Group membership and its adverse psychological effects in \u3cem\u3eThe Ox-Bow Incident\u3c/em\u3e

This paper examines and analyzes the causes and motivations of groups in the book The Ox-Bow Incident

Radical Right Populist Parties in Britain and the Netherlands: Explaining Electoral Success

Radical right-wing populist parties have recently emerged throughout Europe, but the electoral success among these parties is incredibly inconsistent. In the Netherlands, Geert Wilders’ Dutch Party for Freedom (PVV) has become established in the country’s political system, while the British National Party (BNP) and the United Kingdom Independence Party (UKIP) struggle to gain even a single seat in British parliament. Models outlining a formula for the rise and success of populist parties can help explain why some parties achieve an electoral breakthrough and others do not. Researcher of radical right populism Pippa Norris’ model of electoral success is divided into a political demand side that focuses on the public grievances driving these parties, and a political supply side that focuses on internal party activity as well as external factors shaping opportunity structure. This essay compares Britain’s two radical right populist parties, the BNP and the UKIP, with the PVV in the Netherlands, and applies Norris’ framework to explain the greater electoral success of the PVV. It concludes that while Britain and Netherlands are similar in terms of political demand, populist parties have seen more success in the Netherlands because supply-side factors are more favorable

Simulation of the Eemian Greenland ice sheet

This thesis focuses on the simulation of the Greenland ice sheet (GrIS) during the Eemian interglacial period (~125,000 years ago). The warm Eemian summers on Greenland are used as a past analogue for future warmer conditions. The aim of this work is a contribution to the improvement of future sea level rise predictions and to better understand how model uncertainties propagate through the chain of models necessary to simulate ice sheet evolution in past climates. Firstly, the influence of surface mass balance (SMB) models and climate model resolution on the simulation of the Eemian SMB is investigated. The corresponding study shows that both, the selection of the SMB model as well as the climate model resolution are essential for simulating the Eemian SMB, and either of these two factors can have a dominating effect on the results. However, which factor dominates the results depends on the climate state (cold or warm) and particularly the prevailing insolation regime. It is shown that an inclusion of insolation in the selected SMB model is essential for the simulated warm early Eemian conditions. Secondly, the influence of SMB forcing on millennial time scale ice sheet modeling is tested. The simulations with two different SMB forcings reveal a large difference in the evolution of the ice sheet, while ice flow sensitivity tests with changed basal friction and changed ice flow approximation show small differences. Thirdly, regional climate simulations with a full surface energy balance model are analyzed focusing on Greenland surface melt. This analysis shows that all Greenland ice core locations, also GRIP near the summit of Greenland, are affected by surface melt during the Eemian interglacial period. Elevated levels of Eemian surface melt indicate that ice cores might be affected more strongly than previously considered. Therefore, caution needs to be applied when interpreting Greenland ice core records from warm periods such as the Eemian interglacial period. This thesis shows that forcing from a single climate model can lead to a wide range of SMBs and ice sheets. To quantify this large uncertainty, a systematic approach of model intercomparison, similar to what is used to constrain future climate projections, is advised. Climate and SMB model biases and uncertainties need to be explored and outliers rejected, to be able to provide a most likely range for the Eemian GrIS topography and its contribution to sea level

Judicial Practice Makes Perfect: Explaining Asylum Recognition in the European Union

Vast disparities in asylum recognition rates have persisted in the European Union despite legislative efforts to standardize the asylum determination process. National judiciaries play an important role in this process and scholars mostly agree that differences in judicial practice pose a challenge to the harmonization of recognition rates. However, no study has specifically analyzed the relationship between these two variables. The aim of this research is to determine whether differences in judicial practice account for the variation in asylum recognition rates in the EU. To observe these differences, precedent relating to three areas of the refugee determination process is identified in selected EU states. Application of this precedent is then analyzed in order to identify restrictive judicial practices. Analyses reveal that differences in judicial practice impact the outcomes of asylum cases, and therefore recognition rates

A review of hydrophilic silicon wafer bonding

Hydrophilically activated direct wafer bonding is a technique for gluelessly attaching oxide-coated wafers together. This ability is a vital step in the construction of many microelectronic and microelectromechanical (MEMS) devices. In particular this technique is widely used in the production of 3d interconnected devices due to the lack of interlayer

Serotonin 5-HT2A - Dopamin D2-Rezeptor-Heterodimere: Charakterisierung und Funktionelle Untersuchung

A large number of therapeutically used drugs unfold their desired effect by addressing heptahelical, integral membrane receptors, known as G protein-coupled receptors (GPCRs). These act as translators of external signals to the inside of the cell through activation of cytosolic effectors such as G proteins or β-arrestins. When coupled to an activated GPCR, these effector proteins can initiate stimulatory or inhibitory signaling pathways resulting in specific cellular responses. Experimental findings of the last three decades suggest that individual GPCRs are able to physically interact and thus form functionally active receptor complexes, which are termed heterodimers if they consist of two different types of receptors. Characteristically, such receptor assemblies exhibit properties that differ significantly from those of the corresponding monomers.16,17,23,35 It is a fundamental question whether heteromerization of GPCRs, which has mostly been observed and described in vitro, is of physiological or pathophysiological relevance. Demonstrating the existence of GPCR complexes in primary tissue or even in living organisms would be a strong indication that they are likely involved in pharmacologically interesting biochemical processes.218 In the brain, the dopaminergic and the serotonergic pathways overlap mainly in the striatum, cerebral cortex and the basal ganglia. Therefore, both neurotransmitter systems interact and jointly control important neurological tasks such as motor function, mood or cognition. The widely expressed dopamine D2 and serotonin 5-HT2A receptors play central roles in mediating the effects of their innate monoamine ligands. For instance in the therapy of schizophrenia, both receptors are the primary targets of many first-line antipsychotics.129,192 Furthermore, 5-HT2AR-D2R complexes have been identified in transfected cells as well as in different areas of the rat brain and have been shown to signal characteristically due to modulatory crosstalk.44,226–228 Since 5-HT2AR and D2R are key elements in the treatment of neuropsychiatric disorders, the motivation of this thesis was to provide information about the characteristics of 5-HT2AR-D2R signaling and to evaluate whether formation of 5-HT2AR-D2R heteroreceptor complexes contributes to impaired neurological mechanisms. After validating the interaction of 5-HT2AR and D2R, we investigated 5-HT2AR-D2R-expressing cells regarding G protein-mediated signal transduction and the possible influence of ligand-induced β-arrestin recruitment. The results allowed insights into the specificity of the interactions between 5-HT2AR and D2R within a heterocomplex. Finally, the in vivo effect of 5-HT2AR stimulation on the extent of heteromerization of 5-HT2AR and D2R was studied in a rat model. This animal experiment provides preliminary data on whether the genesis of psychosis-like symptoms might be related to the formation of 5-HT2AR-D2R complexes. Serotonin 5-HT2A receptor-mediated calcium signaling of 5-HT2AR-D2R complexes For NTS1R-D2R heterocomplexes, earlier studies performed by Thorsten Schäfer have suggested an inhibitory action of the D2 protomer towards NTS1R-mediated calcium signals.111,112 Inspired by this functional receptor-receptor interaction, we investigated 5-HT2AR-associated, Gq-mediated calcium signaling and a possible regulatory influence of co-expressed D2R in an analogous manner. Single cell fluorescent microscopy of transiently (co-)transfected cells (HT22, HEK293T) allowed to monitor ligand-induced changes in cytosolic calcium concentrations. Irrespective of the type of 5-HT2AR ligand used as a stimulus – either the non-hallucinogenic endogenous agonist serotonin (5-HT) or the hallucinogenic agonist DOI – the calcium response in 5-HT2AR-D2R-expressing cells was not different from that in 5-HT2AR mono-expressing cells (Figure 65). Co-activation or co-inhibition of the D2 protomer did not have an effect on the calcium signal. Whereas D2R has negatively affected the ability of NTS1R to increase intracellular calcium, the results here indicate that in our test systems D2R does not regulate 5-HT2AR-mediated Gq signaling. Based on this comparative approach, we concluded that functional interactions between different GPCRs are highly specific and depend significantly on the composition of the complex. Interestingly, activation of 5-HT2A receptors led to heterogeneous calcium signaling profiles (Figure 65). Especially in hippocampal HT22 cells, but rarely in HEK293T cells, an initial peak calcium response was followed by oscillations of irregular frequency and amplitude. Such calcium spikes were observed for 5-HT2AR independent of the presence, activation or deactivation of D2 receptors suggesting a signaling behavior that is not directly connected to heteromerization of 5-HT2AR and D2R, but seems to be 5-HT2AR-specific, particularly in a neuronal environment. Figure 65. Influence of unoccupied (left) or occupied (center) D2 receptors on 5-HT2A receptor agonist-induced calcium signals in HT22 cells. Representative oscillatory signaling profiles of individual 5-HT2AR-expressing cells (right). BRET-validated interaction of 5-HT2AR and D2R By measuring bioluminescence resonance energy transfer (BRET), we were able to show physical interaction between co-expressed 5-HT2A and D2 receptors. More specifically, in HEK293T cells BRET saturation assays with Rluc8-donor and mVenus-acceptor fusion constructs yielded hyperbolic BRET curves, confirming the specificity of the interaction. Due to low expression levels of the biosensor constructs in HT22 cells, it was difficult to robustly validate 5-HT2AR-D2R interaction there. Since the ligand-induced calcium response was highly similar in 5-HT2AR-D2R-expressing HT22 and HEK293T cells, analysis of the BRET data allowed the conclusion that the absence of modulatory effects is not due to the absence of 5-HT2AR-D2R complexes. Further BRET saturation assays were conducted to investigate the influence of increased concentrations of dopamine (DA) and serotonin, respectively, on the interaction of the protomers. Irrespective of the BRET pair, i.e. which receptor was tagged to the donor or the acceptor protein, the agonists neither increased nor decreased the BRET ratio. The unchanged BRET efficiencies indicate an activation-independent, constitutive formation of 5-HT2AR-D2R heteroreceptor complexes. Figure 66. Saturated BRET curve confirms specific 5-HT2AR-D2R interaction in HEK293T cells (left). Activation of 5-HT2AR (center) and D2R (right), respectively, did not alter the energy transfer between donor and acceptor. Evaluation of Gi protein-mediated signaling of 5-HT2AR-D2R complexes There is evidence that modulatory interactions within GPCR complexes can be bidirectional.97 After no effect of D2R on 5-HT2AR-related Gq signaling could be shown, we focused on evaluating D2R-mediated Gi activation and whether it was influenced by 5-HT2AR. Taking advantage of the cytosolic BRET-biosensor CAMYEL, the inhibition of forskolin stimulated cAMP formation was analyzed in HEK293T cells. When 5-HT2AR and D2R were co-expressed, the maximum cAMP inhibition triggered by dopamine was lowered by co-application of serotonin (Figure 67). In contrast, maximum inhibition of cAMP was increased when D2R was activated by dopamine and 5-HT2AR was blocked by the antagonist ketanserin. For D2R mono-expressing cells, such attenuating or enhancing effects were not observed. These were therefore interpreted as the result of the simultaneous occupation of both protomers of 5-HT2AR-D2R complexes, which in the overall context suggested a negative modulation of the D2R-associated Gi signaling by the 5-HT2A protomer. In presence of YM-254890, a selective inhibitor of Gαq/11 proteins, a mix of 5-HT and DA was not able to decrease the maximum cAMP inhibition anymore (Figure 67). Consequently, active Gαq seems to play a crucial role in mediating negative cooperativity within the 5-HT2AR-D2R heterocomplex. According to cAMP assays performed without forskolin pre-stimulation, Gαs proteins could be precluded to contribute significantly to the inhibitory 5-HT2AR-D2R crosstalk. Figure 67. Dopamine-triggered inhibition of cAMP accumulation in 5-HT2AR-D2R-expressing HEK293T cells influenced by 5-HT2AR co-activation (left), 5-HT2AR co-inhibition (center) and the Gαq inhibitor YM-254890 (right). The experiments investigating release of intracellular calcium and inhibition of cAMP accumulation upon ligand binding provided information about the in vitro properties of 5-HT2AR-D2R complexes. The consequences of 5-HT2AR-D2R heteromerization on G protein-mediated signal transduction, which can be derived from our results, are illustrated in Figure 68. Figure 68. G protein signaling characteristics of 5-HT2AR-D2R heteroreceptor complexes. β-arrestin recruitment to 5-HT2AR-D2R heterocomplexes Apart from the influence of other G protein subtypes on the modified cAMP signal of co-activated 5-HT2AR-D2R heteromers, we subsequently studied a possible relationship with the agonist-stimulated recruitment of β-arrestin-2 to the complex. For this purpose, we used PK-tagged 5-HT2AR and D2R constructs, which allowed the detection of β-arrestin-2 recruitment through enzyme complementation and a subsequent substrate conversion. As expected, in HEK293 cells serotonin receptor agonists induced recruitment of β-arrestin-2 to monomeric 5-HT2A receptors and dopamine receptor agonists to monomeric D2 receptors. Figure 69. Dopamine and serotonin stimulated β-arrestin-2 recruitment to 5-HT2AR-D2R complexes consisting of two functionally active protomers (left and center right) or of one functional and one signaling incompetent protomer (center left and right). Interestingly, we obtained characteristic coupling of β-arrestin-2 in cells that expressed heterodimers consisting of one PK-tagged protomer and one wild-type protomer each (Figure 69). Selective activation of the untagged protomer led to a low, but measurable β-arrestin-2 recruitment to the complex. To further analyze the specificity of the observed β-arrestin-2 recruitment, within the heterocomplexes the wild-type receptors were exchanged by signaling incompetent mutants. Here, activation of the mutant protomer did not direct β-arrestin-2 to the complex. Whether stimulation of the untagged receptor leads to a reaction of β-arrestin-2 with the PK-fragment of the vicinal protomer or whether allosteric interaction within the 5-HT2AR-D2R complex enables asymmetric recruitment, cannot be assessed conclusively. Certainly, functional activity of both protomers appears to be necessary for the observed recruitment of β-arrestin-2 to 5-HT2AR-D2R. Influence of acute serotonergic treatment on the interaction of 5-HT2AR and D2R in the rat brain There are reports that 5-HT2AR and D2R physically and most probably also functionally interact in the central nervous system.228,329 Based on these findings, we subjected Sprague-Dawley rats to single-dose treatment with different serotonin receptor agonists, which was presented in the final part of the thesis. By simulating an acutely enhanced activity of serotonergic transmission, we intended to evaluate whether activation of 5-HT2A receptors affected the formation of 5-HT2AR-D2R heterocomplexes in vivo. Employing the in situ proximity ligation assay (PLA) on fixed rat brain sections of the striatum and the cerebral cortex, the GPCR complexes could be identified, subsequently localized and quantified by confocal microscopy. I performed the PLA and microscopy studies during a research stay in the laboratory of Prof. Kjell Fuxe at Karolinska Institutet in Stockholm. Compared to the vehicle control, application of both the hallucinogenic 5-HT2AR agonist DOI and the non-hallucinogenic 5-HT2AR agonist TCB-2 increased the density of 5-HT2AR-D2R clusters in subcortical and cortical areas with TCB-2 being even slightly more potent (Figure 70). Within striatal regions, DOI, but not TCB-2, enhanced the number of complexes exclusively in the medial part of the nucleus accumbens shell. MDL-100907, a selective 5-HT2AR antagonist, eliminated the DOI effects in the striatum and in the subcortical claustrum, resulting in densities of 5-HT2AR-D2R heterocomplexes that were comparable to the vehicle controls. Therefore, the DOI-mediated increase of 5-HT2AR-D2R complexes in certain areas of the rat brain is related to the activation of 5-HT2A receptors. Figure 70. Quantification of PLA signals in the medial part of the nucleus accumbens shell (left) and the claustrum (right) after acute treatment with 5-HT2AR ligands. According to our findings, 5-HT2AR-D2R heterodimers accumulate in some areas of the brain, where their formation can be additionally stimulated by specific 5-HT2AR activation. The nucleus accumbens shell and the claustrum are involved in the control of emotion and consciousness.341,349 Interestingly, both regions were sensitive to hallucinogenic DOI. Up-regulation of 5-HT2AR-D2R complexes there might contribute to the manifestation of psychotic symptoms by impairing the processing of information essential for normal sensory perception.Viele therapeutisch eingesetzte Arzneistoffe entfalten ihre erwünschte Wirkung über heptahelikale, membranständige Rezeptoren, besser bekannt als G-Protein-gekoppelte Rezeptoren (GPCR). Diese dienen als Überträger externer Signale ins Zellinnere, indem sie zytosolische Effektoren wie G-Proteine oder β-Arrestine aktivieren. Wenn sie an einen aktivierten GPCR gekoppelt sind, können diese Effektorproteine anregende oder hemmende Signalwege in Gang setzen und dadurch spezifische Zellantworten auslösen. Erkenntnisse experimenteller Untersuchungen der letzten drei Jahrzehnte legen nahe, dass individuelle GPCR in der Lage sind, physisch miteinander zu interagieren und so funktionsfähige Rezeptorkomplexe zu bilden, welche man, sofern sie aus zwei unterschiedlichen Rezeptortypen bestehen, als Heterodimere bezeichnet. Typischerweise zeigen derartige Rezeptorverbünde Eigenschaften, die sich wesentlich von denen der entsprechenden Monomere unterscheiden.16,17,23,35 Es ist eine elementare Frage, inwiefern die GPCR-Heteromerisierung, die bisher hauptsächlich in vitro beobachtet und beschrieben wurde, von physiologischer oder pathophysiologischer Relevanz ist. GPCR-Komplexe in Primärgewebe oder sogar in lebenden Organismen nachzuweisen, wäre ein starkes Indiz für deren Beteiligung an pharmakologisch interessanten biochemischen Vorgängen.218 Im Gehirn überschneiden sich die dopaminergen und serotonergen Nervenbahnen vor allem im Striatum, in der Hirnrinde und in den Basalganglien. Aufgrund dessen interagieren beide Neurotransmittersysteme und kontrollieren gemeinsam wichtige neurologische Aufgaben wie die Motorik, die Gemütslage oder die Kognition. Die umfangreich exprimierten Dopamin D2- und Serotonin 5-HT2A-Rezeptoren spielen bei der Vermittlung der Effekte ihrer natürlichen Monoaminliganden eine zentrale Rolle. Bei der Behandlung der Schizophrenie beispielsweise sind die beiden Rezeptoren die Hauptangriffspunkte vieler first-line Antipsychotika.129,192 Darüber hinaus sind 5-HT2AR-D2R-Komplexe in transfizierten Zellen sowie in unterschiedlichen Regionen des Rattenhirns nachgewiesen worden und es hat sich herausgestellt, dass sie aufgrund modulatorischer Wechselwirkungen ein charakteristisches Signalverhalten zeigen.44,226–228 Nachdem 5-HT2AR und D2R wesentliche Komponenten bei der Behandlung neuropsychiatrischer Störungen sind, war es die Motivation dieser Arbeit, Informationen über die Besonderheiten des 5-HT2AR-D2R-typischen Signalverhaltens zu gewinnen und herauszufinden, ob die Bildung von 5-HT2AR-D2R-Heterorezeptorkomplexen zur Dysfunktion neurologischer Mechanismen beiträgt. Nach der Validierung der Interaktion von 5-HT2AR und D2R untersuchten wir 5-HT2AR-D2R-exprimierende Zellen hinsichtlich ihrer G-Protein-vermittelten Signaltransduktion sowie dem möglichen Einfluss Ligand-induzierter β-Arrestin Rekrutierung. Die Ergebnisse gewährten Einblick in die Spezifizität der Interaktionen zwischen 5-HT2AR und D2R innerhalb eines Heterokomplexes. Abschließend wurde in einem Rattenmodell der in vivo Effekt der Stimulation von 5-HT2AR auf das Ausmaß der Heteromerisierung von 5-HT2AR und D2R untersucht. Dieses Tierexperiment stellt erste Daten darüber zu Verfügung, ob die Entwicklung Psychose-ähnlicher Symptome mit der Bildung von 5-HT2AR-D2R-Komplexen in Zusammenhang stehen könnte. Serotonin 5-HT2A-Rezeptor-vermittelte Calciumsignale von 5-HT2AR-D2R-Komplexen Frühere Untersuchungen an NTS1R-D2R-Heterokomplexen, durchgeführt von Thorsten Schäfer, ließen eine inhibitorische Wirkung des D2-Protomers auf NTS1R-vermittelte Calciumsignale vermuten.111,112 Inspiriert durch diese funktionale Rezeptor-Rezeptor-Interaktion untersuchten wir auf dieselbe Art und Weise 5-HT2AR-bezogene, Gq-vermittelte Calciumsignale und einen eventuellen regulatorischen Einfluss co-exprimierter D2R auf diesen Signalweg. Mittels Einzelzellfluoreszenzmikroskopie konnten im Zytosol transient (co-)transfizierter Zellen (HT22, HEK293T) Ligand-induzierte Änderungen der Calciumkonzentrationen beobachtet werden. Unabhängig von der Art des 5-HT2AR-Liganden, der als Stimulus eingesetzt wurde – entweder der nicht-halluzinogene, endogene Agonist Serotonin (5-HT) oder der halluzinogene Agonist DOI –, haben sich die Calciumantworten in 5-HT2AR und D2R co-exprimierenden und 5-HT2AR mono-exprimierenden Zellen nicht unterschieden (Abbildung 64). Eine gleichzeitige Aktivierung oder Inhibition des D2-Protomers hatte keinen Effekt auf das Calciumsignal. Wohingegen D2R die Fähigkeit von NTS1R, intrazelluläres Calcium zu erhöhen, abgeschwächt hat, deuten die Ergebnisse hier an, dass in unseren Testsystemen D2R den 5-HT2AR-vermittelten Gq-Signalweg nicht reguliert. Auf Grundlage dieses vergleichenden Ansatzes kamen wir zu dem Schluss, dass funktionelle Interaktionen zwischen verschiedenen GPCR höchst spezifisch sind und maßgeblich von der Zusammensetzung des Komplexes abhängen. Die Aktivierung der 5-HT2A-Rezeptoren führte interessanterweise zu heterogenen Calciumsignalprofilen (Abbildung 64). Vor allem in den hippokampalen HT22-Zellen, jedoch kaum in HEK293T-Zellen folgten auf den initialen Calciumpeak Oszillationen mit unregelmäßiger Frequenz und Amplitude. Solche Calciumimpulse wurden unabhängig von der Anwesenheit, einer Aktivierung oder Deaktivierung von D2-Rezeptoren beobachtet, was auf ein Signalverhalten deutet, das nicht unmittelbar mit der Heteromerisierung von 5-HT2AR und D2R in Verbindung steht, sondern speziell in einer neuronalen Umgebung spezifisch für 5-HT2AR zu sein scheint. Die Validierung der Interaktion zwischen 5-HT2AR und D2R mittels BRET Durch Messung des Biolumineszenz-Resonanz-Energie-Transfers (BRET) konnten wir zwischen co-exprimierten 5-HT2A- und D2-Rezeptoren physische Interaktionen nachweisen. Genauer gesagt erhielt man in HEK293T-Zellen unter Zuhilfenahme von Rluc8-Donor- und mVenus-Akzeptor-Fusionskonstrukten in BRET-Sättigungs-Assays hyperbolische BRET-Kurven, die die Spezifizität der Interaktion bestätigten. Aufgrund niedriger Expressionslevel der Biosensor-Konstrukte in HT22-Zellen war es mit Schwierigkeiten verbunden, darin die 5-HT2AR-D2R-Interaktion reproduzierbar nachzuweisen. Da jedoch die Ligand-induzierte Calciumantwort in 5-HT2AR-D2R-exprimierenden HT22- und HEK293T-Zellen sehr ähnlich war, ließ die Auswertung der BRET-Daten die Schlussfolgerung zu, dass das Fehlen modulierender Effekte nicht durch die Abwesenheit von 5-HT2AR-D2R-Komplexen bedingt ist. Es wurden weitere BRET-Sättigungs-Assays durchgeführt, um den Einfluss erhöhter Konzentrationen an Dopamin (DA) bzw. Serotonin auf die Interaktion der Protomere zu untersuchen. Weder erhöhten noch verringerten die Agonisten das BRET-Ratio, unabhängig davon, welcher Rezeptor mit dem Donor- oder dem Akzeptorprotein verbunden war. Die unveränderten BRET-Effizienzen deuten auf eine konstitutive Bildung von 5-HT2AR-D2R-Heterorezeptorkomplexen hin, die unabhängig von deren Aktivierung ist. Die Untersuchung Gi-Protein-vermittelter Signale von 5-HT2AR-D2R-Komplexen Es gibt Anhaltspunkte, dass modulatorische Interaktionen innerhalb von GPCR-Komplexen bidirektional gerichtet sein können.97 Nachdem kein Effekt von D2R auf die 5-HT2AR-abhängige Gq-Signaltransduktion gezeigt werden konnte, legten wir den Schwerpunkt auf die Untersuchung der D2R-vermittelten Aktivierung von Gi und ob diese durch 5-HT2AR beeinflusst wurde. Mit Hilfe des zytosolischen BRET-Biosensors CAMYEL wurde in HEK293T-Zellen die Hemmung der Forskolin-stimulierten cAMP-Bildung analysiert. Im 5-HT2AR-D2R-exprimierenden Zustand wurde die durch Dopamin ausgelöste maximale cAMP-Hemmung durch die gleichzeitige Zugabe von Serotonin gesenkt (Abbildung 66). Im Gegensatz dazu wurde die maximale cAMP-Hemmung gesteigert, wenn D2-Rezeptoren durch Dopamin aktiviert und 5-HT2A-Rezeptoren durch den Antagonisten Ketanserin blockiert wurden. In D2R mono-exprimierenden Zellen wurden derartige abschwächende oder verstärkende Effekte nicht beobachtet. Diese wurden daher als Resultat der gleichzeitigen Besetzung beider Protomere von 5-HT2AR-D2R-Komplexen aufgefasst, was im Gesamtzusammenhang betrachtet auf eine negative Beeinflussung der D2R-assoziierten Gi-Signaltransduktion durch das 5-HT2A-Protomer hindeutet. Eine Kombination aus 5-HT und DA war in Gegenwart von YM-254890, einem selektiven Inhibitor von Gαq/11-Proteinen, nicht mehr in der Lage, die maximale cAMP-Inhibition zu verringern (Abbildung 66). Demzufolge scheint aktives Gαq eine entscheidende Rolle dabei zu spielen, die negative Kooperativität innerhalb des 5-HT2AR-D2R-Heterokomplexes zu vermitteln. Gemäß den cAMP-Assays, die ohne Vorsti

Evolution of substrate specificity and protein-protein interactions in three enzyme superfamilies

Superfamilies are a classification system to combine proteins that are related through a common evolutionary origin, share similar sequences, structures, and core reaction mechanisms, but exert different functions. Today, for most superfamilies tens of thousands of sequences and hundreds of structures are known and most of the different functions of their members have been elucidated. Superfamilies thus provide a formal and biologically sensible framework to study evolutionary relationships between proteins. In the present work, the frameworks of three enzyme superfamilies were utilized to get insights into several important aspects of enzyme evolution. The first part of this work addresses the question how enzymatic mono- and bi-functionality have evolved in the superfamily of ribose-binding (βα)8-barrel sugar isomerases. This superfamily contains the homologous enzymes HisA and TrpF, which catalyze similar reactions in histidine and tryptophan biosynthesis, as well as the bi-functional enzyme PriA, which catalyzes both the HisA and TrpF isomerization reactions. HisA and TrpF are ubiquitous in Archaea and Bacteria, whereas PriA is only found in certain Actinobacteria. These species have lost the dedicated TrpF enzyme and PriA is consequently part of both tryptophan and histidine biosynthesis. Much has been speculated on the evolutionary relationship of these enzymes and whether the bi-functionality of PriA is a remnant from ancient evolutionary times or a more recent development in Actinobacteria. Using ancestral sequence reconstruction it was demonstrated in this work that evolutionary ancestors of modern HisA enzymes display bi-functionality, reminiscent of PriA. A detailed enzymatic characterization of three reconstructed HisA ancestors showed that they catalyze not only the HisA but also the TrpF reaction with comparable catalytic efficiencies in vitro. Metabolic complementation experiments with hisA and trpF deficient Escherichia coli strains furthermore demonstrated that the bi-functional HisA ancestors could support both histidine and tryptophan biosynthesis in vivo. By a combination of sequence- and network-based in silicomethods, several modern HisA enzymes were subsequently identified that possess sequence motifs typical for bi-functional PriA enzymes. The enzymatic characterization of three such modern HisA representatives revealed that they are also bi-functional, albeit to a lesser extent, although the respective organisms possess dedicated TrpF enzymes. Thus, the ancestral bi-functionality has pertained for billions of years in HisA enzymes, without any obvious selective pressure. Consequently, a new model for the evolution of HisA, TrpF, and PriA was proposed: The bi-functionality of ancient HisA variants may have played an important role in maintaining early metabolism by supporting both histidine and tryptophan biosynthesis. After the emergence of dedicated TrpF enzymes the bi-functionality of the ancestors became expendable and diminished to the level observed in modern HisA enzymes. However, the inherent bi-functionality of HisA contributed to the robustness of microbial metabolism and made possible to compensate the loss of a dedicated trpF gene in some Actinobacteria. In these organisms, the available bi-functionality of HisA was exploited, selected for, and enhanced, which eventually led to the modern PriA enzymes. The second part of this work deals with the evolution of substrate specificity and secondary metabolic enzymes in a superfamily of chorismate-utilizing enzymes, named MST-superfamily. Chorismate is a central metabolic node molecule and the starting point for the biosynthesis of various important metabolites, including aromatic amino acids, folate, or iron-chelating siderophores. The MST-enzymes catalyze the committed steps of these biosynthetic pathways and are highly similar in sequence, structure, and reaction mechanism. However, the MST-enzymes that are part of primary metabolic pathways employ exclusively ammonia as a nucleophile to aminate chorismate, whereas those that are part of secondary metabolic pathways exclusively employ water as a nucleophile to hydroxylate chorismate. Based on the notion that secondary metabolic enzymes are descendants of primary metabolic ones, it was investigated in this part of this work by which mechanism the transition from primary metabolic to secondary metabolic MSTenzymes went along with a change in nucleophile-specificity from ammonia to water. Initially, network-based, phylogenetic, and structure-based in silicomethods were applied to identify two key amino acids in the nucleophile access channel of the active site that distinguish primary-metabolic/ammonia-utilizing and secondary-metabolic/water-utilizing MST-enzymes. The importance of these key positions was subsequently examined by rationally designing sixteen variants of the MST-enzyme anthranilate synthase, which normally employs ammonia as a nucleophile. The enzymatic characterization of these variants by HPLC-MS showed that the right combination of amino acids at the two key positions indeed resulted in a broadening of nucleophile specificity to also include water. These anthranilate synthase variants hydroxylated chorismate and formed isochorismate with efficiencies comparable to native secondary-metabolic/water-utilizing isochorismate synthases. Moreover, these variants were still able to employ ammonia as a nucleophile and formed their native product anthranilate; hence they were bi-functional. These experiments demonstrated that nucleophile specificity in the MST-superfamily can readily switch from ammonia to water. Moreover, the observed bi-functionality of the anthranilate synthase variants argues that the evolution of secondary metabolic MST-enzymes may have proceeded through bi-functional intermediates. Such metabolic generalists may have allowed for the formation of novel metabolites (isochorismate) while maintaining the formation of important primary metabolic metabolites (anthranilate). This scenario consequently does not a priorirequire gene duplication events and thus precludes negative metabolic effects linked to retaining redundant gene copies. The third part of this work pursues the question how protein-protein interaction specificity is assured in superfamilies of structurally related protein complexes and how the determinants of interaction specificity have evolved. Specific interactions between proteins are vital for almost all cellular functions. This specificity is usually achieved by shape and electrostatic complementarity of protein interfaces. However, the number of different protein folds and interface geometries found in Nature is limited, due to the constraints imposed by efficiently packing hydrogen-bonded secondary structure elements. It is thus a challenging question how interaction specificity is achieved despite structural limitations and how the formation of non-physiological complexes is avoided when several possible interaction partners with similar interface geometries are available. In order to address this problem, initially a comprehensive computational survey of the interface geometries of over 300 bacterial, heteromeric protein complexes and all their homologs of respective superfamilies was performed. This survey revealed that in about 10% of the superfamilies interface geometries vary significantly between related complexes that share homologous subunits. In these cases interfaces were extended by socalled interface add-ons, which typically comprise 10-20 amino acids, form well-defined secondary structure elements, and significantly contribute to complex stability. These characteristics suggested that interface add-ons differentiate between structurally related protein complexes and contribute to interaction specificity through negative design. In order to back this assumption, the case of the interface add-on found in a superfamily of glutamine amidotransferase complexes involved in tryptophan and folate biosynthesis was subsequently analyzed in detail. These complexes comprise synthase and glutaminase subunits that interact to transfer ammonia from glutamine to an acceptor substrate. A subset of synthase subunits exclusively involved in tryptophan biosynthesis contains the interface add-on, whereas it is absent in all other homologous synthase subunits, including those exclusively involved in folate biosynthesis. The comprehensive experimental characterization of 54 combinations of different synthase and glutaminase subunits by chromatographic methods, light scattering, mass spectrometry, and enzyme kinetics demonstrated that the presence or absence of the interface add-on determines interaction specificity. An in silicogenetic profiling of over 15000 archaeal and bacterial genomes together with in vivogrowth assays showed that the interface add-on found in complexes of tryptophan biosynthesis is biologically relevant for preventing cross-interactions with the homologous complexes of folate biosynthesis, which would lead to harmful metabolic cross-talk that negatively affects cellular fitness. It was finally shown by protein design that the evolution of the interface add-on in these complexes most likely proceeded via intermediary complexes with relaxed interaction specificity. In conclusion, this part of this work demonstrates that interface add-ons are evolutionary tools to facilitate interaction specificity in superfamilies of homologous proteins or in cases where a protein has to discriminate between several potential interaction partners that share similar interface geometries. In summary, the presented work leads to an improved understanding of the mechanisms behind the evolution of enzymatic mono- and bi-functionality, emphasizes the importance of generalist, bi- or multi-functional enzymes for the evolution of secondary metabolic pathways, and finally describes a so far overlooked structural tool for the evolutionary specification of protein-protein interactions

COVID-19 policy interventions and fertility dynamics in the context of pre-pandemic welfare support

This paper focuses on nonpharmaceutical interventions (NPIs) to explain fertility dy-namics during the pandemic, while considering countries’ institutional context. Weargue that containment policies disrupted people’s lives and increased their uncer-tainty more in countries with weak welfare support systems, while health-relatedand economic support NPIs mitigated such disruptions much more there, as theywere less expected by citizens. We estimate monthly “excess” crude birth rates (CBRs)and find that countries with low public support—Southern Europe, East Asia, andEastern Europe—experienced larger decreases and less of a rebound in CBRs thancountries with histories of high public spending—Western, Central, and NorthernEurope. However, in low support countries, NPIs are much more strongly associ-ated with excess CBRs—containment NPIs more negatively and health and economicsupport NPIs more positively—with the exception of the one-month lag of contain-ment NPIs, for which the opposite holds. When putting these coefficients into broaderperspective, our findings suggest that the actual implementation of all NPIs takentogether mitigated fertility declines. This is especially the case for low public supportcountries, whereas one might have seen a birth decline even in high support countriesif the NPIs were not implemented