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Little is known about how diet and energy metabolism interact in determination of lifespan under ad libitum feeding. From 12 weeks of age until death, male and female wild-type (WT) and transgenic (TG) mice with increased skeletal muscle mitochondrial uncoupling (HSA-mUCP1 mice) were fed one of three different semisynthetic diets differing in macronutrient ratio: control (high-carbohydrate/low-fat-HCLF) and two high-fat diets: high-carbohydrate/high-fat (HCHF), and low-carbohydrate/high-fat (LCHF). Compared to control and LCHF, HCHF feeding rapidly and significantly increased body fat content in WT. Median lifespan of WT was decreased by 33% (HCHF) and 7% (LCHF) compared to HCLF. HCHF significantly increased insulin resistance (HOMA) of WT from 24 weeks on compared to control. TG mice had lower lean body mass and increased energy expenditure, insulin sensitivity, and maximum lifespan (+10%) compared to WT. They showed a delayed development of obesity on HCHF but reached similar maximum adiposity as WT. TG median lifespan was only slightly reduced by HCHF (-7%) and unaffected by LCHF compared to control. Correlation analyses showed that decreased longevity was more strongly linked to a high rate of fat gain than to adiposity itself. Furthermore, insulin resistance was negatively and weight-specific energy expenditure was positively correlated with longevity. We conclude that (i) dietary macronutrient ratios strongly affected obesity development, glucose homeostasis, and longevity, (ii) that skeletal muscle mitochondrial uncoupling alleviated the detrimental effects of high-fat diets, and (iii) that early imbalances in energy homeostasis leading to increased insulin resistance are predictive for a decreased lifespan.

Supplementary material 1 (DOCX 15Â kb)

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1−/−) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1−/− mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1−/− mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1+/− mice responded to sweet and umami compounds, whereas those in T1R1−/− mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1−/− than in T1R1+/− mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1−/− and T1R1+/− mice. Conditioned taste aversion tests demonstrated that both T1R1−/− and T1R1+/− mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds.

Um die mentale Repräsentation des Energiemanagements bei der manuellen Flugzeugführung zu untersuchen, wurde eine Simulatorkampagne mit n = 12 lizenzierten Verkehrspiloten durchgeführt. Ihre Aufgabe war es, künstlich generierte Höhen- und Geschwindigkeitsablagen auf dem Gleitpfad des Instrumentenlandesystems (ILS) im Endanflug zu korrigieren. Dabei wurde untersucht, ob und inwiefern sie von dem Prinzip des Energieaustauschs Gebrauch machen. Die Ergebnisse zeigen eine wenig einheitlich ausgeprägte Repräsentation des Wissens bezüglich des Energiemanagements. Des Weiteren war zu erkennen, dass nicht die vollen Möglichkeiten des Energieaustausches genutzt wurden. Vielmehr wurden damit lediglich kleine Korrekturen durchgeführt.

Ill., graph. Darst.

Gegensätzliche Vorstellungen existieren wie Geschmacksinformation vom Mund ins Gehirn weitergeleitet, prozessiert und kodiert wird. Die Übertragung von Informationen verschiedener Geschmacksqualitäten geschieht gemäß der labeled-line Theorie auf getrennten neuronalen Bahnen, oder erfolgt gemäß der across fiber pattern Theorie durch die Aktivitätsmuster von Neuronen, die auf alle Geschmacksstimuli reagieren. Weiterhin wird auch eine temporale Kodierung diskutiert. Zur Erforschung dieser Problematik sollten im Rahmen dieser Arbeit zelluläre gustatorische Netzwerke dargestellt werden. Dazu habe ich Mauslinien erzeugt, die im Tas2r131 Bitterrezeptorlocus bzw. im Locus der spezifischen Untereinheit des Umami-rezeptors, Tas1r1, das transsynaptische Markerprotein Gerstenlektin (BL) sowie stationäre, fluoreszierende Markerproteine exprimieren. Während die Fluoreszenz-proteine die Zellen sichtbar machen, die Tas2r131 oder Tas1r1 exprimieren, findet sich das BL-Protein sowohl in diesen Zellen, als auch in solchen, die mit den Ursprungszellen in (synaptischen) Kontakt stehen. Durch RT-PCR-, in-situ-Hybridisierungs- und immunhistochemische Experimente konnte ich den erfolgreichen Knock-out beider Zielgene sowie den funktionellen Knock-in der Expressionskassetten verifizieren. Die Fluoreszenzproteine wurden erwartungsgemäß spezifisch in der Bitter- bzw. Umami-Zellpopulation oraler Geschmacksknospen exprimiert. Das BL war zusätzlich mit allen anderen untersuchten Zelltypen sowie den afferenten Fasern assoziiert, was für seinen nicht-selektiven Transfer innerhalb der Geschmacksknospen spricht. In peripheren Ganglien sowie im zentralen Nervensystem konnte das BL in Neuronen, in denen keine stationären Fluoreszenzproteine und deren mRNAs beobachtet wurden, nachgewiesen werden. Obwohl diese Ergebnisse auf einen Transfer des BLs von den Rezeptorzellen in die Nervenzellen hindeuten, sprechen die zu geringe Anzahl BL positiver Ganglienneurone und die Verteilung BL positiver zentraler Nervenzellen dagegen. Zur Aufklärung dieser Widersprüche habe ich zwei weitere Mauslinien generiert, die neben epitopmarkierten BL-Varianten die Cre-Rekombinase im Tas2r131- bzw. Tas1r1-Locus exprimieren. Die Kreuzung dieser Mäuse mit Reportermäusen führte in den Nachkommen zum sensitiven Nachweis von Tas2r131- bzw. Tas1r1-exprimierenden Neuronen. Auf diese Weise konnte erstmals eindeutig die Expression von Geschmacksrezeptoren im peripheren und zentralen Nervengewebe auf zellulärer Ebene gezeigt werden. Damit kann aufgrund der mangelnden Selektivität des BL-Transfers in der Geschmackknospe sowie der lokalen BL-Expression im Nervengewebe das vorliegende Verfahren zur Aufklärung der funktionellen Kopplung von Geschmacks-rezeptorzellen und gustatorischen Nerven als ungeeignet eingestuft werden. Die Entdeckung der Existenz von peripheren und zentralen Neuronen, die Geschmacks-rezeptoren exprimieren, war überraschend. Zur Aufklärung ihrer Bedeutung können in zukünftigen Untersuchungen die erzeugten Mausmodelle beitragen. Contrary theories exist how taste information is transferred and processed from the oral cavity to the brain. According to the labeled line theory it is assumed that gustatory information of different taste qualities is transferred on separate neuron populations. Otherwise it is proposed by the across fibre pattern theory that taste information is transferred by a specific neuronal activation pattern involving the identical neuronal population. Moreover, a temporal coding of taste information is also under debate. To investigate these neuronal difficulties I generated mouse lines to visualize the cellular gustatory networks. These gene targeted mice express in the locus of either bitter taste receptor Tas2r131 or the umami receptor specific subunit Tas1r1 the transsynaptic marker protein barley lectin (BL) as well as a fluorescent marker protein. Thereby, cells expressing the fluorescent protein identify Tas2r131 or Tas1r1 expressing cells. However, BL protein is present in BL expressing cells and additionally in cells (synaptically) connected with the expressing cells. Using RT-PCR, in situ hybridization and immunhistochemical experiments I confirmed the successful knock out of these taste receptor genes as well as a functional knock in of the targeting cassettes. As expected, the fluorescent proteins were expressed specifically in the bitter or umami cell population within taste buds. In both models, BL associates with all taste bud cell types and with the gustatory fibres. This point to an unselective transfer of BL within taste buds. Therefore, the observed BL-labeled fibers likely received the transsynaptic marker directly from the source cell as well as indirectly following lateral diffusion preventing identification of taste-specific fibers. Moreover, the number of BL-labeled neuronal somata in the ganglia disagrees with that of connected receptor cells. In addition, although numerous neurons in various gustatory areas contain the tracer, the first order central taste neurons do not or only rarely. Even though we did not detect fluorescent cells and Tas2r131 and Tas1r1 expression by in situ hybridization outside the taste buds, we suspect that the extra-taste bud BL originates from local low level gene expression. To follow up this suspicion we engineered mice to efficiently and faithfully express BL and a red fluorescence protein from the Rosa26 locus in Tas2r131 and Tas1r1 cells. This strategy eventually identified Tas2r131 and Tas1r1 expressing cells in taste buds, the gustatory ganglia, brain stem, and various other gustatory and non-gustatory brain areas. Due to the insufficient selectivity of BL-transfer within taste buds and the local expression of taste receptors in the peripheral and central nervous system make this approach inappropriate to trace gustatory pathways reliable from type II taste receptor cells. The discovery of peripheral and central neurons expressing taste receptors was surprising. Investigations using the generated mouse lines will help to elucidate their relevancy in future projects.

Die vorliegende Arbeit beschreibt die Reaktionen von Nitrenen und Dinitrenen, die durch die Photolyse ihrer aromatischen Azide und Bisazide in einer Novolakmatrix generiert wurden. Ein Reaktionsmechanismus wird vorgestellt. Eine Reihe von aromatischen Aziden und Bisaziden wurden synthetisiert und spektroskopisch charakterisiert. Die Vergrößerung des konjugierten -Systems im Molekül durch die Einführung entsprechender Gruppen zwischen den beiden Phenylringen zeigt sich in der bathochromen Verschiebung der langwelligsten Absorptionsbande zur i-Linie (365 nm) der Quecksilberbestrahlungsquelle. Gleichzeitig wird die Quantenausbeute der Photolyse der Azidgruppen in ein Nitren unter Abspaltung von molekularem Stickstoff reduziert. Novolakdimere und Trimere wurden als Modellverbindungen für ein Novolak mit geringem Molekulargewicht synthetisiert. Struktur-Eigenschafts-Beziehungen dieser Oligomere wurden durch den Einsatz von spektroskopischen und chromatographischen Methoden aufgestellt. Die Photolyse aller untersuchten Bisazide erfolgt ausschließlich biphotonisch, d.h. die Abspaltung der beiden Stickstoffmoleküle erfolgt sequentiell durch die Absorption von zwei Photonen. Die Hauptreaktionsprodukte der in der Novolakmatrix photolytisch generierten Nitrene sind erstens, die primären Amine Aminoazid und Diamin. Zweitens, entstehen sekundäre Amine durch die H-Abstraktion der Nitrene an den Methylenbrücken vom Novolakoligomer und der anschließenden Rekombination der gebildeten Imino- und Kohlenstoffradikale. Aus der Wasserstoffabstraktion der Nitrene an den Hydroxygruppen vom Novolak resultiert die Bildung von Chinoniminderivaten. Die Rekombination von Oligomerradikalen führt zu einem Novolak mit erhöhtem Molekulargewicht. Durch die Anwendung von chromatographischen und spektroskopischen Methoden und der Synthese von Modellverbindungen wurde sowohl die Bildung der Aminoazide in der photochemischen Reaktion der Bisazide, als auch die Reaktion der Nitrene an den Hydroxygruppen des Novolakes erstmalig bewiesen. In this thesis experimental investigations of the reactions of aromatic nitrenes and bisnitrenes generated by photolysis of azides and bisazides in a novolak matrix are presented and a reaction mechanism is proposed. Various bisazides and aminoazides were synthesized and characterized using spectroscopic methods. The extension of the conjugated -system in the molecules attained by introducing appropriate linkage groups between the two phenyl rings is reflected by a bathochromic shift of the absorption maximum towards the i-line (365 nm) of the mercury lamp. At the same time the quantum yield of the photolysis of the azido group leading to a nitrene by extrusion of molecular nitrogen (N2) is reduced. Novolak dimers and trimers used as model compounds for low molecular weight novolaks were synthesized. The relationship between structure and properties of these oligomers was established by means of spectroscopic and chromatographic methods. The photolysis of all investigated bisazides exclusively proceeds by a biphotonic mechanism, i.e. the two nitrogen molecules are sequentially extruded by absorption of two photons. Main reaction products of the photochemically generated nitrenes in the novolak matrix are firstly, the corresponding primary amines, aminoazides and diamines. Secondly, secondary amines are formed by hydrogen abstraction of the nitrenes from the methylene bridges and subsequent recombination of the formed imino radical and the carbon radical. The hydrogen abstraction from the hydroxy group of the novolak oligomers results in the formation of quinonimine derivatives. Recombination of the polymer radicals leads to novolaks with increased molecular weight. Applying chromatographic methods and synthesizing model compounds, the formation of aminoazides in the photochemical reaction of bisazides and the reaction of the nitrenes with the hydroxy group of the novolak were evidenced for the first time.

Supplementary material 2 (DOCX 19Â kb)

Nanocomposites based on an organic polymer and inorganic nanocrystals (NCs) represent a class of high impact functional materials able to convey the unique size and shape dependent properties of nano-objects to highly processable resists.[1] In this work, a novel magnetic nanocomposite based on a negative tone epoxy photoresist and magnetic colloidal Fe2O3 NCs has been manufactured for fabricating AFM probes. Epoxy-type photoresist grant superior lithographic performances when patterned by standard near- ultraviolet (UV) optical lithography, providing structures with high aspect-ratio and nearly vertical sidewalls. Such resists are at present employed in optical and micromechanical applications for the fabrication of optical waveguides, microfluidic systems and scanning probes.[2] However, these materials lack of any inherent functionality, e.g. electrical conductivity, luminescence, magnetism, piezoresistivity and dielectricity. Hence, the incorporation of NCs can confer them new properties maintaining the patterning resolutions required for the manufacturing of highly miniaturized devices. These added properties can be interesting for the fabrication of novel micro/nanoelectromechanical systems (MEMS/NEMS). The current challenge consists on incorporating NCs in the photoresist host matrix to add functionality preserving at the same time the polymer photostructurability. The NC addition has relied on the efficient dispersion of pre-synthesized functional nanosized fillers into the pre-made epoxy photoresist, by using a common solvent as recently reported by the authors in the incorporation of highly luminescent CdSe@ZnS NCs in the same epoxy photoresist.[3] Here, the magnetic properties, UV-photostructurability and mechanical characteristics of the nanocomposite have been investigated. The results show that, after the incorporation, the Fe2O3 NCs preserve their magnetism which is conveyed to the photoresist and retained after the UV-lithography process (Figure 1).

Supplementary material 2 (DOCX 19Â kb)

We report on the synthesis, characterization and application of a novel nanocomposite made of a negative tone epoxy based photoresist modified with organic-capped Fe(2)O(3) nanocrystals (NCs). The mechanical properties of the nanocomposite drastically improve upon incorporation of a suitable concentration of NCs in the polymer, without deteriorating its photolithography performance. High aspect ratio 3D microstructures made of the nanocomposite have been fabricated with a uniform surface morphology and with a resolution down to few micrometres. The embedded organic-capped Fe(2)O(3) NCs drastically increase the stiffness and hardness of the epoxy based photoresist matrix, making the final material extremely interesting for manufacturing miniaturized polymer based mechanical devices and systems. In particular, the nanocomposite has been used as structural material for fabricating photoplastic Atomic Force Microscopy (AFM) probes with integrated tips showing outstanding mechanical response and high resolution imaging performance. The fabricated probes consist of straight cantilevers with low stress-gradient and high quality factors, incorporating sharp polymeric tips. They present considerably improved performance compared to pure epoxy based photoresist AFM probes, and to commercial silicon AFM probes.