Podcasts about ddc:570

Mon, 2 May 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19429/ https://edoc.ub.uni-muenchen.de/19429/1/Chakraborty_Sayantan.pdf Chakraborty, Sayantan ddc:570, ddc:500,

Organisms respond to changes in their environment affecting their physiological or ecological optimum by reactions called stress responses. These stress responses may enable the organism to survive by counteracting the consequences of the environ- mental change, the stressor, and usually consist of plastic alterations of traits related to physiology, behaviour, or morphology. In the ecological model species Daphnia, the waterflea, stressors like predators or parasites are known to have an important role in adaptive evolution and have been therefore studied in great detail. However, although various aspects of stress responses in Daphnia have been analysed, molecu- lar mechanisms underlying these traits are not well understood so far. For studying unknown molecular mechanisms, untargeted ‘omics’ approaches are especially suit- able, as they may identify undescribed key players and processes. Recently, ‘omics’ approaches became available for Daphnia. Daphnia is a cosmo- politan distributed fresh water crustacean and has been in research focus for a long time because of its central role in the limnic food web. Furthermore, the responses of this organism to a variety of stressors have been intensively studied e.g. to hypoxic conditions, temperature changes, ecotoxicological relevant substances, parasites or predation. Of these environmental factors, especially predation and interactions with parasites have gained much attention, as both are known to have great influence on the structure of Daphnia populations. In the work presented in this thesis, I characterised the stress responses of Daphnia using proteomic approaches. Proteomics is particularly well suited to analyse bio- logical systems, as proteins are the main effector of nearly all biological processes. However, performing Daphnia proteomics is a challenging task due to high proteolytic activity in the samples, which most probably originate from proteases located in the gut of Daphnia, and are not inhibited by proteomics standard sample pre- paration protocols. Therefore, before performing successful proteomic approaches, I had to optimise the sample preparation step to inhibit proteolytic activity in Daph- nia samples. After succeeding with this task, I was able to analyse stress responses of Daphnia to well-studied stressors like predation and parasites. Furthermore, I stud- ied their response to microgravity exposure, a stressor not well analysed in Daphnia so far. My work on proteins involved in predator-induced phenotypic plasticity is de- scribed in chapter 2 and 3. Daphnia is a textbook example for this phenomenon and is known to show a multitude of inducible defences. For my analysis, I used the system of Daphnia magna and its predator Triops cancriformis. D. magna is known to change its morphology and to increase the stability of its carapace when exposed to the pred- ator, which has been shown to serve as an efficient protection against T. cancriformis predation. In chapter 2, I used a proteomic approach to study predator-induced traits in late-stage D. magna embryos. D. magna neonates are known to be defended against Triops immediately after the release from the brood pouch, if mothers were exposed to the predator. Therefore, the formation of the defensive traits most probably oc- curs during embryonic development. Furthermore, embryos should have reduced protease abundances, as they do not feed inside the brood pouch until release. To study proteins differing in abundance between D. magna exposed to the predator and a control group, I applied a proteomic 2D-DIGE approach, which is a gel based method and therefore enables visual monitoring of protein sample quality. I found differences in traits directly associated with known defences like cuticle proteins and chitin-modifying enzymes most probably involved in carapace stability. In addition, enzymes of the energy metabolism and the yolk protein vitellogenin indicated alterations in energy demand. In chapter 3, I present a subsequent study supporting these results. Here, I analysed responses of adult D. magna to Triops predation at the proteome level using an optimised sample preparation procedure, which was able to generate adult protein samples thereby inhibiting proteolysis. Furthermore, I established a different proteomic approach using a mass-spectrometry based label- free quantification, in which I integrated additional genotypes of D. magna to create a more comprehensive analysis. With this approach, I was able to confirm the results of the embryo study, as similar biological processes indicated by cuticle proteins and vi- tellogenins were involved. Furthermore, additional calcium-binding cuticle proteins and chitin-modifying enzymes and proteins involved in other processes, e.g. protein biosynthesis, could be assigned. Interestingly, I also found evidence for proteins in- volved in a general or a genotype dependent response, with one genotype, which is known to share its habitat with Triops, showing the most distinct responses. Genotype dependent changes in the proteome were also detectable in the study which I present in chapter 4. Here, I analysed molecular mechanisms underlying host-parasite interactions using the well characterised system of D. magna and the bacterial endoparasite Pasteuria ramosa. P. ramosa is known to castrate and kill their host and the infection success is known to depend strongly on the host’s and the para- site’s genotype. I applied a similar proteomic approach as in chapter 3 using label- free quantification, but contrastingly, I did not use whole animal samples but only the freshly shed cuticle. It has been shown, that the genotypic specificity of P. ramosa infection is related to the parasite’s successful attachment to the cuticle of the host and is therefore most probably caused by differences in cuticle composition. Hence, I analysed exuvia proteomes of two different genotypes known to be either suscept- ible to P. ramosa or not. Furthermore, I compared exuvia proteomes of susceptible Daphnia exposed to P. ramosa to a control group for finding proteins involved in the infection process and in the stress response of the host. The proteomes of the different genotypes showed indeed very interesting abundance alterations, connected either to cuticle proteins or matrix metalloproteinases (MMPs). Additionally, the cuticle pro- teins more abundant in the susceptible genotype showed a remarkable increase in predicted glycosylation sites, supporting the hypothesis that P. ramosa attaches to the host’s cuticle by using surface collagen-like proteins to bind to glycosylated cuticle proteins. Most interestingly, in all replicates of the susceptible genotype exposed to P. ramosa, such a collagen-like protein was found in high abundances. Another group of proteins found in higher abundance in the non-susceptible genotype, the MMPs, are also connected to this topic, as they may have collagenolytic characteristics and therefore could interfere with parasite infection. Furthermore, the data indicate that parasite infection may lead to retarded moulting in Daphnia, as moulting is known to reduce the infection success. Contrastingly to the work presented so far, the study described in chapter 5 invest- igated the protein response of Daphnia to a stressor not well studied on other levels, namely microgravity. As gravity is the only environmental parameter which has not changed since life on earth began, organisms usually do not encounter alterations of gravity on earth and cannot adapt to this kind of change. Daphnia has been part of one mission to space, however, responses of the animals to microgravity are not well described so far. In addition, as Daphnia are an interesting candidate organisms for aquatic modules of biological life support systems (BLSS), more information on their response to microgravity is necessary. For this reason, proteomics is an interesting ap- proach, as biological processes not detectable at the morphological or physiological level may become apparent. Therefore, a ground-based method, a 2D-clinostat, was used to simulate microgravity, as studies under real microgravity conditions in space need high technical complexity and financial investment. Subsequently, a proteomic 2D-DIGE approach was applied to compare adult Daphnia exposed to microgravity to a control group. Daphnia showed a strong response to microgravity with abundance alterations in proteins related to the cytoskeleton, protein folding and energy meta- bolism. Most interestingly, this response is very similar to the reactions of a broad range of other organisms to microgravity exposure, indicating that the response to altered gravity conditions in Daphnia follows a general concept. Altogether, the work of my thesis showed a variety of examples of how a proteomic approach may increase the knowledge on stress responses in an organisms not well- established in proteomics. I described both, the analysis of molecular mechanisms underlying well-known traits and the detection of proteins involved in a response not well characterised. Furthermore, I gave examples for highly genotype dependent and also more general stress responses. Therefore, this thesis improves our understanding of the interactions between genotype, phenotype and environment and, moreover, offers interesting starting points for studying the molecular mechanisms underlying stress responses of Daphnia in more detail.

Mon, 11 Apr 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19365/ https://edoc.ub.uni-muenchen.de/19365/1/Carlichi-Witjes_Nadine_M.P.pdf Carlichi-Witjes, Nadine ddc:570, ddc:500, Fakultät für Biologie 0

Fri, 18 Mar 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19411/ https://edoc.ub.uni-muenchen.de/19411/1/Diepold_Rebekka.pdf Diepold, Rebekka ddc:570, ddc:500, Fakultät für Biologie

Eye movements are important to aid vision, and they serve two main functions: to stabilize a moving visual target on the retina and to stabilize gaze during own body movements. Six types of eye movements have been evolved fulfilling this function: saccades, smooth pursuit, vestibulo-ocular reflex, optokinetic response, convergence and gaze holding. In all vertebrates the eyes are moved by six pairs of extraocular muscles that enable horizontal, vertical and rotatory eye movements. The motoneurons of these muscles are located in the oculomotor (nIII), trochlear (nIV) and abducens (nVI) nucleus in the brainstem. Motoneurons of the lateral rectus muscle (LR) in nVI and of the medial rectus muscle (MR) in nIII provide horizontal eye movements, those of inferior oblique (IO) and superior rectus muscle (SR) in nIII upward eye movements. Motoneurons of the superior oblique (SO) and the inferior rectus muscle (IR) in nIII convey downward eye movements. Recently, it was shown that each extraocular muscle is controlled by two motoneuronal groups: 1. Motoneurons of singly innervated muscle fibers (SIF) that lie within the boundaries of motonuclei providing a fast muscle contraction (twitch) and 2. motoneurons of multiply innervated muscle fibers (MIF) in the periphery of motonuclei providing a tonic muscle contraction (non-twitch). Tract-tracing studies indicate that both motoneuronal groups receive premotor inputs from different brainstem areas. A current hypothesis suggests that pathways controlling twitch motoneurons serve to generate eye movements, whereas the non-twitch system is involved in gaze holding. Lesions of inputs to the twitch motoneuron system may lead to supranuclear gaze palsies, whereas impairment of the non-twitch motoneuron system may result in gaze holding deficits, like nystagmus, or strabismus. Up to date only limited data are available about the histochemical characteristics including transmitters to the SIF- (twitch) and MIF (non-twitch) motoneurons. The present study was undertaken to investigate the histochemical profile of inputs to motoneuronal groups of individual eye muscles mediating horizontal and vertical eye movements including the inputs to MIF- and SIF motoneurons. The MIF motoneurons of the IR and MR are located in the periphery dorsolateral to nIII, close to the Edinger-Westphal nucleus (EW), which is known to contain preganglionic cholinergic neurons. Other scientists have found that the EW is composed of urocortin-positive neurons involved in food intake or stress. In order to delineate these different cell populations within the supraoculomotor area dorsal to nIII, a comparative study in different mammals was conducted to locate the cholinergic preganglionic neurons and urocortin-positive neurons. Only then, it became obvious that the cytoarchitecturally defined EW labels different cell populations in different species. In rat, ferret and human the cytoarchitecturally defined EW is composed of urocortin-positive neurons. Only in monkey the EW contains cholinergic preganglionic neurons, which lie close to the MIF-motoneurons of MR and IR in the C-group. In monkey, I performed a systematic study on the histochemical profile and transmitter inputs to the different motoneuron subgroups, including MIF- and SIF motoneurons. Brainstem sections containing prelabelled motoneurons were immunostained for the calcium-binding protein calretinin (CR), gamma-aminobutyric acid (GABA) or glutamate decarboxylase (GAD), glycine transporter 2, glycine receptor 1, and the vesicular glutamate transporters (vGlut) 1 and 2. The study on the histochemical profile of the motoneuron inputs revealed three main results: 1.The inhibitory control of SIF motoneurons for horizontal and vertical eye movements differs. Unlike previous studies in the primate a considerable GABAergic input was found to all SIF motoneuronal groups, but a glycinergic input was confined to motoneurons of the MR mediating horizontal eye movements. 2. The excitatory inputs to motoneurons for upgaze and downgaze differ in their histochemistry. A striking finding was that CR-positive nerve endings were confined to the motoneurons of muscles involved in upgaze, e.g. SR, IO and the levator palpebrae, which elevates the upper eyelid and acts in synchrony with the SR. Since double-immunoflourescence labelling with anti-GAD did not reveal any colocalization of GAD and CR, the CR-input to upgaze motoneurons is considered as excitatory. 3. The histochemistry of MIF- and SIF motoneurons differs only for vGlut1. Whereas SIF- and MIF motoneurons of individual eye muscles do not differ in their GABAergic, glycinergic and vGlut2 input, vGlut1 containing terminals were covering the supraoculomotor area and targeting only MR MIF motoneurons. It is reasonable to assume that the vGlut1 input affects the near response system in the supraoculomotor area, which houses the preganglionic neurons in the EW mediating pupillary constriction and accommodation and the MR MIF motoneurones involved in vergence. The histochemical data in monkey enabled the localization of the corresponding motoneuronal subgroups of individual eye muscles in human with the development of an updated nIII map. Taken together the present work provides new data on the histochemical properties of premotor inputs to motoneuronal groups of the twitch- and non-twitch eye muscle systems in primates. Especially the selective association of CR in premotor upgaze pathways may open the possibility for a targeted research of this system in human post-mortem studies of clinical cases with impairment of upward eye movements, such as progressive supranuclear palsy (PSP) or Niemann-Pick disease (NPC).

Tue, 26 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19125/ https://edoc.ub.uni-muenchen.de/19125/1/Jordan_Felix.pdf Jordan, Felix ddc:570, ddc:500, Fakultät für Biologie

Desmosomen sind spezialisierte Haftstrukturen, die die Stabilisierung des Zellverbundes gegenüber Zug- und Scherkräften gewährleisten. Dazu binden desmosomale Cadherine extrazellulär an Haftmoleküle benachbarter Zellen und sind intrazellulär unter anderem über Desmoplakin (DP) und Plakoglobin (PG) an Keratinfilamenten verankert. Insbesondere für das desmosomale Cadherin Desmoglein 3 (Dsg3), das sowohl innerhalb als auch außerhalb der Desmosomen vorkommt, wurde eine wichtige Bedeutung als Adhäsionsprotein in Keratinozyten nachgewiesen. Trotz ihrer Funktion, Widerstand gegen hohe mechanische Belastungen zu vermitteln, sind Desmosomen dynamische Strukturen, die einem stetigen Umbau unterliegen. Die Notwendigkeit einer genauen Regulierung des desmosomalen Auf- und Abbaus wird durch das Vorkommen zahlreicher vererbbarer und autoimmuner Erkrankungen unterstrichen. In der vorliegenden Arbeit wurden Mechanismen, die der geordneten Assemblierung der Desmosomen und der Disassemblierung nach Störung der desmosomalen Zell-Zell-Haftung unterliegen, untersucht. Im ersten Teil der vorliegenden Studien standen die Vorgänge der Desmosomenbildung in humanen Keratinozyten im Fokus. Adhärenskontakte und deren Zusammenwirken mit Actinfilamenten spielen eine wichtige Rolle in der Ausbildung der Desmosomen. Für die Actin-Bindeproteine Adducin und Cortactin wurde durch siRNA-Interferenzstudien eine essentielle Funktion für die Vermittlung der desmosomalen Zell-Zell-Haftung nachgewiesen. Die siRNA-induzierte Depletion von Adducin verursachte eine Reduktion der zytoskelettal-gebundenen Dsg3-Moleküle, was mit einer reduzierten Membranmobiltät korrelierte. Für Cortactin wurde eine direkte Interaktion mit Dsg3 mittels zweier unabhängiger molekularbiologischer Methoden nachgewiesen. Dies deutet auf eine direkte Rolle des Cortactins in der Regulierung der Desmosomen hin. Die siRNA-induzierte Depletion von E-Cadherin führte zum Verlust der membranständigen Lokalisation von Dsg3 und zu einer verminderten Verankerung der Dsg3-Moleküle innerhalb der zytoskelettalen Proteinfraktion. Es wurde ein Signalkomplex aus extradesmosomalen Dsg3, E-Cadherin und der Tyrosinkinase Src identifiziert, dessen Stabilität durch Src reguliert wurde. Hierbei wurden Dsg3 und E-Cadherin an Tyrosinresten durch Src phosphoryliert, deren Aktivität sowohl für die Inkorporation von Dsg3 in die Desmosomen als auch für die Reifung der Desmosomen zu stabilen Haftkontakten essentiell war. Im zweiten Teil der vorliegenden Arbeit wurden die Prozesse der desmosomalen Disassemblierung nach Inkubation mit Pemphigus vulgaris-Autoantikörpern (PV-IgG) analysiert.PV ist eine etablierte Modellerkrankung zur Untersuchung der Desmosomen-vermittelten Zelladhäsion in Keratinozyten. Die Bindung der gegen Dsg1 und Dsg3 gerichteten PV-IgGs induziert eine Reduktion der Dsg3-Proteinmengen und eine Aktivierung verschiedener Signalwege, u.a. von RhoA und PKC. Da diese Signalwege ebenfalls Adducin regulieren und PV-IgGs eine Umorganisierung des Actin-Zytoskeletts verursachen, die durch exogene Aktivierung von RhoA verhindert wird, wurde das Zusammenspiel von PV-IgGs, RhoA und Adducin untersucht. Die protektive Wirkung der RhoA-Aktivierung auf die Zell-Zell-Haftung und die Verteilung von Dsg3 nach Applikation der PV-IgGs war sowohl von der Expression als auch von der Phosphorylierung von Adducin an Serin726 abhängig. Interessanterweise verursachten PV-IgGs über den Ca2+-Einstrom und über PKC, unabhangig von RhoA, eine schnelle Phosphorylierung von Adducin an Serin726. Die durch den Ca2+-Einstrom- und PKC-vermittelte Phosphorylierung von Adducin könnte somit einen Rettungsmechanismus der Keratinozyten darstellen, der in Reaktion auf die PV-IgG-Bindung einsetzt und die desmosomale Assemblierung induziert. Ferner wurde die reduzierte Verankerung der Keratinfilamente an Desmosomen, ein weiteres Merkmal der PV-Pathogenese, mit der Aktivität von PKC korreliert. Keratinfilamente, die einer dynamischen Regulierung durch p38MAPK unterliegen, lösen sich in Reaktion auf PV-IgGs von den Desmosomen und akkumulieren perinukleär. Dieses Phänomen der Zytokeratin-Retraktion wurde durch Inkubation mit Tandempeptid (TP), das die Transinteraktion von Desmogleinen stärkt, verhindert. Zusammenfassend liefern die in dieser Arbeit gewonnenen Daten neue Erkenntnisse über die Mechanismen des desmosomalen Umsatzes. Adducin und E-Cadherin nehmen eine essentielle Rolle in der Ausbildung und Aufrechterhaltung der desmosomalen Haftstrukturen ein. Untersuchungen der pathogenen Effekte der PV-IgGs unterstreichen die hohe Relevanz eines intakten Actin- und Keratin-Stützgerüsts für die interzelluläre Haftung von Keratinozyten. Diese Befunde könnten in Zukunft auch von medizinischer Relevanz für die Therapie von Pemphigus-Patienten sein.

Super-resolution fluorescence microscopy performed via 3D structured illumination microscopy (3D-SIM) features an 8-fold volumetric resolution improvement over conventional microscopy and is well established on flat, adherent cells. However, blastomeres in mammalian embryos are non-adherent, round and large. Scanning whole mount mammalian embryos with 3D-SIM is prone to failure due to non-adherent embryos moving during scanning and a large distance to the cover glass. The biggest challenge and achievement of this doctorate thesis was the development of a novel method to perform 3D-SIM on mammalian embryos (“3D structured illumination microscopy of mammalian embryos and spermatozoa” published in BMC Developmental Biology). The development and fine-tuning of this method took over two years due to the time-intense generation of embryos and the subsequent two day long embryo staining, embedding and scanning with steps that required novel techniques such as micromanipulation which was not associated with sample preparation prior to this protocol. Problem identification was time-intensive since each of the numerous steps necessary could negatively affect the image quality. This method was fine-tuned during three studies. The first study “Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos” (published in Nucleus) investigates the profound changes of nuclear architecture during cattle preimplantation development of embryos generated by somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Fibroblast nuclei in embryos generated by SCNT go through similar changes in nuclear architecture as embryos generated by IVF. In both embryo types the occurrence of a large, chromatin-free lacuna in the center of nuclei around major embryonic genome activation (EGA) was noted. Similarly, the chromosome territory-interchromatin compartment (CT-IC) model applied to both types of embryos, featuring a lacuna or not, with an enrichment of RNA polymerase II and H3K4me3, a histone modification for transcriptionally competent chromatin, in less concentrated chromatin and an enrichment of H3K9me3, a transcriptionally restrictive histone modification, in more concentrated chromatin. However, large, highly concentrated H3K4me3 and H3K9me3 clusters were noted in both embryo types at chromatin concentrations that did not fit to the model. The chromatin-free lacunas were highly enriched in newly synthesized mRNA. The second study “Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications” (published in PLOS ONE) presents the changes of the nuclear envelope and lamina during bovine preimplantation development. Before major EGA, chromatin-free areas of the nuclear periphery were also free of nuclear pore complexes (NPCs), whereas after major EGA, the entire nuclear periphery was equipped with at least a fine layer of chromatin and associated NPCs. Three types of nuclear invaginations were predominant at different stages. The most common invagination was lamin B and NUP153 positive and was most prominent between the 2-cell and 8-cell stages until the onset of major EGA. Lamin B positive, but NUP153 negative invaginations were most prominent during stages with large nuclear volume and surface reductions. The least common invagination was lamin B negative but NUP153 positive and occurred almost exclusively at the morula stage. RNA-Seq and 3D-SIM data showed large deposits of spliced NUP153 mRNA and cytoplasmic NUP153 protein clusters until shortly after major EGA. NUP153 association with chromatin was initiated at metaphase. The third study “Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development” (published in the Journal of Reproduction and Development) demonstrated that rabbit embryonic nuclei feature a nuclear invagination type containing a large volume of cytoplasm that provides cytoplasmic proximity to nucleoli in addition to the small volume invaginations that were previously observed in bovine nuclei. The underlying mechanism for these two invaginations must differ from each other since small volume invaginations were frequently emanating from large volume invaginations emanating from the nuclear border but large volume invaginations were never emanating from small volume invaginations emanating from the nuclear border. Abundance of import/export competent invaginations featuring NPCs peaked at the 4-cell stage, which is the last stage before a drastic nuclear volume decline and also the last stage before major EGA is initiated at the 8- to 16-cell stage. Import/export incompetent invaginations positive for lamin B but not NUP153 peaked at the 2-cell stage. This was the stage with the largest variability in nuclear volumes. This may hint at an interphase nuclear surface reduction mechanism. Additionally, previously generated but unpublished 3D-FISH data about the localization changes of a stably inserted reporter gene upon activation in cloned bovine embryos was analyzed and documented in the study “Positional changes of a pluripotency marker gene during structural reorganization of fibroblast nuclei in cloned early bovine embryos” (published in Nucleus). This study showed that the stably inserted OCT-4 reporter gene “GOF” in bovine fetal fibroblasts was initially moved towards the nuclear interior in day 2 bovine embryos generated by SCNT of bovine fetal fibroblasts. However, in day 4 SCNT embryos the localization of GOF had moved towards the periphery while it was still activated. Its carrier chromosome territory did not significantly move differently compared with the non-carrier homolog. Constant proximity of GOF to its carrier chromosome territory ruled out a movement by giant loops. In cooperation with the Department of Histology and Embryology of the Ege University (Izmir, Turkey) the destructive effects of cryopreservation on blastomere integrity were analyzed in the study “Ultra-Structural Alterations in In Vitro Produced Four-Cell Bovine Embryos Following Controlled Slow Freezing or Vitrification” (published in Anatomia, Histologia, Embryologia). The cryopreservation method slow freezing caused more damage to blastomeres and to the zona pellucida than its fast freezing alternative vitrification. This was most likely caused by ice crystal formation and the longer exposure to the toxic side effects of cryoprotectants before freezing was complete.

Childhood abuse is one of the major risk factors for the development of adult psychopathology though the response to childhood abuse and other types of early life adversities is not uniform. Genetic predisposition modulates the exposure to environmental factors in form of gene by environment interaction. This has been shown for FKBP5, a modulator of the stress hormone axis, with certain alleles in FKBP5 conferring a higher risk towards PTSD in adulthood in response to childhood abuse. This thesis investigates the potential molecular mechanism behind this gene by environment interaction and delineates an allele-specific demethylation mechanism in response to childhood abuse. In addition, data on genome-wide gene expression and DNA methylation profiles in peripheral blood in response to childhood abuse is presented providing evidence for the hypothesis that childhood trauma leads to a different molecular trajectory towards adult psychopathology compared to adult traumatization. The data presented here contribute to our understanding of the molecular mechanisms underlying gene by environment interactions in psychiatry and the pathophysiology of trauma- and stress-induced psychiatric disorders.

Fri, 15 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19073/ https://edoc.ub.uni-muenchen.de/19073/1/Hoefler_Carolin.pdf Höfler, Carolin ddc:570, ddc:500, Fakultät für Biologie

Tue, 12 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19075/ https://edoc.ub.uni-muenchen.de/19075/1/Knief_Johann_Ulrich.pdf Knief, Johann Ulrich ddc:570, ddc:500, Fakultät für B

Tue, 22 Dec 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19054/ https://edoc.ub.uni-muenchen.de/19054/1/Blohberger_Jan.pdf Blohberger, Jan ddc:570, ddc:500, Fakultät für Biologie

Eukaryotic genomes make use of nucleosomes to considerably reduce their packaging volumes. As a consequence, the underlying DNA is rendered inaccessible. Cells make use of ATP-dependent remodeling factors to disrupt histone-DNA contacts and bring about access to the DNA. ACF1 is the largest regulatory subunit of two nucleosome remodeling factors, namely ACF and CHRAC. These complexes assemble, slide or evenly space nucleosomes on DNA with an ability to sense the linker lengths. However, roles of ACF1 in organizing nucleosomes in vivo and their physiological consequences are largely unclear. To understand the roles of ACF1 on chromatin organization, I compared nucleosome occupancy and transcription profiles in wild-type and ACF1-deficient Drosophila embryos. To further investigate and corroborate these chromatin changes, I performed genomewide mapping of ACF1 using chromatin immunoprecipitation. Nucleosome occupancy was mapped by subjecting DNA obtained from MNase-digested chromatin to deep sequencing and the occupancies were analyzed using advanced analog signal processing methods. We found discontinuous and discrete patches of regularly positioned nucleosomes in wild-type tissue, referred to as ‘regularity regions’. These regions span actively transcribing and silent chromatin domains and show associated variation in the linker lengths across them. A subset of these regions located at sides remote from the transcriptional start sites loses regularity upon ACF1 deletion and show presence of a novel DNA sequence motif. Analyzing nucleosome periodicity by autocorrelation function revealed that nucleosome linker length is longer in ACF1-deficient embryos. Despite profound quantifiable changes in the chromatin organization the RNA expression analyses did not show any major changes. Genomewide localization of ACF1 was studied using by chromatin immunoprecipitation. We observed a strong enrichment of ACF1 along active promoter regions, coinciding strikingly well with another remodeling factor, RSF-1. However, careful analyses using mutant tissues for both proteins demonstrated that the observed enrichments were in fact false positive. We define 3100 genomic sites as false positive ‘Phantom Peaks’ that tend to enrich in the ChIP-seq experiments. By comparing publicly accessible profiles and the Phantom regions, we showed that several ChIP-seq profiles of the epigenetic regulators show strong enrichment along the Phantom Peaks. In conclusion, we identify regions of regularly organized nucleosomes across the genome and show that a subset localized in silent chromatin regions is affected by ACF1 deletion. Moreover, we identified a class of false positive ChIP-seq peaks at active promoters. This list of Phantom Peaks can be used to assess potential false positive signal in a ChIP-seq profile, especially when mutant tissue is not available as a control.

Thu, 26 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18960/ https://edoc.ub.uni-muenchen.de/18960/1/Schaeffner_Marisa.pdf Schäffner, Marisa ddc:570, ddc:500, Fakultät für Bi

Thu, 26 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18938/ https://edoc.ub.uni-muenchen.de/18938/1/Schrode_Nadine.pdf Schrode, Nadine ddc:570, ddc:500, Fakultät für Biologie

Tue, 24 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19033/ https://edoc.ub.uni-muenchen.de/19033/1/Lehnert_Simon.pdf Lehnert, Simon ddc:570, ddc:500, Fakultät für Biologie

Wed, 11 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19085/ https://edoc.ub.uni-muenchen.de/19085/1/Jeltsch_Katharina.pdf Jeltsch, Katharina ddc:570, ddc:500,

Das größte Problem für HIV-infizierte Personen ist die Tatsache, dass die Infektion nicht „geheilt“ werden kann und die Betroffenen ihr Leben lang infiziert bleiben. Deshalb erfordert die HIV Infektion die dauerhafte Anwendung von Therapien, die das Virus an der Replikation hindern und damit die „Viruslast“ im Körper möglichst gering halten. Die optimale Bekämpfung der HIV-Infektion wären Wirkstoffkombinationen die sowohl die Produktion des Virus durch persistent infizierte Reservoirs als auch die Neuinfektion von HIV-Zielzellen unterbinden. Interessanterweise sind einige zelluläre Faktoren bekannt, die in die HIV-Replikation eingreifen und der HIV-Produktion entgegenwirken können. Ein Beispiel für solche HIV-Restriktionsfaktoren sind die Risp/ Fam21 Proteine, die mit dem HIV Rev Protein interagieren und so vermutlich seine regulatorische Funktion hemmen können. Frühere Untersuchungen an persistent HIV-infizierten Astrozyten belegten einen Zusammenhang zwischen der Expressionsstärke von Risp/ Fam21 und der Hemmung der HIV Produktion in diesen Zellen. In dieser Arbeit wurde mit Hilfe der quantitativen PCR Analyse gezeigt, dass risp/ fam21 Gene in unterschiedlichen Stärken in menschlichen Zellen exprimiert werden. Zur Modulation der Risp/ Fam21 Expression in diesen als auch anderen für HIV relevanten Zellen wurde ein lentivirales Vektorsystem etabliert. In akut infizierten T-Zellen wurde kein Einfluss der Risp/ Fam21-Modulation auf die HIV-Infektion gefunden, was die Theorie nahelegt, dass Risp/ Fam21 Proteine nur in persistent infizierten Zellen wie den o.g. Astrozyten eine Aktivität zeigen könnten. Um neue Inhibitoren der akuten HIV-Infektion gesunder Zellen zu identifizieren, wurde die medizinische Heilpflanze Cistus incanus (Ci) im Hinblick auf ihre anti-HIV Aktivität getestet. Bei dieser Pflanze handelt es sich um eine sehr polyphenolreiche Pflanze und Polyphenole stellen eine interessante Klasse an HIV-Inhibitoren dar. In der vorliegenden Arbeit wurde gezeigt, dass Präparate aus Ci die Infektion von Zellen hemmen, indem sie spezifisch an die Virusoberfläche binden und die Anheftung der Viren an die Zielzellen verhindern. Präparate aus Ci inhibieren ein sehr breites Spektrum an verschiedenen HIV-Laborstämmen und –Patientenisolaten.

Diese Arbeit beschäftigt sich mit der Expression des astrozytenspezifischen Enzyms Glutaminsynthetase in Ergänzung zum gliaspezifischen Marker Repo, um Gliazellen, die mit der embryonalen Entwicklung des Zentralkomplexes in Schistocerca gregaria assoziiert sind, zellulär und molekular zu charakterisieren. Der Zentralkomplex ist ein modulares System neuropiler Strukturen im Mittelhirn aller Insekten, und ist in vielen Verhaltensvorgängen wie Laufen, Fliegen, Stridulation und Ernährung involviert. In der Heuschrecke entwickeln sich die Neuropile des Zentralkomplexes im Laufe der Embryogenese und sind zum Zeitpunkt des Schlüpfens funktionsfähig. Trotz großer Kenntnisse neuronaler Aspekte über die Entwicklung des Zentralkomplexes verbleibt die Funktion der Gliazellen unklar. In dieser Arbeit wurde das Expressionsmuster des astrozytenspezifischen Enzyms Glutaminsynthetase (GS) und des gliaspezifischen Homöobox Gens reversed polarity (repo) in Kombination mit der negativen Expression des neuron-spezifischen Markers Meerrettich Peroxidase (HRP) zur Identifizierung glialer Zellen benutzt. Doppelfärbungen zeigen, dass alle GS-positiven Zellen, die mit dem Zentralkomplex assoziiert sind, gleichzeitig Repo-positiv sind. Zum ersten Mal konnte ich durch diese Kombination nicht nur Zellkörper, sondern auch Projektionen (Gliapodien) der Gliazellen sichtbar machen. Während der Embryogenese, also noch vor der Entwicklung des Zentralkomplexes, formen Gliazellen eine zusammenhängende Population, die aus der Pars intercerebralis in die Region der Faserbündel einwandert. Anschließend verteilen sich die Gliazellen neu und umhüllen jedes der einzelnen Module des Zentralkomplexes. Innerhalb der einzelnen Neuropile des Zentralkomplexes sind keine glialen Zellkörper zu finden. Rekonstruktionen einzelner Zellen zeigen Populationen von Gliazellen, die ausgedehnte umhüllende Projektionen um die Neuropile des Zentralkomplexes, wie den Zentralkörper, senden, während eine andere Population von Gliazellen säulenartige Verzweigungen in den Zentralkörper hinein projiziert. Solche Verzweigungen in den Modulen des Zentralkomplexes sind erst nach Fertigstellung der Neuroarchitektur zu erkennen. Daher kann man annehmen, dass diese Verzweigungen auf ein zuvor entstandenes Gerüst von Neuronen oder Tracheen projizieren. Höchstwahrscheinlich sind diese Gliaprojektionen in die Transmitterregulation innerhalb des Neuropils involviert. Da Gliazellen weitreichende Projektionen (Gliapodien) in und um die Mittelhirnneuropile senden, wurden in gefrorenen Hirnschnitten intrazelluläre Injektionen durchgeführt um zu erforschen, ob diese Gliazellen ein zelluläres Netzwerk via Zellkopplung im Verlauf der Embryogenese bilden. Färbungen individueller Zellen, die an vier unterschiedlichen Injektionsstellen um den Zentralkörper lokalisiert sind, zeigen eine Population gekoppelter Zellen, deren Anzahl und räumliche Verteilung stereotypisch für jeden der Injektionspunkte ist. Darüber hinaus sind sie sowohl bei 70%igem wie auch bei einem embryonalen Entwicklungsstand von 100% miteinander vergleichbar. Anschließende immunhistochemische Experimente bestätigen, dass es sich bei den gekoppelten Zellen um astrozytenähnliche Gliazellen handelt. Durch Hinzufügen von n-Heptanol in das Puffermedium wurde die Zellkopplung verhindert. Da die Zellkopplung auch ohne direkten intersomalen Kontakt auftritt, könnten die erheblichen Verzweigungen der Gliapodien, die sich im Laufe der Embryogenese ausbreiten, involviert sein. Durch die Datenerhebung aller Injektionspunkte kann darauf geschlossen werden, dass die Gliazellen, welche den Zentralkörper umrunden, ein Netzwerk gekoppelter Gliazellen bilden, das als Positionierungssystem der sich entwickelnden Neuropile des Zentralkomplexes dient.

The CHO-K1 cell line is the most common expression system for therapeutic proteins in the pharmaceutical industry. Due to the nature of economics, the cell lines and the vector design are subject to constant change to increase product quality and quantity. During the cultivation, the production cell lines are susceptible to decreasing productivity over time. Often the loss of production can be associated with a reduction of copy number and the silencing of transgenes. During cell line development, the most promising cell lines are cultivated in large batch culture. Consequently, the loss of a stable production cell line can be very cost-intensive. For this reason I developed different strategies to avoid a reduced productivity. Instability of production cell lines can be predicted by the degree of CpG methylation of the driving promoter. Considering that the DNA methylation is at the end of an epigenetic cascade and associated with the maintenance of the repressive state, I investigated the upstream signals of histone modifications with the assumption to obtain a higher predictive power of production instability. For this reason I performed a chromatin immunoprecipitation of the histone modifications H3K9me3 and H3K27me3 as repressive signals and H3ac as well as H3K4me3 as active marks. The accumulations of those signals were measured close to the hCMV-MIE at the beginning of the cultivation and were then compared with the loss of productivity over two month. I found that the degree of the H3 acetylation (H3ac) correlated best with the production stability. Furthermore I was able to identify an H3ac threshold to exclude most of the unstable producers. In the second project I aimed to improve the vector design by considering epigenetic mechanisms. To this end I designed on the one hand a target-oriented histone acetyltransferase to enforce an open and active chromatin status at the transgene. On the other hand I point-mutated methylation-susceptible CpGs within the hCMV-MIE to impede the maintenance of inactive heterochromatin formation. Remarkably, the C to G mutation located 179 bp upstream of transcription start site resulted in very stable antibody producing cell lines. In addition, the examination of cell pools expressing eGFP showed that G-179 promoter variants were less prone to a general methylation and gene amplification, which illustrates the dominating effect in epigenetic mechanisms of one single CpG. The last project was performed to localize stable integration sites within the CHO-K1 genome. In so doing I could show that the transfection leads predominantly to integration into inactive regions. Furthermore I identified promising integration sites with a high potential to induce stable expression. However, those results are preliminary and must be viewed with caution. Further examination needs to be done to confirm these results. Considering the results of all three projects, I propose that the interplay of metabolic burden and selection pressure at an early time point of cultivation plays an important role in cell line development. Small alterations of selection pressure can lead to a decisive change of cell properties. Therefore, stable cells are less susceptible than weak producers. The increase of selection pressure leads to compensatory effect by gene amplification in the instable cell lines. The resulting adjustment of productivity masks the truly stable cells, which precludes the selection of the right cell lines. For this reason the selection pressure, the copy number as well as the growth rate should be considered to minimize repressive effects.

Magnetotactic bacteria (MTB) contain nanometer-sized crystals of a magnetic iron mineral enabling directed swimming along geomagnetic field lines. However, although this unique behavior was discovered already 40 years ago, it still has remained poorly understood at the cellular level and the molecular mechanisms responsible for sensing environmental stimuli and transducing signals to the flagellar motors have been unknown. Therefore, the major goal of this thesis was to investigate the swimming behavior of Magnetospirillum gryphiswaldense both at the behavioral and molecular level. Individual motors of tethered M. gryphiswaldense cells were found to rotate both clockwise and counterclockwise with equal speed. Cells swam at speeds of up to 60 µm s-1 and commonly displayed runs of several hundred µm in length. In striking contrast to E. coli, which reorients the cell body between run intervals at random angles, motor switching events caused swimming reversals with reorientation angles close to 180°. The sensory repertoire of M. gryphiswaldense was analyzed by classical macroscopic chemotaxis assays, and aerotaxis was found to be the dominant behavior. In addition to the strong microaerophilic response in oxygen gradients, I observed tactic bands also under anoxic conditions within gradients of the alternative electron acceptor nitrate, suggesting that aerotaxis is part of a general redox or energy taxis mechanism. The aerotactic response of M. gryphiswaldense was furthermore analyzed by recording and tracking single cells under controlled atmospheric conditions in a gas perfusion chamber. Compared to other well-studied bacteria, M. gryphiswaldense displayed unusually low swimming reversal rates (

In order to react to changes within their environment, plants developed a specific signaling network that enables the cells to convert external stimuli including light, abiotic and biotic stress as well as hormones into cellular signals. One example is the influx of calcium, a second messenger stored in apoplasts or internal reservoirs, into the cytosol. This causes changes in the calcium-ion-concentration within the cell that are recognized by specific sensors including Calmodulin and lead to the induction of a cellular signal response. Calcium signals do not only occur in the cytosol, but also appear within the nucleus, chloroplasts, mitochondria as well as peroxisoms (Bachs et al. 1992, Chigri et al. 2005, Kuhn et al. 2009, Dolze et al. 2013). The import of nuclear encoded proteins into the mitochondria is regulated by calcium and Calmodulin at level of the TIM23- and TIM22-complex. This study identified atTim23.2, the pore-forming component of the TIM23-complex, as a Calmodulin-binding protein. Pull-down-assays using Calmodulin-agarose revealed a specific and calcium-dependent binding. Furthermore, in silico analysis identified two potential Calmodulin-binding domains (CaMBD). Topology studies of atTim23.2 demonstrated that the proposed N-terminal CaMBD is located within the intermembrane space, the binding region within the first loops is located in the matrix of the mitochondria. Moreover, a topology of four transmembran domains of the protein could be shown. The recently in the mitochondria identified Calmodulin-like protein CML30 appeared to be a potential binding partner for atTim23.2. CML30 could be indeed detected in the intermembrane space of the mitochondria, but a direct interaction of the two proteins could not have been detected so far. Furthermore, using the split-ubiquitin system proved the ability of atTim23.2 to dimerize which might be responsible for the regulation of opening and closing of the importpore as it was already shown in S.cerevisiae. However, a correlation between the two functions of atTim23.2 to bind Calmodulin as well as to dimerize could not have been confirmed, yet. Nevertheless, the regulation of the pore via the calcium/Calmodulin signaling network could connect the import process of matrix proteins with the stress regulation of the cell.

The genetic basis underlying adaptive evolution is still largely unknown. Adaptive evolution is facilitated by natural selection that acts on the genetic variation present in a population. Favoring some genetic variants over others, natural selection eventually produces adaptations that allow populations to survive in changing or new environments. Populations colonizing new habitats that differ from their original habitat are often confronted with a multitude of novel ecological constraints to which they need to adapt. A well-annotated genome and a diverse genetic toolkit make the fruit fly Drosophila melanogaster an ideal model system for studying the genetics underlying adaptation. As a cosmopolitan species, D. melanogaster has adapted to a wide range of thermal environments. Despite having a tropical origin in southern-central Africa, it has successfully settled in temperate environments around the world. Thermal adaptations that have helped to deal with the greater range and variability in temperature as well as low-temperature extremes have been required to prosper in temperate environments. Chromatin-based gene regulation is known to be disrupted by varying temperatures. Variation in the temperature, at which flies live, result in varying expression levels of Polycomb group (PcG) regulated genes with higher expression at lower temperatures. Chapter 1 and 2 of this thesis aim to answer the question whether this thermosensitivity of PcG regulation has been detrimental for colonizing temperate environments and thus needed to be buffered by natural selection. Thermosensitivity of PcG regulation was observed in different natural populations of D. melanogaster. A lower degree of thermosensitive expression was consistently found for populations from temperate climates when compared to those from the tropics. In Chapter 1, evidence is presented for positive selection acting on the polyhomeotic (ph) gene region to reduce thermosensitivity of PcG regulation in temperate populations from Europe. The targets of selection appear to be single nucleotide polymorphisms (SNPs) in a relatively small cis-regulatory region between the two PcG target genes polyhomeotic proximal (ph-p) and CG3835 that are highly differentiated between European and African populations. Using reporter gene assays, it was demonstrated that these SNPs influence gene expression and that the European alleles confer reduced thermosensitivity of expression in contrast to the African alleles. In Chapter 2, thermosensitivity of another PcG target gene, vestigial (vg), was investigated in six natural populations including four temperate populations from high-altitude Africa and central to high-latitude Europe, and two tropical populations from the ancestral species range. All four temperate populations exhibited a lower degree of thermosensitive expression than the two tropical populations. The underlying mechanisms of increased buffering, however, seem to differ between these temperate populations. Thermal adaptation to temperate environments also includes dealing with low-temperature extremes. Severe cold stress is a main limiting factor imposed on D. melanogaster by temperate climates. Increased cold tolerance in temperate populations is thought to have evolved by natural selection. Cold tolerance is a quantitative trait that appears to be highly polygenic and has been mapped to different quantitative trait loci (QTL) in the genome. In Chapter 3, such a QTL region was fine-mapped to localize causal genes for increased cold tolerance in temperate flies. As a result, brinker (brk) was identified as a new candidate gene putatively involved in cold stress adaptation.

At least four phylogenetically distinct groups of bacteria encode repeat proteins with the common ability to bind specific DNA sequences with a unique but conserved code. Each repeat binds a single DNA base, and specificity is determined by the amino acid residue at position 13 of each repeat. Repeats are typically 33-35 amino acids long. Comparing repeat sequences across all groups reveals that only three positions are hyper-conserved. Repeats are in most cases functionally compatible such that they can be assembled together into a single chimeric array. This functional conformity and inter-compatibility is a result of structural conservation. Repeat arrays of these proteins have been demonstrated or predicted to form almost identical tertiary structures: a right-handed super helix that wraps around the DNA double strand with the base specifying residue of each repeat positioned in the major groove next to its cognate target base. The mechanism of DNA binding is conserved. The first discovered group, providing the name for the rest, are the Transcription Activator Like Effectors (TALEs) of plant-pathogenic Xanthomonas bacteria. The eukaryotic transactivation domain, which lends this group their name, allows them to activate specifically targeted host genes for the benefit of the bacterial invader. The other groups, discovered after the TALEs, are the RipTALs of Ralstonia solanacearum, the Bats of Burkholderia rhizoxinica, and MOrTL1 and MOrTL2 of unknown marine bacteria. Together they are designated TALE-likes. Each designation contains some allusion to the TALEs. The term RipTAL stands for Ralstonia injected proteins TALE-like, the Bats are Burkholderia TALE likes, and the MOrTLs Marine Organism TALE-likes. This unity of terminology belies disunity in the lifestyles of these different bacteria, and the biological roles fulfilled by these proteins. The TALEs have already been researched extensively. The code that describes the relationship between the base specifying residues and their cognate bases is often referred to as the TALE code. This code was deciphered by two groups independently and published in 2009, a year before I began my doctoral work. Since then research into TALEs has not slowed and a great deal has been learnt both about the native biology and biotechnological uses of TALEs. My work has been focused on the other TALE-like groups, none of which had been previously characterized in terms of DNA recognition properties, before I began my work. RipTALs are effector proteins delivered during bacterial wilt disease caused by R. solanacearum strains. This devastating disease affects numerous crop species worldwide. Characterizing the molecular properties of the RipTALs provides a first step towards uncovering their role in the disease. The Bats and MOrTLs are primarily of interest as comparison groups to the TALEs and RipTALs and as sources of sequence diversity for future efforts into TALE repeat engineering. In the introduction of this dissertation, which explores TALE biology, a particular focus will be placed on the DNA binding properties of TALEs and how this can be put to use in TALE technology. After this the RipTALs, Bats and MOrTLs are each introduced, explaining what is known about their provenance and sequence features. The aims of my doctoral work are then listed and expounded in turn. The proximal goal of my doctoral work was to carry out a comparative molecular characterization of each group of non-TALE TALE-likes. In doing so we hoped to gain insights into the principles of TALE-like DNA-binding properties, evolutionary history of the different groups and their potential uses in biotechnology. In the case of the RipTALs this work should begin to unravel the role these proteins play in bacterial wilt disease, as a means to fight this devastating pathogen. The articles I have worked on covering the molecular characterizations of RipTALs, Bats and MOrTLs are then presented in turn. Working together with others I was able to show that repeats from each group of TALE-likes mediate sequence specific DNA binding, revealing a conserved code in each case. This code links position 13 of any TALE-like repeat to a specific DNA base preference in a reliable fashion. I will argue that the TALE-likes represent a fascinating case of conserved structure and function in a diverse sequence space. In addition the TALEs and RipTALs may simply represent one face of the TALE-likes, a protein family mediating as yet unknown biological roles as bacterial DNA binding proteins.

Dendritic integration is a fundamental element of neuronal information processing. So far, few studies have provided a detailed picture of this process, describing the properties of local dendritic activity and its subcellular organization. Here, I used 2-photon calcium imaging in optic flow processing neurons of the blowfly Calliphora vicina to determine the preferred location and direction of local motion cues for small branchlets throughout the entire dendrite. I found a pronounced retinotopic mapping on both the subcellular and the cell population level. In addition, dendritic branchlets residing in different layers of the neuropil were tuned to distinct directions of motion. Within one layer, local preferred directions varied according to the deflections of the ommatidial lattice. Summing the local receptive fields of all dendritic branchlets reproduced the characteristic properties of these neurons’ axonal output receptive fields. These results corroborate the notion that the dendritic morphology of vertical system cells allows them to selectively collect local motion inputs with particular directional preferences from a spatially organized input repertoire, thus forming filters that match global patterns of optic flow. These data illustrate a highly structured circuit organization as an efficient way to hard-wire a complex sensory task.

Physical changes in neuronal connections, dictated by the neuronal network activity, are believed to be essential for learning and memory. Long-term potentiation (LTP) of synaptic transmission has emerged as a model to study activity-driven plasticity. The majority of excitatory contacts between neurons, called synapses, are found on spines, small dendritic protrusions. LTP is known to trigger the formation and stabilization of new dendritic spines in vitro. Similarly, experience-dependent plasticity in vivo is associated with changes in the number and stability of spines. However, to date, the contribution of excitatory synaptogenesis to the enhanced synaptic transmission after LTP remains elusive. Do new spines form functional synapses with the inputs stimulated during LTP induction and thereby follow Hebbian co-activation rules, or do they connect with random partners? Furthermore, at which time-point are de novo spines functionally integrated into the network? I developed an optical approach to stably and exclusively stimulate the axons of a defined channelrhodopsin-2 (ChR2)-transduced subset of CA3 cell in mature hippocampal slice culture over extended periods of time (up to 24h). I continuously monitored synaptic activation and synaptic structure of CA1 cells dendrites using two-photon imaging. To control the dendritic location where LTP and associated spinogenesis were allowed to take place, I globally blocked Na+-dependent action potential firing and directly evoke neurotransmitter release by local light-evoked depolarization of ChR2-expressing presynaptic boutons (in TTX, 4-AP). I induced optical LTP specifically at this location by combining optogenetic activation with chemical pairing (in low [Mg2+]o, high [Ca2+]o, forskolin, and rolipram). Taking advantage of the NMDA-receptor mediated calcium influx during synaptic activation I assessed the formation of functional synapses using the genetically encoded calcium indicator GCaMP6s. I find that optical LTP led to the generation of new spines, decreased the stability of preexisting spines and increased the stability of new spines. Under optical LTP conditions, a fraction of new spines responded to optical presynaptic stimulation within hours after formation. However, the occurrence of the first synaptic calcium response in de novo spines varied considerably, ranging from 8.5 min to 25 h. Most new spines became responsive within 4 h (1.2 ± 0.9 h, mean ± S.D., n = 16 out of 20), whereas the remainder showed their first response only on the second experimental day (18.2 ± 3.7 h). Importantly, new spines generated under optical LTP were more likely to build functional synapses with light-activated, ChR2-expressing axons than spontaneously formed spines (new responsive spines under optical LTP: 64 ± 4 %; control 1: 0%; control 2: 13 ± 4 %; control 3: 11 ± 4 %). Furthermore, new spines that were responsive to optical presynaptic stimulation were less prone to be eliminated after overnight incubation than new spines that failed to respond (% overnight spine survival; 81 ± 3 % new responsive spines; 58 ± 4 % of new unresponsive spines). In summary, the results from my thesis demonstrate that synapses can form rapidly in an input-specific manner.

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

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.

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.

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

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.

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.

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

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.

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

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.

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

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.