Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06

Tue, 22 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18720/ https://edoc.ub.uni-muenchen.de/18720/1/Fang_Chong.pdf Fang, Chong

Bacteria necessitate multiple signal transduction systems to sense the ever-changing environments and mediate the cellular response accordingly. The major bacterial signal transduction systems are one-component system (1CS), two-component system (2CS) and extracytoplasmic function (ECF) σ factor. Compared to 1CSs and 2CSs, ECF σ factors have only been identified much later and therefore the knowledge about their molecular mechanisms and physiological roles is less profound. This thesis mainly focuses on the study of ECF σ factors from the bacterial phyla, Planctomycetes and Actinobacteria.

Fri, 14 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18684/ https://edoc.ub.uni-muenchen.de/18684/1/Maba_Dao_Lamega.pdf Maba, Dao Lamèga

Polarity is a fundamental feature of almost all cells. It generally refers to the asymmetric organization of several cellular components. The plasma membrane, for example, exhibits both a transbilayer and a lateral asymmetry in most eukaryotic cells. Lipids are asymmetrically distributed between the cytoplasmic and the extracellular leaflet of the membrane and segregate laterally together with specific proteins to form dynamic nanoscale assemblies, known as rafts. Polarity can also specifically describe the asymmetric distribution of key molecules within a cell. These molecules, known as polarity determinants, can orient a multitude of specialized cellular functions, such as cell shape, cell division and fate determination. In the framework of this thesis, we aimed to reconstitute essential features of membrane unmixing and cell polarity with a "bottom-up" synthetic biology approach. We worked with both: pure lipid systems, whose unmixing is driven by the asymmetric distribution of lipids in the two leaflets, and a lipid-protein system, whose polarization is instead due to reaction-diffusion mechanisms. In both cases, we used Giant Unilamellar Vesicles (GUVs) and Sup- ported Lipid Bilayers (SLBs) to model biological membranes and employed modern biophys- ical techniques, such as fluorescence correlation spectroscopy, to quantitatively characterize lipid bilayers and protein-lipid interactions. In the pure lipid systems, we first reconstituted membrane transbilayer asymmetry, applying a cyclodextrin-mediated lipid exchange method, which enables us to enrich membranes with lipids of choice. The enrichment of the membrane with sphingomyelin and/or cholesterol triggers the segregation of lipids into two coexisting asymmetric phases both in SLBs and GUVs, whereas exchanging different amounts of phosphatidylglycerol with the outer leaflet of the GUV membranes controls vesicle shape. Tuning the lipid content of model membranes revealed that small changes in the composition of one leaflet affect the overall lipid miscibility of the bilayer and that membrane shape transformations are possible also in absence of a protein machinery and as a consequence of the lipid redistribution in the membrane. In the protein-lipid system, we aimed to reconstitute a minimal polarization system inspired by the C. elegans embryo at one-cell stage, which polarize along the anterior-posterior axis by sorting the PARtitioning defective (PAR) proteins into two distinct cortical domains. In this system polarity is maintained by the mutual inhibition between anterior (aPARs: PAR-3, PAR-6 and PKC-3) and posterior (pPARs: PAR-1, PAR-2 and LGL-1) PARs, which reciprocally antagonize their binding to the cortex, mutually excluding each other. We focused on LGL-1, which acts directly on PAR-6. Submitting LGL-1 to model membranes allowed us to identify a conserved region of the protein that binds negatively-charged membranes and to determine its lipid binding affinity and specificity. Selected LGL-1 mutants were then gen- erated to better understand the electrostatic mechanism involved in the membrane binding. LGL-1 was finally combined with PKC-3 to generate a functional membrane binding switch.

Adulte Weichgewebesarkome (engl. soft tissue sarcoma; STS) werden zu einer Gruppe seltener maligner und teilweise aggressiver Tumoren klassifiziert, die eine Tendenz zur Bildung von hämatogenen Fernmetastasen aufweisen. Die Kombination der Regionalen Hyperthermie mit einer Chemotherapie erwies sich in vorangegangenen Studien als eine vielversprechende Behandlungsoption beim lokalisierten Hochrisiko STS. Es wurde gezeigt, dass eine neoadjuvante Chemotherapie mit Regionaler Hyperthermie bei diesen Sarkomen das Tumoransprechen, das lokale progressionsfreie und das krankheitsfreie Überleben im Vergleich zu einer alleinigen Chemotherapie signifikant verbessert. Auf zellulärer Ebene induziert ein Hitzeschock (HS) bei klinisch relevanten Temperaturen (41,8°C/43°C) unter anderem eine temporäre Defizienz der Homologen Rekombinationsreparatur (HR), einem essentiellen Mechanismus für die fehlerfreie Reparatur von DNA-Doppelstrangbrüchen (DSB). Dies steht im Zusammenhang mit einer hitzeinduzierten proteosomalen Degradierung von BRCA2, einer unerlässlichen Komponente der HR. Trabectedin (Tr) ist eine antiproliferativ wirksame Substanz, die ursprünglich aus dem marinen Tunikat Ecteinascidia turbinata isoliert wurde. Die vielfältigen zytotoxischen Aktivitäten von Tr umfassen neben dem Interferieren mit der aktivierten Transkription und der Modulation der Tumor-Mikroumgebung hauptsächlich die Induktion von DSBs. Seit 2007 wird Tr in der Zweitlinientherapie zur Behandlung refraktärer STS, sowie bei Patienten eingesetzt, bei denen die Erstlinientherapie (Ifosfamid und/oder Doxorubicin) nicht angewendet werden kann. In Anbetracht der hitzeinduzierten Inaktivierung von BRCA2 und den DNA schädigenden Eigenschaften von Tr wurde in dieser Arbeit untersucht, ob und wie die Hyperthermie zu einer Wirkungsverstärkung der zytotoxischen Effekte von Tr beitragen kann. Tr bewirkt in vitro bei Zelllinien unterschiedlicher Sarkomentitäten (U2Os, SW872, SW982) eine dosisabhängige Reduktion des klonogenen Überlebens, das durch einen HS zusätzlich verstärkt wird. Die erhöhte antiproliferative Aktivität von Tr nach einem HS wird als thermale Chemosenitivierung definiert. Zudem konnte durch die Analyse der DNA-Verteilung bei U2Os und SW872 Zellen eine Intensivierung und Verlängerung der Tr-induzierten G2/M-Blockade nachgewiesen werden. Darüber hinaus wurden Zelllinien-spezifische Unterschiede bezüglich einer behandlungsinduzierten Apoptoseinduktion oder Senseszenzantwort identifiziert. SW872 Zellen weisen einen dosis- und temperaturabhängigen Anstieg des Anteiles apoptotischer Zellen auf, der mit einer starken Aktivierung der Effektorcaspasen 3 und 7 einhergeht. Dem entgegen gehen U2Os Zellen in eine ausgeprägte behandlungsinduzierte zelluläre Seneszenz über. Anhand der quantitativen Analyse Tr-induzierter H2AX Foci hat sich ein relevanter Anstieg an DSBs durch eine zusätzliche Hitzeexposition herausgestellt, der eine Beeinträchtigung der BRCA2-vermittelten vollständigen Assemblierung der DNA-Reparaturfoci vermuten lässt. Die Hypothese einer thermalen Chemosensitivierung gegenüber Tr durch eine hitzeinduzierte HR-Defizienz – insbesondere im Rahmen der hitzeinduzierten BRCA2 Degradierung – wurde zudem durch das Ausbleiben der hitzebedingten Verstärkung der Tr-induzierten Zytotoxizität bei BRCA2-defizienten Zellen bekräftigt. Darüber hinaus wurde durch Hochdurchsatzanalysen bestätigt, dass eine hitzevermittelte, erhöhte antiproliferative Aktivität von Tr nach einem Knockdown zahlreicher HR-spezifischer Komponenten ausbleibt. Durch Hochdurchsatzanalysen sowie durch anschließende Validierungsexperimente wurden Proteine identifiziert, die sich als relevant für weitere präklinische und klinische Untersuchungen herausgestellt haben. Die Proteine BRCA1, PARP1 und CHEK1 stellen dabei potentielle molekulare Marker für ein Tumoransprechen auf die Kombinationstherapie von Tr und Hyperthermie dar. Deren Inhibition erwies sich zudem als eine weitere Strategie, um die Effektivität der ursprünglichen Behandlung zusätzlich zu erhöhen. Darüber hinaus wurde die Funktion von FANCD2 als prädiktiver Marker und von ERCC1 als Resistenzmarker für das Therapieansprechen einer alleinigen Tr-Behandlung in vitro bestätigt. Die herausgearbeitete thermale Chemosensitivierung gegenüber Tr mit Hyperthermie durch die induzierte HR-Defizienz mittels passagerer BRCA2 Degradierung (induzierte synthetische Letalität) sowie die Identifizierung weiterer Proteine, deren medikamentöse Inhibition die Effektivität der Kombinationsbehandlung zusätzlich erhöhen könnte, eröffnen neue Möglichkeiten in der Therapie solider Tumoren.

The phylotypic stage, as part of the embryonic period, is the stage where embryos of different species of a phylum show a high degree of similarity. Johann Friedrich Meckel, Karl Ernst von Baer and Ernst Haeckel already described it for vertebrates in the 19th century. They observed that vertebrate embryos pass through a period of morphological similarity. Since then, scientists have researched the field of the phylotypic stage and it was subject of many controversial discussions. The name “phylotypic stage” was coined by Klaus Sander in 1983 and describes not only the stage of the highest similarity but also the stage, typical (characteristic) for a phylum. The following study examines the phylotypic stage of zebrafish (Danio rerio). Looking at different conserving mechanisms like internal constrains and stabilizing selection, different hypothesis and concepts by several researchers were tested. To test if the phylotypic stage is accessible to selection (although it generally is considered a conserved evolutionary stage) I have studied patterns of variation during embryogenesis. I have looked at the phenotypic variance and the number of significant correlations among embryonic traits and described the phylotypic stage as a period characterized by a high number of internal correlations and declining phenotypic variance. Then, I tested if changes in the raising conditions could elicit phenotypic changes. Therefore, zebrafish embryos have been raised under different experimental conditions to see if developmental plasticity can be induced during the early developmental period and if clearly defined modules can be identified. Eggs of zebrafish were raised in: (1) different temperatures; (2) different salinities; and (3) different levels of oxygen concentration. Up to 14 characters of individual embryos were measured during early development, encompassing the phylotypic stage. In particular I found a considerable degree of heterochrony and modularity. Embryos grew slower at lower temperatures and lower oxygen levels. Plasticity was detected in the overall size of the embryo and the size of somites in the oxygen and temperature experiment. The development of the eye and otic vesicle was shifted to a later x stage under severe hypoxia. Thus, eye and otic vesicle could be identified as modules, which can be dissociated from other characters of the developing embryo (heterochrony). Changes in raising condition affect early development of the zebrafish on three levels: (1) developmental rate (2) size and shape, and (3) dissociation of modules. Thus, plasticity and modularity are effective during early embryonic development. Finally I studied the heritability of embryonic traits to examine how inheritance contributes to the stabilization of the phylotypic stage in variable environments. Following the heritabilities of certain traits reveals that the phylotypic stage is not characterized by a certain pattern of decreased heritability and thus decreased additive genetic variance. The results suggest that the phylotypic stage of zebrafish is constrained by multiple internal correlations when embryos are developing in standard conditions. However, under marginal developmental conditions so far ineffective modules become effective and buffer the embryo against disruptive effects of the environment. Patterns of family resemblance are present, indicating an inherited genetic portion of the phylotypic stage. However, under strong environmental influence it is dominated by variation associated with phenotypic plasticity. My general conclusion is that the phylotypic stage is not established because additive genetic variance is exhausted during the early period of vertebrate development but that it is under environmental and genetic influence, thus is accessible to selection. Internal constraints could be identified to stabilize morphology during the phylotypic stage, but a certain degree of phenotypic variation can be observed.

Fri, 24 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18502/ https://edoc.ub.uni-muenchen.de/18502/1/Padula_Anderson_Vinicius.pdf Padula Anderson, Vinicius ddc:570, ddc:500, Fakultät für Biologie 0

Wed, 22 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18599/ https://edoc.ub.uni-muenchen.de/18599/1/Kepert_Ingeborg.pdf Kepert, Ingeborg d

Mon, 6 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18430/ https://edoc.ub.uni-muenchen.de/18430/1/Stuber_Erica.pdf Stuber, Erica

Labile characters, like behaviors, are phenotypes that are expressed repeatedly in the life of an individual. These types of characters allow individuals to adjust their phenotype to various levels of environmental variation, and therefore play a key role in the evolutionary process. Labile phenotypes are distinct because of their multi-level nature; individuals can differ in their average phenotypic expression (causing among-individual variation), but they can also vary their phenotype in each expression (causing within-individual variation). In order to understand the role of labile characters in the evolutionary process it is necessary to acknowledge that variation at each level is caused by different processes. Variation at the among-individual level is caused by genetic or environmental differences having a permanent effect on an individual’s phenotype, whereas variation at the within-individual level is caused by an individual’s adjustment of its phenotype to a changing environment. The implications of these multi layered effects in the expression of labile characters have been acknowledged by different fields of evolutionary ecology, but major areas of evolutionary research do not fully incorporated this idea. The general aim of my thesis was to fully integrate this multi-level nature in the study of the adaptive causes and evolutionary consequences of variation in labile characters. My thesis is composed of five chapters: the first three are conceptual and methodological works aimed at integrating the multi-level nature of labile characters into already existing evolutionary frameworks. The last two chapters describe, as a worked example, how the different levels of variation and covariation between (labile) fertilization related traits affect the evolution of the alternative reproductive strategies in a wild passerine bird (the great tit). The first chapter is a conceptual work focusing on how to define and statistically characterize behavioral characters. We argue that behavioral characters can be studied using the “evolutionary character concept”. This framework was developed to study characters that only vary among individuals (i.e. “fixed characters”); therefore we extended this framework to include characters that also vary within-individuals. The second chapter of the thesis is a methodological work where we proposed a way to quantify multi-level variation in reaction norms, which allows the estimation of repeatability of plasticity. Behavioral ecologists have recently developed theory predicting the ecological conditions where repeatable vs. non-repeatable variation in phenotypic plasticity should evolve. However, there was no methodological framework to estimate repeatability of plasticity. Therefore, we proposed a study design and mixed effect model structure to estimate repeatability of plasticity. To help researchers use the proposed methodology, we developed an R simulation package to estimate bias, precision and accuracy for different sampling designs. The third chapter is an opinion paper that urges researchers to combine theory and methods developed in behavioral ecology and quantitative genetics to study phenotypic variation in a social context. Quantitative geneticists have developed a framework to study social evolution aimed at predicting the evolutionary response to selection of traits affected by the phenotypes of other individuals (the “social environment”). Phenotypes expressed in a social context, also called interactive phenotypes, exhibit a particular evolutionary dynamic because their environmental component is composed of genes and can thus evolve. Despite that fact that the effects of the social environment are commonly mediated by labile characters, this social evolution framework has not fully considered the multi-level nature of labile characters. Therefore, for chapter three we integrated the multi-level nature of labile characters in this social evolution framework. The final two chapters focus, as a worked example, on within-pair and extra-pair reproductive behavior in great tits. For these chapters, we utilized the theoretical and methodological developments of the previous chapters to study the sources of evolutionary constraints on alternative fertilization routes in male great tits. One of the chapters has a more evolutionary perspective, while the other applies a more behavioral ecology view point. In chapter four we studied male extra-pair and within-pair reproduction as interactive phenotypes that are affected by the phenotypes of both the male and the female member of great tit breeding pairs. We showed that male fertilization strategies depend heavily on the phenotype of their female. This social environment effect should influence the evolutionary response to selection of male fertilization strategies, and could partly explain evolutionary stasis, observed in natural populations, in traits so closely linked to fitness. In chapter four we also studied whether trade-offs among- or within-individuals can constrain the phenotypic evolution of male alternative reproductive strategies. We showed that among-male trade-offs between within-pair and extra-pair reproduction could also be a source of evolutionary constrain. In chapter five, we corroborated the existence of trade-offs between alternative reproductive routes by studying whether within-pair and extra-pair fertilizations are obtained at the same time, allowing for the possibility of a trade-off between the two. We found that a male's extra-pair fertilization success is actually higher when it constrains his ability to secure within-pair fertilizations. This result is consistent with our finding that there is indeed a trade-off between extra-pair and within-pair reproduction in this species. The empirical works in this thesis highlight the importance of the social environment as a source of phenotypic variation in the expression of labile traits. But more generally, from the works in this thesis, we can conclude that to fully understand the role of labile characters in the evolutionary process it is necessary to acknowledge their multi-level nature.

Membrane remodeling is a dynamic process that occurs in bacterial cells to facilitate substrate transport and to provide protection to bacteria during environmental stress. In eukaryotic cells, membrane remodeling is carried out by dynamin-like proteins (DLPs). These proteins are involved in diverse membrane-associated functions such as cargo transport via vesicles, cytokinesis, division of cell organelles and resistance to pathogens. DLPs are also conserved in bacterial species; however, their function is still not clearly understood. The genome of B. subtilis contains a gene dynA (ypbR), which encodes a large DLP (136 KDa),DynA, that can tether membranes and induce membrane fusion in vitro. Deletion of dynA in B. subtilis strain 168 fails to produce any observable growth phenotype under standard laboratory conditions. B. subtilis is a soil bacterium and prey to several environmental stress factors to which laboratory strains are normally not exposed. Hence, it was conceivable that DynA might be required when bacteria are exposed to stress. To address this hypothesis, the behavior of DynA was examined under conditions causing membrane-stress, such as exposure to antibiotics and phage infection. A strain lacking dynA showed impaired growth in the presence of sublethal amounts of antibiotics that target the cell membrane and was more sensitive to phage infection compared to wild-type strains. Time-lapse microscopy and fluorescence loss in photobleaching (FLIP) experiments showed that ΔdynA cells have compromised membrane remodeling compared to wild-type strain. In conclusion, all results propose DynA to play a role in protecting the cell membrane under stress conditions. Also, for the first time, it is shown that a bacterial DLP contributes to innate immunity of bacteria. DynA not only has a unique membrane protection function but also distinctive structural features. A single DynA polypeptide contains two dynamin-like subunits, each consisting of a GTPase domain and a dynamin-like stalk region. Both subunits, D1 and D2, share strong intra-molecular cooperativity to facilitate GTPase activity. Here, a combination of mutational analysis and subsequent in vivo and in vitro investigation was applied to further characterise structural assembly and biochemical properties of DynA. Size-exclusion chromatography elucidated that DynA dimerisation requires C-terminal amino acids 591-620. In addition, in vivo localisation, in vitro lipid-binding and GTPase analysis revealed arginine at position 512 of DynA to be a key regulator of GTP hydrolysis as well as lipid-binding. Furthermore, in vivo localisation and bacterial two-hybrid experiments were employed to confirm interaction of DynA with putative interaction partners (YneK, YwpG and YmdA). YneK was found to interact with D1 and YwpG with D1 and D2 individually, whereas YmdA required a full-length DynA (D1+D2) for interaction. Taken together, the results presented here greatly expand on current knowledge regarding functional, biochemical and structural properties of a bacterial dynamin-like protein (BDLP). This thesis not only demonstrates the preserved membrane remodeling function of DLPs in bacteria but also explain their conservation from bacteria to higher-organisms.

Fri, 3 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18473/ https://edoc.ub.uni-muenchen.de/18473/1/Garcia-Verdugo_Rosa.pdf Garcia-Verdugo, Rosa ddc:570, ddc:500, Fakultät für Biologie

Wed, 24 Jun 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18377/ https://edoc.ub.uni-muenchen.de/18377/1/Wirnhier_Susanne.pdf Wirnhier, Susanne ddc:570, ddc:500, Fakultät für

In all cellular systems, the transmission of bulk genetic information during proliferation occurs in the form of chromosomes. The segregation of these entities upon cell division is of pivotal importance for all forms of life. Structural maintenance of chromosomes (SMC)-kleisin complexes are ubiquitous and essential factors that ensure proper organisation and segregation of the genetic material. Aim of this work was to elucidate evolutionary conserved features in the architecture of SMC-kleisin complexes, and to probe these features for functional relevance. We find that two major architectural themes have been constrained by evolution: (I) SMC-kleisin complexes form asymmetric assemblies with a ring-like topology, whereby a kleisin monomer bridges two different binding sites on a SMC dimer, (II) SMC-kleisin complexes form rod-like structures, whereby the SMC proteins of a given dimer are closely juxtaposed in a well-defined manner. Based on these findings, we propose that SMC-kleisin complexes from all domains of life act by a unifying mechanism.

Wed, 20 May 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18319/ https://edoc.ub.uni-muenchen.de/18319/1/Rabus_Max.pdf Rabus, Max ddc:570, ddc:500, Fakultät für Biologie

Mon, 18 May 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18306/ https://edoc.ub.uni-muenchen.de/18306/1/Gritenaite_Dalia.pdf Gritenaite, Dalia ddc:570, ddc:500, Fakultät für Biologie

Epigenetic programming facilitates the adaptation of an organism to changes in the environment through lasting alterations in gene expression that underlie certain physical and behavioral phenotypes. Exposure to adverse events in early postnatal life is known to increase the risk for stress-related psychiatric disorders later on. Our previous studies showed that early-life stress (ELS) in mice caused by periodic infant-mother separation (MS) leads to increased hyperactivity of the HPA axis, reduced glucocorticoid feedback inhibition, and depressive-like behavior. Moreover, our work revealed ELS-induced hypomethylation of the arginine vasopressin (Avp) gene enhancer and pro-opiomelanocortin (Pomc) promoter. The aim of the study was to investigate whether ELS can also lead to epigenetic programming of the mouse glucocorticoid receptor (GR, Nr3c1). GR is a major feedback regulator of the hypothalamic-pituitary adrenal (HPA) stress axis and its expression is regulated by multiple promoters associated with its5’ untranslated first exons. Given the fact that the mouse GR promoter was only partly characterized, we aimed to determine its genomic structure. In addition, tissue distribution and absolute quantification of newly identified alternative first exon transcripts were analysed. Although most of the first exon transcripts were found to be widely expressed, some of them are shown to be differentially regulated by growth factor- and depolarization-induced signaling. In the present work we show also that mice with a history of maternal separation display up-regulated GR mRNA levels. This observation was confined to Crh-producing neurons in the hypothalamic paraventricular nucleus (PVN), which are principal effectors of the stress response. Moreover, elevated levels of GR are shown to be responsible for stronger induction of its downstream target genes (Fkbp5, Sgk1, and DUSP1), which suggests an enhanced transcriptional activity of the GR in ELS mice. This effect is supported by a higher occupancy of the GR at the glucocorticoid response elements (GREs), following corticosterone injection (i.p.). Finally, we report here that an enhanced level of GR expression in ELS mice is accompanied by an increased methylation of specific CpG residues at the CpG island shore region of the GR promoter. These ELS-responsive CpGs comprise a DNA binding site for the transcriptional repressor Yin Yang 1 (YY1). Given the high homology of the mouse and human GR promoter, and the conservation of the YY1 binding site, we conducted a methylation analysis of the hGR CpG island shore region in peripheral tissues and post mortem brain samples. Our findings might serve as a basis for comparing the methylation patterns in tissues from control subjects and patients with stress-related brain disorders.

Sleep characteristics are candidates for predictive biological markers in patients with severe psychiatric diseases, in particular affective disorder and schizophrenia. The genetic components of sleep determination in humans remain, to a large degree, unelucidated. In particular, the heritability of rapid eye movement (REM) sleep and EEG bursts of oscillatory brain activity in Non-REM sleep, i.e. sleep spindles, are of interest. In addition, recent findings suggest a strong role of distinct sleep spindle types in memory consolidation, making it important to identify sleep spindles in slow wave sleep (SWS) and to separate slow and fast spindle localization in the frequency range. However, predictive sleep biomarker research requires large sample sizes of healthy and affected human individuals. Therefore, the present work addressed two questions. The first aim was to optimize data analysis by developing algorithms that allow an efficient and reliable identification of rapid eye movements (REMs) and sleep EEG spindles. In the second part, developed methods were applied to sleep EEG data from a classical twin study to identify genetic effects on tonic and phasic REM sleep parameters, sleep spindles, and their trait-like characteristics. The algorithm for REM detection was developed for standard clinical two channel electrooculographic montage. The goal was to detect REMs visible above the background noise, and in the case of REM saccades to classify each movement separately. In order to achieve a high level of sensitivity, detection was based on a first derivative of electrooculogram (EOG) potentials and two detection thresholds. The developed REM detector was then validated in n=12 polysomnographic recordings from n=7 healthy subjects who had been previously scored visually by two human experts according to standard guidelines. Comparison of automatic REM detection with human scorers revealed mean correlations of 0.94 and 0.90, respectively (mean correlation between experts was 0.91). The developed automatic sleep spindle detector assessed individualized signal amplitude for each channel as well as slow and fast spindle frequency peaks based on the spectral analysis of the EEG signal. The spindle detection was based on Continuous Wavelet Transform (CWT); it localized the exact length of sleep spindles and was sensitive also for detection of sleep spindles intermingled in high amplitude slow wave EEG activity. The automatic spindle detector was validated in n=18 naps from n=10 subjects, where EEG data were scored both visually and by a commercial automatic algorithm (SIESTA). Comparison of our own spindle detector with results from the SIESTA algorithm and visual scoring revealed the correlations of 0.97 and 0.92, respectively (correlation between SIESTA algorithm and visual scoring was 0.90). In the second part of the work, the similarity of given sleep EEG parameters in n=32 healthy monozygotic (MZ) twins was compared with the similarity in n=14 healthy same-gender dizygotic (DZ) twins. The author of the current work did not participate in acquisition of twin study sample. EEG sleep recordings used for the heritability study were collected and already described by Ambrosius et al. (2008). Investigation of REM sleep included the absolute EEG spectral power, the shape of REM power spectrum, the amount and the structural organization of REMs; parameters of Non-REM sleep included slow and fast sleep spindle characteristics as well as the shape of the Non-REM power spectrum in general. In addition to estimating genetic effects, differences in within-pair similarity and night-to-night stability of given parameters were illustrated by intraclass correlation coefficients (ICC) and cluster analysis. A substantial genetic influence on both spectral composition and phasic parameters of REM sleep was observed. A significant genetic variance in spectral power affected delta to high sigma and high beta to gamma EEG frequency bands, as well as all phasic REM parameters with the exception of the REMs occurring outside REM bursts. Furthermore, MZ and DZ twins differed significantly in their within-pair similarity of non-REM and REM EEG spectra morphology. Regarding sleep spindles, statistical analysis revealed a significant genetic influence on localization in frequency range as well as on basic spindle characteristics (amplitude, length, quantity), except in the quantity of fast spindles in stage 2 and whole Non-REM sleep. Basic spindle parameters showed trait-like characteristics and significant differences in within-pair similarity between the twin groups. In summary, the developed algorithms for automatic REM and sleep spindle detection provide several advantages: the elimination of human scorer biases and intra-rater variability, investigation of structural organization of REMs, exact determination of fast and slow spindle frequency for each individual. Algorithms are fully automated and therefore well suited to score REM density and sleep spindles in large patient samples. In the second part of the study, sleep EEG analysis in MZ and DZ twins revealed a substantial genetic determination of both tonic and phasic REM sleep parameters. This complements previous findings of a high genetic determination of the Non-REM sleep power spectrum. Interestingly, smaller genetic effects and lower night-to-night stability were observed for fast spindles, especially in SWS. This is in line with recent hypotheses on the differential function of sleep spindle types for memory consolidation. The results from the presented studies strongly support the application of sleep EEG to identify clinically relevant biomarkers for psychiatric disorders.

Das Einkomponentensystem CadC in Escherichia coli zählt zur Gruppe der ToxR-ähnlichen Transkriptionsregulatoren und aktiviert bei niedrigem pH-Wert die Expression des cadBA-Operons, einem Säure-induzierbaren Lysin-Decarboxylase-System. Transkriptionsregulatoren der ToxR-Familie zeichnen sich durch einen gemeinsamen modularen Aufbau aus und bestehen aus einer periplasmatischen Sensordomäne, einer Transmembranhelix und einer zytoplasmatischen Effektordomäne. Die Signalwahrnehmung, -weiterleitung und -verarbeitung erfolgt bei den ToxR-ähnlichen Transkriptionsregulatoren innerhalb eines einzelnen Proteins. Die molekularen Mechanismen der Reizwahrnehmung durch CadC sind bekannt, die Signalweiterleitung und -verarbeitung im Zytoplasma sind hingegen weitgehend ungeklärt. In CadC ist ein zytoplasmatischer Linker (51 Aminosäuren) essentiell für die Signaltransduktion von der sensorischen Domäne zur DNA-Bindedomäne. Im ersten Teil dieser Arbeit wurde der Mechanismus der Signalweiterleitung von der sensorischen Domäne zur DNA-Bindedomäne untersucht. Mit Hilfe der Kernspinresonanzspektroskopie konnte gezeigt werden, dass die Linkerregion unstrukturiert vorliegt. Im Rahmen einer umfangreichen Mutagenesestudie wurde beobachtet, dass sowohl eine Vielzahl an Aminosäuresubstitutionen (Veränderungen der Ladung, der Rigidität oder der Wahrscheinlichkeit zur Bildung einer α-Helix) als auch die Verlängerung des CadC-Linkers zu keiner funktionellen Beeinträchtigung führte. Jedoch wurde die Signalverarbeitung im Zytoplasma durch Verkürzung des Linkers modifiziert und verursachte ein invertiertes Expressionsprofil des Zieloperons cadBA oder die Entkopplung der Expression vom externen pH. Der Linkerregion in CadC konnte keine Rolle in der Oligomerisierung zugeordnet werden. Unabhängig vom Linker wurde in einer in vivo Interaktionsstudie eine pH-abhängige Interaktion (pH < 6,8) zwischen CadC-Monomeren gezeigt. Im zweiten Teil dieser Arbeit wurde die Röntgenkristallstruktur (2,0 Ångström) und in einem parallelen Ansatz die NMR-Struktur (0,46 backbone RMSD) der zytoplasmatischen Effektordomäne in CadC als erste dreidimensionale Struktur der DNA-Bindedomäne eines ToxR-ähnlichen Regulators aufgeklärt. In der Struktur von CadC1-107 wurde ein „winged Helix-Turn-Helix“-Motiv aus der Familie der OmpR-ähnlichen Transkriptionsregulatoren beobachtet. Im Gegensatz zu der Topologie bereits gelöster OmpR-ähnlichen Regulatoren enthält CadC am Übergang von DNA-Bindedomäne und Linkerregion einen zusätzlichen β-Strang (β-Strang 7), welcher sich stabilisierend auf die DNA-Bindung auswirken könnte. Im dritten Teil dieser Arbeit wurde der DNA-Bindemechanismus von CadC an den cadBA-Promotor untersucht. In in vitro Versuchen zur Bindung von löslichen CadC-Varianten an DNA konnte eine sehr geringe Dissoziationsrate beobachtet werden. Somit ist nicht die Affinität zur DNA sondern die Stimulus-abhängige Interaktion von CadC mit der α-Untereinheit der RNA-Polymerase essentiell für die Aktivierung des cadBA-Operons. Außerdem wurden, basierend auf der Kristallstruktur der DNA-Bindedomäne von CadC Aminosäuresubstitutionen durchgeführt. Die Aminosäure His66 in der Erkennungshelix α3 ist an der Interaktion mit der großen Furche der DNA beteiligt, während die Aminosäuren Lys95 und Arg96 die Interaktion mit der kleinen Furche der DNA vermitteln. Die Ergebnisse dieser Arbeit postulieren ein Modell zur Signalverarbeitung in CadC, in welchem die Signalwahrnehmung im Periplasma zu konformationellen Veränderungen des unstrukturierten CadC-Linkers führt und somit die räumliche Positionierung der DNA-Bindedomänen im CadC-Dimer ermöglicht wird.

Die Strahlenempfindlichkeit des Normalgewebes ist in der humanen Bevölkerung sehr heterogen und kann bislang nicht über prädiagnostische Biomarker charakterisiert werden. Im Rahmen der vorliegenden Arbeit wurde ein Verfahren entwickelt, um die Strahlenempfindlichkeit in lymphoblastoiden Zelllinien von jungen Lungenkrebspatienten in einem Hochdurchsatz Screening-Ansatz zu untersuchen. Fünf Zelllinien mit unterschiedlicher Strahlenempfindlichkeit wurden gewählt, um in einem ungerichteten Versuchsansatz (2D DIGE Methode = two-dimensional difference gel electrophoresis) strahlenspezifische Proteinregulation nach gamma-Bestrahlung (137Cs-Quelle) zu untersuchen. Dabei konnten sowohl neue Proteine, wie z.B. Mcm7und SerpinB9 identifiziert werden, als auch Proteine (Strukturproteine, Chaperone), die bereits in der Literatur in Verbindung mit der zellulären Stressantwort beschrieben wurden. Die 2D DIGE Ergebnisse konnten beispielhaft anhand von vier Kandidatenproteinen im Westernblot validiert werden. Die Untersuchungen zeigten, dass die intraindividuellen Expressionsunterschiede nach gamma-Bestrahlung auf Proteinebene sehr gering waren. Die geringen Expressionsunterschiede konnten jedoch validiert werden. Die Untersuchungen gaben Hinweise darauf, dass die interindividuelle Strahlenantwort sehr unterschiedlich ist. Dies konnte in weiterführenden Experimenten bestätigt werden. Da die Proteinexpression der Regulation durch mikroRNAs unterliegt, wurde in einem weiteren Ansatz eine miRNA Array Analyse durchgeführt. Hier bestätigte sich ebenfalls die Beobachtung aus der 2D Proteinanalyse, dass die Strahlenantwort interindividuell sehr heterogen ist. Die Ergebnisse dieser Arbeit zeigten, dass die Strahlenantwort auf verschiedenen zellulären Ebenen intraindividuell kaum variiert, die interindividuelle Varianz aber sehr groß ist. Diese beobachtete Heterogenität erklärt die Problematik einzelne Biomarker zur Prädiktion der Strahlenempfindlichkeit zu identifizieren.

Mon, 4 May 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18505/ https://edoc.ub.uni-muenchen.de/18505/1/Rabener_Elaine.pdf Rabener, Elaine ddc:570, ddc:500, Fakultät für Biologie

Durch Fehler entstandene tetraploide Zellen sind chromosomal instabil und können zu Zelltransformation führen. Die Beweise verdichten sich, dass die Propagation von tetraploiden Säugetierzellen durch einen p53-vermittelten Arrest eingeschränkt wird; jedoch ist weiterhin unklar, was die Ursache dieses p53-vermittelten Arrests ist. Um die Ursache des p53-vermittelten Arrests zu identifizieren, wurden individuelle Zellen mittels zeitraffender Mikroskopie in Echtzeit verfolgt. Neu entstandene tetraploide Zellen können einen Zellzyklus vollenden, aber die Mehrzahl der Zellen starb oder verharrte in einem Arrest in der folgenden G1-Phase, abhängig davon ob die vorangegangene Mitose fehlerfrei verlief oder nicht. Tochterzellen, denen eine fehlerhafte Mitose voranging, akkumulierten p53 im Zellkern, was zum Zelltod oder einem irreversiblen Zellzyklusarrest führte. Es zeigte sich durch den Anstieg von 8-OHdG, einem Indikator für oxidative DNA Schädigung, dass tetraploide Zellen durch die vermehrten fehlerhaften Mitosen höheren Konzentrationen von reaktiven oxidativen Spezien (ROS) ausgesetzt sind. Der Anstieg von 8-OHdG korrelierte mit der p53-Akkumulation im Zellkern. Da keine vermehrte Phosphorylierung des Histons H2AX (γ-H2AX), ein Marker für DNA-Strangbrüche, detektiert wurde, lässt sich schlussfolgern, dass ROS entscheidend für den p53 vermittelten Arrest verantwortlich sind. Mehrere p53-aktivierende Kinasen wurden mittels RNA Interferenz (RNAi) und chemischer Genetik untersucht, ob sie einen Einfluss auf den Zellzyklusarrest von tetraploiden Zellen haben. Von den getesteten Kinasen hatte nur ATM einen Einfluss auf die Aktivierung von p53 nach fehlerhaften tetraploiden Mitosen. Zwar wird ATM in der Regel durch DNA-Schäden aktiviert, jedoch wurde bereits zuvor gezeigt, dass ATM auch durch erhöhte ROS Konzentrationen aktiviert werden kann. Um die Zusammenhänge des Zellzyklusarrests weiter aufzuklären, wurde ein genomübergreifender esiRNA Screen etabliert, der die Zellproliferation nach induzierter Tetraploidisierung analysiert. Durch Kombination der Zellzyklusanalyse an Hand des DNA-Gehalts zusammen mit den FUCCI-Zellzyklusindikatoren, konnten tetraploide und diploide Zellen nebeneinander mikroskopisch analysiert werden, ohne zuvor tetraploide und diploide Zellen isolieren zu müssen. Dieser neue experimentelle Ansatz ermöglichte die Identifikation von Genen, die spezifisch die Proliferation von tetraploiden Zellen verstärken oder einschränken Im Primärscreen wurden 1159 Gene identifiziert, deren Inhibition die Proliferation einschränken. Weiter wurden 431 Gene identifiziert, deren Inhibition die Proliferation der tetraploiden Zellen verstärken. Von den 431 Genen, deren Inhibition die Proliferation verstärken, wurden 371 Gene einem Konfirmationsscreen unterzogen, in dem 158 der identifizierten 371 Gene bestätigt wurden. Die bioinformatische Analyse der 158 Gene zeigte eine signifikante Anhäufung von Genen, die mit DNA-Replikation, dem kanonischen Wnt-Signalweg oder mit Tumorsignalwegen assoziiert sind. Unter letzteren ist CCDC6 sehr interessant, da dessen Genprodukt durch ATM phosphoryliert wird und nachgeschaltet den Tumorsuppressor 14-3-3σ reguliert. Des weiteren wurden mittels einer Meta Analyse der Ergebnisse des Primärscreens, zusammen mit den Daten aus dem “Project Achilles”, welches genomweit den Effekt von shRNA-vermittelter Geninhibition auf die Proliferation von 108 Krebszelllinien untersuchte, 18 Gene identifiziert, deren Inhibition sowohl die Proliferation von tetraploiden Zellen einschränkt, als auch die Proliferation von Zelllinien hemmt, welche von Krebsarten stammen, die zu meist chromosomale Instabilitäten (CIN) aufweisen. Damit bilden die präsentierten Daten nicht nur eine gute Basis zur Aufklärung des Zellzyklusarrests tetraploider Zellen, sondern auch für die Identifikation neuer potentieller Zielmoleküle, welche benutzt werden können um Tumorerkrankungen mit chromosomaler Instabilität zu behandeln, welche häufig resistent gegen die bislang verfügbaren Behandlungen sind.

Fri, 17 Apr 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18699/ https://edoc.ub.uni-muenchen.de/18699/1/Bellot_Sidonie.pdf Bellot, Sidonie

Thu, 16 Apr 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18966/ https://edoc.ub.uni-muenchen.de/18966/1/Flosdorff_Sandra.pdf Flosdorff, Sandra ddc:570, ddc:500, Fak

Today’s pervasive presence of stress renders stress-related psychiatric disorders (SRPDs), a relevant global health problem. Memory impairment is a major symptom likely mediated by the hippocampus (HIP), a limbic brain region highly vulnerable to stress. Recent evidence suggests that information processing problems within specific neuronal networks might underlie SRPDs. However, the precise functional neurocircuitry that mediates hippocampal CA1 long-term potentiation (LTP), a putative correlate of mammalian learning and memory, remains unknown at present. Furthermore, valuable assays for studying stress and drug effects on polysynaptic activity flow through the classical input/output circuit of the HIP are missing. To engage a circuit-centered approach, voltage-sensitive dye imaging was applied in mouse brain slices. Single pulse entorhinal cortex (EC) to dentate gyrus (DG) input, evoked by perforant path stimulation, entailed strong neuronal activity in the DG, but no distinct neuronal activity in the CA3 and CA1 subfield of the HIP. In contrast, a thetafrequency (5 Hz) stimulus train induced waves of neuronal activity percolating through the entire hippocampal trisynaptic circuit (HTC-waves). Spatially restricted blocking of glutamate release at CA3 mossy fiber synapses caused a complete disappearance of HTC-waves, suggesting frequency facilitation at DG to CA3 synapses the pivotal gating mechanism. In turn, non-theta frequency stimulations (0.2/1/20 Hz) proved much less effective at generating HTC-waves. CA1 long-term potentiation (CA1 LTP) is the best understood form of synaptic plasticity in the brain, but predominantly at the monosynaptic level. Here, HTC-waves comprise high-frequency firing of CA3 pyramidal neurons (>100 Hz), inducing NMDA receptordependent CA1 LTP within a few seconds. Detailed examination revealed the existence of an induction threshold for LTP. Consequently, baseline recordings with a reduced number of HTC-waves were carried out to test the effects of memory enhancing drugs and HPA axis hormones on hippocampal network dynamics. Bath application of caffeine (5 mM), corticosterone (100 nM) and corticotropin-releasing hormone (5 & 50 nM) rapidly boosted HTC-waves. Cognitive processes taking place within the HIP are challenged by stress exposure, but whether and how chronic stress shapes "net" neuronal activity flow through the HIP remains elusive. The HTC-wave assay, refined for group comparisons, revealed that chronic stress markedly lowers the strength of evoked neuronal activity propagation through the hippocampal trisynaptic circuit. In contrast, antidepressants (ADs) of several classes, the mood stabilizer lithium, the anesthetic ketamine, and the neurotrophin brainderived neurotrophic factor amplified HTC-waves. An opposite effect was obtained with the antipsychotic haloperidol and the anxiolytic diazepam. The tested ADs exert this effect at low micromolar concentrations, but not at 100 nM, and nearly always, also not at 500 nM. Furthermore, the AD fluoxetine was found to facilitate LTP of HTC-waves. Finally, pharmacological blockade of the tyrosine-related kinase B receptor abolished fluoxetine effects on HTC-waves. These results highlight a circuit-centered approach suggesting evoked synchronous theta rhythmical firing of EC principal cells as a valuable tool to investigate several aspects of neuronal activity flow through the HIP. The physiological relevance is emphasized by the finding that the resulting HTC-waves, which likely occur during EC theta oscillations, evoke NMDA receptor-dependent CA1 LTP within a few seconds. Furthermore, HTC-waves allow to integrate molecular, cellular and structural adaptations in the HIP, pointing to a monoaminergic neurotransmission-independent, "circuit-level" mechanism of ADs, to balance the detrimental effects of chronic stress on HIP-dependent cognitive abilities.

Wed, 15 Apr 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19357/ https://edoc.ub.uni-muenchen.de/19357/1/Raffler_Johannes.pdf Raffler, Johannes ddc:570, ddc:

Fluorescent protein sensors have gained great importance in research as they exhibit a number of advantages over synthetic dyes. They can be targeted precisely, large populations of cells can be imaged simultaneously, and they allow for chronic imaging approaches. Many of them however still suffer from comparably low signal changes. Improving fluorescent protein sensors can be tedious and time-consuming. For this reason, great efforts have been made not only to improve existing sensors, but also to develop better strategies to improve them. In this work, a novel large-scale bacterial based screening assay was established to complement rational design. Sensor expression, stimulation, and screening in bacteria, as well as the handling of large amounts of data created by such a screening assay were optimized. While the new assay can be adapted for other applications, it is especially well suited for the screening of genetically encoded Ca2+ indicators of the basis of FRET (Förster Resonance Energy Transfer). We used the assay to optimize such sensors, utilizing the Ca2+ binding protein Troponin C fused between the fluorescent proteins ECFP and cpCitrine. The resulting ‘Twitch’ sensor series exhibited a large dynamic range of up to 1000% FRET ratio change, great sensitivity and fast kinetics. In a second approach, we attempted to develop a similar sensor deploying red-shifted fluorescent proteins. To this end, further screening was conducted to optimize the orange fluorescent protein mKOκ for FRET, and a FRET sensor deploying mKOκ. The sensor we developed utilized troponin C and the fluorescent protein Dreiklang (photoswitchable) in addition to mKOκ. It was bright and exhibited a FRET ratio change of approximately 170%. In summary, the screening procedures presented in this thesis, will facilitate the development of a range of genetically encoded biosensors, and were already employed to develop a number of highly effective Ca2+ FRET indicators.

Das Paprika Resistenzgen Bs4C aus Capsicum pubescens vermittelt Resistenz gegenüber Xanthomonas campestris pv. vesicatoria (Xcv)-Stämmen, die den (transcription activator-like) TAL-Effektor AvrBs4 exprimieren. Vorangegangene Arbeiten ließen vermuten, dass AvrBs4 die Expression von Bs4C transkriptionell induziert. In einem “proof of principle”-Experiment, wurde Bs4C unter Verwendung eines RNA-Seq-basierten Ansatzes isoliert. Unter 68 differentiell AvrBs4-induzierten Paprikagenen war jedoch nur eines, das ausschließlich in der resistenten und nicht in der suszeptiblen Akzession induziert war und für das kein Transkript in Abwesenheit von AvrBs4 in der resistenten Akzession nachgewiesen wurde. Kopplungs- und Komplementationsanalysen bestätigten dieses Kandidatengen als das gesuchte Resistenzgen Bs4C. Im Bs4C-Promoter konnte ein Effektorbindeelement (EBE) für AvrBs4 identifiziert werden, das notwendig und ausreichend für die AvrBs4-Bindung an und transkriptionelle Aktivierung von Bs4C ist. Bindungsstudien ließen erkennen, dass zwei Nukleotidpolymorphismen in der korrespondierenden Region der suszeptiblen Akzession eine stark reduzierte Affinität (10fach) gegenüber AvrBs4 bedingen. Außerdem zeigten GUS-Studien, dass der Promoter des suszeptiblen Allels nicht durch AvrBs4 induzierbar ist. Folglich bestimmt ein Substitutionspolymorphismus von zwei Basenpaaren in den Promotoren des resistenten und suszeptiblen Bs4C-Allels über Resistenz oder Suszeptibilität gegenüber AvrBs4-exprimierenden Xanthomonaden. Bs4C kodiert für ein 164-AS großes Protein, das keine Homologie zu Proteinen mit bekannter Funktion aufweist. In silico Proteinstrukturanalysen sagen vier Transmembranhelices in Bs4C vorher und demzufolge stellt es einen neuen Typ von Exekutorproteinen dar, welcher Resistenz gegen TALE-exprimierende Xanthomonas-Stämme vermittelt. Zudem konnten Sequenzanalysen mindestens ein Homolog in C. pubescens und mindestens sieben Homologe in C. annuum identifizieren. Interessanterweise kodieren die meisten von ihnen aufgrund von Nukleotidaustauschen, Leserahmenverschiebungen und Insertions/Deletionspolymorphismen nicht für Volllängen Bs4C-ähnliche Proteine. Folglich könnten diese homologen Sequenzen duplizierte Gene repräsentieren, die nicht mehr funktional sind.

Wed, 1 Apr 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18136/ https://edoc.ub.uni-muenchen.de/18136/1/Sevim_Muezeyyen.pdf Sevim, Muezeyyen ddc:570, ddc:500, Fakultät

Mon, 16 Mar 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18234/ https://edoc.ub.uni-muenchen.de/18234/1/Tomi_Nils-Sebastian.pdf Tomi, Nils-Sebastian ddc:570, ddc:500, Fakultät für Biolog

Fri, 13 Mar 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18103/ https://edoc.ub.uni-muenchen.de/18103/1/Villavicencio_Camila_P.pdf Villavicencio Reyes, Camila Patricia

Fri, 13 Mar 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19309/ https://edoc.ub.uni-muenchen.de/19309/1/Heinz_Gitta.pdf Heinz, Gitta Anne Maren

All living cells are highly organized and exhibit complex cellular machineries facilitating biochemical reactions. Compartmentalization is a prerequisite to allocate an appropriate environment for these processes. In this work, compartments that are involved in Bacillus subtilis membrane organization and cell division were studied. B. subtilis division site selection is dependent on the nucleoid occlusion and the Min system. The B. subtilis Min system consists of four components. MinC is the actual inhibitor of the tubulin homologue FtsZ that is a crucial component of the divisome, forming the so called Z-ring. MinC is bound to the ATPase MinD that is tethered via the adapter protein MinJ to DivIVA. DivIVA senses membrane curvature and was supposed to be stably tethered to the cell poles. Thereby a stable, static DivIVA / MinJDC gradient with minimum concentration at midcell is formed. Using advanced microscopy techniques like single cell time lapse microscopy, fluorescence recovery after photo bleaching and by utilization of photo-activatable / convertible fluorophores we could demonstrate that DivIVA is in vegetative cells recruited from the cell pole to mature septa. These data provide first evidence that the role of the B. subtilis Min system is not to define midcell, but prevents reinitiation of Z-ring constriction after fulfilled division. Utilizing single cell time lapse microscopy we could further demonstrate that proteins crucial to condense the chromosome are vital for correct chromosome segregation during cell division by influencing the replication fork velocity or resolution. As a second compartment B. subtilis flotillin dependent membrane microdomains were studied. These domains are likely scaffolded by the membrane protein flotillin. This protein is pinned to the membrane via a hairpin loop as shown by SNAP–tag labelling experiments. Utilizing the anisotropic dye Laurdan we could further show spectroscopically and microscopically that flotillins prevent condensation of microdomains. Flotillin deletion strains also exhibit a generally more liquid ordered membrane compared to wild type cells. Using co–immunoprecipitation experiments several proteins interacting with flotillin were identified. These interactions were confirmed with microscopical co–localization analysis. B. subtilis flotillin was additionally heterologously purified via affinity chromatography. The purified protein creates large homo–oligomers likely in mega Dalton size. Using truncation mutants it could be shown that flotillin oligomerizes via a flotillin specific domain, namely the PHB domain. Though, contrary to eukaryotic cells, B. subtilis PHB domain does not contribute to lipid binding. However, several cellular machineries that interact with flotillins, as exemplary shown for the secretion machinery, are impaired in their functionality in absence of flotillins. These data provide first evidence that prokaryotic flotillins are elements that scaffold the plasma membrane and thereby provide a lipid environment that is vital for correct functionality of diverse cellular machineries.

Tue, 10 Mar 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18281/ https://edoc.ub.uni-muenchen.de/18281/1/Kim_Jeehee.pdf Kim, Jeehee ddc:570, ddc:500, Fakultät für Biologie

Thu, 5 Mar 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18078/ https://edoc.ub.uni-muenchen.de/18078/1/Guentner_Michaela_R.pdf Güntner, Michaela ddc:570, ddc:

Mon, 2 Mar 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18005/ https://edoc.ub.uni-muenchen.de/18005/1/Reichel_Judith_M.pdf Reichel, Judith ddc:570, ddc:500, Fakultät für Biologie

Bacteria constantly need to monitor their environment and adapt the bacterial group-coordinated behaviour to changing habitats like nutrition alterations or host variations. Commonly cell-cell communication via acyl homoserine lactones (AHLs)is used to synchronise the behaviour of a bacterial population dependent on cell size. This process is referred to as quorum sensing (QS) and predominantly occurs in Gram-negative bacteria. The typical QS system consists of a LuxI-synthase that synthesises AHLs, and a LuxR-type receptor, which then responds to these AHLs. Upon AHL-binding, the LuxR-type receptor regulates the expression of different target genes and thus influences several processes, like biofilm formation, virulence, antibiotic production or cell-cell interaction. Interestingly, many proteobacteria possess additional LuxR homologs, but lack a cognate LuxI-type synthase. Those LuxR-type receptors are referred to as LuxR orphans or LuxR solos and can expand the regulatory QS network. Photorhabdus species are insect pathogenic bacteria, living in symbiosis with entomopathogenic nematodes. They all possess an exceptionally high number of LuxR solos, but lack LuxI homologs and therefore do not produce AHLs. The function of these LuxR solos, their role in cell-cell communication and the identification of their cognate signalling molecules in Photorhabdus species is the main focus of this work. In this thesis a novel signalling molecule used for QS could be identified for the first time in P. luminescens. This novel QS molecule is an α-pyrone named photopyrone (PPY) and produced endogenously by the photopyrone synthase (PpyS). The PPYs are specifically recognized by the LuxR solo regulator PluR, which then activates expression of the pcf (Photorhabdus clumping factor) operon leading to cell clumping of P. luminescens cells. Moreover, the PpyS/PluR quorum sensing system and its induced cell clumping contribute to the overall toxicity of P. luminescens. Furthermore, a second novel signalling molecule sensed by a LuxR solo of Photorhabdus species could be identified besides PPYs. The insect and human pathogenic bacteria P. asymbiotica lacks a PpyS homolog as well as a LuxI homolog, but harbours a pcf operon and a homologue to PluR, which is named PauR. The signalling molecule sensed by the LuxR-type receptor PauR could be identified, which is neither an AHL nor a PPY. PauR recognises a 2,5-dialkylresorcinol (DAR) produced by the DarABC pathway. Upon binding of the cognate signalling molecule, Summary XII PauR activates expression of the pcf operon. This also leads to cell clumping in P. asymbiotica. Furthermore, the DarABC/PauR QS system also contributes to the overall pathogenicity of P. asymbiotica against Galleria mellonella insect larvae. A bioinformatics approach revealed a high number of LuxR solos present in P. temperata and P. asymbiotica like in P. luminescens. Thereby, several conserved motives of amino acids could be identified, which are potentially important for signalbinding and -specificity. Variations in these amino acid motifs are assumed to reflect the overall variety of signals that can be sensed by LuxR solos. Furthermore, the specificity of the two LuxR solos PluR and PauR towards their cognate signalling molecules, PPYs and DARs, respectively, was analysed. Thereby, it could be shown that the previously identified conserved amino acid motives in the signal-binding domain (SBD), the TYDQCS-motif of PluR and the TYDQYI-motif of PauR, are essential but not sufficient for ligand-binding. Similar as for AHLs, it was unclear how the signalling molecules PPYs and DARs can cross the bacterial cell membrane. In the last part of this thesis the import mechanism for the Photorhabdus-specific signalling compounds PPYs and DARs were identified. Initial evidence could be provided that the membrane-integrated transporter FadL is mainly involved in the import of these hydrophobic compounds, and that they are not transported via simple diffusion across the cell membrane, which is assumed for AHLs. In conclusion, the data that is compiled presents two LuxR solos of Photorhabdus species adapted to sense and respond to novel non-AHL signalling molecules used for QS. Therefore, this thesis reveals that cell-cell communication via LuxR-type receptors goes far beyond AHL-signalling in nature.

Tue, 24 Feb 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17996/ https://edoc.ub.uni-muenchen.de/17996/1/Shi_Song.pdf Shi, Song ddc:570, ddc:500, Fakul

Corynebacterium glutamicum is a Gram positive soil bacterium with high industrial importance in ton scale production of amino acids. Apart from that, it becomes more and more important for medical studies, where it serves as model organism due to its close relation to bacteria causing several pathogens such as tuberculosis, diphtheria and leprosy. C. glutamicum, like Mycobacterium tuberculosis, has a distinct cell wall which is composed of a peptidoglycan layer (murein) with covalently bound polysaccharide layers that are capped with mycolic acids. In addition, both organisms have a polar cell wall synthesis machinery which is spatially regulated by DivIVA (Wag31 in M. tuberculosis). The present study shows that DivIVA regulates cell wall synthesis upon direct interaction with the lipid II flippase RodA. RodA determines morphology and growth in C. glutamicum and is localized to the poles and septa. The absence of rodA results in growth defects and cell shape alterations as well as altered lipid II proliferation of the poles (polar cell growth is sustained). DivIVA is furthermore involved in chromosome segregation upon direct interaction with the partitioning ParB protein, which binds to parS sites on the chromosome, thus tethering the replicated nucleoids to the cell poles. Interactions of DivIVA with ParB and RodA were identified in a synthetic in vivo protein-protein interaction assay where fluorescently labeled proteins of interest are expressed in E. coli cells and interaction is analyzed microscopically. A decisive improvement of this assay is the application of FRET, which is more sensitive and allows quantification of interaction. In order to test whether ParB and RodA compete for the same interaction site in DivIVA, we mapped interaction sites of both proteins. It turned out that ParB binds to a middle region of DivIVA, whereas RodA binds to the N-terminal domain of DivIVA where one lysine residue is essential for interaction. To fight bacterial infections, that cause thousands of casualties each year, it is mandatory to understand mechanisms in cellular processes, such as cell division and growth, to find new targets for antibiotic intervention. Unfortunately, bacteria are able to develop resistances against many antibiotics. The mycolic acid or arabinan layer and synthesis machinery are good candidates for new antibiotics. Amongst others, two of them have emerged as useful drugs against M. tuberculosis, ethambutol (EMB) and BTZ043. In this study, we investigated the modes of action and antibiotic susceptibility of C. glutamicum after EMB and BTZ043 treatment. We found that both antibiotics, which target the arabinan synthesis pathway, affect exclusively polar elongation growth, as demonstrated in different staining assays. Interestingly, only 10% of the cells were killed and cells in stationary phase were not affected by EMB or BTZ043. Moreover, we used a chromosomal DivIVA-mCherry fusion and found that DivIVA protein level is drastically increased. The cells show asymmetric recovery after treatment, in which one daughter cell acquires the excess DivIVA whereas the other daughter cell exhibits normal cell growth.

Wed, 18 Feb 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19270/ https://edoc.ub.uni-muenchen.de/19270/1/Hinkofer_Lisa.pdf Hinkofer, Lisa

Fri, 6 Feb 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17957/ https://edoc.ub.uni-muenchen.de/17957/1/Labermaier_Christiana.pdf Labermaier, Christiana ddc:570, ddc:500

Thu, 5 Feb 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19322/ https://edoc.ub.uni-muenchen.de/19322/1/Ehrenberg_Stefanie.pdf Ehrenberg, Stefanie ddc:570, ddc:500, Fakultät für Biol

Behavioural ecologists aim at providing insights into the evolutionary and ecological processes that shape animal behaviour. Mate choice is a decision faced by most animals that can strongly affect an individual’s reproductive success, an important fitness component. This behaviour has therefore the potential to show many adaptations which have been the subject of a vivid research interest over the last decades. Studies on mate choice have typically focused on female preferences for traits that increase a male's overall attractiveness, which supposedly reflects the male’s absolute quality. Preferences for such traits are expected to provide females with benefits such as good paternal care or ‘good genes’ for their offspring. Nevertheless, in some species, individuals actually show little consensus on what represents a high-quality partner. Such individually-specific preferences are poorly understood, but it has been hypothesized that they have evolved to optimise genetic compatibility (to increase offspring viability) or, although rarely mentioned, to optimise behavioural compatibility (to facilitate joint parental care). The zebra finch is a life-time monogamous passerine whose mating preferences for putative quality traits have been widely studied but where no such quality indicator has been repeatedly shown to influence mating decisions. In this species, preferences seem instead largely idiosyncratic. In this thesis, I investigated whether female zebra finches choose genetically and/or behaviourally compatible social mates (Chapters 2 and 3). More precisely, I measured, in a large-scale breeding design, the fitness consequences of free mate choice for compatibility while experimentally controlling for effects of overall quality (Chapter 3). In this system, genetic incompatibility results in embryo mortality and therefore hatching failure, while behavioural incompatibility results in offspring mortality. I therefore expected social mate choice for genetic and/or behavioural compatibility to optimise embryo and offspring survival (Chapter 3). Moreover, I tested whether siblings who grew up together avoid choosing each other as social partner; in other words, if they avoid inbreeding depression by choosing a genetically more compatible partner (Chapter 2). In addition, I performed a meta-analysis of published experiments on zebra finches that allow for different mechanisms of kin discrimination to take place (Chapter 2). Finally, I investigated whether females enhance their extra-pair behaviour when paired to an apparently genetically incompatible male to obtain compatible genes benefits (Chapter 1). Indeed, extra-pair behaviour is largely hypothesized to be an adaptive response that would allow females to compensate for a potentially sub-optimal social mate choice. Specifically, I tested whether female extra-pair mating evolved as a counter strategy when females experience low hatching success with their social partner, that is to say whether female extra-pair mate choice targets fertility benefits and/or compatible genes benefits (Chapter 1). Contrary to all expectations derived from optimality, zebra finches were unable to identify partners with whom they would minimise embryo mortality (Chapter 3), or to recognize unfamiliar kin on the basis of genetic similarity per se to minimize inbreeding depression (meta-analysis of Chapter 2), and did not adjust their extra-pair mating behaviour in response to repeated hatching failure (Chapter 1). This suggests that, in zebra finches, individuals have not evolved any ability (other than avoiding familiar kin, experiment of Chapter 2) to judge genetic compatibility of any kind, despite the adaptive value of such behaviour. Finally, allowing free social mate choice did enhance pair fitness due to direct compatibility benefits (Chapter 3). This thesis provides therefore the first evidence of mate choice for behavioural compatibility. Nevertheless, the adaptive significance of the underlying choosiness remains speculative (Chapter 3). A null model consisting of random non-adaptive genetic variation in sensory systems and variation in phenotypes that are better at stimulating some sensory systems than others might well account for the observed fitness differences between compatible and incompatible pairs. This study highlights that there are limits to adaptation.

Wed, 21 Jan 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17871/ https://edoc.ub.uni-muenchen.de/17871/1/Stingele_Julian.pdf Stingele, Julian ddc:570, ddc:500, Fakultät für Biologie

Fri, 16 Jan 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17888/ https://edoc.ub.uni-muenchen.de/17888/1/Borg_Sarah.pdf Borg, Sarah

In the mammalian genome, cytosine methylation (5mC) plays a central role in the epigenetic regulation of gene expression and has been implicated in a variety of biological processes, including genome stability, imprinting or differentiation. Compared to other epigenetic marks, DNA methylation has been thought to be relatively stable. However, genome-wide loss of 5mC, or DNA demethylation, has been observed in specific developmental stages and in various types of cancer. The discovery of the TET family of enzymes in 2009 was a watershed moment in comprehending the mechanisms of DNA demethylation. TET proteins oxidize 5mC to 5- hydroxymethylcytosine (5hmC), 5-formlycytosine (5fC) and 5-carboxylcytosine (5caC), which not only serve as key intermediates in active DNA demethylation pathways, but can also act as independent epigenetic marks. In this study, various aspects of TET-mediated DNA demethylation have been intensively investigated. Using quantitative mass-spectrometry-based proteomics readers for the different cytosine derivatives in mouse embryonic stem cells (ESCs), neuronal progenitor cells, and adult mouse brain tissue were identified. Readers for these modifications are only partially overlapping and are dynamic during differentiation. Moreover, the oxidized derivatives of 5mC recruit distinct transcription regulators as well as a large number of DNA repair proteins, implicating DNA damage response as the main pathway contributing to active DNA demethylation. To identify additional non-canonical DNA bases, highly sensitive quantitative mass-spectrometry led to the discovery of 5-hydroxymethyluracil (5hmU) in ESCs. Genomic 5hmU is not generated via deamination of 5hmC, as widely suggested, but through direct oxidation of thymine by TET proteins. In addition, screening for specific 5hmU readers identified different transcriptional and epigenetic factors, implicating that this mark has a specific function in ESCs. So far, only little is known how TET enzymes are regulated and how they are modified by posttranslational modifications (PTMs). Mapping TET phosphorylation and glycosylation sites at amino acid resolution revealed that these PTMs are interdependent and mostly occur at regulatory protein regions. Finally, a reporter gene based assay could demonstrate that in vitro methylation causes gene silencing while subsequent oxidation, resulting in DNA demethylation, leads to gene reactivation in vivo. Different knockout and rescue experiments clearly show that oxidation of methylcytosine by TET proteins and subsequent removal by TDG or NEIL glycosylases and the base excision repair pathway results in reactivation of epigenetically silenced genes. In conclusion, this work provides new insights how TET proteins can set DNA modifications, how these oxidized bases are read by various factors and how TET proteins can be posttranslationally modified. Furthermore, removal of 5mC is achieved through TET-mediated oxidation and depends on the activity of specific glycosylases, which leads to gene reactivation.

Mon, 15 Dec 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17850/ https://edoc.ub.uni-muenchen.de/17850/1/Kolinko_Sebastian.pdf Kolinko, Sebastian ddc:570, ddc:500, Fakultät für Biologie

Viele Vogelarten sind monogam, aber es kommt zugleich häufig zu Paarungen außerhalb des Paarbundes (Griffith et al. 2002). In einer großen Zahl von Studien wurde bereits der adaptive Wert der aus diesem Verhalten hervorgehenden „Fremdvaterschaften“, insbesondere für Weibchen, anhand einer Kosten-Nutzen-Analyse auf genetischer Ebene untersucht. Die Ergebnisse dieser Studien haben jedoch bisher nicht zu generalisierbaren Resultaten geführt (Akçay & Roughgarden 2007). Im Gegensatz dazu gibt es relativ wenig Informationen über verhaltensbegründete Kosten und Vorteile von Fremdvaterschaften, obwohl diese ein Verständnis der Mechanismen von Fremdverpaarungen erleichtern können. Die hier vorliegende Studie untersucht, wie zeitliche, räumliche, und soziale Faktoren beeinflussen wann, wo, und warum bestimmte Individuen Nachkommen mit einem anderen als ihrem sozialen Partner zeugen. Ich untersuche dies an der Blaumeise (Cyanistes caeruleus), einer Art, für die genetische Konsequenzen von Fremdvaterschaften bereits detailliert untersucht wurden, bei der die Kenntnisse im Bereich des außerpaarlichen Verhaltens selbst aber noch unvollständig sind. In den ersten beiden Studien stelle ich einen neuartigen Ansatz zur Untersuchung von Fremdvaterschaften vor und validiere diesen. Fremdverpaarungen erfordern als Beteiligte sowohl ein Weibchen als auch ein Männchen und beide können beeinflussen, ob eine Paarung stattfindet. In dem von mir vorgeschlagenen Ansatz werden nicht nur Informationen über das Individuum, sondern auch über potentielle und realisierte außerpaarliche Partner mit eingebunden. Bei Arten wie der Blaumeise, deren Territorien nicht nur in einzelnen, sondern in allen Aspekten des Brutverhaltens (Nahrungssuche, Balz, etc.) genutzt werden, sind Interaktionen räumlich oft auf Individuen aus nahegelegenen Territorien beschränkt. Auch außerpaarliches Verhalten zeigt eine solche Beschränkung. Im vorliegenden Ansatz werden daher räumliche Verhaltensstrukturen explizit in das Modell mit einbezogen. Um solche räumliche Verhaltensinformationen für einen Langzeitdatensatz über Brutverhalten (die hier eingesetzen Daten von Blaumeisen umfassen 12 Jahre und 2 Populationen) zu erhalten, ist es notwendig, im Nachhinein die Position der Territorien der Brutpaare abzuschätzen. In der Ökologie werden zu diesem Zweck immer häufiger Thiessen-Polygone eingesetzt (z.B. Wilkin et al. 2006, Valcu & Kempenaers 2008). Im 1. Kapitel (Chapter 1) belege ich daher zunächst die Gültigkeit einer solchen Abschätzung von Territorien mit Hilfe von Thiessen-Polygonen, basierend of 14 publizierten Studien, für die detaillierten Informationen über Territorien vorliegen. Im 2. Kapitel (Chapter 2) stelle ich dann den neuartigen Ansatz im Detail vor. Anschließend wende ich diesen auf den genannten Langzeitdatensatz an, nutze dabei Thiessen-Polygone als Approximation für die räumliche Beschränkung von außerpaarlichem Verhalten und untersuche gleichzeitig die Korrelation relevanter Parameter mit außerpaarlichem Fortpflanzungserfolg. Meine Resultate bestätigen die Ergebnisse früherer Studien und zeigen, dass dieser Ansatz genutzt werden kann, um Hypothesen zu testen, die mit bisherigen Methoden nicht statistisch robust überprüft werden konnten. Fremdverpaarungen können früh morgens stattfinden und aus einer Studie über die nahe verwandte Kohlmeise (Parus major) geht hervor, dass Weibchen, die ihren Schlafplatz morgens früher verließen, mit größerer Wahrscheinlichkeit außerpaarliche Nachkommen hatten. Im 3. Kapitel (Chapter 3) untersuche ich mit Hilfe von korrelativen Daten über vier Jahre diesen Zusammenhang für Blaumeisen. Darüber hinaus wurden die Aufstehzeiten von Weibchen über zwei Jahre hinweg experimentell manipuliert, um einen direkten kausalen Zusammenhang zwischen Aufstehzeiten und außerpaarlichem Fortpflanzungserfolg zu ergründen. Ich konnte keinen solchen Zusammenhang feststellen, jedoch führte das Experiment interessanterweise in den zwei Jahren zu entgegengesetzten Ergebnissen. Dies könnte ein Hinweis darauf sein, dass das Verhalten von Weibchen am frühen Morgen durchaus Relevanz für Fremdverpaarungen hat, dass jedoch eine Interaktion mit Umwelteinflüssen diese Zusammenhänge beeinflussen kann. Im 4. Kapitel (Chapter 4) beschreibe ich, dass Blaumeisen vor oder während der Legephase der Weibchen immer wieder die Nester fremder Paare besuchen. Für Männchen korrelierte dieses Verhalten auch mit ihrem außerpaarlichen Fortpflanzungserfolg. Es erwies sich nämlich, dass ein Männchen, welches das Nest eines Weibchens besucht, mit deutlich größerer Wahrscheinlichkeit außerpaarliche Nachkommen mit diesem Weibchen zeugt. Im Gegensatz zu einer früheren Studie an Blaumeisen (Kempenaers et al. 1992) zeigt dieses Ergebnis, dass nicht nur das Verhalten des Weibchens, sondern auch das des Männchens einen wichtigen Einfluss auf die außerpaarliche Fortpflanzung haben kann. Im 5. Kapitel (Chapter 5) betrachte ich abschließend das Balzverhalten der Blaumeisen. Dies erfolgt mittels Audioaufnahmen von Rufen, die speziell im Balzkontext eingesetzt werden (Bijnens & Dhondt 1984). Zunächst beschreibe ich das zeitliche Vorkommen dieser Rufe im Laufe der Brutsaison und im Tagesverlauf. Im nächsten Schritt vergleiche ich diese Daten mit früheren Studien, um zu verifizieren, dass Balzrufe tatsächlich als Maß für das Balzverhalten verwendet werden können. Anschließend untersuche ich dann speziell Balzrufe von Männchen, die in einem Kontext auftreten, der nahelegt, dass die Rufe nicht an das soziale Weibchen gerichtet sind. Es ist wahrscheinlich, dass diese (zu großen Teilen) außerpaarliche Balz anzeigen. Es gab keine Korrelation der „außerpaarlichen Balzversuche“ von Männchen mit ihrem außerpaarlichen Fortpflanzungserfolg, jedoch verloren Männchen mit vielen außerpaarlichen Balzversuchen weniger Vaterschaft in ihrem eigenen Nest. Dies könnte darauf hindeuten, dass die Qualität von Blaumeisenmännchen auf Verhaltensebene relevant für den außerpaarlichen Fortpflanzungserfolg ist. In der allgemeinen Einleitung (General Introduction) beschreibe ich detailliert den biologischen Hintergrund dieser fünf Studien. Weiterhin diskutiere ich die Relevanz von Studien im Bereich der Forschung über Fremdvaterschaften, die anstelle der rein genetischen Betrachtung darüber hinaus auch das Verhalten untersuchen. In der allgemeinen Diskussion (General Discussion) erörtere ich die Implikationen dieser fünf Studien für außerpaarliches Verhalten: Das zeitliche Vorkommen von Balz und Paarungen im Tagesverlauf und über die Saison hinweg, das räumliche Vorkommen, die relative Wichtigkeit des Verhaltens von Männchen und Weibchen und welche Faktoren beeinflussen, dass es zu Fremdverpaarungen zwischen einem ganz bestimmten Männchen und Weibchen kommt.

Fri, 12 Dec 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17787/ https://edoc.ub.uni-muenchen.de/17787/1/Toussaint_Emmanuel_F_A.pdf Toussaint, Emmanuel FA

This dissertation addresses the application of the statistical computing language R in the study of evolution and diversification of plants. The topics included range from the worldwide historical biogeography of the cucurbit family and the phylogenetic composition of the Mediterranean Oxalis flora in central Chile to the interplay between population genetics and climatic niche evolution in four Horde- um clades in the Americas. In these studies, I drew on existing methods in R and on java and C programs that could be easily integrated with R. Whenever necessary, I created additional software available in four new R packages. R's features, e.g., intersystem-interfaces, extensibility, reproducibility and advanced graphical capability, proved well suited for evolutionary and phylogenetic research. My coauthors and I addressed the history of Cucurbitaceae, one of the most economically important families of plants, using a multi-gene phylogeny for 114 of the 115 genera and 25 per cent of the 960 species. Worldwide sampling was achieved by using specimens from 30 herbaria. Results reveal an Asian origin of Cucurbitaceae in the Late Cretaceous, followed by the repeated spread of lineages into the African, American and Australian continents via transoceanic long-distance dispersal (LDD). North American cucurbits stem from at least seven range expansions of Central and South American lineages; Madagascar was colonized 13 times, always from Africa; Australia was reached 12 times, apparently always from Southeast Asia. Overall, Cucurbitaceae underwent at least 43 successful LDD events over the past 60 Myr, which would translate into an average of seven LDDs every 10 Myr. These and similar findings from other angiosperms stress the need for an increased tapping of museum collections to achieve extensive geographical sampling in plant phylogenetics. The second study focused on the interplay of population demography with the evolution of ecological niches during or after speciation in Hordeum. While large populations maintain a high level of standing genetic diversity, gene ow and recombination buffers against fast alterations in ecological adaptation. Small populations harbor lower allele diversity but can more easily shift to new niches if they initially survive under changed conditions. Thus, large populations should be more conservative regarding niche changes in comparison to small populations. My coauthors and I used environmental niche modeling together with phylogenetic, phylogeographic and population genetic analyses to infer the correlation of population demography with changes in ecological niche dimensions in 12 diploid Hordeum species from the New World, forming four monophyletic groups. Our analyses found both shifts and conservatism in certain niche dimensions within and among clades. Speciation due to vicariance resulted in three species with no pronounced climate niche differences, while species originating due to long-distance dispersals or otherwise encountering genetic bottlenecks mostly revealed climate niche shifts. Niche convergence among clades indicates a niche-filling pattern during the last 2 Myr in South American Hordeum. We provide evidence that species that did not encounter population reductions mainly show ecoclimatic niche conservatism, while major niche shifts have occurred in species that have undergone population bottlenecks. Our analyses allow the conclusion that population demography influences adaptation and niche shifts or conservatism in South American Hordeum species. Finally, I studied the phylogenetic composition of Oxalis flora of Mediterranean zone of Chile by asking whether in such a species-rich clade xerophytic adaptations arose in parallel, at different times, or simultaneously. Answering this type of question has been a major concern of evolutionary biology over the past few years, with a growing consensus that lineages tend to be conservative in their vegetative traits and niche requirements. Combined nuclear and chloroplast DNA sequences for 112 species of Oxalidales (4900 aligned nucleotides) were used for a fossil-calibrated phylogeny that includes 43 of the 54 species of Chilean Oxalis, and species distribution models (SDMs) incorporating precipitation, temperature, and fog, and the phylogeny were used to reconstruct ancestral habitat preferences, relying on likelihood and Bayesian techniques. Since uneven collecting can reduce the power of SDMs, we compared 3 strategies to correct for collecting effort. Unexpectedly, the Oxalis flora of Chile consists of 7 distant lineages that originated at different times prior to the last Andean uplift pulse; some had features preadapting them to seasonally arid or xeric conditions. Models that incorporated fog and a `collecting activity surface' performed best and identified the Mediterranean zone as a hotspot of Oxalis species as well as lineage diversity because it harbors a mix of ancient and young groups, including insuficiently arid-adapted species. There is no evidence of rapid adaptive radiation.

Postural control during standing and walking is an inherently unstable task requiring the interaction of various biomechanical, sensory, and neurophysiological mechanisms to shape stable patterns of whole-body coordination that are able to counteract postural disequilibrium. This thesis focused on the examination of central aspects of the functional roles of these mechanisms and the modes of interaction between them. A further aim was to determine the conditions of dynamic stability for healthy standing and walking performance as well as for certain balance and gait disorders. By studying movement fluctuations in the walking pattern it could be demonstrated that dynamic stability during walking depends on gait speed and is differentially regulated for the medio-lateral and the fore-aft walking planes. Stability control in the fore-aft walking plane exhibits attractor dynamics typical for a dynamical system. Accordingly, the most stable pattern of movement coordination in terms of minimal fluctuations in the order parameter (i.e., the relative phase between the two oscillating legs) can be observed at the attractor of self-paced walking. Critical fluctuations occur at increasingly non-preferred speeds, indicating a loss of dynamic gait stability close to the speed boundaries of the walking mode. Moreover, stability control during slow walking is critically dependent on sensory feedback control, whereas dynamic stability during fast walking relies mainly on the smooth operation of cerebellar pacemaker regions. Disturbances of sensory and cerebellar locomotor control in certain gait disorders could be further linked to a loss of dynamic gait stability, in particular an increased risk of falls. Furthermore, this thesis examined alterations in the sensorimotor postural control scheme that may trigger the experience of subjective imbalance and vertigo in the conditions of phobic postural vertigo and visual height intolerance. Both conditions are characterized by an inadequate mode of balance regulation featuring increased levels of open-loop balance control and a precipitate integration of closed-loop sensory feedback into the postural control scheme. This inadequate balance control strategy is accompanied by a stiffening of the anti-gravity musculature and is elicited by specific influences of attention and sensory feedback control. The findings of this thesis contribute to the understanding of central sensorimotor mechanisms involved in the control of dynamic postural stability during standing and walking. They further provide relevant information for the differential diagnosis and fall risk estimation of certain balance and gait disorders.