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Chemical pollution is one of the main contributors to the degradation of lotic ecosystems and their biodiversity. Among chemicals driving lotic biodiversity decline are anthropogenic organic micropollutants (AOM), which affect the survival and functioning of freshwater organisms. Continuous exposure of freshwater organisms to AOM leads to adverse effects that sometimes cannot be traced with standard toxicity methods such as standard toxicity testing or biodiversity indices. Among these effects of AOM are selective or mutagenic effects that cause impaired species genetic diversity. Thus, the correlation between different levels of AOM and genetic diversity of species is still poorly understood. However, it can be explored by applying population genetics screening.
In Chapter 1 of this thesis, background information on environmental pollution, genetic screening, and the detection of evolutionary-relevant AOM effects in freshwater organisms are described and the thesis goals are identified. The main goal of the thesis is to study whether AOM exposure occurring in European rivers causes a significant evolutionary footprint in freshwater species and leads to a selection of more tolerant geno-and phenotypes. Therefore, population genetics indices together with high-resolution chemical exposure screening of a widespread indicator invertebrate species, Gammarus pulex (Linnaeus, 1758), living in polluted and pristine European rivers were investigated.
In Chapter 2, the development of a genetic screening method for G. pulex (microsatellites) is described. Due to genetic differentiation and the presence of morphologically cryptic lineages, the available sets of target loci do not enable a reliable population genetic characterization of G. pulex from central Germany. Thus, a novel set of microsatellite loci for a high-precision assessment of population genetic diversity was here applied. Eleven loci were first identified and thereafter amplified in G. pulex from three rivers. The new loci reliably amplified and indicated polymorphisms in the studied amphipods. The amplification resulted in the successful identification of genetically distinct populations of G. pulex from the analyzed rivers. Moreover, the microsatellite loci were amplified in other genetic lineages of G. pulex and another Gammarus species, G. fossarum, promising a broader applicability of the loci in related amphipod species.
In Chapter 3, the effects of AOM on species genetic differentiation and sensitivity to toxic chemicals in a typical central European river with pristine and AOM-polluted sections was investigated. The river’s site-specific concentrations of AOM were assessed by chemical analysis of G. pulex tissue and water samples. To test, whether different levels of AOM in the river select for pollution-dependent genotypes, the genetic structure of G. pulex from the river was analyzed. Finally, the toxicokinetics of and sensitivity to the commonly used insecticide imidacloprid were determined for amphipods sampled at pristine and polluted sections to assess whether various levels of AOM in the river influence sensitivity of G. pulex to imidacloprid. The results indicated that different levels of AOM did not drive genetic divergence of G. pulex within the river but led to an increased sensitivity of exposed amphipods to imidacloprid. The amphipods living in polluted river sections were more sensitive to the insecticide due to chronic exposure to toxic levels of AOM.
In Chapter 4, the relationship between site-specific pollution levels of AOM and genetic diversity parameters of G. pulex was analyzed at the regional scale within six rivers in central Germany. The genetic structure of G. pulex in the studied area was tested for relatedness to the waterway distance between sites. Gammarus pulex genetic diversity parameters, including allelic richness and inbreeding rate, were tested against environmental pollution parameters using linear mixed-effect- and structural-equation models. According to the results, G. pulex genetic diversity parameters were significantly associated with the detected AOM levels. At sites with high concentrations of AOM and toxicity potential G. pulex showed reduced genetic diversity and increased rates of inbreeding. These results suggest that AOM play a major role in shaping the genetic diversity of G. pulex in rivers.
According to the findings presented here, the applied microsatellites can be used to successfully detect changes in genetic patterns in freshwater amphipods facing increased levels of AOM. The findings indicate that levels of AOM representative for European rivers do not lead to the separation of genotypes among G. pulex as the connectivity between sites majorly contributes to species’ genetic structure. However, the chronic exposure to increased levels of toxic AOM leads to a reduction of species genetic diversity and increases the sensitivity of G. pulex to the toxic chemical effects.
Ataxin-2 (human gene symbol ATXN2) acts during stress responses, modulating mRNA translation and nutrient metabolism. Ataxin-2 knockout mice exhibit progressive obesity, dyslipidemia, and insulin resistance. Conversely, the progressive ATXN2 gain of function due to the fact of polyglutamine (polyQ) expansions leads to a dominantly inherited neurodegenerative process named spinocerebellar ataxia type 2 (SCA2) with early adipose tissue loss and late muscle atrophy. We tried to understand lipid dysregulation in a SCA2 patient brain and in an authentic mouse model. Thin layer chromatography of a patient cerebellum was compared to the lipid metabolome of Atxn2-CAG100-Knockin (KIN) mouse spinocerebellar tissue. The human pathology caused deficits of sulfatide, galactosylceramide, cholesterol, C22/24-sphingomyelin, and gangliosides GM1a/GD1b despite quite normal levels of C18-sphingomyelin. Cerebellum and spinal cord from the KIN mouse showed a consistent decrease of various ceramides with a significant elevation of sphingosine in the more severely affected spinal cord. Deficiency of C24/26-sphingomyelins contrasted with excess C18/20-sphingomyelin. Spinocerebellar expression profiling revealed consistent reductions of CERS protein isoforms, Sptlc2 and Smpd3, but upregulation of Cers2 mRNA, as prominent anomalies in the ceramide–sphingosine metabolism. Reduction of Asah2 mRNA correlated to deficient S1P levels. In addition, downregulations for the elongase Elovl1, Elovl4, Elovl5 mRNAs and ELOVL4 protein explain the deficit of very long-chain sphingomyelin. Reduced ASMase protein levels correlated to the accumulation of long-chain sphingomyelin. Overall, a deficit of myelin lipids was prominent in SCA2 nervous tissue at prefinal stage and not compensated by transcriptional adaptation of several metabolic enzymes. Myelination is controlled by mTORC1 signals; thus, our human and murine observations are in agreement with the known role of ATXN2 yeast, nematode, and mouse orthologs as mTORC1 inhibitors and autophagy promoters.
Weiterentwicklung und Evaluation eines drei-dimensionalen Hautmodelles zur pharmakologischen Testung
(2008)
Orthopockenviren sind große DNA-Viren, die im Zytoplasma der Wirtszelle replizieren und für über 200 Proteine kodieren. Sie besitzen ein breites Wirtszellspektrum (host-range) und modulieren auf komplexe Art und Weise zelluläre Prozesse, um ihre Replikation zu gewährleisten. Zu diesem Genus der Familie der Pockenviren gehört auch das modifizierte Vacciniavirus Ankara (MVA). MVA ist ein hoch attenuiertes, replikationsdefizientes Impfvirus, dem im Vergleich zu ursprünglichen Vacciniavirus-Stämmen viele virale Genfunktionen fehlen. Zu diesen verlorengegangenen Genen zählen so genannte host-range-Gene, die für das breite Wirtszellspektrum des Vacciniavirus (VACV) verantwortlich sind, deren molekulare Funktion aber größtenteils unbekannt ist. Diese Arbeit befasste sich zum einen mit der Untersuchung der Rolle der host-range-Gene K1L und C7L in der MVA-Infektion. Zum anderen sollte geprüft werden, ob der im MVA-Genom unvollständige Leserahmen F11L durch Wiederherstellung seiner Funktionalität den Wirtstropismus von MVA erweitern kann. Das Fehlen von K1L und C7L in MVA ist mit dem Verlust der späten viralen Genexpression verbunden. Als mögliche Ursache hierfür wurde in dieser Arbeit die Phosphorylierung des eukaryotischen Translationsinitiationsfaktors 2alpha (eIF2alpha) entdeckt, welche zum Abbruch der Proteinsynthese in der infizierten Zelle führt. Unter den möglichen Kinasen wurde die Proteinkinase R (PKR) als das verantwortliche Schlüsselenzym identifiziert und somit gezeigt, dass das K1- und C7-Protein den anti-viralen PKR-eIF2alpha-Signalweg inhibieren. Es stellte sich heraus, dass die eIF2alpha-Phosphorylierung alleine jedoch nicht für das Fehlen der späten Genexpression verantwortlich ist. Neben dem inhibitorischen Einfluss auf den PKR-eIF2alpha-Signalweg zeigte sich, dass C7 die Aktivierung des NFKB-Signalwegs reduziert, welcher für eine anti-virale Antwort der Wirtszelle wichtig ist. Ein weiterer Ansatz zur Aufklärung der K1- und C7-Funktion bestand darin, zelluläre Interaktionspartner zu identifizieren. Hierbei konnte das heterogene nukleäre Ribonukleoprotein K (HNRPK) als möglicher Interaktionspartner von C7 entdeckt werden. Neben den bisher bekannten host-range-Genen gibt es vermutlich weitere Gene, die den Wirtsbereich des VACV definieren. Das im MVA-Genom defekte F11L-Gen war ein guter Kandidat für eine solche Genfunktion, da es bei der Virionenmorphogenese, Virusausbreitung und der Migration VACV-infizierter Zellen eine Rolle zu spielen scheint. Deshalb wurde ein rekombinantes MVA mit vollständiger F11L-Gensequenz konstruiert und das Wirtszellspektrum dieses Virus untersucht. Die Reparatur des F11L-Gens ermöglichte MVA die Induktion von Zellbewegung nach Infektion, jedoch blieben seine unvollständige Morphogenese und eingeschränkte Vermehrungsfähigkeit in Säugetierzellen unbeeinflusst. F11L hat daher zumindest keine selbstständige Funktion als VACV host-range-Gen. Die Ergebnisse dieser Arbeit sind ein Beitrag zum besseren Verständnis der komplexen Virus-Wirts-Interaktionen des VACV sowie des eingeschränkten Wirtstropismus des Impfvirus MVA.
In the insect brain, the mushroom body is a higher order brain area that is key to memory formation and sensory processing. Mushroom body (MB) extrinsic neurons leaving the output region of the MB, the lobes and the peduncle, are thought to be especially important in these processes. In the honeybee brain, a distinct class of MB extrinsic neurons, A3 neurons, are implicated in playing a role in learning. Their MB arborisations are either restricted to the lobes and the peduncle, here called A3 lobe connecting neurons, or they provide feedback information from the lobes to the input region of the MB, the calyces, here called A3 feedback neurons. In this study, we analyzed the morphology of individual A3 lobe connecting and feedback neurons using confocal imaging. A3 feedback neurons were previously assumed to innervate each lip compartment homogenously. We demonstrate here that A3 feedback neurons do not innervate whole subcompartments, but rather innervate zones of varying sizes in the MB lip, collar, and basal ring. We describe for the first time the anatomical details of A3 lobe connecting neurons and show that their connection pattern in the lobes resemble those of A3 feedback cells. Previous studies showed that A3 feedback neurons mostly connect zones of the vertical lobe that receive input from Kenyon cells of distinct calycal subcompartments with the corresponding subcompartments of the calyces. We can show that this also applies to the neck of the peduncle and the medial lobe, where both types of A3 neurons arborize only in corresponding zones in the calycal subcompartments. Some A3 lobe connecting neurons however connect multiple vertical lobe areas. Contrarily, in the medial lobe, the A3 neurons only innervate one division. We found evidence for both input and output areas in the vertical lobe. Thus, A3 neurons are more diverse than previously thought. The understanding of their detailed anatomy might enable us to derive circuit models for learning and memory and test physiological data.
Fuer die schlechte Prognose von Glioblastompatienten mit einer ueberlebenszeit von 9-15 Monaten (Norden and Wen, 2006) ist vor allem die hohe Invasivitaet dieser Tumore verantwortlich. Nach operativer Entfernung des Haupttumors entstehen aus den verbleibenden invadierten Zellen sekundaere Tumore, die sich mitunter ueber weite Bereiche des Hirns verteilen. Des Weitern sind die hochinvasiven Tumorzellen oft resistent gegen Chemo- und Strahlentherapie (Drappatz et al., 2009; Lefranc et al., 2005). In Maustumormodellen und Pateinten konnte zudem gezeigt werden, dass die neuartige antiangiogenetische Therapie zwar das Tumorwachstum verringert, jedoch die Invasivitaet stark erhoeht. (Norden et al., 2008; Ebos et al., 2009; Paez-Ribes et al., 2009). Ueber die Mechanismen die diese hohen Invasivitaet induzieren, ist bislang nur sehr wenig bekannt. Die durch Reduktion von Blutgefaessen steigende Hypoxie des Tumors foerdert die Expression von Matrix-Metalloproteinasen (MMPs). Dies fuehrt zum Abbau der extrazelluaeren Matrix des umgebenden gesunden Gewebes und beguenstigt dadurch die Tumorzellinvasion (Indelicato et al., 2010; Miyazaki et al., 2008; Shyu et al., 2007). Die Umformung des Aktinzytoskeletts und damit die Mobilitaet von Zellen wird vorwiegend durch ein akkurates Zusammenspeil der Rho GTPasen Rac, Rho und Cdc42, kontrolliert (Ridley et al., 2003). Fuer die Organisation von Axonen im Nervensystem und fuer die Blut- und Lymphgefaessbildung wurde gezeigt, dass die Interaktion der Eph-Rezeptortyrosinkinasen und Ihrer Ephrin-Liganden Signalwege induziert, die in die Regulation dieses Zusammenspiels involviert sind (Egea and Klein, 2007; Makinen et al., 2005; Palmer et al., 2002; Sawamiphak et al., 2010). Des Weiteren zeigt die Analyse der Genloci von Eph-Rezeptoren und Ephrinen in verschieden Hirntumoren eine gehaeufte Deletionen des Ephrin-B2-Gens. Die Quantifizierung von Ephrin-B2 mRNA in diesen Tumoren hat ausserdem ergeben, dass mit zunehmender Malignitaet die Expression von Ephrin-B2 sinkt. Aus diesen Gruenden wurden die Untersuchungen in dieser Arbeit auf die Rolle von Ephrin-B2 anhaengigen Signalwegen in der Glioblastomzellinvasion konzentriert. In einem modifiziertem Boyden-Chamber-Assay konnte gezeigt werden, dass das Ephrin-B2 induzierte EphB4 forward signaling und EphB4 induzierte Ephrin-B2 reverse signaling die Invasivitaet der human Glioblastomzelllinien LN-229, G55 und SNB-19 reduziert. In einem Maustumormodel konnte weiterhin gezeigt werden, dass Ephrin-B2 Knock-Out (KO) Astrozytomzellen, im Vergleich zu Wild-Typ (WT) Zellen, Tumore mit einem groesseren Volumen und einer erhoehten Invasivitaet bilden. Da die Expressionslevel fuer die Ephrin-B2 bindenden Rezeptoren EphA4, EphB1 EphB3 und EphB6 auch im adulten Hirn hoch sind (Hafner et al., 2004), weisen diese in vitro und in vivo Ergebnisse auf eine Tumorsupressorfunktion von Ephrin-B2 hin, die durch repulsive Effekte des Ephrin-B2 reverse signaling vermittelte werden koennten. Dies geht mit Erkenntnissen ueber kolorektale Tumore einher (Batlle et al., 2005). Die in einem Sphaeroid-Invasionsassay mit einer EphB-Rezeptoren freien Umgebung beobachtete verminderte Invasion von Ephrin-B2 WT deutet auf eine zusaetzliche invasionsblockierende Rolle der Ephrin-B2-Eph-Rezeptor Interaktion zwischen benachbarten Tumorzellen hin, wie sie auch in Brusttumoren gefunden wurde (Noren et al., 2006). Es scheint als sei Tumorprogression und Invasion erst moeglich, nachdem die Expression von Ephrin-B2 vermindert wurde. Es konnte weiterhin gezeigt werden, dass in hypoxischen Glioblastomzellen die Ephrin-B2 Expression durch die direkte Bindung des den Transkriptionsfaktors ZEB2 an den Ephrin-B2 Promoter reprimiert wird. In einem Weiteren Maustumormodel konnte gezeigt werden, dass die Blockierung der ZEB2 Expression mittels shRNA und die damit einhergehenden Inhibition der hypoxie induzierten Ephrin-B2 Repression das Wachstum und die Invasivitaet von Glioblastomen verringert. Zusaetzlich wurde gezeigt, dass der Verlust von ZEB2 ausreicht, die durch antiangiogenetische Therapie induzierte stark erhoehte Invasivitaet zu vermeiden. Die in dieser Arbeit gewonnen Erkenntnisse fuehren zu folgendem Modelmechanismus. In kleinen normoxischen Tumoren koennen repulsive Effekte des Ephrin-B2 reverse signalings und EphB forward signalings zwischen Tumorzellen und Zellen des umgebenden Gewebes die Ausbreitung und Invasion des Tumors unterdruecken. Zusaetzlich koennte das Ephrin-B2 induzierte EphB forward signaling zwischen benachbarten Tumorzellen die Mobilitaet der Tumorzellen wie in Brusttumoren inhibieren. Beim Erreichen einer bestimmten Tumorgroesse tritt Hypoxie auf, wodurch HIF-1alpha stabilisiert wird. Dies fuehrt dann zur ZEB2 Expression und leitet die Repression von Ephrin-B2 ein, was wiederum zur erhoehten Tumorzellemobilitaet und im Zusammenspiel mit MMPs zu Invasion fuehren kann. Gleichzeitig werden durch den HIF-induzierten VEGF-Gradienten neue Blutgefaesse rekrutiert. Damit wird der hypoxie-induzierten Invasivitaet entgegengewirkt. Wird mittels antiangiogenetischer Behandlung versucht Tumorprogression entgegenzuwirken, resultiert daraus eine erneut gesteigerte Hypoxie, die dann durch die ZEB2 vermittelte Repression von Ephrin-B2 wieder eine erhoehte Invasivitaet induzieren kann. Das Blockieren der ZEB2 Expression kann dieser durch antiangiogenetischen Behandlung induzierten Invasivitaet entgegenwirken.
Aim: To provide distribution information and preliminary conservation assessments for all species of the pineapple family (Bromeliaceae), one of the most diverse and ecologically important plant groups of the American tropics—a global biodiversity hotspot. Furthermore, we aim to analyse patterns of diversity, endemism and the conservation status of the Bromeliaceae on the continental level in the light of their evolutionary history.
Location: The Americas.
Methods: We compiled a dataset of occurrence records for 3,272 bromeliad species (93.4% of the family) and modelled their geographic distribution using either climate‐based species distribution models, convex hulls or geographic buffers dependent on the number of occurrences available. We then combined this data with information on taxonomy and used the ConR software for a preliminary assessment of the conservation status of all species following Criterion B of the International Union for the Conservation of Nature (IUCN).
Results: Our results stress the Atlantic Forest in eastern Brazil, the Andean slopes, Central America and the Guiana Highlands as centres of bromeliad diversity and endemism. Phylogenetically ancient subfamilies of bromeliads are centred in the Guiana highlands whereas the large radiations of the group spread across different habitats and large geographic area. A total of 81% of the evaluated bromeliad species are Possibly Threatened with extinction. We provide range polygons for 3,272 species, as well as newly georeferenced point localities for 911 species in the novel “bromeliad” r package, together with functions to generate diversity maps for individual taxonomic or functional groups.
Main conclusions: Diversity centres of the Bromeliaceae agreed with macroecological patterns of other plant and animal groups, but show some particular patterns related to the evolutionary origin of the family, especially ancient dispersal corridors. A staggering 2/3rds of Bromeliaceae species might be threatened with extinction, especially so in tropical rain forests, raising concerns about the conservation of the family and bromeliad‐dependent animal species.
PaCATB : a secreted catalase protecting Podospora anserina against exogenous oxidative stress
(2011)
A differential mass spectrometry analysis of secreted proteins from juvenile and senescentPodospora anserina cultures revealed age-related differences in protein profiles. Among other proteins with decreased abundance in the secretome of senescent cultures a catalase, termed PaCATB, was identified. Genetic modulation of the abundance of PaCATB identified differential effects on the phenotype of the corresponding strains. Deletion of PaCatB resulted in decreased resistance, over-expression in increased resistance against hydrogen peroxide. While the lifespan of the genetically modified strains was found to be unaffected under standard growth conditions, increased exogenous hydrogen peroxide stress in the growth medium markedly reduced the lifespan of the PaCatB deletion strain but extended the lifespan of PaCatB over-expressors. Overall our data identify a component of the secretome of P. anserina as a new effective factor to cope with environmental stress, stress that under natural conditions is constantly applied on organisms and influences aging processes.
Divergent selection between ecologically dissimilar habitats promotes local adaptation, which can lead to reproductive isolation (RI). Populations in the Poecilia mexicana species complex have independently adapted to toxic hydrogen sulfide and show varying degrees of RI. Here, we examined the variation in the mate choice component of prezygotic RI. Mate choice tests across drainages (with stimulus males from another drainage) suggest that specific features of the males coupled with a general female preference for yellow color patterns explain the observed variation. Analyses of male body coloration identified the intensity of yellow fin coloration as a strong candidate to explain this pattern, and common-garden rearing suggested heritable population differences. Male sexual ornamentation apparently evolved differently across sulfide-adapted populations, for example because of differences in natural counterselection via predation. The ubiquitous preference for yellow color ornaments in poeciliid females likely undermines the emergence of strong RI, as female discrimination in favor of own males becomes weaker when yellow fin coloration in the respective sulfide ecotype increases. Our study illustrates the complexity of the (partly non-parallel) pathways to divergence among replicated ecological gradients. We suggest that future work should identify the genomic loci involved in the pattern reported here, making use of the increasing genomic and transcriptomic datasets available for our study system.