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.

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 E

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 pheno

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 tw

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.

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.

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, a

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.