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

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.

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

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.

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.

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/18938/ https://edoc.ub.uni-muenchen.de/18938/1/Schrode_Nadine.pdf Schrode, Nadine ddc:570, ddc:500, Fakultät für Biologie

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

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 (

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.

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.

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

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.