On the development of novel cocaine-analogues for in vivo imaging of the dopamine transporter status

The present thesis is concerned with the development of novel cocaine-derived dopamine transporter ligands for the non-invasive exploration of the striatal and extra-striatal dopamine transporter (DAT) in living systems. The presynaptic dopamine transporter acquires an important function within the mediation of dopaminergic signal transduction. Its availability can serve as a measure for the overall integrity of the dopaminergic system. The DAT is upregulated in early Parkinson’s disease (PD), resulting in an increased availability of DAT-binding sites in the striatal DAT domains. Thereby, DAT imaging has become an important routine diagnostic tool for the early diagnosis of PD in patients, as well as for the differentiation of PD from symptomatically similar medical conditions. Furthermore, the dopaminergic system is involved in a variety of psychiatric diseases. In this regard, DAT-selective imaging agents may provide detailed insights into the scientific understanding of the biochemical background of both, the progress as well as the origins of the symptoms. DAT-imaging may also contribute to the determination of the dopaminergic therapeutic response for a given medication and thereby contribute to more convenient conditions for the patient. From an imaging point of view, the former demands a high availability of the radioactive probe to facilitate broad application of the modality, whereas the latter profits from short-lived probes, suitable for multi-injection studies. Therefore, labelling with longer-lived 18F-fluoride and in particular the generator nuclide 68Ga is worthwhile for clinical routine imaging. In contrast, the introduction of a 11C-label is a prerequisite for detailed scientific studies of neuronal interactions. The development of suitable DAT-ligands for medical imaging has often been complicated by the mixed binding profile of many compounds that that interact with the DAT. Other drawbacks have included high non-specific binding, extensive metabolism and slow accumulation in the DAT-rich brain areas. However, some recent examples have partially overcome the mentioned complications. Based on the structural speciality of these leads, novel ligand structures were designed and successfully synthesised in the present work. A structure activity relationship (SAR) study was conducted wherein the new structural modifications were examined for their influence on DAT-affinity and selectivity. Two of the compounds showed improvements in in vitro affinity for the DAT as well as selectivity versus the serotonin transporter (SERT) and norepinephrine transporter (NET). The main effort was focussed on the high-affinity candidate PR04.MZ, which was subsequently labelled with 18F and 11C in high yield. An initial pharmacological characterisation of PR04.MZ in rodents revealed highly specific binding to the target brain structures. As a result of low non-specific binding, the DAT-rich striatal area was clearly visualised by autoradiography and µPET. Furthermore, the radioactivity uptake into the DAT-rich brain regions was rapid and indicated fast binding equilibrium. No radioactive metabolite was found in the rat brain. [18F]PR04.MZ and [11C]PR04.MZ were compared in the primate brain and the plasma metabolism was studied. It was found that the ligands specifically visualise the DAT in high and low density in the primate brain. The activity uptake was rapid and quantitative evaluation by Logan graphical analysis and simplified reference tissue model was possible after a scanning time of 30 min. These results further reflect the good characteristics of PR04.MZ as a selective ligand of the neuronal DAT. To pursue 68Ga-labelling of the DAT, initial synthetic studies were performed as part of the present thesis. Thereby, a concept for the convenient preparation of novel bifunctional chelators (BFCs) was developed. Furthermore, the suitability of novel 1,4,7-triazacyclononane based N3S3-type BFCs for biomolecule-chelator conjugates of sufficient lipophilicity for the penetration of the blood-brain-barrier was elucidated.

Molecular phylogenetic inferences on the position of the Mollusca within the Lophotrochozoa

Die phylogenetische Position der Mollusken innerhalb der Trochozoa sowie die interne Evolution der Klassen der Mollusca sind weitgehend unbekannt und wurden in meiner Arbeit anhand molekularer Merkmale untersucht. Phylogenomische Analysen zeigten in der Vergangenheit eine gute Auflösung für ursprüngliche Speziationsereignisse. Daher wurden hier drei neue EST Datensätze generiert: für Sipunculus nudus (Sipuncula), Barentsia elongata (Kamptozoa) und Lepidochitona cinerea, (Polyplacophora, Mollusca). Zusätzlich wurden gezielt Gene verschiedener Mollusken mittels RT-PCR amplifiziert. rnSowohl Kamptozoen als auch Sipunculiden wurden aufgrund morphologischer Kriterien bisher als mögliche Schwestergruppe der Mollusken gehandelt, aber die hier erzielten Ergebnisse zur Evolution der Hämerythrine, Gen-Anordnungen der mitochondrialen Genome und phylogenetische Analysen der ribosomalen und der mitochondriellen Proteine stützen diese Hypothese nicht. Die Position der Kamptozoa erwies sich hier generell als unbeständig; phylogenomische Analysen deuten eine Nähe zu den Bryozoen an, aber diese Position wird stark durch die Auswahl der Taxa beeinflusst. Dagegen weisen meine Analysen klar auf eine nähere Beziehung zwischen Annelida und Sipuncula hin. Die ribosomalen Proteine zeigen Sipuncula (und Echiura) sogar als Subtaxa der Anneliden. Wie den Mollusken fehlt den Sipunculiden jegliche Segmentierung und meine Ergebnisse legen hier die Möglichkeit des Verlusts dieses Merkmals innerhalb der Anneliden bei den Sipunculiden nahe. Innerhalb der Mollusken wurden die Solenogastren bereits als Schwestergruppe aller rezenten Mollusken vorgeschlagen. Im Rahmen meiner Arbeit wurden von drei verschiedenen Solenogastren-Arten die ersten zuverlässigen 18S rRNA-Sequenzen ermittelt, und es zeigte sich, dass alle bisher veröffentlichten 18S-Sequenzen dieser Molluskenklasse höchst unvollständig oder fehlerhaft sind. rnRibosomale Proteine sind gute phylogenetische Marker und hier wurden die Auswahl und Anzahl dieser Gene für phylogenetische Analysen optimiert. Über Sonden-basierte Detektion wurde eine sampling-Strategie getestet, die im Vergleich mit standard-phylogenomischen Ansätzen zukünftige molekulare Stammbaumrekonstruktionen mit größerem Taxonsampling ermöglicht.rn

Influence of ice crystal habit and cirrus spatial inhomogeneities on the retrieval of cirrus optical thickness and effective radius

Diese Dissertation untersucht den Einfluss von Eiskristallform und räumlicher Inhomogenität von Zirren auf das Retrieval von optischer Wolkendicke und effektivem Eispartikelradius. Zu diesem Zweck werden flugzeuggetragene spektrale Messungen solarer Strahlung sowie solare und langwellige Strahlungstransfersimulationen durchgeführt. Flugzeuggetragene spektrale aufwärtsgerichtete Radianzen (Strahldichten) sind mit dem SMART-Albedometer (Spectral Modular Airborne Radiation measurement sysTem) während des CIRCLE-2 (CIRrus CLoud Experiment-2) Feldexperiments im Mai 2007 gemessen worden. Basierend auf diesen Radianzdaten werden mittels eines Wolkenretrievalalgorithmus optische Wolkendicken und effektive Eispartikelradien anhand von eindimensionalen Strahlungstransferrechnungen bestimmt. Die Auswirkung der Annahme unterschiedlicher Eiskristallformen auf die retrievten Parameter wird durch Variation der Einfachstreueigenschaften der Eispartikel untersucht. Darüber hinaus wird mittels Strahlungstransferrechnungen auch der Einfluss der Eiskristallform auf den Strahlungsantrieb von Eiswolken ermittelt. Die Frage nach dem relativen Einfluss von räumlicher Wolkeninhomogenität und Eiskristallform wird anhand von dreidimensionalen und independent pixel approximation (IPA) Strahlungssimulationen untersucht. Die Analyse basiert auf einer Modelleiswolke, die aus Daten des NASA (National Aeronautics and Space Administration) TC4 (Tropical Composition, Cloud, and Climate Coupling) Feldexperiments im Sommer 2007 in Costa Rica erzeugt wurde. Lokal gesehen können beide Effekte - Eiskristallform und räumliche Eiswolkeninhomogenität - die gleiche Grössenordnung haben und zu einer Unter- bzw. Überschätzung der retrievten Parameter um 40 – 60% führen. Gemittelt über die ganze Wolke ist jedoch der Einfluss der Eiskristallform viel bedeutender als der von räumlichen Inhomogenitäten.

On the inheritance and mechanism of baculovirus resistance of the codling moth, Cydia pomonella (L.)

Das Cydia pomonella Granulovirus (CpGV, Baculoviridae) wird seit Ende der 1980er Jahre als hoch-selektives und effizientes biologisches Bekämpfungsmittel zur Kontrolle des Apfelwicklers im Obstanbau eingesetzt. Seit 2004 wurden in Europa verschiedene Apfelwicklerpopulationen beobachtet die resistent gegenüber dem hauptsächlich angewendeten Isolat CpGV-M aufweisen. Die vorliegende Arbeit befasst sich mit der Untersuchung der Vererbung und des Mechanismus der CpGV Resistenz. Einzelpaarkreuzungen zwischen einem empfindlichen Laborstamm (CpS) und einem homogen resistenten Stamm (CpRR1) zeigten, dass die Resistenz durch ein einziges dominantes Gen, das auf dem Z-Chromosom lokalisiert ist, vererbt wird. Massernkreuzungen zwischen CpS und einer heterogen resistenten Feldpopulation (CpR) deuteten zunächst auf einen unvollständig dominanten autosomalen Erbgang hin. Einzelpaarkreuzungen zwischen CpS und CpR bewiesen jedoch, dass die Resistenz in CpR ebenfalls monogen dominant und geschlechtsgebunden auf dem Z-Chromosom vererbt wird. Diese Arbeit diskutiert zudem die Vor- und Nachteile von Einzelpaarkreuzungen gegenüber Massernkreuzungen bei der Untersuchung von Vererbungsmechanismen. Die Wirksamkeit eines neuen CpGV Isolates aus dem Iran (CpGV-I12) gegenüber CpRR1 Larven, wurde in Bioassays getestet. Die Ergebnisse zeigen, dass CpGV-I12 die Resistenz in allen Larvenstadien von CpRR1 brechen kann und fast so gut wirkt wie CpGV-M gegenüber CpS Larven. Daher ist CpGV-I12 für die Kontrolle des Apfelwicklers in Anlagen wo die Resistenz aufgetreten ist geeignet. Um den der CpGV Resistenz zugrunde liegenden Mechanismus zu untersuchen, wurden vier verschiedene Experimente durchgeführt: 1) die peritrophische Membran degradiert indem ein optischer Aufheller dem virus-enthaltenden Futtermedium beigefügt wurde. Das Entfernen dieser mechanischen Schutzbarriere, die den Mitteldarm auskleidet, führte allerdings nicht zu einer Reduzierung der Resistenz in CpR Larven. Demnach ist die peritrophische Membran nicht am Resistenzmechanismus beteiligt. 2) Die Injektion von Budded Virus in das Hämocoel führte nicht zur Brechung der Resistenz. Folglich die die Resistenz nicht auf den Mitteldarm beschränkt, sondern auch in der Sekundärinfektion wirksam. 3) Die Replikation von CpGV in verschiedenen Geweben (Mitteldarm, Hämolymphe und Fettkörper) von CpS und CpRR1 wurde mittels quantitativer PCR verfolgt. In CpS Larven konnte in allen drei Gewebetypen sowohl nach oraler als auch nach intra-hämocoelarer Infektion eine Zunahme der CpGV Genome in Abhängigkeit der Zeit festgestellt werden. Dagegen konnte in den Geweben aus CpRR1 nach oraler sowie intra-hämocoelarer Infektion keine Virusreplikation detektiert werden. Dies deutet darauf hin, dass die CpGV Resistenz in allen Zelltypen präsent ist. 4) Um zu untersuchen ob ein humoraler Faktor in der Hämolymphe ursächlich an der Resistenz beteiligt ist, wurde Hämolymphe aus CpRR1 Larven in CpS Larven injiziert und diese anschließend oral mit CpGV infiziert. Es konnte jedoch keine Immunreaktion beobachtet und kein Faktor in der Hämolymphe identifiziert werden, der Resistenz induzieren könnte. Auf Grundlage dieser Ergebnisse kann festgestellt werden, dass in resistenten Apfelwicklerlarven die virale Replikation in allen Zelltypen verhindert wird, was auf eine Virus-Zell Inkompatibilität hinweist. Da in CpRR1 keine DNA Replikation beobachtet wurde, wird die CpGV Resistenz wahrscheinlich durch eine frühe Unterbindung der Virusreplikation verursacht.Das früh exprimierte Gen pe38 codiert für ein Protein, das wahrscheinlich für die Resistenzbrechung durch CpGV-I12 verantwortlich ist. Interaktionen zwischen dem Protein PE38 und Proteinen in CpRR1 wurden mit Hilfe des Yeast Two-Hybrid (Y2H) Systems untersucht. Die detektierten Interaktionen sind noch nicht durch andere Methoden bestätigt, jedoch wurden zwei mögliche Gene auf dem Z-Chromosom und eines auf Chromosom 15 gefunden, wie möglicherweise an der CpGV Resistenz beteiligt sind.

Laboratory study on the optical properties of absorbing atmospheric aerosols and field applications

The interaction between aerosols and sun light plays an important role in the radiative balance of Earth’s atmosphere. This interaction is obtained by measuring the removal (extinction), redistribution (scattering), and transformation into heat (absorption) of light by the aerosols; i.e. their optical properties. Knowledge of these properties is crucial for our understanding of the atmospheric system. rn Light absorption by aerosols is a major contributor to the direct and indirect effects on our climate system, and an accurate and sensitive measurement method is crucial to further our understanding. A homebuilt photoacoustic sensor (PAS), measuring at a 532nm wavelength, was fully characterized and its functionality validated for measurements of absorbing aerosols. The optical absorption cross-sections of absorbing polystyrene latex spheres, to be used as a standard for aerosol absorption measurements, were measured and compared to literature values. Additionally, a calibration method using absorbing aerosol of known complex refractive index was presented.rn A new approach to retrieve the effective broadband refractive indices (mbroad,eff) of aerosol particles by a white light aerosol spectrometer (WELAS) optical particle counter (OPC) was achieved. Using a tandem differential mobility analyzer (DMA)-OPC system, the nbroad,eff are obtained for both laboratory and field applications. This method was tested in the laboratory using substances with a wide range of optical properties and it was used in ambient measurements to retrieve the nbroad,eff of biomass burning aerosols in a nationwide burning event in Israel. The retrieved effective broadband refractive indices for laboratory generated scattering aerosols were: ammonium sulfate (AS), glutaric acid (GA), and sodium chloride, all within 4% of literature values. For absorbing substances, nigrosine and various mixtures of nigrosine with AS and GA were measured, as well as a lightly absorbing substance, Suwannee river fulvic acid (SRFA). For the ambient measurements, the calibration curves generated from this method were to follow the optical evolution of biomass burning (BB) aerosols. A decrease in the overall aerosol absorption and scattering for aged aerosols during the day after the fires compared to the smoldering phase of the fires was found. rn The connection between light extinction of aerosols, their chemical composition and hygroscopicity for particles with different degrees of absorption was studied. The extinction cross-section (σext) at 532nm for different mobility diameters was measured at 80% and 90% relative humidity (RH), and at an RH<10%. The ratio of the humidified aerosols to the dry ones, fRHext(%RH,Dry), is presented. For purely scattering aerosols, fRHext(%RH,Dry) is inversely proportional with size; this dependence was suppressed for lightly absorbing ones. In addition, the validity of the mixing rules for water soluble absorbing aerosols is explored. The difference between the derived and calculated real parts of the complex RIs were less than 5.3% for all substances, wavelengths, and RHs. The obtained imaginary parts for the retrieved and calculated RIs were in good agreement with each other, and well within the measurement errors of retrieval from pulsed CRD spectroscopy measurements. Finally, a core-shell structure model is also used to explore the differences between the models, for substances with low growth factors, under these hydration conditions. It was found that at 80% RH and for size parameters less than 2.5, there is less than a 5 % difference between the extinction efficiencies calculated with both models. This difference is within measurement errors; hence, there is no significant difference between the models in this case. However, for greater size parameters the difference can be up to 10%. For 90% RH the differences below a size parameter of 2.5 were up to 7%.rn Finally, the fully characterized PAS together with a cavity ring down spectrometer (CRD), were used to study the optical properties of soot and secondary organic aerosol (SOA) during the SOOT-11 project in the AIDA chamber in Karlsruhe, Germany. The fresh fractal-like soot particles were allowed to coagulate for 28 hours before stepwise coating them with SOA. The single scattering albedo for fresh fractal-like soot was measured to be 0.2 (±0.03), and after allowing the soot to coagulate for 28 hours and coating it with SOA, it increased to 0.71(±0.01). An absorption enhancement of the coated soot of up to 1.71 (±0.03) times from the non-coated coagulated soot was directly measured with the PAS. Monodisperse measurements of SOA and soot coated with SOA were performed to derive the complex refractive index (m) of both aerosols. A complex refractive index of m = 1.471(±0.008) + i0.0(±0.002) for the SOA-αO3 was retrieved. For the compact coagulated soot a preliminary complex refractive index of m = 2.04(+0.21/-0.14) + i0.34(+0.18/-0.06) with 10nm(+4/-6) coating thickness was retrieved.rn These detail properties can be use by modelers to decrease uncertainties in assessing climatic impacts of the different species and to improve weather forecasting.rn

The NreABC system of Staphylococcus carnosus combines nitrate and oxygen sensing by an NreA,NreB sensor complex

Staphylococcus carnosus is a facultative anaerobic bacterium which features the cytoplasmic NreABC system. It is necessary for regulation of nitrate respiration and the nitrate reductase gene narG in response to oxygen and nitrate availability. NreB is a sensor kinase of a two-component system and represents the oxygen sensor of the system. It binds an oxygen labile [4Fe-4S]2+ cluster under anaerobic conditions. NreB autophosphorylates and phosphoryl transfer activates the response regulator NreC which induces narG expression. The third component of the Nre system is the nitrate receptor NreA. In this study the role of the nitrate receptor protein NreA in nitrate regulation and its functional and physiological effect on oxygen regulation and interaction with the NreBC two-component system were detected. In vivo, a reporter gene assay for measuring expression of the NreABC regulated nitrate reductase gene narG was used for quantitative evaluation of NreA function. Maximal narG expression in wild type S. carnosus required anaerobic conditions and the presence of nitrate. Deletion of nreA allowed expression of narG under aerobic conditions, and under anaerobic conditions nitrate was no longer required for maximal induction. This indicates that NreA is a nitrate regulated inhibitor of narG expression. Purified NreA and variant NreA(Y95A) inhibited the autophosphorylation of anaerobic NreB in part and completely, respectively. Neither NreA nor NreA(Y95A) stimulated dephosphorylation of NreB-phosphate, however. Inhibition of phosphorylation was relieved completely when NreA with bound nitrate (NreA•[NO3-]) was used. The same effects of NreA were monitored with aerobically isolated Fe-S-less NreB, which indicates that NreA does not have an influence on the iron-sulfur cluster of NreB. In summary, the data of this study show that NreA interacts with the oxygen sensor NreB and controls its phosphorylation level in a nitrate dependent manner. This modulation of NreB-function by NreA and nitrate results in nitrate/oxygen co-sensing by an NreA/NreB sensory unit. It transmits the regulatory signal from oxygen and nitrate in a joint signal to target promoters. Therefore, nitrate and oxygen regulation of nitrate dissimilation follows a new mode of regulation not present in other facultative anaerobic bacteria.

Targeting neuronal populations by AAV-mediated gene transfer for studying the endocannabinoid system

The cannabinoid type 1 (CB1) receptor is involved in a plethora of physiological functions and heterogeneously expressed on different neuronal populations. Several conditional loss-of-function studies revealed distinct effects of CB1 receptor signaling on glutamatergic and GABAergic neurons, respectively. To gain a comprehensive picture of CB1 receptor-mediated effects, the present study aimed at developing a gain-of-function approach, which complements conditional loss-of-function studies. Therefore, adeno-associated virus (AAV)-mediated gene delivery and Cre-mediated recombination were combined to recreate an innovative method, which ensures region- and cell type-specific transgene expression in the brain. This method was used to overexpress the CB1 receptor in glutamatergic pyramidal neurons of the mouse hippocampus. Enhanced CB1 receptor activity at glutamatergic terminals caused impairment in hippocampus-dependent memory performance. On the other hand, elevated CB1 receptor levels provoked an increased protection against kainic acid-induced seizures and against excitotoxic neuronal cell death. This finding indicates the protective role of CB1 receptor on hippocampal glutamatergic terminals as a molecular stout guard in controlling excessive neuronal network activity. Hence, CB1 receptor on glutamatergic hippocampal neurons may represent a target for novel agents to restrain excitotoxic events and to treat neurodegenerative diseases. Endocannabinoid synthesizing and degrading enzymes tightly regulate endocannabinoid signaling, and thus, represent a promising therapeutic target. To further elucidate the precise function of the 2-AG degrading enzyme monoacylglycerol lipase (MAGL), MAGL was overexpressed specifically in hippocampal pyramidal neurons. This genetic modification resulted in highly increased MAGL activity accompanied by a 50 % decrease in 2-AG levels without affecting the content of arachidonic acid and anandamide. Elevated MAGL protein levels at glutamatergic terminals eliminated depolarization-induced suppression of excitation (DSE), while depolarization-induced suppression of inhibition (DSI) was unchanged. This result indicates that the on-demand availability of the endocannabinoid 2-AG is crucial for short-term plasticity at glutamatergic synapses in the hippocampus. Mice overexpressing MAGL exhibited elevated corticosterone levels under basal conditions and an increase in anxiety-like behavior, but surprisingly, showed no changes in aversive memory formation and in seizure susceptibility. This finding suggests that 2 AG-mediated hippocampal DSE is essential for adapting to aversive situations, but is not required to form aversive memory and to protect against kainic acid-induced seizures. Thus, specific inhibition of MAGL expressed in hippocampal pyramidal neurons may represent a potential treatment strategy for anxiety and stress disorders. Finally, the method of AAV-mediated cell type-specific transgene expression was advanced to allow drug-inducible and reversible transgene expression. Therefore, elements of the tetracycline-controlled gene expression system were incorporated in our “conditional” AAV vector. This approach showed that transgene expression is switched on after drug application and that background activity in the uninduced state was only detectable in scattered cells of the hippocampus. Thus, this AAV vector will proof useful for future research applications and gene therapy approaches.