Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 04/06

Tue, 31 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14761/ https://edoc.ub.uni-muenchen.de/14761/1/Lang_Claudia.pdf Lang, Claudia ddc:570, ddc:500

Mon, 30 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14648/ https://edoc.ub.uni-muenchen.de/14648/1/Berchtold_Doris.pdf Berchtold, Doris ddc:570, ddc:500, Fakultät für Biologie

Mon, 30 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14657/ https://edoc.ub.uni-muenchen.de/14657/1/Ballhorn_Uwe.pdf Ballhorn, Uwe

Anxiety disorders, such as posttraumatic stress disorder (PTSD), are characterized by a high prevalence and debilitating symptoms. However, the current first-line treatment for these conditions, which consists of selective serotonin reuptake inhibitors (SSRIs) and cognitive behavioral therapy, alongside symptomatic treatment with benzodiazepines, does not represent by far a functional solution for all affected patients. Therefore, identifying and characterizing novel candidates for alternative anxiolytic therapies are a crucial focus of psychiatric and neurobiological research. This study focuses on Neuropeptide S (NPS), a newly identified endogenous neuropeptide that has been shown to exert strong anxiolytic effects upon intracerebral injection in rodents. In an approach that combines basic research with incipient clinically relevant application, novel mechanisms and brain targets of NPS-mediated anxiolytic effects were identified, and a noninvasive application procedure also applicable in patients, namely the intranasal administration, was established for the first time for NPS in mouse models. In a first step, the feasibility of intranasal NPS delivery was established in mice using fluorophore-coupled NPS to allow intracerebral tracking. This method permitted for the first time tracking of intranasally applied substances within the brain at a single-cell resolution. These results not only proved the applicability of intranasal NPS administration in the mouse, but also allowed identification and characterization of hitherto undescribed cerebral NPS target cells, which were shown to be most likely exclusively neurons. Moreover, specific uptake of fluorescently labeled NPS in the hippocampus provided the first direct evidence linking this brain region, a well-known major player in the regulation of fear expression, to the NPS circuitry. Further investigation into the functional role of the hippocampus in NPS-elicited anxiolytic effects revealed that local microinjections of NPS into the ventral CA1 (vCA1) region are sufficient to elicit anxiolysis in C57BL6/N mice on the elevated plus maze (EPM). In a second step, behavioral and molecular effects of intranasal NPS treatment were characterized in C57BL/6N mice. Intranasal application of NPS was shown here to produce anxiolytic effects similar to those described by others after intracerebral injection. This finding represents the basis for the implementation of a future NPS-based therapy via nasal sprays in patients suffering from anxiety disorders. Furthermore, the molecular effects of NPS treatment on cerebral protein expression were examined here for the first time. This research led to identification of novel downstream targets of NPS-mediated regulation in the hippocampus and the prefrontal cortex. These new targets include proteins involved in the glutamatergic system and in synaptic plasticity, both of which are known to be dysregulated in anxiety disorders. Finally, the effects of intranasal NPS treatment, hitherto described only in non-pathological animal models, were examined for the first time in mouse models of anxiety disorders, namely the high anxiety behavior (HAB) mice and a mouse model of PTSD. In HAB mice, NPS treatment elicited anxiolytic effects similar to those observed in C57BL/6N mice. In the mouse model of PTSD, NPS counteracted disease-related changes in expression levels of hippocampal synaptic proteins. To sum up, this work expands the current state-of-knowledge concerning the molecular and mechanistic background of NPS-mediated anxiolysis by characterizing the role of the hippocampus in the NPS circuitry and by identifying novel downstream targets of NPS. The data on anxiolytic effects of intranasal NPS treatment especially in mouse models of anxiety disorders furthermore establishes the therapeutic potential of NPS as a novel anxiolytic treatment.

Mon, 30 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15410/ https://edoc.ub.uni-muenchen.de/15410/1/Boenisch_Clemens.pdf Bönisch, Clemens ddc:570, ddc:500, Fakultät für Biologie

Fri, 27 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14668/ https://edoc.ub.uni-muenchen.de/14668/1/Zimmermann_Dennis.pdf Zimmermann, Dennis ddc:570, ddc:

Fri, 27 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14810/ https://edoc.ub.uni-muenchen.de/14810/1/Hess_Julia.pdf Heß, Julia

Mon, 23 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17329/ https://edoc.ub.uni-muenchen.de/17329/1/Wang_Fei.pdf Wang, Fei

Fri, 20 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14587/ https://edoc.ub.uni-muenchen.de/14587/1/Vukajlovic_Marija.pdf Vukajlovic, Marija

Fri, 20 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16427/ https://edoc.ub.uni-muenchen.de/16427/1/Haage_Kristina.pdf Haage, Kristina ddc:570, ddc:500, Fakultä

Wed, 18 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14820/ https://edoc.ub.uni-muenchen.de/14820/1/Naduvilezhath_Lisha.pdf Naduvilezhath, Lisha ddc:570, ddc:500, Fak

Wed, 18 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17865/ https://edoc.ub.uni-muenchen.de/17865/1/Holstein_Norbert.pdf Holstein, Norbert ddc:570, ddc:500, Fakultät für B

Tue, 17 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15332/ https://edoc.ub.uni-muenchen.de/15332/1/Gomes_Rocha_Agostinho.pdf Gomes Rocha, Agostinho Manuel

Tue, 17 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16019/ https://edoc.ub.uni-muenchen.de/16019/1/Mues_Marsilius.pdf Mues, Marsilius ddc:570, ddc:500, Fakultät für Biologie

Mon, 16 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14701/ https://edoc.ub.uni-muenchen.de/14701/1/Mackinnon_Jonathan.pdf Mackinnon, Jonathan AG ddc:570, ddc:500, Fakultät für Bio

Mon, 16 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15331/ https://edoc.ub.uni-muenchen.de/15331/1/Katzmann_Emanuel.pdf Katzmann, Emanuel

Mon, 9 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14597/ https://edoc.ub.uni-muenchen.de/14597/1/Ruecker_Ovidiu.pdf Ruecker, Ovidiu Ludwig ddc:570, ddc:500, F

Wed, 4 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14987/ https://edoc.ub.uni-muenchen.de/14987/1/Loes_Corinna.pdf Loës, Corinna ddc:570, ddc:500, Fakultät für Biologie

Tue, 26 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15266/ https://edoc.ub.uni-muenchen.de/15266/1/Raba_Michael.pdf Raba, Michael ddc:570,

Tue, 26 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16161/ https://edoc.ub.uni-muenchen.de/16161/1/Kreile_Anne_Kristina.pdf Kreile, Anne Kristina ddc:57

Tue, 26 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16185/ https://edoc.ub.uni-muenchen.de/16185/1/Jeshen_Ingrid.pdf Jeshen, Ingrid Karin ddc:570, ddc:500, Fakultät für Biologie

Mon, 25 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14567/ https://edoc.ub.uni-muenchen.de/14567/1/Bultmann_Sebastian.pdf Bultmann, Sebastian ddc:570, ddc:500, Fakultät für Biologie

Plastid transformation is a valuable technique for both basic and applied science. In basic science the technique is used to study chloroplast function. Applied approaches deal with the potential of the plastid for the production of medicinal therapeutics, in most cases vaccine antigens coupled to adjuvants. Adjuvants are used for the trans-mucosal delivery of attached cargoes. Cell penetrating peptides (CPPs) emerged as valuable tools for the delivery of cell-impermeable cargoes across cell barriers more than twenty years ago. Although the exact mechanism of CPP penetration of cells is still discussed, the applied value of CPPs is documented in a number of clinical studies. Recently, scientists working in the CPP field launched a call for an alternative expression platform for CPP fusion peptides / proteins. Only a short time before, CPPs were introduced into plant science and some impressive first results, manipulating plant cells from the “outside”, were achieved. The present study aimed at combining the fields of plastid transformation and CPPs from the “inside”. We report the first expression of CPP fusion proteins in a plant, more precisely in the plastid. The approach focused on three aspects of CPP fusion protein expression in the organelle: (A) the principal feasibility of CPP-fusion protein expression in the plastid, (B) the location of CPP fusion proteins in the plant cell upon plastid-based expression and (C) the use of plastids for the manufacture of CPP fusions to provide an alternative to the bacterial expression system. Nine prominent CPPs were employed in three vector series to investigate these aspects. In vector series I the selected CPPs were fused to the fluorescent protein eGFP to provide an optical read-out; in vector series II, the CPPs were fused to Arabidopsis MYB transcription factor PAP1 to provide a biological read-out and in vector series III, two CPPs were fused to the human enzyme PAH to introduce plant-based CPP fusion protein expression. Taken together, the expression of CPP fusion in the plastid turned out to be feasible. Transplastomic plants reached homoplasmy, produced viable seeds and stably inherited the desired trait to their progenies in a maternal fashion. Only low protein accumulation levels were detected. Pleiotropic effects occured at the low protein accumulation levels observed. Localisation of CPP fusion proteins was shown to be restricted to the plastid. An inability of CPP fusion proteins isolated from vector series I to penetrate protoplasts, young plant tissue and human cell lines was revealed. The value of a plastid-based manufacture of CPP fusion proteins for clinical approaches failed to be demonstrated due to low fusion protein accumulation levels. Bottlenecks of the current study are discussed and suggestions are made to provide a framework for future efforts.

Thu, 21 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15496/ https://edoc.ub.uni-muenchen.de/15496/1/Spira_Felix.pdf Spira, Felix ddc:570, ddc:500, Fakultät für Biolog

Fri, 15 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15185/ https://edoc.ub.uni-muenchen.de/15185/1/Muehl_Bastian.pdf Muehl, Bastian Jan Josef ddc:570, ddc:500, Fakultät für Biologie

Thu, 14 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16018/ https://edoc.ub.uni-muenchen.de/16018/1/Freisinger_Tina.pdf Freisinger, Tina ddc:570, ddc:500, Fakultät für Biologie

Tue, 12 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14511/ https://edoc.ub.uni-muenchen.de/14511/1/Feistel_Susanne.pdf Feistel, Susanne ddc:570, ddc:500, Fakultät für Biologie

Mon, 11 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14428/ https://edoc.ub.uni-muenchen.de/14428/1/Hieber_Carolin.pdf Hieber, Carolin

Mon, 11 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14512/ https://edoc.ub.uni-muenchen.de/14512/1/Mendler_Anna.pdf Mendler, Anna

Fri, 1 Jun 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16380/ https://edoc.ub.uni-muenchen.de/16380/1/Fellerer_Christine.pdf Fellerer, Christine ddc:570, ddc:500, Fakultät für Biolog

The gourd family, Cucurbitaceae, is among the economically most important families of plants, with many crop species that form the basis of multi-million dollar industries. Knowledge of these species’ geographic origin and their closest wild relatives is fundamental to breeding efforts, genetic improvement, and conservation. Surprisingly, these aspects have been unknown or misunderstood for many widely cultivated species, even though plant material that could have been used for broad phylogenetic studies has long been available in herbaria. For the thesis presented here, I focused on the phylogenetic relationships within two clades of Cucurbitaceae that comprise cultivated species: the genus Cucumis, to which cucumber (Cucumis sativus) and melon (Cucumis melo) belong, and the New World Sicyoeae, which contain vegetable pear or chayote (Sicyos (Sechium) edulis) and tacaco (Frantzia tacaco), locally important in Mexico and Costa Rica, and the former also cultivated worldwide. I used a combination of DNA sequence data from up to 175-year old herbarium specimens and molecular phylogenetic methods as well as traditional morphological and ecological data from my own fieldwork in Asia and Australia to infer the phylogenetic relationships among these clades. I also discovered and described several new species, and reconstructed plausible scenarios for the two clades’ geographical unfolding over time. Until recently, only two species of Cucumis, namely cucumber and its closest relative C. hystrix, were thought to be of Asian origin, and melon was thought to have originated in Africa, from where 30 species were known. Using DNA sequences from plastid and nuclear markers for some 100 Cucumis accessions from Africa, Australia, and Asia, I have shown that cucumber and melon both are of Asian (probably Indian) origin and form a clade with 23 previously overlooked species-level relatives in Asia, Australia, and around the Indian Ocean, at least nine of them new to science and some described as part of this thesis. Fieldwork I carried out in Thailand and Australia contributed new knowledge about the life forms and habitats of some of these species and resulted in fertile material essential for the descriptions. My study furthermore revealed that the sister species of melon is the re-discovered C. picrocarpus from Australia. Future breeding efforts and investigations of wild species related to melon and cucumber should therefore concentrate on Asia and Australia, instead of Africa. In my second study group, the Sicyoeae, my aim was to test long-problematic generic boundaries and to reconstruct the history of the tribe’s name-giving genus, Sicyos, which has an exceptional geographical distribution. Using a densely sampled molecular phylogeny that included type species of 23 currently or formerly accepted genera of Sicyoeae, I showed that morphology-based concepts did not result in monophyletic genera, and that species from numerous smaller genera, including chayote, need to be part of Sicyos if monophyly is to be established. Sicyos, in its new circumscription, has a center of distribution in the Neotropics, where c. 50 species occur, but long-distance dispersal has resulted in the group’s presence on Hawaii (where it radiated into 14 species), at least two arrivals on the Galápagos archipelago (but no radiations), and one arrival in Australia and New Zealand, now with three species, two of them new to science. Using molecular clock models, I dated these four trans-Pacific dispersal events, all from the American mainland, to the last 4.5 to 1 million years. The mode of dispersal may have been adherence of the small, spiny fruits to birds, which would fit with the documented occurrence of Sicyos plants near seabird nesting colonies. The rapid diversification on Hawaii may have followed the loss of the fruit spines in the ancestor of the 14 Hawaiian species, leading to lower dispersal ability and faster allopatric speciation in the diverse habitats of the archipelago.

Thu, 24 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15132/ https://edoc.ub.uni-muenchen.de/15132/1/Hintermair_Corinna.pdf Hintermair, Corinna

In plants, long distance transport of sugars from photosynthetic source leaves to sink organs comprises different crucial steps depending on the species and organ types. Sucrose, the main carbohydrate for long distance transport is synthesized in the mesophyll and then loaded into the phloem. After long distance transport through the phloem vessels, sucrose is finally unloaded towards sink organs. Alternatively, sugar can also be transferred to non-plant sinks and plant colonization by heterotrophic organisms increases the sink strength and creates an additional sugar demand for the host plant. These sugar fluxes are coordinated by transport systems. Main sugar transporters in plants comprise sucrose (SUTs) and monosaccharide (MSTs) transporters which constitute key components for carbon partitioning at the whole plant level and in interactions with fungi. Although complete SUTs and MSTs gene families have been identified from the reference Dicot Arabidopsis thaliana and Monocot rice (Oriza sativa), sugar transporter families of the leguminous plant Medicago truncatula, which represents a widely used model for studying plant-fungal interactions in arbuscular mycorrhiza (AM), have not yet been investigated. With the recent completion of the M. truncatula genome sequencing as well as the release of transcriptomic databases, monosaccharide and sucrose transporter families of M. truncatula were identified and now comprise 62 MtMSTs and 6 MtSUTs. I focused on the study of the newly identified MtSUTs at a full family scale; phylogenetic analyses showed that the 6 members of the MtSUT family distributed in all three Dicotyledonous SUT clades; they were named upon phylogenetic grouping into particular clades: MtSUT1-1, MtSUT1-2, MtSUT1-3, MtSUT2, MtSUT4-1 and MtSUT4-2. Functional analyses by yeast complementation and expression profiles obtained by quantitative RT-PCR revealed that MtSUT1-1 and MtSUT4-1 are H+/sucrose symporters and represent key members of the MtSUT family. Conservation of transport capacity between orthologous leguminous proteins, expression profiles and subcellular localization compared to previously characterized plant SUTs indicate that MtSUT1-1 is the main protein involved in phloem loading in source leaves whilst MtSUT4-1 mediates vacuolar sucrose export for remobilization of intracellular reserve. The AM symbiosis between plants and fungi from the phylum Glomeromycota is characterized by trophic exchanges between the two partners. The fungus supplies the autotrophic host with nutrients and thereby promotes plant growth. In return, the host plant provides photosynthate (sugars) to the heterotrophic symbiont. Here, sugar fluxes from plant source leaves towards colonized sink roots in the association between the model leguminous plant M. truncatula and the arbuscular mycorrhizal fungus (AMF) Glomus intraradices were investigated. Sugar transporter candidates from both the plant and fungal partners presenting differential expression profiles using available transcriptomic tools were pinpointed. Gene expression profiles of MtSUTs and sugar quantification analyses upon high and low phosphorus nutrient supply and inoculation by the AMF suggest a mycorrhiza-driven stronger sink in AM roots with a finetuning regulation of MtSUT gene expression. Conserved regulation patterns were observed for orthologous SUTs in response to colonization by glomeromycotan fungi. In parallel, a non-targeted strategy consisting in the development of a M. truncatula - G. intraradices expression library suitable for yeast functional complementation and screening of symbiotic marker genes, similar to the approach that led to the identification of the first glomeromycotan hexose transporter (GpMST1), has been developed in this study. Taken together, with the identification, functional characterization and gene expression pattern of sugar transporter families, a more complete picture of sugar fluxes in the AM symbiosis has begun to emerge. This study opens new perspectives by identifying interesting candidate genes involved in sugar partitioning at both the plant and fungal levels and at the symbiotic interface in the widely used AM symbiosis model between M. truncatula and G. intraradices.

Tue, 22 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14379/ https://edoc.ub.uni-muenchen.de/14379/2/Pichler_Garwin.pdf Pichler, Garwin ddc:570, ddc:500, Fakultät für Biologie

Thu, 10 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15080/ https://edoc.ub.uni-muenchen.de/15080/1/JALAL_ABDULLAH.pdf Jalal, Abdullah ddc:570, ddc:500, Fakultät für Biologie

Tue, 8 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14380/ https://edoc.ub.uni-muenchen.de/14380/1/Widner_Andrae_Regina.pdf Widner-Andrä, Regina Andrea ddc:570, ddc:500, Faku

Wed, 2 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15344/ https://edoc.ub.uni-muenchen.de/15344/1/Uebe_Rene.pdf Uebe, René dd

Allergic asthma has a high prevalence and is characterized by airway inflammation, tissue remodeling and a decline in respiratory function. Although the pathogenesis is well known, the underlying mechanisms are still poorly understood. It is believed that a fine interplay exists between the exposure to environmental stimuli and relatively small changes in expression of several genes with inter-individual variation. As microRNAs (miRNAs) are known to be responsive to environmental exposures and show dysregulated levels in diseased states, their function as regulators of gene expression might be a missing link for the changes seen in asthma. In this project, changes in miRNA expression in a mouse model for allergic asthma were investigated and the interaction with possible target genes was analyzed. Female Balb/c mice were i. p. sensitized with ovalbumin (OVA) followed by aerosol challenge on two consecutive days. An asthmatic phenotype was confirmed by elevated total cell numbers due to a rise in inflammatory cells, as well as increased CCL17 levels in broncho-alveolar lavage (BAL). High titres of OVA-specific serum IgE were measured and lung histopathology revealed infiltration of inflammatory cells with eosinophilia. To study changes in miRNA expression, whole lung RNA was subjected to miRNA-microarray analysis (Exiqon). From 580 screened miRNAs, 319 were found to be expressed, of which 36 were differentially regulated in the allergic asthma group compared to healthy control mice. A second, TaqMan® chemistry based array was performed for validation. Based on the overlap between the two arrays in addition to fold changes and p-values (Exiqon), eight miRNAs were selected for single RT-qPCR measurement. Dysregulated expression of six miRNAs could be confirmed (miRNA-21, -142-3p, -144, -205, -208, -451). Due to relatively low fold changes and in order to monitor possible co-regulation, the top 100 differentially regulated miRNAs from the Exiqon array were included in an in situ target prediction. Applying a “full consensus” approach of five prediction algorithms, 961 putative target genes were identified. Based on the assumption, that target genes harboring multiple miRNA sites might be more relevant, 11 targets containing more than four miRNA binding sites were selected. From these, the transcription factor cAMP-responsive element-binding protein 1 (CREB1) was chosen for further analysis because of its previous association with asthmatic disease. Moreover, four miRNAs (miRNA-17, -22, -144, -181a) were predicted to bind at eight different sites, one of them being miRNA-144, a significantly up-regulated miRNA identified in the initial asthma profile. To experimentally test the functional interaction between CREB1 and the predicted miRNAs, a CREB1 3´-untranslated region (UTR) containing luciferase based reporter plasmid vector was constructed and co-transfected with precursor (pre-) miRNAs into human bronchial epithelial cells. Binding of all four miRNAs could be confirmed by measuring luciferase expression. Furthermore, three of four miRNAs, when transfected alone, were able to down-regulate endogenous CREB1 expression in vitro. In the lung tissue of asthma mice, CREB1 mRNA levels were significantly reduced compared to healthy controls, in contrast to two miRNAs, miRNA-17 and -144, which showed up-regulation. To gain further insight into expression patterns during sensitization and after challenge, expression of CREB1, the two validated binding partners miRNA-17, and -144, as well as the two miRNAs (miRNA-21, -451) with most significant p-values and high fold changes from the Exiqon array were analyzed. Clear expression changes happened after OVA aerosol challenge with CREB1 levels being steadily decreased, whereas all tested miRNAs showed elevated levels at 24 h post challenge, which further intensified after 120 h. This increase resembles measurements of inflammatory cell counts in BAL pointing at a possible origin within this population. In order to test whether findings can be translated into the human situation, miRNA changes in whole blood samples of mice were compared to miRNA patterns in peripheral blood of asthmatic children. In contrast to measurements in lung tissue, all four miRNAs showed markedly decreased expression in murine blood. In human samples this reduction was mirrored and significant for miRNA-144 and -451.

Thu, 26 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14413/ https://edoc.ub.uni-muenchen.de/14413/1/Ohler_Stephan.pdf Ohler, Stephan ddc:570, ddc:500, Fakultät für Biologie

Thu, 26 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15112/ https://edoc.ub.uni-muenchen.de/15112/1/Groll_Bettina.pdf Groll, Bettina ddc:570, ddc:500, Fakultät für Biologie

Wed, 25 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14990/ https://edoc.ub.uni-muenchen.de/14990/1/Wolf_Miriam.pdf Wolf, Miriam ddc:570, ddc:500, Fakultät für Biologie

Tue, 24 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15108/ https://edoc.ub.uni-muenchen.de/15108/1/Apfelbeck_Beate.pdf Apfelbeck, Beate ddc:570, ddc:500, Fakultät

Tue, 24 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15813/ https://edoc.ub.uni-muenchen.de/15813/1/Edle_von_Dall_Armi_Ekaterina.pdf Edle von Dall'Armi, Ekaterina ddc:570, d

Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA virus that infects humans and, after a primary lytic infection, establishes lifelong latency in the sensory neurons of the trigeminal ganglia (TG). HSV 1 latency is accompanied by a chronic immune cell infiltration of the TG, the infiltrate being mainly composed of CD8+ T cells. These T cells are believed to control viral latency, but cellular and viral factors like viral microRNAs are also considered to play a crucial role in the establishment and maintenance of viral latency. In the present work, it was investigated whether the tissue-infiltrating T cells are clonally expanded, which would indicate that these T cells are activated by antigen. By applying complementarity determining region 3 (CDR3) spectratyping and immunohistochemistry, several clonal expansions were identified in the TG-resident T cells. In addition, several T cells were present that seemed to be unspecific bystander T cells. Strikingly, some expanded T cell clones were present in the right and left TG of the same individual. This strongly suggests that similar antigens are present in both TG and that the infiltration of immune cells to the TG is driven by antigen. The morphology of the TG was investigated by immunohistochemistry and in situ hybridiza¬tion. Analysis of the distribution of T cells throughout the TG provided puzzling results: unexpectedly, most neurons surrounded by T cells did not harbour the only known prominent transcript during latency, the latency associated transcript (LAT). Whether these neurons do actually harbour latent virus was addressed by a combination of LAT in situ hybridisation, T cell immunohistochemistry, and single cell analysis of laser microdissected sensory neurons by PCR. This analysis revealed that only LAT+ neurons were harbouring HSV 1 DNA and viral microRNAs. Also, mRNA for a viral gene product was only detected in LAT+ neurons. All analysed LAT– neurons were devoid of viral microRNAs and DNA of HSV 1. DNA of HSV 2 or varicella-zoster virus (VZV) was not detected in any of the excised neurons. Alto¬gether this indicates that in the vast majority of infected human neurons, HSV 1 latency is not directly controlled by T cells, but rather by cellular or viral factors like the miRNAs. Our data suggest that CD8+ T cells only come into action if these mechanisms are overrun.

Early-life stress (ELS) can lead to enduring changes in the structure and function of neural circuits and endocrine pathways, resulting in altered vulnerability thresholds for stress-related disorders such as depression and anxiety. The question addressed in this work was whether epigenetic mechanisms contribute to the long-term programming of altered hypothalamus-pituitary-adrenal axis activity in ELS (maternal separated on postnatal days 1-10) mice. Adrenocorticotropic hormone (ACTH), a key pituitary mediator of the adrenocortical response to stress, is encoded by the proopiomelanocortin (Pomc) gene. Corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) are the main upstream neural regulators of Pomc gene expression and the post-translational processing of its peptidergic products, whereas glucocorticoids, secreted by the adrenals in response to stress, exert negative feedback actions on Pomc synthesis and ACTH secretion. It was shown that Pomc mRNA level is persistently increased in ELS mice and leads to sustained hypersecretion of glucocorticoids. Interestingly, ELS causes a reduction in DNA methylation at a critical regulatory region of the Pomc gene; this occurs with some delay after onset of the stress and persists for up to 1 year. A series of experiments (including reporter-, EMSA-, IHC- and ChIP-assays) supported the concept that the adverse early-life event induces changes in Pomc gene methylation and results in persistently upregulated expression of the Pomc gene. Interestingly, stress-induced changes in DNA-methylation were found to be more pronounced in males than in females, raising the possibility that epigenetic encoding occurs in a sex-specific manner; this may help to explain sex differences in susceptibility to stress-related disorders. Collectively, the results of this study indicate that epigenetic mechanisms can serve to translate environmental cues into stable changes (“cellular memory”) in gene expression in post-mitotic tissues, without the need for alterations in the genetic code.

Mon, 16 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14315/ https://edoc.ub.uni-muenchen.de/14315/1/Werthat_Florian.pdf Werthat, Florian

Im Zellkern einer jeden Zelle besteht eine gewisse Ordnung der darin vorhandenen DNA und Proteine. Diese Ordnung wird unter dem Begriff „Zellkernarchitektur“ zusammengefasst. In der vorliegenden Arbeit ging es um die nähere Betrachtung einiger Aspekte der Zellkernarchitektur. Diese Aspekte betrafen 1. die Anordnung von Genen, 2. die Anordnung von Chromatin mit Hilfe unterschiedlicher Histonmodifikationen und 3. die Anordnungen von Chromosomenabschnitten, die mit komplexen messenger RNA-Sonden hybridisiert werden. Im ersten Teil der vorliegenden Arbeit wurde mittels 3D FISH die dreidimensionale Positionierung von drei auf dem Chromosom 1 lokalisierten Genen in Zellkernen der Burkitt- Lymphom Zelllinie DG75 bestimmt. Diese Zelllinie wurde von Stefan Bohlander zur Verfügung gestellt und enthielt einen induzierbaren episomalen Vektor für das CALM-AF 10 Gen. Messungen der Genexpression, die in der Bohlander Gruppe mit Hilfe eines Affymetrix- Chips durchgeführt wurden, zeigten das die Induktion des Transgens zu genomweiten Veränderungen der Expressionsmuster hunderter Gene in dieser Zelllinie führten. Die für die 3D FISH Experimente ausgewählten Markergene zeigten nach der Induktion eine signifikant veränderte Expression. Dennoch änderte sich die radiale Positionierung dieser Gene, darunter versteht man die mehr innere oder mehr periphere Position der Gene, nicht. Dieses Ergebnis schien zuerst darauf hinzuweisen, dass die Transkriptionsstärke keine bedeutsamer Faktor im Hinblick auf die radiale Positionierung ist. Die Befunde der Affymetrix-Chip Analyse für diese Gene konnten jedoch in einer anschließende Untersuchungen der Genexpression mit Real-Time-PCR nicht bestätigt werden, obwohl der Vergleich von Affymetrix-Chip und Real- Time-PCR Daten insgesamt eine klare Korrelation zwischen den Datensätzen zeigte. Bei Diskrepanzen gehen wir davon aus, dass Real-Time-PCR die zuverlässigeren Ergebnisse liefert. Bei der hier durchgeführten Real-Time-PCR Untersuchung wurden auch die Expressionsstärken aller in einer Nachbarschaft von etwa 1 Mbp um die Markergene annotierten Gene ermittelt. Dieses Fenster wurde gewählt, weil Untersuchungen in der Arbeitsgruppe von Thomas Cremer und anderen Gruppen gezeigt haben, dass ~1 Mbp Chromatindomänen die Basisstruktur der Chromatinorganisation darstellen. Als Maß für die gesamte Genexpression einer Chromatindomäne wurde eine „Total Expression Strength“ (TES) berechnet. Dieser Wert basiert auf den Real-Time-PCR Werten der annotierten Gene und berücksichtigt auch die Länge der ungespleissten RNA, die von einem Gen transkribiert wird. Dabei zeigte sich, dass das Markergen in der Domäne mit dem höchsten TES Wert am weitesten innen im Zellkern lokalisiert ist. Dieser Befund unterstützt Befunde aus der wissenschaftlichen Literatur, dass die radiale Positionierung von individuellen Genen von Eigenschaften der lokalen Umgebung abhängt. Da sich die Nachbarschaft der untersuchten Markergene nicht nur im Hinblick auf die TES Werte sondern auch im Hinblick auf die Dichte der dort annotierten Gene und den GC-Gehalt unterscheidet, bleibt offen, welcher dieser Parameter als Prädiktor für die zu erwartende radiale Position individueller Gene eine entscheidende Rolle spielt. Möglich ist auch, dass alle Parameter zusammenwirken oder dass je nach den speziellen Umständen einer Untersuchung verschiedene Parameter die radiale Positionierung eines Gens bevorzugt beeinflussen. Die Stabilität der radialen Positionierung der Markergene trotz einer genomweiten Veränderung des Genexpressionsmusters nach CALM-AF 10 Induktion stimmt mit Befunden verschiedener Arbeitsgruppe überein, die für einen hohen Grad an räumlicher Stabilität der Chromatinanordnung während der Interphase sprechen; ~1 Mbp Chromatindomänen zeigen dementsprechend meist nur sehr begrenzte lokale Bewergungen (

Fri, 13 Apr 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14264/ https://edoc.ub.uni-muenchen.de/14264/1/Pohl_Sebastian.pdf Pohl, Sebastian ddc:570, ddc:500,

Abstract Considering the globally increasing rate of incidence, Type 2 diabetes mellitus belongs to the most frequent endocrine metabolic disorders today. In addition to insulin resistance of peripheral tissues, this disease is the result of dysfunction of the endocrine pancreas and in particular of the functional failure of β-cells. The progress of therapeutical strategies is based on the research of the underlying mechanisms. The aim of the present dissertation was the analysis of new molecular regulators which might improve our underdstanding of the differentiation of pancreatic islets and the functional maintenance of adult β-cells. The first part of this work concerned the role of the transcription factor Pax6 and especially the role of its transactivation domain (TA) and of its two DNA binding domains, the paired domain (PD) and the homeodomain (HD), in differentiation of pancreatic endocrine cells. By analyzing four different mouse lines with specific mutations in one of these three domains, we found that the PD of Pax6 is essential for differentiation of glucagon producing α-cells. Inactivation of this domain resulted in a phenotype similar to that of Pax6 knockout mice (Pax6-/-) with a near complete absence of glucagon positive α-cells, a markedly reduced number of insulin producing β-cells, and a disorganized islet structure. Mutations of HD or TA showed a less severe pancreatic phenotype. Islets either exhibited no morphological changes or they showed a reduction of α- and β-cells. Intraperitoneal glucose tolerance tests demonstrated the utmost importance of the transcription factor Pax6 for maintenance of normal pancreatic endocrine function in adult animals. In the second part of this study we identified new genes and proteins, respectively, which could play a regulatory role in normal function of β-cells. In particular it was possible to show that Eny2, hitherto a protein only described in yeast or invertebrates like drosophila, is involved in the regulation of insulin secreting vertebrate cells. si-RNA mediated knockdown of Eny2 resulted in markedly increased glucose and incretin-induced insulin secretion. This could be at least in part attributed to a higher glucose-dependent cellular metabolism and an enhanced signal transduction via protein kinase A and is accompanied by elevated levels of intracellular calcium. Taken together, these results indicate that Eny2 functions as a negative regulator of glucose-stimulated and incretin-mediated insulin secretion, at least in vitro. However, a gap of knowledge still remains between the established nuclear functions of Eny2 and the cellular phenotype we observed upon its suppression. Nevertheless, the effects of an Eny2-knockdown are glucose dependent and additive to the incretin signaling. This feature makes this model attractive to obtain new insights in how insulin secretion of β- cells proceeds and how to find new therapeutical strategies to treat type 2 diabetes mellitus.

Golden goal (Gogo) is a cell-surface protein critical for proper synaptic layer targeting of photoreceptors in the Drosophila melanogaster visual system. In collaboration with the seven-transmembrane cadherin Flamingo (Fmi), Gogo mediates both temporal and final layer targeting of R cell axons and its cytoplasmic domain is required. However, it is not known how Gogo activity is regulated. I show in my Dissertation that a conserved tripeptide Tyr-Tyr-Asp (YYD motif) in Gogo cytoplasmic domain is required for photoreceptor axon targeting. Deleting the YYD motif is sufficient to completely abolish Gogo function. I demonstrate that the YYD motif is a phosphorylation site and that mutations in the YYD tripeptide impair synaptic layer targeting. Gogo phosphorylation results in premature axon stopping and dephosphorylation is crucial for the collaboration with Fmi during the final target layer targeting. Therefore, both temporal and final layer targeting strongly depend on Gogo phosphorylation status. Drosophila Insulin Receptor (DInR) has been reported to regulate wiring of photoreceptors in the fly. I show that insulin signaling is a positive regulator of YYD motif phosphorylation in a direct or indirect way. My findings suggest a novel mechanism of the regulation of Gogo activity by phosphorylation which can be induced by insulin signaling. I propose the model that a constant phosphorylation signal is antagonized by a presumably temporal dephosphorylation signal, which creates a permissive signal that could govern developmental timing in axon targeting.

A long-standing question in evolutionary biology concerns the molecular causes underlying adaptive evolution. These can either stem from structural changes in proteins or from changes in the expression patterns of proteins or mature RNAs. Over the last decade, many studies have shown that gene expression changes can have a huge impact on the phenotype of an organism and play an important role in adaptive evolution. A major prerequisite for adaptive evolution to occur at the gene expression level is the presence of expression variation among members of a population. This variation serves as the raw material for adaptive evolution. The genetic causes underlying changes in expression patterns can either be located in cis-regulatory regions of the affected gene, such as transcription factor binding sites, or in trans-regulatory regions, such as transcription factors. Mutations in cis-regulatory elements have relatively few pleiotropic effects and their effects are often additive, thus, cis-regulatory changes are thought to be especially well-suited targets of selection. A major factor influencing gene expression is the sex of an organism. The sex-bias of a gene also influences the pace at which proteins evolve, such that male-biased genes often show more rapid evolution than female-biased or unbiased genes between Drosophila species. Here, we investigated genome-wide gene expression variation in adult females of two populations of D. melanogaster, one from the ancestral species range (Zimbabwe) and one from the derived species range (the Netherlands). We found relatively little expression polymorphism present within the populations and high expression divergence between the populations. More than 500 genes were expressed differentially between the populations. These are candidate genes for those that have undergone adaptive regulatory evolution to the new, derived environment. When comparing our study of female adults to a study investigating male adult flies of the same populations, we found that there is significantly less expression polymorphism in females within the populations but significantly more expression divergence between the populations. Further, there was little overlap in genes that differ in expression between the populations in males and females. This suggests that general differences exist between the sexes in gene expression regulation and that regulatory evolution has been mainly sex-specific. Our findings show that extensive gene expression variation exists in D. melanogaster and further highlight the importance of accounting for sex when investigating gene expression. In order to elucidate the genetic and evolutionary mechanisms that underlie differential gene expression between the populations, we employed a candidate gene approach. Analysis of molecular variation in the coding and upstream regions of several differentially expressed genes in both populations revealed evidence for a recent selective sweep in the European population for the gene CG34330. In the putative promoter region of the gene, there is one indel and one SNP where a derived variant is fixed in the European population, but at low frequency in the African population. These are candidates for those variants that control the expression level of the gene. For another gene, Jon99Ciii, we found evidence for recurrent structural protein evolution acting since the split of D. melanogaster from D. simulans and D. sechellia. However, no evidence for recent regulatory evolution could be found for this gene. Motivated by findings that male-biased genes often evolve faster than both female- and unbiased genes between Drosophila species, we examined the molecular evolution of sex-biased genes and their contribution to within-population polymorphism, between-population divergence and between-species divergence in D. melanogaster and D. ananassae. This was studied on both the DNA-sequence level and the expression level. We found strong purifying selection limiting protein sequence variation within species. In contrast, a high proportion of divergence could be attributed to positive selection. In D. melanogaster, male-biased genes showed the highest fraction of adaptive substitutions, a pattern that was especially pronounced on the X chromosome. In contrast, male-biased genes did not show higher variation within or between populations, suggesting that inter-species divergence is not just a simple extension of inter-population divergence and intra-population variation. For D. ananassae, we did not observe a higher rate of adaptive evolution for male-biased genes, a finding that suggests that the type or strength of selection acting on sex-biased genes differs between lineages. Similarly, on the expression level, we found that sex-biased genes show high expression divergence between species, but low divergence between populations.