit keine effektiven Therapiemöglichkeiten vorliegen. Diese Dissertation untersucht die vorteilhafte Wirkung der Aktivierung des Neuropeptid Y (NPY)-Y2-Rezeptors in dem R6/2 Maus- und PC12 Zellmodell der HD. Die erzielten Ergebnisse zeigten, dass die Aktivierung des Y2 Rezeptors mit intranasalem NPY die motorischen Defizite in R6/2 Mäusen verminderte. Weiterhin führte intranasales NPY oder NPY\(_{13-36}\) zu einer verringerten mHtt Aggregation und Neuroinflammation, zeigte positive Auswirkungen auf das Überleben und vermittelte Neuroprotektion in R6/2 Mäusen. \(\textit {In vitro}\) Versuche zeigten, dass die Behandlung mit NPY oder NPY\(_{13-36}\) vor mHtt-vermittelten neuronalen morphologischen Defekten und Toxizität schützte. Zusammengefasst legen diese Ergebnisse nahe, dass die gezielte Aktivierung des Y2-Rezeptors eine potentielle neuroprotektive Therapie für HD sein könnte.

ALKANAT, MEHMET/0000-0001-8079-3749 WOS: 000425181900010 PubMed: 29405731 In this study, we aimed to explain the role of apelin-13 on body weight, food and water intake with serum leptin, ghrelin, neuropeptid Y (NPY) and peptid YY (PYY) levels in male rat. Thirty-two Sprague-Dawley male rats were used for the study. the rats were injected SP (0.9 %) intraperitoneally (i.p) in the control group and 30 (AP30), 100 (AP100) and 300 (AP300) mu g/kg apelin-13 in the study groups, respectively, 10 min before the transition to dark period, for 10 days. During the experimental period, with light and dark periods of food and water intake, body weights were recorded in rats. Rats were euthanized and serum samples were obtained. in serum samples leptin, ghrelin, NPY and PYY levels were measured with specific ELISA kit. Apelin-13 was increased body weights in all three (AP30, AP100 and AP300) groups compared with the control group. AP100 and AP300 groups had increased food intake in the dark and the cumulative period, but in the light period food intake values were not significantly increased (p > 0.05). As for the value of water intake, compared with the control group, all dose of apelin-13 increased water intake during the dark and the cumulative period. There was no significant change in water intake in the light period. on the other hand, compared with the control group, serum leptin levels were found to increase in the groups administered 100 and 300 mu g/kg of apelin-13 (p < 0.05). Ghrelin levels were found high in all groups treated with apelin-13. Serum levels of NPY decreased only in the 300 mu g/kg apelin-13 treated group (p < 0.05). There was no statistically significant change in levels of PYY (p > 0.05). Apelin-13 increases body weight in rats as well as food and water intake (dark and cumulative period). Additionally, ghrelin can mediate the orexigenic effect of apelin-13 in the regulation of food intake (Fig. 4, Ref. 37). Karadeniz Technical University (KTU) scientific research Project Unit [1024] This project was supported by Karadeniz Technical University (KTU) scientific research Project Unit (Praje No 1024).

Eine depressive Komorbidität bei Patienten mit einer koronaren Herzerkrankung (KHK) bedingt eine Verschlechterung des klinischen Verlaufs mit einem erhöhten Mortalitäts- und Morbiditätsrisiko. Die pathophysiologischen Ursachen für diesen schlechteren klinischen Verlauf sind nicht hinreichend geklärt. Eine mögliche Erklärung bietet das Konzept der Homöostase und Allostase. Dieses Konzept beschreibt den Prozess und die Zusammenhänge, die den Körper in die Lage versetzen, angemessen auf Stress zu reagieren und zu antworten. Eine Fehlanpassung an diesen Regelkreis kann einen Risikofaktor für gesundheitliche Schäden des Körpers darstellen. Die depressive Erkrankung wird schon seit Längerem mit einer Veränderung der Stressreaktion in Verbindung gebracht. Insbesondere die Rolle des Neuropeptid Y (NPY) wird im Zusammenhang mit chronischen und intensiven Stressreaktionen diskutiert. Ziel der vorliegenden Arbeit ist es daher, eine Veränderung der Stressreaktion bei Patienten mit einer KHK und einer depressiven Komorbidität, insbesondere durch Analyse der peripheren sympathiko–adrenergen-medullären–Stressachse (SAM-Achse) und des NPY– Dipeptidylpeptidase 4 (DPP4)–Systems zu detektieren. Zusätzlich soll der Zusammenhang zwischen der kurzwirksamen SAM-Achse und dem neuromodulatorisch wirksamen NPY– DPP4–System herausgearbeitet werden. Mittels eines standardisierten Verfahrens zur experimentellen Induktion von sozialem Stress, Trierer Sozialer Stresstest (TSST), wurden 43 Patienten untersucht. Der TSST beinhaltet eine freie Rede und eine Rechenaufgabe vor einer Jury, die aus bis zu drei Personen besteht. Die Einteilung in die beiden Gruppen, Patienten mit KHK ohne Depression (KHK-Gruppe: 24 Patienten), und Patienten mit KHK und einer depressiven Komorbidität (KHK+D-Gruppe: 19 Patienten), erfolgte anhand der deutschen Version des Hospital Anxiety and Depression Scale (HADS). Die Hormone der SMA–Achse, Noradrenalin, Adrenalin und Dopamin, NPY und DDP4 wurden im venösen Plasma vor dem TSST, direkt nach dem TSST, 5 min, 15 min und 30 min nach dem TSST erhoben. Es ließ sich keine Veränderung der Stressreaktion bei der KHK+D-Gruppe im Vergleich zu der KHK-Gruppe nachweisen. Die Korrelation zwischen den Depressionsummenwerten des HADS und NPY zeigte keine Signifikanz. Entsprechend zeigten sich auch keine nachweisbaren Gruppenunterschiede bezogen auf die Höhe und den Verlauf im NPY–DPP4–System. Eine negative Korrelation trat zwischen NPY und dem State-Fragebogen des State-Trait-Angstinventar (STAI-S) vor dem TSST (r = -0.34, p = .02). Weiterhin lag eine negative Korrelation zwischen NPY und den Angstsummenwerten des HADS vor dem TSST (r = -0.31, p = .04) und 5 min nach dem TSST (r = -0.36, p = .02). Hinsichtlich des Zusammenhangs zwischen der SMA–Achse und dem NPY ergab sich eine positive Korrelation zwischen Noradrenalin und NPY für den Messzeitpunkt 15 min nach TSST (rpb = .37, p = .01). Zusätzlich zeigte sich eine positive Korrelation zwischen den Dopamin und NPY vor dem TSST (rpb = .29, p = .04). Zu dem Messzeitpunkt vor dem TSST (rpb = .38, p = .01) und 15 min nach dem TSST (rpb = .35, p = .02) bestand ebenfalls zwischen den mittleren Konzentrationen der Adrenalin und NPY eine positive Korrelation. Der Body Mass Index (BMI) stellte eine wichtige Moderatorvariable dar. Es zeigte sich eine negative Korrelation zu NPY und dem Messzeitpunkt 5 min nach dem TSST (rpb = -.35, p = .04). Eine veränderte Stressreaktion bei einer KHK und depressiver Komorbidität ließ sich in dem peripheren NPY–DDP4–System nicht bestätigen. Es zeigte sich jedoch ein bedeutsamer Zusammenhang mit ängstlicher Symptomatik. Daraus lässt sich folgern, dass eine ängstliche Symptomatik mit Veränderungen des NPY-DPP4-Systems sowohl in Ruhe als auch unter Stress assoziiert ist und Ängstlichkeit daher eine wichtige Moderatorvariable bei Patienten mit KHK unter Stress darstellen kann. Es zeigt sich erwartungsgemäß, dass unter sozialem Stress Noradrenalin gemeinsam mit NPY ausgeschüttet wird. Zusätzlich verdeutlicht die Korrelation zwischen Adrenalin und NPY unter der Ruhebedingung, dass die Adrenalin und NPY-Ausschüttung auch in Ruhe gemeinsam erfolgt. Diese Befunde verdeutlichten eine Assoziation zwischen der SAM–Achse und NPY sowohl in Ruhe als auch unter Stress bei KHK-Patienten. Der BMI wurde als eine wichtige Moderatorvariable für das NPY–DPP4–System bestätigt. Limitiert sind die Ergebnisse durch die Heterogenität der depressiven Verstimmung der Patienten, durch das Studiendesign in Form einer Querschnittsstudie und die unterschiedlichen Tageszeitpunkte der Messungen.

Over the past three decades, it has been confirmed that the alarming increase in number of obese patients is related to the sleep duration and its quality. Neurohumoral response to short sleep duration and poor sleep quality leads to decreased levels of melatonin, leptin and orexin, asto increased levels of cortisol, ghrelin and neuropeptide Y. Such an inaccurate regulation contributes both to excessive intake of energy-dense diet and to the reduction of energy expenditure during physical activity. The bachelor thesis summarizes recent information about the relationship of sleep and obesity, focusing on hormones and peptides involved in the regulation of energy balance processes, including the posssibility to use their mechanisms for weight control.

Die Aufmerksamkeitsdefizit-/Hyperaktivitätsstörung ist eine der häufigsten psychiatrischen Erkrankung des Kindesalters, die eine hohe Heritabilität aufweist und häufig bis ins Erwachsenenalter persistiert und lebenslang zu sozialen, gesundheitlichen und ökonomischen Problemen führt. Die ADHS tritt bei vielen Patienten in Kombina-tion mit anderen psychiatrischen und nicht-psychiatrischen Erkrankungen auf. In den letzten Jahren rückte zunehmend die häufig zur ADHS komorbid auftretende Adipositas in den Fokus der Forschung. Auf der Suche nach copy number variations in Zusammenhang mit ADHS, wurde eine Duplikation auf Chromosom 7p15 – dem Genlocus des NPY – entdeckt. NPY, ist ein endogenes orexigenes Peptid, welches physiologischerweise die Nahrungsaufnahme stimuliert und neben zahlreichen Effekten, wie Blutdruck- und Knochenregulation, auch in Zusammenhang mit neuropsychiatrischen Erkrankungen gebracht werden konnte. Diese Duplikation auf einem Genort, dessen Produkt für die Regulation von Energiehaushalt und Körpergewicht zuständig ist, bildete die Grundlage, eine Assoziationsuntersuchung zu NPY-Genvarianten und dem Körpergewicht bei Kindern durchzuführen. Untersucht wurden bei 269 an ADHS erkrankten Kindern und 142 gesunden Kontrollkindern die Assoziation zwischen NPY-Genvarianten (rs16147, rs16139, rs5574, rs16124) und ADHS, sowie die Assoziation zwischen NPY-Genvarianten und BMI-Perzentilen bei ADHS. Es ergab sich keine signifikante Assoziation bezüglich der aufgestellten Hypothesen., The attention-deficit/hyperactivity disorder (ADHD) is one of the most frequent psychiatric disorders in children. It is highly heritable, often persists until adulthood and causes social, economic and health problems. While psychiatric comorbidities of ADHD have been extensively explored, in the last decade mounting evidence pointed to a possible association between ADHD and obesity. In search of copy number variations related to ADHD and this new comorbidity, a duplication on chromosome 7p15 – the gene of Neuropeptide Y (NPY) - has been identified. NPY is an orexigenic peptide, which stimulates food intake and it has also been implicated in other psychiatric diseases. Therefor we performed an association study in a case contol study design including 269 children with ADHD and 142 healthy controls. The objectiv of our investigation was to study the association between four single nucleotid polymorphisms of the NPY- gene (rs16147, rs16139, rs5574, rs16124) and ADHD, and the association between the NPY-polymorphisms and the bmi-percentiles in children with ADHD. Results: No association between the polymorphisms and ADHD in children was found. No association between bmi-percentiles and the polymorphisms was found in children with ADHD.

Uvod: Dijabetes uzrokuje promjene funkcije trigeminalnog živca, što se očituje promijenjenim provođenjem osjetnih informacija iz područja lica i mijenjanjem odgovora na bolni podražaj. Kalcij/kalmodulin-ovisna kinaza II alfa (CaMKIIα) može imati važnu ulogu u modulaciji nocicepcijske aktivnosti i plastičnosti osjetnih trigeminalnih neurona. Dijabetesom podržavan upalni odgovor preko medijatora neuropeptida Y (NPY) i vaskularnog endotelnog čimbenika rasta (VEGF) upućuje na moguću ulogu istih u patogenezi dijabetičke neuropatije. Cilj istraživanja bio je istražiti izražaj VEGF i NPY, te promjenu intenziteta izražaja aktivnog oblika enzima CaMKIIα na štakorskom modelu trigeminalnog ganglija (TG) ranog dijabetesa tipa 1 (DM1) i tipa 2 (DM2). Dodatni cilj je bio istražiti distribuciju prema veličini neurona i postojanje razlike u ispitivanim parametrima u neurokemijski specifičnim subpopulacijama osjetnih neurona TG, 2 tjedna i 2 mjeseca nakon indukcije DM1 i DM2. Metode: U pokusu su korišteni Sprague-Dawley štakori muškog spola (mase oko 200 g). Model DM1 izazvan je intraperitonealnim (i.p.) ubrizgavanjem streptozotocina (STZ) u dozi od 55 mg/kg. Model DM2 dobiven je i.p. ubrizgavanjem manje doze STZ (35 mg/kg) i hranjenjem štakora hranom s visokim udjelom masti. Štakori su žrtvovani 2 tjedna i 2 mjeseca nakon induciranja dijabetesa, a zatim je imunohistokemijski analiziran intenzitet izražaja pCaMKIIα, izražaj VEGF i NPY, samostalno, ili u kolokalizaciji s izolektinom B4 (IB4). Rezultati: Oba modela dijabetesa uspješno su inducirana, što je potvrđeno mjerenjem glukoze u plazmi i tjelesne mase. U TG štakora oba DM modela utvrđene su morfološke promjene, koje su se očitovale promjenom dijametra neurona te aktivacijom satelitskih stanica i propadanjem Schwanovih stanica. Dva mjeseca nakon ubrizgavanja STZ, intenzitet izražaja pCaMKIIα se značajno povećao u TG DM1 štakora, isključivo u malim IB4-negativnim neuronima. DM1 model, kao i pretilost (kontrolna DM2 skupina) su rezultirali atrofijom, dok je DM2 model uzrokovao hipertrofiju neurona TG. Porast izražaja NPY u oba modela, u DM1 već nakon 2 tjedna, a DM2 tek nakon 2 mjeseca, zahvatio je prvenstveno nociceptivne male neurone - IB4- i IB4+ subpopulacija. Dva tjedna nakon indukcije, DM1 je uzrokovao porast izražaja VEGF isključivo u populaciji velikih IB4- neurona, dok je DM2 istovremeno rezultirao porastom izražaja VEGF u populaciji IB4+ neurona. Dva mjeseca nakon indukcije obaju modela, izražaj VEGF u neuronima TG štakora se smanjio, posebice u populaciji malih peptidergičkih neurona štakora DM1 modela. Zaključci: Dobiveni rezultati sugeriraju na promjene intenziteta izražaja pCaMKIIα i izražaja VEGF i NPY, upravo u nocicepcijskim neuronima, što ukazuje na njihovu moguću ulogu u patogenezi dijabetičke trigeminalne neuropatije. Promatrane promjene mogu imati kritičnu ulogu u modulaciji nocicepcijske aktivnosti i plastičnosti primarnih osjetnih neurona TG. Dobivene spoznaje doprinose poznavanju patofiziologije trigeminalne dijabetičke neuropatije. Background: DM influences the trigeminal nerve function by changing the pain response and transduction of the orofacial sensory pathways. The activity of calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. Diabetes affects inflammatory response via neuropeptide Y (NPY) and vascular endothelial growth factor (VEGF), which potencialy have relevant role in pathophysiology of diabetic neuropathy. The aim of this study was to investigate the expression of pCaMKIIα, VEGF and NPY in subpopulations of trigeminal ganglion (TG) neurons in rat models of early diabetes type 1 (DM1) and 2 (DM2). Additional aim was to analyze change in the diameter of TG neurons 2 weeks and 2 months after the induction of both DM models. Methods: DM1 model was induced by intraperitoneall (i.p.) injection of streptozotocin (STZ) (55 mg/kg). DM2 rats were fed with the high fat diet (HFD) for two weeks and then received 35 mg/kg of STZ i.p. Two weeks and 2 months after the STZ-diabetes induction, rats were sacrificed and immunohistochemical analysis for detection of pCaMKIIα, VEGF and NPY expression and in addition to double immunoflorescent labelling with isolectin (IB4) was performed in TG. Results: Increased expression of pCaMKIIα, restricted to IB4-negative small-diameter neurons, was seen in TG two months after STZ-DM1 induction. DM1 model, as well as the obesity (control DM2 groups) resulted in the decrease of neuronal diameter, while DM2 model led to neuron hypertrophy in TG. Increased percentage of NPY was observed 2 weeks after DM1 and 2 months post DM2 induction. It was restricted to IB4-negative small-diameter and IB4+ neurons. Two weeks after the induction of DM a percentage increase of VEGF/IB4- large-diameter neurons was observed in DM1 animals while in DM2 animals there was a percentage increase in VEGF/IB4+ neurons. Reduced ratio of VEGF/IB4- small-diameter neurons was observed after two months of DM1 induction. Conclusion: Observed changes may play a critical role in the modulation of nociceptor activity and plasticity of primary sensory trigeminal neurons. The results contribute to understanding of the basic pathophysiology of trigeminal diabetic neuropathy. Modulation of pCaMKIIα, VEGF and NPY expression in specific subpopulations of neurons could be used in therapeutic approaches for diabetic trigeminal neuropathy.

The aim of this study was to investigate the changes in the levels of cystatin C, which protects neurodegeneration in the central nervous system with the inhibition of cysteine protease and by inducing autophagy in the pathogenesis of cerebral vasospasm and levels of vasoconstrictive neuropeptid Y (NPY) in the brain tissue homogenates of rat model of subarachnoid hemorrhage (SAH). Three experimental groups were used: Day 2 and Day 7 groups after SAH, and also a control group. There were seven Wistar albino rats in each group. SAH was accomplished by transclival basilar artery puncture. Rat cystatin C, rat NPY were determined with ELISA in brain tissue homogenates. Day 2 group showed significantly enhanced cystatin C values in comparision with the control group (P=0.048). NPY levels between the Day 2 and Day 7 groups and the control groups were not significantly different (P=0.315). In histopathological examination, there was less neuronal loss in the Day 2 group than in the Day 7 group. Regarding our results, it would be more valuable to measure NPY levels in specific brain areas. The increased cystatin C levels on the second day after SAH is probably a pathophysiologic mechanism to organize protease activity.

Morbus Huntington (MH) ist eine autosomal dominant vererbte, progressive neurodegenerative Erkrankung, deren verantwortliche Mutation eine Verlängerung einer variablen \((CAG)_{n}\)-Sequenz im \(\it {HTT}\)-Gen darstellt. Zwischen der CAG-Blocklänge und dem Auftreten der ersten Symptome bei MH gibt es statistisch eine inverse Korrelation. Dabei erklärt der expandierte CAG-Block jedoch nur 30 bis 70% der Variabilität des Erkrankungsalters von MH-Patienten, wobei noch weitere genetische Faktoren das Erkrankungsalter mitbestimmen. Ein wesentlicher Teil dieser Arbeit bestand deshalb in der Suche von \(\it {modifier}\)-Variationen in Genen, die an NPY-Funktionen beteiligt sind. Zusammengefasst führen die Ergebnisse dieser Arbeit zu der Annahme, dass NPY durch den Y2-Rezeptor neuroprotektiv wirken könnte, um den Verlauf von MH zu verlangsamen. Darüber hinaus könnten die NPY-Analoga als potentielle therapeutische Mittel bei MH von Interesse sein.

1) Hintergrund und Ziele der Studie Sowohl in tierexperimentellen als auch in humanen Studien ist gezeigt worden, dass Adipositas zu einem erhöhten Risiko für eine chronische Niereninsuffizienz führt [20, 28, 47]. Es finden histologische Veränderungen im Sinne einer Obesity Related Glomerulopathy (ORG) [106] und eine niederschwellige systemische Inflammation statt [106, 125]. Dennoch ist es noch weitgehend ungeklärt, ob postnatale Hyperalimentation zu einem Funktionsverlust der Niere führt. Daher ist es das Ziel dieser Arbeit letzteres und eine sich eventuell entwickelnde profibrotische Neigung mit Inflammation sowie eine Veränderung von Signalwegen des Systems zwischen Leptin und dem sympathischen Kotransmitter Neuropeptid Y (NPY), zu untersuchen. 2) Methoden Die postnatale Hyperalimentation wurde durch eine Reduktion der primären Wurfgröße am postnatalen Lebenstag (P) 1 erreicht. Nach 24-stündiger Haltung in metabolischen Käfigen wurden die Tiere am Tag P70 getötet. Aus entnommenem Nierengewebe wurden Polymerase-Kettenreaktionen durchgeführt, um die Genregulation von NPY, seinen Rezeptoren, verschiedenen Adipozytokinen wie Leptin und Interleukin 6 zu untersuchen. Weiter wurden Western Blots von Interleukin-6, Signal transducer and activator of transcription 3 (STAT-3) und Suppressor of cytokine signaling 3 (SOCS-3) durchgeführt. Auch wurde eine Immunhistologie von Kollagen I angefertigt. 3) Ergebnisse und Beobachtungen Veränderungen betrafen nicht nur den Phänotyp der hyperalimentierten Tiere mit erhöhtem Körpergewicht, sondern sie manifestierten darüber hinaus eine eingeschränkte glomeruläre Nierenfunktion mit Veränderung der extrazellulären Matrix durch verstärkte Kollagen I Bildung, erhöhter Expression lokaler proinflammatorischer Zytokine wie IL-6 und des ebenso proinflammatorischen Peptidhormons Leptin. Im Gegensatz dazu war antiinflammatorisch wirkendes IL-10 vermindert. In hyperalimentierten Würfen kam es weiter zu einer verstärkten NPY-Expression mit einer Modifikation der Leptinsignalkaskade über STAT-3, da sowohl der Anteil an phosphoryliertem, also aktiven STAT-3 geringer und der Anteil des unphosphorylierten STAT-3 erhöht waren. Zusätzlich war mehr SOCS-3 messbar. 4) Praktische Schlußfolgerungen Zusammenfassend zeigt die vorgelegte Studie, dass postnatale Hyperalimentation vor allem bei einer Wurfreduktion auf sechs Tiere Veränderungen im renalen Leptin-NPY-System hervorruft, die am postnatalen Lebenstag P70 persistieren. Damit hat eine Programmierung des renalen Leptin-NPY-Systems stattgefunden. Wahrscheinlich kommt es im Rahmen der lokalen Leptinvermehrung zu einer Sympathikusaktivierung mit NPY-Freisetzung, das wiederum als Stimulus für eine IL-6-Sekretion aus zirkulierenden Makrophagen in Frage kommt. Die mögliche Sympathikusaktivierung könnte zur Modifikation der extrazellulären Matrix mit erhöhter Kollagen I Bildung in den Glomeruli beitragen, da Leptin profibrotisch wirkt. Die renale STAT-3/JAK-2 Signaltransduktion ist beeinträchtigt, erkennbar an einer verminderten Phosphorylierung von STAT-3 und erhöhter Messbarkeit von SOCS-3, einem Inhibitor des STAT-3/JAK-2 Weges. Dies deutet auf eine lokale Postrezeptor-Leptinresistenz hin. Alles in allem entsteht so das Bild einer ORG, die durch frühe postnatale Hyperalimentation getriggert wird. Adipositas ist eine Epidemie, die ein erhöhtes Risiko für chronische Niereninsuffizienz birgt. Daher sind diese Ergebnisse von großer Relevanz. 1) Background It has been shown that obesity leads to an increased risk for chronic kidney disease in animals as well as in humans [20, 28, 47]. Histological changes that are characteristic of an obesity related glomerulopathy (ORG) [106] and a low grade systemic inflammation occur [106, 125]. Still it is unknown wether postnatal hyperalimentation leads to a reduction of kidney function. The aim of this study was to determine if postnatal hyperalimentation by litter size reduction has an influence on kidney function, local inflammation and renal fibrosis. Moreover changes in the signal transduction in the interaction of leptin and neuropeptid Y (NPY) should be analysed. 2) Methods Postnatal hyperalimentation was induced by litter size reduction (LSR) on postnatal day (P) 1. After keeping the rats in a metabolic cage for 24 hours they were sacrificed on day P70. Kidney material was used to perform polymerase chain reactions to evaluate gene regulation of NPY and its‘ receptors, different adipocytokines like leptin and interleukin-6 (IL-6). Furthermore, western blots of IL-6, signal transducer and activator of transcription 3 (STAT-3) and Suppressor of cytokine signaling 3 (SOCS-3) and an immunhistologic staining of collagen I were done. 3) Results Rats that were affected from LSR were heavier than animals from the control group. They also showed reduced glomerular kidney function and changes of the extracellular matrix with increased production of collagen I, increased production of proinflammatory cytokines like IL-6 and leptin, whereas anti-inflammatory IL-10 was reduced. In LSR rats NPY-expression was increased together with a modification of leptin signalling via STAT-3: phosphorylated, active STAT-3 was decreased, while unphosphorylated STAT-2 was elevated. Also SOCS-3 levels were higher in LSR animals. 4) Conclusions All in all, this study shows that postnatal hyperalimentation leads to changes in the leptin-NPY-system, especially in litters that were reduced to six animals. These changes persist until P70 which could be due to metabolic programming of the renal leptin-NPY-system. Probably local increase of leptin expression causes activation of the sympathetic nervous system and NPY secretion. NPY may stimulate IL-6 secretion in circulating macrophages. Consistent with leptin’s profibrotic function the activation of the sympathetic nervous system could then change glomerular extracellular matrix composition by increasing collagen I production. The renal STAT-3/JAK-2 pathway is impaired as phosphorylated STAT-3 levels are decreased and SOCS-3 levels are increased. SOCS-3 is a potent inhibitor of STAT-3/JAK-2 signalling. This indicates a local postceptor leptin resistance. Altogether early postnatal hyperalimentation creates an ORG. Obesity is a worldwide epidemic that involves a high risk of chronic renal failure. Because of that the results from this study are highly relevant and further work on this topic is necessary.

Dipeptidyl peptidase 4 (DP 4, EC 3.4.14.5) is a serine protease that hydrolyses dipeptides from the N-termini of peptides with either proline, hydroxyproline or to a lesser extent alanine at the penultimate position. The type II integral membrane glycoprotein is a homodimer and involved peptide metabolism, cell adhesion and T-cell activation. As it inactivates the insulinotropic incretines GLP-1 and GIP, DP 4-inhibitors are used as novel anti-diabetic drugs to enhance insulin secretion from β-cells of the islets of Langerhans. Yet, DP 4 inhibitors have to be selective regarding additional DP 4-like enzymes like dipeptidyl peptidase 8 (DP 8), dipeptidyl peptidase 9 (DP 9) and fibroblast activation protein alpha (FAP) of the DP 4-gene family S9b, as well as dipeptidyl peptidase 2 (DP 2) of the S28 family. These enzymes exhibit DP 4 activity, though they have different sub-cellular localisations and little is known about their functions. While the role of DP 4 in glucose homeostasis has been proven possible paracrine or neuronal roles of DP 4-like enzymes in regulating insulin secretion from islets of Langerhans still had to be examined. Thus, the first part of the study dealt with characterisation of DP 4-like enzymes for the treatment of type 2 diabetes and included isolation of DP 4 isoforms to improve crystallisation conditions of native glycosylated DP 4 as well as the analysis of DP 4-like enzymes in various tissues, particularly in pancreatic islets of Langerhans. The second part of the thesis involved the identification and characterisation of DP 4 as an additional pharmaceutical target for anxiety, depression or schizophrenia, based on altered stress-related behaviour in DP 4-deficient rats, DP 4-/- k.o. mice and upon inhibition of DP 4 in vivo. The study included analysis of DP 4-like enzymes in rat neuronal tissues and human body fluids, identification of target substrates and target organs, inhibitor profile as well as target disease. As to improve crystallisation conditions, a new method was developed to solve the glycosylation-based microheterogeneity of 17 isoforms of purified porcine DP 4. This was achieved by elaborating on the refractionation method of preparative liquid-phase IEF to obtain 2-3 isoforms per fraction that were pure enough to acquire a 1.8 Å crystal structure with a unique tetrameric assembly of two homodimers. Characterisation of porcine islets of Langerhans and pancreas, using activity studies, enzymatic histochemistry, immuno-histochemistry and RT-PCR analysis, revealed that the majority of DP 4-like activity originates from the exocrine tissue. Immunohistochemical staining with anti-DP 4 pAb demonstrated the expression of DP 4 on capillaries surrounding the islets. However, sub-cellular fractionation and histochemical analysis also implied the presence of DP 4-like activity at different sub-cellular localisations. The presence of DP 2 could also be demonstrated in isolated granules and by enzymatic histochemical analysis. As DP 8 and DP 9 are cytosolic and DP 2 is not able to cleave any of the known DP 4 substrates, the results suggest that DP 4 also influences paracrine and neuronal regulation of insulin secretion from the -cells. Besides, a potential drug interaction between anti-diabetic DP 4 inhibitors and anti-hypertensive ACE inhibitors was identified when analysing DP 4-hydrolysis of neuropeptide Y (NPY) in serum due to additional C-terminal truncation by angiotensin converting enzyme (ACE). Thus, a suspected increase of vasocontrictive NPY1-36 by DP4 inhibitor may be compensated by C-terminal inactivation by ACE, but fails if ACE is also blocked. In addition, DP 4 was identified as a potential target for stress-related diseases such as anxiety, depression and schizophrenia. Although DP 2, DP 8 and DP 9 mostly contributed to the overall DP 4-like activity in neuronal tissues, high levels of DP 4 could be detected in the circumventricular organs (CVO). As the CVO’s lack the blood brain barrier, this result implies an important role of DP 4 at the neuroendocrino-immunological interphase, involving both stress axis: hypothalamic-pituitary-adrenal axis and the neuro-sympathico axis. Furthermore, assessing the metabolism of several stress-related neuropeptides in rat neuronal tissues and human body fluids, revealed that NPY is the target substrate of DP 4, though the two novel DP 4 substrates Galanin-like-peptide and rat Orexin B could also be identified. While no N-terminal truncation of NPY by DP 4 could be detected in human cerebrospinal fluids, DP 4-derived NPY3-36 was the major metabolite in human sera and ex vivo unfractionated blood, followed by ACE-like C-terminal truncation. Moreover, circulating NPY was found to be equally metabolised by endothelial DP 4 as well as soluble DP 4. Finally, metabolomic studies of NPY identified seven novel NPY-cleaving enzymes, including the lysosomal cathepsins D, B, L, S and G, tissue kallikrein and ACE. Dipeptidylpeptidase 4 (DP 4, EC: 3.4.14.5) ist eine Serinprotease, die N-terminal Dipeptide von Peptiden mit Prolin, Hydroxyprolin oder in geringerem Maße Alanin an vorletzter Stelle spaltet. Das integrale Typ II Membranglycoprotein ist ein Homodimer und in Peptidstoffwechsel, Zelladhäsion und T-Zellaktivierung involviert. Da es das insulinotropische Inkretin GLP-1 inaktiviert, werden DP 4-Hemmer als neue Antidiabetika zur erhöhten Insulinausschüttung eingesetzt. Die Hemmer müssen jedoch selektiv gegen die DP 4-ähnlichen Enzyme Dipeptidylpeptidase 8 (DP 8), Dipeptidylpeptidase 9 (DP 9) und das Fibroblast Aktivierungsprotein alpha (FAP) aus der DP 4-Gen-spezifischen Familie S9b sowie Dipeptidylpeptidase 2 (DP 2) aus der S28-Familie sein. Diese Enzyme haben ähnliche Aktivitäten, sind aber subzellulär unterschiedlich verteilt. Während die Rolle der DP 4 in der Glukose-Homöostase schon erwiesen ist, galt es eine mögliche parakrine und neuronale Rolle der DP 4-ähnlichen Enzyme in der Regulierung von Insulinausschüttung nachzuweisen. Dabei wurden folgende Aspekte bezüglich Typ-2-Diabetes untersucht: Isolierung der glykosilierten Isoformen der gereinigten DP 4 zur Aufklärung der 3D-Kristallstruktur und Charakterisierung der DP 4-ähnlichen Enzyme in verschiedenen Geweben und den Langerhans‘schen Inseln. Danach wurde die Identifizierung der DP 4 als ein neues pharmazeutisches Behandlungsziel für Angst, Depression und Schizophrenie, welche sich auf Verhaltensänderungen in DP 4-defizienten Ratten, DP 4-/- k.o. Mäusen und Hemmung der DP 4 in vivo unter Stress beziehen, behandelt. Die Studie umfasste eine Analyse der DP 4-ähnlichen Enzyme in neuronalen Geweben und humanen Körperflüssigkeiten, die Identifizierung der Hauptsubstrate und Zielorgane, das Inhibitorprofil sowie die psychiatrischen Indikationen. Eine neue Methode wurde entwickelt, um die 17 Isoformen der gereinigten Schweine-DP 4 in nur 2 bis 3 Isoformen pro Fraktion zu trennen. Dies wurde durch Einführung eines zweiten Refraktionierungsschritts mittels einer präparativen Flüssigphase-IEF erreicht. Eine Kristallstruktur mit einer Auflösung von 1,8 Å und einer tetrameren Anordnung zweier Homodimere wurde erhalten. Die Charakterisierung der Langerhans‘schen Inseln und des Schweinepankreas mittels Aktivitätsstudien, histochemischer Aktivitätsfärbung, Immunhistochemie und RT-PCR offenbarte, dass der größte Anteil der DP 4-Aktivität aus dem exokrinen Gewebe stammt. Eine DP 4-Expression in den Inseln umgebenden Blutgefäßen wurde immunhistochemisch nachgewiesen. Subzelluläre Fraktionierung und histochemische Aktivitätsfärbung zeigten DP 4-ähnliche Enzyme mit unterschiedlicher zellulärer Lokalisierung. DP 2 wurde anhand histochemischer Aktivitätsfärbung und Analyse isolierter Granulae in den Inseln ermittelt. Da sowohl DP 8 als auch DP 9 im Zytosol vorkommen und DP 2 nicht in der Lage ist DP 4-Substrate zu spalten, weisen die vorliegenden Ergebnisse auf zusätzliche parakrine und neuronale Funktionen der DP 4 in der Regulierung der Insulinausschüttung hin. Ferner wurde während einer Serumanalyse des DP 4-Substrates Neuropeptid Y (NPY) eine mögliche Medikamenten-Wechselwirkung zwischen antidiabetischen DP 4- und anihypertensiven ACE-Hemmern detektiert, da ACE zusätzlich NPY C-terminal spaltet. Eine durch DP 4-Hemmer zu erwartende Anreicherung von vasokonstriktiven NPY1-36 wird durch die von ACE verursachte Inaktivierung zu NPY1-34 kompensiert, jedoch wird diese unterbunden, wenn ACE ebenfalls blockiert wird. Außerdem konnte DP 4 als mögliches Behandlungsziel für die Indikationen Depression, Angst und Schizophrenie identifiziert werden. Obwohl in neuronalen Geweben der Anteil der DP 2-, DP 8- und DP 9-Aktivitäten höher war als die der DP 4, wurde eine hohe DP 4-Aktivität und Expression in den zirkumventrikulären Organen (ZVOs) gefunden. Da ZVOs keine Blut-Hirnschranke besitzen, deutet dies auf eine wichtige Rolle der DP 4 in der neuroendokrinen-immunologischen Interphase betreffs der beiden Stressachsen Hypothalamus-Hypophysen-Nebennieren Achse und neurosympathische Achse hin. Bei Untersuchungen des proteolytischen Abbaus mehrerer stressverwandter Neuropeptide in neuronalen Rattengeweben und humanen Körperflüssigkeiten, wurde NPY als das Hauptsubstrat der DP 4 bezüglich stressvermittelter Indikationen identifiziert sowie „Galanin-like peptide“ und Ratten-Orexin B als neue DP 4-Substrate entdeckt. Während im humanen Liquor cerebrospinalis keine DP 4-Trunkierung von NPY nachgewiesen wurde, ist das durch DP 4 gespaltene NPY3-36 der Hauptmetabolit in humanem Serum und Vollblut, gefolgt von der C-terminalen Inaktivierung durch ACE. Ferner wird NPY im Blut gleichermaßen von Endothel-DP 4 und löslicher DP 4 gespalten. Außerdem wurden durch Untersuchungen zum NPY-Metabolismus sieben neue NPY-spaltende Proteasen identifiziert, nämlich die lysosomalen Kathepsine D, B, L, S und G, Kallikrein 1 sowie ACE.

Neuropeptid Y (NPY) izražavaju stanice iz loze osteoblasta. In vitro studije su pokazale da NPY ima negativan učinak na diferencijaciju osteoblasta. Vođeni pretpostavkom da NPY izražen u stanicama loze zrelih osteoblasta može kontrolirati diferencijaciju mezenhimskih progenitorskih stanica istražili smo in vivo ulogu neuropeptida Y kao regulatora osteogeneze. U tu svrhu uzgojili smo transgeničnog miša (Col2.3NPY-ires GFPtopaz). Lančana reakcija polimerazom u stvarnom vremenu (RT-qPCR) i NPY EIA analiza pokazali su pojačani izražaj NPY-a u transgeničnim miševima. Col2.3NPY miševi imali su 25% smanjen udio trabekularne kosti i 7-10% tanje trabekule. Analiza genske ekspresije nije pokazala nikakve razlike u ekspresiji OC, BSP i DMP-1 kod transgeničnih životinja. Col2.3NPY mCOB kulture imale su 6 puta manju površinu mineralizacije te smanjeni izražaj markera osteogeneze. Ovaj mišji model pokazao je da lokalno izražen NPY ima važnu ulogu u regulaciji koštane mase te regulira promjene u koštanoj masi. Svi ovi rezultati pokazali su da periferni signalni put NPY-a ima bitnu ulogu u koštanoj pregradnji. Neuropeptide Y is expressed by osteoblast lineage cells and when administered in vitro can inhibit osteoblast lineage differentiation. We hypothesize that NPY, generated by mature osteoblast lineage cells, can control and modulate differentiation of osteoprogenitor cells. In this study we have evaluated the effects of bone-specific overexpression by generating Col2.3NPY-ires GFPtopaz transgenic mice. RT-qPCR and NPY EIA analysis showed increased expression of NPY in transgenic mice. Col2.3NPY mice had 25% lower trabecular volume and 7-10% lower trabecular thickness. Gene expression analysis in Col2.3NPY mice did not show any changes in expression levels of OC, BSP and DMP-1. Terminal osteoblast differentiation was impaired in Col2.3NPY mCOB cultures as indicated by a 6-fold decrease in mineralized area and decreased expression of all osteogenic markers. Our model has shown that locally expressed NPY plays an important role in regulation of bone mass. All these findings suggest that peripheral mechanism of the NPY signaling is important in bone remodeling.

Acute tryptophan depletion (ATD) Moja-De is a physiological method of reducing central nervous serotonin (5-HT) synthesis in children, adolescents, and adults. It involves the administration of amino acids (AA) within an aqueous suspension lacking in tryptophan (TRP). TRP is an essential AA serving as the physiological precursor of 5-HT. ATD occurs because all relevant AA in the beverage use the same active large neutral amino acid (LNAA) transport system (L1) in the capillary cell plasma membrane to pass the blood-brain barrier. However, to date the use of conventional ATD protocols in children and adolescents was limited due to frequently observed side effects (e.g., vomiting and nausea). In contrast, Moja-De is a weight-adapted protocol that takes a positive correlation between body weight and plasma TRP into account (Kewitz, 2002; Demisch et al., 2002), therefore allowing its use in children and adolescents with fewer side effects. This study’s aim was to investigate the ATD procedure Moja-De by looking at plasma concentrations of TRP and competing LNAA and TRP influx across the blood-brain barrier into the brain on the one hand. On the other hand, the effects of ATD Moja-De on the leptin axis as indexed by Leptin and sOB-R concentrations as well as the free leptin index (FLI, the quotient of leptin and its soluble receptor sOB-R) and on peripheral neuropeptide Y (NPY) concentrations were investigated. The sample consisted of 24 healthy, young participants aged 21 to 30 years (12 males and 12 females). Data were obtained on two different study days for each participant. The participants received an AA beverage at the beginning of each study day, either ATD or a TRP-balanced amino acid load (BAL), using a randomized double-blind within-subject repeated measures crossover design. Participants’ blood samples were taken at baseline before and at 90, 180, and 270 minutes after ATD/BAL intake to assess plasma concentrations of the relevant AA that compete with TRP on the same transport system as well as serum concentrations of Leptin, sOB-R, and NPY. TRP influx across the blood–brain barrier was calculated using Michaelis–Menten kinetics with a correction for multiple substrate competition, indicating a significant decrease in TRP influx into the central nervous system after intake of ATD Moja-De. The decreased TRP influx into the brain suggests a diminished central nervous system availability of the 5-HT precursor TRP and thus, a reduced central nervous 5-HT synthesis, particularly in terms of a decreased substrate availability for Tryptophan hydroxylase 2 (TPH2), the enzyme that limits the 5-HT synthesis rate in the CNS. Overall, the AA beverages were well tolerated amongst all study participants. ATD Moja-De can be considered as a safe and effective method of reducing TRP influx into the brain. It can not only be used in adults, but also in children and adolescents because of its fewer side effects and a body weight adapted dosing regimen. Concerning the ATD Moja-De induced alterations of the leptin axis, there were effects on sOB-R serum concentrations in female participants observed as indexed by reduced sOB-R concentrations in female participants after administration of the ATD-test. This relationship has not been demonstrated before, and previous research on interactions between brain 5-HT and the leptin axis mainly relied on animal models. This effect might indicate a reduced transport of leptin across the blood-brian-barrier. Furthermore, it may suggest an alternative regulatory mechanism of peripheral leptin availability, possibly via immunological mechanisms affecting OB-R- and sOB-R-concentrations. Taking into account that females in general show a lower central nervous synthesis of 5-HT when compared to males (Nishizawa et al., 1997), these gender-related effects with respect to an interaction between the leptin axis and 5-HT are relevant for the understanding of the pathophysiology of eating disorders. The present results do not support an association between ATD administration and the plasma concentrations of leptin, FLI, and NPY in healthy adults. Future research with larger samples including healthy populations and patients with neuropsychiatric disorders which are associated with changes in central nervous 5-HT function as well as simultaneous consideration of other secondary compensatory effects induced by ATD is needed. Administration of the ATD-test Moja-De as a physiological method to lower central nervous 5-HT synthesis allows the involvement of such vulnerable groups because of its safe neurochemical properties and good tolerability.

Zusammenfassung Hintergrund und Ziele Neuropeptid Y (NPY) ist ein weit verbreitetes und viel erforschtes Neuropeptid im zentralen und peripheren Nervensystem. Über vier verschiedene, G-Protein-gekoppelte Rezeptoren (Y1, Y2, Y4, Y5) ist es als Transmitter an der Regulation vieler physiologischer Funktionen wie Essverhalten und Entstehung von Übergewicht, Angstverhalten, Krampfentwicklung, Blutdruck und Entstehung von Abhängigkeit beteiligt. Die Modulation dieser Funktionen ist Effekt vieler psychopharmakologischer Behandlungen, wobei der genaue Wirkmechanismus der Medikamente häufig noch nicht vollständig aufgeklärt ist. Gewichtszunahme, antiepileptische Wirkung oder Senkung der Krampfschwelle, sowie Sedation und Anxiolyse, zählen zu den erwünschten und unerwünschten Wirkungen von Valproinsäure, Clomethiazol und Clozapin, und die Vermutung liegt nahe, dass diese Medikamente Teile ihrer Wirkungen über Modulation von Neuropeptid Y in Verbindung mit seinen vier Rezeptoren entfalten. So ist es das Ziel dieser Arbeit, herauszufinden, ob sich die Genexpression von Neuropeptid Y und seinen Rezeptoren unter Stimulation mit Valproinsäure, Clomethiazol und Clozapin verändert und so zur Aufklärung der molekularen Wirkmechanismen dieser Medikamente beitragen. Material und Methoden Humane Neuroblastomzellen, SH-SY5Y (SY5Y) und humane, embryonale Nierenzellen, HEK-293 (HEK) wurden in Zellkulturexperimenten mit oben genannten Substanzen für je 4, 24 und 48 Stunden stimuliert. Die hierfür geeigneten Stimulationskonzentrationen wurden zuvor mittels eines Zytotoxizitätstests bestimmt. Die Ermittlung des Expressionsniveaus für NPY und seine Rezeptoren erfolgte relativ im Vergleich zu unbehandelten Kontrollen durch eine quantitative PCR auf Ebene der Gentranskription und teilweise folgte im Western-Blot die Analyse der nachfolgenden Translation. Ergebnisse Nach Stimulation mit Valproinsäure zeigten sich in der HEK-Zelllinie signifikante Anstiege der Expression von NPY, Y1, Y2 und Y5. Die Induktion von Y1 ließ sich auch in den SY5Y Zellen zeigen. Clomethiazol zeigte variable Beeinflussung des NPY-Systems; während in den HEK-Zellen eine Inhibition von Y2 und Y5 nachweisbar war, führte es bei den Zellen der Linie SY5Y zu einer Induktion von NPY selbst und Y1 und ebenfalls zu einer Inhibition von Y2. Nach Stimulation mit Clozapin konnte bei der Zelllinie HEK eine verminderte Expression von Y2 festgestellt werden, alle anderen Untersuchungen zeigten keine signifikanten Veränderungen. Schlussfolgerungen Die Ergebnisse unterstützen die Vermutung, dass erwünschte und unerwünschte Wirkungen von Valproinsäure (Sedation, Antikonvulsion, Anxiolyse, Gewichtszunahme) durch Veränderung der Expression von NPY und seinen Rezeptoren mitverursacht werden. Im Gegensatz dazu konnte gezeigt werden, dass Clozapin seine Wirkungen (Sedierung, Gewichtszunahme, Senkung der Krampfschwelle), wenn sie überhaupt durch Veränderungen im NPY-System hervorgerufen werden, nur über die Beeinflussung von Y2 entfalten kann. Die Ergebnisse für Clomethiazol legen nahe, dass nachweisbare Veränderungen der Expression von NPY und seinen Rezeptoren teilweise durch physiologisch stärkere, andere Wirkungen überdeckt werden, da die nachgewiesenen zellulären Veränderungen partiell im Gegensatz zur antikonvulsiven und sedierenden Wirkung stehen. Insgesamt ergibt die Repression von Y2 interessante Interpretationsmöglichkeiten bezüglich der anxiolytischen und auch antikonvulsiven Wirkung von Clomethiazol und der physiologischen Wirkungen von Clozapin. Summary Background and aims Neuropeptide Y is a widely expressed peptide in the central and peripheral nervous system and has been found to be associated with many physiological functions. Acting via four different G-protein coupled receptors (Y1, Y2, Y4, Y5) it is involved in feeding behaviour and the pathology of obesity, anxiety, blood pressure, addiction and convulsion. Psychotropic drugs are designed to modulate those functions, even though little is known about the exact effects on cellular pathways. Gain of weight, epileptic or antiepileptic effects, sedation and anxiolysis are among the effects and side effects of valproic acid, clomethiazole and clozapine, suggesting that these drugs effect NPY and its receptor-system. The aim of the present study was to investigate the effects of valproic acid, clomethiazole and clozapine on gene expression of NPY and its receptors, which is a step towards understanding the drugs‘ mechanisms of action. Material and methods Human neuroblastoma cells, SH-SY5Y (SY5Y) and human embryonic kidney cells, HEK-293 (HEK) were stimulated with the above-named substances for 4, 24 and 48 hours. The employed drug-concentrations for incubation were determined using cytotoxicity-tests. The gene expression level of NPY and its receptors was measured by relative quantification using real-time dye-based PCR and the protein expression was analysed for selected genes using western blots. Results Stimulation with valproic acid showed a significant increase of gene expression of NPY, Y1, Y2 and Y5 in HEK-cells. Induction of Y1 was also seen in SY5Y-cells. Clomethiazole showed various effects on the NPY system, inhibiting the expression of Y2 and Y5 in HEK but increasing the expression of NPY and Y1 in SY5Y and also inhibiting Y2. Stimulation with clozapine showed only a decrease in expression for Y2 in HEK and no other significant results. Conclusions The results give evidence, that the effects and side effects of valproic acid (sedation, antiepileptic effects, anxiolysis, gain of weight) are correlated with modulation in gene-expression of NPY and its receptors. It could be shown that clozapine can only unfold its physiological effects (sedation, gain of weight, epileptic effects), if at all via affecting the NPY-system, through repression of Y2. Since the data of clomethiazole ist partly contrary to its physiological effects (sedation, anticonvulsion), the detectable and significant changes in gene-expression of the NPY-system are probably covered from other, physiologically stronger mechanisms. However, there are interesting ways of interpreting the repression of Y2 for the anxiolytic and also antiepileptic effects of clomethiazole and for the physiological effects of clozapine.

Die Amygdala ist ein Kernkomplex, der dicht von serotonergen Afferenzen innerviert wird. Sowohl bei Tieren als auch beim Menschen spielen Interaktionen zwischen dem serotonergen System und der Amygdala bei der Verarbeitung von Reizen, die mit Angst oder Stress assoziiert sind, eine zentrale Rolle. Genetische Variationen im serotonergen System und/oder dauerhafter Stress können dazu führen, dass diese Verarbeitungsprozesse fehlerhaft ablaufen, wodurch Verhaltensanormalitäten bzw. die Entstehung psychiatrischer Erkrankungen begünstigt werden. Die Zielneurone der serotonergen Transmission in der Amygdala, die molekularen Mechanismen möglicher Interaktionen und strukturelle Konsequenzen der Störungen dieser Interaktionen sind jedoch bis zum heutigen Zeitpunkt noch nicht vollständig bekannt. Daher bestand ein Ziel der vorliegenden Arbeit darin, den Einfluss eines Ungleichgewichts im serotonergen System (5-Htt KO) sowie von wiederholtem, sozialem Stress auf die neuronale Morphologie der Amygdala zu analysieren und Zielneurone serotonerger Afferenzen zu identifizieren und zu charakterisieren, um die neuronalen Netzwerke der Emotionsverarbeitung besser verstehen zu können. Um vom 5-Htt–Genotyp abhängige und stressbedingte neuromorphologische Veränderungen zu untersuchen, wurden dreidimensionale Rekonstruktionen von Neuronen der laterobasalen Amygdala von männlichen, adulten Wildtyp (WT)- und 5-Htt KO-Mäusen angefertigt und bezüglich verschiedener morphologischer Parameter ausgewertet. An den Pyramidenzellen wurden nur geringfügige Veränderungen der dendritischen Komplexität, jedoch, im Vergleich zu WT-Mäusen, eine wesentliche Erhöhung der Dornendichte an spezifischen dendritischen Kompartimenten bei gestressten WT-Mäusen, sowie nicht gestressten und gestressten 5-Htt KO-Mäusen nachgewiesen. Im Vergleich zu nicht gestressten WT–Mäusen war die dendritische Dornendichte aller anderen Gruppen gleichermaßen erhöht. Die Sternzelle, zeigten bezüglich der untersuchten Parameter keine morphologischen Veränderungen auf. Eine besondere Subpopulation der Interneurone stellen die NeuropeptidY (NPY)–Neurone der laterobasalen Amygdala dar, da sie in diesen Nuclei anxiolytisch wirken. Es gibt nur wenige Anhaltspunkte darüber, durch welche Systeme NPY–Neurone moduliert werden. Da sowohl NPY–Neurone in der laterobasalen Amygdala als auch das serotonerge System an angstregulierenden Prozessen beteiligt sind, sollte im zweiten Teil der vorliegenden Arbeit untersucht werden, ob es sich bei diesen Neuronen um Zielstrukturen des serotonergen Systems handelt. Mittels licht- und elektronenmikroskopischer Analysen wurden synaptische Kontakte zwischen serotonergen Afferenzen und NPY-immunreaktiven Neuronen in der laterobasalen Amygdala von Ratten verifiziert. Da der funktionelle Einfluss der serotonergen Innervation auf diese Zielneurone von deren Serotoninrezeptor (5-HTR)-Ausstattung abhängt, wurden Koexpressionsanalysen von NPY mRNA mit den mRNAs verschiedener 5-HTR durchgeführt. Die Analysen ergaben, dass NPY mRNA–reaktive Neurone in der laterobasalen Amygdala 5-HT1A und 5-HT2C, jedoch nicht 5-HT3 mRNA koexprimieren. Die in der vorliegenden Arbeit erzielten Resultate liefern neue Erkenntnisse über den Einfluss des serotonergen Systems auf die laterobasale Amygdala von Mäusen und Ratten. Bei den Veränderungen der dendritischen Dornendichte nach sozialen Stresserfahrungen könnte es sich um neuroadaptive bzw. kompensatorische Mechanismen der Pyramidenzellen handeln, die WT-Mäusen eine Anpassung an sich ändernde, negative Umweltbedingungen ermöglicht. Die erhöhte Dornendichte könnte dabei die Ausbildung eines „emotionalen Gedächtnisses“ repräsentieren, das eine flexible Verhaltensantwort auf ein erneutes Auftauchen von Gefahr erlaubt. Eine solche Modulation der Erregbarkeit der laterobasalen Amygdala könnte beispielsweise über eine situationsentsprechende Hemmung des Outputs der Pyramidenzellen durch differentiell aktive inhibitorische Netzwerke erfolgen. Eine differentielle Aktivierung kann z. B. über unterschiedliche Rezeptorausstattungen, wie es in der Subpopulation der NPY–Neurone in der vorliegenden Arbeit nachgewiesen wurde, erfolgen. Das erhöhte angstähnliche Verhalten der 5-Htt KO-Mäuse nach wiederholtem Stress könnte mit der Unfähigkeit zusammenhängen, in entsprechenden Situationen durch Neubildung von Dornen zu reagieren, da die Dornendichte bei diesen Tieren schon unter stressarmen Umweltbedingungen ihr Maximum erreicht hat. Sowohl Fehlfunktionen der neuronalen Plastizität als auch mögliche Fehlfunktionen der differentiellen Inhibierung der Pyramidenzellen durch Interneurone, die durch genetische Variationen und/oder Stress bedingt sein können, könnten eine „offene Tür“ repräsentieren, die zu manifesten Auffälligkeiten im Verhalten bei Tieren führt bzw. auch zur Entstehung bestimmter psychiatrischer Erkrankungen beim Menschen beiträgt., The amygdala is a heterogenous nuclear complex, which belongs to the limbic system and recieves a dense serotonergic innervation. In animals and in humans, interactions between the serotonergic system and the amygdala play a key role in emotional stimulus processing, especially for anxiety- and stress–related stimuli. Genetic variations within the serotonergic system and/or chronic stress can cause dysregulation of these mechanisms, which promote abnormal behavior and development of psychiatric disorders, respectively. Up to now, the target neurons of serotonergic neurotransmission in the amygdala, the molecular mechanisms of possible interactions and morphological consequences of dysregulation of these interactions are not investigated completely, yet. Thus, one aim of this thesis was to analyze the impact of an imbalance within the serotonergic system (serotonin transporter knock out, 5-Htt KO) and repeated social stress on neuronal morphology in the amygdala and to identify and characterize target neurons of serotonergic afferents. This will help to get a better understanding of the neuronal networks associated with emotional stimulus processing. To investigate 5-Htt genotype–dependent and stress–related neuromorphological changes, three-dimensional reconstructions of pyramidal cells and stellate interneurons of the laterobasal amygdala of male, adult wildtype (WT) and 5-HttKO mice were carried out and different morpholgical parameter were analyzed. Pyramidal cells displayed only subtle changes in dendritic complexity but a significant increase in spine density on specific dendritic compartments in stressed WT mice and naïve and stressed 5-HttKO mice, compared to naïve WT mice. In all groups, the increase in spine density was, compared to WT mice, similar. Stellate interneurons lacked detectable genotype–dependent and stress–related morphological differences. NeuropeptideY (NPY) neurons represent a unique and special interneuronal subpopulation in the laterobasal amygdala due to their anxiolytic effects in these nuclei. There is much information about the molecular effects of NPY via specific receptors and how NPY can regulate emotion–related states, especially anxiety–like behavior. However, only few indications are available, by which systems NPY neurons are modulated themselves. Since both NPY neurons in the laterobasal amygdala and the serotonergic system are involved in anxiety–regulating processes, the task of the second part of the present thesis was to investigate if these neurons were targets of the serotonergic system. Applying light and electron microscopic methods, perisomatic synaptic contacts between serotonergic afferents and NPY-immunoreactive neurons were identified in the laterobasal amygdala of rats. As the functional impact of the serotonergic innervation significantly depends on the serotonin receptor (5-HTR) equipment of the target neurons, coexpression of NPY mRNA and different 5-HTR mRNAs was studied. For this purpose, a novel variation of in situ hybridization, which allows specific coexpression analyses of NPY mRNA with low-abundant mRNAs, was developed and established. The results show that NPY mRNA–reactive neurons in the laterobasal amygdala coexpress 5-HT1A and 5-HT2C, but lack 5-HT3 mRNA. In the present thesis, detailed, new data on the impact of the serotonergic system on the laterobasal amygdala of mice and rats were obtained. The changes in dendritic spine density after repeated stress could represent neuroadaptive or compensatory mechanisms of these neurons, which enable WT mice to adapt to negative environmental changes. The increase in dendritic spine density could stand for the formation of an “emotional memory“ facilitating quick and flexible behavioral reactions upon re-appearing danger. This modulation of the laterobasal amygdala´s excitability could occur, for instance, via a specific inhibition of pyramidal output by differentially activated inhibitory networks. Such a differential activation can result from a variable receptor equipment, as shown for NPY neurons in the laterobasal amygdala in the present thesis. The increased anxiety–like behavior of 5-HttKO mice after repeated social stress could be due to the unability of these mice to further increase spine density in corresponding situations, since spine density already reached the maximum level in a low-stress environment. Both dysfunction of neuronal plasticity and possible dysfunction of the differential inhibition of pyramidal cells via interneurons presumably caused by genetic variations and/or stressful life events could represent an “open door“ facilitating abnormal behavior in animals or development of certain psychiatric disorders in humans.

Title: Acipimox during Short-Term Exercise Exerts A Negative Feedback of Growth Hormone on Ghrelin Secretion in Patients with Bulimia Nervosa and in Healthy Women: The Role of Lipolysis Objective: Eating disorders, such as bulimia nervosa (BN) and anorexia nervosa (AN), are characterized by abnormal eating behavior. The main features of BN are binge-eating and inappropriate compensatory methods to prevent weight gain. The appetite-modulating peptide ghrelin is secreted by the stomach and shows a strong release of growth hormone (GH). A potential GH-ghrelin feedback loop between stomach and the pituitary has been recently reported. Acipimox (Aci), an analogue of nicotinic acid, inhibits lipolysis in adipose tissue (AT) and reduces plasma glycerol and free fatty acids (FFA) levels. Exercise and Aci are stimulators of GH secretion. We suppose that a negative feedback from increased GH levels during exercise may play a role in reducing plasma ghrelin levels. We surmised that altered baseline activity and exercise-induced activation of the sympathetic nervous system (SNS) results in excessive stimulation of lipolysis associated with negative energy balance and may lead to abnormal AT metabolism in patients with BN. Disruption of the gut-brain-AT axis might be involved in the pathogenesis of BN. The...

Hintergrund und Ziele: Adipozytokine spielen in der Pathogenese humaner Adipositas eine wesentliche Rolle. Des Weiteren sind sie mit kardiovaskulären bzw. metabolischen Risikofaktoren assoziiert. Das Ziel unserer Studie war es, die Genexpression von Omentin, Visfatin und Neuropeptid Y (NPY) in humanem Fettgewebe Erwachsener zu bestimmen. Ferner erhoben wir klinische Parameter und untersuchten die Konzentrationen von NPY im Plasma und Kortisol im Serum, um letztere mit Urinsteroiden bei adipösen und normalgewichtigen Erwachsenen und adipösen Kindern zu vergleichen. Methoden: Unsere Studienpopulation bestand aus 133 adipösen und normalgewichtigen Erwachsenen sowie aus 56 adipösen Kindern. Mithilfe von Real-time-PCR bestimmten wir die Expression von Omentin, Visfatin und NPY in subkutanem und viszeralem Fettgewebe von 62 erwachsenen adipösen und normalgewichtigen Probanden, die sich einer elektiven laparoskopischen Operation unterzogen hatten. Darüber hinaus bestimmten wir klinische Parameter, NPY-Plasmakonzentrationen (mittels Radioimmunoassay), Serumkortisolkonzentrationen (mittels ECLIA) und Kortisolmetabolite im Urin (mittels GC-MS). Ergebnisse: In viszeralem Fettgewebe konnten wir eine höhere Omentin-Genexpression feststellen als in subkutanem Fettgewebe. Die Visfatin-Genexpression war in subkutanem Fettgewebe adipöser Patienten reduziert (p

Uvod. Upalne bolesti crijeva grupa su kroničnih autoimunih bolesti gastrointestinalnog sustava nedovoljno poznate etiopatologije. Dosadašnja istraživanja ukazuju na povezanost dipeptidil-peptidaze IV (DPP IV/CD26) i molekularnih mehanizama uključenih u procese nastanka i cijeljenja upale. Pokazano je da ova molekula ima vrlo važnu ulogu u razgradnji neuroimunoendokrinih medijatora uključenih u održavanje integriteta sluznice, kao i nastanak upalnih promjena. Cilj istraživanja. Ovo se istraživanje temelji na pretpostavci da DPP IV/CD26 ima značajnu neuroimunomodulacijsku ulogu, obzirom da je biološka aktivnost mnogih neuropeptida i interleukina pod izravnim utjecajem ove molekule. Cilj ovog istraživanja bio je ispitati povezanost DPP IV/CD26 i Crohnove bolesti, praćenjem profila medijatora neuroimunološkog odgovora tijekom nastanka i cijeljenja kemijski induciranog kolitisa u CD26 deficijentnom i divljem tipu miša. Materijal i metode. Model Crohnove bolesti u miša induciran je rektalnom, intraluminalnom aplikacijom etanolne otopine trinitrobenzensulfonske kiseline (TNBS). Kako bi ispitali in vivo učinke DPP IV/CD26 u razvoju i cijeljenju upalnih promjena, kolitis je izazvan u CD26 deficijentnom (CD26-/-) te divljem tipu miša soja C57BL/6, istog spola i dobi. Kontrolne skupine predstavljale su životinje odgovarajućeg soja kojima je pod istim uvjetima rektalno aplicirana sterilna fiziološka otopina, odnosno otopina etanola. Razvoj, intenzitet i cijeljenje kolitisa praćeno je u različitim vremenskim intervalima temeljem kliničkih i histoloških parametara na sistemskoj i lokalnoj razini, u oba soja pokusnih životinja. Stupanj oštećenja debelog crijeva pratio se određivanjem makroskopskih promjena, patohistološkom analizom, određivanjem mikroskopskog indeksa oštećenja te histomorfometrijskom analizom parafinskih tkivnih rezova. Aktivnost DPP IV/CD26 u C57BL/6 soja miša te aktivnost DPP IV/CD26-sličnih enzima u CD26-/- životinja u serumu, mozgu i debelom crijevu određivana je spektrofotometrijski. Proteinski izražaj DPP IV/CD26 u mozgu i debelom crijevu C57BL/6 životinja određivan je tehnikom Western blot. Sistemske (serumske) i lokalne (u ekstraktima mozga i debelog crijeva) koncentracije neuropeptida Y (NPY) i vazoaktivnog intestinalnog peptida (VIP) određivane su specifičnim imunoenzimskim testovima (ELISA), a njihov proteinski izražaj u mozgu i debelom crijevu tehnikom Western blot. Koncentracije proupalnog interleukina-6 i protuupalnog interleukina-10 u cirkulaciji te u ekstraktima navedenih tkiva također su određivane specifičnim imunoenzimskim testovima (ELISA). Rezultati. Tijekom razvoja kemijski induciranog TNBS-kolitisa u C57BL/6 i CD26-/- soju životinja dolazi do kliničke manifestacije bolesti, statistički značajnih promjena tjelesne mase, mase jetre i slezene, makroskopskih, patohistoloških i histomorfometrijskih promjena debelog crijeva uz značajan porast mikroskopskog indeksa oštećenja, čime je potvrđena uspostava modela kolitisa. Upalne promjene u obliku ulceracija u CD26-/- životinja bile su lokalizirane na površinski sloj, uz određeni broj žarišnih transmuralnih promjena. Za razliku od CD26-/-, kod C57BL/6 životinja prisutna je difuzna upala koja zahvaća cijelu cirkumferenciju crijeva. Kod ciljanih imunobiokemijskih parametara (enzimske aktivnosti, koncentracije neuropeptida, koncentracije interleukina) utvrđene su statistički značajne promjene i na sistemskoj i na lokalnoj razini u oba soja pokusnih životinja kojima je induciran kolitis u odnosu na kontrolne skupine, ali i između ispitivanih sojeva životinja. Aktivnost DPP IV u C57BL/6 životinja statistički je značajno snižena u serumu, mozgu i debelom crijevu životinja kojima je induciran kolitis, u odnosu na kontrolnu skupinu. U akutnoj fazi kolitsa, u CD26-/- životinjama utvrđene su statistički značajne razlike u promjenama koncentracija NPY-a i VIP-a te IL-6 i IL-10 u cirkulaciji i na lokalnoj razini (u mozgu i debelom crijevu), u odnosu na skupine C57BL/6 životinja. Koncentracije protuupalnih medijatora IL-10 i VIP-a u CD26-/- životinja statistički su značajno više i sistemski i lokalno, dok C57BL/6 životinje sadrže veće koncentracije NPY-a u mozgu i debelom crijevu. Zaključak. TNBS-kolitis (engl. Crohn-like colitis) uspostavljen je u oba soja miša, što je potvrđeno statistički značajnim promjenama kliničkih i histoloških parametara na lokalnoj i sistemskoj razini, s odgovarajućim razlikama u patohistološkim upalnim promjenama. Tijekom razvoja i cijeljenja kolitisa u CD26-/- životinja dolazi do statistički značajnih promjena sistemskih i tkivnih koncentracija NPY-a, VIP-a, IL-6 i IL-10 u odnosu na C57BL/6 životinje. Dobiveni rezultati ukazuju da se upalne promjene na razini debelog crijeva reflektiraju promjenama ispitivanih imunobiokemijskih parametara u mozgu. Rezultati ovog istraživanja ukazuju da DPP IV/CD26 posjeduje i neuroimunomodulacijska svojstva tijekom razvoja i cijeljenja upalne bolesti crijeva u miša. Introduction. Inflammatory bowel diseases represent a group of chronic autoimmune diseases of the gastrointestinal tract which exact etiopathogenesis is unknown. Previous studies suggested an association between dipeptidyl peptidase IV (DPP IV/CD26) and molecular mechanisms involved in the process of intestinal inflammation. It has been shown that this molecule plays an important role in the breakdown of neuroimmunoendocrine mediators involved in maintaining the integrity of intestinal mucosa and the occurrence of inflammatory events. Objectives. This research is based on the hypothesis that DPP IV/CD26 possesses an important neuroimmunomodulative role, given that the biological activity of many neuropeptides and interleukins is directly influenced by this molecule. The aim of this study was to investigate the causative connections between DPP IV/CD26 and Crohn's disease, by monitoring the profile of mediators involved in the neuroimmune responses during emergence and course of chemically induced colitis in CD26 deficient and wild type mice. Materials and methods. The model of Crohn's disease in mice was induced by rectal, intraluminal application of trinitrobenzensulfonic acid (TNBS) ethanol solution. In order to examine the in vivo properties of DPP IV/CD26 in the development and healing of inflammation, colitis was induced in CD26 deficient (CD26-/-) and wild type mouse strain C57BL/6, of same gender and age. The control group was represented by the appropriate animal strain which has been rectally applied, under same conditions, sterile physiological solution or ethanol solution. Colitis development, intensity and healing were followed at different time intervals based on clinical and histological parameters at the systemic and local levels in both strains of experimental animals. The degree of colonic mucosa damage was assessed by evaluation of macroscopic changes, histopathological analysis, microscopic indices of damage, and histomorphometric analysis of paraffin tissue sections at different time intervals during the development and healing of inflammatory changes. DPP IV/CD26 activity in C57BL/6 mouse strain and the activity of DPP IV/CD26-like enzymes in CD26 deficient animals in serum, brain and large intestine were determined spectrophotometrically. Protein expression of DPP IV/CD26 in brain and colon of C57BL/6 mice was determined by Western blot technique. Systemic (serum) and local (in brain and colon protein extracts) concentrations of neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were determined by specific immunoenzymatic tests (ELISA), and their protein expressions by Western blot technique. Concentrations of the proinflammatoty interleukin 6 (IL-6) and antiinflammatory interleukin 10 (IL-10) in circulation and in brain and colon tissue extracts were also determined by specific immunoenzymatic tests (ELISA). Results. The development of chemically induced colitis in C57BL/6 and CD26 deficient mice leads to clinical manifestations of the disease, statistically significant changes in body weight, liver and spleen weights, macroscopic and histological changes in the colon, which confirms the establishment of a model of colitis. During the development and healing of colitis, CD26 molecule deficiency resulted in histological changes in the manifestation of inflammatory process: in CD26 deficient animals inflammatory changes were mainly localized, with transmural inflammatory changes, while in C57BL/6 animals a diffuse inflammation that affects the whole intestinal circumference could be found. Targeted immunochemical parameters (enzyme activities, concentrations of neuropeptides, interleukins concentrations) resulted in statistically significant changes at the systemic and local levels in both strains of experimental animals, in mice with induced colitis compared to controls, but also between mice strains. DPP IV activity in C57BL/6 animals was statistically significantly decreased in serum, brain and colon in animals with induced colitis, compared to the control group. In the acute phase of colitis, in CD26-/- animals, statistically significant differences in changes in concentrations of NPY and VIP and IL-6 and IL-10 in circulation and at the local level (in brain and colon) were found, compared to C57BL/6 animals. Concentrations of anti-inflammatory mediators IL-10 and VIP were statistically significantly higher in serum and at local levels in CD26-/- animals, while C57BL/6 animals had higher concentrations of NPY in brain and colon. Conclusion. The TNBS-colitis (Crohn-like colitis) has been established in both mouse strains, as confirmed by statistically significant changes in clinical and histological parameters at local and systemic level, with corresponding differences in histological inflammatory changes. During the development and healing of colitis in CD26-/- animals, significant changes in systemic and tissue concentrations of NPY, VIP, IL-6 and IL-10 occurred, compared to C57BL/6 animals. The results obtained in this study indicate that inflammatory changes in immunobiochemical parameters at the level of the colon reflect in changes of those parameters in the brain. The results of this study indicate that DPP IV/CD26 possesses also neuroimmunomodulative properties during development and healing of inflammatory bowel disease in mice.

Uvod. Upalne bolesti crijeva grupa su kroničnih autoimunih bolesti gastrointestinalnog sustava nedovoljno poznate etiopatologije. Dosadašnja istraživanja ukazuju na povezanost dipeptidil-peptidaze IV (DPP IV/CD26) i molekularnih mehanizama uključenih u procese nastanka i cijeljenja upale. Pokazano je da ova molekula ima vrlo važnu ulogu u razgradnji neuroimunoendokrinih medijatora uključenih u održavanje integriteta sluznice, kao i nastanak upalnih promjena. Cilj istraživanja. Ovo se istraživanje temelji na pretpostavci da DPP IV/CD26 ima značajnu neuroimunomodulacijsku ulogu, obzirom da je biološka aktivnost mnogih neuropeptida i interleukina pod izravnim utjecajem ove molekule. Cilj ovog istraživanja bio je ispitati povezanost DPP IV/CD26 i Crohnove bolesti, praćenjem profila medijatora neuroimunološkog odgovora tijekom nastanka i cijeljenja kemijski induciranog kolitisa u CD26 deficijentnom i divljem tipu miša. Materijal i metode. Model Crohnove bolesti u miša induciran je rektalnom, intraluminalnom aplikacijom etanolne otopine trinitrobenzensulfonske kiseline (TNBS). Kako bi ispitali in vivo učinke DPP IV/CD26 u razvoju i cijeljenju upalnih promjena, kolitis je izazvan u CD26 deficijentnom (CD26-/-) te divljem tipu miša soja C57BL/6, istog spola i dobi. Kontrolne skupine predstavljale su životinje odgovarajućeg soja kojima je pod istim uvjetima rektalno aplicirana sterilna fiziološka otopina, odnosno otopina etanola. Razvoj, intenzitet i cijeljenje kolitisa praćeno je u različitim vremenskim intervalima temeljem kliničkih i histoloških parametara na sistemskoj i lokalnoj razini, u oba soja pokusnih životinja. Stupanj oštećenja debelog crijeva pratio se određivanjem makroskopskih promjena, patohistološkom analizom, određivanjem mikroskopskog indeksa oštećenja te histomorfometrijskom analizom parafinskih tkivnih rezova. Aktivnost DPP IV/CD26 u C57BL/6 soja miša te aktivnost DPP IV/CD26-sličnih enzima u CD26-/- životinja u serumu, mozgu i debelom crijevu određivana je spektrofotometrijski. Proteinski izražaj DPP IV/CD26 u mozgu i debelom crijevu C57BL/6 životinja određivan je tehnikom Western blot. Sistemske (serumske) i lokalne (u ekstraktima mozga i debelog crijeva) koncentracije neuropeptida Y (NPY) i vazoaktivnog intestinalnog peptida (VIP) određivane su specifičnim imunoenzimskim testovima (ELISA), a njihov proteinski izražaj u mozgu i debelom crijevu tehnikom Western blot. Koncentracije proupalnog interleukina-6 i protuupalnog interleukina-10 u cirkulaciji te u ekstraktima navedenih tkiva također su određivane specifičnim imunoenzimskim testovima (ELISA). Rezultati. Tijekom razvoja kemijski induciranog TNBS-kolitisa u C57BL/6 i CD26-/- soju životinja dolazi do kliničke manifestacije bolesti, statistički značajnih promjena tjelesne mase, mase jetre i slezene, makroskopskih, patohistoloških i histomorfometrijskih promjena debelog crijeva uz značajan porast mikroskopskog indeksa oštećenja, čime je potvrđena uspostava modela kolitisa. Upalne promjene u obliku ulceracija u CD26-/- životinja bile su lokalizirane na površinski sloj, uz određeni broj žarišnih transmuralnih promjena. Za razliku od CD26-/-, kod C57BL/6 životinja prisutna je difuzna upala koja zahvaća cijelu cirkumferenciju crijeva. Kod ciljanih imunobiokemijskih parametara (enzimske aktivnosti, koncentracije neuropeptida, koncentracije interleukina) utvrđene su statistički značajne promjene i na sistemskoj i na lokalnoj razini u oba soja pokusnih životinja kojima je induciran kolitis u odnosu na kontrolne skupine, ali i između ispitivanih sojeva životinja. Aktivnost DPP IV u C57BL/6 životinja statistički je značajno snižena u serumu, mozgu i debelom crijevu životinja kojima je induciran kolitis, u odnosu na kontrolnu skupinu. U akutnoj fazi kolitsa, u CD26-/- životinjama utvrđene su statistički značajne razlike u promjenama koncentracija NPY-a i VIP-a te IL-6 i IL-10 u cirkulaciji i na lokalnoj razini (u mozgu i debelom crijevu), u odnosu na skupine C57BL/6 životinja. Koncentracije protuupalnih medijatora IL-10 i VIP-a u CD26-/- životinja statistički su značajno više i sistemski i lokalno, dok C57BL/6 životinje sadrže veće koncentracije NPY-a u mozgu i debelom crijevu. Zaključak. TNBS-kolitis (engl. Crohn-like colitis) uspostavljen je u oba soja miša, što je potvrđeno statistički značajnim promjenama kliničkih i histoloških parametara na lokalnoj i sistemskoj razini, s odgovarajućim razlikama u patohistološkim upalnim promjenama. Tijekom razvoja i cijeljenja kolitisa u CD26-/- životinja dolazi do statistički značajnih promjena sistemskih i tkivnih koncentracija NPY-a, VIP-a, IL-6 i IL-10 u odnosu na C57BL/6 životinje. Dobiveni rezultati ukazuju da se upalne promjene na razini debelog crijeva reflektiraju promjenama ispitivanih imunobiokemijskih parametara u mozgu. Rezultati ovog istraživanja ukazuju da DPP IV/CD26 posjeduje i neuroimunomodulacijska svojstva tijekom razvoja i cijeljenja upalne bolesti crijeva u miša.

Eine tierexperimentelle Studie hatte nach epikardialer Defibrillation mit >30 Joule einen Anstieg des systemischen Gefäßwiderstandes gezeigt. Zur Überprüfung der Ergebnisse beim Menschen wurden 37 herzchirurgische Patienten (koronare Bypassoperation und/oder Aortenklappenersatz) nach kardioplegischem Herzstillstand in 3 Gruppen untersucht: Patienten, die mit niedriger (10 J) oder hoher (50 J) Energie defibrilliert wurden und solche die keine Defibrillation benötigten. Es wurde sowohl der systemische Gefäßwiderstand als auch die Konzentration vasoaktiver Substanzen (Noradrenalin, Neuropeptid Y, Endothelin-1 und aktives Renin) zentralvenös und im Sinus coronarius nach Defibrillation bestimmt. Ergebnis der Studie war, dass die Defibrillation weder auf den Gefäßwiderstand noch auf die Konzentration der gemessenen Substanzen einen signifikanten Einfluß hatte A recent study with dogs had shown an increase of total peripheral resistance after epicardial defibrillation with shock energies >30 Joule. To verify these findings in daily clinical routine we examined 37 patients after cardiplegic arrest during coronary bypass surgery or aortic valve replacement. Patients were divided into 3 groups: defibrillation with low energy (10J), high energy (50J) and no defibrillation (spontaneous rhythm). We monitored systemic vascular resistance as well as plasma concentrations of important vasoconstrictors (noradrenaline, neuropeptide Y, endothelin-1 and active renin) taken from a central vein and from the coronary sinus after defibrillation. In conclusion defibrillation (and the choice of the energy) had no significant influence on total peripheral resistance and didn´t lead to an increased outflow of one of the measured vasoconstrictors.

Ispitivana je koncentracija leptina, neuropeptida Y, kortizola, hormona rasta, i insulina u krvi u cirkadijanom ritmu 19 bolesnika liječenih ponavljanim hemodijalizama prije i poslije primjene eritropoetina.Također je ispitivana povezanost leptina s pokazateljima uhranjenosti dijaliziranih bolesnika, te s duljinom i kakvoćom dijalize.

Primarni oblik u kojem se skladišti potencijalna kemijska energija u organizmu je masno tkivo odnosno trigliceridi. Količina triglicerida u masnom tkivu je kumulativna suma razlika nastala tijekom vremena, između unosa energije (hrana) i utroška energije (potrebe za metabolizam u mirovanju, za unos i obradu hrane te tjelesnu aktivnost). Iako homeostatski mehanizmi drže ovu razliku vrlo blizu nule, vrlo male neravnoteže tijekom dugog vremenskog perioda imaju veliki kumulativni učinak. Pretilosti pridonose i mnogi genetski čimbenici, ali nijedan od njih nije sam po sebi dovoljan za veliki razmjer rizika. Povećana sklonost pretilosti rezultat je višestrukih kombinacija genetskih i okolišnih čimbenika, te čimbenika ponašanja.

Altres ajuts: DGICYT/PM92/0071 Altres ajuts: CIRIT/GRQ94-2005 Hem revisat els sistemes neuroreguladors centrals implicats en 1'ansietat, fent un èmfasi especial en aquell que tradicionalment s'hi ha considerat més relacionat, el sistema GABAèrgic. Es descriuen els principals moduladors de 1'acció GABAèrgica i es discuteix la importància de la composició molecular del complex GABAA/Bz/Cl- en determinar la seva resposta a les manipulations farmacolbgiques. A mes, es fa un resum dels diversos intents realitzats des de línies de recerca diferents per trobar possibles substàncies endògenes que actuïn als receptors benzodiazepinics. Pel que fa a altres sistemes de neuroregulació implicats en 1'ansietat, es revisen les evidencies a favor del paper de la serotonina, la colecistocinina, el factor d'alliberament de la corticotropina, el glutamat i el neuropèptid Y. Finalment es parla de 1'existència de diferències individuals en la susceptibilitat ansiosa i dels intents d'abordar-les mitjançant 1'estudi de la possible contribució genètica en soques de rosegadors que difereixen en la reactivitat emocional.