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This thesis is divided into three parts with the main goal allocating novel antimicrobial compounds that could be used as future antibiotics. The first part aimed to evaluate the potential of plant suspension cultures for the production of antimicrobial proteins. The extracellular, intracellular and cell wall bound fractions of seven heterotrophic and photomixotrophic plant cell suspension cultures treated with nine different elicitors were tested for the elicitor dependent production of antimicrobial proteins. Bioactivities were tested against a selected panel of human isolates including Gram-positive and Gram-negative bacteria as well as fungi using the disc diffusion assay. The intracellular fractions of elicited cell cultures were more active than extracellular fractions while the cell wall bound fractions showed lowest activities. Among the 21 fractions tested, the intracellular fraction of Lavendula angustifolia elicited with DC3000 was most active against Candida maltosa. The second most active fraction was the intracellular fraction of Arabidopsis thaliana elicited with salicylic acid which was moreover active against all test strains. The antimicrobial activity of elicited Arabidopsis thaliana cell cultures was tested by bioautography to locate the antimicrobial proteins in the crude extract. The intracellular fraction of photomixotrophic Arabidopsis thaliana cells elicited with salicylic acid was selected for further gel filtration chromatography on S-200 column leading to the purification of one 19 kDa antimicrobially active protein, designated, AtAMP. Our findings suggest that elicited plant cell cultures may present a new promising alternative source of antimicrobial proteins. The second part comprises the isolation of actinomycetes associated with marine sponges and testing the bioactivities of new species for further investigations. Actinobacterial communities of eleven taxonomically different sponges that had been collected from offshore Ras Mohamed (Egypt) and from Rovinj (Croatia) were investigated by a culture-based approach using different standard media for isolation of actinomycetes and media enriched with aqueous sponge extract to target rare and new actinomycete species. Phylogenetic characterization of 52 representative isolates out of 90 based on almost complete sequences of genes encoding 16S rRNA supported their assignment to 18 different actinomycete genera. Altogether 14 putatively new species were identified based on sequence similarity values below 98.2% to other strains in the NCBI database. The use of M1 agar amended with aqueous sponge extract yielded a putative new genus related to Rubrobacter which highlighting the need for innovative cultivation protocols. Biological activity testing showed that five isolates were active against Gram-positives only, one isolate was active against Candida albicans only and one isolate showed activity against both groups of pathogens. Moreover, the antiparasistic activity was documented for four isolates. These results showed a high diversity of actinomycetes associated with marine sponges as well as highlighted their potential to produce anti-infective agents. The third part of the thesis focused on the isolation and structure elucidation of new bioactive compounds. Streptomyces strain RV15 recovered from sponge Dysidea tupha, was selected for further chemical analysis by virtue of the fact that it exhibited the greatest antimicrobial potential against Staphylococcus aureus as well as Candida albicans among the all tested strains. Moreover, members of the genus Streptomyces are well known as prolific producers of interesting pharmacologically active metabolites. Chemical analysis of the methanolic crude extract using different chromatographic tools yielded four new compounds. The structures of the new compounds were spectroscopically elucidated to be four new cyclic peptides, namely, cyclodysidins A-D. Their bioactivity was tested against different proteases, bacteria and Candida as well as tumor cell lines. The compounds did not show any significant activities at this point.
Terrestrial actinomycetes are noteworthy producers of a multitude of antibiotics, however the marine representatives are much less studied in this regard. In this study, 90 actinomycetes were isolated from 11 different species of marine sponges that had been collected from offshore Ras Mohamed (Egypt) and from Rovinj (Croatia). Phylogenetic characterization of the isolates based on 16S rRNA gene sequencing supported their assignment to 18 different actinomycete genera representing seven different suborders. Fourteen putatively novel species were identified based on sequence similarity values below 98.2% to other strains in the NCBI database. A putative new genus related to Rubrobacter was isolated on M1 agar that had been amended with sponge extract, thus highlighting the need for innovative cultivation protocols. Testing for anti-infective activities was performed against clinically relevant, Gram-positive (Enterococcus faecalis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, fungi (Candida albicans) and human parasites (Leishmania major, Trypanosoma brucei). Bioactivities against these pathogens were documented for 10 actinomycete isolates. These results show a high diversity of actinomycetes associated with marine sponges as well as highlight their potential to produce anti-infective agents.
Malaria still persists as one of the deadliest infectious disease in addition to AIDS and tuberculosis. lt is a leading cause of high mortality and morbidity rates in the developing world despite of groundbreaking research on global eradication of the disease initiated by WHO, about half a century ago. Lack of a commercially available vaccine and rapid spread of drug resistance have hampered the attempts of extinguishing malaria, which still leads to an annual death toll of about one million people. Resistance to anti-malarial compounds thus renders search for new target proteins imperative. The kinome of the human malaria parasite Plasmodium falciparum comprises representatives of most eukaryotic protein kinase groups, including kinases which regulate proliferation and differentiation processes. Several reports till date have suggested involvement of parasite kinases in the human host and as well as in the mosquito vector. Kinases essential for life cycle stages of the parasite represent promising targets for anti-malarial compounds thus, provoking characterization of additional malarial kinases. Despite extensive research on most plasmodial enzymes, very little information is available regarding the four identified members of the cyclin dependent kinase like kinase (CLK) family. Thus, the present thesis dealt with the functional characterization of four members of the PfCLK kinase family of the parasite denoted as PfCLK-1/Lammer, PfCLK-2, PfCLK-3 and PfCLK-4 with a special focus on the first two kinases. Additionally, one Ca2+/Calmodulin dependent putative kinase-related protein, PfPKRP, presumed to be involved in sexual stage development of the parasite, was investigated for its expression in the life cycle of the parasite. In other eukaryotes, CLK kinases regulate mRNA splicing through phosphorylation of Serine/Arginine-rich proteins. Transcription analysis revealed abundance of PfCLK kinase genes throughout the asexual blood stages and in gametocytes. By reverse genetics approach it was demonstrated that all four kinases are essential for completion of the asexual replication cycle of P. falciparum. PfCLK 1/Lammer possesses two nuclear localization signals and PfCLK-2 possesses one of these signals upstream of the C-terminal catalytic domains. Protein level expression and sub-cellular localization of the two kinases was determined by generation of antiserum directed against the kinase domains of the respective kinase. Indirect immunofluorescence, Western blot and electron microscopy data confirm that the kinases are primarily localized in the parasite nucleus, and in vitro assays show that both enzymes are associated with phosphorylation activity. Finally, mass spectrometric analysis of co immunoprecipitated proteins shows interactions of the two PfCLK kinases with proteins, which have putative nuclease, phosphatase or helicase functions. PfPKRP on the other hand is predominantly expressed during gametocyte differentiation as identified from transcriptional analysis. Antiserum directed against the catalytic domain of PfPKRP detected the protein expression profile in both asexual and gametocyte parasite lysates. Via immunofluorescence assay, the kinase was localized in the parasite cytoplasm in a punctuated manner, mostly in the gametocyte stages. Reverse genetics resulted in the generation of PfPKRP gene-disruptant parasites, thus demonstrating that unlike CLK kinases, PfPKRP is dispensable for asexual parasite survival and hence might have crucial role in sexual development of the parasite. On one hand, characterization of PfCLK kinases exemplified the kinases involved in parasite replication cycle. Successful gene-disruption and protein expression of PfPKRP kinase on the other hand, demonstrated a role of the kinase in sexual stage development of the parasite. Both kinase families therefore, represent potential candidates for anti-plasmodial compounds.
Traumatic brain injury, a leading cause of death and disability, is a result of an outside force causing mechanical disruption of brain tissue and delayed pathogenic events which collectively exacerbate the injury. These pathogenic injury processes are poorly understood and accordingly no effective neuroprotective treatment is available so far. Experimental models are essential for further clarification of the highly complex pathology of traumatic brain injury towards the development of novel treatments. Among the rodent models of traumatic brain injury the most commonly used are the weight-drop, the fluid percussion, and the cortical contusion injury models. As the entire spectrum of events that might occur in traumatic brain injury cannot be covered by one single rodent model, the design and choice of a specific model represents a major challenge for neuroscientists. This review summarizes and evaluates the strengths and weaknesses of the currently available rodent models for traumatic brain injury.
Chlamydia trachomatis is an obligate intracellular pathogenic bacterium that has been refractory to genetic manipulations. Although the genomes of several strains have been sequenced, very little information is available on the gene structure of these bacteria. We used deep sequencing to define the transcriptome of purified elementary bodies (EB) and reticulate bodies (RB) of C. trachomatis L2b, respectively. Using an RNAseq approach, we have mapped 363 transcriptional start sites (TSS) of annotated genes. Semiquantitative analysis of mapped cDNA reads revealed differences in the RNA levels of 84 genes isolated from EB and RB, respectively. We have identified and in part confirmed 42 genome- and 1 plasmid-derived novel non-coding RNAs. The genome encoded non-coding RNA, ctrR0332 was one of the most abundantly and differentially expressed RNA in EB and RB, implying an important role in the developmental cycle of C. trachomatis. The detailed map of TSS in a thus far unprecedented resolution as a complement to the genome sequence will help to understand the organization, control and function of genes of this important pathogen.
In order to survive, organisms avoid threats and seek rewards. Classical conditioning is a simple model to explain how animals and humans learn associations between events that allow them to predict threats and rewards efficiently. In the classical conditioning paradigm, a neutral stimulus is paired with a biologically significant event (the unconditioned stimulus – US). In virtue of this association, the neutral stimulus acquires affective motivational properties, and becomes a conditioned stimulus (CS+). Defensive responses emerge for pairings with an aversive US (e.g., pain), and appetitive responses emerge for pairing with an appetitive event (e.g., reward). It has been observed that animals avoid a CS+ when it precedes an aversive US during a training phase (CS+ US; forward conditioning); whereas they approach a CS+ when it follows an aversive US during the training phase (US CS+; backward conditioning). These findings indicate that the CS+ acquires aversive properties after a forward conditioning, whereas acquires appetitive properties after a backward conditioning. It is thus of interest whether event timing also modulates conditioned responses in such an opponent fashion in humans, who are capable of explicit cognition about the associations. For this purpose, four experiments were conducted in which a discriminative conditioning was applied in groups of participants that only differed in the temporal sequence between CS+ onset and US onset (i.e., the interstimulus interval – ISI). During the acquisition phase (conditioning), two simple geometrical shapes were presented as conditioned stimuli. One shape (CS+) was always associated with a mild painful electric shock (i.e., the aversive US) and the other one (CS-) was never associated with the shock. In a between-subjects design, participants underwent either forward or backward conditioning. During the test phase (extinction), emotional responses to CS+ and CS- were tested and the US was never presented. Additionally, a novel neutral shape (NEW) was presented as control stimulus. To assess cognitive components, participants had to rate both the valence (the degree of unpleasantness or pleasantness) and the arousal (the degree of calmness or excitation) associated with the shapes before and after conditioning. In the first study, startle responses, an ancestral defensive reflex consisting of a fast twitch of facial and body muscles evoked by sudden and intense stimuli, was measured as an index of stimulus implicit valence. Startle amplitude was potentiated in the presence of the forward CS+ whilst attenuated in the presence of the backward CS+. Respectively, the former response indicates an implicit negative valence of the CS+ and an activation of the defensive system; the latter indicated an implicit positive valence of the CS+ and an activation of the appetitive system. In the second study, the blood-oxygen level dependent (BOLD) response was measured by means of functional magnetic resonance imaging (fMRI) to investigate neural responses after event learning. Stronger amygdala activation in response to forward CS+ and stronger striatum activation in response to backward CS+ were found in comparison to CS-. These results support the notion that the defensive motivational system is activated after forward conditioning since the amygdala plays a crucial role in fear acquisition and expression. Whilst the appetitive motivational system is activated after backward conditioning since the striatum plays a crucial role in reward processing. In the third study, attentional processes underlying event learning were observed by means of steady-state visual evoked potentials (ssVEPs). This study showed that both forward and backward CS+ caught attentional resources. More specifically, ssVEP amplitude was higher during the last seconds of forward CS+ that is just before the US, but during the first seconds of backward CS+ that is just after the US. Supposedly, attentional processes were located at the most informative part of CS+ in respect to the US. Participants of all three studies rated both forward and backward CS+ more negative and arousing compared to the CS-. This indicated that event timing did not influence verbal reports similarly as the neural and behavioral responses indicating a dissociation between the explicit and implicit responses. Accordingly, dual process theories propose that human behavior is determined by the output of two systems: (1) an impulsive implicit system that works on associative principles, and (2) a reflective explicit system that functions on the basis of knowledge about facts and values. Most importantly, these two systems can operate in a synergic or antagonistic fashion. Hence, the three studies of this thesis congruently suggest that the impulsive and the reflective systems act after backward association in an antagonistic fashion. In sum, event timing may turn punishment into reward in humans even though they subjectively rate the stimulus associated with aversive events as being aversive. This dissociation might contribute to understand psychiatric disorders, like anxiety disorders or drug addiction.
The human gut is home for thousands of microbes that are important for human life. As most of these cannot be cultivated, metagenomics is an important means to understand this important community. To perform comparative metagenomic analysis of the human gut microbiome, I have developed SMASH (Simple metagenomic analysis shell), a computational pipeline. SMASH can also be used to assemble and analyze single genomes, and has been successfully applied to the bacterium Mycoplasma pneumoniae and the fungus Chaetomium thermophilum. In the context of the MetaHIT (Metagenomics of the human intestinal tract) consortium our group is participating in, I used SMASH to validate the assembly and to estimate the assembly error rate of 576.7 Gb metagenome sequence obtained using Illumina Solexa technology from fecal DNA of 124 European individuals. I also estimated the completeness of the gene catalogue containing 3.3 million open reading frames obtained from these metagenomes. Finally, I used SMASH to analyze human gut metagenomes of 39 individuals from 6 countries encompassing a wide range of host properties such as age, body mass index and disease states. We find that the variation in the gut microbiome is not continuous but stratified into enterotypes. Enterotypes are complex host-microbial symbiotic states that are not explained by host properties, nutritional habits or possible technical biases. The concept of enterotypes might have far reaching implications, for example, to explain different responses to diet or drug intake. We also find several functional markers in the human gut microbiome that correlate with a number of host properties such as body mass index, highlighting the need for functional analysis and raising hopes for the application of microbial markers as diagnostic or even prognostic tools for microbiota-associated human disorders.
This thesis consists of three major chapters, each of which has been separately published or under the process for publication. The first chapter is about anatomical characterization of the mushroom body of adult Drosophila melanogaster. The mushroom body is the center for olfactory learning and many other functions in the insect brains. The functions of the mushroom body have been studied by utilizing the GAL4/UAS gene expression system. The present study characterized the expression patterns of the commonly used GAL4 drivers for the mushroom body intrinsic neurons, Kenyon cells. Thereby, we revealed the numerical composition of the different types of Kenyon cells and found one subtype of the Kenyon cells that have not been described. The second and third chapters together demonstrate that the multiple types of dopaminergic neurons mediate the aversive reinforcement signals to the mushroom body. They induce the parallel memory traces that constitute the different temporal domains of the aversive odor memory. In prior to these chapters, “General introduction and discussion” section reviews and discuss about the current understanding of neuronal circuit for olfactory learning in Drosophila.
Regulation of pathogen-inducible volatile compounds in Arabidopsis and their role in plant defense
(2010)
Plants are constantly attacked by pathogenic microbes. As a result, they have evolved a plethora of constitutive and inducible defense responses to defend against attempted pathogen infection. Although volatile organic compounds have been implicated in plant defense, direct evidence of their function in plant resistance is still lacking. I have examined the role of VOCs in Arabidopsis defense against the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. maculicola. The obtained results show that the vegetative parts of Arabidopsis produces and emits the volatile phenylpropanoid MeSA and three kinds of terpenoids, (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), alpha-ionon and beta-farnesen, upon avirulent and virulent P. syringae inoculation. Whereas the most abundant volatiles, MeSA and TMTT, are already produced at early stages of infection in the compatible and incompatible interaction, enhanced emission of alpha-ionon and beta-farnesen can only be detected in later stages of the compatible interaction. It was revealed that pathogen-induced synthesis of TMTT in Arabidopsis requires the JA signaling pathway but occurs independently of SA defense signaling. Similarly, the production of MeSA is dependent on JA signaling but not on the SA defense signaling pathway. Furthermore, production of MeSA is dependent on the function of ISOCHORISMATE SYNTHASE1, which produces its precursor SA. Upon inoculation with avirulent P. syringae, endogenously produced JA activates the JA signalling pathway to mediate MeSA and TMTT synthesis. By contrast, in the compatible Arabidopsis-Psm interaction, production of MeSA predominantly depends on the P. syringea the virulence factor coronatine, which activates JA downstream signaling. To learn more about the role of inducible VOCs in plant defense responses, I have identified an Arabidopsis T-DNA insertions line with a defect in the TERPENE SYNTHASE4 (TPS4) gene. Emission profiles from this mutant revealed that the induced production of TMTT but not of alpha-ionone, beta-farnesene or MeSA are abolished, demonstrating that TPS4 specifically regulates the P. syringae-induced synthesis of TMTT in Arabidopsis. The lack of TMTT in tps4 mutants, however, does not affect plant defense responses and resistance induction against P. syringae. This excludes a role of the terpenoid as an effective phytoalexin in Arabidopsis leaves against the bacterial pathogen. Moreover, tps4 mutant plants are still able to mount a SAR response, excluding a signaling function of TMTT during SAR. An important aim of our studies was to address the defensive role of MeSA, the major VOC emitted from P. syringae-inoculated Arabidopsis leaves. MeSA has been recently proposed as a critical long distance signal in the development of SAR. I found that two independent T-DNA insertions lines with defects in expression of the pathogen-inducible SA methyl transferase gene BSMT1 are completely devoid of pathogen-induced production of MeSA. However, bsmt1 mutant plants are capable to increase the level of SA in systemic, non-infected leaves of Arabodopsis and develop SAR like wild-type plants upon local P. syringae-inoculation. Thus, MeSA does not function as a critical SAR signal in Arabidopsis. Further experiments showed that SA accumulation in distant leaves occurs due to de novo synthesis through isochorismate synthase. In addition, we also ruled out a critical defensive role of MeSA at inoculation sites, because bsmt1 mutants are able to build up SA-dependent defense responses and local resistance in a wild-type-like manner. The conversion of SA to MeSA and subsequently emission of MeSA from the plant might help the plant to detoxify an excess of SA. This process is regulated by the JA pathway and might be one means to mediate negative crosstalk between JA and SA signaling. Moreover, the COR-triggered conversion of SA to MeSA and emission of the volatile methyl ester could be a way by which virulent P. syringae is able to attenuate the SA-defense pathway.
Für die Lösung der quantenmechanischen Bewegungsgleichungen, die komplexe, molekulare Systeme beschreiben, sind effiziente und verlässliche Näherungsverfahren erforderlich. Die Dichtefunktionaltheorie (DFT) stellt für die Behandlung der Elektronenwechselwirkung in vielen Fällen den besten Kompromiss zwischen Effizienz und Genauigkeit dar. Im Rahmen der DFT wird die gesamte nicht-klassische Elektron-Elektron-Wechselwirkung im so genannten Austausch-Korrelationsfunktional angenähert. Viele solcher Näherungen sind semi-empirischer Natur, andere wurden ausschließlich von physikalischen Überlegungen abgeleitet. In globalen Hybridfunktionale wird ein konstanter Anteil der integrierten DFT-Austauschenergiedichte durch exakten Austausch aus der Hartree-Fock Näherung ersetzt. Das populärste Funktional B3LYP enthält 20 % exakten Austausch und mehrere empirische Parameter. Der optimale Prozentsatz hängt allerdings sehr stark von den zu berechnenden Systemen und molekularen Eigenschaften ab. Eine Lösung dieses Problems sollten lokale Hybridfunktionale liefern, in denen die Beimischung der exakten Austauschenergiedichte über eine lokale Mischfunktion (LMF) gesteuert wird und daher positions- und molekülabhängig ist. In dieser Arbeit wird ein semi-empirischer Ansatz für die Entwicklung neuer lokaler Hybridfunktionale verfolgt: während die Energiedichten unverändert aus etablierten Näherungen zum Austauschkorrelationsfunktional übernommen werden, stehen parametrisierte LMFs im Zentrum der Untersuchungen. Die verschiedenen LMFs beinhalten neben mindestens einem empirischen Parameter eine Variable die vom Quotienten der von-Weizsäcker kinetischen Energiedichte und der korrelierten kinetischen Energiedichte (sogenannte t-LMFs) bzw. dem reduzierten Dichtegradienten (bezeichnet als t-LMFs) abhängt. Weitere LMFs werden durch zusätzliche Berücksichtigung der Spinpolarisation erhalten. Alle Parameter werden an Atomisierungsenergien bzw. Reaktionsbarrieren bekannter molekularer Testsätze gefittet. Durch Visualisierung der LMFs können zusätzlich Einblicke in den physikalischen Hintergrund und in Möglichkeiten der Weiterentwicklung gewonnen werden. Es wurde beispielsweise beobachtet, dass entlang einer gedehnten Bindung höhere Werte der LMF und damit größere Beimischungen exakter Austauschenergie in Übergangszuständen einhergehen. Dieser Effekt ist für t-LMFs am ausgeprägtesten und korreliert mit besseren Ergebnissen für Reaktionsbarrieren mit lokalen Hybridfunktionalen, die auf einer t-LMF basieren. Bis auf wenige Ausnahmen leiten sich die lokalen Hybridfunktionale in dieser Arbeit aus dem Austausch- und Korrelationsfunktional der lokalen Dichtenäherung (LSDA) ab und enthalten keine Gradientenkorrektur im Sinne der GGA (generalized gradient approximation). Die neuen Funktionale wurden zunächst nicht-selbstkonsistent in eine Entwicklerversion des quantenchemischen Programmpaketes Turbomole implementiert. Das bedeutet, für gegebene Molekülorbitale bzw. eine gegeben Elektronendichte kann lediglich die Gesamtenergie berechnet werden. Dies ist eine anerkannte Näherung, die vor allem für die Optimierung der Parameter eine große Zeitersparnis darstellt. Um letztlich orbitalabhängige, molekulare Eigenschaften berechnen zu können wird neben der Gesamtenergie auch noch das zugehörige lokale Hybridpotential benötigt. Für die selbstkonsistente Implementierung wird die funktionale Ableitung der Austauschkorrelationsenergie nach den Orbitalen bestimmt. Daraus resultierend müssen neben den üblichen lokalen Austauschkorrelationspotentialtermen auch Integrale berechnet werden, die das mit der LMF gewichtete nicht-lokale exakte Austauschpotential enthalten. Die entsprechenden Terme kann man, genauso wie die exakte Austauschenergiedichte an sich, nicht analytisch berechnen. Früheren Ansätzen folgend wurden sie in der vorliegenden Arbeit in einer Basissatzentwicklung angenähert, wobei der Einfachheit halber die atomaren Basisfunktionen verwendet wurden. Um die Genauigkeit dieser sogenannten RI (resolution of the identity)-Näherung validieren zu können und auch schon im Hinblick auf die Anpassung einer Hilfsbasis, wurde darüber hinaus die numerische Berechnung aller Integrale, die das exakte Austauschpotential und die entsprechende Energiedichte enthalten, implementiert. Unter Verwendung der RI-Näherung ist der Rechenaufwand lokaler Hybride vergleichbar mit dem globaler Hybridfunktionale: Während die formale Skalierung in Abhängigkeit der Systemgröße gleich ist, ergab sich ein etwas höherer Vorfaktor für die lokalen Hybride. Verschiedene Literaturbekannte Testsätze mit Atomisierungsenergien, Reaktionsbarrieren, Dissoziationsenergien oder Gleichgewichtsabständen, die teilweise einige Schwächen bisheriger Dichtefunktionalnäherungen aufdecken, wurden berücksichtigt. Für die 223 Atomisierungsenergien des G3 Testsatzes stellen alle unsere Funktionale eine signifikante Verbesserung gegenüber B3LYP dar. Atomisierungsenergien sind insofern ein sensibler Test, da alle Bindungen gebrochen werden und Fehlerkompensation eine untergeordnete Rolle spielt. Vor allem lokale Hybridfunktionale, deren LMFs neben der kinetischen Energiedichte explizit von der Spinpolarisation abhängen, lieferten hervorragende Resultate. Obwohl im Vergleich zu Atomisierungsenergien für die korrekte Berechnung von Reaktionsbarrieren im Allgemeinen mehr exakter Austausch benötigt wird, sind unsere Funktionale auch für zwei Testsätze mit jeweils 38 Reaktionsbarrieren besser als B3LYP. Zwar kann mit einem globalen Hybrid mit 50 % exaktem Austausch eine geringere Abweichung von den Richtwerten erzielt werden, aber ein solches Funktional ist für thermochemische Daten unzureichend. Hier wurde erstmals gezeigt, dass lokale Hybridfunktionale ohne Gradientenkorrektur sowohl für Thermochemie als auch für Kinetik zufrieden stellende Ergebnisse liefern können. Das Dissoziationsverhalten symmetrischer Radikalkationen stellt für die hier diskutierten Dichtefunktionale nach wie vor eine Herausforderung dar: Die Dissoziationsenergien von sieben Modellsystemen werden mit unseren Funktionalen stark überschätzt und Gleichgewichtsabstände unterschätzt. Insgesamt sind die Werte nur marginal besser als mit B3LYP. Neben Eigenschaften von Hauptgruppenverbindungen wurden zudem Übergangsmetalldimere und -monohydride untersucht. Für erstere ist eine gute Beschreibung dynamischer sowie statischer Elektronenkorrelation ausschlaggebend. In den Hydriden andererseits dominiert mit gängigen Dichtefunktionalen die unphysikalische Selbstwechselwirkung eines Elektrons mit sich selbst. Für die 3d-Übergangsmetalldimere sind die getesteten Funktionale genauso gut wie B3LYP und für die Hydride etwas besser. Atomare s-d Transferenergien von 3d Übergangsmetallen verbleiben auch für unsere lokalen Hybridfunktionale, die insgesamt schlechtere Ergebnisse erzielen als B3LYP, noch problematisch. Das hierfür geeignetste lokale Hybridfunktional basiert auf einer s-LMF und beinhaltet LYP Korrelation. Für die isotropen Hyperfeinkopplungskonstanten (HFCCs) kleiner Hauptgruppenverbindungen wurden zufriedenstellende Ergebnisse (ähnlich wie B3LYP) mit einem t-LMF basierten lokalen Hybrid erzielt. Die RI Näherung zum lokalen Hybridpotential wurde dem numerisch exakten Potential für die Berechnung von Gesamtenergien, isotrope HFCCs und Orbitalenergien für verschiedene Basissätze gegenübergestellt. Wie erwartet ist der Fehler für Gesamtenergien mit der RI-Näherungen vergleichsweise gering, vor allem relativ zu den verbleibenden Abweichungen von experimentellen Energien. Der Vergleich der mittleren absoluten Abweichung von experimentellen Werten für 26 isotrope HFCCs zeigt sogar für mittelgroße und kontrahierte IGLO Basissätze nur geringe Unterschiede zwischen dem RI-Potential und dem numerisch exakten lokalen Hybridpotential. Die Analyse der HFCCs einzelner Moleküle und der Orbitalenergien des CN Moleküls offenbart allerdings, dass Ungenauigkeiten aufgrund der RI-Näherung hier eine größere Rolle spielen, vor allem wenn zu kleine atomare Basissätze verwendet werden. Von den untersuchten lokalen Hybriden stellen sich einige als hervorragende Kandidaten für die Berechnung thermochemischer und kinetischer Eigenschaften heraus. Jeweils unterschiedliche Funktionale erzielen darüber hinaus mit den besten bekannten Funktionalen vergleichbare Ergebnisse für isotrope Hyperfeinkopplungskonstanten und ausgewählte Eigenschaften kleiner Übergangsmetallverbindungen. Die in dieser Arbeit präsentierten lokalen Hybridfunktionale stellen daher einen wichtigen Schritt in der Entwicklung universeller Näherungen zum Austauschkorrelationsfunktional dar. Zur akkuraten Beschreibung molekularer Eigenschaften von Übergangsmetallkomplexen und dem Dissoziationsverhalten von Radikal-Kation-Dimeren neben Thermochemie und Kinetik, werden in Zukunft wohl komplexere LMFs benötigt. Um konkurrenzfähige lokale Hybride mit gradientenkorrigierter Austausch- und Korrelationsenergiedichte zu entwickeln, müssen darüber hinaus weitere Studien zum Einfluss des abweichenden Eichursprungs der miteinander kombinierten Austauschenergiedichten durchgeführt werden. Eine andere Möglichkeit ist die Entwicklung speziell abgestimmter Korrelationsfunktionale für lokale Hybride. Außerdem sollte die Qualität der RI-Näherung zum lokalen Hybridpotential detaillierter untersucht werden. Hierfür könnten zum Beispiel Ionisierungsenergien und Elektronenaffinitäten herangezogen werden. Um zusätzliche Abweichungen oder sogar fälschlicherweise "zu gute" Ergebnisse bei Validierungsrechnungen zu vermeiden, sollten Hilfsbasen für die Entwicklung des nicht-lokalen exakten Austauschpotentials implementiert und optimiert werden. Einer der nächsten Implementierungsschritte sollte auch Gradienten bezüglich der Kernkoordinaten beinhalten, um die Validierung der neuen lokalen Hybridfunktionale auf Strukturoptimierungen auszuweiten.
The genus Borrelia belongs to the Spirochaetes phylum which is far related to Gram negative bacteria. This phylum possesses a characteristic long helically coiled shape with lengths that vary from 5 to 250 μm. Other pathogens as Treponema and Leptospira which cause syphilis and leptospirosis, also belong to the Spirochaetes. Borrelia itself is the causative agent of two human diseases, the Lyme disease and relapsing fever. Borreliae are pathogenic bacteria which cycle between their arthropod vector, in most cases a tick, and a mammal host, very often small rodents. This complex life cycle requires an extraordinary protein up- and down-regulation in order to survive in such different organisms and avoid their immunologic systems. Lyme disease is a multisystemic disease that can affect different organs like skin, joints and nervous system. A red rash with concentric rings, called erythema migrans is a distinctive manifestation that allows clinical diagnosis. It appears after the bite of an infected tick and spreads out to diameters that can reach 15 cm. Relapsing fever is characterized by sudden recurrent fever peaks accompanied with chills, headache, muscle and joint pain and nausea. Both diseases are easily treated with antibiotics in early infection stages. Borrelia species possess a small genome. Many of their genes are related with virulence and the adaptation to the different hosts. The absence of genes in Borrelia involved in the biosynthesis of amino acids, fatty acids or nucleotide is very remarkable. This metabolic deficiency makes Borrelia species dependent on substances produced by the host. The first step in nutrient uptake is accomplished by porins. Bacterial porins are water-filled channels that facilitate the transport of essential molecules through the outer membrane. Four porins have been described in Borrelia up to this point. P66, P13 and Oms28 have been found in Borrelia burgdorferi while Oms38 was discovered in relapsing fever spirochetes. P66 is a singular porin with an extremely high single channel conductance of 11 nS. P13 is a small protein with an α-helical secondary structure which does not fit into the general porin model. The function of Oms28 as a porin has been questioned recently due to its periplasmic membrane-associated location. Finally, Oms38 is a specific porin for dicarboxilates with homologues in Lyme disease species. The aim of this thesis was to broaden the knowledge of the P66 and P13 porins described in the genus Borrelia. Both differ in structure and size from the general Gram negative porin model and could be highly involved in specific tasks in the genus Borrelia. In the first project of this thesis, the presence and pore forming capacity of P66 was studied in several Borrelia species including members of the relapsing fever group. P66 is the best studied porin in Borrelia with a dual function as porin and adhesin. This knowledge is restricted to B. burgdorferi and little or nothing is known about homologues in other Borrelia species. Therefore, three Lyme disease and three relapsing fever species were chosen as representative agents of the genus and the pore forming activity of their P66 homologues was studied. Five out of the six homologues exhibited a similar single channel conductance in a range from 9 to 11 nS. All of them showed no selectivity for cations or anions, and they were voltage dependent starting at different voltages from 30 to 70 mV. Only in the case of the B. hermsii homologue no pore forming activity could be established. It remains unclear if the lack of activity was due to an evolutionary loss of its porin function or to a higher sensibility to the detergents used for purification. In another project, the controversial P66 pore diameter of B. burgdorferi was analyzed with an empirical method. In a former study, the diameter of the P66 channel was estimated to be 2.6 nm based on theoretical considerations. This diameter is rather large and could impair the outer membrane protective function. Different non-electrolytes were used to study the P66 pore diameter indicating a 1.8 nm entrance diameter and a 0.8 nm inner constriction. In addition, the blockage of the channel with some of those non-electrolytes disclosed an oligomeric organization formed by approximately eight independent channels. Such a structure has not been observed so far in any other living organism and could be exclusive of Borrelia or spirochetes. The third project of this thesis deal with the recombinant production of a B. burgdorferi protein with immunogenic potential. This protein might be used to develop new diagnosis tests and therapeutic treatments. P13 is an outer membrane protein present in LD and RF species and it does not have any other known bacterial homologue. These facts make of P13 a good candidate to be used as a therapeutic target. For such purpose, P13 was cloned in two organisms. First, in Escherichia coli were two different constructs were designed to establish the role of a periplasmic cleaved C-terminus. Second, in a virus based vector delivered by Agrobacterium tumefaciens into tobacco plant cells. The vector replicates inside the plant cells spreading the infection to adjacent cells and at the same time producing the recombinant protein. This second expression method should enable the production of large amounts of the recombinant protein reducing time and costs. The last project of this thesis looked into the outer membrane complexome of B. burgdorferi focusing on the P13 and P66 porin complexes. Blue Native Page and second dimension SDS Page were the technique chosen for this purpose. P66 could be shown to be the only protein involved in the formation of the 11 nS pore which complex is probably formed by eight monomers. It was also possible to divide this complex in two halves with approximately half the molecular weight and a conductance of 5.5 nS. In the case of the P13 complex, a possible association with the lipoprotein OspC was revealed. The gel extraction of the P13 complex and its test with the Back Lipid Bilayer assay exhibited a 0.6 nS activity. This is in high contrast with the 3.5 nS activity previously described for this protein. To sum up, P66 is a porin present in many Borrelia species including not only LD but also RF species and which homologues show similar biophysical properties. The diameter of this pore is smaller than previously thought and it has molecular weight sieving properties. In the case of P13, its recombinant procurement will allow the use of P13 as a diagnostic and therapeutic target. The possible association with OspC could facilitate to unravel in future experiments the function of this intriguing protein.
Avian pathogenic Escherichia coli (APEC) represent a subset of the so-called extraintestinal pathogenic Escherichia coli (ExPEC) pathotype that can cause various extraintestinal infections in humans and animals. APEC are the causative agent of localized colibacillosis or systemic infection in poultry. In this latter case, the syndrome starts as an infection of the upper respiratory tract and develops into a systemic infection. Generally, ExPEC are characterized by a broad variety of virulence-associated factors that may contribute to pathogenesis. Major virulence factors, however, that clearly define this pathotype, have not been identified. Instead, virulence-associated genes of ExPEC and thus also of APEC could be used in a mix-and-match-fashion. Both pathotypes could not be clearly distinguished by molecular epidemiology, and this suggested a hypothetical zoonotic risk caused by APEC. Accordingly, the main scientific question of this study was to characterize common traits as well as differences of APEC and human ExPEC variants that could either support the possible zoonotic risk posed by these pathogenic E. coli strains or indicate factors involved in host specificity. Comparative genomic analysis of selected APEC and human ExPEC isolates of the same serotype indicated that these variants could not be clearly distinguished on the basis of (i) general phenotypes, (ii) phylogeny, (iii) the presence of typical ExPEC virulence genes, and (iv) the presence of pathoadaptive mutations. Allelic variations in genes coding for adhesins such as MatB and CsgA or their regulators MatA and CsgD have been observed, but further studies are required to analyze their impact on pathogenicity. On this background, the second part of this thesis focused on the analysis of differences between human ExPEC and APEC isolates at the gene expression level. The analysis of gene expression of APEC and human ExPEC under growth conditions that mimick their hosts should answer the question whether these bacterial variants may express factors required for their host-specificity. The transcriptomes of APEC strain BEN374 and human ExPEC isolate IHE3034 were compared to decipher whether there was a specific or common behavior of APEC and human ExPEC, in response to the different body temperatures of man (37°C) or poultry (41°C). Only a few genes were induced at 41 °C in each strain relative to growth at 37 °C. The group of down-regulated genes in both strains was markedly bigger and mainly included motility and chemotaxis genes. The results obtained from the transcriptome, genomic as well as phenotypic comparison of human ExPEC and APEC, supports the idea of a potential zoonotic risk of APEC and certain human ExPEC variants. In the third part of the thesis, the focus was set on the characterization of Mat fimbriae, and their potential role during ExPEC infection. Comparison of the mat gene cluster in K-12 strain MG1655 and O18:K1 isolate IHE3034 led to the discovery of differences in (i) DNA sequence, (ii) the presence of transcriptional start and transcription factor binding sites as well as (iii) the structure of the matA upstream region that account for the different regulation of Mat fimbriae expression in these strains. A negative role of the H-NS protein on Mat fimbriae expression was also proven at 20 °C and 37 °C by real-time PCR. A major role of this fimbrial adhesin was demonstrated for biofilm formation, but a significant role of Mat fimbriae for APEC in vivo virulence could not yet be determined. Interestingly, the absence of either a functional matA gene or that of the structural genes matBCDEF independently resulted in upregulation of motility in E. coli strains MG1655 and IHE3034 by a so far unknown mechanism. In conclusion, the results of this thesis indicate a considerable overlap between human and animal ExPEC strains in terms of genome content and phenotypes. It becomes more and more apparent that the presence of a common set of virulence-associated genes among ExPEC strains as well as similar virulence gene expression patterns and phylogenetic backgrounds indicate a significant zoonotic risk of avian-derived E. coli isolates. In addition, new virulence factors identified in human ExPEC may also play a role in the pathogenesis of avian ExPEC.
Thermodynamics of Competitive Molecular Channel Transport: Application to Artificial Nuclear Pores
(2010)
In an analytical model channel transport is analyzed as a function of key parameters, determining efficiency and selectivity of particle transport in a competitive molecular environment. These key parameters are the concentration of particles, solvent-channel exchange dynamics, as well as particle-in-channel- and interparticle interaction. These parameters are explicitly related to translocation dynamics and channel occupation probability. Slowing down the exchange dynamics at the channel ends, or elevating the particle concentration reduces the in-channel binding strength necessary to maintain maximum transport. Optimized in-channel interaction may even shift from binding to repulsion. A simple equation gives the interrelation of access dynamics and concentration at this transition point. The model is readily transferred to competitive transport of different species, each of them having their individual in-channel affinity. Combinations of channel affinities are determined which differentially favor selectivity of certain species on the cost of others. Selectivity for a species increases if its in-channel binding enhances the species’ translocation probablity when compared to that of the other species. Selectivity increases particularly for a wide binding site, long channels, and fast access dynamics. Recent experiments on competitive transport of in-channel binding and inert molecules through artificial nuclear pores serve as a paradigm for our model. It explains qualitatively and quantitatively how binding molecules are favored for transport at the cost of the transport of inert molecules.
Introduction: Chronic nonbacterial osteomyelitis (CNO) is an inflammatory disorder of unknown etiology. In children and adolescents CNO predominantly affects the metaphyses of the long bones, but lesions can occur at any site of the skeleton. Prospectively followed cohorts using a standardized protocol in diagnosis and treatment have rarely been reported. Methods: Thirty-seven children diagnosed with CNO were treated with naproxen continuously for the first 6 months. If assessment at that time revealed progressive disease or no further improvement, sulfasalazine and short-term corticosteroids were added. The aims of our short-term follow-up study were to describe treatment response in detail and to identify potential risk factors for an unfavorable outcome. Results: Naproxen treatment was highly effective in general, inducing a symptom-free status in 43% of our patients after 6 months. However, four nonsteroidal anti-inflammatory drug (NSAID) partial-responders were additionally treated with sulfasalazine and short-term corticosteroids. The total number of clinical detectable lesions was significantly reduced. Mean disease activity estimated by the patient/physician and the physical aspect of health-related quality of life including functional ability (global assessment/childhood health assessment questionnaire and childhood health assessment questionnaire) and pain improved significantly. Forty-one percent of our patients showed radiological relapses, but 67% of them were clinically silent. Conclusions: Most children show a favorable clinical course in the first year of anti-inflammatory treatment with NSAIDs. Relapses and new radiological lesions can occur at any time and at any site in the skeleton but may not be clinically symptomatic. Whole-body magnetic resonance imaging proved to be very sensitive for initial and follow-up diagnostics.
We develop a purchasing portfolio method by integrating a company view, a market-based view and a process view, aggregated in a 3-dimensional portfolio cube. Top management typically takes another view on purchasing issues than purchasing itself. Furthermore, it seems crucial to include the process view, since strategies have to be executed and organisational design features to support these strategies have to be compatible with purchasing processes. This integrated approach seems more complete compared to single, 2-dimensional portfolio methods.
The construction of mound-shaped nests by ants is considered as a behavioral adaptation to low environmental temperatures, i.e., colonies achieve higher and more stables temperatures than those of the environment. Besides the well-known nests of boreal Formica wood-ants, several species of South American leaf-cutting ants of the genus Acromyrmex construct thatched nests. Acromyrmex workers import plant fragments as building material, and arrange them so as to form a thatch covering a central chamber, where the fungus garden is located. Thus, the degree of thermoregulation attained by the fungus garden inside the thatched nest largely depends on how the thatch affects the thermal relations between the fungus and the environment. This work was aimed at studying the thermoregulatory function of the thatched nests built by the grass-cutting ant Acromyrmex heyeri Forel (Hymenoptera: Formicidae: Myrmicinae). Nest and environmental temperatures were measured as a function of solar radiation on the long-term. The thermal diffusivity of the nest thatch was measured and compared to that of the surrounding soil, in order to assess the influence of the building material on the nest’s thermoregulatory ability. The results showed that the average core temperature of thatched nests was higher than that of the environment, but remained below values harmful for the fungus. This thermoregulation was brought about by the low thermal diffusivity of the nest thatch built by workers with plant fragments, instead of the readily-available soil particles that have a higher thermal diffusivity. The thatch prevented diurnal nest overheating by the incoming solar radiation, and avoided losses of the accumulated daily heat into the cold air during the night. The adaptive value of thatching behavior in Acromyrmex leaf-cutting ants occurring in the southernmost distribution range is discussed.
Interleukin-18 (IL-18) is a proinflammatory cytokine of the interleukin-1 family which is upregulated after cerebral ischemia. The functional role of IL-18 in cerebral ischemia is unknown. In the present study, we compared infarct size in IL-18 knock-out and wild-type mice 24 hours and 48 hours after 1-hour transient middle cerebral artery occlusion (tMCAO). Moreover, the functional outcome was evaluated in a modified Bederson score, foot fault test and grip test. There were no significant differences in infarct size or functional outcome tests between wild-type and IL-18 knock-out mice. These data indicate that the early inflammatory response to cerebral ischemia does not involve IL-18, in contrast to other interleukin-1 family members such as interleukin-1.
Cysteines play important roles in the biochemistry of many proteins. The high reactivity, redox properties, and ability of the free thiol group to coordinate metal ions designate cysteines as the amino acids of choice to form key catalytic components of many enzymes. Also, cysteines readily react with reactive oxygen and nitrogen species to form reversible oxidative thiol modifications. Over the last few years, an increasing number of proteins have been identified that use redox-mediated thiol modifications to modulate their function, activity, or localization. These redox-regulated proteins are central players in numerous important cellular processes. First aim of this study was to discover nitric oxide (NO) sensitive proteins in E. coli, whose redox-mediated functional changes might explain the physiological alterations observed in E. coli cells suffering from NO-stress. To identify E. coli proteins that undergo reversible thiol modifications upon NO-treatment in vivo, I applied a differential thiol trapping technique combined with two-dimensional gel analysis. 10 proteins were found to contain thiol groups sensitive to NO-treatment. Subsequent genetic studies revealed that the oxidative modifications of AceF & IlvC are, in part, responsible for the observed NO-induced growth inhibition. Noteworthy, the majority of identified protein targets turned out to be specifically sensitive towards reactive nitrogen species. This oxidant specificity was tested on one NO-sensitive protein, the small subunit of glutamate synthase. In vivo and in vitro activity studies demonstrated that glutamate synthase rapidly inactivates upon nitric oxide treatment but is resistant towards other oxidative stressors. These results imply that reactive oxygen and nitrogen species affect distinct physiological processes in bacteria. The second aim of my study was to identify redox-sensitive proteins in S. cerevisiae and to use their redox state as in vivo read-out to assess the role of oxidative stress during the eukaryotic aging process. I first determined the precise in vivo thiol status of almost 300 yeast proteins located in the cytosol and sub-cellular compartments of yeast cells using a highly quantitative mass spectrometry based thiol trapping technique, called OxICAT. The identified proteins can be clustered in four groups: 1) proteins, whose cysteine residues are oxidation resistant; 2) proteins with structurally or functionally important cysteine modifications 3) proteins with highly oxidation-sensitive active site cysteines, which are partially oxidized in exponentially growing yeast cells due to their exquisite sensitivity towards low amounts of ROS; 4) proteins that are reduced in exponentially growing cells but harbor redox-sensitive cysteine(s) that affect the catalytic function of the protein during oxidative stress. These oxidative stress sensitive proteins were identified by exposure of yeast cells to sublethal concentrations of H2O2 or superoxide. It was shown that the major targets of peroxide- and superoxide-mediated stress in the cell are proteins involved in translation, glycolysis, TCA cycle and amino acid biosynthesis. These targets indicate that cells rapidly redirect the metabolic flux and energy towards the pentose phosphate pathway in an attempt to ensure the production of the reducing equivalent NADPH to counterattack oxidative stress. These results reveal that the quantitative assessment of a protein’s oxidation state is a valuable tool to identify catalytically active and redox-sensitive cysteine residues. The OxICAT technology was then used to precisely determine extent and onset of oxidative stress in chronologically aging S. cerevisiae cells by utilizing the redox status of proteins as physiological read-out. I found that chronological aging yeast cells undergo a global collapse of the cellular redox homeostasis, which precedes cell death. The onset of this collapse appears to correlate with the yeast life span, as caloric restriction increases the life span and delays the redox collapse. These results suggest that maintenance of the redox balance might contribute to the life expanding benefits of regulating the caloric intake of yeast. Clustering analysis of all oxidatively modified proteins in chronological aging yeast revealed a subset of proteins whose oxidative thiol modifications significantly precede the general redox collapse. Oxidation of these early target proteins, which most likely results in a loss of their activity, might contribute to or even cause the observed loss of redox homeostasis (i.e., thioredoxin reductase) in chronologically aging yeast. These studies in aging yeast expand our understanding how changes in redox homeostasis affect the life span of yeast cells and confirm the importance of oxidative thiol modifications as key posttranslational modifications in pro- and eukaryotic organisms.
Background: Dendritic cells (DC) can act tolerogenic at a semi-mature stage by induction of protective CD4+ T cell and NKT cell responses. Methodology/Principal Findings: Here we studied the role of the co-inhibitory molecule B7-H1 (PD-L1, CD274) on semimature DC that were generated from bone marrow (BM) cells of B7-H12/2 mice and applied to the model of Experimental Autoimmune Encephalomyelitis (EAE). Injections of B7-H1-deficient DC showed increased EAE protection as compared to wild type (WT)-DC. Injections of B7-H12/2 TNF-DC induced higher release of peptide-specific IL-10 and IL-13 after restimulation in vitro together with elevated serum cytokines IL-4 and IL-13 produced by NKT cells, and reduced IL-17 and IFN-c production in the CNS. Experiments in CD1d2/2 and Ja2812/2 mice as well as with type I and II NKT cell lines indicated that only type II NKT cells but not type I NKT cells (invariant NKT cells) could be stimulated by an endogenous CD1d-ligand on DC and were responsible for the increased serum cytokine production in the absence of B7-H1. Conclusions/Significance: Together, our data indicate that BM-DC express an endogenous CD1d ligand and B7-H1 to ihibit type II but not type I NKT cells. In the absence of B7-H1 on these DC their tolerogenic potential to stimulate tolerogenic CD4+ and NKT cell responses is enhanced.
Cooperation is beneficial for social groups and is exemplified in its most sophisticated form in social insects. In particular, eusocial Hymenoptera, like ants and honey bees, exhibit a level of cooperation only rarely matched by other animals. To assure effective defense of group members, foes need to be recognized reliably. Ants use low-volatile, colony-specific profiles of cuticular hydrocarbons (colony odor) to discriminate colony members (nestmates) from foreign workers (non-nestmates). For colony recognition, it is assumed that multi-component colony odors are compared to a neuronal template, located in a so far unidentified part of the nervous system, where a mismatch results in aggression. Alternatively, a sensory filter in the periphery of the nervous system has been suggested to act as a template, causing specific anosmia to nestmate colony odor due to sensory adaptation and effectively blocking perception of nestmates. Colony odors are not stable, but change over time due to environmental influences. To adjust for this, the recognition system has to be constantly updated (template reformation). In this thesis, I provide evidence that template reformation can be induced artificially, by modifying the sensory experience of carpenter ants (Camponotus floridanus; Chapter 1). The results of the experiments showed that template reformation is a relatively slow process taking several hours and this contradicts the adaptation-based sensory filter hypothesis. This finding is supported by first in-vivo measurements describing the neuronal processes underlying template reformation (Chapter 5). Neurophysiological measurements were impeded at the beginning of this study by the lack of adequate technical means to present colony odors. In a behavioral assay, I showed that tactile interaction is not necessary for colony recognition, although colony odors are of very low volatility (Chapter 2). I developed a novel stimulation technique (dummy-delivered stimulation) and tested its suitability for neurophysiological experiments (Chapter 3). My experiments showed that dummy-delivered stimulation is especially advantageous for presentation of low-volatile odors. Colony odor concentration in headspace was further increased by moderately heating the dummies, and this allowed me to measure neuronal correlates of colony odors in the peripheral and the central nervous system using electroantennography and calcium imaging, respectively (Chapter 4). Nestmate and non-nestmate colony odor elicited strong neuronal responses in olfactory receptor neurons of the antenna and in the functional units of the first olfactory neuropile of the ant brain, the glomeruli of the antennal lobe (AL). My results show that ants are not anosmic to nestmate colony odor and this clearly invalidates the previously suggested sensory filter hypothesis. Advanced two-photon microscopy allowed me to investigate the neuronal representation of colony odors in different neuroanatomical compartments of the AL (Chapter 5). Although neuronal activity was distributed inhomogeneously, I did not find exclusive representation restricted to a single AL compartment. This result indicates that information about colony odors is processed in parallel, using the computational power of the whole AL network. In the AL, the patterns of glomerular activity (spatial activity patterns) were variable, even in response to repeated stimulation with the same colony odor (Chapter 4&5). This finding is surprising, as earlier studies indicated that spatial activity patterns in the AL reflect how an odor is perceived by an animal (odor quality). Under natural conditions, multi-component odors constitute varying and fluctuating stimuli, and most probably animals are generally faced with the problem that these elicit variable neuronal responses. Two-photon microscopy revealed that variability was higher in response to nestmate than to non-nestmate colony odor (Chapter 5), possibly reflecting plasticity of the AL network, which allows template reformation. Due to their high variability, spatial activity patterns in response to different colony odors were not sufficiently distinct to allow attribution of odor qualities like ‘friend’ or ‘foe’. This finding challenges our current notion of how odor quality of complex, multi-component odors is coded. Additional neuronal parameters, e.g. precise timing of neuronal activity, are most likely necessary to allow discrimination. The lower variability of activity patterns elicited by non-nestmate compared to nestmate colony odor might facilitate recognition of non-nestmates at the next level of the olfactory pathway. My research efforts made the colony recognition system accessible for direct neurophysiological investigations. My results show that ants can perceive their own nestmates. The neuronal representation of colony odors is distributed across AL compartments, indicating parallel processing. Surprisingly, the spatial activity patterns in response to colony are highly variable, raising the question how odor quality is coded in this system. The experimental advance presented in this thesis will be useful to gain further insights into how social insects discriminate friends and foes. Furthermore, my work will be beneficial for the research field of insect olfaction as colony recognition in social insects is an excellent model system to study the coding of odor quality and long-term memory mechanisms underlying recognition of complex, multi-component odors.
Surveys by the Universities of Wuerzburg and Berlin, starting in the 1970´s have revealed the existence of palaeolakes in remote areas in Niger. Initial research has shown that the sediments found are suitable for reconstructing its late quaternary palaeoenvironment. Although a high number of investigations focused on the succession of climatological conditions in the Central Sahara, some uncertainties still exist as the results show discontinuities and mostly are of low temporal and spatial
resolution.
Two expeditions in 2005 and 2006 headed to the northeastern parts of Niger to investigate the known remains of palaeolakes and search some new and undetected ones. Samples were taken at several study sites in order to receive a complete picture of the Late Quaternary environmental settings and to produce high-resolution proxies for palaeoclimate modelling.
The most valuable and best-investigated study site is the sebkha of Seggedim, where a core of 15 meters length could be extracted which revealed a composition of high-resolution sections. Stratigraphical, structural and geochemical investigations as well as the analysis of thin sections allow the characterization of different environmental conditions from Early to Mid Holocene. Driven by climate and hydrogeological influence, the water body developed from a water pond of several metres depth within a stable, grass and shrub vegetated landscape, to an alternating freshwater lake in a more dynamic environmental setting. Radiocarbon dates set the beginning of the stage at about 10.6 ka cal BP, with an exceptionally stable regime to 6.6 ka cal BP (at 12.6 metres’ depth), when a major change in the sedimentation regime of the basin is recorded in the core. Increased erosion, likely due to decreased vegetation cover within the basin, led to the siltation/filling of the lake within a few hundred years and the subsequent development of a sebkha/salt pan due to massive evaporation. Due to the lack of dateable material in the upper core section, the termination of the lake stage and the onset of the subsequent sebkha stage cannot be determined precisely but can be narrowed to a period around 6 ka BP.
The results obtained from the core are compared with those from terrestrial and lacustrine sediments from outside the depression, situated a few hundred kilometres further to the north. These supplementary study sites are required to validate the information obtained from the coring. Within the plateau landscape of Djado, Mangueni and Tchigai, two depressions and a valley containing lacustrine deposits, were investigated for palaeoenvironmental reconstruction. Depending on modifying local factors, these sediment archives were of shorter existence than IX the lake, but reveal additional information about the landscape dynamics from Early to Mid Holocene.
A damming situation within a small tributary at Enneri Achelouma led to lacustrine sedimentation conditions at Early Holocene in the upper reaches of the valley. The remnants of the lacustrine accumulations show distinct changes in the environmental conditions within the small catchment, as the archive immediately responded to local climate-induced changes of precipitation. Radiocarbon dating of the deposited sediments revealed ages from 8780 ± 260 cal a BP to 9480 ± 80 cal a BP.
The sites of Yoo Ango and Fabérgé show a completely different sedimentation milieu as they consist of basins within the foothills of the Tchigai. The study sites show increased catchment sizes, probably extending towards the Tchigai massif and are most likely influenced by groundwater charge. The widespread occurrence of wind shaped relicts and the limited amount of lacustrine remnants indicate a generally high aeolian activity in both areas. Only in wind sheltered spots, parts of the lacustrine sequences were preserved, that show ages spanning from Early to Mid Holocene (9440 ± 140 cal a BP – 6810 ±140 cal a BP) and give additional evidence of fires from pre-LGM periods. Although intensively weathered, all profiles indicate distinct changes in the sedimentation conditions by alternating geochemical values and the mineralogical composition.
The information obtained from the records investigated in this work confirms the heterogeneity of reconstructed environmental succession in the Central Sahara. The Mid Holocene rapid (within decades) and uniform development from more humid to extremely arid environmental conditions cannot be confirmed for the Central Sahara. In addition, a division of Early and Mid Holocene wet periods cannot be confirmed, either. Actually, the evidences obtained from the palaeoenvironmental reconstructions revealed major variations in the timing and extend of lacustrine and aeolian periods. Evidently, a transitional time has existed between 7 to 5 ka BP where alternating influences prevailed. This is indicated by the varying sedimentation conditions in the Seggedim depression as well as the evidence of soil properties on a fossil dune, with a time of deposition dated to 6200 ± 400 cal a BP and the removal of lacustrine Sediments at the Seeterrassental at Mid Holocene. In respect to provide a complete picture of landscape succession and to avoid misinterpretation, the investigation of several dissimilar spots within a designated study area is prerequisite for further investigations.
In the molecular structure of the title compound, C34H58B2N2, each B atom of the diborane(4) is connected to one dimethylamino group and one Tip ligand (Tip = 2,4,6-triisopropylphenyl). These findings indicate that the increased steric demand of the Tip groups exerts influence solely on the B—B separation but not on the overall geometry of the title compound.
Since the beginning, central banks have used a wide range of instruments to achieve their ultimate purpose of price stability. One measure in the authorities toolbox is a foreign exchange market intervention. The discussion about this instrument has come a long way. So far, the discussion relied mainly on industrialized countries' experiences. The negative outcomes of most studies with respect to the effectiveness of the intervention tool, opened up a discussion, whether interventions should be used by the authorities to manage exchange rate aspects. Consequently, the question about the dynamics of foreign exchange market interventions is now open to the subject-matter of developing and emerging market countries. Monetary policy in those countries often constitutes an active management of exchange rates. However, the basic discussions about intervention dynamics have had one essential drawback. Neither the primary literature of industrialized countries nor studies dealing with developing countries have considered the fact that intervention purposes and the corresponding effects are likely to vary over time. This thesis is designed to provide the reader with essential issues of central bank interventions, and aims to give further, as well as new contributions, in terms of empirical research on interventions in emerging markets. The main objectives of this study are the analysis of central bank intervention motives, and the corresponding effects on exchange rates in emerging markets. The time dependency of both issues is explicitly considered, which states a novelty in academic research of central bank interventions. Additionally, the outcomes are discussed against the background of underlying economic and monetary policy fundamentals. This could well serve as a starting point for further research.
Background: High mobility group A (HMGA) proteins regulate gene transcription through architectural modulation of chromatin and the formation of multi-protein complexes on promoter/enhancer regions. Differential expression of HMGA variants has been found to be important for distinct differentiation processes and deregulated expression was linked to several disorders. Here we used mouse C2C12 myoblasts and C2C12 cells stably over-expressing HMGA1a-eGFP to study the impact of deregulated HMGA1 expression levels on cellular differentiation. Results: We found that induction of the myogenic or osteogenic program of C2C12 cells caused an immediate down-regulation of HMGA1. In contrast to wild type C2C12 cells, an engineered cell line with stable overexpression of HMGA1a-eGFP failed to differentiate into myotubes. Immunolocalization studies demonstrated that sustained HMGA1a-eGFP expression prevented myotube formation and chromatin reorganization that normally accompanies differentiation. Western Blot analyses showed that elevated HMGA1a-eGFP levels affected chromatin composition through either down-regulation of histone H1 or premature expression of MeCP2. RT-PCR analyses further revealed that sustained HMGA1a expression also affected myogenic gene expression and caused either down-regulation of genes such as MyoD, myogenin, Igf1, Igf2, Igfbp1-3 or up-regulation of the transcriptional repressor Msx1. Interestingly, siRNA experiments demonstrated that knock-down of HMGA1a was required and sufficient to reactivate the myogenic program in induced HMGA1a over-expressing cells. Conclusions: Our data demonstrate that HMGA1 down-regulation after induction is required to initiate the myogenic program in C2C12 cells. Sustained HMGA1a expression after induction prevents expression of key myogenic factors. This may be due to specific gene regulation and/or global effects on chromatin. Our data further corroborate that altered HMGA1 levels influence the expression of other chromatin proteins. Thus, HMGA1 is able to establish a specific chromatin composition. This work contributes to the understanding of how differential HMGA1 expression is involved in chromatin organization during cellular differentiation processes and it may help to comprehend effects of HMGA1 over-expression occurring in malign or benign tumours.
Summary: I previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nNOS) inhibited L-type Ca2+-channels and decreased myocardial contractility1 (Burkard N. et al. (2007). Circ Res 100, 32-44). However, nNOS has multiple targets within the cardiac myocyte and it is possible that interesting biological functions of this protein remain to be elucidated. In this study, I showed that nNOS overexpression has a cardioprotective effect after ischemia-reperfusion injury by inhibiting mitochondrial function and reducing the generation of reactive oxygen species (ROS). The effect of conditional nNOS overexpression in cardiac myocytes in ischemiareperfusion injury was assessed. Ischemia-reperfusion injury in WT mice resulted in nNOS accumulation in the mitochondria. Similary, transgenic nNOS overexpression caused nNOS abundance in mitochondria. Electron microscopy of mouse myocardium from nNOS overexpressing mice showed that after induction of its expression, nNOS is additionally localised in mitochondria. nNOS translocation into mitochondria was dependent on HSP90. Ischemia-reperfusion experiments in isolated hearts showed a cardioprotective effect of nNOS overexpression (30min post-ischemia, LVDP 27.0±2.5mmHg in non-induced animals vs. 45.2±1.9mmHg in nNOS overexpressing mice, n=12, p<0.05). Consistently with this finding, in vivo the infarct size within the area at risk was significantly decreased in nNOS overexpressing mice compared to non-induced animals (36.6±8.4 relative % vs. 61.1±2.9 relative %, n=12, p<0.05). nNOS overexpression also caused a significant increase in mitochondrial nitrite levels accompanied by a decrease of cytochrome c oxidase activity (72.0±8.9units/ml in nNOS overexpressing mice vs. 113.2±17.1units/ml in non-induced mice, n=12, p<0.01) resulting in an inhibition of mitochondrial function. Accordingly, O2-consumption (MVO2) in isolated heart muscle stripes was decreased in nNOS overexpressing mice, already under resting conditions (0.016±0.0015 vs. 0.024±0.006ml[O2] x mm-3 x min-1, n=13, p<0.05). Additionally, this study showed that the ROS concentration was significantlydecreased in hearts of nNOS overexpressing mice compared to non-induced animals (6.14±0.685 vs. 14.53±1.7μM, n=8, p<0.01). Application of different inhibitors, Western Blot analysis and activity assays showed that the lower ROS concentration in nNOS overexpressing mice was caused by inhibition of the xanthine oxidoreductase (XOR) activity by the increased abundance of nNOS expression. In summary, this study demonstrated that the conditional transgenic overexpression of nNOS resulted in myocardial protection after ischemia-reperfusion injury. Besides reduction of myocardial Ca2+-overload after reperfusion this might be caused by inhibition of mitochondrial function through nNOS, which reduced myocardial oxygen consumption already under baseline conditions (Burkard N. conditionally accepted by
This thesis is concerned with the statistical physics of various systems far from thermal equilibrium, focusing on universal critical properties, scaling laws and the role of fluctuations. To this end we study several models which serve as paradigmatic examples, such as surface growth and non-equilibrium wetting as well as phase transitions into absorbing states. As a particular interesting example of a model with a non-conventional scaling behavior, we study a simplified model for pulsed laser deposition by rate equations and Monte Carlo simulations. We consider a set of equations, where islands are assumed to be point-like, as well as an improved one that takes the size of the islands into account. The first set of equations is solved exactly but its predictive power is restricted to the first few pulses. The improved set of equations is integrated numerically, is in excellent agreement with simulations, and fully accounts for the crossover from continuous to pulsed deposition. Moreover, we analyze the scaling of the nucleation density and show numerical results indicating that a previously observed logarithmic scaling does not apply. In order to understand the impact of boundaries on critical phenomena, we introduce particle models displaying a boundary-induced absorbing state phase transition. These are one-dimensional systems consisting of a single site (the boundary) where creation and annihilation of particles occur, while particles move diffusively in the bulk. We study different versions of these models and confirm that, except for one exactly solvable bosonic variant exhibiting a discontinuous transition with trivial exponents, all the others display a non-trivial behavior, with critical exponents differing from their mean-field values, representing a universality class. We show that these systems are related to a $(0+1)$-dimensional non-Markovian model, meaning that in nonequilibrium a phase transition can take place even in zero dimensions, if time long-range interactions are considered. We argue that these models constitute the simplest universality class of phase transition into an absorbing state, because the transition is induced by the dynamics of a single site. Moreover, this universality class has a simple field theory, corresponding to a zero dimensional limit of direct percolation with L{\'e}vy flights in time. Another boundary phenomena occurs if a nonequilibrium growing interface is exposed to a substrate, in this case a nonequilibrium wetting transition may take place. This transition can be studied through Langevin equations or discrete growth models. In the first case, the Kardar-Parisi-Zhang equation, which defines a very robust universality class for nonequilibrium moving interfaces, is combined with a soft-wall potential. While in the second, microscopic models, in the corresponding universality class, with evaporation and deposition of particles in the presence of hard-wall are studied. Equilibrium wetting is related to a particular case of the problem, corresponding to the Edwards-Wilkinson equation with a potential in the continuum approach or to the fulfillment of detailed balance in the microscopic models. In this thesis we present the analytical and numerical methods used to investigate the problem and the very rich behavior that is observed with them. The entropy production for a Markov process with a nonequilibrium stationary state is expected to give a quantitative measure of the distance form equilibrium. In the final chapter of this thesis, we consider a Kardar-Parisi-Zhang interface and investigate how entropy production varies with the interface velocity and its dependence on the interface slope, which are quantities that characterize how far the stationary state of the interface is away from equilibrium. We obtain results in agreement with the idea that the entropy production gives a measure of the distance from equilibrium. Moreover we use the same model to study fluctuation relations. The fluctuation relation is a symmetry in the large deviation function associated to the probability of the variation of entropy during a fixed time interval. We argue that the entropy and height are similar quantities within the model we consider and we calculate the Legendre transform of the large deviation function associated to the height for small systems. We observe that there is no fluctuation relation for the height, nevertheless its large deviation function is still symmetric.
Sucrose- and H+-Dependent Charge Movements Associated with the Gating of Sucrose Transporter ZmSUT1
(2010)
Background: In contrast to man the majority of higher plants use sucrose as mobile carbohydrate. Accordingly protondriven sucrose transporters are crucial for cell-to-cell and long-distance distribution within the plant body. Generally very negative plant membrane potentials and the ability to accumulate sucrose quantities of more than 1 M document that plants must have evolved transporters with unique structural and functional features. Methodology/Principal Findings: To unravel the functional properties of one specific high capacity plasma membrane sucrose transporter in detail, we expressed the sucrose/H+ co-transporter from maize ZmSUT1 in Xenopus oocytes. Application of sucrose in an acidic pH environment elicited inward proton currents. Interestingly the sucrose-dependent H+ transport was associated with a decrease in membrane capacitance (Cm). In addition to sucrose Cm was modulated by the membrane potential and external protons. In order to explore the molecular mechanism underlying these Cm changes, presteady-state currents (Ipre) of ZmSUT1 transport were analyzed. Decay of Ipre could be best fitted by double exponentials. When plotted against the voltage the charge Q, associated to Ipre, was dependent on sucrose and protons. The mathematical derivative of the charge Q versus voltage was well in line with the observed Cm changes. Based on these parameters a turnover rate of 500 molecules sucrose/s was calculated. In contrast to gating currents of voltage dependentpotassium channels the analysis of ZmSUT1-derived presteady-state currents in the absence of sucrose (I =Q/t) was sufficient to predict ZmSUT1 transport-associated currents. Conclusions: Taken together our results indicate that in the absence of sucrose, ‘trapped’ protons move back and forth between an outer and an inner site within the transmembrane domains of ZmSUT1. This movement of protons in the electric field of the membrane gives rise to the presteady-state currents and in turn to Cm changes. Upon application of external sucrose, protons can pass the membrane turning presteady-state into transport currents.
Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice
(2010)
Background: Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Results: Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund’s adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1b), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1b. The increase of the antiinflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-1b, and IL-10 following CFA, overall corroborating the inhibitor data. Conclusion: These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.
The overarching goal of this research was to explore accurate methods of mapping irrigated crops, where digital cadastre information is unavailable: (a) Boundary separation by object-oriented image segmentation using very high spatial resolution (2.5–5 m) data was followed by (b) identification of crops and crop rotations by means of phenology, tasselled cap, and rule-based classification using high resolution (15–30 m) bi-temporal data. The extensive irrigated cotton production system of the Khorezm province in Uzbekistan, Central Asia, was selected as a study region. Image segmentation was carried out on pan-sharpened SPOT data. Varying combinations of segmentation parameters (shape, compactness, and color) were tested for optimized boundary separation. The resulting geometry was validated against polygons digitized from the data and cadastre maps, analysing similarity (size, shape) and congruence. The parameters shape and compactness were decisive for segmentation accuracy. Differences between crop phenologies were analyzed at field level using bi-temporal ASTER data. A rule set based on the tasselled cap indices greenness and brightness allowed for classifying crop rotations of cotton, winter-wheat and rice, resulting in an overall accuracy of 80 %. The proposed field-based crop classification method can be an important tool for use in water demand estimations, crop yield simulations, or economic models in agricultural systems similar to Khorezm.
The main aim of this thesis was the synthesis and structural characterization of penta and hexacoordinate silicon(IV) complexes. In the course of these studies, the neutral pentacoordinate silicon(IV) complexes 38, 39, 43−48, 54 and 55 were prepared. Furthermore, the neutral hexacoordinate silicon(IV) complexes 33−36, 49, 50, 52, 53, 56−62, 63, 64 and 65 were synthesized. All compounds were characterized by elemental analyses, NMR spectroscopy in solution (1H, 13C, 15N, 29Si) and in the solid-state (13C, 15N, 29Si VACP/MAS NMR), as well as single-crystal X-ray diffraction (except 45, 47−49, 52, 53 and 63).
In the framework of this thesis, new UV-patternable organic-inorganic hybrid polymers with higher refractive indices than reported in the literature for photonic applications were developed and studied with respect to their chemical structure, their optical properties, and their ability of being patterned by 1PP and 2PP. Particularly with 2PP, one could create 3D structures using the novel hybrid materials. The materials were prepared from hydrolysis and polycondensation reactions of · organo-alkoxysilanes and titanium alkoxide precursors, modified with and without CL and organo-alkoxysilanes precursors, and · organo-alkoxysilanes, titanium alkoxide and organophosphorus precursors. The major scope of this work was to increase the refractive index of ORMCER® materials based on only organo-alkoxysilanes. Thus, the parameters which influence the refractive index were investigated thoroughly. In particular, the synthesis parameters such as the introduction of titanium alkoxide and its concentration, the organo-alkoxysilanes, the catalyst concentration, the solvent used, but, also the processing parameters such as, the UV exposure dose, initiator concentration, and developer were investigated.
Synthesis and Characterization of an Oligo(Phenylene Ethynylene)-Based Perylene Bisimide Foldamer
(2010)
The present work is part of the currently only rudimentary understanding of the structure-property relationships in the self-assembly of pi-conjugated organic molecules. Such structures may reveal favorable photophysical and semiconducting properties due to the weak non-covalent pi-pi interactions between the monomer units. The specific mutual orientation of the dyes is known to evoke individual functional properties for the condensed matter, however, the related electronic processes are still not well-understood and further enhancements of functional properties are seldom triggered by rational design. The pi-pi self-assembly structures of perylene bisimide (PBI) dyes are promising, versatile materials for organic electronic devices and have been elected for this thesis as an archetype aggregate system to investigate the dye-dye interactions in more detail. In cooperation with experts in the field of spectroscopy and theory the development of reliable routines towards a better understanding of the origins of the functional properties may be feasible, and, on a longer time-line, such knowledge may enable optimization of functional organic materials. Having designed such structures entailed the challenge of developing feasible synthesis strategies, and to actually generate the targeted molecules by synthesis. Several synthesis approaches were conducted until finally a perylene bisimide foldamer was obtained based on a Sonogashira co-polymerization reaction. After purification and enrichment of the larger-sized species by means of semi-preparative gel permeation chromatography (GPC) the average size of an octamer (8500 Da) species was determined by analytical GPC. The low polydispersity index (PD) of 1.1 is indicative of a sharp size distribution of the oligomers. This average size was confirmed by performing diffusion ordered NMR spectroscopy (DOSY). Furthermore, MALDI-TOF mass analysis substantiated the structural integrity of the co-polymerization product. Solvent-dependent UV/vis spectroscopic investigations demonstrated that intramolecular PBI aggregates are reversibly formed, indicating that this oligomer is able to fold and unfold in the intended manner upon changing external conditions. In the unfolded states, the PBI moieties are closely arranged due to the short OPE bridges (< 2.4 nm), which is expressed by an exciton coupling interaction of the dyes and therefore the characteristic monomer absorption pattern of the PBI chromophore cannot be obtained in the unfolded states. More interestingly, the folded state revealed a pronounced aggregate spectrum of the PBIs, however, striking differences in the shape of the absorption spectrum compared to our previously investigated PBI self-assembly were obtained.
Arabidopsis thaliana (A.th.) mesophyll cells play a pivotal role in the regulation of the drought stress response. The signaling & transport components involved in drought stress regulation within lipid rafts of the plasma membrane were investigated by DRM isolation from highly purified plasma membranes. Detergent treatment with Brij-98 and Triton X-100 resulted in a total of 246 DRM proteins which were identified by nano HPLC-MS/MS. The majority of these proteins could be isolated by Triton X-100 treatment (78.5 %) which remains the ”golden” standard for the isolation of DRMs. Comparing in-gel and in-solution digestion approaches disclosed additional protein identifications for each method but the in-gel approach clearly delivered the majority of the identified proteins (81.8 %). Functionally, a clear bias on signaling proteins was visible – almost 1/3 of the detected DRM proteins belonged to the group of kinases, phosphatases and other signaling proteins. Especially leucine-rich repeat receptor-like protein kinases and calcium-dependent protein kinases were present in Brij-98 & Triton X-100 DRMs, for instance the calcium-dependent protein kinase CPK21. Another prominent member of DRMs was the protein phosphatase 2C 56, ABI1, which is a key regulator of the ABA-mediated drought stress response in A.th. The lipid raft localization of the identified DRM proteins was confirmed by sterol-depletion with the chemical drug MCD. Proteins which depend upon a sterol-rich environment are depleted from DRMs by MCD application. Especially signaling proteins exhibited a strong sterol-dependency. They represented the vast majority (41.5 %) among the Triton X-100 DRM proteins which were no longer detected following MCD treatment. AtRem 1.2 & 1.3 could be shown to be sterol-dependent in mesophyll cells as well as two CPKs (CPK10 & CPK21) and the protein phosphatase ABI1. AtRem 1.2 & 1.3 could be proven to represent ideal plant lipid raft marker proteins due to their strong presence in Triton X-100 DRMs and dependency upon a sterol-rich environment. When fluorescence labeled AtRem 1.2 & 1.3 were transiently expressed in A.th. leaves, they localized to small, patchy structures at the plasma membrane. CPK21 was an intrinsic member of Triton X-100 DRMs and displayed extreme susceptibility to sterol-depletion by MCD in immunological and proteomic assays. Calcium-dependent protein kinases (CPKs) have already been studied to be involved in drought stress regulation, for instance at the regulation of S-type anion channels in guard cells. Hence, further transient expression studies with the anion channel SLAH3, protein kinase CPK21 and its counterpart, protein phosphatase ABI1 were performed in Nicotiana benthamiana. Transient co-expression of CPK21 and the anion channel SLAH3, a highly mesophyll- specific homologue of the guard cell anion channel SLAC1, resulted in a combined, sterol-dependent localization of both proteins in DRMs. Supplementary co-expression of the counterpart protein phosphatase ABI1 induced dislocation of SLAH3 from DRMs, probably by inactivation of the protein kinase CPK21. CPK21 is known to regulate the anion channel SLAH3 by phosphorylation. ABI1 dephosphorylates CPK21 thus leading to deactivation and dislocation of SLAH3 from DRMs. All this regulative events are taking place in DRMs of A.th. mesophyll cells. This study presents the first evidence for a lipid raft-resident protein complex combining signaling and transport functions in A.th. Future perspectives for lipid raft research might target investigations on the lipid raft localization of candidate DRM proteins under presence of abiotic and biotic stress factors. For instance, which alterations in the DRM protein composition are detectable upon exogenous application of the plant hormone ABA? Quantitative proteomics approaches will surely increase our knowledge of the post-transcriptional regulation of gene activity under drought stress conditions.
The Marketing Effect
(2010)
Migration of immune cells to the target organ plays a key role in autoimmune disorders like multiple sclerosis (MS). However, the exact underlying mechanisms of this active process during autoimmune lesion pathogenesis remain elusive. To test if pro-inflammatory and regulatory T cells migrate via a similar molecular mechanism, we analyzed the expression of different adhesion molecules, as well as the composition of infiltrating T cells in an in vivo model of MS, adoptive transfer experimental autoimmune encephalomyelitis in rats. We found that the upregulation of ICAM-I and VCAM-I parallels the development of clinical disease onset, but persists on elevated levels also in the phase of clinical remission. However, the composition of infiltrating T cells found in the developing versus resolving lesion phase changed over time, containing increased numbers of regulatory T cells (FoxP3) only in the phase of clinical remission. In order to test the relevance of the expression of cell adhesion molecules, animals were treated with purified antibodies to ICAM-I and VCAM-I either in the phase of active disease or in early remission. Treatment with a blocking ICAM-I antibody in the phase of disease progression led to a milder disease course. However, administration during early clinical remission aggravates clinical symptoms. Treatment with anti-VCAM-I at different timepoints had no significant effect on the disease course. In summary, our results indicate that adhesion molecules are not only important for capture and migration of pro-inflammatory T cells into the central nervous system, but also permit access of anti-inflammatory cells, such as regulatory T cells. Therefore it is likely to assume that intervention at the blood brain barrier is time dependent and could result in different therapeutic outcomes depending on the phase of CNS lesion development.
Background: In populations of most social insects, gene flow is maintained through mating between reproductive individuals from different colonies in periodic nuptial flights followed by dispersal of the fertilized foundresses. Some ant species, however, form large polygynous supercolonies, in which mating takes place within the maternal nest (intranidal mating) and fertilized queens disperse within or along the boundary of the supercolony, leading to supercolony growth (colony budding). As a consequence, gene flow is largely confined within supercolonies. Over time, such supercolonies may diverge genetically and, thus, also in recognition cues (cuticular hydrocarbons, CHC’s) by a combination of genetic drift and accumulation of colony-specific, neutral mutations. Methodology/Principal Findings: We tested this hypothesis for six supercolonies of the invasive ant Anoplolepis gracilipes in north-east Borneo. Within supercolonies, workers from different nests tolerated each other, were closely related and showed highly similar CHC profiles. Between supercolonies, aggression ranged from tolerance to mortal encounters and was negatively correlated with relatedness and CHC profile similarity. Supercolonies were genetically and chemically distinct, with mutually aggressive supercolony pairs sharing only 33.1%617.5% (mean 6 SD) of their alleles across six microsatellite loci and 73.8%611.6% of the compounds in their CHC profile. Moreover, the proportion of alleles that differed between supercolony pairs was positively correlated to the proportion of qualitatively different CHC compounds. These qualitatively differing CHC compounds were found across various substance classes including alkanes, alkenes and mono-, di- and trimethyl-branched alkanes. Conclusions: We conclude that positive feedback between genetic, chemical and behavioural traits may further enhance supercolony differentiation through genetic drift and neutral evolution, and may drive colonies towards different evolutionary pathways, possibly including speciation.
Protein phosphatases can be classified into at least three major families based on amino acid sequences at their active sites. A newly emerging phosphatase family contains the active site sequence DXDX(T/V), and belongs to the haloacid dehalogenase (HAD) superfamily of hydrolases, a ubiquitous and evolutionarily conserved enzyme family. Although the existence of 58 human HAD enzymes has been predicted by database analysis, our understanding of their biological functions remains rudimentary.By database mining amd phylogenetic analysis of human HAD phosphatases, we have found a marked increase in cell area of spreading cells, as well as accelerated cell spreading onfibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration. a previously unidentified enzyme with homology to Chronophin, a cytoskeletal regulatory HAD phosphatase. We have cloned and characterized this novel enzyme and named it AUM,for actin remodeling, ubiquitously expressed, magnesium-dependent HAD phosphatase. By Northern blot, real-time PCR and Western blot analysis, we show that AUM is broadly expressed in all major human and mouse tissues with highest levels found in testis. Using immunohistochemistry, we can show that AUM is specifically expressed in maturing germ cells and that its expression peaks during spermiogenesis. To characterize the substrate preference of AUM, we have conducted an in vitro phosphatase substrate screen with 720 phosphopeptides derived from human phosphorylation sites. AUM exclusively dephosphorylates phosphotyrosine (pTyr)-containing peptides. Furthermore, only 17 pTyr peptides (~2% of all pTyr peptides investigated) acted as AUM substrates, indicating a high degree of substrate specificity. Putative AUM substrates include proteins involved in cytoskeletal dynamics and tyrosine kinase signaling.In accordance with the phosphopeptide screen, phosphatase overlay assays employing whole-cell extracts of pervanadate-treated HeLa cells show that AUM dephosphorylates only a limited number of tyrosyl-phosphorylated proteins.The role of AUM for cellular signaling was investigated in response to epidermal growth factor (EGF) stimulation in a spermatogonial cell line (GC-1 spg). The overexpression of AUM reduces, whereas the RNAi-mediated depletion of endogenous AUM increases EGF inducedtyrosine phosphorylation, including changes in the phosphorylation of the EGF receptor itself. Interestingly, in vitro kinase/phosphatase assays with purified Src and AUM indicate that AUM can activate Src, which in turn phosphorylates and inactivates AUM. Although it is at present unclear how Src and AUM regulate each other, our initial findings suggests that AUM enhances Src kinase activity independently of its phosphatase activity, whereas Src diminishes AUM phosphatase activity in a kinase dependent manner. On a cellular level, AUM-depleted cells are characterized by altered actin cytoskeletal dynamics and adhesion, as indicated by stabilized actin filaments, enlarged focal adhesions,a marked increase in cell area of spreading cells, as well as accelerated cell spreading on fibronectin. Taken together, we have identified and characterized AUM as a novel member of the emerging family of aspartate-dependent protein tyrosine phosphatases. Our findings implicate AUM as an important regulator of Src-dependent cytoskeletal dynamics during cell adhesion and migration.
BACKGROUND: Recently, members of the two-pore domain potassium channel family (K2P channels) could be shown to be involved in mechanisms contributing to neuronal damage after cerebral ischemia. K2P3.1-/- animals showed larger infarct volumes and a worse functional outcome following experimentally induced ischemic stroke. Here, we question the role of the closely related K2P channel K2P9.1. METHODS: We combine electrophysiological recordings in brain-slice preparations of wildtype and K2P9.1-/- mice with an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional impact of K2P9.1 in stroke formation. RESULTS: Patch-clamp recordings reveal that currents mediated through K2P9.1 can be obtained in slice preparations of the dorsal lateral geniculate nucleus (dLGN) as a model of central nervous relay neurons. Current characteristics are indicative of K2P9.1 as they display an increase upon removal of extracellular divalent cations, an outward rectification and a reversal potential close to the potassium equilibrium potential. Lowering extracellular pH values from 7.35 to 6.0 showed comparable current reductions in neurons from wildtype and K2P9.1-/- mice (68.31 +/- 9.80% and 69.92 +/- 11.65%, respectively). These results could be translated in an in vivo model of cerebral ischemia where infarct volumes and functional outcomes showed a none significant tendency towards smaller infarct volumes in K2P9.1-/- animals compared to wildtype mice 24 hours after 60 min of tMCAO induction (60.50 +/- 17.31 mm3 and 47.10 +/- 19.26 mm3, respectively). CONCLUSIONS: Together with findings from earlier studies on K2P2.1-/- and K2P3.1-/- mice, the results of the present study on K2P9.1-/- mice indicate a differential contribution of K2P channel subtypes to the diverse and complex in vivo effects in rodent models of cerebral ischemia.
In this study poplar trees have been examined under different stress conditions. Apart from the detailed descriptions above two main conclusions might be drawn: i) A small plant like Arabidopsis thaliana is highly susceptible to stress situations that might become life-threatening compared to a tree that has extremely more biomass at its disposal. Such an organism might be able to compensate severe stress much longer than a smaller one. It seems therefore reasonable that a crop like Arabidopsis reacts earlier and faster to a massive threat. ii) In poplar both tested stress responses seemed to be regulated by hormones. The reactions to abiotic salt stress are mainly controlled by ABA, which also has a strong impact upon cold and drought stress situations. The term commonly used for ABA is “stress hormone” and is at least applicable to all abiotic stresses. In case of herbivory (biotic stress), jasmonic acid appears to be the key-player that coordinates the defence mechanism underlying extrafloral nectary and nectar production. Thus the presented work has gained a few more insights into the complex network of general stress induced processes of poplar trees. Future studies will help to understand the particular role of the intriguing indirect defence system of the extrafloral nectaries in more detail.
The Apologetic revisited: Exonerating Luke from an Ancestral Exegetical and Theological Burden
(2010)
The trend in the scholarship of Luke has been that of presenting Luke as a theologian interested in the survival of Christianity in the Roman world. Becuase of this aim, he seems to overlook the wrongdoings of the Powerful in his time inorder not to endanger the peace of Christianity. However, the intention of this work is to show the defiance and fearlessness of Luke in his dealings with the Rich and the Powerful. He never compromised with the basic teachings of Christianity. A second proper look opens the critical dynamics of his Gospel and the acts, beginning with the Magnificat running through the angelic annunciation scene and the temptation and ending with the punishment of Herod Agrippa in the Acts. The reader beholds a hitherto unknown Luke, who operates within a particular critical stance to the Powerful.
Reactive oxygen species (ROS) are continuously generated in cells and are involved in physiological processes including signal transduction but also their damaging effects on biological molecules have been well described. A number of reports in the literature implicate excessive oxidative stress and/or inadequate antioxidant defense in the pathogenesis of cancer, atherosclerosis, chronic and age related disorders. Several studies have indicated that activation of the renin-angiotensin-aldosterone-system can lead to the formation of ROS. Epidemiological studies have revealed higher renal cell cancer incidences and also higher cancer mortalities in hypertensive individuals. Recently, our group has shown that perfusion of the isolated mouse kidney with Ang II or treatment of several cell lines with Ang II leads to formation of DNA damage and oxidative base modifications. Here, we tried to scrutinize the pathway involved in genotoxicity of Ang II. We confirmed the genotoxicity of Ang II in two kidney cell lines of human origin. Ang II treatment led to the production of superoxide anions which we could hinder when we used the membrane permeable superoxide dismutase (SOD) mimetic TEMPOL. One of the enzymes which is activated in the cells after Ang II treatment and is able to produce ROS is NADPH oxidase. We demonstrated the activation of NADPH oxidase in response to Ang II by upregulation of its p47 subunit using RT-PCR. Also, pPhosphorylation of p47 subunit of NADPH oxidase after Ang II treatment was enhanced. Using two inhibitors we showed that NADPH oxidase inhibition completely prevents DNA damage by Ang II treatment. To differentiate between Nox2 and Nox4 isoforms of NADPH oxidase subunits in the genotoxicity of Ang II, we performed siRNA inhibition and found a role only for Nox4, while Nox2 was not involved. Next, we investigated PKC as a potential activator of NADPH oxidase. We showed that PKC becomes phosphorylated after Ang II treatment and also that inhibition of PKC hinders Ang II from damaging the cells. Our results from using several inhibitors of different parts of the pathway revealed that PKC activation in this pathway is dependent on the action of PLC on membrane phospholipids and production of IP3. IP3 binds to its receptor at endoplasmic reticulum (ER), opening a channel which allows calcium efflux into the cytoplasm. In this manner, both ER calcium stores and extracellular calcium cooperate so that Ang II can exert its genotoxic effect. PLC is activated by AT1R stimulation. We could also show that the genotoxicity of Ang II is mediated via AT1R signaling using the AT1R antagonist candesartan. In conclusion, here we have shown that Ang II is able to damage genomic damage in cell lines of kidney origin. The observed damage is associated with production of ROS. A decrease in Ang II-induced DNA damage was observed after inhibition of G-proteins, PLC, PKC and NADPH oxidase and interfering with intra- as well as extracellular calcium signaling. This leads to the following preliminary model of signaling in Ang II-induced DNA damage: binding of Ang II to the AT1 receptor activates PLC via stimulation of G-proteins, resulting in the activation of PKC in a calcium dependent manner which in turn, activates NADPH oxidase. NADPH oxidase with involvement of its Nox4 subunit then produces reactive oxygen species which cause DNA damage. Dopamine content and metabolism in the peripheral lymphocytes of PD patients are influenced by L-Dopa administration. The PD patients receiving a high dose of L-Dopa show a significantly higher content of dopamine in their lymphocytes compared to PD patients who received a low dose of L-Dopa or the healthy control. Central to many of the processes involved in oxidative stress and oxidative damage in PD are the actions of monoamine oxidase (MAO), the enzyme which is responsible for the enzymatic oxidation of dopamine which leadsing to production of H2O2 as a by-product. We investigated whether dopamine oxidation can cause genotoxicity in lymphocytes of PD patents who were under high dose L-Dopa therapy and afterward questioned the occurrence of DNA damage after dopamine treatment in vitro and tried to reveal the mechanism by which dopamine exerts its genotoxic effect. The frequency of micronuclei in peripheral blood lymphocytes of the PD patients was not elevated compared to healthy age-matched individuals, although the formation of micronuclei revealed a positive correlation with the daily dose of L-Dopa administration in patients who received L-Dopa therapy together with dopamine receptor agonists. In vitro, we describe an induction of genomic damage detected as micronucleus formation by low micromolar concentrations in cell lines with of different tissue origins. The genotoxic effect of dopamine was reduced by addition of the antioxidants TEMPOL and dimethylthiourea which proved the involvement of ROS production in dopamine-induced DNA damage. To determine whether oxidation of dopamine by MAO is relevant in its genotoxicity, we inhibited MAO with two inhibitors, trans-2-phenylcyclopropylamine hydrochloride (PCPA) and Ro 16-6491 which both reduced the formation of micronuclei in PC-12 cells. We also studied the role of the dopamine transporter (DAT) and dopamine type 2 receptor (D2R) signaling in the genotoxicity of dopamine. Inhibitors of the DAT, GBR-12909 and nomifensine, hindered dopamine-induced genotoxicity. These results were confirmed by treatment of MDCK and MDCK-DAT cells, the latter containing the human DAT gene, with dopamine. Only MDCK-DAT cells showed elevated chromosomal damage and dopamine uptake. Although stimulation of D2R with quinpirole in the absence of dopamine did not induce genotoxicity in PC-12 cells, interference with D2R signaling using D2R antagonist and inhibition of G-proteins, phosphoinositide 3 kinase and extracellular signal-regulated kinases reduced dopamine-induced genotoxicity and affected the ability of DAT to take up dopamine. Furthermore, the D2R antagonist sulpiride inhibited the dopamine-induced migration of DAT from cytosol to cell membrane. Overall, the neurotransmitter dopamine causes DNA damage and oxidative stress in vitro. There are also indications that high dose L-Dopa therapy might lead to oxidative stress. Dopamine exerts its genotoxicity in vitro upon transport into the cells and oxidization oxidation by MAO. Transport of dopamine by DAT has the central role in this process. D2R signaling is involved in the genotoxicity of dopamine by affecting activation and cell surface expression of DAT and hence modulating dopamine uptake. We provided evidences for receptor-mediated genotoxicity of two compounds with different mechanism of actions. The involvement of these receptors in many human complications urges more investigations to reveal whether abnormalities in the endogenous compounds-mediated signaling can play a role in the initiation of new conditions like carcinogenesis.
We study reachability matrices R(A, b) = [b,Ab, . . . ,An−1b], where A is an n × n matrix over a field K and b is in Kn. We characterize those matrices that are reachability matrices for some pair (A, b). In the case of a cyclic matrix A and an n-vector of indeterminates x, we derive a factorization of the polynomial det(R(A, x)).
Cardiovascular disease is the most common mortality risk in the industrialized world. Myocardial infarction (MI) results in the irreversible loss of cardiac muscle, triggering pathophysiological remodelling of the ventricle and development of heart failure. Insufficient myocardial capillary density within the surviving myocardium after MI has been identified as a critical event in this process, although the underlying molecular signalling pathways of cardiac angiogenesis are mechanistically not well understood. The discovery of microRNAs (miRNAs, miRs), small non-coding RNAs with 19-25 nucleotides in length, has introduced a new level of the regulation of cardiac signalling pathways. MiRNAs regulate gene expression post-transcriptionally by binding to their complementary target messenger RNAs (mRNAs) and represent promising therapeutic targets for gene therapy. Here, it is shown that cardiac miR-24 is primarily expressed in cardiac endothelial cells and upregulated following MI in mice and hypoxic conditions in vitro. Enhanced miR-24 expression induces endothelial cell apoptosis and impairs endothelial capillary network formation. These effects on endothelial cell biology are at least in part mediated through targeting of transcription factor GATA2, histone deacetylase H2A.X, p21-activated kinase PAK4 and Ras p21 protein activator RASA1. Mechanistically, target repression abolishes respective and secondary downstream signalling cascades. Here it is shown that endothelial GATA2 is an important mediator of cell cycle, apoptosis and angiogenesis at least in part by regulation of cytoprotective heme oxygenase 1 (HMOX1). Moreover, additional control of endothelial apoptosis is achieved by the direct miR-24 target PAK4. Its kinase function is essential for anti-apoptotic Bad phosphorylation in endothelial cells. In a mouse model of MI, blocking of endothelial miR-24 by systemic administration of a specific antagonist (antagomir) enhances capillary density in the infarcted heart and preserves cardiac function. The current findings indicate miR-24 to act as a critical regulator of endothelial cell apoptosis and angiogenesis. Modulation of miR-24 may be potentially a suitable strategy for therapeutic intervention in the setting of ischemic heart diseases.
Members of the RAF protein kinase family are key regulators of diverse cellular processes. The need for isoform-specific regulation is reflected by the fact that all RAFs not only display a different degree of activity but also perform isoform-specific functions at diverse cellular compartments. Protein-protein-interactions and phosphorylation events are essential for the signal propagation along the Ras-RAF-MEK-ERK cascade. More than 40 interaction partners of RAF kinases have been described so far. Two of the most important regulators of RAF activity, namely Ras and 14-3-3 proteins, are subject of this work. So far, coupling of RAF with its upstream modulator protein Ras has only been investigated using truncated versions of RAF and regardless of the lipidation status of Ras. We quantitatively analyzed the binding properties of full-length B- and C-RAF to farnesylated H-Ras in presence and absence of membrane lipids. While the isolated Ras-binding domain of RAF exhibit a high binding affinity to both, farnesylated and nonfarnesylated H-Ras, the full-length RAF kinases demonstrate crucial differences in their affinity to Ras. In contrast to C-RAF that requires carboxyterminal farnesylated H-Ras for interaction at the plasma membrane, B-RAF also binds to nonfarnesylated H-Ras in the cytosol. For identification of the potential farnesyl binding site we used several fragments of the regulatory domain of C-RAF and found that the binding of farnesylated H-Ras is considerably increased in the presence of the cysteine-rich domain of RAF. In B-RAF a sequence of 98 amino acids at the extreme N terminus enables binding of Ras independent of its farnesylation status. The deletion of this region altered Ras binding as well as kinase properties of B-RAF to resemble C-RAF. Immunofluorescence studies in mammalian cells revealed essential differences between B- and C-RAF regarding the colocalization with Ras. In conclusion, our data suggest that that B-RAF, in contrast to C-RAF, is also accessible for nonfarnesylated Ras in the cytosolic environment due to its prolonged N terminus. Therefore, the activation of B-RAF may take place both at the plasma membrane and in the cytosolic environment. Furthermore, the interaction of RAF isoforms with Ras at different subcellular sites may also be governed by the complex formation with 14-3-3 proteins. 14-3-3 adapter proteins play a crucial role in the activation of RAF kinases, but so far no information about the selectivity of the seven mammalian isoforms concerning RAF association and activation is available. We analyzed the composition of in vivo RAF/14-3-3 complexes isolated from mammalian cells with mass spectrometry and found that B-RAF associates with a greater variety of 14-3-3 proteins than C- and A-RAF. In vitro binding assays with purified proteins supported this observation since B-RAF showed highest affinity to all seven 14-3-3 isoforms, whereas C-RAF exhibited reduced affinity to some and A-RAF did not bind to the 14-3-3 isoforms epsilon, sigma, and tau. To further examine this isoform specificity we addressed the question of whether both homo- and heterodimeric forms of 14-3-3 proteins participate in RAF signaling. By deleting one of the two 14-3-3 isoforms in Saccharomyces cerevisiae we were able to show that homodimeric 14-3-3 proteins are sufficient for functional activation of B- and C-RAF. In this context, the diverging effect of the internal, inhibiting and the activating C-terminal 14-3-3 binding domain in RAF could be demonstrated. Furthermore, we unveil that prohibitin stimulates C-RAF activity by interfering with 14-3-3 at the internal binding site. This region of C-RAF is also target of phosphorylation as part of a negative feedback loop. Using tandem MS we were able to identify so far unknown phosphorylation sites at serines 296 and 301. Phosphorylation of these sites in vivo, mediated by activated ERK, leads to inhibition of C-RAF kinase activity. The relationship of prohibitin interference with 14-3-3 binding and phosphorylation of adjacent sites has to be further elucidated. Taken together, our results provide important new information on the isoform-specific regulation of RAF kinases by differential interaction with Ras and 14-3-3 proteins and shed more light on the complex mechanism of RAF kinase activation.
Basal cell carcinoma (BCC) is the most common neoplasm in the Caucasian population. Only a fraction of BCC exhibits pigmentation. Lack of melanocyte colonization has been suggested to be due to p53-inactivating mutations in the BCC cells interfering with the p53-proopiomelanocortin pathway and the production of alpha melanocyte-stimulating hormone in the tumor. To evaluate this, we determined tumor pigmentation as well as expression of melan-A and of p53 in 49 BCC tissues bymeans of immunohistochemistry. As expected, we observed a positive relation between tumor pigmentation and melan-A positive intratumoral melanocytes.Melanocyte colonization and, to a lesser extent, p53 overexpression showed intraindividual heterogeneity in larger tumors. p53 overexpression, which is indicative of p53 mutations, was not correlated to melanocyte colonization of BCC. Sequencing of exon 5–8 of the p53 gene in selected BCC cases revealed that colonization by melanocytes and BCC pigmentation is neither ablated by p53 mutations nor generally present in BCCs with wild-type p53.
The Enterobacteriaceae comprise a large number of clinically relevant species with several individual subspecies. Overlapping virulence-associated gene pools and the high overall genome plasticity often interferes with correct enterobacterial strain typing and risk assessment. Array technology offers a fast, reproducible and standardisable means for bacterial typing and thus provides many advantages for bacterial diagnostics, risk assessment and surveillance. The development of highly discriminative broad-range microbial diagnostic microarrays remains a challenge, because of marked genome plasticity of many bacterial pathogens. Results: We developed a DNA microarray for strain typing and detection of major antimicrobial resistance genes of clinically relevant enterobacteria. For this purpose, we applied a global genome-wide probe selection strategy on 32 available complete enterobacterial genomes combined with a regression model for pathogen classification. The discriminative power of the probe set was further tested in silico on 15 additional complete enterobacterial genome sequences. DNA microarrays based on the selected probes were used to type 92 clinical enterobacterial isolates. Phenotypic tests confirmed the array-based typing results and corroborate that the selected probes allowed correct typing and prediction of major antibiotic resistances of clinically relevant Enterobacteriaceae, including the subspecies level, e.g. the reliable distinction of different E. coli pathotypes. Conclusions: Our results demonstrate that the global probe selection approach based on longest common factor statistics as well as the design of a DNA microarray with a restricted set of discriminative probes enables robust discrimination of different enterobacterial variants and represents a proof of concept that can be adopted for diagnostics of a wide range of microbial pathogens. Our approach circumvents misclassifications arising from the application of virulence markers, which are highly affected by horizontal gene transfer. Moreover, a broad range of pathogens have been covered by an efficient probe set size enabling the design of high-throughput diagnostics.
The phylum Tardigrada consists of about 1000 described species to date. The animals live in habitats within marine, freshwater and terrestrial ecosystems allover the world. Tardigrades are polyextremophiles. They are capable to resist extreme temperature, pressure or radiation. In the event of desiccation, tardigrades enter a so-called tun stage. The reason for their great tolerance capabilities against extreme environmental conditions is not discovered yet. Our Funcrypta project aims at finding answers to the question what mechanisms underlie these adaption capabilities particularly with regard to the species Milnesium tardigradum. The first part of this thesis describes the establishment of expressed sequence tags (ESTs) libraries for different stages of M. tardigradum. From proteomics data we bioinformatically identified 144 proteins with a known function and additionally 36 proteins which seemed to be specific for M. tardigradum. The generation of a comprehensive web-based database allows us to merge the proteome and transcriptome data. Therefore we created an annotation pipeline for the functional annotation of the protein and nucleotide sequences. Additionally, we clustered the obtained proteome dataset and identified some tardigrade-specific proteins (TSPs) which did not show homology to known proteins. Moreover, we examined the heat shock proteins of M. tardigradum and their different expression levels depending on the actual state of the animals. In further bioinformatical analyses of the whole data set, we discovered promising proteins and pathways which are described to be correlated with the stress tolerance, e.g. late embryogenesis abundant (LEA) proteins. Besides, we compared the tardigrades with nematodes, rotifers, yeast and man to identify shared and tardigrade specific stress pathways. An analysis of the 50 and 30 untranslated regions (UTRs) demonstrates a strong usage of stabilising motifs like the 15-lipoxygenase differentiation control element (15-LOX-DICE) but also reveals a lack of other common UTR motifs normally used, e.g. AU rich elements. The second part of this thesis focuses on the relatedness between several cryptic species within the tardigrade genus Paramacrobiotus. Therefore for the first time, we used the sequence-structure information of the internal transcribed spacer 2 (ITS2) as a phylogenetic marker in tardigrades. This allowed the description of three new species which were indistinguishable using morphological characters or common molecular markers like the 18S ribosomal ribonucleic acid (rRNA) or the Cytochrome c oxidase subunit I (COI). In a large in silico simulation study we also succeeded to show the benefit for the phylogenetic tree reconstruction by adding structure information to the ITS2 sequence. Next to the genus Paramacrobiotus we used the ITS2 to corroborate a monophyletic DO-group (Sphaeropleales) within the Chlorophyceae. Additionally we redesigned another comprehensive database—the ITS2 database resulting in a doubled number of sequence-structure pairs of the ITS2. In conclusion, this thesis shows the first insights (6 first author publications and 4 coauthor publications) into the reasons for the enormous adaption capabilities of tardigrades and offers a solution to the debate on the phylogenetic relatedness within the tardigrade genus Paramacrobiotus.