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This study investigated patterns of arthropod community organisation and the processes structuring these communities on a range of different tree species in a natural West African savannah (Comoé National Park, Côte d'Ivoire). It described and analysed patterns of arthropod distribution on the level of whole communities, on the level of multiple-species interactions, and on the level of individual insect species. Community samples were obtained by applying (i) canopy fogging for mature individuals of three tree species (Anogeissus leiocarpa, Burkea africana, Crossopteryx febrifuga) and (ii) a modified beating technique allowing to sample the complete arthropod communities of the respective study plants for medium-sized (up to 3 m) individuals of two other species (Combretum fragrans, Pseudocedrela kotschyi). General information on ant-plant interactions was retrieved from ant community comparisons of the mature savannah trees. In addition, ant-ant, ant-plant and ant-herbivore interactions were studied in more detail considering the ant assemblages on the myrmecophilic tree Pseudocedrela kotschyi. Herbivore-plant interactions were investigated on a multiple-species level (interrelationships between herbivores and Pseudocedrela trees) and on a species level (detailed studies of interrelationships between herbivorous beetles and caterpillars and the host tree Combretum fragrans). The studies on individual herbivore species were complemented by a study on an abundant ant species, clarifying not only the relationship between host plant and associated animal but allowing also to look at interactive (competitive) aspects of community organisation. The study demonstrated for the first time that (i) the structure of beetle communities on tropical trees can be strongly dependent on the host tree species, (ii) individual trees can host specific arthropod communities whose characteristic structure is stable over years and is strongly determined by the individual tree's attributes, (iii) ants can express a pronounced fidelity to single leaves as foraging area and can thereby determine distribution patterns of other ants, (iv) intraspecifically variable palatability of plants for insect herbivores can be stable over years and can influence the distribution of herbivores that can distinguish between individual hosts according to palatability and (v) intraspecific host plant change can positively affect fitness of herbivores if host plant quality is variable. In general, the present study contributes to our knowledge of anthropogenically unaltered processes affecting community assembly in a natural environment. The fundamental understanding of these processes is crucial for the identification of anthropogenic alterations and the establishment of sustainable management measures. The study points out the important role local factors can play for the distribution of organisms and thereby for community organisation. It emphasises the relevance of small scale heterogeneity of the abiotic and biotic environment to biodiversity and the need to consider these factors for development of effective conservation and restoration strategies.
Sand ramps have been (and still are) neglected in geomorphological research. Only recently any awareness of their potential of being a major source of palaeoenvironmental information, thanks to their multi-process character, has been developed. In Namibia, sand ramps were terra incognita. This study defines, classifies and systematizes sand ramps, investigates the formative processes and examines their palaeoenvironmental significance. The study region is located between the coastal Namib desert and the Great Escarpment, between the Tiras Mountains to the north and the Aus area to the south. Two lines of work were followed: geomorphological and sedimentological investigations in the field, assisted by interpretation of satellite images, aerial photographs and topographic maps, and palaeopedological and sedimentological analytical work in the laboratory. Two generations of sand ramps could be identified. The older generation, represented by a single sand ramp within the study region, is characterized by the presence of old basal sediments. The bulk of the sand ramps is assigned to the young generation, which is divided into three morpho-types: in windward positions voluminous ramps are found, in leeward positions low-volume ramps exist, either of very high or very low slope angle. The most distinct characteristic of sand ramp sediments is their formation by interacting aeolian deposition and fluvial slope wash. The last period of deposition, which shaped all the entire young sand ramps, but also the upper part of the old ramp, is suggested to have occurred after c. 40 ka BP, implying a highly dynamic climatic system during that time, with seasonal aridity and low-frequency, but high-intensity rainfall. A phase of environmental stability followed, most likely around 25 ka BP, supporting growth of vegetation, stabilization and consolidation of the sediments as well as soil formation. Subsequently, the profile was truncated and a desert pavement formed, under climatic conditions comparable to those of the present semi-desert. The ramps were then largely cut off from the bedrock slopes, implying a change towards higher ecosystem variability. As the final major process, recent and modern aeolian sands accumulated on the upper ramp slopes. A luminescence date for the recent sand places their deposition at about 16 ka BP, close to the Last Glacial Maximum. Regarding the source of the sands, a local origin is proposed. For the sand ramp of the old generation the "basic cycle" of initial deposition, stabilization and denudation occurred twelve times, including a phase of calcrete and/or root-cast formation in each of them, adding up to around 60 changes in morphodynamics altogether. At least nine of these cycles took place between 105 ka BP and the LGM, indicating that the general cooling trend during the Late Pleistocene was subject to a high number of oscillations of the environmental conditions not identified before for southern Namibia. Due to the high resolution obtained by the study of sand ramp sediments, but also due to the very special situation of the study area in a desert margin, 100 km from the South Atlantic and in the transition zone between summer and winter rainfall, correlation with stratigraphies (of mostly lower resolution) established for different regions in southern Africa did not appear promising. In conclusion, sand ramps generally serve as a valuable tool for detailed deciphering of past morphodynamics and thereby palaeoenvironmental conditions. For south-west Namibia, sand ramps shed some more light on the Late Quaternary landscape evolution.
The Skeleton Coast forms part of the Atlantic coastline of NW Namibia comprising several ephemeral rivers, which flow west-southwest towards the Atlantic Ocean. The area is hyper-arid with less than 50 mm average annual rainfall and a rainfall variability of 72%. Therefore, the major catchment areas of the rivers are about 100-200 km further inland in regions with relatively high annual rainfall of 300-600 mm. The coastal plain in the river downstream areas is characterized by a prominent NNW trending, 165 km long belt of 20-50 m high, locally compound, barchanoid and transverse dunes. This dune belt, termed Skeleton Coast Erg, starts abruptly with a series of barchans and large compound dunes 15 km north of the Koigab River and extends from 2-5 km inland sub-parallel to the South Atlantic margin of NW Namibia over a width of 3-20 km. As the SSE-NNW trending dune belt is oriented perpendicular to river flow, the dunefield dams and interacts with the west-southwestward flowing ephemeral river systems. This study focused on three main topics: 1) investigation and classification of the Koigab Fan, 2) the investigation of the Cenozoic succession in the Uniabmond area and 3) comparative studies of fluvio-aeolian interaction between five ephemeral rivers and the Skeleton Coast Erg. Sedimentological and geomorphological investigations show that the Koigab Fan represents a yet undocumented type of a braided fluvial fan system, which operates in an arid climatic, tropical latitude setting, is dominated by ephemeral mixed gravel/sand braided rivers, lacks significant vegetation on the fan surface, has been relatively little affected by human activity, is a perfect study site for recording various types of fluvio-aeolian interaction and thereby acts additionally as a model for certain Precambrian and Early Palaeozoic fan depositional systems deposited prior to the evolution of land plants. The Cenozoic succession in the Uniabmond area consists of three major unconformity-bounded units, which have been subdivided into the Red Canyon, the Whitecliff, and the Uniabmond Formation. The Tertiary Red Canyon Fm. is characterized by continental reddish sediments documenting an alluvial fan and braided river to floodplain depositional environment. The Whitecliff Fm. displays a wide variety of continental and marine facies. This formation provides the possibility to examine fluvio-aeolian interactions and spectacular, steep onlap relationships towards older sediments preserved in ancient seacliffs. The Whitecliff Fm. has been subdivided into four sedimentary cycles, which resulted from sea level changes during the Plio- to Middle Pleistocene. The following Uniabmond Fm. provides a unique insight into the depositional history of the NW Namibian coast during the Last Pleistocene glacial cycle. The formation has been subdivided into four units, which are separated by unconformities controlled by sea level changes. Unit 1 represents deposits of an Eemian palaeo-beach. The overlying Units 2-4 build up the sedimentary body of the Uniab Fan, again a braided river dominated fan, which is nowadays degraded and characterized by deeply incised valleys, deflation surfaces and aeolian landforms. The Uniabmond Fm. is overlain by the dunes of the Skeleton Coast Erg, whose development is related to the Last Glacial Maximum (LGM). The damming of river flow by aeolian landforms has been previously recognized as one of several principal types of fluvio-aeolian interaction. Five ephemeral rivers (from S to N: Koigab, Uniab, Hunkab, Hoanib, Hoarusib), which variously interact with the Skeleton Coast Erg, were chosen for the purpose of this study to consider the variability of parameters within these fluvio-aeolian systems and the resulting differences in the effectiveness of aeolian damming. The fluvio-aeolian interactions between the rivers and the dune field are controlled by the climate characteristics and the geology of the river catchment areas, the sediment load of the rivers, their depositional architecture, the longitudinal river profiles as well as the anatomy of the Skeleton Coast Erg. Resulting processes are 1) aeolian winnowing of fluvially derived sediments and sediment transfer into and deposition in the erg; 2) dune erosion during break-through resulting in hyperconcentrated flow and intra-erg mass flow deposits; 3) the development of extensive flood-reservoir basins caused by dune damming of the rivers during flood; 4) interdune flooding causing stacked mud-pond sequences; and 5) the termination of the erg by more frequent river floods.
Summary Myelin protein zero (P0) is a key myelin component in maintaining the integrity and functionality of the peripheral nervous system. Mutated variants are the cause for several disabilitating peripheral neuropathies such as Charcot-Marie-Tooth disease or Dejerine –Sotas syndrome. Using P0 knockout mice - a mouse model for these diseases - together with their wt counterparts on C57BL/6 background we studied the shaping of the T-cell repertoire specific for P0 in the presence and in the absence of this protein during the ontogeny of T-cells. Our approach was to use a series of overlapping 20-mer peptides covering the entire amino acid sequence of P0. This series of P0 peptides was employed for epitope mapping of the H2-Ab restricted T cell response. Thus, P0 peptide 5 (P0 41-60) in the extracellular domain of P0 was identified as the main immunogenic peptide. The immunogenic peptide containing the core immunodominant determinant in the P0 sequence was employed in studies of tolerance, revealing a highly reactive P0 specific T-cell repertoire in P0 ko mice while in wt mice the high avidity repertoire was inactivated in order to ensure self tolerance. In wild type and heterozygous P0 mice tolerance is not dependent on gene dosage. P0 is a tissue specific antigen whose expression is limited to myelinating Schwann cells. The classical view on tolerance to tissue specific antigens attributed this role to peripheral mechanisms. Driven by the finding that intrathymic expression of tissue-specific antigens is a common occurrence, we confirmed that “promiscuous” expression on thymic stroma holds true also for myelin P0. In addition, using bone marrow chimeras we investigated the capacity of bone marrow derived cells versus nonhematopoietic cells to induce tolerance towards P0. Our findings show that bone marrow derived cells although tolerogenic to some degree are not sufficient to mediate complete tolerance. P0 expression on cells with origin other than bone marrow showed to be sufficient and necessary to induce sound tolerance. We identified one cryptic (P0 peptide 8) and two subdominant epitopes (P0 petides 1, and 3). P0 peptide 8 was reactive in both wt and P0 ko mice. Peptides 1 and 3 were immunogenic in P0 ko but not in wt mice. Several P0 peptides including the immunogenic peptide 5 were involved in direct and adoptive transfer EAN studies. None of them induced clinical signs of EAN. Immunization with P0 peptide 3 did induce inflammation of the peripheral nerves reflected by the infiltration of macrophages and CD3 positive cells. More studies involving highly P0 specific T-cell lines are needed to characterize the P0 induced EAN. Our findings may have direct implications for secondary autoimmunity and inflammation in peripheral nerves developing after correcting the P0 genetic defect by gene therapy in aforementioned diseases.
Best disease (OMIM 153700) is an early-onset, autosomal dominant maculopathy characterized by egg yolk-like lesions in the central retina. The disease gene, the vitelliform macular dystrophy gene type 2 (VMD2), encodes a 585-aa VMD2 transmembrane protein, termed bestrophin. The protein is predominantly expressed on the basolateral side of the retinal pigment epithelium (RPE) and is thought to be involved in the transport of chloride ions. Bestrophin as well as three closely related VMD2-like proteins (VMD2L1-L3) contain multiple putative transmembrane (TM) domains and an invariant tripeptide (RFP) motif in the N-terminal half of the protein. This and the tissue-restricted expression to polarized epithelial cells are typical features of the VMD2 RFP-TM family. Best disease is predominantly caused by missense mutations, clustering in four distinct „hotspots“ in the evolutionary highly conserved N-terminal region of the protein. To further augment the spectrum of mutations and to gain novel insights into the underlying molecular mechanisms, we screened VMD2 in a large cohort of affected patients. In total, nine novel VMD2 mutations were identified, raising the total number of known Best disease-related mutations from 83 to 92. Eight out of nine novel mutations are hotspot-specific missense mutations, underscoring their functional/structural significance and corroborating the dominant-negative nature of the mutations. Of special interest is a one-basepair deletion (Pro260fsX288) encoding a truncated protein with a deletion of an important functional domain (TM domain four) as well as the entire C-terminal half of bestrophin. For the first time, a nonsense mutation leading to a 50 % non-functional protein has been identified suggesting that on rare occassions Best disease may be caused by haploinsufficiency. Molecular diagnostics strongly requires a reliable classification of VMD2 sequence changes into pathogenic and non-pathogenic types. Since the molecular pathomechanism is unclear at present, the pathogenicity of novel sequence changes of VMD2 are currently assessed in light of known mutations. We therefore initiated a publicly accessible VMD2 mutation database (http://www.uni-wuerzburg.de/humangenetics/vmd2.html) and are collecting and administrating the growing number of mutations, rare sequence variants and common polymorphisms. Missense mutations may disrupt the function of proteins in numerous ways. To evaluate the functional consequences of VMD2 mutations in respect to intracellular mislocalization and/or protein elimination, a set of molecular tools were generated. These included the establishment of an in vitro COS7 heterologous expression assay, the generation of numerous VMD2 mutations by site-directed mutagenesis as well as the development of bestrophin-specific antibodies. Surprisingly, membrane fractionation/Western blot experiments revealed no significant quantitative differences between intact and mutant bestrophin. Irrelevant of the type or location of mutation, incorporation of mutant bestrophin to the membraneous fraction was observed. Thus, impaired membrane integration may be ruled out as causative pathomechanism of Best disease consistent with a dominant-negative effect of the mutations. In a different approach, efforts were directed towards identifying and characterizing the VMD2 RFP-TM protein family in mouse. While clarification of the genomic organization of murine Vmd2 was required as basis to generate Vmd2-targeted animals (see below), the study of closely related proteins (Vmd2L1, Vmd2L2 and Vmd2L3) may provide further clues as to the function of bestrophin. For this, biocomputational as well as RT PCR analyses were performed. Moreover, the novel genes were analyzed by real time quantitative RT PCR, displaying predominant expression in testis, colon and skeletal muscle of Vmd2, Vmd2L1 and Vmd2L3 transcripts, respectively as well as in eye tissue. Interestingly, neither an ORF was determined for murine Vmd2L2 nor was the transcript present in a panel of 12 mouse tissues, suggesting that murine Vmd2L2 may represent a functionally inactive pseudogene. The murine Vmd2L3 gene, as its human counterpart, is a highly differentially spliced transcript. Finally, generating mouse models of Best disease will provide essential tools to investigate the pathophysiology of bestrophin in vivo. We have initiated the generation of two different mouse lineages, one deficient of Vmd2 (knock-out) and the other carrying a human disease-related mutation (Tyr227Asn) in the orthologous murine gene (knock-in). Genetic engineering of both constructs has been achieved and presently, four ES clones harboring the homologous recombination event (Vmd2+/-) have been isolated and are ready for the subsequent steps to generate chimeric animals. The resulting mouse lineages will represent two key models to elucidate the functional role of bestrophin in Best disease, in RPE development and physiology.
A distinguishing feature of eukaryotic cells is the spatial separation of the site of mRNA synthesis (nucleus) from the site of mRNA function (cytoplasm) by the nuclear envelope. As a consequence, mRNAs need to be actively exported from the nucleus to the cytoplasm. At the time when this study was initiated, both human TAP and yeast Mex67p had been proposed to play a role in this process. Work presented in this thesis (section 2.1) revealed that TAP and Mex67p belong to an evolutionarily conserved family of proteins which are characterized by a conserved modular domain organization. This family was termed nuclear export factor (NXF) family. While the yeast genome encodes only one NXF protein (Mex67p), the genomes of higher eukaryotes encode several NXF proteins. There are two nxf genes in C. elegans and A. gambiae, four in D. melanogaster, and at least four in H. sapiens and M. musculus. It was unclear whether, apart from TAP and Mex67p, other members of this family would also be involved in mRNA export. In the first part of this thesis (2.1), several human NXF members were tested for a possible function in nuclear mRNA export. They were analyzed for their interaction with RNA, nucleoporins and other known TAP partners in vitro, and tested for their ability to promote nuclear export of a reporter mRNA in vivo. Using these assays, human NXF2, NXF3 and NXF5 were all shown to interact with the known NXF partner p15. NXF2 and NXF5 were also found to bind directly to RNA, but only NXF2 was able to bind directly to nucleoporins and to promote the nuclear export of an (untethered) reporter mRNA. Thus NXF2 possesses many and NXF3 and NXF5 possess some of the features required to serve as an export receptor for cellular mRNAs. As NXF2 and NXF3 transcripts were mainly found in testis, and the closest orthologue of NXF5 in mouse has the highest levels of expression in brain, these NXF members could potentially serve as tissue-specific mRNA export receptors. In the second part of this work (2.2), the role of different Drosophila NXF proteins and other export factors in mRNA export was investigated using double-stranded RNA interference (RNAi) in Drosophila Schneider cells. Three of the four predicted Drosophila NXF members (NXF1-3) were found to be expressed in this cell line and could be targeted by RNAi. Depletion of endogenous NXF1 inhibited growth and resulted in the nuclear accumulation of polyadenylated RNA. Fluorescence in situ hybridization revealed that export of both heat shock and non-heat shock mRNAs, including intron-containing and intronless mRNAs, was inhibited. Depleting endogenous NXF2 or NXF3 had no apparent phenotype. These results suggested that NXF1 (but not NXF2-NXF4) mediates the export of bulk mRNA in Drosophila cells. We and others have shown that human NXF proteins function as heterodimers bound to the small protein p15. Accordingly, silencing of Drosophila p15 resulted in a block of mRNA export which was indistinguishable from the export inhibition seen after targeting NXF1. These observations indicated that neither NXF1 nor p15 can promote export in the absence of the other subunit of the heterodimer. NXF1:p15 heterodimers are implicated in late steps of mRNA export, i.e. in the translocation of mRNP export cargoes across the nuclear pore complex. The mechanism by which NXF1:p15 dimers are recruited to the mRNA is unclear. A protein that is thought to play a role in this process is the putative RNA helicase UAP56. Similar to NXF1 and p15, UAP56 was shown to be essential for mRNA export in Drosophila. UAP56 is recruited cotranscriptionally to nascent transcripts and was suggested to facilitate the interaction of NXF1:p15 with mRNPs. Even though both NXF1:p15 heterodimers and UAP56 had been implicated in general mRNA export, it was unclear whether there are classes of mRNAs that require NXF1:p15, but not UAP56 or vice versa. It was also unclear what fraction of cellular mRNAs is exported by NXF1:p15 dimers and UAP56, and whether mRNAs exist that reach the cytoplasm through alternative routes, i.e. by recruiting other export receptors. To address these issues we performed a genome-wide analysis of nuclear mRNA export pathways using microarray technology (2.2.2). We analyzed the relative abundance of nearly half of the Drosophila transcriptome in the cytoplasm of Drosophila Schneider cells depleted of different export factors by RNAi. We showed that the vast majority of transcripts were underrepresented in the cytoplasm of cells depleted of NXF1, p15 or UAP56 as compared to control cells. Only a small number of mRNAs were apparently not affected by the depletions. These observations, together with the wide and similar effects on mRNA levels caused by the depletion of NXF1, p15 or UAP56, indicate that these proteins define the major mRNA export pathway in these cells. We also identified a small subset of mRNAs which appeared to be exported by NXF1:p15 dimers independently of UAP56. In contrast, no significant changes in mRNA expression profiles were observed in cells depleted of NXF2 or NXF3, suggesting that neither NXF2 nor NXF3 play an essential role in mRNA export in Drosophila Schneider cells. Crm1 is a transport receptor implicated in the export of a variety of non-mRNA and protein cargoes. In addition, human Crm1 has been suggested to be involved in the export of a specific mRNA species, serving as a "specialized" mRNA export receptor. A role of human Crm1 in the export of bulk mRNA is considered unlikely. We analyzed the role of Drosophila Crm1 in mRNA export by inhibiting Crm1 with the drug leptomycin B in Schneider cells. Subsequent microarray analysis demonstrated that the inactivation of Crm1 resulted in decreased cytoplasmic levels of less than 1% of all mRNAs, indicating that Crm1 is indeed not a major mRNA export receptor. The genome-wide analysis also revealed a feedback loop by which a block to mRNA export triggers the upregulation of genes involved in this process. This thesis also includes two sections describing projects in which I participated during my Ph.D., but which were not the main focus of this thesis. In section 2.3, the role of the different TAP/NXF1 domains in nuclear mRNA export is discussed. Section 2.4 describes results that were obtained as part of a collaboration using the RNAi technique in Schneider cells to study the function of Cdc37.
The extracellular matrix within connective tissues represents a structural scaffold as well as a barrier for motile cells, such as invading tumor cells or passenger leukocytes. It remains unclear how different cell types utilize matrix-degrading enzymes for proteolytic migration strategies and, on the other hand, non-proteolytic strategies to overcome 3D fibrillar matrix networks. To monitor cell migration, a 3D collagen model in vitro or the mouse dermis in vivo were used, in combination with time-lapse video-, confocal- or intravital multiphoton-microscopy, and computer-assisted cell tracking. Expression of proteases, including several MMPs, ADAMs, serine proteases and cathepsins, was shown by flow cytometry, Western blot, zymography, and RT-PCR. Protease activity by migrating HT-1080 fibrosarcoma cells resulting in collagenolysis in situ and generation of tube-like matrix defects was detected by three newly developed techniques:(i) quantitative FITC-release from FITC-labelled collagen, (ii) structural alteration of the pyhsical matrix structure (macroscopically and microscopically), and (iii) the visualization of focal in situ cleavage of individual collagen fibers. The results show that highly invasive ollagenolytic cells utilized a spindle-shaped "mesenchymal" migration strategy, which involved beta1 integrindependent interaction with fibers, coclustering of beta1 integrins and matrix metalloproteinases (MMPs) at fiber bundling sites, and the proteolytic generation of a tube-like matrix-defect by MMPs and additional proteases. In contrast to tumor cells, activated T cells migrated through the collagen fiber network by flexible "amoeboid" crawling including a roundish, elliptoid shape and morphological adaptation along collagen fibers, which was independent of collagenase function and fiber degradation. Abrogation of collagenolysis in tumor cells was achieved by a cocktail of broad-spectrum protease inhibitors at non-toxic conditions blocking collagenolysis by up to 95%. While in T cells protease inhibition induced neither morphodynamic changes nor reduced migration rates, in tumor cells a time-dependent conversion was obtained from proteolytic mesenchymal to non-proteolytic amoeboid migration in collagen lattices in vitro as well as the mouse dermis in vivo monitored by intravital microscopy. Tumor cells vigorously squeezed through matrix gaps and formed constriction rings in regions of narrow space, while the matrix structure remained intact. MMPs were excluded from fiber binding sites and beta1 integrin distribution was non-clustered linear. Besides for fibrosarcoma cells, this mesenchymal-toameboid transition (MAT) was confirmed for epithelial MDA-MB-231 breast carcinoma cells. In conclusion, cells of different origin exhibit significant diversity as well as plasticity of protease function in migration. In tumor cells, MAT could respresent a functionally important cellular and molecular escape pathway in tumor invasion and migration.
Summary Background: In a previous study, nitrate reductase (NR, EC 1.6.6.1) from leaves of Ricinus communis L. showed different regulatory properties from most other higher plants NR's by an unusually strong Mg2+-sensitivity, a different pH-activity profile and only little ATP-dependent inactivation. The aim of this work was to elucidate the deviating properties of Ricinus NR in more details, from both molecular and physiological aspects. For that purpose, the NR gene from R. communis was cloned, expressed heterologously and characterized. Results: The deduced protein sequence showed that Ricinus NR shared high similarity with other NRs, apart from the N-terminal region. In the N-terminal region, the Ricinus NR possesses an acidic stretch which is conserved only in higher plants. Within the Moco-binding domain the Ricinus NR contained few amino acid residues which were unique in comparison with 17 plant NRs, including His103, Gln123, Val266 and Ala284 where other NRs possess asparagine, arginine, aspartate and praline. In the Dimer interface and Hinge 1 regions, the Ricinus NR also had some unique residues like Asn460 and Ala498 where other NRs have isoleucine and glycine instead. The Ricinus NR possesses an Arg482 which provides an additional predicted Trypsin cleavage site within 481KRHK484 (while most of plant-NRs possess KPHK). Additionally, the Ricinus NR contains a serine phosphorylation site (Ser-526) within the potential 14-3-3 binding motif 523KSVS*TP528, which is a common characteristic of nitrate reductases. In the C-Terminus of Ricinus NR a sequence 886CGPPP890 confirmed that Ricinus NR is a NADH-specific enzyme. Functional Ricinus NR protein was expressed in Pichia pastoris and compared with the features of Arabidopsis NR2 synthesized by the same expression system (AtNR2). The recombinant Ricinus NR (RcNR) itself was unresponsive to the incubation with MgATP, and so was AtNR2. As yeast extracts might lack factors required for NR regulation, desalted leaf extracts containing NR kinases and 14-3-3s were prepared from 4-day darkened (and therefore NR-free) leaves of Arabidopsis (ADL), spinach (SDL) and Ricinus (RDL), and added to the assay of RcNR and AtNR2 to check for ATP-dependent inactivation and Mg2+-sensitivity. When RcNR was combined with the NR-free extracts described above, it's unusually high Mg2+-sensitivity was restored only by incubation with RDL, but it remained unresponsive to ATP. In contrast, AtNR2 became inactive when incubated with the protein mixtures and ATP. It is obvious that one or some factors existing in RDL could interact with RcNR and therefore provide its high Mg2+-sensitivity. Interestingly, incubation of AtNR2 with different NR-free leaf extracts gave a significant activation of the enzyme activities, both in Mg2+ and EDTA, which were not observed in the case of RcNR. Moreover, using ammonium sulfate to fractionation the RDL revealed that about 0.2 mg of the protein factor(s) from 0-35% of ammonium sulfate precipitation was sufficient to provide the maximum inhibition of the RcNR. Conclusions: The insensitivity to ATP appears an inherent property of Ricinus NR, whereas the high Mg2+-sensitivity depends on one or several factors in Ricinus leaves. This as yet unknown factor(s) was boiling-sensitive and could be precipitated by ammonium sulfate. It appeared to interact specifically with recombinant Ricinus-NR to provide the Mg2+-sensitivity of the authentic leaf enzyme. Presumably, there is also a positive regulatory factor(s) for nitrate reductase existing in the leaves of higher plants.
Plants have evolved an elaborate system to cope with a variety of biotic and abiotic stresses. Typically, under stress conditions an appropriate defense response is invoked which is accompanied by changes in the metabolic status of the plant. Photosynthesis is downregulated and sucrose is imported into the tissue, which provides a faster and more constant flux of energy and carbon skeletons to perform the defense response. Interestingly, these processes are co-ordinately regulated and the signal transduction chains underlying these cellular programs appear to share at least some common elements. Both the induction of sink metabolism and defense response is dependent on signal transduction pathways involving protein phosphorylation. Furthermore, regulation of extracellular invertase (INV) and phenylalanine ammonia lyase (PAL) which are markers for sink metabolism and defense response is preceded by the transient activation of MAP kinases. In depth analysis of MAP kinase activation by partial purification led to the discovery that, depending on the stimulus, different subsets of MAP kinases are activated. This differential MAPK activation is likely to possess a signal encoding function. In addition, the partial purification of MAP kinases was found to be suitable to address specific cellular functions to individual MAP kinase isoenzymes. By this way, LpWIPK was identified as the major MAP kinase activity induced after stimulation of tomato cells with different elicitors. LpWIPK is thus considered as a key regulator of defense response together with sink induction in tomato. A study using nonmetabolisable sucrose analogs revealed that the regulation of photosynthesis is not directly coupled to this signal transduction pathway since it is independent of MAP kinase activation. Nonetheless, downregulation is induced by the same stimuli that induce the defense response and sink metabolism and it will therefore be interesting to uncover the branch points of this signalling network in the future. MAP kinases are not only central components regulating the response to biotic stresses. In addition to e.g. pathogens, MAP kinases are as well involved in signal transduction events invoked by abiotic stresses like cold and drought. In a recent study, we could show that a MAP kinase is activated by heat stress, under conditions a plant will encounter in nature. This previously unknown MAP kinase is able to specifically recognise the heat stress transcription factor HsfA3 as a substrate, which supports a role of this MAP kinase in the regulation of the heat stress response. Moreover, the observation that HsfA3 is phosphorylated by the heat activated MAP kinase in vitro provides a promising basis to identify HsfA3 as the first physiological substrate of a plant MAP kinase. Intracellular protons have been implicated in the signal transduction of defense related signals. In a study using Chenopodium rubrum cells, we could show that cytosolic changes in pH values do not precede the regulation of the marker genes INV and PAL. Depending on the stimulus applied, cytosolic acidification or alkalinisation can be observed, which excludes a role for protons as signals in this pathway. Together with the concomitant changes of the pH value of the extracellular space, these variations can thus be considered as terminal part of the defense response itself rather than as a second messenger. WRKY transcription factors have only recently been identified as indirect targets of a central plant MAP kinase cascade. In addition, the identification of cognate binding sites in the promoters of INV and PAL supports a role for these proteins in the co-ordinate regulation of defense response and sink induction. A novel elicitor responsive WRKY transcription factor, LpWRKY1, was cloned from tomato and characterised with respect to its posttranslational modification. This immediate early transcription factor is transiently induced upon pathogen attack and the induction is dependent on phosphorylation. Furthermore, it was shown for the first time with respect to WRKY transcription factors, that LpWRKY1 is phosphorylated in vivo. Analysis of the role of this phosphorylation by in gel assays using recombinant WRKY protein as the substrate revealed two protein kinases that are transiently activated during the defense response to phosphorylate LpWRKY1. This data demonstrates that WRKY proteins require phosphorylation to modulate their DNA binding or transactivating activity.
Two isoforms of human CD23 (CD23a and CD23b) have been described. They differ by only 6-7 residues in the N-terminal cytoplasmic tail. CD23a is restrictively expressed on B-cells while CD23b is inducible on B-cells, as well as monocytes, eosinophils, macrophages and a variety of other cell types, after IL-4 stimulation. The two isoforms seems to have different functions. CD23a appears to be the isoform associated with endocytosis of IgE immune complexes and mediating antigen presentation on B-cells. CD23b has a phagocytosis motif and seems to be involved in the phagocytosis of IgE-coated particles, cytokine release and the generation of superoxides. Previous studies indicate that the two isoforms connect to different signal transduction pathways. Comparing the cells that express only one or both CD23 isoforms suggests that CD23b is involved in upregulating cAMP and iNOS, whereas CD23a mediates an increase in intracellular calcium. In the main part of the study we investigated how the CD23a B-cell specific expression is regulated. Pax-5 is a B-cell restricted transcription factor with an essential role in early and late B-cell development. Putative Pax-5 binding sites have been predicted in the CD23a proximal promoter. Analyses of the CD23a promoter revealed three putative Pax-5 binding sites with more than 50% homology to the consensus sequence. One of these sites, named CD23-1 can compete a high affinity Pax-5 binding site or can directly bind Pax-5 protein in electrophoretic mobility shift assays. Introducing mutations into this site abrogates the binding. A different approach, in which overlapping peptides covering the length of the CD23a promoter were tested in competition assays against a high affinity binding site, also revealed CD23-1 as the only site that directly binds Pax-5 protein. Expression of Pax-5 in 293 cells resulted in a 7-fold activation of a CD23a core promoter construct. Co-transfection together with STAT6 showed that Pax-5 cooperates with this transcription factor in enhancing the level of transcription of a CD23a extended promoter construct. Most importantly, ectopic expression of Pax-5 in the monocytic cell line U-937 that regularly expresses only the CD23b isoform enabled a significant CD23a expression after stimulation with IL-4 and PMA. Our results suggest that Pax-5 is a key regulator of the B-cell restricted expression of the CD23a isoform. In the second part of the project, we used a yeast two-hybrid system (CytoTrapTM from Stratagene) in order to look for cytoplasmic interaction partners for the CD23 receptor. The system was established in order to reach a high efficiency of transformation and different bait vector constructs were made. The screening was performed using a human spleen library cloned in the target vector of the system. The first bait constructs used (pSosCD23a and pSosCD23b) expressed the very short (22 amino acids) cytoplasmic tails of the isoforms at the C-terminal end of the fusion protein (human SOS). Improved bait constructs, (pSosCD23a+Linker and pSos CD23b+Linker) expressed the cytoplasmic tail of CD23a/b at the N-terminal side of the human SOS and had in consequence the N-terminal part free as a bait, as it occurs in vivo. A flexible linker region separated the fusion proteins in order to make the small amino acid bait chain more obvious. Approximately three million library clones were screened with these various constructs. No “true positive” interaction was detected. A relatively high number of “false positive” clones were obtained and checked in another two-hybrid system. A new bait construct, in which the tyrosine residue in the cytoplasmic tail of CD23a was replaced by a glutamic acid residue will be used for future screening. The system was also used in order to test the interaction between CD23 and p59fyn, a member of the Src family of protein kinases that was mentioned to associate with CD23a. No interaction was detected by using the CytoTrap two-hybrid system. In conclusion, the key result of the study demonstrates that Pax-5 is a main regulator of the B-cell specific expression of the CD23a isoform. In addition, a two-hybrid system was established and employed in order to look for cytoplasmic interaction partners for CD23.