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Soluble guanylyl cyclase (sGC) is the best established receptor for nitric oxide (NO) and regulates a great number of important physiological functions. Surprisingly, despite the wellappreciated roles of this enzyme in regulation of vascular tone, smooth muscle cell proliferation, platelet aggregation, renal sodium secretion, synaptic plasticity, and other functions, extremely little is known about the regulation of sGC activity and protein levels. To date, the only well-proven physiologically relevant sGC regulator is NO. In the present study, some additional possibilities for sGC regulation were shown. Firstly, we evaluated the ability of different NO donors to stimulate sGC. Significant differences in the sGC stimulation by SNP and DEA/NO were found. DEA/NO stimulated sGC much stronger than did SNP. Interestingly, no correlation between the sGC protein and maximal activity distribution was found in rat brain regions tested, suggesting the existence of some additional regulatory mechanisms for sGC. The failure of SNP to stimulate sGC maximally might be one of the reasons why the lack of correlation between the distribution of sGC activity and proteins in brain was not detected earlier. Prolonged exposure of endothelial cells to NO donors produced desensitization of the cGMP response. This desensitization cannot be explained by increased PDE activity, since PDE inhibitors were not able to prevent the NO donor-induced decrease of the maximal cGMP response in endothelial cells. The failure of SH-reducing agents to improve the cGMP response after its desensitization by NO suggests that a SH-independent mechanism mediates NO effects. Demonstration that the potency of the recently described activator of oxidized (heme-free) sGC, BAY58-2667, to stimulate sGC increases after prolonged exposure of the cells to an NO donor, DETA/NO, suggests that oxidation of heme may be a reason for NOinduced desensitization of sGC and decrease in sGC protein level. Indeed, the well-known heme-oxidizing agent ODQ produces a dramatic decrease in sGC protein levels in endothelial cells and BAY58-2667 prevents this effect. Although the mechanism of sGC activation and stabilization by BAY58-2667 is unknown, this substance is an interesting candidate to modulate sGC under conditions where sGC heme iron is oxidized. Very little is known about regulation of sGC by intracellular localization or translocation between different intracellular compartments. In the present study, an increase in sGC sensitivity to NO under membrane association was demonstrated. Treatment of isolated lung with VEGF markedly increased sGC in membrane fractions of endothelial cells. Failure of VEGF to stimulate sGC membrane association in cultured endothelial cells allows us to propose a complex mechanism of regulation of sGC membrane association and/or a transient character of sGC membrane attachment. A very likely mechanism for the attachment of sGC to membranes is via sGCinteracting proteins. These proteins may participate also in other aspects of sGC regulation. The role of the recently described sGC interaction partner, Hsp90, was investigated. Shortterm treatment of endothelial cells with an Hsp90 inhibitor does not affect NO donor or calcium ionophore-stimulated cGMP accumulation in the cells. However, inhibition of Hsp90 results in a rapid and dramatic decrease in sGC protein levels in endothelial cells. These effects were unrelated to changes in sGC transcription, since inhibition of transcription had much slower effect on sGC protein levels. In contrast, inhibitors of proteasomes abolished the reduction in sGC protein levels produced by an Hsp90 inhibitor, suggesting involvement of proteolytic degradation of sGC proteins during inhibition of Hsp90. All these data together suggest that Hsp90 is required to maintain mature sGC proteins. In conclusion, in the present study it was demonstrated that multiple mechanisms are involved in the regulation of sGC activity and its sensitivity to NO. Oxidation of sGC heme by NO seems to be one of the mechanisms for negative regulation of sGC in the presence of high or prolonged stimulation with NO. Another possible means of regulating sGC sensitivity to NO is via the intracellular translocation of the enzyme. It has been also demonstrated here that attachment of sGC to the membrane fraction results in an apparent increase in the enzyme sensitivity to NO. Additionally, Hsp90 was required to maintain sGC protein in endothelial and other cell types. However, we could not find any acute affect of Hsp90 on sGC activity, as reported recently. All these findings demonstrate that the regulation of sGC activity and protein level is a much more complex process than had been assumed earlier.
There is substantial interest in the identification of genes underlying susceptibility to complex human diseases because of the potential utility of such genes in disease prediction and therapy. The complex age-related macular degeneration (AMD) is a prevalent cause of legal blindness in industrialized countries and predominantly affects the elderly population over 75 years of age. Although vision loss in AMD results from photoreceptor cell death in the central retina, the initial pathogenesis likely involves processes in the retinal pigment epithelium (RPE) (Liang and Godley, 2003). The goal of the current study was to identify and characterize genes specifically or abundantly expressed in the RPE in order to determine more comprehensively the transcriptome of the RPE. In addition, our aim was to assess the role of these genes in AMD pathogenesis. Towards this end, a bovine cDNA library enriched for RPE transcripts was constructed in-house using a PCR-based suppression subtractive hybridization (SSH) technique (Diatchenko et al., 1996, 1999), which normalizes for sequence abundance and achieves high enrichment for differentially expressed genes. CAP3 (Huang and Madan, 1999) was used to assemble the high quality sequences of all the 2379 ESTs into clusters or singletons. 1.2% of the 2379 RPE-ESTs contains vector sequences and was excluded from further analysis. 5% of the RPE-ESTs showed homology to multipe chromosomes and were not included in further assembly process. The rest of the ESTs (2245) were assembled into 175 contigs and 509 singletons, which revealed approximately 684 unique genes in the dataset. Out of the 684, 343 bovine RPE transcripts did not align to their human orthologues. A large fraction of clones were shown to include a considerable 3´untranslated regions of the gene that are not conserved between bovine and human. It is the coding regions that can be conserved between bovine and human and not the 3’ UTR (Sharma et al., 2002). Therefore, more sequencing from the cDNA library with reclustering of those 343 ESTs together with continuous blasting might reveal their human orthologoues. To handle the large volume of data that the RPE cDNA library project has generated a highly efficient and user-friendly RDBMS was designed. Using RDBMS data storage can be managed efficiently and flexibly. The RDBMS allows displaying the results in query-based form and report format with additional annotations, links and search functions. Out of the 341 known and predicted genes identified in this study, 2 were further analyzed. The RPE or/and retina specificity of these two clones were further confirmed by RT-PCR analysis in adult human tissues. Construction of a single nucleotide polymphism (SNP) map was initiated as a first step in future case/control association studies. SNP genotyping was carried out for one of these two clones (RPE01-D2, now known as RDH12). 12 SNPs were identified from direct sequencing of the 23.4-kb region, of which 5 are of high frequency. In a next step, comparison of allele frequencies between AMD patients and healthy controls is required. Completion of the expression analysis for other predicted genes identified during this study is in progress using real time RT-PCR and will provide additional candidate genes for further analyses. This study is expected to contribute to our understanding of the genetic basis of RPE function and to clarify the role of the RPE-expressed genes in the predisposition to AMD. It may also help reveal the mechanisms and pathways that are involved in the development of AMD or other retinal dystrophies.
The study deals with the area of the allosteric modulation of the muscarinic M2 receptors. The allosteric modulators have an influence on binding of orthosteric ligands (agonists and antagonists) to the classical orthosteric binding site of the muscarinic M2-receptors. The modulators are able to enhance (positive cooperativity) or decrease (negative cooperativity)the affinity of ligands to the orthosteric binding site. The allosteric binding site is located at the entrance of the receptor binding pocket. It is less conserved than the orthosteric binding site which is located in a narrow cavity created by the seven transmembrane domains. Consequently, development of subtype selective allosteric ligands is easier than subtypeselective muscarinic agonists or antagonists. Furthermore, subtype selectivity can be achieved by differently cooperative interactions between the allosteric and orthosteric ligand at different receptor subtypes. For example, the allosteric modulators that are positively cooperative with ACh at M1 receptors and neutrally cooperative at the other receptor subtypes could be beneficial for treatment of the Alzheimer’s disease. Bisquaternary analogues of the Strychnos alkaloid caracurine V are among the most potent allosteric modulators of muscarinic M2-receptors. The very rigid ring skeleton comprises the pharmacophoric elements of two positively charged nitrogens at an approximate distance of 10 surrounded by two aromatic ring systems in a distinct spatial arrangement. Owing to the close structural relationship of caracurine V salts to the strong muscle relaxants toxiferine and alcuronium, they are likely to exhibit neuromuscular blocking activity, which would limit their usefulness as research tools and make the therapeutical use impossible. Reduction of the caracurine V ring skeletons to structural features responsible for good allosteric potency could possibly lead to compounds with negligible neuromuscular blocking activity and very high affinity to the allosteric binding site at M2 receptor. Thus, the aim of this study was to synthesize and pharmacologically evaluate analogues of a novel heterocyclic ring system, which comprises the pharmacophoric elements mentioned previously. The key step of the synthesis of the desired 6,7,14,15-tetrahydro[1,5]diazocino[1,2-a:6,5-a]-diindole ring system (6) involved the intermolecular double N-alkylation of the bromoethylindole (5), which was prepared from the known indolyl methylacetate (3) by reduction of the ester group to alcohol and subsequent substitution by bromine. 3 could be prepared in three steps involving N,N-dibenzylation of tryptamine followed by introduction of the dimethyl malonate moiety at C-2 of indole ring and a subsequent demethoxycarbonylation. The total synthesis of 6,7,14,15-tetrahydro[1,5]diazocino[1,2-a:6,5-a]diindole ring system (6) is shown in Scheme 24. In order to examine the influence of the length of the side-chain on muscarinic activity,exchange of the ethylamine moieties of 14 by the methylamino groups was planned. This should be accomplished by dimerization of the unsubstituted 2-bromoethylindole (32), and subsequent Mannich aminomethylation of the resulting unsubstituted pentacyclic ring. The total synthesis of the 6,7,14,15-tetrahydro-15aH-azocino[1,2-a:6,5-b]diindole ring system(35) is shown in Scheme 25. 32 was prepared from indole-2-carboxylic acid in six steps involving reduction of the acid to the corresponding alcohol 26, benzoylation of 26 followed by nucleophilic substitution with KCN, hydrolysis of the cyanide 28 to indolyl acetic acid 29,reduction of 29 to the corresponding alcohol 30, and finally bromination of 30 to give the bromide 32. Since dimerization attempts of 32 provided only 2-vinylindole (33), the tosylate 34 was used as starting material for the intermolecular alkylation to give exclusively an isomeric pentacyclic ring system, 7,14,15-tetrahydro-15aH-azocino[1,2-a:6,5-b]diindole (35). The formation of the novel, asymmetric ring skeleton can be explained by the ambident nucleophilic character of the indolyl anion that can be alkylated either at nitrogen or at C-3 of indole ring. 35 was subjected to a Mannich reaction to give 2,13-dimethylaminoalkylated product 37 as well as small amounts of the 13-monosubstituted compound (36). The geometry of novel ring systems 6 was elucidated by means of NMR spectroscopy and semiempirical calculations. The diazocinodiindole ring skeleton of 6 exists in chloroform solution at room temperature in a twisted-boat conformation, as indicated by 600 MHz ROESY experiment, vicinal coupling constants within the eight-membered ring, and AM1 calculations. In order to obtain potent allosteric ligands, the new heterocycles 6 and 37 were quarternized with methyliodide to the corresponding ammonium salts 14 and 38, respectively. Additionally, the N,N -diallylsalts of 37 (compound 39) was prepared. The allosteric effect of 14, 38, and 39 on the dissociation of the orthosteric radioligand [3H]Nmethylscopolamine([3H]NMS) and their effects on [3H]NMS equilibrium binding were studied in homogenates of porcine heart ventricles. The concentration of an allosteric agent for a half-maximum effect on orthosteric ligand dissociation (EC50,diss) corresponds to a 50 % occupancy of the liganded receptors by the respective allosteric test compounds. Due to the presence of two benzyl groups on each nitrogen in the side chains of 14, its binding affinity can be best compared with that of N,N -dibenzylcaracurinium V dibromide (EC50,diss = 69 nM). Compound 14 exhibited the comparable affinity to N,N -dibenzylcaracurinium V dibromide with EC50,diss = 54 nM. This result suggested that replacement of the bulky benzyl groups of 14 by smaller substitutents will probably increase the allosteric potency, since dimethyl- and diallylcaracurinium salts showed a 5-fold increase of binding affinity relative to the dibenzyl analogue. Even though the new azocinodiindole ring system of 38 and 39, is not included in the caracurine V ring skeleton, it comprises the essentially pharmacophoric elements of allosteric potency. Due to the different spatial arrangements of the aromatic rings, as well as to different internitrogen distances in both ring systems, compound 38 and 39 exhibited 4-fold lower M2 binding affinity (EC50,diss = 35 and 48 nM, respectively) than the corresponding caracurine V analogues. This study deals with the synthesis of the first representative (Compound 6) of a novel pentacyclic ring system derived from caracurine V. The high allosteric potency of its dimethyl analogue reveals the [1,5]diazocino[1,2-a:6,5-a]-diindole ring system as a new promising lead structure for allosteric modulators of muscarinic M2 receptors. Future research will be focused on structural modifications of the new ring system in order to increase the affinity to the muscarinic receptors. Furthermore, the binding affinities of the new synthesized compounds to the muscle type of nicotinic ACh-receptor should reveal structural features responsible for the muscarinic/nicotinic selectivity.
The mammalian Vasodilator Stimulated Phosphoprotein (VASP) is a founding member of the Ena/VASP family of proteins that includes Drosophila Enabled (ena), the mammalian Ena homologue (Mena) and the Ena-VASP-like protein (Evl). VASP was initially discovered and characterized as a substrate for cGMP- and cAMP-dependent protein kinases (cGKs and cAKs). Ena/VASP proteins are involved in Actin-filament formation, plasma membrane protrusion, acceleration of Actin-based motility of Listeria and the establishment of cell-cell adhesion. Moreover, Ena/VASP proteins have been implicated as inhibitory factors in repulsive axon guidance and inhibition of plasma membrane activity and random motility in fibroblast. In order to study the physiological function of VASP, VASP-deficient mice had been generated in the laboratory by homologous recombination. VASP-/- mice showed hyperplasia of megakaryocytes in the bone marrow and spleen and a two-fold increase in thrombin- and collagen-induced platelet activation. To further investigate the cellular function of VASP, I established cardiac fibroblast cell lines derived from both wild type and VASP-/- mice. Both cell lines presented similar growth rates and normal contact dependent-growth inhibition but showed differences in morphology, migration and adhesion. Adherent VASP-/- cells, despite normal Mena and Evl expression levels, were highly spread. VASP-/- cells covered about twice the substrate surface area as wild type cells, while the cell volumes were unchanged. This shape difference suggests that VASP is involved in the regulation of spreading. Since the small GTPases Rac and Cdc 42 and their effector p21-activated kinase (Pak) are key regulators of lamellipodia formation and cell spreading, I analyzed this signalling pathway in VASP-/- cells stimulated with Platelet Derived Growth Factor-BB (PDGF-BB) or fetal calf serum. In wild type cells Rac and Pak were rapidly and transiently activated by PDGF or serum; however, in the absence of VASP both Rac and Pak activation was dramatically prolonged. The Rac/Pak pathway is known to play an essential role in cell motility. VASP deficient cells showed compromised migration and reorientation in a wound healing assay, probably due to enhanced Rac activity. The spreading phenotype, compromised migration and the effect observed on the Rac and Pak activities were reverted in VASP-/- cells stably transfected with full lenght human VASP, indicating a VASP dependent modulation of the Rac/Pak pathway and Rac/Pak regulated processes. Moreover, adhesion and detachment of VASP-deficient cells were significantly slower when compared to wild type cells. Preincubation of VASP+/+ cells with a cGMP analog accelerated adhesion. This acceleration did not take place in the VASP-/- cells, suggesting a VASP dependent effect. The second part of this work focused on VASP function in platelets. On the one hand I investigated the possibility of VASP-dependent Rac regulation in mouse platelets. Murine platelets are a good model for studying Rac regulation since they express high levels of VASP but not Mena/Evl and since VASP-deficient platelets show an increased platelet activation. Rac was activated by platelet agonists which was inhibited by preincubation with cGMP and cAMP analogs. Initial results which need to be extended showed that the cGMPcaused inhibition of Rac activation was VASP-dependent. Finally, in vivo platelet adhesion (platelet-vessel wall interactions) was studied using VASP-deficient mice. These studies demonstrated in-vivo that VASP down regulates platelet adhesion to the vascular wall under both physiological and pathophysiological conditions.
In the work here presented four distinctly different problems were investigated. The first problem was an investigation into the degradation of Dichloroethylene (DCE) and 1,1-bis (p-Chlorophenyl)-2-dichloroethylene (DDE) utilising pure bacterial cultures. The second investigation dealt with the degradation of DDE and polychlorinated Biphenyl’s (PCB’s) utilising anaerobic sediments and soils from New Zealand. The third investigation worked on the Granulation of anaerobic River-sediments in Upflow Anaerobic Sludge Blanket (UASB) Reactors. The last investigation describes the commissioning of an industrial aerobic Wastewater Treatment Plant and the Implementation of biological Nitrogen- and Phosphate removal in this Wastewater Treatment Plant. Since the chemical Structure of DCE and DDE have certain similarities, Bacteria that were capable of degrading DCE, were tested here, whether they would also be able to degrade DDE utilising a co-metabolic pathway. In the experiments the aerobic bacteria Methylosinus trichosporium and Mycobacterium vaccae and the anaerobic bacteria Acetobacterium woodii and Clostridium butyricum were used. Approximately 60% of the added DCE was degraded by M. vaccae, while M. trichosporium degraded approximately 50%. A. woodii and C. butyricum degraded 40% and 30% respectively of the added DCE. Further experiments with these cultures and DDE lead to a microbial degradation of DDE to an extent of 34.6% for M. vaccae, 14.1% for C. butyricum, 2.2% for A. woodii and 10.5% for M. trichosporium. Additional experiments, utilising [14C]-DDE, showed that the DDE had not been degraded but were attached to the bacterial cells. The second investigation utilised anaerobic soils and sediments from New Zealand to study the anaerobic co-metabolic degradation of DDE and PCB’s. The soils and sediments originated from the River Waikato, from Wastewater Ponds in Kinleith, Marine-Sediments from Mapua, and a variety of soils comtaminated with Pentachlorophenyl (PCP). The cultures from these soils and sediments were raised on a variety of Carbon- and Energy-sources. Beside DDE, Aroclor 1260, and a mix of four pure PCB-Congeneres (one Tetra-, one Hexa, one Hepta- and one Deca-Chlorobiphenyl) were used to test for the reductive dechlorination. The cultivation process of the baceria lasted six months. Samples of the cultures were taken after zero, three and six months. These samples were tested for the increase of cell-protein, the degradation of carbon- and energy-sources, and the removal of the added polychlorinated chemicals. The organochlorines were analysed using reversed phase HPLC and FID-GC. When a change in the Chromatogram was detected the respective cultures were further analysed using ECD-GC and GC-MS. The results showed that the culutres grew under these conditions, but no degradation of DDE and the PCB-Mix could be detected, and only small changes in the composition/chromatograms of Aroclor 1260 were found. The third investigation worked on the Granulation of River-Sediments in UASB-Reactors. Sediments from the River Waikato in New Zealand and the River Saale in Germany were used. In both cases the Granulation process was successful, which was demonstrated by microscopic comparisons of the Sediments and the resulting Granules. The two main bacterial cultures detected were Methanosarcina- and Methanothrix-like cultures. The main carbon- and energy-source was Lactic Acid, which was used at a concentration of 21,8 g COD/L. The Granulation-Process was a combination of using high a COD-Concentration combined with a low Volumetric Loading-Rate. Comparisons of the specific degradation-rates of a variety of carbon- and energy-sources between the Sediments and the Granules, showed no increased degradation rates in regard to the same cell-mass, but the increased bio-mass in the Granules allowed for higher degradation-rates within the UASB-reactors. The fourth investigation describes the commissioning of an industrial Wastewater Treatment Plant for a Dairy-Site in Edendale, Southland, New Zealand. This Plant consists of a DAF-Unit (Dissolved Air Flotation), two Extended Aeration Lagoons with Activated Sludge and two Clarifiers, one for the Activated Sludge and the second for the dosing of Aluminium-Sulphate and the removal of Phosphat-Sulphate. Biological processes for the removal of carbon- and energy-sources were optimised and biological processes for the reduction of Nitrogen- and Phosphate-Concentrations within the wastewater were implemented and optimised. Bilogical removal rates for COD of 95% and above, for Nitrogen of 85-92% and Phosphate of 64-83% were achieved.
Fanconi anemia (FA) is a genetically and phenotypically heterogenous autoso- mal recessive disease associated with chromosomal instability, progressive bone marrow failure, typical birth defects and predisposition to neoplasia. The clinical phenotype is similar in all known complementation groups (FA-A, FA-B, FA-C,FA-D1, FA-D2, FA-E, FA-F and FA-G). The cellular phenotype is characterized by hypersensitivity to DNA crosslinking agents (MMC,DEB), which is exploited as a diagnostic tool. Alltogether, the FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. FANCA, FANCC, FANCE, FANCF and FANCG interact in a nuclear complex upstream of FANCD2. Complementation group FA-D1 was recently shown to be due to biallelic mutations in the human breast cancer gene 2 (BRCA2). After DNA damage, the nuclear complex regulates monoubiquitylation of FANCD2, result- ing in targeting of this protein into nuclear foci together with BRCA1 and other DNA damage response proteins. The close connection resp. identity of the FA genes and known players of the DSB repair pathways (BRCA1, BRCA2, Rad51) firmly establishs an important role of the FA gene family in the maintenance of genome integrity. The chapter 1 provides a general introduction to the thesis describing the current knowledge and unsolved problems of Fanconi anemia. The following chapters represent papers submitted or published in scientific literature. They are succeeded by a short general discussion (chapter 7). Mutation analysis in the Fanconi anemia genes revealed gene specific mutation spectra as well as different distributions throughout the genes. These results are described in chapter 1 and chapter 2 with main attention to the first genes identified, namely FANCC, FANCA and FANCG. In chapter 2 we provide general background on mutation analysis and we report all mutations published for FANCA, FANCC and FANCG as well as our own unpublished mutations until the year 2000. In chapter 3 we report a shift of the mutation spectrum previously reported for FANCC after examining ten FA-patients belonging to complementation group C. Seven of those patients carried at least one previously unknown mutation, whereas the other three patients carried five alleles with the Dutch founder mu- tation 65delG and one allele with the Ashkenazi founder mutation IVS4+4A>T, albeit without any known Ashkenazi ancestry. We also describe the first large deletion in FANCC. The newly detected alterations include two missense mu- tations (L423P and T529P) in the 3´-area of the FANCC gene. Since the only previously described missense mutation L554P is also located in this area, a case can be made for the existence of functional domain(s) in that region of the gene. In chapter 4 we report the spectrum of mutations found in the FANCG gene com- piled by several laboratories working on FA. As with other FA genes, most muta- tions have been found only once, however, the truncating mutation, E105X, was identified as a German founder mutation after haplotype analysis. Direct compar- ison of the murine and the human protein sequences revealed two leucine zipper motifs. In one of these the only identified missense mutation was located at a conserved residue, suggesting the leucine zipper providing an essential protein-protein interaction required for FANCG function. With regard to genotype-phenotype correlations, two patients carrying a homozygous E105X mutation were seen to have an early onset of the hematological disorder, whereas the missense mutation seems to lead to a disease with later onset and milder clinical course. In chapter 5 we explore the phenomenon of revertant mosaicism which emerges quite frequently in peripheral blood cells of patients suffering from FA. We de- scribe the types of reversion found in five mosaic FA-patients belonging to com- plementation groups FA-A and FA-C. For our single FA-C-patient intragenic crossover could be proven as the mechanism of self-correction. In the remaining four patients (all of them being compound heterozygous in FANCA), either the paternal or maternal allele has reverted back to WT sequence. We also describe a first example of in vitro phenotypic reversion via the emergence of a compensat- ing missense mutation 15 amino acids downstream of the constitutional mutation explaining the MMC-resistance of the lymphoblastoid cell line of this patient. In chapter 6 we report two FA-A mosaic patients where it could be shown that the spontaneous reversion had taken place in a single hematopoietic stem cell. This has been done by separating blood cells from both patients and searching for the reverted mutation in their granulocytes, monocytes, T- and B-lymphocytes as well as in skin fibroblasts. In both patients, all hematopoietic lineages, but not the fibroblasts, carried the reversion, and comparison to their increase in erythrocyte and platelet counts over time demonstrated that reversion must have taken place in a single hematopoietic stem cell. This corrected stem cell then has been able to undergo self-renewal and also to create a corrected progeny, which over time repopulated all hematopoietic lineages. The pancytopenia of these patients has been cured due to the strong selective growth advantage of the corrected cells in vivo and the increased apoptosis of the mutant hematopoietic cells.
In vitro and in vivo studies on the activating platelet collagen receptor glycoprotein VI in mice
(2003)
The work summarized here focused on the characterization of the murine platelet collagen receptor glycoprotein (GP) VI and was performed to evaluate its potential as an antithrombotic target. The first mAb against (mouse) GPVI, JAQ1, was generated and used to demonstrate that GPVI requires the FcRgamma-chain for its expression and function and that this receptor is the central molecule in collagen-induced platelet activation. Blocking the major collagen binding site on GPVI with JAQ1 revealed the presence of a second activatory epitope within collagen. Additionally, the collagen receptor integrin alpha2beta1 was found to be required for activation via this second pathway but not to be essential for collagen-induced activation of normal platelets. In studies with mice expressing reduced levels of the GPVI-FcRgamma-complex, differential responses to GPVI ligands were observed. Most importantly, the striking difference between platelet responses to collagen and the GPVI specific synthetic collagen related peptide (CRP) confirmed the supportive role of other collagen receptor(s) on platelets. Irrespective of yet undefined additional receptors, studies with mice deficient in GPVI (FcRgamma-chain) or alpha2beta1 showed that GPVI, but not alpha2beta1 is essential for platelet-collagen interaction. Based on these results, the model of platelet attachment to collagen was revised establishing GPVI as the initial activating receptor which upregulates the activity of integrins, thus enabling firm attachment of platelets to the ECM. While the mAb JAQ1 had only limited inhibitory effects on collagen-induced activation in vitro, its in vivo application to mice resulted in completely abolished platelet responses to collagen and the GPVI specific agonists CRP and convulxin. This effect was found to be due to antibody-induced irreversible down-regulation of GPVI on circulating platelets for at least two weeks. Further studies revealed that GPVI depletion occurs independently of the targeted epitope on the receptor and does not require the divalent form of IgG as it was also induced by mAbs (JAQ2, JAQ3) or the respective Fab fragments directed against epitopes distinct from the major collagen binding site. The internalization of GPVI in vivo resulted in a long-term protection of the mice from lethal collagen-dependent thromboembolism whereas it had only moderate effects on the bleeding time, probably because the treatment did not affect other activation pathways. These results establish GPVI as a potential pharmacological target for the prevention of ischemic cardiovascular diseases and may open the way for a completely new generation of antithrombotics.
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.
The Church is mandated by Jesus Christ to continue the mission for which he was sent into the world. The mission of Christ, which consists in “bringing the good news to the poor, proclaiming liberty to captives, restoring sight to the blind, setting the downtrodden free and the proclamation of the Lord’s year of favour” , remains the fundamental basis of the missionary and evangelising vocation of the Church. She has a message to proclaim and that message is the proclamation of making the kingdom of God present in the lives of the people. Through the ages the Church has responded to this command of the Lord to evangelise, using various methods according to different situations and times. Dialogue is a conditio sine qua non in the Church’s evangelisation. By con-voking the Second Vatican Council, Pope John gave special attention to the Church’s self-knowledge, that is the knowledge of her nature and vocation as well as the realisation of the necessity of dialogue in the Church’s pursuit of Church unity and healthy relationships with non-Christian religions and bod-ies. Besides the emphases on the importance of dialogue in the Church’s exe-cution of her mission and apostolate of building up the people of God, evi-dence from the human sciences portray the indispensable and invaluable roles of dialogue and communication in a globalised world.
Elevation of intracellular cAMP in T lymphocytes, induced by agents such as IL-1α, prostaglandins or forskolin, inhibits Th1-type cytokine production but stimulates Th2-type cytokine production. The signaling pathway engaged in cAMP-mediated induction of Th2 lymphokines remains obscure and therefore my doctoral work was focused on the elucidation of cAMP pathway in primary Th lymphocytes. While forskolin treatment of EL-4 cells led both to an activation of Th2 lymphokines and inhibition of Th1 lymphokines, ectopic expression of catalytically active PKA stimulated Th2 lymphokines but failed to inhibit Th1 lymphokine expression. Thus, the PKA activity is selectively involved in the stimulation of Th2 lymphokine expression whereas other cAMP-dependent pathway(s) appears to downregulate Th1 lymphokines. By investigating different types of primary murine Th cells, it was found that active PKA enhanced IL-5 expression only in Th0 and Th2 but not in Th1 cells. This is likely due to the different levels of GATA-3 whose expression is high in Th2, moderate in Th0 and very low in Th1 cells. Ectopic expression of GATA-3 in Th1 cells induced Th2 lymphokine expression which could be further enhanced by increased cAMP levels or PKA activity. Investigations on the role of increased cAMP levels on Th2 lymphokines in D10 cells, a Th2-type cell line, led to the conclusion that elevated cAMP concentrations do not stimulate PKA but p38 activity which, through phosphorylation of GATA-3, appeared to induce IL-5 and IL-13 expression (Chen et al., 2000). While focusing on primary Th lymphocytes, it was observed that expression of the catalytic subunit α of PKA is sufficient for optimal IL-5 expression in primary Th0 cells. In addition, downregulation of IL-5 production in primary Th2 cells by the treatment with low concentrations of H-89, a PKA specific inhibitor, as well as by the ectopic expression of a negatively acting version of regulatory PKA subunit I demonstrates that active PKA plays an important role in IL-5 gene regulation. These findings using different types of primary CD4+ T lymphocytes, including Th2 cells, the one likely to represent the native IL-5 producers in vivo, demonstrates that the adenylyl cyclase/cAMP/PKA signaling pathway plays an important role in IL-5 gene expression in primary Th2 cells. Thus the importance of cAMP/PKA signaling pathway in Th2 effector function was established during this doctoral research work.
Zars and co-workers were able to localize an engram of aversive olfactory memory to the mushroom bodies of Drosophila (Zars et al., 2000). In this thesis, I followed up on this finding in two ways. Inspired by Zars et al. (2000), I first focused on the whether it would also be possible to localize memory extinction.While memory extinction is well established behaviorally, little is known about the underlying circuitry and molecular mechanisms. In extension to the findings by Zars et al (2000), I show that aversive olfactory memories remain localized to a subset of mushroom body Kenyon cells for up to 3 hours. Extinction localizes to the same set of Kenyon cells. This common localization suggests a model in which unreinforced presentations of a previously learned odorant intracellularly antagonizes the signaling cascades underlying memory formation. The second part also targets memory localization, but addresses appetitive memory. I show that memories for the same olfactory cue can be established through either sugar or electric shock reinforcement. Importantly, these memories localize to the same set of neurons within the mushroom body. Thus, the question becomes apparent how the same signal can be associated with different events. It is shown that two different monoamines are specificaly necessary for formation of either of these memories, dopamine in case of electric shock and octopamine in case of sugar memory, respectively. Taking the representation of the olfactory cue within the mushroom bodies into account, the data suggest that the two memory traces are located in the same Kenyon cells, but in separate subcellular domains, one modulated by dopamine, the other by octopamine. Taken together, this study takes two further steps in the search for the engram. (1) The result that in Drosophila olfactory learning several memories are organized within the same set of Kenyon cells is in contrast to the pessimism expressed by Lashley that is might not be possible to localize an engram. (2) Beyond localization, a possibible mechanism how several engrams about the same stimulus can be localized within the same neurons might be suggested by the models of subcellular organisation, as postulated in case of appetitive and aversive memory on the one hand and acquisition and extinction of aversive memory on the other hand.
Living apart together
(2002)
Cohesiveness between members of a social unit is a defining characteristic of animal social organization. Dispersed social organizations, where members of a social unit spend the main part of their activity period apart, have only recently been distinguished from cohesive social organizations and are still poorly understood with respect to their ecological basis and reproductive consequences. The general goal of this dissertation was to study the three components of the social system of fork-marked lemurs (Phaner furcifer), a small nocturnal primate from Madagascar living in dispersed pairs. First, I characterise their social organization, focusing on behavioural mechanisms of cohesion between pair partners. Second, through application of van Schaik's ecological model, I investigate predictions about the ecological basis of female intra-sexual avoidance, male-female social relationships and the determinants of differential female reproductive success. Finally, I analyse behavioural and genetic aspects of the mating system to test a recent hypothesis that proposes high extra-pair paternity in dispersed primate pairs resulting from constraints on male mate guarding. The study was conducted in Kirindy Forest in Madagascar between September 1998 and April 2001 during three field seasons for a total of 20 months. During more than 1400 hours of focal animal protocols, I sampled year-round data on space use, feeding ecology, time budgets, and social behaviour of all adults and three subadults of 8 families, complemented by simultaneous focal follows of both pair partners, year-round information on sleeping site use, measures on food abundance in each territory, morphological measurements, and DNA-microsatellite data for seven newly discovered polymorphic loci. Across eight social units and three breeding seasons, pairs were the prevailing grouping pattern (18 of 21 family years). Most pairs were stable for more than three mating seasons and used well defined stable territories. Although both pair partners used the same territory in a fairly similar fashion, average distance between pair partners was 100m, which was far considering that many territories measure only 200m in diameter. Pair partners spent only about 20% of activity time in less than 25m distance of each other and shared a sleeping site on average only every third day. Females were found to be dominant over their partner as well as over neighbouring males in all behavioural contexts. Most important food resources were exudates of a small number of tree species. Major food resources were distributed in small, defendable patches characterized by fast depletion and rapid renewal. In accordance with the ecological model, this led to strong within-group contest and scramble competition and weak between-group contest competition over food, as indicated by a positive dominance effect and a negative group size effect on female physical condition. Female reproductive success was determined mainly by family size. Paternity likelihood and exclusion analyses revealed that four out of seven offspring were most likely sired by an extra-pair male. Behaviour during the mating season implied that females as well as males take an active part in obtaining extra-pair copulations and that males try to guard their mates. Dispersed social organization in itself, i.e. low cohesion between pair partners, cannot explain high extra-pair paternity. I propose instead that several other factors common to most primates living in dispersed pairs constrain mate guarding and lead to high EPP. The ecological settings determine the mode of food competition and have shaped the social system of fork-marked lemurs in several ways. Intense within-group competition for food may have ultimately led to female intra-sexual avoidance and range exclusivity which represents an evolutionary precursor of pair-living. Although it remains elusive why females ultimately associated with single males, patterns of within-group contest competition for food explain why pair partners avoid each other during nocturnal activity. The limited number of food resources that is used in repetitive fashion and incomplete knowledge about the pair partners position explain why pair partners meet relatively often and why most encounters involve agonistic conflict. Rigid feeding itineraries characteristic of exudate feeders are likely to pose high costs to offspring dispersing to unfamiliar areas. Feeding ecology can, therefore, explain why parents tolerate delayed natal dispersal despite a negative effect on actual female reproductive success. In conclusion, the present study successfully applied existing socio-ecological theory to a new area of research, refined a recent evolutionary model and contributed important comparative data to our understanding of dispersed pairs in particular and primate and animal societies in general.
Originally renowned for their spectacular epigaeic raids, army ants have captured scientific attention for almost two centuries. They now belong to one of the best studied group of ants. However, most of our knowledge about army ants was derived from the study of the minority of specialized, epigaeicly active species. These species evolved probably rather recently from hypogaeic ancestors. The majority of army ant species still leads a hypogaeic life and is almost completely unknown in its entire sociobiology. It thus remained speculative, whether the assumed 'general' characteristics of army ants represent an adaptation to epigaeic activity or apply also to the majority of hypogaeic species. Based on the recent observation that the hypogaeic Asian army ant Dorylus (Dichthadia) laevigatus recruits predictably to palm oil baits, I developed and tested an oil-baiting method for the study of hypogaeic (army)ants. Prior to my study, nothing was known about the sociobiology of the assumed rare D. laevigatus. Throughout my work, I showed D. laevigatus to be very common and abundant in a wide range of habitats in West-Malaysia and on Borneo. Investigating its foraging behavior, I revealed D. laevigatus to differ from epigaeicly active species in several ways. Never demonstrated for any of the epigaeic species, D. laevigatus established stable trunk trail systems. Such a trail system contradicted the perception of army ant foraging, which was believed to be characterized by raids with constantly alternating trail directions. The trunk trail system further enabled a near omnipresence of D. laevigatus within its foraging area, which was also believed to be atypical for an army ant. Raids differed in structure and composition of participating workers from those of epigaeic species. Also, bulky food sources could be exploited over long periods of time. The foraging system of D. laevigatus resembled in several ways that of e.g. leaf-cutter and harvester ants. Likewise contrary to the assumptions, D. laevigatus had a wide food spectrum and showed only little effect on local arthropod communities, even falling itself prey to other ants. Strong aggressive behavior was observed only towards ant species with similar lifestyles, enabling me to provide the first detailed documentation of interspecific fights between two sympatric Dorylus species. Similar to foraging habits or ecological impact, nothing was known about colony size and composition, nesting habits, or worker polymorphism for D. laevigatus or any other hypogaeic Dorylus species prior to my work. By observing and eventually excavating a colony, I showed D. laevigatus to have a much smaller colony size and to lack the large sized workers of epigaeic Dorylus species. Similar to epigaeic Dorylinae, I showed D. laevigatus to have a non-phasic brood production, to emigrate rarely, and to alter its nest form along with habitat conditions. Detailed morphological and geographical descriptions give an impression of the Asian Dorylus species and are expected to aid other researchers in the difficult species identification. The genetic analysis of a male collected at a light trap demonstrated its relation to D. laevigatus. Confirming the male and queen associations, D. laevigatus is now one of five Dorylus species (out of a total of 61), for which all castes are known. In cooperation with D. Kistner, I provide a morphological and taxonomical description of nine Coleopteran beetles associated with D. laevigatus. Behavioral observations indicated the degree of their integration into the colony. The taxonomic position of the beetles further indicated that D. laevigatus emigrated from Africa to Asia, and was accompanied by the majority of associated beetles. The diversity of D. laevigatus guests, which included a number of unidentified mites, was rather low compared to that of epigaeic species. Overall, I demonstrated the developed baiting containers to effectively enable the study of hypogaeic ants. I showed several other hypogaeic ant species to be undersampled by other methods. Furthermore, the method enabled me to documented a second hypogaeic Dorylus species on Borneo. A detailed description of this species' morphology, ecology, and interactions with D. laevigatus is provided. My study indicated D. laevigatus to be an ecologically important species, able to influence soil structure and organisms of tropical regions in many ways. Relating the observed traits of D. laevigatus to epigaeicly active species, I conclude that our assumption of 'general' army ant behavior is erroneous in several aspects and needs to be changed. The oil-baiting method finally provides a tool enabling the location and study of hypogaeic (army)ant species. This opens a broad field for future studies on this cryptic but nonetheless important group of ants.
The number of males in animal groups is an essential determinant of male and female reproductive strategies. Females may benefit from living with several males, whereas males generally strive to monopolize a group of females. Due to male intrasexual competition, the sex ratio of groups of anthropoid primates is generally female-biased. Gregarious Malagasy lemurs deviate from theoretical expectations derived from sexual selection theory and from patterns found among anthropoids because they live in relatively small groups with an even or male-biased adult sex ratio and lack sexual dimorphism. The aim of this thesis was to investigate sex-specific reproductive strategies relating to the unusual group composition of redfronted lemurs (Eulemur fulvus rufus) by combining behavioral, demographic and endocrinological data. In the first of a set of four studies I investigated the applicability of non-invasive endocrine measurements for monitoring ovarian function in wild redfronted lemur females in order to evaluate the degree of estrus synchrony. Further, I tested the prediction that males living in multi-male groups rely on indirect mechanisms of intrasexual competition, such as physiological suppression of testicular function. Several possible benefits gained from living with many males have been proposed and the hypothesis that additional males improve social thermoregulation was tested in the third study. Finally, I examined the proximate determinants of the unusual sex ratio within groups, the variation in the adult sex ratio as well as possible social benefits of the high number of males for both sexes. The study was conducted in Kirindy Forest, Madagascar, between April 1999 and July 2000. I recorded >3000 hours of focal animal data on social and sexual behavior of all adult members of five groups. Additionally, >2200 fecal samples of males and females were collected for subsequent hormone analysis using enzymeimmunoassay (EIA). Further, I analyzed demographic data from seven Eulemur fulvus rufus groups collected between 1996 and 2002. The analyses of fecal estrogen and progestogen excretion in wild and captive females revealed that monitoring ovarian function is principally possible in redfronted lemurs, as demonstrated by the analysis of samples from captive females. Characterization of ovarian cycles in wild females, however, was not possible, because of a high day-to-day variability in excreted hormones. Nevertheless, the study provided reliable information on gestation and cycle length as well as endocrine changes associated with gestation. Additionally, I established a method for prenatal sex determination using maternal fecal samples collected during late gestation. The excretion pattern of androgens in samples of males revealed no differences between dominant and subordinate males, indicating that dominant males did not suppress the endocrine function of subordinate rivals. High frequencies of matings in combination with large testes size suggest that male reproductive competition relies at least partly on sperm competition. Females did not benefit from the high number of males in their groups in terms of improved thermoregulation because surplus males did not participate frequently in huddling groups with females. Analysis of the demographic data revealed that birth and mortality rates were not sex-biased and that males migrated considerably more frequently than females, providing no proximate explanation for the unusual sex ratio. Females in this study may proximately regulate group composition by synchronizing their fertile periods, which were inferred indirectly from the temporal distribution of births within groups. Both males and females benefit from the high number of co-resident males because reduced male group size seemed to be the main predictor of take-over rate, and thus, infanticide risk. The results of these studies suggest that certain life history traits (fast maturation, short inter-birth intervals) may ultimately determine the high number of males and the lack of single-male groups seen in redfronted lemurs. An accelerated male life history may facilitate joint group transfers and take-overs of male coalitions without a transitional time outside bisexual groups. Because males and females both benefit from a high number of males the conflict of interests between the sexes is considerably defused.
In my Ph.D. thesis "On the geometry and parametrization of almost invariant subspaces and observer theory" I consider the set of almost conditioned invariant subspaces of fixed dimension for a given fixed linear finite-dimensional time-invariant observable control system in state space form. Almost conditioned invariant subspaces were introduced by Willems. They generalize the concept of a conditioned invariant subspace requiring the invariance condition to hold only up to an arbitrarily small deviation in the metric of the state space. One of the goals of the theory of almost conditioned invariant subspaces was to identify the subspaces appearing as limits of sequences of conditioned invariant subspaces. An example due to {\"O}zveren, Verghese and Willsky, however, shows that the set of almost conditioned invariant subspaces is not big enough. I address this question in a joint paper with Helmke and Fuhrmann (Towards a compactification of the set of conditioned invariant subspaces, Systems and Control Letters, 48(2):101-111, 2003). Antoulas derived a description of conditioned invariant subspaces as kernels of permuted and truncated reachability matrices of controllable pairs of the appropriate size. This description was used by Helmke and Fuhrmann to construct a diffeomorphism from the set of similarity classes of certain controllable pairs onto the set of tight conditioned invariant subspaces. In my thesis I generalize this result to almost conditioned invariant subspaces describing them in terms of restricted system equivalence classes of controllable triples. Furthermore, I identify the controllable pairs appearing in the kernel representations of conditioned invariant subspaces as being induced by corestrictions of the original system to the subspace. Conditioned invariant subspaces are known to be closely related to partial observers. In fact, a tracking observer for a linear function of the state of the observed system exists if and only if the kernel of that function is conditioned invariant. In my thesis I show that the system matrices of the observers are in fact the corestrictions of the observed system to the kernels of the observed functions. They in turn are closely related to partial realizations. Exploring this connection further, I prove that the set of tracking observer parameters of fixed size, i.e. tracking observers of fixed order together with the functions they are tracking, is a smooth manifold. Furthermore, I construct a vector bundle structure for the set of conditioned invariant subspaces of fixed dimension together with their friends, i.e. the output injections making the subspaces invariant, over that manifold. Willems and Trentelman generalized the concept of a tracking observer by including derivatives of the output of the observed system in the observer equations (PID-observers). They showed that a PID-observer for a linear function of the state of the observed system exists if and only if the kernel of that function is almost conditioned invariant. In my thesis I replace PID-observers by singular systems, which has the advantage that the system matrices of the observers coincide with the matrices appearing in the kernel representations of the subspaces. In a second approach to the parametrization of conditioned invariant subspaces Hinrichsen, M{\"u}nzner and Pr{\"a}tzel-Wolters, Fuhrmann and Helmke and Ferrer, F. Puerta, X. Puerta and Zaballa derived a description of conditioned invariant subspaces in terms of images of block Toeplitz type matrices. They used this description to construct a stratification of the set of conditioned invariant subspaces of fixed dimension into smooth manifolds. These so called Brunovsky strata consist of all the subspaces with fixed restriction indices. They constructed a cell decomposition of the Brunovsky strata into so called Kronecker cells. In my thesis I show that in the tight case this cell decomposition is induced by a Bruhat decomposition of a generalized flag manifold. I identify the adherence order of the cell decomposition as being induced by the reverse Bruhat order.
This work presents the analysis, 3D modeling and interpretation of gravity and aeromagnetic data of Jordan and Middle East. The potential field data delineate the location of the major faults, basins, swells, anticlines, synclines and domes in Jordan. The surface geology of Jordan and the immediate area east of the Rift is dominated by two large basins, the Al-Jafr basin in the south and the Al-Azraq-Wadi as Sirhan basin to the northeast. These two basins strike southeast-northwest and are separated by an anticlinal axis, the Kilwah-Bayir swell. The Karak Wadi El Fayha fault system occurs along the western flank of the swell. The Swaqa fault occurs on the southwest hinge of Al-Azraq basin and the Fuluq fault occurs on its northeast hinge. In the south west of Jordan, Wadi Utm-Quwaira and Disi-Mudawara fault zones are shown clearly in the aeromagnetic and gravity maps. The previous major faults are well correlated with the structural map of Jordan published by Bender (1968). 3D modeling of gravity data in the Dead Sea basin (DSB) was used together with existing geological and geophysical information to give a complete structural picture of the basin. The 3D models of the DSB show that the internal structure of the Dead Sea basin (DSB) is controlled by longitudinal faults and the basin is developed as a full graben bounded by sub-vertical faults along its long sides. In the northern planes of the 3D model, the accumulation of Quaternary (salt and marl) and Mesozoic (pre-rift) sediments are thinner than in the central and southern planes of the model. In the northern planes, the thickness of the Quaternary sediments is about 4 km, 5 km in the southern planes and it exceeds 8 km in the central planes of the DSR. The thickness of the pre-rift sediments reaches 10-12 km in the northern and southern planes and exceeds 15 km in the central planes of the DSR. The planes of the 3D models show that the depth to the crystalline basement under the eastern shoulders of the DSR is shallower than those beneath the western shoulders. It is about 3-5 km beneath the eastern shoulders and 7-9 km under the western shoulder of the DSR. The gravity anomaly maps of residual and first derivative gravity delineate the subsurface basins of widely varying size, shape, and depth along the Rift Valley. The basins are created by the combination of the lateral motion along a right-tending step over and normal faulting along the opposite sides. Al Bakura basin occupies the upper Jordanian River valley and extends into the southern Tiberias Lake. Bet Shean basin to the south of Al Bakura basin plunges asymmetrically toward the east. The Damia basin, comprising the central Jordan Valley and Jericho areas to the north of the Dead Sea is shallow basin (~600-800m deep). The Lisan basin is the deepest basin in the Rift. The 3D gravity models indicate a maximum of ~12 km of basin fill. Three basins are found in Wadi Araba area, Gharandal, Timna (Qa'-Taba) and Aqaba (Elat) basin. The three basins become successively wider and deeper to the south. The three regional gravity long E-W profiles (225 km) from the Mediterranean Sea crossing the Rift Valley to the east to the Saudi Arabia borders, show the positive correlation between topography and free air anomaly and strong negative Bouguer anomaly under the central part of the Dead Sea Basin (DSB) and normal regional Bouguer anomaly outside of the DSB in the transform valley. Depth to the top of the bedrock in the under ground of Jordan was calculated from potential field data. The basement crops out in the south west of Jordan and becomes deeper to northwards and eastwards to be about ~ 8 km below ground surface in the Risha area.
The first goal of this study was to develop cell lines with a stable expression of bio-fluorescent topo II and topo I. This was successfully achieved using a bicistronic vector system. Control experiments showed that proteins of expected size were expressed, and that GFP-tagged topos I, IIa, and IIb were active in the cells and fully integrated in the endogenous pools of the enzymes. These cell-lines provided a novel tool for investigating the cell biology of human DNA topoisomerases. Our most important finding was, that both types of mammalian topoisomerases are entirely mobile proteins that are in continuous and rapid flux between all compartments of the nucleus and between the cytososl and the chromosomes of mitotic cells. This was particularly surprising with regard to topo II, which is considered to be a structural component of the nuclear matrix and the chromosome scaffold. We must conclude that if this was the case, then these architectural structures appear to be much more dynamic than believed until now. In this context it should also be mentioned, that the alignment of topo II with the central axes of the chromosome arms, which has until now been considered a hall-mark of the enzyme’s association with the chromosomal scaffold, is not seen in vivo and can be demonstrated to be to some extent an artefact of immunohistochemistry. Furthermore, we show that the two isoforms of topo II (a and b) have a different localisation during mitotic cell division, supporting the general concept that topo II functions at mitosis are exclusively assigned to the a-form, whereas at interphase the two isoenzymes work in concert. Despite unrestricted mobility within the entire nuclear space, topoisomerases I and II impose as mostly nucleolar proteins. We show that this is due to the fact that in the nucleoli they are moving slower than in the nucleoplasm. The decreased nucleolar mobility cannot be due to DNA-interactions, because compounds that fix topoisomerases to the DNA deplete them from the nucleoli. Interestingly, the subnucleolar distribution of topoisomerases I and II was complementary. The type II enzyme filled the entire nucleolar space, but excluded the fibrial centers, whereas topo I accumulated at the fibrial centers, an allocation directed by the enzyme’s N-terminus. During mitosis, it also mediates association with the nucleolar organising regions of the acrocentric chromosomes. Thus, topo I stays associated with the rDNA during the entire cell-cycle and consistently colocalizes there with RNA-polymerase I. Finally, we show that certain cancer drugs believed to act by stabilising covalent catalytic DNA-intermediates of topoisomerases, do indeed immobilize the enzymes in living cells. Interestingly, these drugs do not target topoisomerases in the nucleoli but only in the nucleoplasm.
Transforming-Growth-Factor-beta1 (TGF-b1) is a multifunctional cytokine that regulates cell growth and differentiation in many types of cells. TGF-b1 is especially known to exert a variety of regulatory functions in the immune system, such as T cell differentiation and T cell function. Signal transduction of TGF-b1 is mediated by phosphorylation of receptorassociated Smad proteins (R-Smads). R-Smads are phosphorylated by the activated type I receptor, which is itself phosphorylated by the high affinity type II receptor upon ligand binding. The phosphorylated R-Smads then associate with Co-Smads. Heterooligomers of R- and Co-Smads translocate into the nucleus where they regulate transcription of target genes in concert with other transcription factors such as CBP/p300 or AP-1. Recent findings suggest that the pleiotropic effects of TGF-b1 are conferred by crosstalks to other signal transduction pathways such as the MAP-kinases or the STAT-pathway. Here we describe the effect of long-term exposure to TGF-b1 on the effector function of differentially stimulated primary murine splenocytes and purified primary murine CD8+ cytotoxic T cells. Long-term exposure to TGF-b1 results in non-responsiveness to TGF-b1- induced Smad2 phosphorylation. This is seen either by no phosphorylation or sustained phosphorylation of Smad2. Furthermore, we observed a strong correlation between sustained Smad2 phosphorylation and resistance to TGF-b1 mediated growth inhibition. In contrast, splenocyte cultures strongly growth inhibited by TGF-b1 showed no Smad2 phosphorylation. Lytic activity of these cultures, however, was found to be suppressed regardless of proliferation properties and Smad2 phosphorylation pattern. We also describe that a functional MEK-1 pathway is a prerequisite for rendering murine splenocytes unresponsive to TGF-b1 mediated growth inhibition, and that inhibition of the MEK-1 cascade alters the Smad2 phosphorylation pattern. In addition, we show that resistance to TGF-b1 mediated growth inhibition correlates with the activation of the JNK pathway. However, the resistant phenotype was found unable to be reverted upon administration of exogeneous IFNg and/or aCD28 antibody. In human or mouse T cell lines, however, the described correlation between the type of stimulation and TGF-b growth resistance or growth sensitivity is not present. Thus, this correlation is specific for primary T cells. We also cloned a chimeric dominantnegative TGF-b receptor which is coupled to a suicide gene, in order to render T cells resistant to TGF-b mediated effects.These findings shed light on how TGF-b1 mediates its immunosuppressive role, and may help to gain knowledge of averting these TGF-b1 effects in the course of tumor therapy.
The thesis consists of two major parts. The first part contains a theoretical-experimental study of confocal micro-Raman spectroscopy on hybrid polymer coatings and an application of this spectroscopic method on PDMS-membranes. The theoretical-experimental study includes the application of a model that describes the influence of the refraction effect on the focus length on confocal Raman experiments, and the development of a new model that additionally takes into account the effect of diffraction on the focus dimensions. A parallel comparison between these two theoretical approaches and experimental data has been also drawn and a better agreement between theory and experiment was observed, when both refraction and diffraction effects were considered. Further, confocal resonance micro-Raman spectroscopy has been applied to characterise the diffusion processes of pharmacologically relevant molecules (b-carotene dissolved in dimethylsulfoxide) through a polydimethylsiloxane (PDMS)-membrane. The diffusion rate as a function of the measurement depth and diffusion time as well as the concentration gradient under a steady flux have been determined. The measurements shown that the confocal micro-Raman technique is a powerful tool to investigate the kinetics of diffusion processes within a membrane before the steady state has been reached. The second part of the thesis contains infrared and Raman spectroscopic studies of copper and iron doped B2O3-Bi2O3 glass systems. These studies were performed to obtain specific data regarding their local structure and the role played by dopant ions on boron and bismuthate units. The changes of B2O3 and Bi2O3 structural units due to the relaxation of the amorphous structure, which was induced in these samples by the thermal treatment, were also evidenced.
The thesis contains two major parts. The first part deals with structural investigations on different coordination compounds performed by using infrared absorption and FT-Raman spectroscopy in combination with density functional theory calculations. In the first section of this part the starting materials Ph2P-N(H)SiMe3 and Ph3P=NSiMe3 and their corresponding [(MeSi)2NZnPh2P-NSiMe3]2 and Li(o-C6H4PPh2NSiMe3)]2·Et2O complexes have been investigated in order to determine the influence of the metal coordination on the P–N bond length. In the next section the vibrational spectra of four hexacoordinated silicon(IV) and germanium(IV) complexes with three symmetrical bidentate oxalato(2-) ligands have been elucidated. Kinetic investigations of the hydrolysis of two of them, one with silicon and another one with germanium, have been carried out at room temperature and at different pH values and it was observed that the hydrolysis reaction occurs only for the silicon compound, the fastest reaction taking place at acidic pH. In the last section of this part, the geometric configurations of some hexacoordinated silicon(IV) complexes with three unsymmetrical bidentate hydroximato(2-) ligands have been determined. The second part of the thesis contains vibrational investigations of some biologically active molecules performed by means of Raman spectroscopy together with theoretical simulations. The SER spectra of these molecules at different pH values have also been analysed and the adsorption behaviour on the metal surface as well as the influence of the pH on the molecule-substrate interaction have been established.
Transplantation is now firmly established as a therapeutic approach to extend and improve the life of patients in the final stages of organ failure. It has been demonstrated that transplantation between genetically non-identical individuals leads to the activation of the recipient’s alloimmune response as a major determinant of transplant outcome. T cell recognition of foreign MHC molecules plays a key role in initiating and sustaining allograft rejection. To prevent the risk of rejection, patients are given immunosuppressive drugs, which are non-specific and have major side-effects (infections, malignancies). It has been shown that the alloreactive T cells specifically recognize donor MHC-derived peptides. This implies that it may be possible to develop antigen-specific strategies in order to modulate the alloimmune response by peptide analogues and specifically altered peptide ligands. The purpose of this study was to explore the potential of “recipient-adapted” analogues from the dominant MHC class I peptide to modulate the alloimmune response. Beside the significant role of donor dominant determinants in the rejection process, we tested seven 13-to-24-mer peptides from the Wistar-Furth MHC class I molecule (WF, RT1.Au) for their possible immunogenicity in a fully MHC-mismatched WF to Lewis (LEW, RT1l) rat strain combination. Secondly, the immunodominant allopeptide was selected to generate analogues in order to investigate their modulatory capacity. All peptides were tested in vitro in a standard proliferation assay and in vivo using a heterotopic heart transplantation model. Our findings show that five peptides (P1-P5) were able to induce specific T cell proliferation in LEW responders. Furthermore, we found a hierarchical distribution of the determinants: peptide P1 as a good candidate for the immunodominant determinant, while P2, P3, P4, and P5 as subdominant epitopes and the other two peptides, P6 and P7, as non-immunogenic determinants of WF MHC class I molecule. Furthermore, the dominance of P1 was confirmed by the strong proliferation induced after immunization with a mixture of peptides in the presence of P1. This hierarchical distribution of the proliferative response correlated with the cytokine production. Peptide P1, comprising only 3 allogeneic amino acids (L5, L9, and T10) induced the strongest T cell proliferation and produced high levels of cytokines, especially IL-2 and IFN-g. In addition, the immunodominance of peptide P1 was confirmed by the significant reduction in the allograft survival time in comparison to the non-immunized control animals. Since the TCR Vß repertoire of rejected graft-infiltrating cells in rejected allografts was similar to the profile observed after in vitro restimulation of P1-primed T cells, we concluded that peptide P1 is able to activate the alloreactive T cell population. Our results demonstrate the particular role of the dominant peptide P1 (residues 1-19) in the allograft rejection in WF to LEW rat strain combination. In the second set of experiments, we investigated the fine specificity of the dominant peptide P1-activated T cells using peptide analogues from P1. The “recipient-adapted” analogues were designed by changing the allogeneic RT1.Au amino acids (L5, L9, T10) one-by-one with the correspondent syngeneic RT1.Al amino acids (M5, D9, I10) in the sequence of peptide P1. The six peptide analogues (A1.1-A1.6) consisting of either one or two allogeneic amino acids were able to induce a specific T cell proliferative response and cytokine production. Analogue A1.5 with only one allogeneic amino acid (L5) was of particular interest because it induced a low T cell proliferation and high cytokine levels, especially IL-4 and IL-10. In addition, immunization with A1.5 did not influence the allograft survival time in comparison to the non-immunized LEW recipients. A1.5 was the only analogue able to down-regulate the proliferation of P1-primed T cells. Our results reveal that A1.5 is an MHC competitor as confirmed by the in vitro MHC competition assay and the inhibition of the negative effect of P1 on the allograft survival time when recipients were immunized with a mixture of P1 and A1.5. These findings suggest that it is possible to design peptide analogues, such as A1.5, which do not stimulate the dominant peptide P1-specific T cell population and even more, are able to block its presentation in the MHC molecule. In all, the results indicate that the specific suppression of indirect allorecognition can be achieved by using peptide analogues of the dominant allopeptide.
Transforming growth factor-ß (TGF-ß) is a multifunctional cytokine that is engaged in regulating versatile cellular processes that are pivotal for development and homeostasis of most tissues in multicellular organisms. TGF-ß signal transduction is initially propagated by binding of TGF-ß to transmembrane serine/threonine kinase receptors, designated TßRI and TßRII. Upon activation, the receptors phosphorylate Smad proteins which serve as downstream mediators that enter the nucleus and finally trigger transcriptional responses of specific genes. During the past years, it became evident that signaling cascades do not proceed in a linear fashion but rather represent a complex network of numerous pathways that mutually influence each other. Along these lines, members of the TGF-ß superfamily are attributed to synergize with neurotrophins. Together, they mediate neurotrophic effects in different populations of the nervous system, suggesting that an interdependence exists between TGF-ßs on the one hand and neurotrophins on the other. In the present work, the crosstalk of NGF and TGF-ß/Smad signaling pathways is characterized in rat pheochromocytoma cells (PC12) which are frequently used as a model system for neuronal differentiation. PC12 cells were found to be unresponsive to TGF-ß due to limiting levels of TßRII. However, stimulation with NGF results in initiation of Smad-mediated transcription independent of TGF-ß. Binding of NGF to functional TrkA receptors triggers activation of Smad3. This NGF-dependent Smad activation occurs by a mechanism which is different from being induced by TGF-ß receptors in that it provokes a different phosphorylation pattern of R-Smads. Together with an inferior role of TßRI, Smad3 is proposed to serve as a substrate for cellular kinases other than TßRI. Based on the presented involvement of components of both, the MAPK/Erk and the TAK1/MKK6 cascade, signal mediators of these pathways rank as candidates to mediate direct activation of Smad3. Smad3 is subsequently translocated to the nucleus and activates transcription in a Smad4-dependent manner. Negative regulation is provided by Smad7 which was found to act as a potent inhibitor of Smad signaling not only in TGF-ß- but also in NGF-mediated cascades. The potential of NGF to activate the Smad pathway independent of TGF-ß might be of special importance in regulating expression of genes that are essential for the development and function of neuronal cells or of other NGF-sensitive cells, in particular those which are TGF-ß-resistant.
This theses deals with the syntheses and the coordination behaviour of polyimidosulfur anions like S(NR)32–, S(NR)42–, RS(NR)2– or RS(NR)3–, the nitrogen analogues of the well known oxo-anions SO32–, SO42–, RSO2– and RSO3–. The first aim was the synthesis of a triimidosulfite with three different NR-substituents, a so called asymmetrical triimidosulfite. In all reactions, that have been carried out to obtain a triimidosulfite with three (or two) different residues at nitrogen, the final product was always the dilithium sulfide adduct. The syntheses of chiral alkylenediimidosulfites was successful. Similar to Corey’s S-ylides (R2(O)S+––CR2) and Wittig’s phosphonium ylides (R3P+––CR2) these molecules contain a positively charged sulfur atom next to a carbanionic centre. The structures of the alkylenediimidosulfites are not influenced by the different substituents at nitrogen and carbon, respectively. In each case a doublecubic structure is received. The first members of a completely new class of compounds were synthesised: the aryl-bis-(diimidosulfinates). In this compounds two SN2 units are connected via a heteroaromatic linker, containing a potential donor centre in metal coordination. They represent, like the known alkyldiimidosulfinates, dipodal monoanionic ligands. In the field of sulfur (VI) chemistry the syntheses of aryltriimidosulfonates were successful. Hitherto it was believed, that only spatial less demanding lithium organics could be added to a S=N double bond in S(NtBu)3. This assumption was confirmed by the fact that methyl- and phenylacetylene-triimidosulfonate were the only known alkylsulfonates. Nevertheless, the addition of several lithiumheteroarenes to sulfurtriimide worked without difficulties. If the shape of the nucleophile permits to slot in between the NtBu substituents and to approach the electrophilic sulfur in the sulfurtriimide from the side rather than in an orthogonal angle, the addition reaction works smoothly. Although the steric demand of the tris(tert.-butyl)triimidosulfonate unit is very high, the synthesis of thiophene-bis-(triimidosulfonate) worked. The sulfonate moieties function as dipodal ligands.
Characterization of memories and ignorant (S6KII) mutants in operant conditioning in the heat-box
(2002)
Learning and memory processes of operant conditioning in the heat-box were analysed. Age, sex, and larval desity were not critical parameters influencing memory, while low or high activity levels of flies were negatively correlated with their performance. In a search for conditioning parameters leading to high retention scores, intermittent training was shown to give better results than continuous training. As the memory test is the immediate continuation of the conditioning phase just omitting reinforcement, we obtain a memory which consists of two components: a spatial preference for one side of the chamber and a stay-where-you-are effect in which the side preference is contaminated by the persistence of heat avoidance. Intermittent training strengthens the latter. In the next part, memory retention was investigated. Flies were trained in one chamber and tested in a second one after a brief reminder training. With this direct transfer, memory scores reflect an associative learning process in the first chamber. To investigate memory retention after extended time periods, indirect transfer experiments were performed. The fly was transferred to a different environment between training and test phases. With this procedure an after-effect of the training was still observed two hours later. Surprisingly, exposure to the chamber without conditioning also lead to a memory effect in the indirect transfer experiment. This exposure effect revealed a dispositional change that facilitates operant learning during the reminder training. The various memory effects are independent of the mushroom bodies. The transfer experiments and yoked controls proved that the heat-box records an associative memory. Even two hours after the operant conditioning procedure, the fly remembers that its position in the chamber controls temperature. The cAMP signaling cascade is involved in heat-box learning. Thus, amnesiac, rutabaga, and dunce mutants have an impaired learning / memory. Searching for, yet unknown, genes and signaling cascades involved in operant conditioning, a Drosophila melanogaster mutant screen with 1221 viable X-chromosome P-element lines was performed. 29 lines with consistently reduced heat avoidance/ learning or memory scores were isolated. Among those, three lines have the p[lacW] located in the amnesiac ORF, confirming that with the chosen candidate criteria the heat-box is a useful tool to screen for learning and /or memory mutants. The mutant line ignP1 (8522), which is defective in the gene encoding p90 ribosomal S6 kinase (S6KII), was investigated. The P-insertion of line ignP1 is the first Drosophila mutation in the ignorant (S6KII) gene. It has the transposon inserted in the first exon. Mutant males are characterized by low training performance, while females perform well in the standard experiment. Several deletion mutants of the ignorant gene have been generated. In precise jumpouts the phenotype was reverted. Imprecise jumpouts with a partial loss of the coding region were defective in operant conditioning. Surprisingly, null mutants showed wild-type behavior. This might indicate an indirect effect of the mutated ignorant gene on learning processes. In classical odor avoidance conditioning, ignorant null mutants showed a defect in the 3-min, 30-min, and 3-hr memory, while the precise jumpout of the transposon resulted in a reversion of the behavioral phenotype. Deviating results from operant and classical conditioning indicate different roles for S6KII in the two types of learning.
We consider homogeneous spaces G/H with the same rational homotopy as a product of a 1-sphere and a (m+1)-sphere. We show that these spaces have also the rational cohomology of such a sphere product if H is connected and if the quotient has dimension m+2. Furthermore, we prove that if additionally the fundamental group of G/H is cyclic, then G/H is locally a product of a 1-torus and ofA/H, where A/H is a simply connected rational cohomology (m+1)-sphere (and hence classified). If H fails to be connected, then with U as the connected component of H the G-action on the covering space G/U of G/H has connected stabilizers, and the results apply to G/U. To show that under the assumptions above every natural number may be realized as the order of the group of connected components of H we calculate the cohomology of certain homogeneous spaces. We also determine the rational cohomology of the fibre bundle U-->G-->G/U if G/H meets the assumptions above. This is done by considering the respective Leray-Serre spectral sequence. The structure of the cohomology of U-->G-->G/U then gives a second proof for the structure of compact connected Lie groups acting transitively on spaces with the rational homotopy of a product of a 1-sphere and a (m+1)-sphere. Since a quotient of a homogeneous space with the same rational homotopy or cohomology as a product of a 1-sphere and a (m+1)-sphere is not simply connected, there often arises the question whether or not a considered fibre bundle or fibration is orientable. A large amount of space will therefore be given to the problem of showing that certain fibrations are orientable. For compact connected (m+2)-manifolds with cyclic fundamental groups and with the rational homotopy of a product of a 1-sphere and a (m+1)-sphere we show the following: if a connected Lie group acts transitively on the manifold, then the maximal compact subgroups are either transitive, or their orbits are simply connected rational cohomology spheres of codimension 1. Homogeneous spaces with the same rational cohomology or homotopy as a a product of a 1-sphere and a (m+1)-sphere play a role in the study of different types of geometrical objects. They appear for example as focal manifolds of isoparametric hypersurfaces with four distinct principal curvatures. Further examples of such spaces are the point spaces and the line spaces of compact connected generalized quadrangles. We determine the isometry groups of isoparametric hypersurfaces with 4 principal curvatures of multiplicities 1 and m which are transitive on the focal manifold with non-trivial fundamental group. Buildings were introduced by Jacques Tits to give interpretations of simple groups of Lie type. They are a far-reaching generalization of projective spaces, in particular a generalization of projective planes. There is another generalization of projective planes called generalized polygons. A projective plane is the same as a generalized triangle. The generalized polygons are also contained in the class of buildings: they are the buildings of rank 2. To compact quadrangles one can assign a pair of natural numbers called the topological parameters of the quadrangles. We treat the case k=1. It turns out that there are no other point-transitive compact connected Lie groups for (1,m)-quadrangles than the ones for the real orthogonal quadrangles. Furthermore, we solve the problem of three infinite series of group actions which Kramer left as open problems; there are no quadrangles with the homogeneous spaces in question as point spaces (up to maybe a finite number of small parameters in one of the three series).
Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease associated with autoantibodies to type VII collagen, the major constituent of anchoring fibrils. Previous attempts to demonstrate the blister inducing potential of autoantibodies to this protein have failed. To address this question, we used an in vitro model involving cryosections of human skin incubated with patients’ autoantibodies and leukocytes from healthy donors. We show that sera from 14 out of 16 EBA patients, in contrast to sera from healthy controls, induced dermal-epidermal separation in the cryosections. The level of the experimentally induced split localizes to the lamina lucida of the dermal-epidermal junction. Recruitment and activation of neutrophils at the dermal-epidermal junction was necessary for split induction, whereas mononuclear cells were not required. Importantly, patients’ autoantibodies affinity-purified against a recombinant form of the non-collagenous 1 (NC1) domain of type VII collagen retained their blister-inducing capacity, while patients’ IgG that was depleted of reactivity to type VII collagen lost this ability. Monoclonal antibody LH7.2 to the NC1 domain of type VII collagen also induced subepidermal splits in the cryosections; F(ab’)2 fragments of autoantibodies to type VII collagen were not pathogenic. These findings demonstrate the capacity of autoantibodies to type VII collagen to trigger an Fcg-dependent inflammation leading to split formation in cryosections of human skin.
The hematopoietic-specific Rho-family GTP exchange factor (GEF) Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells. Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, I show that Vav-1 is also expressed in murine NK cells. Vav-1-/- mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as cells from wild type mice. Unexpectedly, IL-2-activated Vav-1-/- NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1-/- NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, these results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK cell natural cytotoxicity. Vav-2, a protein homologous to Vav-1 has also been implicated in NK cell functions. However, NK cells from Vav-2-/- mice have normal cytotoxic activities and NK cells that lack both Vav-1 and Vav-2 exhibit similar defect as Vav-1-/- cells. Thus Vav-2 has no apparent function in the development and the activation of NK cells. Although NK cell development is normal in Vav-1-/- mice, their numbers of NKT cells were dramatically diminished. Furthermore, NKT cells from Vav-1 mutant mice failed to produce IL-4 and IFNg following in vivo CD3 stimulation. A similar loss of NKT cells was observed in Vav-1-/-Vav-2-/- mice, but not in Vav-2-/- mice, suggesting that only Vav-1, and not Vav-2, is an essential regulator of NKT cell development and NK cell cytotoxicity. Similar to Vav-1, Lsc is a Rho GEF that is expressed specifically in the hematopoietic system. It contains a regulator of G-protein signaling (RGS) domain which negatively regulates the Ga12 and Ga13 subunits of G-protein coupled receptors (GPCRs). This study shows that NK and NKT cell development are normal in Lsc-/- mice. However, NK cells from mutant mice display enhanced cytotoxic responses towards a panel of tumor cells. These data implicate for the first time a RGS-containing Rho GEF in cytotoxic responses and suggest that Lsc down-modulate NK cell activation.
OMB and ORG-1
(2002)
Members of the T-box gene family encode transcription factors that play key roles during embryonic development and organogenesis of invertebrates and vertebrates. The defining feature of T-box proteins is an about 200 aa large, conserved DNA binding motif, the T domain. Their importance for proper development is highlighted by the dramatic phenotypes of T-box mutant animals. My thesis was mainly focused on two Drosophila T-box genes, optomotor-blind (omb) and optomotor-blind related 1 (org-1), and included (i) a genetic analysis of org-1 and (ii) the identification of molecular determinants within OMB and ORG-1 that confer functional specificity. (i) Genetic analysis of org-1 initially based on a behavioral Drosophila mutant, C31. C31 is a X-linked, recessive mutant and was mapped to 7E-F, the cytological region of org-1. This pleiotropic mutant is manifested in walking defects, structural aberrations in the central brain, and "held-out" wings. Molecular analysis revealed that C31 contains an insertion of a 5' truncated I retrotransposon within the 3' untranslated transcript of org-1, suggesting that C31 might represent the first org-1 mutant. Based on this hypothesis, we screened 44.500 F1 female offspring of EMS mutagenized males and C31 females for the "held-out" phenotype, but failed to isolate any C31 or org-1 mutant, although this mutagenesis was functional per se. Since we could not exclude the possibility that our failure is due to an idiosyncracy of C31, we intended not to rely on C31 in further genetic experiments and followed a reverse genetic strategy . All P element lines cytologically mapping to 7E-7F were characterized for their precise insertion sites. 13 of the 19 analyzed lines had P element insertions within a hot-spot 37 kb downstream of org-1. No P element insertions within org-1 could be identified, but several P element insertions were determined on either side of org-1. The org-1 nearest insertions were used for local-hop experiments, in which we associated 6 new genes with P insertions, but failed to target org-1. The closest P elements are still 10 kb away from org-1. Subsequently, we employed org-1 flanking P elements to induce precise deletions in 7E-F spanning org-1. Two org-1 flanking P elements were brought together on a recombinant chromosome. Remobilization of P elements in cis configuration frequently results in deletions with the P element insertion sites as deficiency endpoints. In a first attempt, we expected to identify deficiencies by screening for C31 alleles. 8 new C31 alleles could be isolated. The new C31 chromosomes, however, did not carry the desired deletion. Molecular analysis indicated that C31 is not caused by aberrations in org-1, but by mutations in a distal locus. We repeated the P element remobilization and screened for the absence of P element markers. 4 lethal chromosomes could be isolated with a deletion of the org-1 locus. (ii) The consequences of ectopic org-1 were analyzed using UAS-org-1 transgenic flies and a number of different Gal4 driver lines. Misexpression of org-1 during imaginal development interfered with the normal development of many organs and resulted in flies with a plethora of phenotypes. These include a homeotic transformation of distal antenna (flagellum) into distal leg structures, a strong size reduction of the legs along their proximo-distal axis, and stunted wings. Like ectopic org-1, ectopic omb leads to dramatic changes of normal developmental pathways in Drosophila as well. dpp-Gal4/ UAS-omb flies are late pupal lethal and show an ectopic pair of wings and largely reduced eyes. GMR-Gal4 driven ectopic omb expression in the developing eye causes a degeneration of the photoreceptor cells, while GMR-Gal4/ UAS-org-1 flies have intact eyes. Hence, ectopic org-1 and omb induce profound phenotypes that are qualitatively different for these homologous genes. To begin to address the question where within OMB and ORG-1 the specificity determinants reside, we conceptionally subdivided both proteins into three domains and tested the relevance ofthese domains for functional specificity in vivo. The single domains were cloned and used as modules to assemble all possible omb-org-1 chimeric trans- genes. A method was developed to determine the relative expression strength of different UAS-transgenes, allowing to compare the various transgenic constructs for qualitative differences only, excluding different transgene quantities. Analysis of chimeric omb-org-1 transgenes with the GMR-Gal4 driver revealed that all three OMB domains contribute to functional specificity.
In this thesis, I examined honey bee nectar foraging with emphasis on the communication system. To document how a honey bee colony adjusts its daily nectar foraging effort, I observed a random sample of individually marked workers during the entire day, and then estimated the number and activity of all nectar foragers in the colony. The total number of active nectar foragers in a colony changed frequently between days. Foraging activity did not usually change between days. A honey bee colony adjusts its daily foraging effort by changing the number of its nectar foragers rather than their activity. I tested whether volatiles produced by a foraging colony activated nectar foragers of a non-foraging colony by connecting with a glass tube two colonies. Each colony had access to a different green house. In 50% of all experiments, volatile substances from the foraging colony stimulated nectar foragers of the non-foraging colony to fly to an empty feeder. The results of this study show that honey bees can produce a chemical signal or cue that activates nectar foragers. However, more experiments are needed to establish the significance of the activating volatiles for the foraging communication system. The brief piping signal of nectar foragers inhibits forager recruitment by stopping waggle dances (Nieh 1993, Kirchner 1993). However, I observed that many piping signals (approximately 43%) were produced off the dance floor, a restricted area in the hive where most waggle dances are performed. If the inhibition of waggle dances would be the only function of the brief piping signal, tremble dancers should produce piping signals mainly on the dance floor, where the probability to encounter waggle dancers is highest. To therefore investigate the piping signal in more detail, I experimentally established the foraging context of the brief piping signal, characterized its acoustic properties, and documented for the first time the unique behavior of piping nectar foragers by observing foragers throughout their entire stay in the hive. Piping nectar foragers usually began to tremble dance immediately upon their return into the hive, spent more time in the hive, more time dancing, had longer unloading latencies, and were the only foragers that sometimes unloaded their nectar directly into cells instead of giving it to a nectar receiver bee. Most of the brief piping signals (approximately 99%) were produced by tremble dancers, yet not all tremble dancers (approximately 48%) piped. This suggests that piping and tremble dancing have related, but not identical functions in the foraging system. Thus, the brief piping signals may not only inhibit forager recruitment, but have an additional function both on and off the dance floor. In particular, the piping signal might function 1. to stop the recruitment of additional nectar foragers, and 2. as a modulatory signal to alter the response threshold of signal receivers to the tremble dance. The observation that piping tremble dancers often did not experience long unloading delays before they started to dance gave rise to a question. A forager’s unloading delay provides reliable information about the relative work capacities of nectar foragers and nectar receivers, because each returning forager unloads her nectar to a nectar receiver before she takes off for the next foraging trip. Queuing delays for either foragers or receivers lower foraging efficiency and can be eliminated by recruiting workers to the group in shortage. Short unloading delays indicate to the nectar forager a shortage of foragers and stimulate waggle dancing which recruits nectar foragers. Long unloading delays indicate a shortage of nectar receivers and stimulate tremble dancing which recruits nectar receivers (Seeley 1992, Seeley et al. 1996). Because the short unloading delays of piping tremble dancers indicated that tremble dancing can be elicited by other factors than long unloading delays, I tested whether a hive-external stimulus, the density of foragers at the food source, stimulated tremble dancing directly. The experiments show that tremble dancing can be caused directly by a high density of foragers at the food source and suggest that tremble dancing can be elicited by a decrease of foraging efficiency either inside (e.g. shortage of receiver bees) or outside (e.g. difficulty of loading nectar) the hive. Tremble dancing as a reaction to hive-external stimuli seems to occur under natural conditions and can thus be expected to have some adaptive significance. The results imply that if the hive-external factors that elicit tremble dancing do not indicate a shortage of nectar receiver bees in the hive, the function of the tremble dance may not be restricted to the recruitment of additional nectar receivers, but might be the inhibition or re-organization of nectar foraging.
Low-molecular mass natural products from bacteria, fungi, plants and marine organisms exhibit unique structural diversity which are of interest for the identification of new lead structures for medicinals and agrochemicals. In the search for bioactive compounds from marine sponges and sponge-associated fungi, this research work resulted to the isolation of twenty-six compounds, eight of which are new metabolites. The sponges were collected from the Indo-pacific regions, particularly those from Indonesian and Philippine waters, as well as those from the Mediterranean Sea near the island of Elba in Italy. A combination of the chemically- and biologically-driven approach for drug discovery was employed, wherein extracts were screened for antibacterial, antifungal and cytotoxic activities. In addition to the bioassay-guided approach to purify the compounds responsible for the activity of the extract, TLC, UV and MS were also used to isolate the chemically most interesting substances. Hence, purified compounds which are not responsible for the initial bioscreening activity may have a chance to be evaluated for other bioactivities. Enumerated below are the compounds which have been isolated and structurally elucidated and whose bioactivities have been further characterized. 1. The extract of the fungus Cladosporium herbarum associated with the sponge Callyspongia aerizusa afforded seven structurally related polyketides, including two new twelve-membered macrolides: pandangolide 3 and 4, and a new acetyl congener of the previously isolated 5-hydroxymethyl-2-furoic acid. The two furoic acid analogues isolated were found to be responsible for the antimicrobial activity of the extract. The isolation of the known phytotoxin Cladospolide B from Cladosporium herbarum, which was originally known from Cladosporium cladosporioides and C. tenuissimum, indicates the possibility that Cladospolide B may be a chemotaxonomic marker of particular Cladosporium species. 2. The extract of the fungus Curvularia lunata associated with the Indonesian sponge Niphates olemda yielded three compounds, namely the new antimicrobially-active anthraquinone lunatin, the known bisanthraquinone cytoskyrin A, and the known plant hormone abscisic acid. The co-occurrence of the two structurally-related anthraquinones suggests that the monomeric lunatin may be a precursor in the biosynthesis of the bisanthraquinone cytoskyrin A. 3. The fungus Penicillium spp. associated with the Mediterranean sponge Axinella verrucosa yielded six compounds, namely the known antifungal griseofulvin and its less active dechloro analogue; the known toxin oxaline; and the known cytotoxic metabolite communesin B and its two new congeners communesin C and D. The new communesins were less active than communesin B in the brine-shrimp lethality test. 4. An unidentified fungus which was also isolated from the same Mediterranean sponge Axinella verrucosa as Penicillium spp. yielded the known compound monocerin which has been reported to possess phytotoxic and insecticidal activities. 5. The fungus Aspergillus flavus associated with the Philippine sponge Hyrtios aff. reticulatus yielded the known toxin a-cyclopiazonic acid. 6. The Indonesian sponge Agelas nakamurai yielded four bromopyrrole alkaloids namely the new compound 4-bromo-pyrrole-2-carboxylic acid, and the known compounds: 4-bromo-pyrrole-2-carboxamide, mukanadin B and mukanadin C. All of the four compounds except mukanadin B were found to be antimicrobially-active. Bromopyrrole alkaloids are well-known metabolites of the genus Agelas and are proven to play an important role in the chemical defense of the sponge against predation from fishes. 7. The Indonesian sponge Jaspis splendens yielded three known substances which are known for their antiproliferative activities, namely the depsipeptides jaspamide (jasplakinolide), and its derivatives jaspamide B and jaspamide C.
Normoxic and anoxic metabolism of Nicotiana tabacum transformants lacking root nitrate reductase
(2002)
The aim of this work was to find out whether and how nitrate reduction in roots would facilitate survival of hypoxic and anoxic (flooding)-phases. For that purpose, we compared the response of roots of hydroponically grown tobacco wildtype (Nicotiana tabacum cv. Gatersleben) and of a transformant (LNR-H) with no nitrate reductase (NR) in the roots but almost normal NR in leaves (based on a nia2-double mutant). As an additional control we used occasionally a 35S-transformant of the same nia2-double mutant, which on the same genetic background constitutively expressed NR in all organs. In some cases, we also compared the response of roots from WT plants, which had been grown on tungstate for some time in order to completely suppress NR activity. The following root parameters were examined: 1) Growth and morphology 2) Root respiration rates and leaf transpiration 3) Metabolite contents in roots (ATP, hexosemonophosphates, free sugars, starch, amino acids, total protein) 4) Inorganic cation and anion contents 5) Lactate and ethanol production 6) Extractable LDH-and ADH-activities 7) Cytosolic pH values (by 31P-NMR) 8) NO Cation and anion contents of roots from WT and LNR-H were only slightly different, confirming that these plants would be better suited for our purposes than the widely used comparison of nitrate-versus ammonium-grown plants, which usually show up with dramatic differences in their ion contents. Normoxia: LNR-H-plants had shorter and thicker roots than WT with a lower roots surface area per leaf FW. This was probably the major cause for the significantly lower specific leaf transpiration of LNR-H. WT-roots had lower respiration rates, lower ATP-and HMP-contents, slightly lower sugar- and starch contents and somewhat lower amino acid contents than LNR-H roots. However, total protein/FW was almost identical. Obviously the LNR-H transformants did not suffer from N-defciency, and their energy status appeared even better than that of WT-roots. Data from the 35S-transformant were similar to those of WT. This indicates that the observed differences between WT and LNR-H were not due to unknown factors of the genetic nia2-background, but that they could be really traced back to the presence resp. absence of nitrate reduction. Anoxia: Under short-term anoxia (2h) LNR-H plants, but not WT-plants exhibited clear symptoms of wilting, although leaf transpiration was lower with LNR-H. Reasons are not known yet. LNR-H roots produced much more ethanol (which was excreted) and lactate compared to WT, but extractable ADH and LDH activities, were not induced by anoxia. However, the LDH activity background was twice as high as that of the WT troughout the time period studied. Tungstate-treated WT-roots also gave higher fermentation rates than normal WT roots. Sugar- and HMP-contents remained higher in LNR-H roots than in WT. NR in WT roots was activated under anoxia and roots accumulated nitrite, which was also released to the medium. 31P-NMR spectroscopy showed that LNR-H- roots, in spite of their better energy status, acidified their cytosol more than WT roots. Conclusions: Obviously nitrate reduction affects - by as yet unknown mechanisms - root growth and morphology. The much lower anoxic fermentation rates of WT-roots compared to LNR-H roots could not be traced back to an alternative NADH consumption by nitrate reduction, since NR activity was too low for that. An overall estimation of H+-production by glycolysis, fermentation and nitrate reduction (without nitrite reduction, which was absent under anoxia) indicated that the stronger cytosolic acidification of anoxic LNR-H roots was based on their higher fermentation rates. Thus, nitrate reduction under anoxia appears advantageous because of lower fermentation rates and concomitantly lower cytosolic acidification. However, it remained unclear why fermentation rates were so different. Perspective: Preliminary experiments had indicated that WT-roots produced more nitric oxide (NO) under anoxia than LNR-H-roots. Accordingly, we suggest that nitrate reduction, beyond a merely increased NADH-consumption, would lead to advantageous changes in metabolism, eventually via NO-production, which is increasingly recognized as an important signaling compound regulating many plant functions.
The classification of isoparametric hypersurfaces in spheres with a homogeneous focal manifold is a project that has been started by Linus Kramer. It extends results by E. Cartan and Hsiang and Lawson. Kramer does most part of this classification in his Habilitationsschrift. In particular he obtains a classification for the cases where the homogeneous focal manifold is at least 2-connected. Results of E. Cartan, Dorfmeister and Neher, and Takagi also solve parts of the classification problem. This thesis completes the classification. We classify all closed isoparametric hypersurfaces in spheres with g>2 distinct principal curvatures one of whose multiplicities is 2 such that the lower dimensional focal manifold is homogeneous. The methods are essentially the same as in Kramer's 'Habilitationsschrift'. The cohomology of the focal manifolds in question is known. This leads to two topological classification problems, which are also solved in this thesis. We classify simply connected homogeneous spaces of compact Lie groups with the same integral cohomology ring as a product of spheres S^2 x S^m and m odd on the one hand and a truncated polynomial ring Q[a]/(a^m) with one generator of even degree and m > 1 as its rational cohomology ring on the other hand.
In the various groups of social bees, different systems of communication about food sources occur. These communication systems are different solutions to a common problem of social insects: efficiently allocating the necessary number of workers first to the task of foraging and second to the most profitable food sources. The solution chosen by each species depends on the particular ecological circumstances as well as the evolutionary history of that species. For example, the outstanding difference between the bumble bee and the honey bee system is that honey bees can communicate the location of profitable food sources to nestmates, which bumble bees cannot. To identify possible selection pressures that could explain this difference, I have quantified the benefits of communicating location in honey bees. I show that these strongly depend on the habitat, and that communicating location might not benefit bees in temperate habitats. This could be due to the differing spatial distributions of resources in different habitats, in particular between temperate and tropical regions. These distributions may be the reason why the mostly temperate-living bumble bees have never evolved a communication system that allows them to transfer information on location of food sources, whereas most tropical social bees (all honey bees and many stingless bees) are able to recruit nestmates to specific points in their foraging range. Nevertheless, I show that in bumble bees the allocation of workers to foraging is also regulated by communication. Successful foragers distribute in the nest a pheromone which alerts other bees to the presence of food. This pheromone stems from a tergite gland, the function of which had not been identified previously. Usage of a pheromone in the nest to alert other individuals to forage has not been described in other social insects, and might constitute a new mode of communicating about food sources. The signal might be modulated depending on the quality of the food source. Bees in the nest sample the nectar that has been brought into the nest. Their decision whether to go out and forage depends not only on the pheromone signal, but also on the quality of the nectar they have sampled. In this way, foraging activity of a bumble bee colony is adjusted to foraging conditions, which means most bees are allocated to foraging only if high-quality food sources are available. In addition, foraging activity is adjusted to the amount of food already stored. In a colony with full honeypots, no new bees are allocated to foraging. These results help us understand how the allocation of workers to the task of food collection is regulated according to external and internal nest conditions in bumble bees.
S=N versus S+-N-
(2002)
The main aim of this thesis was to characterise structurally four sulfur-nitrogen compounds in terms of their experimental electron density distribution: Sulfurdiimide S(NtBu)2 (I), sulfurtriimide S(NtBu)3 (II), methyl(diimido)sulfinic acid H(NtBu)2SMe (III) and methylene-bis(triimido)sulfonic acid CH2{S(NtBu)2(HNtBu)}2 (IV). The electron density was determined by multipole refinements on high-resolution X-ray data at low temperatures. The refined densities were analysed by means of Bader’s theory of ‘Atoms in Molecules’ to get information about the bonding types (shared/ closed shell), bond strengths, and the extent of polarisation. The distributions of the static deformation densities, which already showed the most important electronical features as lone-pairs and bonding densities, were calculated for all compounds. The spatial distributions provided a first impression about the bonding properties. The nitrogen lone-pair densities were found to be inclined towards the electropositive sulfur atoms. In II, III and IV the spatial distributions already suggested sp3 hybridisation of the nitrogen atoms. In I gradual differences between the E/Z and Z/Z oriented NtBu groups were visualised. The charge density distribution was analysed along the bond paths, which showed some of the S,N bonds to be considerably bent. In the central part of the thesis detailed topological analyses of the electron density distributions were performed. All BCPs and the related electronical properties as the electron density, the negative Laplacian, the eigenvalues of the Hessian matrix, and several values, which can be deduced from these, were calculated. Due to the low number of comparable published compounds, internal scaling facilitated by III and IV led to system-specific ranking of the S-N and S-C bonds in terms of bond type (shared vs. closed shell), bond order, and bond strength. To quantify bond polarisation a criterion was developed which relates shifts in the BCPs to electron transfer from the electropositive to the electronegative bonding partner. The distributions of the Laplacian were determined for all S-E (E = N, C) bonds because of their fundamental importance for the classification of atomic interactions. Furthermore, the spatial distribution of the negative Laplacian with respect to all important bonds was determined around the central sulfur and nitrogen atoms. The analyses led to detailed information about the S,N interactions. A calculation of the reactive surfaces where the Laplacian equals zero revealed possible reaction pathways of nucleophilic attacks to the central sulfur atoms. All nitrogen atoms in H(NtBu)2SMe (III) as well as in CH2{S(NtBu)2(HNtBu)}2 (IV) are predominantly sp3 hybridised. The S,N bonds should therefore be formulated as S+–N– single bonds, strengthened and shortened by electrostatic reinforcement. In S(NtBu)2 (I) the sp2 hybridisation of the nitrogen atoms was verified. All topological criteria unearthed the inequality of the formally equivalent S=N double bonds. The differences were assigned to the molecular E/Z conformation in the solid state. Interaction between the in-plane lone-pair density of the nitrogen and the sulfur atom located at the same side causes the non-bonding charge concentration at the sulfur atom to be dislocated into the second S–N bond. The existence of a delocalised 3-centres-2-electrons system within the planar SN2 core was assumed to be formed by non-hybridised p-orbitals. An effective delocalisation was found to be possibly disturbed by a weak intermolecular S...S interaction. The interpretation of the S,N interaction in S(NtBu)3 (II) was not straightforward, since the electron density distribution showed both, indicators for multiple bonding as well as for sp3 hybridisation of the nitrogen atoms, which verifies the formulation of a S+–N– bonding mode. The bonding situation in S(NtBu)3 was identified as an intermediate state between that of a delocalised 4-centres-6-electrons system formed by non-hybridised p-orbitals within the planar SN3 unit and that of a S+–N– system.
A quantitative model of groundwater flows contributing to the Goblenz state water scheme at the north-western fringe of the Kalahari was developed within this study. The investigated area corresponds to the Upper Omatako basin and encompasses an outer mountainous rim and sediments of the Kalahari sand desert in the centre. This study revealed the eminent importance of the mountainous rim for the water balance of the Kalahari, both in terms of surface and ground water. A hydrochemical subdivision of groundwater types in the mountain rim around the Kalahari was derived from cluster analysis of hydrochemical groundwater data. The western and south-western secondary aquifers within rocks of the Damara Sequence, the Otavi Mountain karst aquifers of the Tsumeb and Abenab subgroups as well as the Waterberg Etjo sandstone aquifer represent the major hydrochemical groups. Ca/Mg and Sr/Ca ratios allowed to trace the groundwater flow from the Otavi Mountains towards the Kalahari near Goblenz. The Otavi Mountains and the Waterberg were identified as the main recharge areas showing almost no or only little isotopic enrichment by evaporation. Soil water balance modelling confirmed that direct groundwater recharge in hard-rock environments tends to be much higher than in areas covered with thick Kalahari sediments. According to the water balance model average recharge rates in hard-rock exposures with only thin sand cover are between 0.1 and 2.5 % of mean annual rainfall. Within the Kalahari itself very limited recharge was predicted (< 1 % of mean annual rainfall). In the Upper Omatako basin the highest recharge probability was found in February in the late rainfall season. The water balance model also indicated that surface runoff is produced sporadically, triggering indirect recharge events. Several sinkholes were discovered in the Otavi Foreland to the north of Goblenz forming short-cuts to the groundwater table and preferential recharge zones. Their relevance for the generation of indirect recharge could be demonstrated by stable isotope variations resulting from observed flood events. Within the Kalahari basin several troughs were identified in the pre-Kalahari surface by GIS-based analyses. A map of saturated thickness of Kalahari sediments revealed that these major troughs are partly saturated with groundwater. The main trough, extending from south-west to north-east, is probably connected to the Goblenz state water scheme and represents a major zone of groundwater confluence, receiving groundwater inflows from several recharge areas in the Upper Omatako basin. As a result of the dominance of mountain front recharge the groundwater of the Kalahari carries an isotopic composition of recharge at higher altitudes. The respective percentages of inflow into the Kalahari from different source areas were determined by a mixing-cell approach. According to the mixing model Goblenz receives most of its inflow (70 to 80 %) from a shallow Kalahari aquifer in the Otavi Foreland which is connected to the Otavi Mountains. Another 15 to 10 % of groundwater inflow to the Kalahari at Goblenz derive from the Etjo sandstone aquifer to the south and from inflow of a mixed component. In conclusion, groundwater abstraction at Goblenz will be affected by measures that heavily influence groundwater inflow from the Otavi Mountains, the Waterberg, and the fractured aquifer north of the Waterberg.
In north-western Namibia the fills of the Karoo-Etendeka depositories can be subdivided into (1) a Carboniferous-Permian, (2) a Triassic-Jurassic and (3) a Cretaceous megasequence, each recording extensional periods related to successive rifting phases in the evolving South Atlantic. The tectonic environment of the depositories in north-western Namibia changes successively from the coast towards the continental interior, which is reflected by the facies distribution and the position of time-stratigraphic gaps. Close to the present-day coastline synsedimentary listric faults, trending parallel to the South Atlantic rift (N-S), caused the formation of wedge shaped sediment bodies. Here, the Karoo Supergroup is only represented by the Permian succession in the Huab area. A hiatus within the Permian can be recognised by the correlation with the main Karoo Basin in South Africa and the Brazilian Paraná Basin. This stratal gap correlates with a pre-Beaufort Group unconformity in the main Karoo Basin that might be related to an orogenic pulse in the Cape Fold Belt. The Permian succession itself is unconformably overlain by the Lower Cretaceous Etendeka Group. This hiatus extending from the Upper Permian to the Lower Cretaceous has probably been induced by a combination of rift shoulder uplift and additional crustal doming associated with Etendeka flood volcanism. The enhanced tectonism during the Early Cretaceous controlled accommodation space for the alluvial-fluvial and aeolian deposits of the lower Etendeka Group. Disconformities within those deposits and the overlying lava succession attribute to distinct phases of tectonic and volcanic activity heralding the South Atlantic breakup. Towards the south-east, the Karoo succession becomes successively more complete. In the vicinity of Mt. Brandberg Early Triassic strata (Middle Omingonde Formation) follow disconformably above the Upper Permian/Lowermost Triassic Doros Formation. The sedimentation there was essentially controlled by the SW-NE trending Damaraland Uplift. South of the Damaraland Uplift the SW-NE trending Waterberg-Omaruru Fault zone is interpreted as a sinistral oblique-slip fault that compartmentalised the South Atlantic rift. This fault controlled accommodation space of the entire Triassic Omingonde Formation and the Early Jurassic Etjo Formation in its associated pull-apart and transtension structures. A locally well developed angular unconformity defines a hiatus between the two formations. Correlation with the main Karoo Basin in South Africa confirms that this gap is of a regional extent and not only a local, fault induced feature. Furthermore, it might also correlate with an orogenic pulse of the Cape Fold Belt. In general, the Mesozoic megasequences record the long-lived history of the southern Atlantic rift evolution. Rifting has been controlled by orogenic pulses derived from the Samfrau active margin throughout the Mesozoic. The associated intracratonic E-W extension caused the formation of grabens and conjugated oblique-slip zones. The generation of voluminous flood basalts marks the climax of intracratonic extension that was accompanied by enhanced uplift of the rift shoulders.
Clinical evaluation of novel methods to determine dialysis parameters using conductivity cells
(2002)
Eine die letzten beiden Jahrzehnte anhaltende Diskussion über die erforderliche Dosis Dialyse, die ein Patient benötigt, ergab, daß der Harnstoff-basierte Kt/V-Wert signifikant mit der Morbidität von ‚end stage renal deasease' (ESRD)- Patienten korreliert. Wenn auch nicht vollständig akzeptiert, so scheint es doch zunehmende Übereinkunft zwischen Nephrologen, daß eine angemessene Dialysebehandlung von Patienten ohne Restdiurese im Rahmen eines 3x4 Stunden pro Woche- Schemas mindestens einen Kt/V-Wert von 1.2 bis 1.3 ergeben sollte, um auf lange Sicht die Lebensqualität zu sichern und die Morbidität und Mortalität niedrig zu halten. K ist die vom Dialysesystem erbrachte Clearance, t die Behandlungszeit und V das Harnstoff-Verteilungsvolumen, welches dem Gesamt-Körperwasser nahezu gleicht. Kt/V wird in der Dosiseinheit (ml Medikament pro ml Körperwasser) ausgedrückt und daher auch häufig als Dialyse-‚Dosis' bezeichnet, auch wenn darüber wegen der Zusammenfassung in nur einer Harnstoff-korrelierten Zahl konträr diskutiert wird. Diese Arbeit besitzt eine eher technische Prämisse und möchte sich nicht an dieser Diskussion beteiligen. Sie möchte dem interessierten Nephrologen lediglich eine patientenfreundliche, präzise, kostenneutrale und leicht zu handhabende technische Lösung an die Hand geben, um kontinuierlich die in Kt/V ausgedrückte Dialysedosis zu überwachen. Natürlich ist damit auch die Hoffnung verbunden, daß die Langzeit-Sterblichkeit verringert werden kann, wenn eine flächendeckende, zeitnahe Erfolgskontrolle der Dialyse ermöglicht wird. Die gewählte technische Lösung basiert auf der Äquivalenz der Diffusionskoeffizienten von gelöstem Harnstoff und Natriumchlorid. Es ist das zentrale Anliegen dieser Studie, festzustellen, ob das Diffusionsverhalten von NaCl und Harnstoff beim Durchtritt durch die Membran des Dialysefilters gleich ist. Der entscheidende Vorteil, der das Verfahren so leicht handhabbar macht, besteht darin, daß NaCl-Konzentrationen sehr genau durch die ohnehin in großer Zahl in Dialysegeräten verwendeten Leitfähigkeitsmeßzellen bestimmt werden können. Um die NaCl-Massenbilanz über den Dialysefilter zu bestimmen, benötigt man lediglich eine weitere Meßzelle, die stromab des Filters zu installieren ist. Die Messung von Harnstoff, die indirekt über die enzymatische Zerlegung in Ammonium-Ionen und das anschließende Erzeugen eines hoch zu verstärkenden elektrischen Potentials an einer Membran geschieht, ist komplizierter. Zudem ist eine geschlossene Kühlkette für das Enzym sicher zu stellen. Um eine leitfähigkeitsbasierte technische Lösung abzusichern, wurden zwei klinische Studien durchgeführt. In der ersten Studie wurde die Leitfähigkeit in Form von konstanten Stufenprofilen variiert. Sie wurde ausgehend von der Grundlinie für 7 min erhöht und anschließend für 7 min erniedrigt. Das Prinzip einer solchen Messung wurde erstmals 1982 in einer Patentschrift beschrieben. In einer Sequenz von 494 solchen Messungen in 206 automatisch aufgezeichneten Dialysesitzungen an 22 Patienten wurde gefunden, daß sich die Harnstoff- Clearance elektrolytisch mit einer Genauigkeit von -1.46+/-4.75% (Fehler+/-Standardabweichung) messen ließ. Die Messung von Kt/V gemäß dem Ein-Kompartment-Modell ergab eine ähnliche Genauigkeit von 2.88+/-4.15%. Obgleich diese Ergebnisse in Übereinstimmung mit anderen Studien stehen, wurde ein Effekt bemerkt, der nicht in Einklang mit der zunächst bestehenden Theorie zu bringen war. Dieser Effekt besteht darin, daß die Genauigkeit der elektrolytischen Clearancemessung vom beim Patienten vorhandenen Harnstoff-Verteilungsvolumen abhängig war. Weiter war es nicht bedeutungslos, welchen Teil des dreiteiligen Stufenprofils man zur Auswertung heranzog: Den Grundlinie-Hoch- Übergang, den von der Grundlinie zum Niedrig-Nieveau oder den Hoch-Niedrig- Übergang. Dies deutete auf einen Mangel an theoretischem Verständnis hin. Eine genaue weitere Untersuchung führte zu dem Ergebnis, daß unerwünschter NaCl-Transfer vom und zum Patienten die Ursache für die Abhängigkeit vom Verteilungsvolumen war. Es wurde daraufhin die Theorie dahingehend erweitert, daß dieser Effekt korrekt und plausibel beschrieben werden konnte. Aus dieser Erweiterung ergab sich die neue Forderung, den NaCl-Transfer bei der Messung weitestgehend zu minimieren. Die Benutzung von Stufenprofilen stellte hier jedoch an sich eine Limitierung dar, da in der zum Einnehmen eines stabilen Zustandes erforderlichen Zeit zuviel NaCl über die Membran transferiert wurde. Die Konsequenz war, von den Stufenprofilen auf kurze, dynamische Leitfähigkeitsboli überzugehen, die erlaubten, die NaCl-Gabe auf das Maß zu verringern, welches aufgrund der technischen Auflösung erforderlich war. Hierzu mußten jedoch die notwendigen mathematischen Algorithmen neu zugeschnitten werden. Nach diesem Schritt wurde eine weitere klinische Studie gestartet, die den Zweck verfolgte, das neue Verfahren am Patienten zu verifizieren. In dieser Studie mit 10 Patienten und 93 Dialysesitzungen, 264 Stufenprofil- und 173 Bolus-Dialysancemesssungen wurde gefunden, daß die Bolus-Messungen ihre zugehörigen blutseitigen Referenzmessungen mit außergewöhnlicher Genauigkeit von 0.06+/-4.76% trafen. Student's t-Test für gepaarte Daten ergab, daß sich die Datensätze nicht signifikant unterschieden (p=0.87). Die blutseitige Kt/V-Referenz auf der Basis des equilibrierten Einkompartment-Modells mit variablem Volumen wurde mit 5.32+/-3.9% getroffen, wobei eine Korrelation von 0.98 erzielt wurde. Die verbleibende Differenz von 5.32% wird der Vernachlässigung der Harnstoff-Erzeugung während der Messung zugeschrieben. Auch das Stufenprofil zeigte trotz seiner Abhängigkeit vom Verteilungsvolumen gegenüber dem gleichen Modell einen mittleren Fehler von 0.05+/-5% bei einer Korrelation von 0.96. Jedoch konnte es die Vernachlässigung der Harnstoff-Erzeugung nicht korrekt abbilden. Die kontinuierlich aufgenommenen Daten wurden auch nach dem 2-pool Modell untersucht, welches auch die Harnstoff-Erzeugung enthält sowie eine innere Kompartimentierung des Patienten annimmt und damit die tatsächlichen Verhältnisse besser beschreibt. Danach weicht das Bolus-Meßprinzip -3.04+/-14.3% von der Referenz ab (n.s.,p=0.13). Die relativ hohe Standardabweichung wird mit der Komplexität des Modells erklärt. Weiter ist aus der Theorie zum Na-Transfer eine vereinfachende Methode zur Messung des Na-Verteilungsvolumens abgeleitet worden. Diese Methode wurde in-vitro gegen ein Behältnis mit einer bekannten Menge an Dialysat geprüft. Es wurde ein mittlerer Fehler von -19.9+/-34% gefunden. Die Korrelation war 0.92 (n.s., p=0.916). Die gleiche Prüfung fand in-vivo gegen das Harnstoff-Verteilungsvolumen statt und ergab einen mittleren Fehler von -7.4+/-23.2% (r=0.71, n.s., p=0.39). Es hat den Anschein, als würden sich gemäß der Theorie der Dilution die Verteilungsvolumina für Natrium und für Harnstoff scheinbar nur wenig unterscheiden, obwohl sie sich absolut natürlich deutlich unterscheiden. In Anbetracht der erheblichen Vereinfachungen, die bei der Ableitung dieses Ansatzes gemacht wurden, scheint es ausgesprochen ermutigend, auf diesem Weg möglicherweise ein Verfahren entwickeln zu können, welches auf rein elektrolytischem Wege nun nicht mehr nur K, sondern auch V und damit alle zur Quantifizierung gesuchten Größen analytisch ermitteln kann. Weiter wurde im Rahmen dieser Arbeit anhand der analytisch ermittelten Volumina verglichen, ob man mit Hilfe anthropometrischer Formeln zur Ermittlung des Harnstoff-Verteilungsvolumens zu einer guten Abschätzung kommen kann. Es wurde gefunden, daß man das Ergebnis der Watson-Formel um ca. 13% vermindern kann und dann zu einem recht guten Wert für das tatsächliche Harnstoff-Verteilungsvolumen gelangt. Dies ist jedoch mit Vorsicht und Erfahrung zu tun, da sich damit Kt/V rechnerisch zum Nachteil des Patienten verändert. Auch ein elektrolytisches Verfahren mit empirischen Komponenten zur Ermittlung des Plasma-Natriums des Patienten wurde erprobt und konnte mit einer Genauigkeit von 4.3+/-1.2% den Laborwert vorhersagen. Zusammenfassend kann gesagt werden, daß sich im Rahmen dieser Arbeit die leitfähigkeitsbasierten Methoden zur Messung von einigen wichtigen Dialyseparametern als sehr nützlich für die klinische Praxis erwiesen haben und zudem mit keinem Zusatzaufwand für die Beteiligten verbunden sind. Das Ergebnis dieser Arbeit ist mittlerweile in größeren Stückzahlen in frei erhältliche Dialysegeräte implementiert worden. Die Erfahrung der ersten Zeit zeigt, daß das verwendete Prinzip von den Klinikern gut angenommen wird.
Previous work on Jurassic bivalves from the Iberian Range is reviewed, whereby emphasis is placed on Callovian-Kimmeridgian species. The taxonomy, distribution pattern and ecology of the bivalve fauna occurring in Middle and Upper Jurassic rocks of the Aragonian Branch of the Iberian Range have been analysed. For this purpose 14 sections and 5 additional outcrops, selected according to the abundance of bivalves, were measured in detail and sampled. The rocks studied belong to the Chelva, Yátova, Sot de Chera and Loriguilla formations of Callovian-Kimmeridgian age. The distribution of species of bivalves is given for each section. More than 3000 specimens of bivalves representing 83 species that belong to 46 genera and subgenera of the subclasses Palaeotaxodonta, Pteriomorphia, Isofilibranchia. Palaeoheterodonta, Heterodonta and Anomaldesmata have been used for the taxonomic analysis. One species is new: Plagiostoma fuersichi from the Callovian of the Chelva Fm. The autecology (trophic group and life habit) of each bivalve has been discussed. 49 samples of four sections habe been selected for a quantitative palaeoecological analysis of the bivalve fraction of the benthic fauna. Five bivalve associations and two assemblages are recognised by a Q-mode hierarchical cluster analysis (Ward method). The main environmental factors controlling bivalve associations are thought to be substrate, water energy and distribution of organic matter. The bivalves exhibit a distinct spatial and temporal distribution pattern within the Aragonian Branch. Four of the bivalve associations occur in the Upper Oxfordian (Sot de Chera Fm) and one association in the Lower Callovian (Chelva Fm). In the Sot de Chera and Loriguilla formations, the abundance of bivalves decreases from NW to SE i.e., from relatively close to the shore line towards the distal-most part of the carbonate platform. In the Chelva Fm. bivalves are abundant in the Ariño region, interpreted as a palaeogeographic high. The distribution of bivalves might have been largely controlled by the availability of nutrients.
The present studies which have been performed in the work-group C-2 (Prof. W. Kiefer) within the program of the Sonderforschungsbereichs 347, deal with the FT-Raman and –IR spectroscopy on new organometallic complexes, synthesized in the work-groups B-2 (Prof. W. Malisch), B-3 (Prof. W. A. Schenk), D-1 (Prof. H. Werner) and D-4 (Prof. D. Stalke). The FT-Raman spectra recorded at 1064 nm led to very useful and interesting information. Furthermore, the DFT calculations which are known to offer promise of obtaining accurate vibrational wavenumbers, were successfully used for the assignment of the vibrational spectra. For the first time it has been possible to ascribe exactly the n(RhC) stretching mode in the vinylidene rhodium(I) complex trans-[RhF(=C=CH2)(PiPr3)2] by using isotopic substitution, in conjunction with theoretical calculations. This is also true for the complexes trans-[RhF(CO)(PiPr3)2], trans-[RhF(C2H4)(PiPr3)2], trans-[RhX(=C=CHPh)(PiPr3)2] (X = F, Cl, Br, I, Me, PhCºC) and trans-[RhX(CN-2,6-xylyl)(PiPr3)2] (X = F, Cl, Br, I, CºCPh). In addition, the comparison between the n(RhC) wavenumbers of the complexes trans-[RhF(=13C=13CH2)(PiPr3)2] and trans-[RhF(CO)(PiPr3)2], containing the isoelectronic ligands 13C=13CH2 and CO, which have the same reduced mass, indicated that the Rh-C bond is stronger in the carbonyl than in the vinylidene complex. Besides, the n(RhF) stretching mode, which has been observed at higher wavenumbers in the FT-Raman and -IR spectra of trans-[RhF(CO)(PiPr3)2], showed that the carbonyl ligand is a better p-acceptor and a less effective s-donor than the vinylidene one. Moreover, the comparison of the n(CºC) and n(Rh-C) modes from the FT-Raman spectrum of the complexes trans-[Rh(CºCPh)(L)(PiPr3)2] (L = C=CHPh, CO, CN-2,6-xylyl) point out that the p-acceptor ability of the ligand trans to CºCPh should rise in the order C=CH2 < CO < CN-2,6-xylyl £ C=CHPh. The investigated sensitivity of the n(RhC), n(CC), n(CO) and n(CN) vibrational modes to the electronic modifications occuring in the vinylidene, carbonyl, ethylene and isonitrile complexes, should allow in the future the examination of the p-acceptor or p-donor properties of further ligands. Likewise, we were able to characterize the influence of various X ligands on the RhC bond by using the n(RhC) stretching mode as a probe for the weakening of this. The calculated wavenumbers of the n(RhC) for the vinylidene complexes trans-[RhX(=C=CHR)(PiPr3)2], where R = H or Ph, suggested that the strength of the Rh=C bond increases along the sequence X = CºCPh < CH3 < I < Br < Cl < F. For the series of carbonyl compounds trans-[RhX(CO)(PiPr3)2], where X = F, Cl, Br and I, analogous results have been obtained and confirmed from the model compounds trans-[RhX(CO)(PMe3)2]. Since, the calculated vibrational modes for the ethylene complex trans-[RhF(C2H4)(PiPr3)2] were in good agreement with the experimental results and supported the description of this complex as a metallacyclopropane, we were interested in getting more information upon this class of compounds. In this context, we have recorded the FT-Raman and -IR spectra of the thioaldehyde complexes mer-[W(CO)3(dmpe)(h2-S=CH2)] and mer-[W(CO)3(dmpe)(h2-S=CD2)] which have been synthezised by B-3. The positions of the different WL vibrational modes anticipated by the DFT calculations, were consistent with the experimental results. Indeed, the analysis of the band shifts in the FT-Raman and –IR spectra of the isotopomer mer-[W(CO)3(dmpe)(h2-S=CD2)] confirmed our assignment. The different stereoisomers of complex mer-[W(CO)3(dmpe)(h2-S=CH2)] were investigated too, since RMN and IR-data have shown that complex mer-[W(CO)3(dmpe)(h2-S=CH2)] lead in solution to an equilibrium. Since the information on the vibrational spectra of the molybdenum and tungsten complexes Cp(CO)2M-PR2-X (M = Mo, W; R = Me, tBu, Ph; X = S, Se) is very scarce, we extended our research work to this class of compounds. We have tried to elucidate the bonding properties in these chalcogenoheterocycle complexes by taking advantage of the mass effect on the different metal atoms (W vs. Mo). Thus, the observed band shifts allowed to assign most of the ML fundamental modes of these complexes. This project and the following one were a cooperation within the work-group B-2. The Raman and IR spectra of the matrix isolated photoproducts expected by the UV irradiation of the iron silyl complex Cp(CO)2FeSiH2CH3 have been already reported by Claudia Fickert and Volker Nagel in their PhD-thesis. Since no exact assignment was feasible for these spectra, we were interested in the study of the reaction products created by irradiation of the carbonyl iron silyl complex Cp(CO)2FeCH2SiH3. Although the calculated characteristic vibrational modes of the metal ligand unit for the various photoproducts are significantly different in constitution, they are very similar in wavenumbers, which did not simplify their identification. However, the theoretical results have been found to be consistent with the earlier experimental results. Finally, the last part of this thesis has been devoted to the (2-Py)2E- anions which exhibit a high selectivity toward metal-coordination. All di(2-pyridyl) amides and -phosphides which were synthesized by D-4, coordinate the R2Al+ fragment via both ring nitrogen atoms. This already suggests that the charge density in the anions is coupled into the rings and accumulated at the ring nitrogen atoms, but the Lewis basicity of the central nitrogen atom in Et2Al(2-Py)2N is still high enough to coordinate a second equivalent AlEt3 to form the Lewis acid base adduct Et2Al(2-Py)2NAlEt3. Due to the higher electronegativity of the central nitrogen atom in Me2Al(2-Py)2N, Et2Al(2-Py)2N and Et2Al(2-Py)2NAlEt3, compared to the bridging two coordinated phosphorus atom in Me2Al(2-Py)2P and Et2Al(2-Py)2P, the di(2-pyridyl)amide is the hardest Lewis base. In the phosphides merely all charge density couples into the rings leaving the central phosphorus atom only attractive for soft metals. These results were confirmed by using DFT and MP2 calculations. Moreover, a similar behaviour has been observed and described for the benzothiazolyl complex [Me2Al{Py(Bth)P}], where complementary investigations are to be continued. The DFT calculations carried out on the model compounds analysed in these studies supply very accurate wavenumbers and molecular geometries, these being in excellent agreement with the experimental results obtained from the corresponding isolated complexes.
The presented work shows the analysis of the correlation between the spatial and temporal expression pattern of NtAQP1 and its function in water relation in planta. In situ immunological studies indicated NtAQP1-protein accumulation in the root exodermis and endodermis, in the cortex, close to vascular bundles, in the xylem parenchyma and in cells of the stomatal cavities. The aquaporin was also found to be abundant in longitudinal cell-rows in the petioles. Expression studies with generated transgenic plants (Ntaqp1-promoter::gus or luc) confirmed the Ntaqp1 accumulation in the root, stem and petioles but also revealed further localization in pollen grains, adventitious roots and leaf glandular hairs. Ntaqp1-expression was induced during growth processes, like stem bending after gravistimulation or photostimulation, seed germination and hypocotyl elongation as well as during the comparatively fast circadian leaf movement. The expression was further stimulated by phytohormones, especially gibberellic acid (GA) and osmotic stress. Further analysis displayed a diurnal and even circadian expression of Ntaqp1 in roots and petioles. The functional analysis of the aquaporin was accomplished by reverse genetics and biophysical studies. The antisense technique was used to reduce NtAQP1-expression in tobacco plants. The antisense (AS) plants exhibited a severe reduction of Ntaqp1-mRNA, less reduction of the highly homologous NtPIP1a RNA and no effect on expression of other aquaporin family genes (PIP2, TIP). The function of NtAQP1 at the cellular level was investigated by a newly developed experimental setup to record the osmotically induced increase in protoplast volume. The reduction of NtAQP1 by the antisense expression decreased the overall cellular waterpermeability Pos for more than 50 %. Function of NtAQP1 at the whole plant level was e.g. measured by the “high-pressure flow meter method”. Those measurements revealed that the root hydraulic conductivity per unit root surface area (KRA) of roots from the AS-lines was reduced by more than 50 %. KRA displayed a strong diurnal and circadian variation with a maximum in the middle of the light period, similar to the expression pattern of Ntaqp1 in roots. Gas exchange-, stem (Ystem) and leaf (Yleaf) water potential measurement gave dissimilar values in AS and control plants under well-watered conditions. Under a water-limiting environment the Y of AS-plants remained at more negative water values, even though a further decrease in transpiration of AS-plants was detected. Quantitative analysis displayed a much stronger wilting reaction in the AS than in the control plants. Quantitative studies of the leaf movement in AS compared to control plants exhibited a dramatic reduction in velocity and also in the extent of the process. The following conclusions can be drawn. NtAQP1 was expressed at sites of anticipated high water fluxes from and to the apoplast or symplast. Additionally, the specific distribution pattern and temporal expression of NtAQP1 in petioles and the bending stem strongly indicate a role in transcellular movement of water. The reduction of NtAQP1 by the antisense expression decreased the overall cellular Pos. Conclusively, NtAQP1-function increases membrane water permeability of tobacco root protoplasts. The decrease of the specific root hydraulic conductivity (KRA) was in the same order of magnitude as the mean cellular water permeability reduction, indicating that aquaporin expression is essential in maintaining a natural root hydraulic conductance. Reduction of KRA in AS plants might be the first definitive proof that the pathway of water uptake from the root surface to the xylem involves passage across membranes. The absence of NtAQP1 resulted in a water stress signal, causing a certain stomatal closure. NtAQP1 seems to contribute to water stress avoidance in tobacco. NtAQP1 plays an essential role in fast plant movements and transcellular water shift.
Network planning has come to great importance during the past decades. Today's telecommunication, traffic systems, and logistics would not have been evolved to the current state without careful analysis of the underlying network problems and precise implementation of the results obtained from those examinations. Graphs with node and arc attributes are a very useful tool to model realistic applications, while on the other hand they are well understood in theory. We investigate network design problems which are motivated particularly from applications in communication networks and logistics. Those problems include the search for homogeneous subgraphs in edge labeled graphs where either the total number of labels or the reload cost are subject to optimize. Further, we investigate some variants of the dial a ride problem. On the other hand, we use node and edge upgrade models to deal with the fact that in many cases one prefers to change existing networks rather than implementing a newly computed solution from scratch. We investigate the construction of bottleneck constrained forests under a node upgrade model, as well as several flow cost problems under a edge based upgrade model. All problems are examined within a framework of multi-criteria optimization. Many of the problems can be shown to be NP-hard, with the consequence that, under the widely accepted assumption that P is not equal to NP, there cannot exist efficient algorithms for solving the problems. This motivates the development of approximation algorithms which compute near-optimal solutions with provable performance guarantee in polynomial time.
The thesis looks at the question asking for the computability of the dot-depth of star-free regular languages. Here one has to determine for a given star-free regular language the minimal number of alternations between concatenation on one hand, and intersection, union, complement on the other hand. This question was first raised in 1971 (Brzozowski/Cohen) and besides the extended star-heights problem usually refered to as one of the most difficult open questions on regular languages. The dot-depth problem can be captured formally by hierarchies of classes of star-free regular languages B(0), B(1/2), B(1), B(3/2),... and L(0), L(1/2), L(1), L(3/2),.... which are defined via alternating the closure under concatenation and Boolean operations, beginning with single alphabet letters. Now the question of dot-depth is the question whether these hierarchy classes have decidable membership problems. The thesis makes progress on this question using the so-called forbidden pattern approach: Classes of regular languages are characterized in terms of patterns in finite automata (subgraphs in the transition graph) that are not allowed. Such a characterization immediately implies the decidability of the respective class, since the absence of a certain pattern in a given automaton can be effectively verified. Before this work, the decidability of B(0), B(1/2), B(1) and L(0), L(1/2), L(1), L(3/2) were known. Here a detailed study of these classes with help of forbidden patterns is given which leads to new insights into their inner structure. Furthermore, the decidability of B(3/2) is proven. Based on these results a theory of pattern iteration is developed which leads to the introduction of two new hierarchies of star-free regular languages. These hierarchies are decidable on one hand, on the other hand they are in close connection to the classes B(n) and L(n). It remains an open question here whether they may in fact coincide. Some evidence is given in favour of this conjecture which opens a new way to attack the dot-depth problem. Moreover, it is shown that the class L(5/2) is decidable in the restricted case of a two-letter alphabet.
In this thesis we investigate near-isomorphism classes and isomorphism classes of almost completely decomposable groups. In Chapter 2 we introduce the concept of almost completely decomposable groups and sum up their most important facts. A local group is an almost completely decomposable group with a primary regulator quotient. A uniform group is a rigid local group with a homocyclic regulator quotient. In Chapter 3 a weakening of isomorphism, called type-isomorphism, appears. It is shown that type-isomorphism agrees with Lady's near-isomorphism. By the Main Decomposition Theorem and the Primary Reduction Theorem we are allowed to restrict ourselves on clipped local groups, namely groups without a direct rank-one summand. In Chapter 4 we collect facts of matrices over commutative rings with an identity element. Matrices over the local ring (Z / p^e Z) of residue classes of the rational integers modulo a prime power play an important role. In Chapter 5 we introduce representing matrices of finite essential extensions. Here a normal form for local groups is found by the Gauß algorithm. Uniform groups have representing matrices in Hermite normal form. The classification problems for almost completely decomposable groups up to isomorphism and up to near-isomorphism can be rephrased as equivalence problems for the representing matrices. In Chapter 6 we derive a criterion for the representing matrices of local groups in Gauß normal form. In Chapter 7 we formulate the matrix criterion for uniform groups. Two representing matrices in Hermite normal form describe isomorphic groups if and only if the rest blocks of the representing matrices are T-diagonally equivalent. Starting from a fixed near-isomorphism class in Chapter 8 we investigate isomorphism classes of uniform groups. We count groups and isomorphism classes. In Chapter 9 we specialize on uniform groups of rank 2r with a regulator quotient of rank r such that the rest block of the representing matrix is invertible and normed.
Complexity and Partitions
(2001)
Computational complexity theory usually investigates the complexity of sets, i.e., the complexity of partitions into two parts. But often it is more appropriate to represent natural problems by partitions into more than two parts. A particularly interesting class of such problems consists of classification problems for relations. For instance, a binary relation R typically defines a partitioning of the set of all pairs (x,y) into four parts, classifiable according to the cases where R(x,y) and R(y,x) hold, only R(x,y) or only R(y,x) holds or even neither R(x,y) nor R(y,x) is true. By means of concrete classification problems such as Graph Embedding or Entailment (for propositional logic), this thesis systematically develops tools, in shape of the boolean hierarchy of NP-partitions and its refinements, for the qualitative analysis of the complexity of partitions generated by NP-relations. The Boolean hierarchy of NP-partitions is introduced as a generalization of the well-known and well-studied Boolean hierarchy (of sets) over NP. Whereas the latter hierarchy has a very simple structure, the situation is much more complicated for the case of partitions into at least three parts. To get an idea of this hierarchy, alternative descriptions of the partition classes are given in terms of finite, labeled lattices. Based on these characterizations the Embedding Conjecture is established providing the complete information on the structure of the hierarchy. This conjecture is supported by several results. A natural extension of the Boolean hierarchy of NP-partitions emerges from the lattice-characterization of its classes by considering partition classes generated by finite, labeled posets. It turns out that all significant ideas translate from the case of lattices. The induced refined Boolean hierarchy of NP-partitions enables us more accuratly capturing the complexity of certain relations (such as Graph Embedding) and a description of projectively closed partition classes.
Starfree regular languages can be build up from alphabet letters by using only Boolean operations and concatenation. The complexity of these languages can be measured with the so-called dot-depth. This measure leads to concatenation hierarchies like the dot-depth hierarchy (DDH) and the closely related Straubing-Thérien hierarchy (STH). The question whether the single levels of these hierarchies are decidable is still open and is known as the dot-depth problem. In this thesis we prove/reprove the decidability of some lower levels of both hierarchies. More precisely, we characterize these levels in terms of patterns in finite automata (subgraphs in the transition graph) that are not allowed. Therefore, such characterizations are called forbidden-pattern characterizations. The main results of the thesis are as follows: forbidden-pattern characterization for level 3/2 of the DDH (this implies the decidability of this level) decidability of the Boolean hierarchy over level 1/2 of the DDH definition of decidable hierarchies having close relations to the DDH and STH Moreover, we prove/reprove the decidability of the levels 1/2 and 3/2 of both hierarchies in terms of forbidden-pattern characterizations. We show the decidability of the Boolean hierarchies over level 1/2 of the DDH and over level 1/2 of the STH. A technique which uses word extensions plays the central role in the proofs of these results. With this technique it is possible to treat the levels 1/2 and 3/2 of both hierarchies in a uniform way. Furthermore, it can be used to prove the decidability of the mentioned Boolean hierarchies. Among other things we provide a combinatorial tool that allows to partition words of arbitrary length into factors of bounded length such that every second factor u leads to a loop with label u in a given finite automaton.
A completely decomposable group is a direct sum of subgroups of the rationals. An almost completely decomposable group is a torsion free abelian group that contains a completely decomposable group as subgroup of finite index. Tight subgroups are maximal subgroups (with respect to set inclusion) among the completely decomposable subgroups of an almost completely decomposable group. In this dissertation we show an extended version of the theorem of Bezout, give a new criterion for the tightness of a completely decomposable subgroup, derive some conditions under which a tight subgroup is regulating and generalize a theorem of Campagna. We give an example of an almost completely decomposable group, all of whose regulating subgroups do not have a quotient with minimal exponent. We show that among the types of elements of a coset modulo a completely decomposable group there exists a unique maximal type and define this type to be -the- coset type. We give criteria for tightness and regulating in term of coset types as well as a representation of the type subgroups using coset types. We introduce the notion of reducible cosets and show their key role for transitions from one completely decomposable subgroup up to another one containing the first one as a proper subgroup. We give an example of a tight, but not regulating subgroup which contains the regulator. We develop the notion of a fully single covered subset of a lattice, show that V-free implies fully single covered, but not necessarily vice versa, and we define an equivalence relation on the set of all finite subsets of a given lattice. We develop some extension of ordinary Hasse diagrams, and apply the lattice theoretic results on the lattice of types and almost completely decomposable groups.
Priority task of the thesis was to replace oxygen atoms in sulfur oxoanions SOnm– or imido groups in sulfur polyimido anions S(NR)nm– isoelectronically by R2C-methylene groups. This would open a wide avenue to new target molecules containing a formally double bonded carbon next to formally double bonded nitrogen atoms in highly charged sulfur-centred anions like S(CR2)x(NR)ym–. They clearly are reminiscent to sulfur ylides. Both, alkylendiimidosulfites and alkylentriimidosulfates are accessible via deprotonaton of the corresponding alkyldiimidosulfinates and alkyltriimidosulfonates with methyllithium. The reactivity of the novel compounds is dominated by the carbanionic centre. Addition reactions to another SN formal doubble bond are feasible and are leading to the yet unknown imidoanalogues compounds alkyl-bis-(diimidosulfinates) and alkyl-bis-(triimidosulfonates).
Implications of Advanced Glycation Endproducts in Oxidative Stress and Neurodegenerative Disorders
(2001)
The reactions of reducing sugars with primary amino groups are the most common nonenzymatic modifications of proteins. Subsequent rearrangements, oxidations, and dehydrations yield a heterogeneous group of mostly colored and fluorescent compounds, termed "Maillard products" or advanced glycation end products (AGEs). AGE formation has been observed on long-lived proteins such as collagen, eye lens crystalline, and in pathological protein deposits in Alzheimer's (AD) and Parkinson's disease (PD) and dialysis-related amyloidosis. AGE-modified proteins are also involved in the complications of diabetes. AGEs accumulate in the the ß-amyloid plaques and neurofibrillary tangles (NFT) associated with AD and in the Lewy bodies characteristic of PD. Increasing evidence supports a role for oxidative stress in neurodegenerative disorders such as AD and PD. AGEs have been shown to contribute towards oxidative damage and chronic inflammation, whereby activated microglia secrete cytokines and free radicals, including nitric oxide (NO). Roles proposed for NO in the pathophysiology of the central nervous system are increasingly diverse and range from intercellular signaling, through necrosis of cells and invading pathogens, to the involvement of NO in apoptosis. Using in vitro experiments, it was shown that AGE-modified bovine serum albumin (BSA-AGE) and AGE-modified ß-amyloid, but not their unmodified proteins, induce NO production in N-11 murine microglia cells. This was mediated by the receptor for AGEs (RAGE) and upregulation of the inducible nitric oxide synthase (iNOS). AGE-induced enzyme activation and NO production could be blocked by intracellular-acting antioxidants: Ginkgo biloba special extract EGb 761, the estrogen derivative, 17ß-estradiol, R-(+)-thioctic acid, and a nitrone-based free radical trap, N-tert.-butyl-*-phenylnitrone (PBN). Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), common precursors in the Maillard reaction, were also tested for their ability to induce the production of NO in N-11 microglia. However, no significant changes in nitrite levels were detected in the cell culture medium. The significance of these findings was supported by in vivo immunostaining of AD brains. Single and double immunostaining of cryostat sections of normal aged and AD brains was performed with polyclonal antibodies to AGEs and iNOS and monoclonal antibodies to Aß and PHF-1 (marker for NFT) and reactive microglia. In aged normal individuals as well as early stage AD brains (i.e. no pathological findings in isocortical areas), a few astrocytes showed co-localisation of AGE and iNOS in the upper neuronal layers of the temporal (Area 22) and entorhinal (Area 28, 34) cortices compared with no astrocytes detected in young controls. In late AD brains, there was a much denser accumulation of astrocytes co-localised with AGE and iNOS in the deeper and particularly upper neuronal layers. Also, numerous neurons with diffuse AGE but not iNOS reactivity and some AGE and iNOS-positive microglia were demonstrated, compared with only a few AGE-reactive neurons and no microglia in controls. Finally, astrocytes co-localised with AGE and iNOS as well as AGE and ß-amyloid were found surrounding mature but not diffuse ß-amyloid plaques in the AD brain. Parts of NFT were AGE-immunoreactive. Immunohistochemical staining of cryostat sections of normal aged and PD brains was performed with polyclonal antibodies to AGEs. The sections were counterstained with monoclonal antibodies to neurofilament components and a-synuclein. AGEs and a-synuclein were colocalized in very early Lewy bodies in the substantia nigra of cases with incidental Lewy body disease. These results support an AGE-induced oxidative damage due to the action of free radicals, such as NO, occurring in the AD and PD brains. Furthermore, the involvement of astrocytes and microglia in this pathological process was confirmed immunohistochemically in the AD brain. It is suggested that oxidative stress and AGEs participate in the very early steps of Lewy body formation and resulting cell death in PD. Since the iNOS gene can be regulated by redox-sensitive transcription factors, the use of membrane permeable antioxidants could be a promising strategy for the treatment and prevention of chronic inflammation in neurodegenerative disorders.
This study has focused on hydrogeological and hydrochemical settings of the Northern Namibian Kalahari Catchment which is the Namibian part of the Makgadikgadi-Kalahari-Catchment. Recharge has been the subject of process-understanding, quantification and regionalisation. Within the semiarid study area a bimodal surface constitution is prominent: hardrocks areas allow for fast infiltration along karsts and joints, whereas areas covered by unconsolidated sediments receive minor diffuse recharge and locally some preferred flow path recharge develops along shrinkage cracks and rootlets. Five substratum classes have been soil physically studied: Pans and vleis, brown to red soils, dune sand, soil with an aeolian influence, and calcrete. Aeolian sands are most promising for the development of direct diffuse recharge. Recharge by preferred flow might occur in all soil classes either due to joints in calcrete or structures and rootlets in soils. All soil classes contribute to indirect recharge because even the dune sand allows, albeit very locally, the generation of runoff. The occurrences of recharge through the unconsolidated soil and the hardrocks have been confirmed by hydrograph interpretation and by a study of hydrochemical data which identified groundwater of flood water and flood water after soil passage composition. Other prominent hydrochemical processes in the Kalahari are associated with the carbonate-equilibrium-system, mixing with highly mineralised water that is either sulphate (central area) or chloride dominated (fringe area) and development of sodium hydrogencarbonate water types. The latter is mostly generated by feldspar weathering. Variations of the hydrochemical compositions were observed for shallow groundwaters. They do not only reflect the recharge amount but also the recharge conditions, e.g. a wetter year is allowing more vegetation which increases the hydrogencarbonate content. Inverse determination of recharge by the chloride mass balance method gives recharge amounts between 0.2 and locally more than 100 mm/a. The least favoured recharge conditions are found for Kalahari covered areas, the largest amount occurs in the Otavi area. The distribution of recharge areas within the catchment is rather complex and regionalisation of recharge for the entire catchment was done by a forward approach using satellite images and by an inverse approach using hydrochemical data. From the inverse hydrochemical approach a basin-wide balanced recharge amount of 1.39 mm/a is achieved. The forward approach gave a basin-wide figure of 0.88 (minimum assumption) to 4.53 mm/a (maximum assumption). A simplistic groundwater flow model confirmed the results from the minimum recharge regionalisation by satellite images and the result from the hydrochemical approach. Altogether a mutually verified basin-wide recharge figure of ca. 1 mm/a turns out.
The social organization of insect colonies has long fascinated naturalists. One of the main features of colony organization is division of labor, whereby each member of the colony specializes in a subset of all tasks required for successful group functioning. The most striking aspect of division of labor is its plasticity: workers switch between tasks in response to external challenges and internal perturbations. The mechanisms underlying flexible division of labor are far from being understood. In order to comprehend how the behavior of individuals gives rise to flexible collective behavior, several questions need to be addressed: We need to know how individuals acquire information about their colony's current demand situation; how they then adjust their behavior according; and which mechanisms integrate dozens or thousands of insect into a higher-order unit. With these questions in mind I have examined two examples of collective and flexible behavior in social bees. First, I addressed the question how a honey bee colony controls its pollen collection. Pollen foraging in honey bees is precisely organized and carefully regulated according to the colony's needs. How this is achieved is unclear. I investigated how foragers acquire information about their colony's pollen need and how they then adjust their behavior. A detailed documentation of pollen foragers in the hive under different pollen need conditions revealed that individual foragers modulate their in-hive working tempo according to the actual pollen need of the colony: Pollen foragers slowed down and stayed in the hive longer when pollen need was low and spent less time in the hive between foraging trips when pollen need of their colony was high. The number of cells inspected before foragers unloaded their pollen load did not change and thus presumably did not serve as cue to pollen need. In contrast, the trophallactic experience of pollen foragers changed with pollen need conditions: trophallactic contacts were shorter when pollen need was high and the number and probability of having short trophallactic contacts increased when pollen need increased. Thus, my results have provided support for the hypothesis that trophallactic experience is one of the various information pathways used by pollen foragers to assess their colony's pollen need. The second example of collective behavior I have examined in this thesis is the control of nest climate in bumble bee colonies, a system differing from pollen collection in honey bees in that information about task need (nest climate parameters) is directly available to all workers. I have shown that an increase in CO2 concentration and temperature level elicits a fanning response whereas an increase in relative humidity does not. The fanning response to temperature and CO2 was graded; the number of fanning bees increased with stimulus intensity. Thus, my study has evidenced flexible colony level control of temperature and CO2. Further, I have shown that the proportion of total work force a colony invests into nest ventilation does not change with colony size. However, the dynamic of the colony response changes: larger colonies show a faster response to perturbations of their colony environment than smaller colonies. Thus, my study has revealed a size-dependent change in the flexible colony behavior underlying homeostasis. I have shown that the colony response to perturbations in nest climate is constituted by workers who differ in responsiveness. Following a brief review of current ideas and models of self-organization and response thresholds in insect colonies, I have presented the first detailed investigation of interindividual variability in the responsiveness of all workers involved in a collective behavior. My study has revealed that bumble bee workers evidence consistent responses to certain stimulus levels and differ in their response thresholds. Some consistently respond to low stimulus intensities, others consistently respond to high stimulus intensities. Workers are stimulus specialists rather than task specialists. Further, I have demonstrated that workers of a colony differ in two other parameters of responsiveness: response probability and fanning activity. Response threshold, response probability and fanning activity are independent parameters of individual behavior. Besides demonstrating and quantifying interindividual variability, my study has provided empirical support for the idea of specialization through reinforcement. Response thresholds of fanning bees decreased over successive trials. I have discussed the importance of interindividual variability for specialization and the collective control of nest climate and present a general discussion of self-organization and selection. This study contributes to our understanding of individual behavior and collective structure in social insects. A fascinating picture of social organization is beginning to emerge. In place of centralized systems of communication and information transmission, insect societies frequently employ mechanisms based upon self-organization. Self-organization promises to be an important and unifying principle in physical, chemical and biological systems.
For many active volcanoes all over the world a civil protection program, normally combined with hazard maps, exists. Optimising of hazard maps and the associated hazard assessment implies a detailed knowledge of the volcanostratigraphy, because the deposits provoke information on the potential behaviour during a new activity cycle. Pyroclastic deposits, however, may vary widely in thickness and distribution over very short lateral distances. High resolution characterisation of single strata often cannot be archived, if solely sedimentological and geochemical methods are used. Gamma-ray measurements taken in the field combined with grain-size depended magnetic susceptibility measurements made in the laboratory are used in this work to optimise the resolution of volcanostratigraphic investigations. The island of Vulcano is part of the Aeolian Archipelago sited of the northern coast of Sicily. La Fossa cone is the active centre of Vulcano, where fumarolic and seismic activity can be observed. The cone was built up during the last 6,000 years, whereby the last eruption period is dated to historic times (1888-1890). For the tuff cone La Fossa the most likely volcanic hazards are the emplacement of pyroclastic deposits as well as gas hazards (especially SOx and CO2), due to this the detailed knowledge of the stratigraphy is mandatory. Most of the population resides in Vulcano Porto and the nearby sited peninsula of Vulcanello, which are highly endangered locations for a future eruption scenario. Measurements, made in standard outcrops, allow a characterisation of the successions Punte Nere, Tufi Varicolori, Palizzi, Commenda, and Cratere Attuale. A discrimination of all successions by solely one of the methods is rarely possible. In some cases, however, the combination of the methods leads to clear results. It can also be noticed that the exposition as well as the sedimentation type (wet-surge or dry-surge deposits) affect the measurements. In general it can be assumed that the higher the magma is evolved the higher the g -ray values and the lower the susceptibility values. Measurements from the Wingertsberg (Laacher See deposits, Eifel, W-Germany) show clearly that a higher degree of magma evolution correlates with lower susceptibility and higher gamma-ray values. Variations of the values can be observed not only by the change of the degree of magmatic evolution but also by the inhomogeneous deposition conditions. Particularly the gamma-ray measurements show lower values for the wet-surge deposits than for the dry-surge deposits, even though the erupted material has the same geochemical composition. This can be explained especially by reactions inside of the moist eruption cloud and short-time after deposition, when easily soluble elements like K, U, and Th can be leached by these aggressive fluids. Even extended exposition and high water content can provoke depletion of various elements within the complete or parts of the outcrop, too. If the deposits are affected by a fumarolic activity especially the susceptibility values show significant variations, whereas in general extreme low values are observed. Contamination of deposits also can occur, if they are overlain by weathered deposits of higher concentration of K, U, and Th. Weathering and mobilisation within the upper deposits can generate an element enrichment within the lower deposits. In general the element ratios of the barried underlying deposits are less affected than the exposed ones. After gauging the values of the well defined succession for standard outcrops undefined outcrops were measured. These outcrops are not clearly classified by sedimentological and geochemical methods, thus a correlation with the combined geophysical methods is useful. In general the combination of the methods allows a correlation, although in some cases more than one interpretation is possible. But in connection with time marker horizons as well as sedimentological features an interpretation is feasible. These situations show that a classification solely based on geophysical methods is possible for many cases but, if the volcanic system is more complex, a combination with sedimentological and geochemical methods may be needed. The investigations on Vulcano, documented in this work, recommend a re-interpretation of the dispersial of some successions of La Fossa cone, especially the presumption that Tufi Varicolori only exist inside of the Caldera of La Fossa. As a consequence the eruption and energy model especially for Tufi Varicolori have to be reviewed.
The coast of Aqaba and the Aqaba region (Jordan) were investigated on their hydrogeo-ecosystem. The results of the research were translated into digits to build a geo-spatial data base. The fillings of the graben aquifer receive indirect type of recharge through the side wadis which drain the highlands. Surface water balance was modeled for a period of 20 years of daily climate records using MODBIL program which attributes direct recharge to wet years only. The hydrodynamic fresh water/seawater interface in the coastal zones was investigated by applying vertical geoelectric surveys and models of several methods to confirm its coincidence with the aquifer’s flow amounts, where human impacts in terms of over-pumping allowed more encroachment of seawater into land, and unintended recharge which led to seaward interface migration. A groundwater balance and solute transport were approached by developing a flow model from the hydrogeological and hydrochemical data. The nature of soil cover and aquifer whose physical properties enhance human impacts indicated the vulnerability of groundwater to pollution. This certainly threatens the marine ecology which forms the sink where the in-excess flow ends. The constructed digital background was exported into GIS to sub-zone the study area in terms of the aquifer’s vulnerability to pollution risks using DRASTIC index. However, it was unable to meet all geo-spatial factors that proved to have significant impacts on the vulnerability. Consequently, a comprehensive index -SALUFT- was developed. This suggests the suitable land use units for each zone in the light of vulnerability grades aiming at protecting the available groundwater resources.
The understanding of the mechanisms underlying the establishment and maintenance of the extraordinary biodiversity in tropical forests is a major challenge for modern biology. In this context, epiphytes are presumed to play an important role. To investigate the biological reality of this persistent yet insufficiently investigated notion, I conducted the present study. The main questions I intended to clarify were: (1) do epiphytes affect arthropod abundance and diversity in tropical tree crowns? and (2) what might be the driving forces behind this potential influence? I studied the arthropod fauna of 25 tree crowns bearing different epiphyte assemblages, and the resident fauna of 90 individual epiphytes. I also quantified the mitigating influence of epiphytes on the microclimate in tree crowns. In total, more than 277,000 arthropods were collected and about 700 morphospecies determined. Epiphytes had a significant moderating influence on canopy microclimate (Chapter 3), both at various microsites within a tree crown and among tree crowns with different epiphyte growth. On hot dry season days, they provided microsites with lower temperatures and reduced evaporative water loss compared to epiphyte-free spaces within the same tree crown. Quantitative sampling of the arthropods inhabiting three different epiphyte species provided compelling evidence for the specificity of epiphyte-associated faunas (Chapter 4). Epiphytes proved to be microhabitats for a diverse and numerous arthropod fauna, and different epiphyte species fostered both taxonomically and ecologically very distinct arthropod assemblages: among epiphyte hosts, the inhabitant faunas showed remarkably little species overlap, and guild composition differed strongly. In the subsequent chapters I investigated if this pronounced effect scaled up to the level of entire tree crowns. Arthropods were captured with three different trap types to obtain an ample spectrum of the canopy fauna (Chapter 2). Four tree categories were classified, three of which were dominated by a different species of epiphyte, and an epiphyte-free control group. On a higher taxonomic level, there were no detectable effects of epiphytes on the fauna: the ordinal composition was similar among tree categories and indifferent of the amount of epiphytes in a tree crown (Chapter 5). I examined three focal groups (ants, beetles and spiders) on species level. The diversity and abundance of ants was not influenced by the epiphyte load of the study trees (Chapter 6). Although many species readily used the epiphytes as nesting site and shelter, they seemed to be highly opportunistic with respect to their host plants. Likewise, the species richness and abundance of beetles, as well as their guild composition were entirely unaffected by the presence of epiphytes in the study trees (Chapter 7). Focusing on herbivorous beetles did not alter these results. Spiders, however, were strongly influenced by the epiphyte assemblages of the host trees (Chapter 8). Overall spider abundance and species richness did not differ among trees, but particular families and guilds exhibited marked differences in abundance between the tree categories. Most remarkable were the substantial differences in spider species composition across trees with different epiphyte assemblages. Conclusion Thus, the prevalent notion that epiphytes positively influence arthropod diversity in tropical canopies seems justified, but not without reservation. Whether an influence of epiphytes on the fauna was discernible depended greatly on (1) the scale of the investigated system: clear faunal distinctions at the microhabitat level were absent or much more subtle at the level of tree crowns. (2) the focal taxa: different arthropod orders allowed for completely different statements concerning the importance of epiphytes for canopy fauna. I therefore recommend a multitaxon approach for the investigation of large-scale ecological questions. In conclusion, I resume that epiphytes are associated with a species-specific inhabiting fauna,and that epiphytes impose an influence on certain, but not all, taxa even at the level of entire tree crowns. Although I could only hypothesize about the potential causes for this influence, this study provided the first comprehensive investigation of the role of epiphytes in determining arthropod abundance and diversity in tropical tree crowns.
The Gram-negative, spiral-shaped, microaerophilic bacterium Helicobacter pylori is the causative agent of various disorders of the upper gastrointestinal tract, such as chronic superficial gastritis, chronic active gastritis, peptic ulceration and adenocarcinoma. Although many of the bacterial factors associated with disease development have been analysed in some detail in the recent years, very few studies have focused so far on the mechanisms that regulate expression of these factors at the molecular level. In an attempt to obtain an overview of the basic mechanisms of virulence gene expression in H. pylori, three important virulence factors of this pathogen, representative of different pathogenic mechanisms and different phases of the infectious process, are investigated in detail in the present thesis regarding their transcriptional regulation. As an essential factor for the early phase of infection, including the colonisation of the gastric mucosa, the flagella are analysed; the chaperones including the putative adhesion factors GroEL and DnaK are investigated as representatives of the phase of adherence to the gastric epithelium and persistence in the mucus layer; and finally the cytotoxin associated antigen CagA is analysed as representative of the cag pathogenicity island, which is supposed to account for the phenomena of chronic inflammation and tissue damage observed in the later phases of infection. RNA analyses and in vitro transcription demonstrate that a single promoter regulates expression of cagA, while two promoters are responsible for expression of the upstream divergently transcribed cagB gene. All three promoters are shown to be recognised by RNA polymerase containing the vegetative sigma factor sigma 80. Promoter deletion analyses establish that full activation of the cagA promoter requires sequences up to -70 and binding of the C-terminal portion of the alpha subunit of RNA polymerase to an UP-like element located between -40 and -60, while full activation of the major cagB promoter requires sequences upstream of -96 which overlap with the cagA promoter. These data suggest that the promoters of the pathogenicity island represent a class of minimum promoters, that ensure a basic level of transcription, while full activation requires regulatory elements or structural DNA binding proteins that provide a suitable DNA context. Regarding flagellar biosynthesis, a master transcriptional factor is identified that regulates expression of a series of flagellar basal body and hook genes in concert with the alternative sigma factor sigma 54. Evidence is provided that this regulator, designated FlgR (for flagellar regulatory protein), is necessary for motility and transcription of five promoters for seven basal body and hook genes. In addition, FlgR is shown to act as a repressor of transcription of the sigma 28-regulated promoter of the flaA gene, while changes in DNA topology are shown to affect transcription of the sigma 54-regulated flaB promoter. These data indicate that the regulatory network that governs flagellar gene expression in H. pylori shows similarities to the systems of both Salmonella spp. and Caulobacter crescentus. In contrast to the flagellar genes which are regulated by three different sigma factors, the three operons encoding the major chaperones of H. pylori are shown to be transcribed by RNA polymerase containing the vegetative sigma factor sigma 80. Expression of these operons is shown to be regulated negatively by the transcriptional repressor HspR, a homologue of a repressor protein of Streptomyces spp., known to be involved in negative regulation of heat shock genes. In vitro studies with purified recombinant HspR establish that the protein represses transcription by binding to large DNA regions centered around the transcription initiation site in the case of one promoter, and around -85 and -120 in the case of the the other two promoters. In contrast to the situation in Streptomyces, where transcription of HspR-regulated genes is induced in response to heat shock, transcription of the HspR-dependent genes in H. pylori is not inducible with thermal stimuli. Transcription of two of the three chaperone encoding operons is induced by osmotic shock, while transcription of the third operon, although HspR-dependent, is not affected by salt treatment. Taken together, the analyses carried out indicate that H. pylori has reduced its repertoire of specific regulatory proteins to a basic level that may ensure coordinate regulation of those factors that are necessary during the initial phase of infection including the passage through the gastric lumen and the colonisation of the gastric mucosa. The importance of DNA topology and/or context for transcription of many virulence gene promoters may on the other hand indicate, that a sophisticated global regulatory network is present in H. pylori, which influences transcription of specific subsets of virulence genes in response to changes in the microenvironment.
Pollinating insects exhibit a complex behavior while foraging for nectar and pollen. Many studies have focused on ultimate mechanisms of this behavior, however, the sensory-perceptual processes that constrain such behavior have rarely been considered. In the present study I used bumblebees (Bombus terrestris), an important pollinating insect, to investigate possible sensory constraints on foraging behavior. Additionally, I survey inter-individual variation in the sensory capabilities and behavior of bumblebees caused by the pronounced size polymorphism among members of a single colony. In the first chapter I have focused on the sensory-perceptual processes that constrain the search for flowers. I measured search time for artificial flowers of various sizes and colors, a key variable defining the value of a prey type in optimal foraging theory. When flowers were large, search times correlate well with the color contrast of the targets with their green foliage-type background, as predicted by a model of color opponent coding using inputs from the bee's UV, blue, and green receptors. Targets which made poor color contrast with their backdrop, such as white, UV-reflecting ones, or red flowers, take longest to detect, even though brightness contrast with the background is pronounced. When searching for small targets, bumblebees change their strategy in several ways. They fly significantly slower and closer to the ground, so increasing the minimum detectable area subtended by an object on the ground. In addition they use a different neuronal channel for flower detection: instead of color contrast, they now employ only the green receptor signal for detection. I related these findings to temporal and spatial limitations of different neuronal channels involved in stimulus detection and recognition. Bumblebees do not only possess species-specific sensory capacities but they also exhibit inter-individual differences due to size. Therefore, in the next two chapters I have examined size-related effects on the visual and olfactory system of Bombus terrestris. Chapter two deals with the effect of scaling on eye architecture and spatial resolving power of workers. Foraging efficiency in bees is strongly affected by proficiency of detecting flowers. Both floral display size and bee spatial vision limit flower detection. In chapter one I have shown that search times for flowers strongly increases with decreasing floral display size. The second factor, bee spatial vision, is mainly limited by two properties of compound eyes: (a) the interommatidial angle Çå and (b) the ommatidial acceptance angle Çá. When a pollinator strives to increase the resolving power of its eyes, it is forced to increase both features simultaneously. Bumblebees show a large variation in body size. I found that larger workers with larger eyes possess more ommatidia and larger facet diameters. Large workers with twice the size of small workers (thorax width) have about 50 per cent more ommatidia, and a 1.5 fold enlarged facet diameter. In a behavioral test, large and small workers were trained to detect the presence of a colored stimulus in a Y-maze apparatus. The stimulus was associated with a sucrose reward and was presented in one arm, the other arm contained neither stimulus nor reward. The minimum visual angle a bee is able to detect was estimated by testing the bee at different stimuli sizes subtending angles between 30° and 3° on the bee’s eye. Minimum visual detection angles range from 3.4° to 7.0° among tested workers. Larger bumblebees are able to detect objects subtending smaller visual angles, i.e. they are able to detect smaller objects than their small conspecifics. Thus morphological and behavioral findings indicate an improved visual system in larger bees. Beside vision, olfaction is the most important sensory modality while foraging in bees. Bumblebees utilize species-specific odors for detecting and identifying nectar and pollen rich flowers. In chapter three I have investigated the olfactory system of Bombus terrestris and the effect of scaling on antennal olfactory sensilla and the first olfactory neuropil in the bumblebee brain, the antennal lobes. I found that the pronounced size polymorphism exhibited by bumblebees also effects their olfactory system. Sensilla number (I measured the most common olfactory sensilla type, s. placodea), sensilla density, volume of antennal lobe neuropil and volume of single identified glomeruli correlate significantly with worker’s size. The enlarged volume of the first olfactory neuropil in large individuals is caused by an increase in glomeruli volume and coarse neuropil volume. Additionally, beside an overall increase of brain volume with scaling I found that the olfactory neuropil increases disproportionately compared to a higher order neuropil, the central body. The data predict a higher odor sensitivity in larger bumblebee workers. In the last chapter I have addressed the question if scaling alters foraging behavior and rate in freely foraging bumblebees. I observed two freely foraging B. terrestris colonies and measured i) trip number, ii) trip time, iii) proportion of nectar trips, and iv) nectar foraging rate of different sized foragers. In all observation periods large foragers exhibit a significantly higher foraging rate than small foragers. None of the other three foraging parameters is affected by workers’ size. Thus, large foragers contribute disproportionately more to the current nectar influx of their colony. To summarize, this study shows that understanding the mechanisms of visual information processing and additionally comprising inter-individual differences of sensory capabilities is crucial to interpret foraging behavior of bees.
B-cells of the rheumatoid synovial tissue are a constant part of and, in some histopathological subtypes, the dominant population of the inflammatory infiltrate, located in the region of tissue destruction. The pattern of B-cell distribution and the relationship to the corresponding antigen-presenting cells (follicular dendritic reticulum cells: FDCs) show a great variety. B-cells may exhibit (i) a follicular organization forming secondary follicles; (ii) follicle-like patterns with irregularly formed FDC networks, and (iii) a diffuse pattern of isolated FDCs. Molecular analysis of immunoglobulin VH and VL genes from human synovial B-cell hybridomas and synovial tissue demonstrates somatic mutations due to antigen activation. The FDC formations in the synovial tissue may therefore serve as an environment for B-cell maturation, which is involved in the generation of autoantibodies. An autoantibody is defined as "pathogenic" if it fulfills the Witebsky-Rose-Koch criteria for classical autoimmune diseases: definition of the autoantibody; induction of the disease by transfer of the autoantibody; and isolation of the autoantibody from the disease-specific lesion. B-cells from rheumatoid synovial tissue show specificity for FcIgG, type II collagen, COMP, sDNA, tetanus toxoid, mitochondrial antigens (M2), filaggrin and bacterial HSPs. The contributions of these antigens to the pathogenesis of RA are still hypothetical. A possible contribution could derive from crossreactivity and epitope mimicry: due to crossreaction, an antibody directed originally against a foreign infectious agent could react with epitopes from articular tissues, perpetuating the local inflammatory process. The characteristic distribution pattern, the localisation within the area of tissue destruction, the hypermutated IgVH and IgVL genes, and their exclusive function to recognize conformation-dependent antigens suggest a central role for B-cells in the inflammatory process of rheumatoid arthritis. Therefore, the analysis of synovial B-cell hybridomas and experimental expression of synovial IgVH and IgVL genes will help to characterise the antigens responsible for the pathogenesis of rheumatoid arthritis. In the present study 55 IgVH genes amplified from 3 different anatomical regions of a RA patient were analysed adding further information on synovial B-cell maturation and recirculation in RA. This analysis demonstrated somatically mutated IgVh genes in all different regions with amino acid deletions and mixed IgVh molecules, suggesting the existence of a novel pathway to generate (auto)antibody specificities. The comparison of amino acid sequences of amplified genes belonging to the VH1 family (with predominantly the same germline counterpart) exhibited a strong homology, indicating an apparently conserved mutational pattern. This suggests that the number of antigens activating B-cells in the different locations is restricted. The most striking result was the finding of clonally related sequences in different anatomical regions indicating a recirculation of activated B-cells between the different affected joints. Also in the present study a synovial B-cell hybridoma was analyzed for its specific recognition of cartilage antigens. A heptameric peptide of cartilage oligomeric protein (COMP) could be defined as the target structure. The IgVH-gene (IgHV4-59*01) of the IgG2l hybridoma has somatically mutated genes with high R/S values in the CDR regions (9:2). Thus, indicating that this hybridoma originates from a synovial B-cell which has been antigen activated/selected for its affinity. To analyse the presence of the clonotypic IgHV4-59*01 sequences in other cases of RA and osteoarthritis (OA) synovitis, primers specific for the CDR3 rearrangement of this hybridoma were used. The clonotypic and clone related sequences (98 per cent ± 1 per cent homology) could only be detected in synovitis of RA cases but not in OA cases indicating that this B-cell is specific to RA synovitis. The identified heptameric peptide of COMP was used in a peptide ELISA to analyse whether there is a specific binding in RA serum samples. Serum samples (IgG) from RA patients (n=22) showed a significant higher efficiency to the COMP heptamer than the OA sera (n=24) and the age matched healthy controls (n=20) (for both p<1x10-4, Students t-test). The specificity of this B-cell hybridoma may therefore be defined as RA specific. Since COMP is restricted to cartilage and tendons which are organs specifically affected in RA this COMP specific autoantibody represents the first organ specific autoantibody in RA. The IgG2 COMP specific autoantibody with somatically mutated IgVH genes is different from germline encoded, antigen clearing IgM autoantibodies and may therefore be directly involved as an "arthritogenic autoantibody" in cartilage and tendons destruction by complement activation.
There are ample sand dune and sand sheets in the Texas Rolling Plains, U.S.A. Their varied location, morphology and paleosol content pointed to differnces in their historical develpment throughout the Holocene. Younger dunes, so called fence line dunes have been identified as remnants of unsound agricultural practices which just recently formed at the beginning of this century. Correspondingly soils were eroded, in parts, down to the C-horizon in some of these areas. More mature sand were dated with the radiocarbon method and identified having formed during the Altithermal warming period. This study identifies major eolian anthropogenic and climatic reactivation and stabilisation phases in the Rolling Plains of Texas during the Holocene, but also ties them into the existing Southern High Plains and Great Plains climatic record. This study also researched the reasons for the regional and local sand reactivation phases and contributes to the eolian history in the Great Plains region. The outline of this dissertation is oriented towards a comprehensive regional approach in cultural and physical geography. Chapter 1 covers the physiographic setting of the Rolling Plains region including geology, geomorphology, climate and vegetation. Here the prerequisites for eolian activity in the area are explained, followed by the criteria for the selection of the individual study sites. In chapter 2 selected dune fields and sand sheets are introduced. Chapter 3 outlines the methodology as a combination of field research, laboratory analysis and remote sensing techniques, along with a brief interpretation of their application and success rate. Chapter 4 investigates interactive processes between the cultural development and the physical landscape of the region. The next 4 chapters are focusing on research results and interpretation. Chapter 5 interprets the youngest eolian episodes resulting from the cultural de-velopment of the area, including a description and definition of so called "fenceline dunes" and "shinnery motts". Other dunes with very young buried horizons are also described in this chapter, and a comparison with outcrops in the Nebraska Sand Hills is performed. Chapter 6 interprets short-term, cyclic, drought related sand reactivations several hundred years ago by means of a Post Oak (Quercus stellata) tree ring record as established by STAHLE and CLEAVELAND (1988). In chapter 7 older Holocene reactivation cycles are introduced, investigating the idea of the existence of a warmer period, previously named the Altithermal, which so far has only been identified in the Southern High Plains. The last chapter (8) includes a brief statement of the study’s purpose along with the summary and discussion of results presented. This chapter will end with further implications of this research.
In the experiments presented in this work, third-order, time-resolved spectroscopy was applied to the disentanglement of nuclear and electronic degrees of freedom in polyatomic molecules. The motivation for approaching this problem was given by the decisive role that the coupling of nuclear and electronic dynamics plays in the mechanism of photochemical reactions and photobiological processes. In order to approach this complex problem, different strategies within the framework of time-resolved, four-wave mixing spectroscopy were developed that allowed for the dynamic as well as the energetic aspects of vibronic coupling in non-radiative transitions of polyatomic molecules to be addressed. This was achieved by utilizing the influence of optical as well as Raman resonances on four-wave mixing processes. These resonance effects on third-order, optical processes allow for a high selectivity to be attained with respect to the interrogation of specific aspects of molecular dynamics. The development of different strategies within the framework of time-resolved, four-wave mixing spectroscopy for addressing the problem of vibronic coupling began with the experiments on gaseous iodine. This simple, well investigated molecular system was chosen in order to unambiguously characterize the effect of Raman resonances on four-wave mixing processes. A time-resolved degenerative four-wave mixing (DFWM) experiment was carried out on gaseous iodine that allowed for the dynamics of coherent Stokes Raman scattering (CSRS) as well as a coherent anti-Stokes Raman scattering (CARS) to be observed parallel to the dynamics of a DFWM process at different spectral positions of the FWM signal. Here, the state-selectivity of these different FWM processes manifests itself in the vibrational wave packet dynamics on different electronic potentials of iodine. It could be shown that Raman resonances determine the selectivity with which these FWM processes prepare and interrogate nuclear dynamics in different electronic states. With the insight gained into the relevance of Raman resonant processes in FWM spectroscopy, an experimental scheme was devised that utilizes this effect to selectively interrogate the dynamics of a specific vibrational mode within a polyatomic molecule during a radiationless electronic transition. Here, a CARS process was employed to selectively probe specific vibrational modes of a molecular system by variably tuning the energy difference between the lasers involved in the CARS process to be in Raman resonance with the vibrational energy spacing of a particular vibrational mode. Using this aspect of a tunable resonance enhancement within a CARS scheme, this optical process was incorporated in a time-resolved pump-probe experiment as a mode-selective probe mechanism. This type of experimental configuration, that employs four pulsed laser fields, was classified as a pump-CARS scheme. Here, a laser pulse independent of the CARS process initiates the molecular dynamics that are interrogated selectively with respect to the vibrational mode of the system through the simultaneous interaction of the three pulsed fields involved in the CARS process. Time-resolution on a femtosecond timescale is achieved by introducing a time delay between the independent pump laser and the laser pulses of the CARS process. The experimental configuration of a pump-CARS scheme was applied to the study of the nuclear dynamics involved in the radiationless electronic transition between the first excited singlet state (S1) and the electronic ground state (S0) of all-trans-b-carotene. The mode-selective CARS probe allowed for the characteristic timescale with which specific vibrational modes are repopulated in the S0 state to be determined. From the varying repopulation times of specific vibrational modes, a mechanism with which the full set of vibrational states of the S0 potential are repopulated subsequent to the internal conversion process could be postulated. Most importantly, the form of nuclear motion that primarily funnels the population between the two electronic states could be identified as the C=C symmetric symmetric stretch mode in the polyene backbone of b-carotene. With this, the reaction coordinate of this radiationless electronic transition could be identified. The experiment shows, that the CARS probe is capable of determining the nuclear motion coupled to a radiationless electronic transition in complex polyatomic systems. The S1/S0 internal conversion process in b-carotene was further investigated with time-resolved transient gratings. Here, the energetic aspects of a non-adiabatic transition was addressed by determining the influence of the vibrational energy on the rate of this internal conversion. In order to compare the rate of internal conversion taking place out of vibrational ground state modes versus this transition initiating out of vibrationally hot modes, the strategy of shifting the probe mechanism in the transient grating scheme to spectral positions within and out of the red flank of the S1 absorption profile was pursued. The interrogation of different vibrational states was verified by determining the degree of vibrational cooling, taking place parallel to the internal conversion process. With this strategy, it could be shown that vibrationally hot states contribute to the internal conversion with a higher rate than vibrational ground state modes. In summary, different third-order, optical processes in the framework of time-resolved FWM were applied to the study of non-adiabatic dynamics in polyatomic molecules. By utilizing the effect of optical as well as Raman resonances on different FWM processes, it could be shown that third-order, time-resolved spectroscopy is a powerful tool for gaining insight into complex molecular dynamics such as vibronic coupling. The experiments presented in this work showed that the CARS process, as a mode-selective probe in time-resolved experiments, is capable of disentangling nuclear and electronic dynamics.
Thin, pyroclastic marker beds are preserved in argillaceous units of the Dwyka Group in southern Nambia and South Africa which are the earliest witnesses of volcanism in Karoo-equivalent strata of southern Africa. The aim of this study is to present the field appearance of these marker beds, to characterise their mineralogy, geochemistry and heavy mineral contents and to present new radiometric age data from their juvenile zircons. Carboniferous-Permian Karoo deposits in the Aranos Basin of southern Namibia include the glacially dominated, Carboniferous Dwyka Group and the shelf sediments of the overlying Permian Ecca Group. The Dwyka Group can be subdivided into four upward-fining deglaciation sequences, each capped by relatively fine-grained glaciolacustrine or glaciomarine deposits. The uppermost part of the second deglaciation sequence comprises a thick fossiliferous mudstone unit, referred to as the ”Ganigobis Shale Member”. An abundance of marine macro- and ichnofossils as well as extrabasinally derived ashfall tuff beds characterise the more than 40 m thick mudstones and provide the basis for an integrated high-resolution biostratigraphic and tephrostratigraphic framework. The Ganigobis Shale Member contains remains of paleoniscoid fishes, bivalves, gastropods, scyphozoa, crinoid stalks, sponges and sponge spicules, radiolaria, coprolites and permineralised wood. These mostly marine body and trace fossils record the extent of the first of a series of marine incursions into the disintegrating Gondwanan interior as early as the Carboniferous. Within the Ganigobis Shale Member 21 bentonitic tuff beds displaying a thickness of 0.1 and 2.0 cm were determined which in part can be traced laterally over tens of kilometres indicating an ashfall derivation. Further bentonitic tuff beds of the Dwyka Group were detected in cut banks of the Orange River near Zwartbas in the Karasburg Basin (southern Namibia). The 65 tuff beds vary between 0.1 and 4.0 cm in thickness. Due to a similar fossil content and age of the background deposits, the tuff beds are thought to have originated from the same source area as those from the Aranos Basin. Thin-sections reveal the derivation of the tuff beds as distal fallout ashes produced by explosive volcanic eruptions. The matrix consists of a micro- to cryptocrystalline clay mineral-quartz mixture. Rare fragments of splinter quartz, completely recrystallized ash-sized particles of former volcanic glass and few apatite and zircon grains are the only juvenile components. The tuff beds contain as non-opaque, juvenile heavy minerals mostly zircon, apatite, monazite and sphene but also biotite, garnet, hornblende and tourmaline. Geochemical analyses point to an original, intermediate to acid composition of the tuff samples. LREE enrichment and Eu-anomalies show that the parent magma of the tuff beds was a highly evolved calc-alkaline magma. Tectonomagmatic discrimination diagrams point to a volcanic arc setting. Bedding characteristics and the lack of any Carboniferous-Permian volcanic successions onshore Namibia makes an aeolian transport of the ash particles over larger distances likely. Siliceous ashes could thus have been transported by prevailing south-westerly winds from arc-related vents in South America to southern Africa. A second, more local source area could have been located in an intracontinental rift zone along the western margin of southern Africa which is indicated by north-south directed ice-flow directions in the Late Carboniferous. SHRIMP-based age determinations of juvenile magmatic zircons separated from the tuff beds allow a new time calibration of Dwyka Group deglaciation sequences II - IV and the Dwyka/Ecca boundary. Zircons of the Ganigobis Shale Member yield SHRIMP-ages of 302-300 Ma. This dates the uppermost part of the second deglaciation sequence in southern Namibia to the Late Carboniferous (Gzelian) and provides a minimum age for the onset of Karoo-equivalent marine deposition. The age of the uppermost argillaceous part of the third deglaciation sequence (297 Ma) was determined from zircons of a tuffaceous bed sampled in a roadcut in the Western Cape Province, South Africa. The deposits correlate with the Hardap Shale Member in the Aranos Basin of southern Namibia which are part of much more widespread Eurydesma transgression. The age of the Dwyka/Ecca boundary was determined by SHRIMP-measurements of juvenile zircons from two tuff beds of the basal Prince Albert Formation sampled in the Western Cape Province (South Africa). The zircons revealed ages of 289 - 288 Ma which date the Dwyka/Ecca boundary at about 290 Ma. According to these ages, deglaciation sequences II-IV lasted for 5 Ma on average.
This study investigates the foraging behaviour of grass-cutting ants, Atta vollenweideri, with specific consideration of the following issues: (a) cutting behaviour and the determination of fragment size, (b) the effect of load size on transport economics, (c) division of labour and task-partitioning. Grass-cutting ants, Atta vollenweideri, harvest grass fragments that serve as substrate for the cultivation of a symbiotic fungus. Foragers were observed to cut grass fragments across the blade, thus resulting in longish, rectangular-shaped fragments in contrast to the semicircular fragments of leaf-cutting ants. Cutting was very time-consuming: In tough grasses like the typical grassland species Paspallum intermedium and Cyperus entrerrianus, cutting times lasted up to more than 20 minutes per fragment and roughly half of all initiated cutting attempts were given up by the ants. Foragers harvesting the softer grass Leersia hexandra were smaller than those foraging on the hard grasses. Fragment size determination and the extent of size-matching between ant body size and fragment size was investigated regarding possible effects of tissue toughness on decision-making and as a function of the distance from the nest. Tissue toughness affected decision-making such that fragment width correlated with ant body mass for the hard grass but not for the soft one, suggesting that when cutting is difficult, larger ants tend to select wider grasses to initiate cutting. The length of the fragments cut out of the two grass species differed statistically, but showed a large overlap in their distribution. Distance from the nest affected load size as well as the extent of size-matching: Fragments collected directly after cutting were significantly larger than those carried on the trail. This indicates that fragments were cut once again on their way to the nest. Size-matching depended on the trail sector considered, and was stronger in ants sampled closer to the nest, suggesting that carriers either cut fragments in sizes corresponding to their body mass prior transport, or transferred them to nestmates of different size after a short carrying distance. During transport, a worker takes a fragment with its mandibles at one end and carries it in a more or less vertical position. Thus, load length might particularly affect maneuverability, because of the marked displacement of the gravitational center. Conversely, based on the energetic of cutting, workers might maximise their individual harvesting rate by cutting long grass fragments, since the longer a grass fragment, the larger is the amount of material harvested per unit cutting effort. I therefore investigated the economics of load transport by focusing on the effects of load size (mass and length) on gross material transport rate to the nest. When controlling for fragment mass, both running speed of foragers and gross material transport rate was observed to be higher for short fragments. In contrast, if fragment mass was doubled and length maintained, running speed differed according to the mass of the loads, with the heavier fragments being transported at the lower pace. For the sizes tested, heavy fragments yielded a higher transport rate in spite of the lower speed of transport, as they did not slow down foragers so much that it counterbalanced the positive effects of fragment mass on material transport rate. The sizes of the fragments cut by grass-cutting ants under natural conditions therefore may represent the outcome of an evolutionary trade-off between maximising harvesting rate at the cutting site and minimising the effects of fragment size on material transport rates. I investigated division of labour and task partitioning during foraging by recording the behaviour of marked ants while cutting, and by monitoring the transport of fragments from the cutting until they reached the nest. A. vollenweideri foragers showed division of labour between cutting and carrying, with larger workers cutting the fragments, and smaller ones transporting them. This division was absent for food sources very close to the nest, when no physical trail was present. Along the trail, the transport of fragment was a partitioned task, i.e., workers formed bucket brigades composed of 2 to 5 carriers. This sequential load transport occurred more often on long than on short trails. The first carriers of a bucket brigade covered only short distances before dropping their fragments, turned back and continued foraging at the same food source. The last carriers covered the longest distance. There was no particular location on the trail for load dropping , i.e., fragments were not cached. I tested the predictions of two hypotheses about the causes of bucket brigades: First, bucket brigades might occur because of load-carriage effects: A load that is too big for an ant to be carried is dropped and carried further by nestmates. Second, fragments carried by bucket brigades might reach the nest quicker than if they are transported by a single carrier. Third, bucket brigades might enhance information flow among foragers: By transferring the load a worker may return earlier back to the foraging site and be able to reinforce the chemical trail, thus recruitment. In addition, the dropped fragment itself may contain information for unladen foragers about currently harvested sources and may enable them to choose between sources of different quality. I investigated load-carriage effects and possible time-saving by presenting ants with fragments of different but defined sizes. Load size did not affect frequency of load dropping nor the distance the first carrier covered before dropping, and transport time by bucket brigades was significantly longer than by single carriers. In order to study the information transfer hypothesis, I presented ants with fragments of different attractivity but constant size. Ants carrying high-quality fragments would be expected to drop them more often than workers transporting low-quality fragments, thus increasing the frequency of bucket brigades. My results show that increasing load quality increased the frequency of bucket brigades as well as it decreased the carrying distance of the first carrier. In other words, more attractive loads were dropped more frequently and after a shorter distance than less attractive ones with the first carriers returning to the foraging site to continue foraging. Summing up, neither load-carriage effects nor time-saving caused the occurrence of bucket brigades. Rather, the benefit might be found at colony level in an enhanced information flow.
Division of reproductive labour in societies represents a topic of interest in evolutionary biology at least since Darwin. The puzzle of how helpers can be selected for, in spite of their reduced fertility has found an explanation in the kin selection theory: workers can overcome the cost of helping and of forgiving direct reproduction by rearing sufficiently related individuals. However, in the Hymenoptera, little is known on the proximate mechanisms that regulate the division of labour in colonies. Our knowledge is based on several "primitive" ants from the subfamily Ponerinae and two highly eusocial Hymenoptera species. In the former, the dominance hierarchies allowing for the establishment of individuals as reproductives are well understood. In contrast, the pheromonal mechanisms that help maintain their reproductive status are not understood. Similarly in "higher" ants, pheromonal regulation mechanisms of worker reproduction by queens remain largely unknown. The aim of this study is to determine the modalities of production, distribution and action, as well as the identity of the queen pheromones affecting worker reproduction in the ant Myrmecia gulosa. This species belongs to the poorly studied subfamily Myrmeciinae, which is endemic to the Australian region. The subfamily represents, together with the Ponerinae, the most "primitive" ants: their morphology is close to that of the hypothetical ancestor of ants, and the specialisation of queens is weaker than that of "higher" ants. Simple regulation mechanisms were therefore expected to facilitate the investigation. The first step in this study was to characterise the morphological specialisation of queens and workers, and to determine the differences in reproductive potential associated with this specialisation. This study contributes to our understanding of the link between regulation of division of reproductive labour and social complexity. Furthermore, it will help shed light on the reproductive biology in the poorly known subfamily Myrmeciinae. Queens were recognised by workers on the basis of cuticular as well as gland extracts or products. What is the exact function of the multiple pheromones identified and how they interact remains to be determined. This could help understand why queen "signal" in a "primitive" ant with weakly specialised queens such as M. gulosa appears to be as complex as in highly eusocial species. Primer pheromones act on workers? physiology and have long-term effect. Whether workers of M. gulosa reproduce or not is determined by the detection of a queen pheromone of this type. Direct physical contact with the queen is necessary for workers to detect this pheromone. Thus, the colony size of M. gulosa is compatible with a simple system of pheromone perception by workers based on direct physical contact with the queen. When prevented from establishing physical contact with their queen, some workers start to reproduce and are policed by nestmates. The low volatility of the cuticular hydrocarbons (CHCs), their repartition over the entire cuticle and the existence of queen and worker specific CHC profiles suggest that these chemicals constitute a queen pheromone. Importance of HC versus non-HC compounds was confirmed by bioassaying purified fraction of both classes of chemicals. This study demonstrates for the first time that purified HCs indeed are at the basis of the recognition of reproductive status. This supports the idea that they are also at the basis of the recognition of queens by their workers. As CHCs profiles of workers and queens become similar with acquisition of reproductive status, they represent honest fertility markers. These markers could be used as signals of the presence of reproductives in the colonies, and represent the basis of the regulation of division of reproductive labour.
A significant relatedness is of fundamental importance for the evolution and maintenance of social life (kin selection theory, Hamilton 1964a,b). Not only kin selection itself, but also more complex evolutionary theories make predictions on the occurrence of conflict and co-operation in animal societies. They all depend on the genetic relationships among individuals. Therefore, the study of unrelated, co-operating individuals provides a unique opportunity to critically test predictions based on these evolutionary theories. Using allozyme electrophoresis, the study species Pachycondyla villosa was found to represent three different species. Young queens in one of these species, provisionally called Pachycondyla cf. inversa, may co-operate during colony founding (pleometrosis). Approximately 50 per cent of all founding colonies collected near Itabuna, Brazil, consisted of two to five founding queens. Queens of P. cf. inversa have to forage for food (semi-claustral founding), and in founding associations only one queen specialised for this risky task. A microsatellite study showed that nestmate queens were typically not related. How can a division of labour be achieved, where one individual performs risky tasks to the favour of another individual to which it is not related? In contrast to the predictions made by group selectionists, this study provided clear evidence that the division of labour among co-foundresses of P. cf. inversa results from social competition: Co-foundresses displayed aggressive interactions and formed dominance hierarchies which predominantly served to force subordinates to forage. The frequency of queen antagonism increased with the duration since food was last added to the foraging arena. The social status was not, or only weakly associated with the reproductive status: As predicted by the reproductive skew theory, all foundresses laid eggs at similar rates, though the subordinate may be harassed during egg laying and occasionally, some of her eggs may be eaten by the dominant. The differential oophagy presumably was also reflected in a microsatellite study of foundress associations, which was conducted shortly after the first workers emerged: Here, the co-foundresses occasionally contributed unequally to the colony’s workers. Conflicts among workers or between workers and queens, e.g. over the division of labour or sex ratio, strongly depend on the genetic relationships among members of a colony. The number of two to five co-founding queens in polygynous colonies of P. cf. inversa, and the lack of relatedness among them, should lead to a decrease in the relatedness of workers. However, nestmate workers were closely related. Furthermore, worker relatedness may decrease as several queens were found to be multiply inseminated. Inbreeding coefficients were significantly different from zero in both queens and workers. No evidence for a geographical substructuring of the population was found. The deviation from random mating presumably was probably due to small, localised nuptial flights. Virgin queens do not mate near their natal nest and disperse before founding colonies. The analysis of cuticular hydrocarbons obtained from live queens revealed consistent differences between the patterns of cuticular hydrocarbons of queens with high vs. low rank: only high-ranking queens showed considerable amounts of cuticular pentadecane (n-C15) and heptadecene (n-C17:1). The presence of the two substances apparently was not associated with reproductive status. It is not yet known, if the two substances indeed serve to communicate high social status in P. cf. inversa. In experimentally assembled associations of two founding queens, queens engaged in aggressive interactions which already within one to twenty minutes resulted in stable dominance hierarchies. The queens attacking first usually won the contest and became dominant. Nest ownership at least for a couple of days did not influence the outcome of dominance interactions in the laboratory experiments, whereas queen body size apparently played an important role: In all eight trials, the larger queen became dominant. However, dominant queens from natural foundress associations were on average not larger than subordinates, suggesting that in the field, resident asymmetries might override size asymmetries only after a more prolonged period of nest ownership. Sequencing of the COI/COII region of mitochondrial DNA displayed sufficient variability for the study of the sociogenetic structure of the secondarily polygynous ant Pachycondyla obscuricornis: Six different haplotypes could be distinguished among six workers of different colonies from one study population in Costa Rica. The variability of other methods which were established (RFLPs, microsatellites, allozymes, and multilocus DNA fingerprinting) was too low for a further study on the genetic structure in P. obscuricornis.
In the present study, a new gene cluster of Listeria monocytogenes EGD containing three internalin genes was identified and characterized. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the class of large, surface-bound internalins. Each of these proteins contains a signal peptide, two regions of repeats (Leucine-rich repeats and B repeats), an inter-repeat region and a putative cell wall anchor sequence containing the sorting motiv LPXTG. PCR analysis revealed the presence of the inlGHE gene cluster in most L. monocytogenes serotypes. A similar gene cluster termed inlC2DE localised to the same position on the chromosome was described in a different L. monocytogenes EGD isolate. Sequence comparison of the two clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and a 5'-portion of inlD. The genes inlG, inlH and inlE seem to be transcribed extracellularly and independent of PrfA. To study the function of the inlGHE gene cluster several in-frame deletion mutants were constructed which lack the genes of the inlGHE cluster individually or in combination with other inl genes. When tested in the mouse model, the inlGHE mutant showed a significant reduction of bacterial counts in liver and spleen in comparison to the wild type strain, indicating that the inlGHE gene cluster plays an important role in virulence of L. monocytogenes. The ability of this mutant to invade non-phagocytic cells in vitro was however two- to three-fold higher than that of the parental strain. To examine whether deletion of the single genes from the cluster has the same stimulatory effect on invasiveness as deletion of the complete gene cluster, the single in-frame deletion mutants inlG, inlH and inlE were constructed. These mutants were subsequently reverted to the wild type by introducing a copy of the corresponding intact gene into the chromosome by homologous recombination using knock-in plasmids. To determine a putative contribution of InlG, InlH and InlE in combination with other internalins to the entry of L. monocytogenes into mammalian cells, the combination mutants inlA/GHE, inlB/GHE, inlC/GHE, inlA/B/GHE, inlB/C/GHE, inlA/C and inlA/C/GHE were constructed. Transcription of the genes inlA, inlB and inlC in these mutants was studied by RT-PCR. Deletion of inlGHE enhances transcription of inlA and inlB, but not of inlC. This enhancement is not transient but can be observed at different time-points of the bacterial growth curve. Deletion of inlA also increases transcription of inlB and vice-versa. In contrast, the amounts of inlA and inlB transcripts in the single deletion mutants inlG, inlH and inlE were similar to those from the wild type.
A central objective of many ecophysiological investigations is the establishment of mechanistic explanations for plant distributions in time and space. The important, albeit mostly ignored, question arises as to the nature of the organisms that should be used as representative in pertinent experiments. I suggest that it is essential to use a “demographic approach” in physiological ecology, because physiological parameters such as photosynthetic capacity (PC, determined under non-limiting conditions with the oxygen electrode) may change considerably with plant size. Moreover, as shown for nine epiphyte species covering the most important taxonomic groups, the intraspecific variability in PC was almost always higher than the interspecific variability when comparing only large individuals. In situ studies with the epiphytic bromeliad V. sanguinolenta revealed that besides physiological parameters (such as PC) almost all morphological, anatomical and other physiological leaf parameters studied changed with plant size as well. Likewise, important processes proved to be size-dependent on whole-plant level. For example, long-term water availability was clearly improved in large specimens compared to smaller conspecifics due to the increased efficiency of the tanks to bridge rainless periods. As model calculations on whole-plant level for V. sanguinolenta under natural conditions have shown photosynthetic leaf carbon gain as well as respiratory losses of heterotrophic plant parts scaled with plant size. The resulting area related annual carbon balances were similar for plants of varying size, which corresponded to observations of size-independent (and low) relative growth rates in situ. Under favorable conditions in the greenhouse, however, small V. sanguinolenta exhibited surprisingly high relative growth rates, similar to annuals, which clearly contradicts the prevalent, but barely tested notion of epiphytes as inherently slow growing plants and simultaneously illustrates the profound resource limitations that epiphytes are subjected to in the canopy of a seasonal rain forest. From habitat conditions it seems that size-related differences in water availability are the driving force behind the observed size-dependent ecophysiological changes: the larger an epiphyte grows the more independent it is with regard to precipitation patterns. In conclusion, the results strongly emphasize the need to treat plant size as an important source of intraspecific variability and thus urge researchers to consider plant size in the design of ecophysiological experiments with vascular epiphytes.
Safer without Sex?
(1999)
Highly eusocial insect societies, such as all known ants, are typically characterized by a reproductive division of labor between queens, who are inseminated and reproduce, and virgin workers, who engage in foraging, nest maintenance and brood care. In most species workers have little reproductive options left: They usually produce haploid males by arrhenotokous parthenogenesis, both in the queenright and queenless condition. In the phylogenetically primitive subfamily Ponerinae reproductive caste dimorphism is much less pronounced: Ovarian morphology is rather similar in queens and workers, which additionally retain a spermatheca. In many ponerine species workers mate and may have completely replaced the queen caste. This similarity in reproductive potential provides for the evolution of diverse reproductive systems. In addition, it increases the opportunity for reproductive conflicts among nestmates substantially. Only in a handful of ant species, including Platythyrea punctata, workers are also able to rear diploid female offspring from unfertilized eggs by thelytokous parthenogenesis. The small ponerine ant P. punctata (Smith) is the only New World member of the genus reaching as far north as the southern USA, with its center of distribution in Central America and the West Indies. P. punctata occurs in a range of forest habitats including subtropical hardwood forests as well as tropical rain forests. In addition to queens, gamergates and thelytokous workers co-occur in the same species. This remarkable complexity of reproductive strategies makes P. punctata unique within ants and provides an ideal model system for the investigation of reproductive conflicts within the female caste. Colonies are usually found in rotten branches on the forest floor but may also be present in higher strata. Colonies contained on average 60 workers, with a maximum colony size of 148 workers. Queens were present in only ten percent of the colonies collected from Florida, but completely absent both from the populations studied in Barbados and Puerto Rico. Males were generally rare. In addition, morphological intermediates between workers and queens (so-called intercastes) were found in 16 colonies collected in Florida. Their thorax morphology varied from an almost worker-like to an almost queen-like thorax structure. Queen and intercaste size, however, did not differ from those of workers. Although workers taken from colonies directly after collection from the field engaged in aggressive interactions, nestmate discrimination ceased in the laboratory suggesting that recognition cues used are derived from the environment. Only one of six queens dissected was found to be inseminated but not fertile. Instead, in most queenless colonies, a single uninseminated worker monopolized reproduction by means of thelytokous parthenogenesis. A single mated, reproductive worker (gamergate) was found dominating reproduction in the presence of an inseminated alate queen only in one of the Florida colonies. The regulation of reproduction was closely examined in ten experimental groups of virgin laboratory-reared workers, in which one worker typically dominated reproduction by thelytoky despite the presence of several individuals with elongated, developing ovaries. In each group only one worker was observed to oviposit. Conflict over reproduction was intense consisting of ritualized physical aggression between some nestmates including antennal boxing, biting, dragging, leap and immobilization behaviors. The average frequency of interactions was low. Aggressive interactions allowed to construct non-linear matrices of social rank. On average, only five workers were responsible for 90 percent of total agonistic interactions. In 80 percent of the groups the rate of agonistic interactions increased after the experimental removal of the reproductive worker. While antennal boxing and biting were the most frequent forms of agonistic behaviors both before and after the removal, biting and dragging increased significantly after the removal indicating that agonistic interactions increased in intensity. Once a worker obtains a high social status it is maintained without the need for physical aggression. The replacement of reproductives by another worker did however not closely correlate with the new reproductive's prior social status. Age, however, had a profound influence on the individual rate of agonistic interactions that workers initiated. Especially younger adults (up to two month of age) and callows were responsible for the increase in observed aggression after the supersedure of the old reproductive. These individuals have a higher chance to become reproductive since older, foraging workers may not be able to develop their ovaries. Aggressions among older workers ceased with increasing age. Workers that already started to develop their ovaries should pose the greatest threat to any reproductive individual. Indeed, dissection of all experimental group revealed that aggression was significantly more often directed towards both individuals with undeveloped and developing ovaries as compared to workers that had degenerated ovaries. In all experimental groups reproductive dominance was achieved by callows or younger workers not older than four month. Age is a better predictor of reproductive dominance than social status as inferred from physical interactions. Since no overt conflict between genetical identical individuals is expected, in P. punctata the function of agonistic interactions in all-worker colonies, given the predominance of thelytokous parthenogenesis, remains unclear. Physical aggression could alternatively function to facilitate a smooth division of non-reproductive labor thereby increasing overall colony efficiency. Asexuality is often thought to constitute an evolutionary dead end as compared with sexual reproduction because genetic recombination is limited or nonexistent in parthenogenetic populations. Microsatellite markers were developed to investigate the consequences of thelytokous reproduction on the genetic structure of four natural populations of P. punctata. In the analysis of 314 workers taken from 51 colonies, low intraspecific levels of variation at all loci, expressed both as the number of alleles detected and heterozygosities observed, was detected. Surprisingly, there was almost no differentiation within populations. Populations rather had a clonal structure, with all individuals from all colonies usually sharing the same genotype. This low level of genotypic diversity reflects the predominance of thelytoky under natural conditions in four populations of P. punctata. In addition, the specificity of ten dinucleotide microsatellite loci developed for P. punctata was investigated in 29 ant species comprising four different subfamilies by cross-species amplification. Positive amplification was only obtained in a limited number of species indicating that sequences flanking the hypervariable region are often not sufficiently conserved to allow amplification, even within the same genus. The karyotype of P. punctata (2n = 84) is one of the highest chromosome numbers reported in ants so far. A first investigation did not show any indication of polyploidy, a phenomenon which has been reported to be associated with the occurrence of parthenogenesis. Thelytokous parthenogenesis does not appear to be a very common phenomenon in the Hymenoptera. It is patchily distributed and restricted to taxa at the distant tips of phylogenies. Within the Formicidae, thelytoky has been demonstrated only in four phylogenetically very distant species, including P. punctata. Despite its advantages, severe costs and constraints may have restricted its rapid evolution and persistence over time. The mechanisms of thelytokous parthenogenesis and its ecological correlates are reviewed for the known cases in the Hymenoptera. Investigating the occurrence of sexual reproduction in asexual lineages indicates that thelytokous parthenogenesis may not be irreversible. In P. punctata the occasional production of sexuals in some of the colonies may provide opportunity for outbreeding and genetic recombination. Thelytoky can thus function as a conditional reproductive strategy. Thelytoky in P. punctata possibly evolved as an adaptation to the risk of colony orphanage or the foundation of new colonies by fission. The current adaptive value of physical aggression and the production of sexuals in clonal populations, where relatedness asymmetries are virtually absent, however is less clear. Quite contrary, thelytoky could thereby serve as the stepping stone for the subsequent loss of the queen caste in P. punctata. Although P. punctata clearly fulfills all three conditions of eusociality, the evolution of thelytoky is interpreted as a first step in a secondary reverse social evolution towards a social system more primitive than eusociality.
Many polymorphisms are linked to alternative reproductive strategies. In animals, this is particularly common in males. Ant queens are an important exception. The case of ant queen size dimorphisms has not been studied in sufficient detail, and thus this thesis aimed at elucidating causes and consequences of the different size of small (microgynous) and large (macrogynous)ant queens using the North American ant species Leptothorax rugatulus as a model system. Employing neutral genetic markers, no evidence for a taxonomically relevant separation of the gene pools of macrogynes and microgynes was found. Queens in polygynous colonies were highly related to each other, supporting the hypothesis that colonies with more than one queen commonly arise by secondary polygyny, i.e. by the adoption of daughter queens into their natal colonies. These results and conclusions are also true for the newly discovered queen size polymorphism in Leptothorax cf. andrei. Several lines of evidence favor the view that macrogynes predominantly found their colonies independently, while microgynes are specialized for dependent colony founding by readoption. Under natural conditions, mother and daughter size are highly correlated and this is also true for laboratory colonies. However, the size of developing queens is influenced by queens present in the colony. Comparing populations across the distribution range, it turns out that queen morphology (head width and ovariole number) is more differentiated among populations than worker morphology (coloration, multivariate size and shape), colony characteristics (queen and worker number per colony) or neutral genetic variation. Northern and southern populations differed consistently which indicates the possibility of two different species. The queen size dimorphism in L. rugatulus did neither influence the sex ratio produced by a colony, nor its ratio of workers to gynes. However, the sex ratio covaried strongly across populations with the average number of queens per colony in accordance with sex ratio theory. At the colony level, sex ratio could not be explained by current theory and a hypothesis at the colony-level was suggested. Furthermore, queen body size has no significant influence on the amount of reproductive skew among queens. Generally, the skew in L. rugatulus is low, and supports incomplete control models, rather than the classic skew models. In eight of fourteen mixed or microgynous colonies, the relative contributions of individual queens to workers, gynes and males were significantly different. This was mainly due to the fact that relative body size was negatively correlated with the ratio of gynes to workers produced. This supports the kin conflict over caste determination hypothesis which views microgyny as a selfish reproductive tactic.
Biofilm production is an important step in the pathogenesis of S. epidermidis polymer-associated infections and depends on the expression of the icaADBC operon leading to the synthesis of a polysaccharide intercellular adhesin (PIA). The PIA represents a sugar polymer consisting of ß-1,6 linked N-acetyl glucosaminoglycans and mediates the intercellular adherence of the bacteria to each other and the accumulation of a multilayered biofilm. Epidemiological and experimental studies strongly suggest that PIA-production and subsequently biofilm formation contributes significantly to the virulence of specific S. epidermidis strains. This work aimed on the investigation of external factors regulating the ica expression in S. epidermidis. For this purpose, a reporter gene fusion between the ica promoter and the beta-galactosidase gene lacZ from E. coli was constructed and integrated into the chromosome of an ica positive S. epidermidis clinical isolate. The reporter gene fusion was used to investigate the influence of external factors and of sub-MICs of different antibiotics on the ica expression. It was shown that the S. epidermidis biofilm formation is growth phase dependent with a maximum expression in the late logarithmic and early stationary growth phase. The optimal expression was recorded at 42 °C at a neutral pH ranging from 7.0 to 7.5. The glucose content of the medium was found to be essential for biofilm formation, since concentrations of 1.5 to 2 per cent glucose induced the ica expression. In addition, external stress factors as high osmolarity (mediated by 3 to 5 per cent sodium chloride), and sub-lethal concentrations of detergents, ethanol, hydrogene peroxide, and urea significantly enhanced the biofilm production. Subinhibitory concentrations of tetracyline, the semisynthetic streptogramin quinupristin/dalfopristin and the streptogramin growth promoter virginiamycin were found to enhance the ica expression 8 to 11-fold, respectively, whereas penicillin, oxacillin, gentamicin, clindamycin, vancomycin, teicoplanin, ofloxacin, and chloramphenicol had no effects. A weak induction was recorded for sub-MICs of erythromycin. Both quinupristin/ dalfopristin and tetracyline exhibited a strong postexposure effect on the S. epidermidis ica expression, respectively, even when the substances were immediately removed from the growth medium. The results were confirmed by Northern blot analysis of the ica transcription and quantitative analysis of biofilm formation in a colorimetric assay. Expression of the icaprom::lacZ reporter gene plasmid in Bacillus subtilis and S. epidermidis revealed that the ica induction by sub-MICs of streptogramins and tetracycline might depend on unidentified regulatory elements which are specific for the staphylococcal cell. In contrast, the activation by external stress signals seems to be mediated by factors which are present both in Staphylococci and in Bacillus subtilis. Construction and analysis of an agr-mutant in a biofilm-forming S. epidermidis strain excluded the possibility that the Agr-quorum-sensing system significantly contributes to the ica expression in the stationary growth phase. However, clear evidence was provided that in S. aureus the ica transcription depends on the expression of the alternative transcription factor sigmaB, which represents a global regulator of the stress response in S. aureus as well as in B. subtilis. For this purpose, a sigB knockout mutant had been constructed in a biofilm-forming S. aureus. This mutant showed a markedly decrease of the ica transcription and biofilm-production, whereas a complement strain carrying the sigB gene on an expression vector completely restored the biofilm-forming phenotype of the S. aureus wild type. Southern blot analysis indicated that the the sigB gene is also present in S. epidermidis and Northern analyses of the sigB and the ica transcription revealed that both genes are activated under identical conditions (i. e. in the stationary growth phase and by external stress factors) suggesting a similar regulatory pathway as in S. aureus. However, since neither in S. aureus nor in S. epidermidis the ica promoter has obvious similiarities to known SigB-dependent promotoer sequences it is tempting to speculate that the ica activation is not directely mediated by SigB, but might be indirectely controlled by other SigB-dependent regulatory elements which remain to be elucidated.
The transmission of proliferative and developmental signals from activated cell-surface receptors to initiation of cellular responses in the nucleus is synergically controlled by the coordinated action of a diverse set of intracellular signalling proteins. The Ras/Raf/MEK/MAPK signalling pathway has been shown to control the expression of genes which are crucial for the physiological regulation of cell proliferation, differentiation and apoptosis. Within this signalling cascade, the Raf protein family of serine/threonine kinases serves as a central intermediate which connects to many of other signal transduction pathways. To elucidate the signalling functions of the different Raf kinases in motoneurons during development, the expression, distribution and subcellular localization of Rafs in the spinal cord and the facial nucleus in brainstem of mice at various embryonic and postnatal stages were investigated. Moreover, we have investigated the intracellular redistribution of Raf molecules in isolated motoneurons from 13 or 14 day old mouse embryos, after addition or withdrawal of neurotrophic factors to induce Raf kinases activation in vitro. Furthermore, in order to investigate the potential anti-apoptotic function of Raf kinases on motoneurons, we isolated motoneurons from B-raf-/- and c-raf-1-/- mouse embryos and analysed the survival and differentiation effects of neurotrophic factors in motoneurons lacking B-Raf and c-Raf-1. We provide evidence here that all three Raf kinases are expressed in mouse spinal motoneurons. Their expression increases during the period of naturally occurring cell death of motoneurons. In sections of embryonic and postnatal spinal cord, motoneurons express exclusively B-Raf and c-Raf-1, but not A-Raf, and subcellularly Raf kinases are obviously colocalized with mitochondria. In isolated motoneurons, most of the B-Raf or c-Raf-1 immunoreactivity is located in the perinuclear space but also in the nucleus, especially after activation by addition of CNTF and BDNF in vitro. We found that c-Raf-1 translocation from the cytosol into the nucleus of motoneurons after its activation by neurotrophic factors is a distinct event. As a central finding of our study, we observed that the viability of isolated motoneurons from B-raf but not c-raf-1 knockout mice is lost even in the presence of CNTF and other neurotrophic factors. This indicates that B-Raf but not c-Raf-1, which is still present in B-raf deficient motoneurons, plays a crucial role in mediating the survival effect of neurotrophic factors during development. In order to prove that B-Raf is an essential player in this scenario, we have re-expressed B-Raf in mutant sensory and motor neurons by transfection. The motoneurons and the sensory neurons from B-raf knockout mouse which were transfected with exogenous B-raf gene revealed the same viability in the presence of neurotrophic factors as primary neurons from wild-type mice. Our results suggest that Raf kinases have important signalling functions in motoneurons in mouse CNS. In vitro, activation causes redistribution of Raf protein kinases, particularly for c-Raf-1, from motoneuronal cytoplasm into the nucleus. This redistribution of c-Raf-1, however, is not necessary for the survival effect of neurotrophic factors, given that B-raf-/- motor and sensory neurons can not survive despite the presence of c-Raf-1. We hypothesize that c-Raf-1 nuclear translocation may play a direct role in transcriptional regulation as a consequence of neurotrophic factor induced phosphorylation and activation of c-Raf-1 in motoneurons. Moreover, the identification of target genes for nuclear translocated c-Raf-1 and of specific cellular functions initiated by this mechanism awaits its characterization.
The Mouthparts of Ants
(2001)
Ant mandible movements cover a wide range of forces, velocities and precision. The key to the versatility of mandible functions is the mandible closer muscle. In ants, this muscle is generally composed of distinct muscle fiber types that differ in morphology and contractile properties. Volume proportions of the fiber types are species-specific and correlate with feeding habits. Two biomechanical models explain how the attachment angles are optimized with respect to force and velocity output and how filament-attached fibers help to generate the largest force output from the available head capsule volume. In general, the entire mandible closer muscle is controlled by 10-12 motor neurons, some of which exclusively supply specific muscle fiber groups. Simultaneous recordings of muscle activity and mandible movement reveal that fast movements require rapid contractions of fast muscle fibers. Slow and accurate movements result from the activation of slow muscle fibers. Forceful movements are generated by simultaneous co-activation of all muscle fiber types. For fine control, distinct fiber bundles can be activated independently of each other. Retrograde tracing shows that most dendritic arborizations of the different sets of motor neurons share the same neuropil in the suboesophageal ganglion. In addition, some motor neurons invade specific parts of the neuropil. The labiomaxillary complex of ants is essential for food intake. I investigated the anatomical design of the labiomaxillary complex in various ant species focusing on movement mechanisms. The protraction of the glossa is a non muscular movement. Upon relaxation of the glossa retractor muscles, the glossa protracts elastically. I compared the design of the labiomaxillary complex of ants with that of the honey bee, and suggest an elastic mechanism for glossa protraction in honey bees as well. Ants employ two different techniques for liquid food intake, in which the glossa works either as a passive duct (sucking), or as an up- and downwards moving shovel (licking). For collecting fluids at ad libitum food sources, workers of a given species always use only one of both techniques. The species-specific feeding technique depends on the existence of a well developed crop and on the resulting mode of transporting the fluid food. In order to evaluate the performance of collecting liquids during foraging, I measured fluid intake rates of four ant species adapted to different ecological niches. Fluid intake rate depends on sugar concentration and the associated fluid viscosity, on the species-specific feeding technique, and on the extent of specialization on collecting liquid food. Furthermore, I compared the four ant species in terms of glossa surface characteristics and relative volumes of the muscles that control licking and sucking. Both probably reflect adaptations to the species-specific ecological niche and determine the physiological performance of liquid feeding. Despite species-specific differences, single components of the whole system are closely adjusted to each other according to a general rule.
Tropical rain forests and coral reefs are usually regarded as the epitome of complexity and diversity. The mechanisms, however, that allow so many species to coexist continuously, still need to be unraveled. Earlier equilibrium models explain community organization with a strict niche separation and specialization of the single species, achieved mainly by interspecific competition and consecutive resource partitioning. Recent non-equilibrium or stochastic models see stochastic factors ("intermediate disturbances") as more important. Such systems are characterized by broad niche overlaps and an unpredictable species composition. Mechanisms of coexistence are most interesting where species interactions are strongest and species packing is highest. This is the case within a functional group or guild where species use similar resources. In this project a community of seven closely related leaf beetle species (Chrysomelidae: Cassidinae) was investigated which coexist on a common host plant system (fam. Convovulaceae) in a tropical moist savanna (Ivory Coast, Comoé-Nationalpark). A broad overlap in the seasonal phenology of the leaf beetle species stood in contrast to a distinct spatial niche differentiation. The beetle community could be separated in a savanna-group (host plant: Ipomoea) and in a river side group (host plant: Merremia). According to a correspondence analysis the five species at the river side, using a common host plant, Merremia hederacea, proved to be predictable in their species composition. They showed a small scale niche differentiation along the light gradient (microhabitats). Laboratory studies confirmed differences in the tolerance towards high temperatures (up to 50°C in the field). Physiological trade-offs between phenology, microclimate and food quality seem best to describe patterns of resource use of the beetle species. Further a phylogeny based on mt-DNA sequencing of the beetle community was compared to its ecological resource use and the evolution of host plant use was reconstructed
Listeriae are Gram positive, facultative, saprophytic bacteria capable of causing opportunistic infections in humans and animals. This thesis presents three separate lines of inquiries that can lead to the eventual convergence of a global view of Listeria as pathogen in the light of evolution, genomics, and function. First, we undertook to resolve the phylogeny of the genus Listeria with the goal of ascertaining insights into the evolution of pathogenic capability of its members. The phylogeny of Listeriae had not yet been clearly resolved due to a scarcity of phylogenetically informative characters within the 16S and 23S rRNA molecules. The genus Listeria contains six species: L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. grayi; of these, L. monocytogenes and L. ivanovii are pathogenic. Pathogenicity is enabled by a 10-15Kb virulence gene cluster found in L. seeligeri, L. monocytogenes and L. ivanovii. The genetic contents of the virulence gene cluster loci, as well as some virulence-associated internalin loci were compared among the six species. Phylogenetic analysis based on a data set of nucleic acid sequences from prs, ldh, vclA, vclB, iap, 16S and 23S rRNA genes identified L. grayi as the ancestral branch of the genus. This is consistent with previous 16S and 23S rRNA findings. The remainder 5 species formed two groupings. One lineage represents L. monocytogenes and L. innocua, while the other contains L. welshimeri, L. ivanovii and L. seeligeri, with L. welshimeri forming the deepest branch within this group. Deletion breakpoints of the virulence gene cluster within L. innocua and L. welshimeri support the proposed tree. This implies that the virulence gene cluster was present in the common ancestor of L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri; and that pathogenic capability has been lost in two separate events represented by L. innocua and L. welshimeri. Second, we attempted to reconstitute L. innocua of its deleted virulence gene cluster, in its original chromosomal location, from the L. monocytogenes 12 Kb virulence gene cluster. This turned out particularly difficult because of the limits of genetic tools presently available for the organism. The reconstitution was partially successful. The methods and approaches are presented, and all the components necessary to complete the constructs are at hand for both L. innocua and the parallel, positive control of L. monocytogenes mutant deleted of its virulence gene cluster. Third, the sequencing of the entire genome of L. monocytogenes EGDe was undertaken as part of an EU Consortium. Our lab was responsible for 10 per cent of the labor intensive gap-closure and annotation efforts, which I helped coordinate. General information and comparisons with sister species L. innocua and a close Gram positive relative Bacillus subtilis are presented in context. The areas I personally investigated, namely, sigma factors and stationary phase functions, are also presented. L. monocytogenes and L. innocua both possess surprisingly few sigma factors: SigA, SigB, SigH, SigL, and an extra-cytoplasmic function type sigma factor (SigECF). The stationary phase genes of L. monocytogenes is compared to the well-studied, complex, stationary phase networks of B. subtilis. This showed that while genetic competence functions may be operative in unknown circumstances, non-sporulating Listeria opted for very different approaches of regulation from B. subtilis. There is virtually no overlap of known, stationary phase genes between Listeria and Gram negative model organism E. coli.
Cofilin
(1999)
This study has identified cofilin, an actin binding protein, as a control element in the reorganization of the actin cytoskeleton which is highly relevant for T lymphocyte activation. Cofilin is regulated in its activity by reversible phosphorylation which is inducible by stimulation through accessory receptors such as CD2 and CD28. First it could be demonstrated that accessory receptor triggering induces the transient association of cofilin with the actin cytoskeleton and that only the dephosphorylated form of cofilin possesses the capacity to bind cytoskeletal actin in vivo. PI3-kinase inhibitors block both the dephosphorylation of cofilin and its association with the actin cytoskeleton. Importantly, cofilin, actin, PI3-kinase and one of its substrates, namely phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) which can bind to cofilin, co-localize within CD2-receptor caps. The cofilin/F-actin interaction has been identified as a crucial regulatory element for receptor cap formation and the strength of signal transduction. To this end, appropriately designed cell permeable non-toxic peptides that are homologous to actin binding motifs of the human cofilin sequence were introduced into untransformed human peripheral blood T lymphocytes. These peptides competitively and dose dependently inhibit the activation induced interaction of cofilin with the actin cytoskeleton in vivo. By this approach it was possible to study, for the first time, the functional consequences of this interaction in immunocompetent T cells. The present data demonstrate that inhibition of the actin/cofilin interaction in human T lymphocytes by means of these cofilin derived peptides abolishes receptor cap formation and strongly modulates functional T cell responses such as T cell proliferation, interleukin-2 production, cell surface expression of CD69, gIFN production, and CD95L expression. Importantly, receptor independent activation by PMA and calcium ionophore circumvents these peptide produced inhibitory effects on lymphocyte stimulation and places the cofilin/actin interaction to a proximal step in the cascade of signaling events following T cell activation via surface signals. The present results are novel since as yet no information existed regarding the molecular elements which link cell surface receptor stimulation directly to the resulting reorganization of the actin cytoskeleton.
Distinct juvenile behaviour differences, changes in adult sizes and reproductive capacity and a long reproductive period triggered the working hypothesis of two alternative life-cycle strategies favouring aestivation or immediate reproduction. The hypothesis for the life-cycles of Hyperolius nitidulus that differed from the commonly assumed reproductive strategy for this species was confirmed by the results of this study. Aestivated juveniles start to mature at the beginning of the rainy season and reproduce subsequently. Their tadpoles grow until metamorphosis and either reproduce in this same season, in which case their offspring aestivates (one year - two generations), or they delay reproduction to the following year and aestivate themselves (one year - one generation). Juveniles trying to reproduce as fast as possible will invest in growth and differentiation and show no costly adaptations to aestivation, while juveniles delaying reproduction to the following rainy season will be well adapted to dry season conditions. Indirect evidence for the existence of a second generation was found in all three investigation years: adult size decreased abruptly towards the end of the rainy season, mainly due to the arrival of very small individuals, and clutch size decreased abruptly. Also at the end of the rainy season juveniles had two behavioural types: one hiding on the ground and clearly avoiding direct sunlight and another sitting freely above ground showing higher tolerance towards dry season conditions (high air temperatures and low humidity). Skin morphology differed between the types showing many more purine crystals in a higher order in the dry-season adapted juveniles. The final proof for the existence of a second generation came with the recapture of individuals marked as juveniles when they left the pond. The 45 recaptured frogs definitely came back to the pond to reproduce during the same season in 1999. Second generation frogs (males and females) were significantly smaller than the rest of all adults and egg diameter was reduced. Clutch size did not differ significantly. It was found that females did not discriminate against second generation males when coming to the ponds to reproduce. Second generation males had a similar chance to be found in amplexus as first generation males. Indirect and direct evidence for a second generation matched very well. The sudden size decrease in adults occurred just at the time when the first marked frogs returned. The observation that freshly metamorphosed froglets were able to sit in the sun directly after leaving the water led to the assumption that the decision whether to aestivate or to reproduce already happens during the frogs' larval period. Water chemistry and the influence of light was investigated to look for the factors triggering the decision, but only contaminated water increased the number of juveniles ready for aestivation. Whether the life history polymorphism observed in Hyperolius nitidulus is due to phenotypic plasticity or genetic polymorphism is still not known. Despite this uncertainty, there is no doubt that the optimal combination of different life histories is profitable and may be a reason for the wide range and high local abundance of Hyperolius nitidulus.
The study examines the sensory ecology of CO2 perception in leaf-cutting ants. It begins with the ecological role of CO2 for leaf-cutting ants. Inside the subterranean nests of Atta vollenweideri large amounts of CO2 are produced by the ants and their symbiotic fungus. Measurements in field nest at different depths revealed that CO2 concentrations do not exceed 2 per cent in mature nests. These findings indicate effective ventilation even at depths of 2 m. Small colonies often face the situation of reduced ventilation when they close their nest openings as a measure against flooding. A simulation of this situation in the field as well as in the laboratory revealed increasing CO2 concentrations causing reduced colony respiration which ultimately might limit colony success. Wind-induced ventilation is the predominant ventilation mechanism of the nests of Atta vollenweideri, shown by an analysis of external wind and airflow in the channels. The mound architecture promotes nest ventilation. Outflow channels have their openings in the upper, central region and inflow channels had their openings in the lower, peripheral region of the nest mound. Air is sucked out through the central channels, followed by a delayed inflow of air through the peripheral channels. The findings support the idea that the nest ventilation mechanism used by Atta vollenweideri resembles the use of Bernoulli’s principle in Venturi Tubes and Viscous Entrainment. CO2 is important in a second context besides microclimatic control. A laboratory experiment with Atta sexdens demonstrated that leaf-cutting ants are able to orientate in a CO2 gradient. Foragers chose places with higher CO2 concentration when returning to the nest. This effect was found in all homing foragers, but it was pronounced for workers carrying leaf fragments compared to workers without leaf fragments. The findings support the hypothesis that CO2 gradients are used as orientation cue inside the (dark) nest to find suited fungus chambers for unloading of the leaf fragments. After the importance of CO2 in the natural history of the ants has thus been demonstrated, the study identifies for the first time in Hymenoptera type and location of the sensory organ for CO2 perception. In Atta sexdens a single neuron associated with the sensilla ampullacea was found to respond to CO2. Since it is the only neuron of this sensillum, the sensillum characters can be assumed to be adapted for CO2 perception. A detailed description of the morphology and the ultrastructure allows a comparison with sensilla for CO2 perception found in other insects and provides more information about sensillum characters and their functional relevance. The CO2 receptor cells respond to increased CO2 with increased neural activity. The frequency of action potentials generated by the receptor cell shows a phasic-tonic time course during CO2 stimulation and a reduced activity after stimulation. Phasic response accomplished with a reduced activity after stimulation results in contrast enhancement and the ability to track fast fluctuations in CO2 concentration. The neurons have a working range of 0 to 10 per cent CO2 and thus are able to respond to the highest concentrations the ants might encounter in their natural environment. The most exciting finding concerning the receptor cells is that the CO2 neurons of the leaf-cutting ants do not adapt to continuous stimulation. This enables the ants to continuously monitor the actual CO2 concentration of their surroundings. Thus, the sensilla ampullacea provide the ants with the information necessary to orientate in a CO2 gradient (tracking of fluctuations) as well as with the necessary information for microclimatic control (measuring of absolute concentrations).
The identification of NRAGE
(2001)
The inhibitor of apoptosis proteins (IAPs) have been shown to interact with a growing number of intracellular proteins and signalling pathways in order to fulfil their anti-apoptotic role. In order to investigate in detail how the avian homologue ITA interfered with both TNF induced apoptosis and the NGF mediated differentiation in PC12 cells, a two hybrid screen was performed with a PC12 library using ITA as a bait. The screen resulted in the identification of several overlapping fragments of a previously unknown gene. The complete cDNA for this gene was isolated, the analysis of which revealed a high homology with a large family of tumour antigens known as MAGE (melanoma associated antigens). This newly identified member of the MAGE family, which was later named NRAGE, exhibited some unique characteristics that suggested for the first time a role in normal cellular physiology for this protein family. MAGE proteins are usually restricted in their expression to malignant or tumour cells, however NRAGE was also expressed in terminally differentiated adult tissue. NRAGE also interacted with the human XIAP in direct two-hybrid tests. The interactions observed in yeast cells were confirmed in mammalian cell culture, employing both coimmunoprecipitation and mammalian two-hybrid methods. Moreover, the results of the coimmunoprecipitation experiments indicated that this interaction requires the RING domain. The widely studied 32D cell system was chosen to investigate the effect of NRAGE on apoptosis. NRAGE was stably transduced in 32D cells, and found to augment cell death induced by the withdrawal of Interleukin-3. One reason for this reduced cell viability in NRAGE expressing cells could be the binding of endogenous XIAP, which occurred inducibly after growth factor withdrawal. Interestingly, NRAGE was able to overcome the protection afforded to 32D cells by the exogenous expression of human Bcl-2. Thus NRAGE was identified during this research doctorate as a novel pro-apoptotic, IAP-interacting protein, able to accelerate apoptosis in a pathway independent of Bcl-2 cell protection.
Cardiovascular disease is the leading cause of mortality in both men and women in the Western world. Earlier observations have pointed out that pre-menopausal women have a lower risk of developing cardiovascular disease than age-matched men, with an increase in risk after the onset of menopause. This observation has directed the attention to estrogen as a potential protective factor in the heart. So far the focus of research and clinical studies has been the vascular system, leaving the current knowledge on the role of estrogen in the myocardium itself rather scarce. Functional estrogen receptor-alpha as well as -beta have recently been identified in the myocardium, making the myocardium an estrogen target organ. The focus of this thesis was 1) to investigate the role of estrogen and estrogen receptors in modulating myocardial gene expression both in vivo in an animal model for cardiac hypertrophy (spontaneously hypertensive rats; SHR), as well as in vitro in isolated neonatal cardiomyocytes, 2) to investigate the mechanisms of the rapid induction of an estrogen target gene, the early growth response gene-1 (Egr-1) and 3) to initiate the search for novel estrogen target genes in the myocardium. 1) The effects of estrogen on the expression of one of the major myocardial specific contractile proteins, the alpha-myosin heavy chain (alpha-MHC) have been investigated. In ovarectomised animals treated either with 17beta-estradiol alone or in combination with a specific estrogen receptor antagonist, ICI 182780, it was shown that both alpha-MHC mRNA and protein were upregulated by estrogen in an estrogen receptor specific manner. The in vivo results were confirmed in vitro in isolated neonatal cardiomyocytes which showed that estrogen has a direct action on the myocardium potent enough to upregulate the expression of alpha-MHC. Furthermore it was shown that the alpha-MHC promoter is induced by estrogen in an estrogen receptor-dependent manner and first investigations into the mechanisms involved in this upregulation identified Egr-1 as a potential transcription factor which, upon induction by estrogen, drives the expression of the alpha-MHC promoter. 2) Previously it was shown that Egr-1 is rapidly induced by estrogen in an estrogen receptor-dependent manner which was mediated via 5 serum response elements (SREs) in the promoter region and surprisingly not via the estrogen response elements (EREs). In this study it was shown that estrogen-treatment of cardiomyocytes resulted in the recruitment of serum response factor (SRF), or an antigenically related protein, to the SREs in the Egr-1 promoter, which was specifically inhibited by the estrogen receptor antagonist ICI 182780. Transfection experiments showed that estrogen induced a heterologous promoter consisting only of 5 tandem repeats of the c-fos SRE in an ER-dependent manner, which identified SREs as promoter elements able to confer an estrogen response to target genes. 3) Potentially new target genes regulated by estrogen in vivo were analysed using hearts of ovarectomised animals as well as ovarectomised animals treated with estrogen. Analyses of cDNA microarray filters containing 1250 known genes identified 24 genes that were modified by estrogen in vivo. Among these genes, some might have potentially important functions in the heart and further analyses of these genes will create a more global picture of the role and function of estrogen in the myocardium. Taken together, the results showed that estrogen does have a direct action on the myocardium both by regulating the expression of myocardial specific genes in vivo, as well as exerting rapid non-nuclear effects in cardiac myocytes. It was shown that SREs in the promoter region of genes can confer an estrogen response to genes identifying SREs as important elements in regulation of genes by estrogen. Furthermore, 24 potentially new estrogen targets were identified in the myocardium, contributing to the general understanding of estrogen action in the myocardium.
Unique functions of DNA topoisomerase IIalpha and IIbeta have been suggested. A human cell line which carries a homozygeous mutation of the nuclear localization sequence of the topoisomerase IIalpha gene expresses the isoform outside the nucleus at the onset of mitosis. At mitosis topoisomerase IIbeta diffused away from the chromatin despite the nuclear lack of the IIalpha-form. Chromosome condensation and disjunction was performed with the aid of cytosolic topoisomerase IIalpha which bound to the mitotic chromatin with low affinity. Consequently an increased rate of nondisjunction is observed in these cells. It is concluded that high affinity chromatin binding of topoisomerase IIalpha is essential for chromosome condensation/disjunction and that topoisomerase IIbeta does not adopt these functions. A centrosomal protein was recognized by topoisomerase IIalpha. This topoisomerase IIalpha-like protein resembles a modified form of topoisomerase IIalpha with an apparent size of 205 kDa compared to 170 kDa. The expression of the protein is constant in all stages of the cell cycle and it appears in proliferating as well as in resting cells. If there is not sufficient topoisomerase IIalpha present at mitosis the centrosomal proteins might adopt the function and a mitotic catastrophe in the cells could therefore be prevented.
Monolayer or suspension cell cultures are of only limited value as experimental models for human cancer. Therefore, more sophisticated, three-dimensional culture systems like spheroid cultures or histocultures are used, which more closely mimic the tumor in individual patients compared to monolayer or suspension cultures. As tissue culture or tissue engineering requires more sophisticated culture, specialized in vitro techniques may also improve experimental tumor models. In the present work, a new miniaturized hollow-fiber bioreactor system for mammalian cell culture in small volumes (up to 3 ml) is characterized with regard to transport characteristics and growth of leukemic cell lines (chapter 2). Cell and medium compartment are separated by dialysis membranes and oxygenation is accomplished using oxygenation membranes. Due to a transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60 and REH were cultivated up to densities of 3.5 x 107/ml without medium change or manipulation of the cells. Growth and viability of the cells in the bioreactor were the same or better, and the viable cell count was always higher compared to culture in Transwellâ plates. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with the flow rate. Influx of low molecular weight substances in the cell compartment could be regulated by variation of the concentration in the medium compartment. Thus, time dependent concentration profiles (e.g. pharmacokinetic profiles of drugs) can be realized as illustrated using glucose as a model compound. Depending on the molecular size cut-off of the membranes used, added growth factors like GM-CSF and IL-3 as well as factors secreted from the cells are retained in the cell compartment for up to one week. Second, a method for monitoring cell proliferation the hollow-fiber bioreactor by use of the Alamar BlueTM dye was developed (chapter 3). Alamar BlueTM is a non-fluorescent compound which yields a fluorescent product after reduction e.g. by living cells. In contrast to the MTT-assay, the Alamar BlueTM-assay does not lead to cell death. However, when not removed from the cells, the Alamar BlueTM dye shows a reversible, time- and concentration-dependent growth inhibition as observed for leukemic cell lines. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows to discriminate between cell densities of 105, 106 and 107 of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 106 cells/ml, in particular. In conclusion, the Alamar BlueTM-assay combined with the hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system. In chapter 4 the use of the hollow-fiber bioreactor as a tool for toxicity testing was investigated, as current models for toxicity as well as efficacy testing of drugs in vitro allow only limited conclusions with regard to the in vivo situation. Examples of the drawbacks of current test systems are the lack of realistic in vitro tumor models and difficulties to model drug pharmacokinetics. The bioreactor proved to be pyrogen free and is steam-sterilizable. Leukemic cell lines like HL-60 and primary cells such as PHA-stimulated lymphocytes can be grown up to high densities of 1-3 x 107 and analyzed during growth in the bioreactor by light-microscopy. The cytostatic drug Ara-C shows a dose-dependent growth inhibition of HL-60 cells and a dose-response curve similar to controls in culture plates. The bioreactor system is highly flexible since several systems can be run in parallel, soluble drugs can be delivered continuously via a perfusion membrane and gaseous compounds via an oxygenation membrane which also allows to control pO2 and pH (via pCO2) during culture in the cell compartment. The modular concept of the bioreactor system allows realization of a variety of different design properties, which may lead to an improved in vitro system for toxicity testing by more closely resembling the in vivo situation. Whereas several distinct advantages of the new system have been demonstrated, more work has to be done to promote in vitro systems in toxicity testing and drug development further and to reduce the need for animal tests.
Priority tasks of the present thesis were to generate various enantiopure C-3-substituted pyroglutamates as well as C-3-substituted glutamates, and furthermore to ameliorate the serious drawback of the bad atom-economy in the reaction sequence of previously published silylether-mediated procedures. To meet these requirements, the ortho ester functionality (OBO ester) developed by Corey was introduced. According to the plan of synthesis, the starting material, non-racemic (S)-pyroglutamic acid, was converted to the corresponding oxetane ester via a DCC-mediated esterification. The latter was N-protected to provide N-acceptor substituted pyroglutamic acid oxetane esters (Acceptor=Boc,Cbz,CO2Me). After rearrangement with boron trifluoride, the ortho ester derivatives (Acceptor=Cbz,CO2Me) were at hand and exclusively the N-Cbz derivative was converted to the corresponding alpha,beta-unsaturated lactam via a syn-elimination reaction. The formation of the C-3-substituted ortho ester compounds (R=methyl,ethyl,butyl,allyl,phenyl,4-chlorophenyl,biphenyl,naphthyl) was performed via a copper-mediated conjugate addition to the alpha,beta-enone system of the N-Cbz-alpha,beta-unsaturated lactam. The OBO functionality hence was envisaged to support perfect trans selectivity in this cuprate addition to the Michael system of the N-Cbz-alpha,beta-unsaturated lactam. Spectroscopic NMR-data, on the basis of 1H-, 13C- and DEPT spectra, proved the assumption that the C-3-substituted ortho ester derivatives exclusively are trans-configurated, i.e. the alkyl derivatives (R=methyl,ethyl,butyl,allyl) are (2S,3S)-configurated and the aryl derivatives (R=phenyl,4-chlorophenyl,biphenyl,naphthyl) are (2S,3R)-configurated). The C-3-substituted ortho ester derivatives were completely deprotected to yield the C-3-substituted pyroglutamates (R=ethyl,phenyl,4-chlorophenyl,naphthyl). Finally, ring opening reaction via route A-2 lead to the desired enantiopure C-3-substituted glutamates. Alternatively, latter preferably were reacted via route A-1 to yield the C-3-substituted glutamates (R=methyl,ethyl,butyl,phenyl,4-chlorophenyl,naphthyl). Their (2S,3R)-configuration (R=aryl) and (2S,3S)-configuration (R=alky), respectively, unambiguously was proved on the basis of available spectroscopic NMR-data. To ensure this assumption, diastereomeric (2S,3R)-3-methyl glutamic acid (i.e. cis-configurated) examplarily was synthesized via route A-3 and spectroscopic NMR-data was compared to that of (2S,3S)-3-methyl glutamic acid (i.e. trans-configurated). Conclusively, there can be recorded the fact that the serious drawback of the bad atom-economy in the reaction sequence previously used can be circumvented by the introduction of the OBO functionality, so the concept of an improved atom-economy is achieved. Additionally, in comparison to the silyl-ether-mediated synthesis, the OBO functionality provided crystalline ortho ester derivatives, which facilitated their purification as well as characterization.
This study investigates mechanisms and consequences of sexual selection in a polygynous population of dusky warblers Phylloscopus fuscatus, breeding near Magadan in the Russian Far East. In particular, the study focuses on individual variation in the reproductive behaviours of both females and males. The mating system of this population is characterised by facultative polygyny (17 per cent of the males mated with more than one female), and by an outstandingly high rate of extra-pair paternity (45 per cent of the offspring was not sired by the social partner of the female). The occurrence of polygyny is best explained by the ‘polygyny-threshold model’ (PTM). A novel finding of this study is that female mating decisions follow a conditional strategy. First-year females that have no prior breeding experience prefer monogamy over territory quality, while older females more often mate polygynously. I argue that the costs of receiving no male help may be higher for inexperienced females, while the benefits of having a free choice between territories may be higher for individuals that know which territories had the highest breeding success in previous years. Furthermore, I find support for the existence of two female mating strategies. The ‘emancipated’ female which is not dependent on male help, is free to choose the best territory and the best copulation partners. The ‘help-dependent’ female, in contrast, is bound to find a partner who is willing to assist her with brood care, thus she will have to accept territories and genetic fathers of lesser quality. The most unexpected finding on female mating behaviours is that this dichotomy between emancipated and help-dependent females is accompanied by morphological specialisation, which indicates that there is genetic variation underlying these female mating strategies. Male mating behaviours are characterised by competition for ownership of the best territories and by advertisement of male quality to females, as these are the factors which largely determine male reproductive success. Male success in obtaining copulations depended on the quality of their song, a fact that explains why males spend most of the daytime singing during the period when females are fertile. Individuals that were able to maintain a relatively high sound amplitude during rapid frequency modulations were consistently preferred by females as copulation partners. Studies of physiological limitations on sound production suggest that such subtle differences in male singing performance can provide an honest reflection of male quality. The present study is the first to indicate that females may judge the quality of a male’s song by his performance in sound production. Quality of song was also related to winter survival, which suggests that females can enhance the viability of their offspring by seeking extra-pair fertilisations from good singers (good-genes hypothesis). In general, the present study demonstrates that a complete understanding of avian mating systems is not possible without a detailed analysis of alternative behavioural strategies and of how individuals adjust their reproductive tactics according to their individual needs and abilities.
Beans, roots and leaves
(2001)
The author presents the first detailed review of the pharmacological therapy of parkinsonism from ancient times until the near present (1980). It is not clear whether parkinsonism as it is now defined – a progressive neurodegenerative disorder of the basal ganglia characterized by sharply reduced striatal dopamine levels, particularly in the striatum – has always affected a significant minority of aged persons, but suggestive evidence to this effect in the older literature is reviewed. The major discussion commences, however, with the administration of various plant alkaloids to parkinsonian patients in the second half of the 19th century. Antiparkinsonian therapy since this time may be divided into a number of phases: 1. The employment of alkaloids derived from solanaceous plants: initially hyoscyamine, then hyoscine/scopolamine and atropine. The discovery and characterization of these alkaloids, and the gradual recognition that other pharmacologically useful solanaceous alkaloids (such as duboisine) were identical with one or other of these three compounds, is discussed. 2. With the outbreak of encephalitis lethargica following the First World War, parkinsonian patient numbers increased dramatically, leading to a multiplicity of new directions, including the use of another solanaceous plant, stramonium, of extremely high atropine doses, and of harmala alkaloids. 3. The so-called “Bulgarian treatment” was popularized in western Europe in the mid-1930s. It was also a belladonna alkaloid-based therapy, but associated with greater efficacy and fewer side effects. This approach, whether as actual plant extracts or as defined combinations of belladonna alkaloids, remained internationally dominant until the end of the 1940s. 4. Synthetic antiparkinsonian agents were examined following the Second World War, with the aim of overcoming the deficiencies of belladonna alkaloid therapy. These agents fell into two major classes: synthetic anticholinergic (= antimuscarinic) agents, such as benzhexol, and antihistaminergic drugs, including diphenhydramine. These agents were regarded as more effective than plant-based remedies, but certainly not as cures for the disease. 5. A complete change in direction was heralded by the discovery in 1960 of the striatal dopamine deficit in parkinsonism. This led to the introduction of L-DOPA therapy for parkinsonism, the first approach directed against an identified physiological abnormality in the disorder. 6. Subsequent developments have thus far concentrated on refinement or supplementation of the L-DOPA effect. Recent attempts to develop neuroprotective or -restorative approaches are also briefly discussed. The thesis also discusses the mechanisms by which the various types of antiparkinsonian agent achieved their effects, and also the problems confronting workers at various periods in the design and assessment of novel agents. The impact of attitudes regarding the etiology and nature of parkinsonism, particularly with regard to symptomatology, is also considered. Finally, the history of antiparkinsonian therapy is discussed in context of the general development of both clinical neurology and fundamental anatomical, physiological and biochemical research. In particular, the deepening understanding of the neurochemical basis of central nervous system function is emphasized, for which reason the history of dopamine research is discussed in some detail. This history of antiparkinsonian therapy also illustrates the fact that the nature of experimental clinical pharmacology has markedly changed throughout this period: No longer the preserve of individual physicians, it is now based firmly on fundamental laboratory research, the clinical relevance of which is not always immediately apparent, and which is only later examined in (large scale) clinical trials. It is concluded that antiparkinsonian therapy was never irrational or without basis, but has always been necessarily rooted in current knowledge regarding neural and muscular function. The achievements of L-DOPA therapy, the first successful pharmacological treatment for a neurodegenerative disorder, derived from the fruitful union of the skills and contributions of different types by laboratory scientists, pharmacologists and clinicians.
T cell activation is supposed to require two signals via engagement of the TCR and a costimulatory molecule. However, the signaling cascade of costimulatory molecules has remained elusive. Here, I provide evidence that CD44 supports proliferation as well as apoptosis mainly, if not exclusively, by enhancing signal transduction via the TCR/CD3 complex. Blockade of CD44 interferes with mounting of an immune response. This has been demonstrated by the significantly decreased IL-2 production of a T helper line, when stimulated in the presence of a competing CD44 receptor globulin. To evaluate the underlying mechanism, CD44 was cross-linked by an immobilized antibody (IM7). Cross-linking of CD44 induces proliferation of peripheral T cells and apoptosis of thymocytes and a T helper line in the presence of subthreshold levels of anti-CD3. CD44-induced proliferation was accompanied by an upregulation of the activation markers CD25 and CD69 and an increased cytokine production. TCR-mediated apoptosis was accompanied by an upregulation of CD95 ligand and CD95 receptor, which could be greatly enhanced by costimulation via CD44. On the level of signal transduction, coligation of CD44 with CD3 resulted in a strong and sustained increase of early tyrosine phosphorylation events and upregulated downstream signal transduction pathways, such as the ras/ERK and the JNK signaling cascades. These pleiotropic effects of CD44 are due to its involvement in the most proximal events in TCR signaling, as demonstrated by a strong increase in the phosphorylation of the TCR z-chain and ZAP-70. Notably, cross-linking of CD44 was binding-site dependent and was only effective when supporting colocalization of the TCR/CD3 complex and CD44. Cross-linking of CD44 via immobilized IM7 also induced profound changes in cell morphology, characterized by strong adhesion, spreading and development of surface extensions, which were dependent on a functional tubulin and actin cytoskeleton. These cytoskeletal rearrangements were mediated by rac1, a small GTPase of the rho subfamily, and src-family kinases, two of which, fyn and lck, were found to be associated with CD44. By cross-linkage of CD44 these kinases were redistributed into so called lipid rafts. It is supposed that for T cell activation a relocation of the TCR/CD3 complex into the same membrane microdomains is required. The data are interpreted in the sense that the costimulatory function of CD44 relies on its cooperativity with the TCR. Most likely by recruitment of phosphokinases CD44 significantly lowers the threshold for the initiation of signaling via the TCR. The requirement for immobilized anti-CD44, the necessity for neighbouring anti-CD3 and the dependence on the binding site of CD44 strongly suggest that the costimulatory mechanism involves cytoskeletal rearrangements, which facilitate recruitment and redirection of src-family protein kinases in glycolipid enriched membrane microdomains.
The present thesis reports on four years of field research on stingless bee ecology in Sabah, Malaysia. Hereby, it was the main focus to evaluate the effect of selective logging for timber extraction on communities of bees, and to elucidate causative relationships involved in regulating bee populations. Included were background studies on resource use (3.1, 3.2, 3.3) and nesting biology (3.4) as well as comparative studies on stingless bee diversity and abundance in logged and unlogged lowland rainforest sites (4.1, 4.2). Stingless bees proved to be generalist foragers that used a large range of plant species as pollen sources. Nevertheless, different species of bees had rather distinct pollen diets, a findind that was independent of fluctuations in flowering activity in the habitat. At one particular point in time colonies of one species (Trigona collina)collected mold spores (Rhizopus sp.) as a pollen surrogate. In order to obtain low-effort estimates of meliponine pollen sources a new method was developed: Trapping of bee garbage (with funnel traps) and the quantitative analysis of pollen in garbage samples. Pollen in bee garbage reflected pollen import with a certain time lag and could therefore be used for an assessment of long-term pollen foraging (see below). The majority of stingless bee nests (275 nests of 12 species) were found in cavities in trunks or under the bases of large, living canopy trees. Nest trees mostly belonged to commercial species and were of the correct size and (partly) timber quality to warrant harvesting. It was estimated that roughly one third of stingless bee nests in an given forest area would be killed during a selective logging operation. Besides causing direct mortality, logging may also indirectly affect bee populations by reducing the availability of potential nest sites (trees). However, in a comparison of primary and differentially logged forest sites (10 to 30 years after logging) no effect of the degree of disturbance on meliponine nest density was found. Instead, the variation in nest density (0 to 16.2 nest/ha) was best explained by differences in the available floral resources (assessed by analysis of pollen in bee garbage). Bee populations in forest edge situations were favored: there was a positive correlation between nest density and the proportion of external non-forest pollen (e.g. from crop plants, road edge vegetation, mangroves) in the bees’ diet. The highest nest density was found in a site bordering the mangroves in Sandakan Bay. Here, the mangrove tree Rhizophora apiculata represented a extraordinary large fraction of the pollen volume. Presumably, external pollen sources effectively supplement bee diets at times when little flowering occurs inside the forest, thus increasing overall bee carrying-capacity. The idea of differential pollen limitation was strengthened by direct measurements of pollen import and foraging activity over a period of five months. Both were elevated in colonies in a site with high bee density. It is concluded that the abundance of stingless bees in forests in Sabah is chiefly dependent on the local availability of food resources. Hereby, bee populations strongly benefit from edge effects and increased habitat diversity. Although direct negative effects of selective logging are strongly indicated by a close association of bee nests with commercial trees, no clear effects were detected in regenerating forests ten to 30 years after logging.
The MEK5/ ERK5 kinase module is a relatively new discovered mitogen-activated protein kinase (MAPK) signalling pathway with a poorly defined physiological function. Since ERK5 and its upstream activator MEK5 are abundant in skeletal muscle a function of the cascade during muscle differentiation was examined. ERK5 becomes activated upon induction of differentiation in mouse myoblasts. The selective activation of the pathway results in promoter activation of differentiation-specific genes, such as the cdk-inhibitor p21 gene, the myosin light chain (MLC1A) gene, or an E-box containing promoter element, where myogenic basic-helix-loop-helix proteins such as MyoD or myogenin bind. Moreover, myogenic differentiation is completely blocked, when ERK5 expression is inhibited by antisense RNA. The effect can be detected also on the expression level of myogenic determination and differentiation markers such as p21, MyoD and myogenin. Another new finding is that stable expression of ERK5 in C2C12 leads to differentiation like phenotype and to increased p21 expression levels under growth conditions. These results provide first evidence that the MEK5/ERK5 MAP kinase cascade is critical for early steps of muscle cell differentiation.
Food borne pathogens that cause systemic disease must cross the intestinal barrier. Many of these pathogens, eg Salmonella typhimurium and Shigella flexneri, use M cells, found only within the follicle associated epithelium (FAE) that overlies Peyer’s patches and other lymphoid follicles, to enter the host. This study is primarily an investigation into the interaction of S. typhimurium and Listeria monocytogenes with the intestinal epithelium, representing the early stage of an infection.
The mode of action of phloretin and its analogs on the permeability of natural membranes for neutral and charged molecules, such as urea, glucose and chloride has been characterized 25 years ago. In contrast to signal molecules with primary effects on transport systems of natural membranes, phloretin also affects model membranes, i.e., artificial membranes, which do not contain proteins. Since the dipole potential reducing effect of phloretin on mono- and bilayers has been found, it became clear that its primary effect must be a biophysical one: phloretin adsorbs to lipid layers and changes biophysical parameters of these layers. The aim of this work was the characterization of the interaction between the surface-active molecule phloretin and artificial lipid layers. We were able to describe structural and functional parameters of the model systems mono- and bilayer as functions of one or few variables. One of these parameters, the dipole potential, measured as a function of the aqueous phloretin concentration, allowed a critical examination of the Langmuir adsorption model that has been postulated for the interaction between phloretin and lipid layers. Surface pressure versus area per lipid molecule isotherms and surface (dipole) potential change versus area per lipid molecule isotherms, measured at lipid monolayers, allowed a structural description of the phloretin-lipid interaction: phloretin integrates into monolayers dependent on the surface pressure and the phase state of the lipid. Calorimetric measurements confirmed the integration of phloretin into membranes because of the strong decrease of the phase transition temperature, but they also showed that the cooperativity of phase transition is hardly affected, even at very high amounts of phloretin in the membrane. Obviously the interaction between phloretin and lipids is restricted to the head groups, an integration into the hydrocarbon layer is unlikely. 2H NMR measurements with spherical unilamellar vesicles of headgroup-deuterated lipid showed changed quadrupolar splittings indicating the interaction between phloretin and headgroups of the lipids.
Large parts of the tropical lowland rain forests of Sabah (Malaysia) were transformed into secondary forests due to heavy logging. Additionally the remaining forest remnants are isolated from each other by large scale oil palm plantations. Biodiversity patterns and responses of the community of leaf litter ants were studied in anthropogenically disturbed habitats and primary forests of different size. In logged over forests, only 70 per cent of the species of a primary forest were present even 25 years after timber extraction. The ant communities were thinned and could be described by a lower species density producing lower species numbers and a different community composition. The similarity in species number and community composition between logged over forests of different degrees of disturbance was explained by source-sink dynamics within a heterogeneous forest matrix. Rain forest fragments displayed even higher reductions in species density, numbers and diversity due to a more pronounced thinning effect. Even forest isolates exceeding 4 000 ha in size did not support more than 50 per cent of the species of the leaf litter ant community of a contiguous primary rain forest. Additionally, an increase in tramp species was recorded with decreasing size of the forest fragments, leading to a very different community composition. Regarding the leaf litter ant community, the remaining rain forest fragments of Sabah are effectively isolated by a barrier of oil palm plantation, now stretching all over the lowlands of the east coast. Only 13 species, which belonged to the forest ant community in highly disturbed areas were collected in these plantations. Some of the 10 other species of the highly reduced ground-dwelling ant community in the plantations are known as invasive tramp species, forming large exclusive territories. Correlative evidence and a field experiment implied, that leaf litter humidity, volume and temperature affect the distribution and community composition of forest leaf litter ant species. The smaller primary forests and the most disturbed logged over forests in this study revealed higher temperatures and lower humidity levels and a reduction in leaf litter volume compared to a large primary forest or forests affected by a lower impact of timber harvesting. If the pattern for leaf litter ants is confirmed for other taxa, the implications for any efficient management design aiming to preserve the majority of the biodiversity of the country are tremendous and current concepts need rethinking.
Most natural learning situations are of a complex nature and consist of a tight conjunction of the animal's behavior (B) with the perceived stimuli. According to the behavior of the animal in response to these stimuli, they are classified as being either biologically neutral (conditioned stimuli, CS) or important (unconditioned stimuli, US or reinforcer). A typical learning situation is thus identified by a three term contingency of B, CS and US. A functional characterization of the single associations during conditioning in such a three term contingency has so far hardly been possible. Therefore, the operational distinction between classical conditioning as a behavior-independent learning process (CS-US associations) and operant conditioning as essentially behavior-dependent learning (B-US associations) has proven very valuable. However, most learning experiments described so far have not been successful in fully separating operant from classical conditioning into single-association tasks. The Drosophila flight simulator in which the relevant behavior is a single motor variable (yaw torque), allows for the first time to completely separate the operant (B-US, B-CS) and the classical (CS-US) components of a complex learning situation and to examine their interactions. In this thesis the contributions of the single associations (CS-US, B-US and B-CS) to memory formation are studied. Moreover, for the first time a particularly prominent single association (CS-US) is characterized extensively in a three term contingency. A yoked control shows that classical (CS-US) pattern learning requires more training than operant pattern learning. Additionally, it can be demonstrated that an operantly trained stimulus can be successfully transferred from the behavior used during training to a new behavior in a subsequent test phase. This result shows unambiguously that during operant conditioning classical (CS-US) associations can be formed. In an extension to this insight, it emerges that such a classical association blocks the formation of an operant association, which would have been formed without the operant control of the learned stimuli. Instead the operant component seems to develop less markedly and is probably merged into a complex three-way association. This three-way association could either be implemented as a sequential B-CS-US or as a hierarchical (B-CS)-US association. The comparison of a simple classical (CS-US) with a composite operant (B, CS and US) learning situation and of a simple operant (B-US) with another composite operant (B, CS and US) learning situation, suggests a hierarchy of predictors of reinforcement. Operant behavior occurring during composite operant conditioning is hardly conditioned at all. The associability of classical stimuli that bear no relation to the behavior of the animal is of an intermediate value, as is operant behavior alone. Stimuli that are controlled by operant behavior accrue associative strength most easily. If several stimuli are available as potential predictors, again the question arises which CS-US associations are formed? A number of different studies in vertebrates yielded amazingly congruent results. These results inspired to examine and compare the properties of the CS-US association in a complex learning situation at the flight simulator with these vertebrate results. It is shown for the first time that Drosophila can learn compound stimuli and recall the individual components independently and in similar proportions. The attempt to obtain second-order conditioning with these stimuli, yielded a relatively small effect. In comparison with vertebrate data, blocking and sensory preconditioning experiments produced conforming as well as dissenting results. While no blocking could be found, a sound sensory preconditioning effect was obtained. Possible reasons for the failure to find blocking are discussed and further experiments are suggested. The sensory preconditioning effect found in this study is revealed using simultaneous stimulus presentation and depends on the amount of preconditioning. It is argued that this effect is a case of 'incidental learning', where two stimuli are associated without the need of reinforcement. Finally, the implications of the results obtained in this study for the general understanding of memory formation in complex learning situations are discussed.
The proventriculus regulates the food passage from crop to midgut. As the haemolymph provides a constantly updated indication of an insect’s nutritional state, it is assumed that the factor controlling the proventri-culus activity is to be found in the haemolymph. The purpose of this doctoral thesis was to investigate how output (metabolic rate), input (food quality and food quantity) and internal state variables (haemolymph osmolarity and haemolymph sugar titer) affect each other and which of these factors controls the activity of the proventriculus in the honeybee. Therefore free-flying foragers were trained to collect con-trolled amounts of different sugar solutions. Immediately after feeding, metabolic rates were measured over different periods of time, then crop-emptying rates and haemolymph sugar titers were measured for the same individual bees. Under all investigated conditions, both the sugar transport rates through the proventriculus and the haemolyph sugar titers depended mainly on the metabolism. For bees collecting controlled amounts of 15 per cent, 30 per cent or 50 per cent sucrose solution haemolymph trehalose, glucose and fructose titers were constant for metabolic rates from 0 to 4.5 mlCO2/h. At higher metabolic rates, trehalose concentration decreased while that of glucose and fructose increased with the exception of bees fed 15 per cent sucrose solution. As the supply of sugar from the crop via the proventriculus was sufficient to support even the highest metabolic rates, the observed pattern must result from an upper limit in the capacity of the fat body to synthesise trehalose. The maximal rate of conversion of glucose to trehalose in the fat body was therefore calculated to average 92.4 µg glucose/min. However, for bees fed 15 per cent sucrose solution both the rate of conversion of glucose to trehalose and the rate of sugar transport from the crop to the midgut were limited, causing an overall decrease in total haemolymph sugar titers for metabolic rates higher than 5 mlCO2/h. Haemolymph sucrose titers were generally low but increased with increasing metabolic rates, even though sucrose was not always detected in bees with high metabolic rates. Though foragers were able to adjust their sugar transport rates precisely to their metabolic rates, a fixed surplus of sugars was transported through the proventriculus under specific feed-ing conditions. This fixed amount of sugars increased with increasing concentration and in-creasing quantity of fed sugar solution, but decreased with progressing time after feeding. This fixed amount of sugars was independent of the metabolic rates of the bees and of the molarity and viscosity of the fed sugar solution. As long as the bees did not exhaust their crop content, the haemolymph sugar titers were unaffected by the sugar surplus, by the time after feeding, by the concentration and by the viscosity of fed sugar solution. When bees were fed pure glucose (or fructose) solutions, un-usually little fructose (or glucose) was found in the haemolymph, leading to lower total haemolymph sugar titers, while the trehalose titer remained unaffected. In order to investigate the mechanisms underlying the regulation of the honeybee proven-triculus, foraging bees were injected either with metabolisable (glucose, fructose, trehalose), or non-metabolisable sugars (sorbose). Bees reacted to injections of metabolisable sugars with reduced crop-emptying rates, but injection of non-metabolisable sugars had no influence on crop emptying. Therefore it is concluded that the proventriculus regulation is controlled by the concentration of metabolisable compounds in the haemolymph, and not by the haemo-lymph osmolarity. A period of 10min was enough to observe reduced crop emptying rates after injections. It is suggested that glucose and fructose have an effect on the proventriculus activity only via their transformation to trehalose. However, when the bees were already in-jected 5min after feeding, no response was detectable. In addition it was investigated whether the overregulation is the result of feed-forward regulation for the imminent take-off and flight. In a first experiment, we investigated whether the bees release an extra amount of sugar solution very shortly before leaving for the hive. In a second experiment, it was tested whether the distance covered by the bees might have an influence on the surplus amount released prior to the take-off. In a third experiment, it was investigated if walking bees fail to release this extra amount of sugars, as they do not have to fly. Though we were not able to demonstrate that the overregulation is the result of feed-forward regulation for the imminent take-off and flight, it is conceivable that this phenome-non is a fixed reaction in foragers that can not be modulated. To investigate whether regulated haemolymph sugar titers are also observed in honeybee foragers returning from natural food sources, their crop contents and haemolymph sugar titers were investigated. While the quantity of the collected nectar was without influence on the haemolymph sugar titers, foragers showed increasing haemolymph sugar titers of glucose, fructose and sucrose with increasing sugar concentration of the carried nectar. In contrast no relationship between crop nectar concentrations and haemolymph trehalose titers was observed. We are sure that the regulation of food passage from crop to midgut is controlled by the trehalose titer. However, under some conditions the balance between consumption and income is not numerically exact. This imprecision depends on the factors which have an impact on the foraging energetics of the bees but are independent of those without influence on the foraging energetics. Therefore we would assume that the proventriculus activity is modulated by the motivational state of the bees.
Different transgenes that can be expressed in neurons to kill or block them were compared. Tetanus neurotoxin blocked chemical synapses very efficiently. Synapses consisting of a chemical and an electrical component were blocked more reliably by expressing a human inwardly rectifying potassium channel. To gain temporal control over neuronal function, three genetic tools have been investigated. None of the systems is without drawbacks, however, the recombination induced tetanus neurotoxin expression is a promising approach. The knowledge gained from the comparative methodological study was used to investigate the role of neurons in sensory systems in processing different sensory informations. Receptor neurons sensitive for chemical or mechanical stimuli were correlated to specific olfactory behaviors or locomotor tasks. The main topic of this thesis is the much discussed question of which neurons are involved in motion processing in the visual system of flies. Neither L2 nor L4 neurons in the first visual neuropil are essential for motion-detection. The results indicate that maybe motion is detected by the network of amacrine cells (a). The vertical motion-sensitive VS cells in the lobula plate are not necessary for behavioral responses to vertical motion. This finding implies that the lack of VS cells in the structural mutant optomotor blind is not causally related to the altered responses to motion stimuli. Other abnormalities in optomotor blind are responsible for this behavioral phenotype. This work shows the potential of the described methods in studying information processing in the Drosophila brain. Groups of neurons were correlated to complex behavioral responses and theories about information processing were tested by behavioral experiments with transgenic flies. The refinement of the genetic tools to interfere with neuronal function will make the Drosophila brain an even better model to study information processing in nervous systems.