Homotrimeric P2X3 and heteromeric P2X2/3 receptors are present in sensory ganglia and participate in pain perception. In order to develop pharmacological antagonists for these receptors, it is important to clarify which amino acid (AA) residues constitute the agonist binding pouch as well as to learn the stoichiometry of the receptor subunits forming a heteromeric receptor. We expressed the homomeric human (h)P2X3 receptor or its mutants in HEK293 cells and measured the ATP-induced responses by the whole-cell patch-clamp method. For the binding-site mutants, all conserved and some non-conserved AAs in the four nucleotide binding segments (NBSs) of the P2X3 subunit were sequentially replaced by alanine. Especially the positively charged AAs Lys and Arg appeared to be of critical importance for the agonist effects. We concluded that groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the P2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor. We were also interested to find out, whether two heteromeric receptors (P2X2/3 and P2X2/6), where P2X2 combines with two different partners, have an obligatory subunit stoichiometry of 1:2 or whether the subunit stoichiometry may be variable. For this purpose we used non-functional P2X2, P2X3 and P2X6 subunit-mutants to investigate the composition of heteromeric P2X2/3 and P2X2/6 receptors. The subunit stoichiometry of P2X2/3 and P2X2/6 was found to be 1:2 and 2:1, respectively. Thus, recognitions sites between P2X2 and its partners rather than random association may govern the subunit compositions of the receptor trimers.:Index of contents Introductory remarks „Wissenschaftlicher Anteil des Promovenden an der Publikation“ „Bibliographische Beschreibung“ I. Introduction Pain as a sensory quality Neuronal circuitry for pain processing and sensation in the PNS and CNS Transformation of thermal, mechanical and chemical stimuli into electrical activity by nociceptors; nociceptor-targeted therapeutic approaches Release mechanisms for nucleotides and their fate in the extracellular space Nucleotide receptor-types ATP-sensitive P2 receptors and pain-sensation References II. Scientific background and aims of my thesis ATP binding-sites of P2X3 receptors; subunit composition of P2X2/3 and P2X2/6 heteromeric receptors The aims of the present work III. Publications IV. Summary and conclusions Amino acid residues constituting the agonist binding site of the human P2X3 receptor ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors „Eigenständigkeitserklärung“ Curriculum vitae Acknowledgements

No / A novel high-performance liquid chromatography (HPLC) assay for the estimation of formoterol in urine samples was developed and validated. A solid phase extraction (SPE) using Oasis HLB was optimised to isolate formoterol from a urine matrix followed by HPLC with UV detection. This extraction procedure concentrated the final analyte forty times so that UV detection can be used to determine even a low concentration of formoterol in urine samples. The urinary assay was performed in accordance with FDA and ICH regulations for the validation of bioanalytical samples. The samples were injected onto a C18 Spherisorb® (250 mm x 4.6 mm x 5 ¿m) analytical column maintained at 30 °C. The mobile phase consisted of 5 mM of potassium dihydrogen orthophosphate buffer (adjusted to pH 3 with ortho phosphoric acid):acetonitrile (ACN) (70:30, v/v), and the formoterol peak was detected at wavelength 214 nm. The extraction recovery of formoterol from the urine sample was >95%. The calibration curve was linear (r2=0.99) over formoterol concentrations ranging from 1.5 to 25 ng/mL (n=6). The method had an accuracy of >92% and intra and inter-day precision CV% of <3.9% and <2.2%, respectively, at three different concentrations low, medium and high (10, 15, 20 ng/mL). The limit of quantification (LOQ) for formoterol was found to be 1.50 ng/mL. The accuracy and precision at the LOQ level were 95% and %CV <3.7% (n = 10), respectively. The method reported is simple, reliable, precise, and accurate and has the capacity to be used for determination of formoterol in urine samples.

Severe perinatal hypoxia represents a substantial brain injury in human newborns. This Diploma thesis is focused on long-term motor outcome of laboratory rat after moderate perinatal hypoxia. We described some behavioral test for detection motor development and presented the influence of perinatal hypoxia on central nervous system. We also discussed an effect of agonists and antagonists of adenosine A1 receptor in brain. The aim of an experimental part was an evaluation of long-term motor behavior in rats affected by perinatal hypoxia. To cause perinatal hypoxia we put pregnant female rats to a hypoxic (10% O2) normobaric room in 11th day of their gestation. The pregnant female rats stayed in hypoxic room until they gave a birth and 6 more days after birth with their litters. For classification of motor development we used battery of tests of motor coordination. These tests correspond to the level of development of the rat. Then a group of rats with perinatal hypoxia was treated by a single administration of an agonist of adenosine A1 receptor 2-chloro-N(6)- cyclopentyladenosin (CCPA) in postnatal day 14. The animals affected by perinatal hypoxia show motor deficits in 3 from 4 selected behavioral tests. Otherwise, this motor behavior was no longer detected in young adults. The rats affected by...

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Major depressive disorder (MDD) is a mood disorder majorly responsible for disability and mortality worldwide. With a lifetime prevalence of 15-20%, it is the main cause of functional impairment in Western societies as well as the fourth most debilitating illness in the world. Although the pathophysiology of MDD is not yet fully understood, some evidence that suggest the presence of a neuroanatomical deficiency have given rise to the theory of a specific imbalance in the monoamine neurotransmitters noradrenaline (NA) and/or serotonin (5-HT) levels in the brain. Overall, the various classes of antidepressant agents that have been developed to increase monoamine levels on the basis of this proposal have been successful. However, facts relating to prevalent escalation in the illness and recurring episodes of depression point towards a need to enhance clinical treatment. Most conventional antidepressants such as selective serotonin reuptake inhibitors (SSRI) and selective serotonin and noradrenaline inhibitors (SNRI) pose problems in symptomatic improvement. These include therapeutic lag, safety and tolerability issues, making more than 30% patients with MDD unable to reach adequate relief. In this respect, the action mechanism has moved beyond conventional SSRI and lead to the introduction of vilazodone, a novel antidepressant with an additional 5-HT1A partial agonist profile argued to be of potential benefit for a greater efficacy, faster onset of action and better tolerability. Using secondary data, this project aimed to evaluate the role of vilazodone as a SPARI-drug in the overall clinical treatment of MDD as well as its potential in addressing some of the most common obstacles in antidepressant treatment. Study results proved vilazodone’s efficacy to be superior to placebo. Patients across all studies showed significant improvement in depressive symptoms measured in MADRS and HAMD17. Vilazodone was also shown to be generally safe and tolerable but was not positively distinguished from placebo with regards to adverse effects. An overall, meaningful improvement in depressive symptoms was demonstrated in vilazodone, which reinforces its merit as an important treatment option for patients with MDD.

Aufgabe der elektrophysiologischen Studie zur Charakterisierung der neuronalen hypothalamischen Plastizität beim Haushuhn war es, den Einfluss des Alters sowie GABAerger Substanzen auf die Feuerrate und die Temperatursensitivität (thermischer Koeffizient: TC) von Hypothalamusneuronen mittels extrazellulärer Ableitungen in Hirnschnitten zu untersuchen. Im Vergleich zu adulten Vögeln und Säugetieren wurde bei juvenilen Hühnern eine hohe neuronale Kältesensitivität nachgewiesen, die offensichtlich eine spezifische Eigenschaft juveniler Vögel ist. Die Ontogenese der neuronalen hypothalamischen Thermosensitivität ist deutlich artspezifisch. Einige Neurone wiesen eine inherente Kältesensitivität auf. Eine mögliche zentrale Rolle kältesensitiver Neurone im Rahmen der Thermoregulation juveniler Hühner wurde postuliert. Muscimol und Baclofen hemmen signifikant die Feuerrate der Hypothalamusneurone, unabhängig von der jeweiligen Thermosensitivität. Demgegenüber bewirken Bicucullin und CGP35348 einem Anstieg der Feuerrate. Nur bei kältesensitiven Neuronen wurde der TC signifikant durch GABAB-Rezeptor-Liganden verändert (signifikant erhöht durch Baclofen und durch CGP35348 gehemmt). Der Effekt von Muscimol und Baclofen auf Feuerrate und TC wurde durch Co-Perfusion mit einer 10-fach höheren Konzentration der entsprechenden Antagonisten Bicucullin und CGP35348 aufgehoben. Der wesentliche GABAerge Einfluss auf thermosensitive und –insensitive Hypothalamusneurone ist mit dem bei Säugetieren nachgewiesenen vergleichbar. Der einzige Unterschied betrifft die GABAB-Rezeptor vermittelte Änderung des TC. Beim Hühnerküken betraf dies die kältesensitiven und beim Säugetier die wärmesensitiven Neurone. Der grundlegende Mechanismus der GABAergen Beeinflussung thermosensitiver und –insensitiver Neurone scheint einen älteren evolutionären Ursprung zu haben. Eine funktionelle Rolle GABAerger Substanzen im Rahmen der zentralen Kontrolle der Körpertemperatur beim Vogel ist möglich. / In the present electrophysiological studies, characterization of neuronal hypothalamic plasticity in the chicken aims to investigate the influence of age during development by extracellular recordings. High neuronal cold sensitivity has been found in juvenile chicken in contrast to adult mammals and birds. High hypothalamic cold sensitivity seems to be a specific characteristic feature in juvenile birds. Between species a species specificity of the early development of neuronal hypothalamic thermosensitivity could be clearly demonstrated. Existence of inherent nature to a certain degree suggests a possible thermoregulatory role of cold-sensitive neurons in chicken. The effects of the GABAergic substances on neuronal tonic activity (firing rate) and temperature sensitivity (temperature coefficient) in hypothalamic neurons have been examined. Muscimol and baclofen in equimolar concentrations significantly inhibited tonic activity, regardless of their type of thermosensitivity. In contrast bicuculline and CGP 35348 increased firing rate. Temperature coefficient was significantly changed by ligands of GABAB receptors, restricted to cold-sensitive neurons. The TC was significantly increased by baclofen and significantly decreased by CGP 35348. Effects of muscimol and baclofen on firing rate and TC were prevented by co-perfusion of appropriate antagonists bicuculline and CGP 35348, respectively in tenfold higher concentration. Thus the main effects of GABA in chicken are similar with that described in mammals. The only difference is in respect of the GABAB receptors mediated change restricted to cold-sensitive neurons in chicken but in mammals only seen in warm-sensitive neurons. However, the results indicate that the fundamental mechanism of GABAergic influence in chicken are conserved during evolution. The response of hypothalamic neurons to temperature changes suggest a possible functional role of GABAergic substances in the control of body temperature in birds.

Type 2 diabetes is still rapidly on the rise today, affecting 10.5% of individuals in the United States between the ages 45 to 64 and 18.4% of those between the ages of 65 to 74. In the past two decades, type 2 diabetes has doubled in all age groups. Many adults with type 2 diabetes experience difficulty managing their blood sugars, which can result in a range of further complications. One of the newest treatment options on the market today is a glucagon-like peptide-1 (GLP-1) receptor agonist, Bydureon. Similar to Byetta, Bydureon has a main ingredient of exenatide. It offers once a week dosing as opposed to twice-a-day, which may be more appealing to patients. The purpose of this study was to examine the efficacy of a newly FDA released medication, Bydureon, once weekly dosage in adults with type 2 diabetes. A descriptive, comparative, retrospective study of 35 patients evaluated efficacy by examining Hgb A1C and body mass index in adults with type 2 diabetes at baseline and 3 months after Bydureon was prescribed. Data were collected by a chart review of records in a primary care practice. Results demonstrated a statistically significant difference between baseline to 3 month means in both Hgb A1C (t (34)= -3.05, p=.0044) and BMI (t (34) = -2.86, p = .0072) for patients using Bydureon. Health care providers need to individualize the patients’ plans of care to address multifactorial areas of their diabetes care and provide them with an opportunity to successfully meet their goals. Practitioners must be knowledgeable about the treatment options available, including the newer GLP-1 receptor agonist, Bydureon and its efficacy for adults with type 2 diabetes.