Refine
Is part of the Bibliography
- yes (345) (remove)
Year of publication
- 2011 (345) (remove)
Document Type
- Journal article (211)
- Doctoral Thesis (118)
- Preprint (10)
- Conference Proceeding (2)
- Master Thesis (2)
- Book (1)
- Report (1)
Language
- English (345) (remove)
Keywords
- Medizin (15)
- Expression (9)
- Activation (8)
- Cancer (7)
- Quran (7)
- Koran (6)
- Taufliege (6)
- Text Mining (6)
- Apoptosis (5)
- Biene (5)
Institute
- Theodor-Boveri-Institut für Biowissenschaften (67)
- Graduate School of Life Sciences (29)
- Physikalisches Institut (27)
- Neurologische Klinik und Poliklinik (19)
- Rudolf-Virchow-Zentrum (19)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (17)
- Institut für Molekulare Infektionsbiologie (16)
- Medizinische Klinik und Poliklinik I (15)
- Institut für Psychologie (13)
- Institut für Virologie und Immunbiologie (12)
Sonstige beteiligte Institutionen
no abstract available
This thesis concerned the quantification of cell adhesion molecules (CAM) in and on thin hydrogel films as surface modification of biomaterials. The established and well characterized, per se inert NCO-sP(EO-stat-PO) hydrogel system which allows the easy and reproducible bioactivation with peptides was used as basis for this thesis. Two methods can be used to functionalize the coatings. Ligands can either be mixed into the prepolymer solution in prior to layer formation (mix-in method), or freshly prepared coatings can be incubated with ligand solution (incubation method). Divided into three major parts, the first part of the thesis dealt with the concentration of ligands in the bulk hydrogel, whereas the second part of the thesis focused on the surface sensitive quantification of CAMs at the biointerface. The results were correlated with cell adhesion kinetics. The third part of this thesis investigated the biochemical and the structural mimicry of the extracellular matrix (ECM). ECM proteins were presented via sugar-lectin mediated binding and cell behavior on these surfaces was analyzed. Cell behavior on three-dimensional fibers with identical surface chemistry as the coatings in the previous sections of the thesis was analyzed and correlated with the amount of peptide used for bioactivation. Overall, the main question of this work was ‘How much?’ regarding maximal as well as optimal ligand concentrations for controlled cell-hydrogel interactions. The focus in the first practical part of this thesis was to analyze the amount of ligands in NCO-sP(EO-stat-PO) hydrogels using classical quantification methods. Coatings in 96-well plates as well as on glass were functionalized with GRGDS and 125I-YRGDS for radioisotopic detection (Chapter 3). Using the incubation method for functionalization, a maximal ligand binding using peptide concentrations of 600 µg/mL could be determined. When functionalization was introduced via the mix-in method, a clear tendency for higher ligand concentrations with increasing ligand to prepolymer ratio was observed, but no maximal ligand binding could be detected with a ligand to prepolymer ratio of 2/1 being the highest ratio investigated. This ratio of 2/1 was not exceeded to ensure that complete crosslinking of the hydrogel was not affected. In Chapter 4, a fluorinated amino acid and an iodinated peptide were immobilized to the hydrogels using the mix-in method and were detected by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). In these measurements, maximal ligand binding was detected for a ligand to prepolymer ratio of 1/1. Higher ligand to prepolymer ratios did not result in any significant increase in ligand concentrations in the surface near regions of the crosslinked hydrogels. To address the question of how many ligands were actually accessible for cell interaction at the interface, surface sensitive quantification methods were applied in the second part of this thesis. For the quantification with surface plasmon resonance (SPR) and surface acoustic wave technology (SAW) (Chapter 5), the hydrogel coating procedure needed to be transferred onto cystamine functionalized gold surfaces. Characterization with ellipsometry and atomic force microscopy (AFM) revealed inhomogeneous cystamine binding to the activated surfaces, which resulted in inhomogeneous coatings. Nevertheless, it could be shown that SPR as well as SAW were suitable methods for the surface sensitive quantification of the ligand concentration on NCO-sP(EO-stat-PO) hydrogels. Non-functionalized coatings resisted non-specific serum as well as streptavidin (SA) adsorption. Coatings functionalized with biocytin and GRGDSK-biotin introduced specific SA binding that was dependent on the biotin concentration at the surface. Additionally, enzyme linked immunosorbent assay (ELISA) and enzyme linked lectin assay (ELLA) (Chapter 6) were applied to coatings in 96-well plates and on glass. Coatings were functionalized with the model molecule biocytin, the biotinylated peptide GRGDSK-biotin, the ECM protein fibronectin (FN), as well as the carbohydrates N-acetylglucosamine (GlcNAc) and N-acetyllactosamine (LacNAc). All ligands could be successfully detected with antibodies or SA via ELISA or ELLA. Maximal GRGDSK-biotin binding to the hydrogel coatings on glass was achieved at a peptide to prepolymer ratio of 1/5, which was used as reference value in Chapter 8. Last but not least, cell adhesion (Chapter 7) was quantified depending on the GRGDS concentration on hydrogel coatings on glass. Maximal adhesion of primary human dermal fibroblast (HDF) was observed at GRGDS to prepolymer ratios of 1/5, when adherent cells were counted on life cell images. Quantification of adherent cells using the CASY® cell counter revealed maximal HDF adhesion at molar ligand to prepolymer ratios of 1/2. However, cell vitality detected by intracellular enzyme activities was not dependent on the GRGDS concentration. Cells which managed to adhere were vital regardless of the amount of ligands present. Additionally, adhesion of fibroblasts from the murine cell line NIH L929 was analyzed by counting on life cell images. These cells, being much smaller than the HDF cells, needed higher GRGDS to prepolymer ratios (2/1) for proper cell adhesion. All quantification methods applied to analyze hydrogels which were functionalized by the mix-in method in Chapter 3, 4, 6 and 7, were compared in Chapter 8. Radiodetection gave information about the ligand concentrations throughout the whole hydrogel and no maximal amount of ligands could be detected when increasing the peptide to prepolymer ratio. In contrast, XPS and TOF-SIMS which only penetrated the surface near regions of the coating, a maximal ligand binding to the hydrogel was detected for 1/1 ratios. SPR and SAW were not included in this comparison, as the coatings on gold need to be optimized first. The two surface sensitive quantification methods (ELISA and HDF adhesion) could give information about the quantity of peptide which was sterically available for SA or cell binding. With these methods, maximal SA and cell binding was detected at ratios of 1/5. These results underline the importance of carefully compare the different methods. Beside ligand quantification on hydrogels, the third part of this thesis was concerned with the biochemical and structural mimicry of the ECM by advanced ECM engineering to design biomimetic biomaterials that are better accepted by cells and tissue. The subject of Chapter 9 was the biomimetic and flexible presentation of the ECM protein FN. FN was attached via sugar-lectin mediated binding to NCO-sP(EO-stat-PO) hydrogels. The build-up of the covalently immobilized sugar poly-N-acetyllactosamine (polyLacNAc), the subsequent non-covalent binding of the fungal galectin His6CGL2, and FN could be elegantly proven by fluorescent staining on coatings which were functionalized with the sugar by micro contact printing (MCP). Further experiments were carried out on build-ups, where polyLacNAc was immobilized on the hydrogel by incubation. Optimal parameters for the layer build-up were determined by ELLA/ELISA. Only the complete build-up induced proper adhesion of HDFs. Compared to tissue culture polystyrene (TCPS), cells adhered and spread faster on the biomimetic surfaces. The flexible presentation of FN allowed HDFs to rearrange homogenously immobilized FN into fibrillar structures, which seemed not to be possible when FN was adsorbed on glass or covalently bound directly to the hydrogel coatings. This new approach of a flexible and biomimetic presentation of an ECM protein allows new ways to design biomaterials with best possible cell-material interactions. The work described in Chapter 10 focused on the structural mimicry of the fibrous ECM structures by electrospinning of synthetic, bioactive, and degradable fibers. Poly(D,L-lactide-co-glycolide) (PLGA) and NCO-sP(EO-stat-PO) were electrospun out of one solution in an easy one-step preparation resulting in fibers with an ultrathin inert hydrogel layer at the surface. By adding GRGDS to the solution prior to electrospinning, specifically interacting fibers could be obtained. In comparison to PLGA, the adsorption of bovine serum albumin (BSA) could be reduced by 99.2%. As a control, the non-active peptide GRGES was immobilized to the fiber. These fibers did not allow cell adhesion, showing that the integrity of the hydrogel coated fibers was not affected by the immobilization of peptides. HDF adhesion was obtained by functionalization with GRGDS, leading to the adhesion, spreading, and proliferation of HDFs. Also mesenchymal stem cells (MSC) could adhere to GRGDS functionalized fibers. Additionally, for ligand quantification, the ELISA technique was successfully transferred to fiber substrates. To highlight the potential of the approaches for the biochemical and structural mimicry of the ECM, the sugar polyLacNAc was immobilized on the PLGA/sP(EO-stat-PO) fibers followed by the subsequent layer build-up with His6CGL2 and FN. These fibers triggered HDF adhesion.
In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs), which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF open reading frame (ORF) collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publically available GATEWAY®-compatible ORF collections. (1) The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex) library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2) A high-throughput microtiter plate based protoplast trans activation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta.
Dendritic cell tumors are extremely rare neoplasms arising from antigen-presenting cells of the immune system. We report a case of a 69-year-old man with an unremarkable medical history who presented with a 2-months history of a gradually enlarging painless, firm, mobile, 2 × 2-cm swelling at the caudal pole of the left parotid gland without systemic symptoms. Histologically, the tumor consisted of a spindle cell proliferation in an intraparotideal lymph node. Based on the histopathologic, immunohistochemical and electron microscopic findings, a dendritic cell tumor, not otherwise specified (NOS) in an intraparotideal lymph node was diagnosed. The patient underwent complete tumor resection, and is currently free of disease, 2 years after surgery. These extremely rare tumors must be distinguished from other more common tumors in the salivary glands. Awareness that dendritic cell tumors may occur in this localization, careful histologic evaluation and ancillary immunohistochemical and electron microscopical analyses should allow for recognition of this entity. Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1614859498581601.
Over the last years, evidence has accumulated suggesting that by systematically reducing the amount of dietary carbohydrates (CHOs) one could suppress, or at least delay, the emergence of cancer, and that proliferation of already existing tumor cells could be slowed down. This hypothesis is supported by the association between modern chronic diseases like the metabolic syndrome and the risk of developing or dying from cancer. CHOs or glucose, to which more complex carbohydrates are ultimately digested, can have direct and indirect effects on tumor cell proliferation: first, contrary to normal cells, most malignant cells depend on steady glucose availability in the blood for their energy and biomass generating demands and are not able to metabolize significant amounts of fatty acids or ketone bodies due to mitochondrial dysfunction. Second, high insulin and insulin-like growth factor (IGF)-1 levels resulting from chronic ingestion of CHO-rich Western diet meals, can directly promote tumor cell proliferation via the insulin/IGF1 signaling pathway. Third, ketone bodies that are elevated when insulin and blood glucose levels are low, have been found to negatively affect proliferation of different malignant cells in vitro or not to be usable by tumor cells for metabolic demands, and a multitude of mouse models have shown antitumorigenic properties of very low CHO ketogenic diets. In addition, many cancer patients exhibit an altered glucose metabolism characterized by insulin resistance and may profit from an increased protein and fat intake. In this review, we address the possible beneficial effects of low CHO diets on cancer prevention and treatment. Emphasis will be placed on the role of insulin and IGF1 signaling in tumorigenesis as well as altered dietary needs of cancer patients.
Bacterial glucuronidase as general marker for oncolytic virotherapy or other biological therapies
(2011)
Background: Oncolytic viral tumor therapy is an emerging field in the fight against cancer with rising numbers of clinical trials and the first clinically approved product (Adenovirus for the treatment of Head and Neck Cancer in China) in this field. Yet, until recently no general (bio)marker or reporter gene was described that could be used to evaluate successful tumor colonization and/or transgene expression in other biological therapies. Methods: Here, a bacterial glucuronidase (GusA) encoded by biological therapeutics (e.g. oncolytic viruses) was used as reporter system. Results: Using fluorogenic probes that were specifically activated by glucuronidase we could show 1) preferential activation in tumors, 2) rena l excretion of the activated fluorescent compounds and 3) reproducible detection of GusA in the serum of oncolytic vaccinia virus treated, tumor bearing mice in several tumor models. Time course studies revealed that reliable differentiation between tumor bearing and healthy mice can be done as early as 9 days post injection of the virus. Regarding the sensitivity of the newly developed assay system, we could show that a single infected tumor cell could be reliably detected in this assay. Conclusion: GusA therefore has the potential to be used as a general marker in the preclinical and clinical evaluation of (novel) biological therapies as well as being useful for the detection of rare cells such as circulating tumor cells
Retinoic acid pathway activity in Wilms tumors and characterization of biological responses in vitro
(2011)
Background: Wilms tumor (WT) is one of the most common malignancies in childhood. With current therapy protocols up to 90% of patients can be cured, but there is still a need to improve therapy for patients with aggressive WT and to reduce treatment intensity where possible. Prior data suggested a deregulation of the retinoic acid (RA) signaling pathway in high-risk WT, but its mode of action remained unclear. Results: The association of retinoid signaling and clinical parameters could be validated in a large independent tumor set, but its relevance in primary nephrectomy tumors from very young children may be different. Reduced RA pathway activity and MYCN overexpression were found in high risk tumors as opposed to tumors with low/ intermediate risk, suggesting a beneficial impact of RA especially on advanced WT. To search for possible modes of action of retinoids as novel therapeutic options, primary tumor cell cultures were treated in vitro with all-trans-RA (ATRA), 9cis-RA, fenretinide and combinations of retinoids and a histone deacetylase (HDAC) inhibitor. Genes deregulated in high risk tumors showed opposite changes upon treatment suggesting a positive effect of retinoids. 6/7 primary cultures tested reduced proliferation, irrespective of prior RA signaling levels. The only variant culture was derived from mesoblastic nephroma, a distinct childhood kidney neoplasm. Retinoid/HDAC inhibitor combinations provided no synergistic effect. ATRA and 9cis-RA induced morphological changes suggestive of differentiation, while fenretinide induced apoptosis in several cultures tested. Microarray analysis of ATRA treated WT cells revealed differential expression of many genes involved in extracellular matrix formation and osteogenic, neuronal or muscle differentiation. The effects documented appear to be reversible upon drug withdrawal, however. Conclusions: Altered retinoic acid signaling has been validated especially in high risk Wilms tumors. In vitro testing of primary tumor cultures provided clear evidence of a potential utility of retinoids in Wilms tumor treatment based on the analysis of gene expression, proliferation, differentiation and apoptosis.
Background: Hybridization can have complex effects on evolutionary dynamics in ants because of the combination of haplodiploid sex-determination and eusociality. While hybrid non-reproductive workers have been found in a range of species, examples of gene-flow via hybrid queens and males are rare. We studied hybridization in East African army ants (Dorylus subgenus Anomma) using morphology, mitochondrial DNA sequences, and nuclear microsatellites. Results: While the mitochondrial phylogeny had a strong geographic signal, different species were not recovered as monophyletic. At our main study site at Kakamega Forest, a mitochondrial haplotype was shared between a “Dorylus molestus-like” and a “Dorylus wilverthi-like” form. This pattern is best explained by introgression following hybridization between D. molestus and D. wilverthi. Microsatellite data from workers showed that the two morphological forms correspond to two distinct genetic clusters, with a significant proportion of individuals being classified as hybrids. Conclusions: We conclude that hybridization and gene-flow between the two army ant species D. molestus and D. wilverthi has occurred, and that mating between the two forms continues to regularly produce hybrid workers. Hybridization is particularly surprising in army ants because workers have control over which males are allowed to mate with a young virgin queen inside the colony.
In common with most viruses, measles virus (MV) relies on the integrity of the cytoskeleton of its host cells both with regard to efficient replication in these cells, but also retention of their motility which favors viral dissemination. It is, however, the surface interaction of the viral glycoprotein (gp) complex with receptors present on lymphocytes and dendritic cells (DCs), that signals effective initiation of host cell cytoskeletal dynamics. For DCs, these may act to regulate processes as diverse as viral uptake and sorting, but also the ability of these cells to successfully establish and maintain functional immune synapses (IS) with T cells. In T cells, MV signaling causes actin cytoskeletal paralysis associated with a loss of polarization, adhesion and motility, which has been linked to activation of sphingomyelinases and subsequent accumulation of membrane ceramides. MV modulation of both DC and T cell cytoskeletal dynamics may be important for the understanding of MV immunosuppression at the cellular level.
Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin’s pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20–40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin’s lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.
Reconstruction of the extensor mechanism is essential for good extremity function after endoprosthetic knee replacement following tumor resection. Only a few biological methods have been able to reliably restore a functional extensor mechanism, but they are often associated with significant complication rates. Reattachment of the patellar tendon to the prosthesis using an alloplastic patellar ligament (Trevira cord) can be an appropriate alternative. In vivo and in vitro studies have already shown that complete fibrous ingrowth in polyethylene chords can be seen after a period of six months. However, until now, no biomechanical study has shown the efficacy of an alloplastic cord and its fixation device in providing sufficient stability and endurance in daily life-activity until newly formed scar tissue can take over this function. In a special test bench developed for this study, different loading regimes were applied to simulate loads during everyday life. Failure loads and failuremodes were evaluated. The properties of the cord were compared before and after physiological conditioning. It was shown that rubbing was the mode of failure under dynamic loading. Tensile forces up to 2558N did not result in material failure. Thus, using an artificial cord together with this fixation device, temporary sufficient stable fixation can be expected.
The wardrobe malfunction—an unanticipated exposure of bodily parts in the public—has become a prevailing issue in concerts, shows and other celebrity events that is reliably reported by the media. The internet as the fastest source for celebrity gossip allows measuring the impact of such wardrobe malfunctions on the public in-terest in a celebrity. This measurement in turn allows conclusions about intention, motivation, and internet be-haviour of a wide variety of internet users. The present study exemplifies the use of an innovative non-reactive measure of active interest—the Search Volume Index—to assess the impact of a variety of internet-related phe-nomena, including wardrobe malfunctions. Results indicate that interest in a celebrity increases immediately af-ter such an event and stays at a high level for about three weeks (the wardrobe plateau). This special form of ce-lebrity gossip thus meets a constant interest of a substantial proportion of internet users.
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.
Background: The role of serotonin (5-hydroxytrptamine, 5-HT) in the modulation of pain has been widely studied. Previous work led to the hypothesis that 5-hydroxyindolacetic acid (5-HIAA), a main metabolite of serotonin, might by itself influence pain thresholds. Results: In the present study, we investigated the role of 5-HIAA in inflammatory pain induced by intraplantar injection of complete Freund’s adjuvant (CFA) into the hind paw of mice. Wild-type mice were compared to mice deficient of the 5-HT transporter (5-HTT-/- mice) using behavioral tests for hyperalgesia and high-performance liquid chromatography (HPLC) to determine tissue levels of 5-HIAA. Wild-type mice reproducibly developed thermal hyperalgesia and paw edema for 5 days after CFA injection. 5-HTT-/- mice treated with CFA had reduced thermal hyperalgesia on day 1 after CFA injection and normal responses to heat hereafter. The 5-HIAA levels in spinal cord and sciatic nerve as measured with HPLC were lower in 5-HTT-/- mice than in wild-type mice after CFA injection. Pretreatment of wild-type mice with intraperitoneal injection of para-chlorophenylalanine (p-CPA), a serotonin synthesis inhibitor, resulted in depletion of the 5-HIAA content in spinal cord and sciatic nerve and decrease in thermal hyperalgesia in CFA injected mice. The application of exogenous 5-HIAA resulted in potentiation of thermal hyperalgesia induced by CFA in 5-HTT-/- mice and in wild-type mice pretreated with p- CPA, but not in wild-type mice without p-CPA pretreatment. Further, methysergide, a broad-spectrum serotonin receptor antagonist, had no effect on 5-HIAA-induced potentiation of thermal hyperalgesia in CFA-treated wildtype mice. Conclusion: Taken together, the present results suggest that 5-HIAA plays an important role in modulating peripheral thermal hyperalgesia in CFA induced inflammation, probably via a non-serotonin receptor mechanism.
Early onset sepsis due to group B streptococcus leads to neonatal morbidity, increased mortality, and long-term neurological deficencies. Interaction between septicemic GBS and confluent monolayers of human coronary artery endothelial cells (HCAECs) was analyzed by genome wide expression profiling. In total, 124 genes were differentially expressed (89 upregulated, 35 downregulated) based on a more than 3-fold difference to control HCAEC. Regulated genes are involved in apoptosis, hemostasis, oxidative stress response, infection, and inflammation. Regulation of selected genes and proteins identified in the gene array analysis was confirmed by Real-time RT-PCR assay (granulocy te chemotactic protein 2), ELISA (urokinase, cyclooxygenase 2, granulocyte chemotactic protein 1), and western blotting (Heme oxygenase1, BCL2 interacting protein) at various time points between 4 and 24 hours. These results indicate that GBS infection might influence signalling pathways leading to impaired function of the innate immune system and hemorrhagic and inflammatory complications during GBS sepsis.
Background: Intraarticular distal radius fractures can be treated with many methods. While internal fixation with angle stable implants has become increasingly popular, the use of bone graft substitutes has also been recommended to address comminution zones and thus increase stability. Whether a combination of both methods will improve clinical outcomes was the purpose of the study Methods: The study was thus conducted as a prospective randomized clinical trial. 39 patients with unilateral, intraarticular fractures of the distal radius were included and randomized to 2 groups, one being treated with internal fixation only, while the second group received an additional bone graft substitute. Results: There was no statistical significance between both groups in functional and radiological results. The occurrence of complications did also not show statistical significance. Conclusions: No advantage of additional granular bone graft substitutes could be seen in this study. Granular bone graft substitutes do not seem to provide extra stability if dorsal angle stable implants are used. Dorsal plates have considerable complication rates such as extensor tendon ruptures and development of CRPS.
Background: Investigation of the expression of an intestinal stem cell marker in esophageal adenocarcinomas (EAC) with and without Barrett’s Esophagus (BE), with respect to a cancer stem cell (CSC) hypothesis. Materials and methods: Expression of a putative intestinal stem cell marker LgR5 was analyzed in esophageal cancer specimen (n = 70: 41 EAC with BE, 19 EAC without BE, and n = 10 esophageal squamous-cell carcinomas, ESCC) and in the adenocarcinoma cell line OE-33. Ki-67 and Cdx-2 were co-labelled with LgR5 in double staining experiments. Immunhistochemical expression results were confirmed by RT-PCR and correlated with tumor stage and five-year survival rates. Results: LgR5was found expressed in 35 of 41 (85%) EAC with BE and in 16 of 19 (81%) EAC without BE. By contrast, LgR5 was not found to be expressed in ESCC. Quantification of immunolabeling showed 15% LgR5+ cells in EAC with BE, 32% LgR5+ cells in adjacent BE and 13% in EAC without BE. Immunofluorescence double staining experiments with LgR5 and Ki-67 revealed a subpopulation (~5%) of proliferating LgR+/Ki-67+ cells. On mRNAlevel, expression of LgR5 was higher in BE in comparison to EAC (p = 0.0159). High levels of LgR5 expression in BE associated EAC were associated with poorer survival in univariate analysis. Conclusion: The stem cell marker LgR5 is expressed in EAC, irrespective of association with BE, and appears to have negative impact on survival. The subset of proliferating LgR5+ cells (<5%) might resemble rapidly cycling CSCs, which needs to be substantiated in further investigations.
Background: Xenobiotics represent an environmental stress and as such are a source for antibiotics, including the isoquinoline (IQ) compound IQ-143. Here, we demonstrate the utility of complementary analysis of both host and pathogen datasets in assessing bacterial adaptation to IQ-143, a synthetic analog of the novel type N,C-coupled naphthyl-isoquinoline alkaloid ancisheynine. Results: Metabolite measurements, gene expression data and functional assays were combined with metabolic modeling to assess the effects of IQ-143 on Staphylococcus aureus, Staphylococcus epidermidis and human cell lines, as a potential paradigm for novel antibiotics. Genome annotation and PCR validation identified novel enzymes in the primary metabolism of staphylococci. Gene expression response analysis and metabolic modeling demonstrated the adaptation of enzymes to IQ-143, including those not affected by significant gene expression changes. At lower concentrations, IQ-143 was bacteriostatic, and at higher concentrations bactericidal, while the analysis suggested that the mode of action was a direct interference in nucleotide and energy metabolism. Experiments in human cell lines supported the conclusions from pathway modeling and found that IQ-143 had low cytotoxicity. Conclusions: The data suggest that IQ-143 is a promising lead compound for antibiotic therapy against staphylococci. The combination of gene expression and metabolite analyses with in silico modeling of metabolite pathways allowed us to study metabolic adaptations in detail and can be used for the evaluation of metabolic effects of other xenobiotics.
Mammalian Sun1 belongs to an evolutionarily conserved family of inner nuclear membrane proteins, which are known as SUN domain proteins. SUN domain proteins interact with KASH domain partners to form bridging complexes, so-called LINC complexes, that physically connect the nuclear interior to the cytoskeleton. LINC complexes are critical for nuclear integrity and play fundamental roles in nuclear positioning, shaping and movement. The mammalian genome codes for at least five different SUN domain proteins used for the formation of a number of different LINC complexes. Recently, we reported on the identification of everal Sun1 isoforms, which tremendously enlarges the alternatives to form functional LINC complexes. We now confirmed that Sun1 actually exists in at least seven distinct splice variants. Besides that, we observed that expression of individual Sun1 isoforms remarkably depends on the cell type, suggesting a cell type-specific adaption of Sun1 dependent LINC complexes to specific cellular and physiological requirements.
We herein present a patient with delayed-type allergic hypersensitivity against prilocaine leading to spreading eczematous dermatitis after subcutaneous injections for local anesthesia with prilocaine. Prilocaine allergy was proven by positive skin testing and subcutaneous provocation, whereas the evaluation of other local anesthetics - among them lidocaine, articaine and mepivacaine - did not exhibit any evidence for cross-reactivity. Interestingly, our patient repeatedly tolerated strictly deep subcutaneous injection of prilocaine in provocation testing while patch and superficial subcutaneous application mounted strong allergic responses. We hypothesize, that lower DC density in deeper cutaneous compartments and/or different DC subsets exhibiting distinct functional immunomodulatory properties in the various layers of the skin may confer to the observed absence of clinical reactivity against prilocaine after deep subcutaneous injection. The term compartment allergy indicates that the route of allergen administration together with the targeted immunologic environment orchestrates on the immunologic outcome: overt T-cell mediated allergy or clinical tolerance.
Popliteal cysts, or Baker cysts, are considered rare in children and may exhibit particular features, as compared with adults. We studied data from80 paediatric patients with 55 Baker cysts, examined over a period of 7 years, and correlated clinical presentation with findings on ultrasonography and MRI. Prevalence of popliteal cysts was 57% in arthritic knees, 58% with hypermobility syndrome, and 28% without risk factors. Only one patient had a trauma history and showed an ipsilateral cyst. Mean cyst volume was 3.4 mL; cysts were larger in boys. Patients with arthritis had echogenic cysts in 53%. Cyst communication with the joint space was seen in 64% on ultrasonography and 86% on MRI. In conclusion, Baker cysts are a common finding in a clinically preselected paediatric population. Children with Baker cysts should be assessed for underlying arthritis and inherited joint hypermobility, while sporadic Baker cysts appear to be common, as well.
On the Fragility Index
(2011)
The Fragility Index captures the amount of risk in a stochastic system of arbitrary dimension. Its main mathematical tool is the asymptotic distribution of exceedance counts within the system which can be derived by use of multivariate extreme value theory. Thereby the basic assumption is that data comes from a distribution which lies in the domain of attraction of a multivariate extreme value distribution. The Fragility Index itself and its extension can serve as a quantitative measure for tail dependence in arbitrary dimensions. It is linked to the well known extremal index for stochastic processes as well the extremal coefficient of an extreme value distribution.
Understanding the mechanisms of fragmentation within silicate melts is of great interest not only for material science, but also for volcanology, particularly regarding molten fuel coolant-interactions (MFCIs). Therefore edge-on hammer impact experiments (HIEs) have been carried out in order to analyze the fracture dynamics in well defined targets by applying a Cranz-Schardin highspeed camera technique. This thesis presents the corresponding results and provides a thorough insight into the dynamics of fragmentation, particularly focussing on the processes of energy dissipation. In HIEs two main classes of cracks can be identified, characterized by completely different fracture mechanisms: Shock wave induced “damage cracks” and “normal cracks”, which are exclusively caused by shear-stresses. This dual fracture situation is taken into account by introducing a new concept, according to which the crack class-specific fracture energies are linearly correlated with the corresponding fracture areas. The respective proportionality constants - denoted “fracture surface energy densities” (FSEDs) - have been quantified for all studied targets under various constraints. By analyzing the corresponding high speed image sequences and introducing useful dynamic parameters it has been possible to specify and describe in detail the evolution of fractures and, moreover, to quantify the energy dissipation rates during the fragmentation. Additionally, comprehensive multivariate statistical analyses have been carried out which have revealed general dependencies of all relevant fracture parameters as well as characteristics of the resulting particles. As a result, an important principle of fracture dynamics has been found, referred to as the “local anisotropy effect”: According to this principle, the fracture dynamics in a material is significantly affected by the location of directed stresses. High local stress gradients cause a more stable crack propagation and consequently a reduction of the energy dissipation rates. As a final step, this thesis focusses on the volcanological conclusions which can be drawn on the basis of the presented HIE results. Therefore fragments stemming from HIEs have been compared with natural and experimental volcanic ash particles of basaltic Grimsvötn and rhyolitic Tepexitl melts. The results of these comparative particle analyses substantiate HIEs to be a very suitable method for reproducing the MFCI loading conditions in silicate melts and prove the FSED concept to be a model which is well transferable to volcanic fragmentation processes.
The material system of interest in this thesis are II-VI-semiconductors. The first part of this thesis focuses on the formation of self-assembled CdSe-based quantum dots (QD) on ZnSe. The lattice constants of ZnSe and CdSe differ as much as about 7\% and therefore a CdSe layer grown on top of ZnSe experiences a huge strain. The aspired strain relief constitutes in the self-assembly of QDs (i.e. a roughened layer structure). Additionally, this QD layer is intermixed with Zn as this is also a possibility to decrease the strain in the layer. For CdSe on ZnSe, in Molecular Beam Epitaxy (MBE), various QD growth procedures were analysed with respect to the resulting Cd-content of the non-stoichiometric ternary (Zn,Cd)Se. The evaluation was performed by Raman Spectroscopy as the phonon frequency depends on the Cd-content. The second part of the thesis emphasis on the interface properties of n-ZnSe on n-GaAs. Different growth start procedures of the ZnSe epilayer may lead to different interface configurations with characteristic band-offsets and carrier depletion layer widths. The analysis is mainly focused on the individual depletion layer widths in the GaAs and ZnSe. This non-destructive analysis is performed by evaluating the Raman signal which comprises of phonon scattering from the depleted regions and coupled plasmon-phonon scattering from regions with free carriers.
Single-molecule microscopy is one of the decisive methodologies that allows one to clarify cellular signaling in both spatial and temporal dimentions by tracking with nanometer precision the diffusion of individual microscopic particles coupled to relevant biological molecules. Trajectory analysis not only enables determination of the mechanisms that drive and constrain the particles motion but also to reveal crucial information about the molecule interaction, mobility, stoichiometry, all existing subpopulations and unique functions of particular molecules. Efficacy of this technique depends on two problematic issues the usage of the proper fluorophore and the type of biochemical attachment of the fluorophore to a biomolecule. The goal of this study was to evolve a highly specific labeling method suitable for single molecule tracking, internalization and trafficking studies that would attain a calculable 1:1 fluorophore-to-receptor stoichiometry. A covalent attachment of quantum dots to transmembrane receptors was successfully achieved with a techinque that amalgamates acyl carrier protein (ACP) system as a comparatively small linker and coenzyme A (CoA)-functionalized quantum dots. The necessity of optimization of the quantum dot usage for more precise calculation of the membrane protein stoichiometries in larger assemblies led to the further study in which methods maximizing the number of signals and the tracking times of diverse QD types were examined. Next, the optimized techniques were applied to analyze behavior of interleukin-5 β-common chain receptor (IL-5Rβc) receptors that are endogenously expressed at low level on living differentiated eosinophil-like HL-60 cells. Obtained data disclosed that perused receptors form stable and higher order oligomers. Additionally, the mobility analysis based on increased in number (>10%) uninterrupted 1000-step trajectories revealed two patterns of confined motion. Thereupon methods were developed that allow both, determination of stoichiometries of cell surface protein complexes and the acquisition of long trajectories for mobility analysis. Sequentially, the aforementioned methods were used to scrutinize on the mobility, internalization and recycling dynamics characterization of a G protein-coupled receptor (GPCRs), the parathyroid hormone receptor (PTHR1) and several bone morphogenetic proteins (BMPs), a member of the TGF-beta superfamily of receptors. These receptors are two important representatives of two varied membrane receptor classes. BMPs activate SMAD- and non-SMAD pathways and as members of the transforming growth factor β (TGF-β) superfamily are entailed in the regulation of proliferation, differentiation, chemotaxis, and apoptosis. For effective ligand induced and ligand independent signaling, two types of transmembrane serine/threonine kinases, BMP type I and type II receptors (BMPRI and BMPRII, respectively) are engaged. Apparently, the lateral mobility profiles of BMPRI and BMPRII receptors differ markedly, which determinate specificity of the signal. Non-SMAD signaling and subsequent osteoblastic differentiation of precursor cells particularly necessitate the confinement of the BMP type I receptor, resulting in the conclusion that receptor lateral mobility is a dominative mechanism to modulate SMAD versus non-SMAD signaling during differentiation. Confined motion was also predominantly observed in the studies devoted to, entailed in the regulation of calcium homeostasis and in bone remodeling, the parathyroid hormone receptor (PTHR1), in which stimulation with five peptide ligands, specific fragments of PTH: hPTH(1–34), hPTHrP(107–111)NH2; PTH(1–14); PTH(1–28) G1R19, bPTH(3–34), first four belonging to PTH agonist group and the last to the antagonist one, were tested in the wide concentration range on living COS-1 and AD293 cells. Next to the mobility, defining the internalization and recycling rates of the PTHR1 receptor maintained in this investigation one of the crucial questions. Internalization, in general, allows to diminish the magnitude of the receptor-mediated G protein signals (desensitization), receptor resensitization via recycling, degradation (down-regulation), and coupling to other signaling pathways (e.g. MAP kinases). Determinants of the internalization process are one of the most addressed in recent studies as key factors for clearer understanding of the process and linking it with biological responses evoked by the signal transduction. The internalization of the PTH-receptor complex occurs via the clathrin-coated pit pathway involving β-arrestin2 and is initiated through the agonist occupancy of the PTHR1 leading to activation of adenylyl cyclase (via Gs), and phosphatidylinositol-specific phospholipase Cβ (via Gq). Taken together, this work embodies complex study of the interleukin-5 β-common chain receptor (IL-5Rβc) receptors, bone morphogenetic proteins (BMPs) and the parathyroid hormone receptor with the application of single-molecule microscopy with the newly attained ACP-quantum dot labeling method and standard techniques.
Magnetic resonance imaging (MRI) is a medical imaging method that involves no ionizing radiation and can be used non-invasively. Another important - if not the most important - reason for the widespread and increasing use of MRI in clinical practice is its interesting and highly flexible image contrast, especially of biological tissue. The main disadvantages of MRI, compared to other widespread imaging modalities like computed tomography (CT), are long measurement times and the directly resulting high costs. In the first part of this work, a new technique for accelerated MRI parameter mapping using a radial IR TrueFISP sequence is presented. IR TrueFISP is a very fast method for the simultaneous quantification of proton density, the longitudinal relaxation time T1, and the transverse relaxation time T2. Chapter 2 presents speed improvements to the original IR TrueFISP method. Using a radial view-sharing technique, it was possible to obtain a full set of relaxometry data in under 6 s per slice. Furthermore, chapter 3 presents the investigation and correction of two major sources of error of the IR TrueFISP method, namely magnetization transfer and imperfect slice profiles. In the second part of this work, a new MRI thermometry method is presented that can be used in MRI-safety investigations of medical implants, e.g. cardiac pacemakers and implantable cardioverter-defibrillators (ICDs). One of the major safety risks associated with MRI examinations of pacemaker and ICD patients is RF induced heating of the pacing electrodes. The design of MRI-safe (or MRI-conditional) pacing electrodes requires elaborate testing. In a first step, many different electrode shapes, electrode positions and sequence parameters are tested in a gel phantom with its geometry and conductivity matched to a human body. The resulting temperature increase is typically observed using temperature probes that are placed at various positions in the gel phantom. An alternative to this local thermometry approach is to use MRI for the temperature measurement. Chapter 5 describes a new approach for MRI thermometry that allows MRI thermometry during RF heating caused by the MRI sequence itself. Specifically, a proton resonance frequency (PRF) shift MRI thermometry method was combined with an MR heating sequence. The method was validated in a gel phantom, with a copper wire serving as a simple model for a medical implant.
Cross-striated muscles enable higher animals to perform directed movements and to create mechanical force. The cells of heart and skeletal muscles consist of myofibrils, serial arrays of the smallest contractile subunits, the sarcomeres. Main components of the sarcomeres are the thin and thick filaments, large protein assemblies consisting of mainly actin (thin filaments) and myosin (thick filaments), whose energy-dependent interaction is responsible for the contraction of sarcomeres and so of the whole muscle. The thin filaments are anchored in the sarcomere bordering Z-discs, while the thick filaments are anchored in the M-bands, traverse structures in the sarcomere center. Electron-microscopic studies revealed that the M-bands consist of regular, lattice-like structures that appear to cross-link the thick filaments. A number of proteins could be identified by immune-fluorescence and biochemical binding studies to be present and interact with each other in the M-bands. These data have been integrated into preliminary models of the M-bands. Detailed knowledge of how these proteins interact with each other in the center of the sarcomeres is, however, largely missing. The current study focuses on the structural characterization of the interactions between the titin, myomesin-1, obscurin and obscurin-like 1 (OBSL1), modular filamentous proteins interacting with each other in the M-bands. The high-resolution crystal structure of the titin M10 – OBSL1 Ig1 complex was solved. The structure and additional biophysical data show that titin and OBSL1 as well as titin and obscurin form stable binary complexes through the formation of a small intermolecular ß-sheet. In contrast to previously characterized intermolecular assemblies of sarcomeric proteins, this sheet is formed between parallel non- homologous ß-strands of the interaction partners. The investigation of disease-related variants of the M10 domain by biophysical methods did not allow to draw unambiguous conclusions on a direct connection between impaired OBSL1/obscurin binding and disease development. Two out of four known M10 variants have effects on the correct domain folding and so interfere with the ability to bind obscurin/OBSL1. The two other known variants displayed however only minor effects on fold and binding affinities. It should therefore be further elucidated whether a direct connection between impaired complex formation and disease development exists. -I- Abstract A direct interaction between titin and myomesin-1 could not be confirmed in vitro. Possible explanations for the different results are discussed. While the consequences of the inability of both proteins to interact are unclear, the further characterization of the putative interacting parts of titin and myomesin-1 led to the discovery of two new potential sites of self-assembly on M-band titin and myomesin-1. The crystal structure of titin M4 showed that this domain can form dimeric assemblies through the formation of a disulfide bridge and an intermolecular metal binding site between residues that are unique to this domain. On myomesin-1, in addition to the described C-terminal interaction site, a potential second site of self-assembly was found in its central Fn3-domain segment. The interacting site was mapped to the predicted Fn3 domain My5. The crystal structure of the domain in its dimeric form showed that the interaction is mediated by a mechanism that has previously not been observed in sarcomeric proteins. Two My5 interact with each other by the mutual exchange of an N-terminal ß-strand which complements the Fn3 fold on the binding partner. This type of interaction can be interpreted as misfolding. However, the position of the interacting domain and its mode of interaction allowed the postulation of a model of how myomesin-1 could be integrated in the M-bands. This model is in good agreement with the electron-microscopic appearance of the M-bands.
Background: Tumor patients exhibit an increased peripheral demand of fatty acids and protein. Contrarily, tumors utilize glucose as their main source of energy supply. Thus, a diet supplying the cancer patient with sufficient fat and protein for his demands while restricting the carbohydrates (CHO) tumors thrive on, could be a helpful strategy in improving the patients’ situation. A ketogenic diet (KD) fulfills these requirements. Therefore, we performed a pilot study to investigate the feasibility of a KD and its influence on the quality of life of patients with advanced metastatic tumors. Methods: Sixteen patients with advanced metastatic tumors and no conventional therapeutic options participated in the study. The patients were instructed to follow a KD (less than 70 g CHO per day) with normal groceries and were provided with a supply of food additives to mix a protein/fat shake to simplify the 3-month intervention period. Quality of life [assessed by EORTC QLQ-C30 (version 2)], serum and general health parameters were determined at baseline, after every two weeks of follow-up, or after drop out. The effect of dietary change on metabolism was monitored daily by measuring urinary ketone bodies. Results: One patient did not tolerate the diet and dropped out within 3 days. Among those who tolerated the diet, two patients died early, one stopped after 2 weeks due to personal reasons, one felt unable to stick to the diet after 4 weeks, one stopped after 6 and two stopped after 7 and 8 weeks due to progress of the disease, one had to discontinue after 6 weeks to resume chemotherapy and five completed the 3 month intervention period. These five and the one who resumed chemotherapy after 6 weeks report an improved emotional functioning and less insomnia, while several other parameters of quality of life remained stable or worsened, reflecting their very advanced disease. Except for temporary constipation and fatigue, we found no severe adverse side effects, especially no changes in cholesterol or blood lipids. Conclusions: These pilot data suggest that a KD is suitable for even advanced cancer patients. It has no severe side effects and might improve aspects of quality of life and blood parameters in some patients with advanced metastatic tumors.
Background: Although literature provides support for cognitive behavioral therapy (CBT) as an efficacious intervention for social phobia, more research is needed to improve treatments for children. Methods: Forty four Caucasian children (ages 8-14) meeting diagnostic criteria of social phobia according to the Diagnostic and Statistical Manual of Mental Disorders (4th ed.; APA, 1994) were randomly allocated to either a newly developed CBT program focusing on cognition according to the model of Clark and Wells (n = 21) or a wait-list control group (n = 23). The primary outcome measure was clinical improvement. Secondary outcomes included improvements in anxiety coping, dysfunctional cognitions, interaction frequency and comorbid symptoms. Outcome measures included child report and clinican completed measures as well as a diagnostic interview. Results: Significant differences between treatment participants (4 dropouts) and controls (2 dropouts) were observed at post test on the German version of the Social Phobia and Anxiety Inventory for Children. Furthermore, in the treatment group, significantly more children were free of diagnosis than in wait-list group at post-test. Additional child completed and clinician completed measures support the results. Discussion: The study is a first step towards investigating whether CBT focusing on cognition is efficacious in treating children with social phobia. Future research will need to compare this treatment to an active treatment group. There remain the questions of whether the effect of the treatment is specific to the disorder and whether the underlying theoretical model is adequate. Conclusion: Preliminary support is provided for the efficacy of the cognitive behavioral treatment focusing on cognition in socially phobic children. Active comparators should be established with other evidence-based CBT programs for anxiety disorders, which differ significantly in their dosage and type of cognitive interventions from those of the manual under evaluation (e.g. Coping Cat).
This work deals with nonlinear optics with wavefront controlled ultra-short laser pulses. The effects studied are self-phase modulation due to filamentation of ultra-short laser pulses and high-order harmonic generation in a jet of noble gas. Additionally, a way to optimize the spectral brilliance of the high-order harmonic source is studied by measuring the spectrum and wavefront of the generated XUV beam.
This thesis investigated the potential of Compressed Sensing (CS) applied to Magnetic Resonance Imaging (MRI). CS is a novel image reconstruction method that emerged from the field of information theory. The framework of CS was first published in technical reports in 2004 by Candès and Donoho. Two years later, the theory of CS was published in a conference abstract and two papers. Candès and Donoho proved that it is possible, with overwhelming probability, to reconstruct a noise-free sparse signal from incomplete frequency samples (e.g., Fourier coefficients). Hereby, it is assumed a priori that the desired signal for reconstruction is sparse. A signal is considered “sparse“ when the number of non-zero elements is significantly smaller than the number of all elements. Sparsity is the most important foundation of CS. When an ideal noise-free signal with few non-zero elements is given, it should be understandably possible to obtain the relevant information from fewer Fourier coefficients than dictated by the Nyquist-Shannon criterion. The theory of CS is based on noise-free sparse signals. As soon as noise is introduced, no exact sparsity can be specified since all elements have signal intensities that are non-zero. However, with the addition of little or moderate noise, an approximate sparsity that can be exploited using the CS framework will still be given. The ability to reconstruct noisy undersampled sparse MRI data using CS has been extensively demonstrated. Although most MR datasets are not sparse in image space, they can be efficiently sparsified by a sparsifying transform. In this thesis, the data are either sparse in the image domain, after Discrete Gradient transformation, or after subtraction of a temporally averaged dataset from the data to be reconstructed (dynamic imaging). The aim of this thesis was to identify possible applications of CS to MRI. Two different algorithms were considered for reconstructing the undersampled sparse data with the CS concept. The Nonlinear Conjugate Gradient based technique with a relaxed data consistency constraint as suggested by Lustig et al. is termed Relaxed DC method. An alternative represents the Gradient or Steepest Descent algorithm with strict data consistency and is, therefore, termed the Strict DC method. Chapter 3 presents simulations illustrating which of these two reconstruction algorithms is best suited to recover undersampled sparse MR datasets. The results lead to the decision for the Strict DC method as reconstruction technique in this thesis. After these simulations, different applications and extensions of CS are demonstrated. Chapter 4 shows how CS benefits spectroscopic 19F imaging at 7 T, allowing a significant reduction of measurement times during in vivo experiments. Furthermore, it allows highly resolved spectroscopic 3D imaging in acceptable measurement times for in vivo applications. Chapter 5 introduces an extension of the Strict DC method called CS-CC (CS on Combined Coils), which allows efficient processing of sparse undersampled multi-coil data. It takes advantage of a concept named “Joint Sparsity“, which exploits the fact that all channels of a coil array detect the same sparse object weighted with the coil sensitivity profiles. The practical use of this new algorithm is demonstrated in dynamic radial cardiac imaging. Accurate reconstructions of cardiac motion in free breathing without ECG triggering were obtained for high undersampling factors. An Iterative GRAPPA algorithm is introduced in Chapter 6 that can recover undersampled data from arbitrary (Non-Cartesian) trajectories and works solely in the Cartesian plane. This characteristic makes the proposed Iterative GRAPPA computationally more efficient than SPIRiT. Iterative GRAPPA was developed in a preceding step to combine parallel imaging with CS. Optimal parameters for Iterative GRAPPA (e.g. number of iterations, GRAPPA kernel size) were determined in phantom experiments and verified by retrospectively undersampling and reconstructing a radial cardiac cine dataset. The synergistic combination of the coil-by-coil Strict DC CS method and Iterative GRAPPA called CS-GRAPPA is presented in Chapter 7. CS-GRAPPA allows accurate reconstruction of undersampled data from even higher acceleration factors than each individual method. It is a formulation equivalent to L1-SPIRiT but computationally more efficient. Additionally, a comparison with CS-CC is given. Interestingly, exploiting joint sparsity in CS-CC is slightly more efficient than the proposed CS-GRAPPA, a hybrid of parallel imaging and CS. The last chapter of this thesis concludes the findings presented in this dissertation. Future applications expected to benefit from CS are discussed and possible synergistic combinations with other existing MR methodologies for accelerated imaging are also contemplated.
When there is an imbalance between reactive oxygen species (ROS) and endogenous antioxidants (glutathione (GSH), superoxide dismutase (SOD), catalase etc.) the oxidative stress is increased and results in the oxidation of lipids, proteins and DNA. Although oxidation of lipids and proteins may also accumulates with age, only DNA oxidation leads to altered genomic information. As one pathway for increased ROS production, many endogenous and exogenous substances activate NADPH oxidase (NOX) enzyme and produce ROS. p47phox is a cytosolic organizer protein which plays an important role in NOX activation. Angiotensin II (Ang II) is an example for an endogenous compound which causes ROS through NOX activation. Rosuvastatin is an example for a drug with antioxidative capacity (upregulation of endogenous antioxidants). It is a lipid lowering drug which also reduces an elevated level of angiotensin II type 1 receptor (AT1R). Commonly, oxidative stress is elevated in ageing and age related diseases (eg. Parkinson’s disease (PD)). The aim of the present study was to investigate the role of NOX derived ROS induced oxidative DNA damage and the influence of ROS in ageing and age related diseases, using different in vitro and in vivo models.
There is evidence that pheromones are communicative signals in animals. However, the existence and function of human pheromones are still under discussion. During the last years several substances have been labeled as putative human pheromones and especially 4,16–androstadien-3-one (androstadienone), found in male and female sweat, became subject of intense investigation. In contrast to common odors androstadienone presumably modulates human physiological and psychological reactions. Data suggest that androstadienone might influence the processing of visual cues, specifically faces or affective stimuli, via projections from the fusiform gyrus and the amygdala. Moreover, attentional processes may be modulated, which is supported by explicit and implicit behavioral data. This thesis includes three experimental studies examining effects of androstadienone exposure on behavioral and cortical reactions to visual and emotional stimuli. The main hypotheses were that androstadienone might influence human behavior to and perception of visual cues. The first study sought to clarify androstadienone effects on attention-related reactions as well as on behavioral tendencies. Motoric approach-avoidance reactions in response to happy and angry facial expressions were investigated in 30 women and 32 men. Participants either inhaled androstadienone or a control solution, without knowing the real content, while performing the following task: they had to push away or to pull towards them a joystick as fast as possible in reaction to either an angry or a happy cartoon face, which was presented on a computer screen. Results showed that androstadienone modulated the participant´s task performance by accelerating the reaction speed compared to the control compound. Faster reactions were observed particularly when reacting to angry faces but not when reacting to happy faces. This might be explained by the finding that human body odors, the source of androstadienone, were found to activate the human fear system, i.e. modulating fear-related attentional processes. Therefore, the quicker reaction towards angry faces with exposure to androstadienone could be due to an enhanced allocation of attentional resources towards fear-related cues like angry faces. Results also showed that androstadienone enhanced men´s approach tendency towards faces independent of emotional expressions. This observation might be explained by androstadienone´s former shown ability to improve attractiveness ratings of other persons. In this regard, the endogenous odor might enhance evaluations of faces in men and, thus, might improve their willingness to approach social stimuli. In contrast to men, women already showed in the control condition higher approach tendency towards faces. Therefore, androstadienone might rather maintain than enhance the approach score in women. In the second study event-related brain potentials (ERPs) triggered by social and non-social visual stimuli were investigated by means of electroencephalography. In a double-blind between-subjects design 51 women participated. Twenty-eight women inhaled androstadienone, whereas 23 women inhaled a control solution. Four different picture categories, i.e. real faces, pictures with couples, pictures with social and non-social scenes, each including three different valence categories, i.e. positive, negative and neutral, should clarify the stimulus type or context androstadienone is acting on. The androstadienone compared to the control odor did not influence brain responses significantly. Explorative analyses, however, suggested that androstadienone influences the processing of faces. While in the control group angry faces elicited larger P300 amplitudes than happy faces, the androstadienone group showed similar P300 amplitudes concerning all emotional expressions. This observation tentatively indicates that the endogenous odor might indeed affect the neuronal responses to emotional facial stimuli, especially late components reflecting evaluative processes. However, this observation has to be verified and further investigated, in particular whether androstadienone caused reduced responses to angry faces or enhanced responses to happy faces. The third study investigated androstadienone effects on face processing especially in men. ERPs elicited by happy, angry and neutral cartoon faces, which were presented on a computer screen, were measured while 16 men, not knowing the applicated odor, inhaled either androstadienone or a control solution. Exposure to androstadienone significantly increased later neuronal responses, the P300 amplitude. This belated component of the ERP reflects attention allocation and evaluative processes towards important stimuli. Therefore, androstadienone might facilitate central nervous face processing by enhancing attention towards these stimuli. In sum, the current results corroborate the notion of androstadienone as an active social chemosignal. In minute amounts and not detectable as an odor it influenced cortical and motoric reactions. Therefore, it might be concluded that androstadienone indeed affects cognitive functions like attentional processes and in turn affects our behavior. The current results further support the notion that androstadienone acts like a human modulator pheromone, namely modulating ongoing behavior or a psychological reaction to a particular context, changing stimulus sensitivity, salience and sensory-motor integration. However, these conclusions remain tentative until further replication takes place, best in ecologically valid environments. Furthermore, one has to keep in mind that the current studies could not replicate several previous findings and could not verify some hypotheses assuming communicative effects of androstadienone. Thus, the main assumption of this thesis that androstadienone is an active chemosignal is still challenged. Also, whether the term “pheromone” is indeed suitable to label androstadienone remains open.
Replication-competent oncolytic viral therapies have shown great promise preclinically and in clinical trials for the treatment of various cancers. They are able to preferentially and selectively propagate in cancer cells, consequently destroying tumor tissue via cell lysis, while leaving noncancerous tissues unharmed. Currently, biopsy is the gold standard for monitoring of viral tumor colonization and oncolysis. This may be feasible in preclinical or early clinical trials; however, a noninvasive method facilitating ongoing monitoring of viral therapy is needed for human studies. The tracking of viral delivery could give clinicians the ability to assess the biodistribution of oncolytic viruses to ensure safety and correlation with treatment efficacy. This work centers on the construction and testing of a VACV strain, GLV-1h153, carrying the human sodium iodide symporter (hNIS) as a marker gene for non-invasive tracking of virus by imaging. Thus, this project aimed to help develop imaging techniques for use in clinical trials of oncolytic viral therapy. Further, the feasibility and effectiveness of virally induced targeted radiotherapy as an anti-cancer strategy was also investigated. hNIS is an intrinsic plasma membrane protein which mediates the active transport and concentration of iodide in the thyroid gland and some extra-thyroidal tissues. It is also one of several human genes currently being used as reporters in preclinical studies and has already been used in clinical studies for imaging viral replication in prostate cancer. hNIS gene transfer via viral vector may allow infected tumor cells to concentrate several carrier-free radionuclide probes such as Iodide-124 (124I), Iodide-131 (131I), and 99m-Technecium Pertechtenate (99mTcO4), which have long been approved for human use. hNIS also has the advantage of being of human origin thus minimizing immunogenicity, and its transporter based system allows intracellular signal amplification. GLV-1h153 was tested in pancreatic adenocarcinoma cell line PANC-1. GLV-1h153 infected, replicated within, and killed PANC-1 cells in cell culture as efficiently as GLV-1h68 and provided dose-dependent levels of hNIS transgene expression in infected cells. Immunofluorescence detected successful transport of the protein to the cell membrane prior to cell lysis, which enhanced dose and time-dependent intracellular uptake of 131I. In vivo, GLV-1h153 was as safe and effective as GLV-1h68 in regressing pancreatic cancer xenografts. Tumor infection by virus was confirmed via optical imaging and histology. GLV-1h153 further facilitated deep tissue imaging of virus replication in tumors via Iodide-124I positron emission tomography (PET) as well as 99mTcO4-mediated gamma scintigraphy. This was possible with both intratumoral and intravenous injection of the virus with radiouptake retained as long as 24 and 48 hours after radiotracer injection. PET image quantitation of radiouptake in tumors was found to correlate well with tissue radiouptake counts. Autoradiography of GLV-1h153-infected tumors revealed a need for presence of virus (visualized with green fluorescent protein expression), viable tissue, and adequate blood flow to enhance radiouptake in tumors. Dosimetric analysis of uptake in infected tumors displayed potential for therapeutic doses of radiotherapy to be delivered systemically to tumors. When GLV-1h153 was combined with 131I for treatment, a modest additive effect was seen as compared to GLV-1h153 alone. Therefore, GLV-1h153 is a promising new candidate for treating pancreatic cancer and noninvasively imaging viral therapy. These findings warrant further investigation into possible long term monitoring of viral therapy, as well as synergistic or additive effects of radioiodine combined with this novel treatment and imaging modality.
In this work the epitaxial growth and characterization of the half-metallic ferromagnet NiMnSb was presented. NiMnSb crystallizes in the C1b structure which is similar to the zinc blende structure from widely used III-V semiconductors. One special property of NiMnSb is the theoretical 100% spin-polarization at the Fermi edge. This makes it a perfect candidate for spintronic experiments and the material of choice for building novel spintronic devices. Another important topic in this work were the magnetic properties of NiMnSb, especially the low magnetic damping of the grown thin films. All grown layers were fabricated with the technique of MBE. The layer stacks for all different experiments and devices were grown on InP substrate in (001) or (111)B orientation. Before the NiMnSb layer a buffer layer of undoped (In,Ga)As was grown. Additional for some samples on InP(111)B, a Si doped (In,Ga)As layer was grown on top of the undoped (In,Ga)As layer. The dopant concentration of this n-doped layer was determined by ETCH-CV. All layers were investigated by structural and the NiMnSb layer additional by magnetic properties. For the structural investigation the in-situ technique RHEED and ex-situ tool HRXRD were used. RHEED observations showed a good quality of the grown buffer and half-metallic ferromagnet layers on both orientations. These results were strengthened by the HRXRD measurement. The vertical lattice constant could be determined. The received value of a(NiMnSb_vertical) = 5.925 Å for NiMnSb on InP(001) is in good agreement to the value a(NiMnSb_Lit) = 5.903 Å found in literature [Cas55]. For NiMnSb on InP(111)B a vertical lattice constant of a(NiMnSb_vertikal) = 6.017 Å could be determined. The horizontal lattice constant of the buffer and the half-metallic ferromagnet layer could be determined as the same of the substrate. For NiMnSb this conclusion is only valid up to a thickness of ≈40nm. To increase this maximum thickness, NiMnSb samples were grown on InP(001) substrates and capped with Ti/Au layers. Afterwards a reciprocal space map of the (533) reflex was drawn with GIXRD at the synchrotron beamline BW2 of HASYLAB [Kum07]. It has been shown that the critical thickness is more than doubled by depositing a Ti/Au capping directly after growth of NiMnSb without breaking the ultrahigh vacuum (UHV). The magnetic properties were determined with FMR experiments and SQUID measurements. The received magnetic damping parameter α from a 40nm thick NiMnSb layer on InP(001) could be determined to 3.19e−3 along [1-10]. The resulting line width of our NiMnSb layers on InP(001) is more than 4.88 times smaller than measured before [Hei04]. Another result is the direction dependence of the damping. It has been measured that the difference of the damping is changed by more than 42% when rotating the applied field by 45° from [1-10] to [100].With SQUID we measured a saturation magnetization of a 40nm thick NiMnSb layer as 4µB. NiMnSb layers on InP(111)B substrate where also measured with FMR with a surprising result. These layers not only showed a decreasing in the anisotropy field with increasing thickness but also an uniaxial anisotropy. This behaviour can be explained with defects on these samples. With an AFM triangle-like defects were measured. These defects originated from the buffer layer and influenced the magnetic properties. Another part of this work is dedicated to the behaviour of NiMnSb at temperatures around 80K. With our samples, no phase transition can be observed in the data of the Hall, anomalous Hall term and resistivity. The last part of this work discusses different spintronic devices build with our NiMnSb layers. In a first device the magnetization acts on the current. This Giant Magneto Resistance (GMR) device consisted of InP:S(001) - 180nm undoped (In,Ga)As - 40nm NiMnSb - 10nm Cu - 6nm NiFe - 10nm Ru in current perpendicular to plane (CPP) geometry. We received a Magneto-Resistance-Ratio of 3.4%. In a second device the current acts on the magnetization and makes use of the spin torque phenomena. This so called Spin Torque Oscillator (STO) emitted frequencies in the GHz range (13.94GHz - 14.1GHz). The last fabricated device is based on the magnetic vortex phenomena. For switching the core polarity the gyrotropic frequencies f + = 254MHz f − = 217MHz and a total static magnetic field of only mµ0H = 65mT were necessary. The reversal efficiency has been determined as better than 99% [Lou09].
While beneficial sponge-microbe associations have received much attention in recent years, less effort has been undertaken to investigate the interactions of sponges with potentially pathogenic microorganisms. Thus, the aim of this study was to examine two selected Caribbean disease conditions, termed “Sponge Orange Band” and “Sponge White Patch”, via ecological and molecular methods. Sponge Orange Band (SOB) disease affects the prominent Caribbean barrel sponge Xestospongia muta that is counted among the high-microbial-abundance (HMA) sponges, whereas Sponge White Patch (SWP) disease affects the abundant rope sponge Amphimedon compressa that belongs to the low-microbial-abundance (LMA) sponges. I have documented for both Caribbean sponge diseases a disease progression going along with massive tissue destruction as well as loss of the characteristic microbial signatures. Even though new bacteria were shown to colonize the bleached areas, the infection trials revealed in both cases no indication for the involvement of a microbial pathogen as an etiologic agent of disease leaving us still in the dark about the cause of Sponge Orange Band as well as Sponge White Patch disease.
Pluripotency describes the ability of stem cells to form every cell type of the body.. Pluripotent stem cells are e.g. embryonic stem cells (ESCs), but also the so called induced pluripotent stem cells (IPS cells), that are generated by reprogramming differentiated somatic cells into a pluripotent state. Furthermore, it has been shown that spermatogonia (SG) derived from adult testes of mouse or human are pluripotent. Because of their ability to differentiate into every somatic cell type, pluripotent stem cells have a unique status in research and regenerative medicine. For the latter, they offer a valuable opportunity to replace destroyed tissues or organs. For basic research, stem cells represent a useful system to study differentiation or developmental processes that are difficult to access in the physiological situation e.g. during embryogenesis. Both applications, however, require methods that allow efficient and directed differentiation of stem cells into defined specialized cell types. This study first aims to investigate the differentiation potential of SG derived from the teleost fish medaka (Oryzias latipes). My results demonstrate that medaka SG are able to form different somatic cell types, namely adipocytes, melanocytes, osteoblasts, and neurons. This indicates that medake SG have retained a broad differentiation potential suggesting that pluripotency is not restricted to mouse and human SG but might be conserved among vertebrates. Next, I wanted to establish a differentiation method that is solely based on ectopic expression of genes known to be essential for the formation of certain somatic cell types – so called master regulators (MRs). My findings show that ectopic expression of the melanocyte-specific transcription factor mitf-m that has previously been shown to induce differentiation of medaka ESCs into pigment cells resulted in the formation of the same cell type in medaka SG. This approach could be used to generate other somatic cell types. Thus, ectopic expression of the MRs cbfa1 and mash1 in MF-SG was sufficient to induce differentiation into osteoblasts and neurons, respectively. Interestingly, these differentiation processes included the activation of genes that are expressed earlier during embryogenesis than the differentiation-inducing MR. Furthermore, my findings show that the approach of MR-induced differentiation can be transferred to mammalian stem cell systems. Ectopic expression of the neural transcription factor ngn2 was sufficient to induce efficient and rapid differentiation of neurons in mouse ESCs. This differentiation process also included the induction of genes that in vivo are activated at earlier stages that ngn2. By generating a transgenic cell line allowing induction of ectopic ngn2 expression, it was possible to obtain a relatively pure culture of functional neurons. Ngn2-induced differentiation did not require any additional signals and occurred even under pluripotency promoting conditions. Moreover, ectopic expression of ngn2 did also induce the formation of cells with neuronal morphology in IPS cells indicating that MR-induced differentiation is operative in different stem cell types. Furthermore, protein transduction of Ngn2 into mouse ESCs also resulted in a neuronal differentiation process up to the appearance of neural precursor cells. Last, my results show that MR-induced differentiation can also be used to generate other cell types than neurons from mouse ESCs. Myoblasts and macrophage-like cells were generated by ectopic expression of the MRs myoD and cebpa, respectively. Using transgenic cell lines enabling induction of MR expression it was possible to obtain mixed cultures with two different differentiation processes occurring in parallel. Altogether this study shows that ectopic expression of single genes is sufficient to induce directed differentiation of stem cells into defined cell types. The feasibility of this approach was demonstrated for different MRs and consequently different somatic cell types. Furthermore, MR induced differentiation was operative in different stem cell types from fish and mouse. Thus, one can conclude that certain genes are able to define cell fates in in vitro stem cell systems and that this cell fate defining potential appears to be a conserved feature in vertebrates. These findings therefore provide new insights in the role of MRs in cell commitment and differentiation processes. Furthermore, this study presents a new method to induce directed differentiation of stem cells that offers several advantages regarding efficiency, rapidness, and reproducibility. MR-induced differentiation therefore represents a promising tool for both stem cell research and regenerative medicine.
This study was conducted to determine the influence of different stress factors on the honeybee Apis mellifera. The investigation was motivated by previous experiments that suggested the existence of an unspecific defense mechanism causing a generalized change of flight behavior after the onset of different diseases. This mechanism is thought to impede the ability of flight bees to return to their respective colonies thereby removing the disease from the colony over time. During the last years, the existence of such a “suicidal behavior” was supported by further studies. Thus, an unnoticed, potentially highly effective defense mechanism of social insects was revealed whose spectrum of activity and physiological basics require further investigation. Suggesting that the reaction by the bees is unspecific to different diseases as well as to other potential stress factors, this study was designed to investigate the influence of pathogens, insecticides, and different brood rearing temperatures on different parameters like lifespan, foraging activity, and foraging trip duration of worker bees.
The nicotinic acetylcholine receptor of skeletal muscle is one of the best-investigated synaptic proteins and often serves as model for the entire family of pentameric ligand gated ion channels (pLGICs). Receptors of this superfamily share a common architecture. After binding the agonist the characteristic C-loop structure closes around the ligand-binding site and triggers a wave of conformational changes that spread through the protein and finally result in the opening of the channel gate. As shown before, high-resolution single channel data can hardly be described by simple kinetic mechanisms (Parzefall et al., 1998, Hallermann et al., 2005). Recent advances in the field of kinetic modelling on receptor currents demonstrate that the introduction of additional short lived shut states in kinetic schemes enhances the quality of estimates of reaction rates. The additional shut states that immediately follow ligand bound states in the mechanism are suggested to resemble the closing movement of the C-loop (Lape et al., 2008; Mukhtasimova et al., 2009). It has not been described yet whether and how the structural differences of the 2 binding sites of the receptor influence the opening behaviour. To address this question, high-resolution single channel recordings, in combination with agonists that are known to exhibit different binding site selectivity, were performed. Thereby, a detailed description of the binding site dependent generation of channel currents is possible. At the embryonic mouse-muscle receptor used in this study the ligand binding sites are located at the α-γ and α-δ subunit interfaces. By allocation of opening characteristics to the α-δ and α-γ sites it is possible to show the binding site dependent activation of distinct kinetic states. Furthermore, it will be shown that the recently introduced short-lived shut states are sufficient to describe high-resolution single channel data. Finally an enhanced kinetic mechanism based on the ‘primed states’ model, published in 2009 by Mukhtasimova et al., will be presented. In this model the structurally diverse α-δ and α-γ binding sites elicit different kinetic channel characteristics. Thus the complex high-resolution kinetic characteristics of the embryonic receptor can be described coherently.
Background: Parkinson’s disease (PD) is characterized at the cellular level by a destruction of neuromelanin (NM)-containing dopaminergic cells and a profound reduction in striatal dopamine. It has been shown recently that antimelanin antibodies are increased in sera of Parkinson patients, suggesting that NM may act as an autoantigen. In this study we tested whether NM is being recognized by dendritic cells (DCs), the major cell type for inducing Tand B-cell responses in vivo. This recognition of NM by DCs is a prerequisite to trigger an adaptive autoimmune response directed against NM-associated structures. Results: Murine DCs were treated with NM of substantia nigra (SN) from human subjects or with synthetic dopamine melanin (DAM). DCs effectively phagocytized NM and subsequently developed a mature phenotype (CD86high/MHCIIhigh). NM-activated DCs secreted the proinflammatory cytokines IL-6 and TNF-a. In addition, they potently triggered T cell proliferation in a mixed lymphocyte reaction, showing that DC activation was functional to induce a primary T cell response. In contrast, DAM, which lacks the protein and lipid components of NM but mimics the dopamine-melanin backbone of NM, had only very little effect on DC phenotype and function. Conclusions: NM is recognized by DCs in vitro and triggers their maturation. If operative in vivo, this would allow the DC-mediated transport and presentation of SN antigens to the adaptive immune system, leading to autoimmmunity in susceptible individuals. Our data provide a rationale for an autoimmune-based pathomechanism of PD with NM as the initial trigger.
The pathogenic yeast Candida albicans can develop resistance to the widely used antifungal agent fluconazole, which inhibits ergosterol biosynthesis, by the overexpression of genes encoding multidrug efflux pumps or ergosterol biosynthesis enzymes. Zinc cluster transcription factors play a central role in the transcriptional regulation of drug resistance. Mrr1 regulates the expression of the major facilitator MDR1, Tac1 controls the expression of the ABC transporters CDR1 and CDR2, and Upc2 regulates ergosterol biosynthesis (ERG) genes. Gain-of-function mutations in these transcription factors result in constitutive overexpression of their target genes and are responsible for fluconazole resistance in many clinical C. albicans isolates. The transcription factor Ndt80 contributes to the drug-induced upregulation of CDR1 and ERG genes and also binds to the MDR1 and CDR2 promoters, suggesting that it is an important component of all major transcriptional mechanisms of fluconazole resistance. However, we found that Ndt80 is not required for the induction of MDR1 and CDR2 expression by inducing chemicals. CDR2 was even partially derepressed in ndt80D mutants, indicating that Ndt80 is a repressor of CDR2 expression. Hyperactive forms of Mrr1, Tac1, and Upc2 promoted overexpression of MDR1, CDR1/CDR2, and ERG11, respectively, with the same efficiency in the presence and absence of Ndt80. Mrr1- and Tac1-mediated fluconazole resistance was even slightly enhanced in ndt80D mutants compared to wild-type cells. These results demonstrate that Ndt80 is dispensable for the constitutive overexpression of Mrr1, Tac1, and Upc2 target genes and the increased fluconazole resistance of strains that have acquired activating mutations in these transcription factors.
Background: To perform a systematic review and meta-analysis on cytokine levels in patients with fibromyalgia syndrome (FMS). Methods: Through December 2010 we systematically reviewed the databases PubMed, MEDLINE, and PsycINFO and screened the reference lists of 22 review articles for suitable original articles. Original articles investigating cytokines in patients with FMS were included. Data were extracted by two independent authors. Differences of the cytokine levels of FMS patients and controls were summarized by standardized mean differences (SMD) using a random effects model. Study quality was assessed applying methodological scores: modified Center of Evidence Based Medicine, Newcastle-Ottawa-Scale, and Würzburg Methodological Quality Score. Results: Twenty-five articles were included investigating 1255 FMS patients and 800 healthy controls. Data of 13/25 studies entered meta-analysis. The overall methodological quality of studies was low. The results of the majority of studies were not comparable because methods, investigated material, and investigated target cytokines differed. Systematic review of the selected 25 articles revealed that FMS patients had higher serum levels of interleukin (IL)-1 receptor antagonist, IL-6, and IL-8, and higher plasma levels of IL-8. Meta-analysis of eligible studies showed that FMS patients had higher plasma IL-6 levels compared to controls (SMD = -0.34 [-0.64, -0.03] 95% CI; p = 0.03). The majority of investigated cytokines were not different between patients and controls. Conclusions: The pathophysiological role of cytokines in FMS is still unclear. Studies of higher quality and with higher numbers of subjects are needed.
Background: Hemostasis is a critical and active function of the blood mediated by platelets. Therefore, the prevention of pathological platelet aggregation is of great importance as well as of pharmaceutical and medical interest. Endogenous platelet inhibition is predominantly based on cyclic nucleotides (cAMP, cGMP) elevation and subsequent cyclic nucleotide-dependent protein kinase (PKA, PKG) activation. In turn, platelet phosphodiesterases (PDEs) and protein phosphatases counterbalance their activity. This main inhibitory pathway in human platelets is crucial for countervailing unwanted platelet activation. Consequently, the regulators of cyclic nucleotide signaling are of particular interest to pharmacology and therapeutics of atherothrombosis. Modeling of pharmacodynamics allows understanding this intricate signaling and supports the precise description of these pivotal targets for pharmacological modulation. Results: We modeled dynamically concentration-dependent responses of pathway effectors (inhibitors, activators, drug combinations) to cyclic nucleotide signaling as well as to downstream signaling events and verified resulting model predictions by experimental data. Experiments with various cAMP affecting compounds including antiplatelet drugs and their combinations revealed a high fidelity, fine-tuned cAMP signaling in platelets without crosstalk to the cGMP pathway. The model and the data provide evidence for two independent feedback loops: PKA, which is activated by elevated cAMP levels in the platelet, subsequently inhibits adenylyl cyclase (AC) but as well activates PDE3. By multi-experiment fitting, we established a comprehensive dynamic model with one predictive, optimized and validated set of parameters. Different pharmacological conditions (inhibition, activation, drug combinations, permanent and transient perturbations) are successfully tested and simulated, including statistical validation and sensitivity analysis. Downstream cyclic nucleotide signaling events target different phosphorylation sites for cAMP- and cGMP-dependent protein kinases (PKA, PKG) in the vasodilator-stimulated phosphoprotein (VASP). VASP phosphorylation as well as cAMP levels resulting from different drug strengths and combined stimulants were quantitatively modeled. These predictions were again experimentally validated. High sensitivity of the signaling pathway at low concentrations is involved in a fine-tuned balance as well as stable activation of this inhibitory cyclic nucleotide pathway. Conclusions: On the basis of experimental data, literature mining and database screening we established a dynamic in silico model of cyclic nucleotide signaling and probed its signaling sensitivity. Thoroughly validated, it successfully predicts drug combination effects on platelet function, including synergism, antagonism and regulatory loops.
Background: Data on complications in children with seasonal influenza virus infection are limited. We initiated a nation-wide three-year surveillance of children who were admitted to a paediatric intensive care unit (PICU) with severe seasonal influenza. Methods: From October 2005 to July 2008, active surveillance was performed using an established reporting system for rare diseases (ESPED) including all paediatric hospitals in Germany. Cases to be reported were hospitalized children < 17 years of age with laboratory-confirmed influenza treated in a PICU or dying in hospital. Results: Twenty severe influenza-associated cases were reported from 14 PICUs during three pre-pandemic influenza seasons (2005-2008). The median age of the patients (12 males/8 females) was 7.5 years (range 0.1-15 years). None had received vaccination against influenza. In 14 (70%) patients, the infection had been caused by influenza A and in five (25%) by influenza B; in one child (5%) the influenza type was not reported. Patients spent a median of 19 (IQR 12-38) days in the hospital and a median of 11 days (IQR 6-18 days) in the PICU; 10 (50%) needed mechanical ventilation. Most frequent diagnoses were influenza-associated pneumonia (60%), bronchitis / bronchiolitis (30%), encephalitis / encephalopathy (25%), secondary bacterial pneumonia (25%), and ARDS (25%). Eleven (55%) children had chronic underlying medical conditions, including 8 (40%) with chronic pulmonary diseases. Two influenza A- associated deaths were reported: i) an 8-year old boy with pneumococcal encephalopathy following influenza infection died from cerebral edema, ii) a 14-year-old boy with asthma bronchiale, cardiac malformation and Addison’s disease died from cardiac and respiratory failure. For nine (45%) patients, possibly permanent sequelae were reported (3 neurological, 3 pulmonary, 3 other sequelae). Conclusions: Influenza-associated pneumonia and secondary bacterial infections are relevant complications of seasonal influenza in Germany. The incidence of severe influenza cases in PICUs was relatively low. This may be either due to the weak to moderate seasonal influenza activity during the years 2005 to 2008 or due to underdiagnosis of influenza by physicians. Fifty % of the observed severe cases might have been prevented by following the recommendations for vaccination of risk groups in Germany.
Background: Gene function analysis of the obligate intracellular bacterium Chlamydia pneumoniae is hampered by the facts that this organism is inaccessible to genetic manipulations and not cultivable outside the host. The genomes of several strains have been sequenced; however, very little information is available on the gene structure and transcriptome of C. pneumoniae. Results: Using a differential RNA-sequencing approach with specific enrichment of primary transcripts, we defined the transcriptome of purified elementary bodies and reticulate bodies of C. pneumoniae strain CWL-029; 565 transcriptional start sites of annotated genes and novel transcripts were mapped. Analysis of adjacent genes for cotranscription revealed 246 polycistronic transcripts. In total, a distinct transcription start site or an affiliation to an operon could be assigned to 862 out of 1,074 annotated protein coding genes. Semi-quantitative analysis of mapped cDNA reads revealed significant differences for 288 genes in the RNA levels of genes isolated from elementary bodies and reticulate bodies. We have identified and in part confirmed 75 novel putative non-coding RNAs. The detailed map of transcription start sites at single nucleotide resolution allowed for the first time a comprehensive and saturating analysis of promoter consensus sequences in Chlamydia. Conclusions: The precise transcriptional landscape as a complement to the genome sequence will provide new insights into the organization, control and function of genes. Novel non-coding RNAs and identified common promoter motifs will help to understand gene regulation of this important human pathogen.
Background: “Negative affect” is one of the major migraine triggers. The aim of the study was to assess attentional biases for negative affective stimuli that might be related to migraine triggers in migraine patients with either few or frequent migraine and healthy controls. Methods: Thirty-three subjects with frequent migraine (FM) or with less frequent episodic migraine, and 20 healthy controls conducted two emotional Stroop tasks in the interictal period. In task 1, general affective words and in task 2, pictures of affective faces (angry, neutral, happy) were used. For each task we calculated two emotional Stroop indices. Groups were compared using one-way ANOVAs. Results: The expected attentional bias in migraine patients was not found. However, in task 2 the controls showed a significant attentional bias to negative faces, whereas the FM group showed indices near zero. Thus, the FM group responded faster to negative than to positive stimuli. The difference between the groups was statistically significant. Conclusions: The findings in the FM group may reflect a learned avoidance mechanism away from affective migraine triggers.
As pattern recognition receptor on dendritic cells (DCs), DC-SIGN binds carbohydrate structures on its pathogen ligands and essentially determines host pathogen interactions because it both skews T cell responses and enhances pathogen uptake for cis infection and/or T cell trans-infection. How these processes are initiated at the plasma membrane level is poorly understood. We now show that DC-SIGN ligation on DCs by antibodies, mannan or measles virus (MV) causes rapid activation of neutral and acid sphingomyelinases followed by accumulation of ceramides in the outer membrane leaflet. SMase activation is important in promoting DC-SIGN signaling, but also for enhancement of MV uptake into DCs. DCSIGN-dependent SMase activation induces efficient, transient recruitment of CD150, which functions both as MV uptake receptor and microbial sensor, from an intracellular Lamp-1+ storage compartment shared with acid sphingomyelinase (ASM) within a few minutes. Subsequently, CD150 is displayed at the cell surface and co-clusters with DC-SIGN. Thus, DCSIGN ligation initiates SMase-dependent formation of ceramide-enriched membrane microdomains which promote vertical segregation of CD150 from intracellular storage compartments along with ASM. Given the ability to promote receptor and signalosome co-segration into (or exclusion from) ceramide enriched microdomains which provide a favorable environment for membrane fusion, DC-SIGN-dependent SMase activation may be of general importance for modes and efficiency of pathogen uptake into DCs, and their routing to specific compartments, but also for modulating T cell responses.
Background: Successful cooperation depends on reliable identification of friends and foes. Social insects discriminate colony members (nestmates/friends) from foreign workers (non-nestmates/foes) by colony-specific, multi-component colony odors. Traditionally, complex processing in the brain has been regarded as crucial for colony recognition. Odor information is represented as spatial patterns of activity and processed in the primary olfactory neuropile, the antennal lobe (AL) of insects, which is analogous to the vertebrate olfactory bulb. Correlative evidence indicates that the spatial activity patterns reflect odor-quality, i.e., how an odor is perceived. For colony odors, alternatively, a sensory filter in the peripheral nervous system was suggested, causing specific anosmia to nestmate colony odors. Here, we investigate neuronal correlates of colony odors in the brain of a social insect to directly test whether they are anosmic to nestmate colony odors and whether spatial activity patterns in the AL can predict how odor qualities like ‘‘friend’’ and ‘‘foe’’ are attributed to colony odors. Methodology/Principal Findings: Using ant dummies that mimic natural conditions, we presented colony odors and investigated their neuronal representation in the ant Camponotus floridanus. Nestmate and non-nestmate colony odors elicited neuronal activity: In the periphery, we recorded sensory responses of olfactory receptor neurons (electroantennography), and in the brain, we measured colony odor specific spatial activity patterns in the AL (calcium imaging). Surprisingly, upon repeated stimulation with the same colony odor, spatial activity patterns were variable, and as variable as activity patterns elicited by different colony odors. Conclusions: Ants are not anosmic to nestmate colony odors. However, spatial activity patterns in the AL alone do not provide sufficient information for colony odor discrimination and this finding challenges the current notion of how odor quality is coded. Our result illustrates the enormous challenge for the nervous system to classify multi-component odors and indicates that other neuronal parameters, e.g., precise timing of neuronal activity, are likely necessary for attribution of odor quality to multi-component odors.
The diversity of species is striking, but can be far exceeded by the chemical diversity of compounds collected, produced or used by them. Here, we relate the specificity of plant-consumer interactions to chemical diversity applying a comparative network analysis to both levels. Chemical diversity was explored for interactions between tropical stingless bees and plant resins, which bees collect for nest construction and to deter predators and microbes. Resins also function as an environmental source for terpenes that serve as appeasement allomones and protection against predators when accumulated on the bees’ body surfaces. To unravel the origin of the bees’ complex chemical profiles, we investigated resin collection and the processing of resin-derived terpenes. We therefore analyzed chemical networks of tree resins, foraging networks of resin collecting bees, and their acquired chemical networks. We revealed that 113 terpenes in nests of six bee species and 83 on their body surfaces comprised a subset of the 1,117 compounds found in resins from seven tree species. Sesquiterpenes were the most variable class of terpenes. Albeit widely present in tree resins, they were only found on the body surface of some species, but entirely lacking in others. Moreover, whereas the nest profile of Tetragonula melanocephala contained sesquiterpenes, its surface profile did not. Stingless bees showed a generalized collecting behavior among resin sources, and only a hitherto undescribed species-specific ‘‘filtering’’ of resin-derived terpenes can explain the variation in chemical profiles of nests and body surfaces fromdifferent species. The tight relationship between bees and tree resins of a large variety of species elucidates why the bees’ surfaces contain a much higher chemodiversity than other hymenopterans.
Background: The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients ,25 years is representative for HIVDR in the rest of the therapy-naive population. Methods and Findings: HIVDR was determined in 88 sequentially enrolled ART-naive patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged, 25 years and 68 patients were aged 25–63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072–0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients .25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095–0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma. Conclusions: ART-naive patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naive population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naive HIV-infected population.
Background: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date. Methodology/Principal Findings: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5–8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53. Conclusions/Significance: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level.
Organic farming is one of the most successful agri-environmental schemes, as humans benefit from high quality food, farmers from higher prices for their products and it often successfully protects biodiversity. However there is little knowledge if organic farming also increases ecosystem services like pest control. We assessed 30 triticale fields (15 organic vs. 15 conventional) and recorded vascular plants, pollinators, aphids and their predators. Further, five conventional fields which were treated with insecticides were compared with 10 non-treated conventional fields. Organic fields had five times higher plant species richness and about twenty times higher pollinator species richness compared to conventional fields. Abundance of pollinators was even more than one-hundred times higher on organic fields. In contrast, the abundance of cereal aphids was five times lower in organic fields, while predator abundances were three times higher and predator-prey ratios twenty times higher in organic fields, indicating a significantly higher potential for biological pest control in organic fields. Insecticide treatment in conventional fields had only a short-term effect on aphid densities while later in the season aphid abundances were even higher and predator abundances lower in treated compared to untreated conventional fields. Our data indicate that insecticide treatment kept aphid predators at low abundances throughout the season, thereby significantly reducing top-down control of aphid populations. Plant and pollinator species richness as well as predator abundances and predator-prey ratios were higher at field edges compared to field centres, highlighting the importance of field edges for ecosystem services. In conclusion organic farming increases biodiversity, including important functional groups like plants, pollinators and predators which enhance natural pest control. Preventative insecticide application in conventional fields has only short-term effects on aphid densities but long-term negative effects on biological pest control. Therefore conventional farmers should restrict insecticide applications to situations where thresholds for pest densities are reached.
We investigate transport measurements on all II-VI semiconductor resonant tunneling diodes (RTDs). Being very versatile, the dilute magnetic semiconductor (DMS) system (Zn,Be,Mn,Cd)Se is a perfect testbed for various spintronic device designs, as it allows for separate control of electrical and magnetic properties. In contrast to the ferromagnetic semiconductor (Ga,Mn)As, doping ZnSe with Mn impurities does not alter the electrical properties of the semiconductor, as the magnetic dopant is isoelectric in the ZnSe host.
For the realization of a programmable logic device, or indeed any nanoscale device, we need a reliable method to probe the magnetization direction of local domains. For this purpose we extend investigations on the previously discovered tunneling anisotropic magneto resistance effect (TAMR) by scaling the pillar size from 100 µm down to 260 nm. We start in chapter 4 with a theoretical description of the TAMR effect and show experimental data of miniaturized pillars in chapter 5. With such small TAMR probes we are able to locally sense the magnetization on the 100 nm scale. Sub-micron TAMR and anisotropic magneto resistance (AMR) measurements of sub-millimeter areas show that the behavior of macroscopic (Ga,Mn)As regions is not that of a true macrospin, but rather an ensemble average of the behavior of many nearly identical macrospins. This shows that the magnetic anisotropies of the local regions are consistent with the behavior extracted from macroscopic characterization. A fully electrically controllable read-write memory device out the ferromagnetic semiconductor (Ga,Mn)As is presented in chapter 6. The structure consists of four nanobars which are connected to a circular center region. The first part of the chapter describes the lithography realization of the device. We make use of the sub-micron TAMR probes to read-out the magnetization state of a 650 nm central disk. Four 200 nm wide nanobars are connected to the central disk and serve as source and drain of a spin-polarized current. With the spin-polarized current we are able to switch the magnetization of the central disk by means of current induced switching. Injecting polarized holes with a spin angular momentum into a magnetic region changes the magnetization direction of the region due to the p-d exchange interaction between localized Mn spins and itinerant holes. The magnetization of the central disk can be controlled fully electrically and it can serve as one bit memory element as part of a logic device. In chapter 7 we discuss the domain wall resistance in (Ga,Mn)As. At the transition from nanobars to central disk we are able to generate 90° and 180° domain walls and measure their resistance. The results presented from chapter 5 to 7 combined with the preexisting ultracompact (Ga,Mn)As-based memory cell of ref. [Papp 07c] are the building blocks needed to realize a fully functioning programmable logic device. The work of ref. [Papp 07c] makes use of lithographically engineered strain relaxation to produce a structure comprised of two nanobars with mutually orthogonal uniaxial easy axes, connected by a narrow constriction. Measurements showed that the resistance of the constriction depends on the relative orientation of the magnetization in the two bars. The programmable logic device consists of two central disks connected by a small constriction. The magnetization of the two central disks are used as the input bits and the constriction serves as the output during the logic operation. The concept is introduced in the end of chapter 6 and as an example for a logic operation an XOR gate is presented. The functionality of the programmable logic scheme presented here can be straightforwardly extended to produce multipurpose functional elements, where the given geometry can be used as various different computational elements depending on the number of input bits and the chosen electrical addressing. The realization of such a programmable logic device is shown in chapter 8, where we see that the constriction indeed can serve as a output of the logic operation because its resistance is dependent on the relative magnetization state of both disks. Contrary to ref. [Papp 07c], where the individual magnetic elements connected to the constriction only have two non-volatile magnetic states, each disk in our scheme connected to the constriction has four non-volatile magnetic states. Switching the magnetization of a central disk with an electrical current does not only change the TAMR read-out of the respective disk, it also changes the resistance of the constriction. The resistance polar plot of the constriction maps the relative magnetization states of the individual disks. The presented device design serves as an all-electrical, all-semiconductor logic element. It combines a memory cell and data processing in a single monolithic paradigm.
Urinary tract infection (UTI) is one of the most serious health problems worldwide. It accounts for a million hospital visits annually in the United States. Among the many uropathogenic bacteria, uropathogenic Escherichia coli (UPEC) is the most common causative agent of UTI. However, not all E. coli that inhabit the urinary tract can cause UTI. Some of them thrive for long periods of time in the urinary bladder without causing overt symptoms of infection. This carrier state is called asymptomatic bacteriuria (ABU). E. coli ABU isolates can live in the host without inducing host response due to deletions, insertions and point mutations in the genome leading to the attenuation of virulence genes. They therefore behave in the same way as commensals. Since bacteria that inhabit the urinary tract are said to originate from the lower intestinal tract and ABU behave in a similar way as commensals, this study compared various phenotypic and genotypic characteristics of ABU and commensal E. coli fecal isolates. The two groups did not show a strict clustering with regards to phylogenetic lineage since there appears to be overlaps in their distribution in some clonal complexes. In addition, it was observed that the UPEC virulence genes were more frequently inactivated in ABU than in fecal isolates. Hence, ABU tend to have less functional virulence traits compared to the fecal isolates. The ABU model organism E. coli 83972 which is known not only for its commensal behavior in the urinary bladder but its ability to outcompete other bacteria in the urinary tract is currently being used as prophylactic treatment in patients who have recurrent episodes of UTI at the University Hospital in Lund, Sweden. The pilot studies showed that upon deliberate long-term colonization of the patients with E. coli 83972, they become protected from symptomatic UTI. In this study, the phenotypic and genotypic characteristics of eight re-isolates taken from initially asymptomatically colonized patients enrolled in the deliberate colonization study who reported an episode of symptoms during the colonization period were investigated. Two out of the eight re-isolates were proven to be a result of super infection by another uropathogen. Six re-isolates, on the other hand, were E. coli 83972. The urine re-isolates confirmed to be E. coli 83972 were phenotypically heterogeneous in that they varied in colony size as well as in swarming motility. Four of these re-isolates were morphologically homogenous and similar to the parent isolate E. coli 83972 whereas one of them appeared phenotypically heterogenous as a mixture of smaller and normal-sized colonies. Still another re-isolate phenotypically resembled small colony variants. Meanwhile, three of the six re-isolates did not differ from the parent isolate with regards to motility. On the other hand, three exhibited a markedly increased motility compared to the parent isolate. Transcriptome analysis demonstrated the upregulation of a cascade of genes involved in flagellar expression and biosynthesis in one of the three motile re-isolates. However, upon further investigation, it was found out that the expression of flagella had no effect on bacterial adhesion to host cells in vitro as well as to the induction of host inflammatory markers. Thus, this implies that the increased motility in the re-isolates is used by the bacteria as a fitness factor for its benefit and not as a virulence factor. In addition, among the various deregulated genes, it was observed that gene regulation tends to be host-specific in that there is no common pattern as to which genes are deregulated in the re-isolates. Taken together, results of this study therefore suggest that the use of E. coli 83972 for prophylactic treatment of symptomatic UTI remains to be very promising.
In this thesis I studied psychological aspects in the behaviour of Drosophila, and especially Drosophila larvae. After an introduction where I present the general scientific context and describe the mechanisms of olfactory perception as well as of classical and operant conditioning, I present the different experiments that I realised during my PhD. Perception The second chapter deals with the way adult Drosophila generalise between single odours and binary mixtures of odours. I found that flies perceive a mixture of two odours as equally similar to the two elements composing it; and that the intensity as well as the physico-chemical nature of the elements composing a mixture affect the degree of generalisation between this mixture and one of its elements. These findings now call for further investigation on the physiological level, using functional imaging. Memory The third chapter presents a series of experiments in Drosophila larvae in order to define some characteristics of a new protocol for classical aversive learning which involves associating odours with mechanical disturbance as a punishment. The protocol and the first results should open new doors for the study of classical conditioning in Drosophila larvae, by allowing the comparison between two types of aversive memory (gustatory vs. mechanical reinforcement), including a comparison of their neurogenetic bases. It will also allow enquiries into the question whether these respective memories are specific for the kind of reinforcer used. Agency The fourth chapter documents our attempts to establish operant memory in Drosophila larvae. By analysing the first moments of the test, I could reveal that the larvae modified their behaviour according to their previous operant training. However, this memory seems to be quickly extinguished during the course of the test. We now aim at repeating these results and improving the protocol, in order to be able to systematically study the mechanisms allowing and underlying operant learning in Drosophila larvae. In the fifth chapter, I use the methods developed in chapter four for an analysis of larval locomotion. I determine whether larval locomotion in terms of speed or angular speed is affected by a treatment with the “cognitive enhancer” Rhodiola rosea, or by mutations in the Synapsin or SAP47 genes which are involved in the formation of olfactory memory. I also characterize the modifications induced by the presence of gustatory stimuli in the substrate on which the larvae are crawling. This thesis thus brings new elements to the current knowledge of Drosophila
By the end of the year 2011, both the CMS and ATLAS experiments at the Large Hadron Collider have recorded around 5 inverse femtobarns of data at an energy of 7 TeV. There are only vague hints from the already analysed data towards new physics at the TeV scale. However, one knows that around this scale, new physics should show up so that theoretical issues of the standard model of particle physics can be cured. During the last decades, extensions to the standard model that are supposed to solve its problems have been constructed, and the corresponding phenomenology has been worked out. As soon as new physics is discovered, one has to deal with the problem of determining the nature of the underlying model. A first hint is of course given by the mass spectrum and quantum numbers such as electric and colour charges of the new particles. However, there are two popular model classes, supersymmetric models and extradimensional models, which can exhibit almost equal properties at the accessible energy range. Both introduce partners to the standard model particles with the same charges and thus one needs an extended discrimination method. From the origin of these partners arises a relevant difference: The partners constructed in extradimensional models have the same spin as their standard model partners while in Supersymmetry they differ by spin 1/2.\\ These different spins have an impact on the phenomenology of the two models. For example, one can exploit the fact that the total cross sections are affected, but this requires a very good knowledge of the couplings and masses involved. Another approach uses angular distributions depending on the particle spins. A prevailing method based on this idea uses the invariant mass distribution of the visible particles in decay chains. One can relate these distributions to the spin of the particle mediating the decay since it reflects itself in the highest power of the invariant mass $\sff$ of the adjacent particles. In this thesis we first study the influence of higher than dimension 4 operators on spin determination in such decay chains. We write down the relevant dimension 5 and 6 operators and calculate their contributions to the invariant mass distribution. We discuss how they affect the determination of spin and couplings.\\ We then address two scenarios which do not involve decay chains in the usual sense. In three body decays, the method pointed out above cannot be applied since it can only be used if the mediating particle is produced on-shell. For off-shell decays, which are important e.g. in split-Supersymmetry or split-Universal Extra Dimensions, the narrow width approximation cannot be made which previously led to the simple relation between spin and the highest power of $\sff$. We work out a strategy for these three body decays that can distinguish between the different spin scenarios. The method relies on the fact that the differential decay width $d\Gamma /d\sff$ can be rewritten in this limit as a global phase space function and a polynomial in $\sff$. The coefficients in this polynomial are functions of masses and couplings and we show that they have distinct signs or ratios depending on the spins involved in the decay. We test the strategy in a series of Monte Carlo studies and discuss the influence of the intermediate particle's mass. In the last part we consider a topology with very short decay chains. Again one cannot use the relation between spin and invariant mass. We investigate one variable that has been invented for the discrimination of Supersymmetry and Universal Extra Dimensions in the high energy limit which reduces the problem to the underlying production process. We show how this variable can also be used in new physics scenarios where the high energy limit is not a viable approximation. We include all possible spin scenarios with renormalizable interactions and study in detail the influence of the involved masses and couplings on the discrimination power of this variable. We find for example that the scenario containing the supersymmetric case is well distinguishable from most other spin scenarios.
Although age is one of the most salient and fundamental aspects of human faces, its processing in the brain has not yet been studied by any neuroimaging experiment. Automatic assessment of temporal changes across faces is a prerequisite to identifying persons over their life-span, and age per se is of biological and social relevance. Using a combination of evocative face morphs controlled for global optical flow and functional magnetic resonance imaging (fMRI), we segregate two areas that process changes of facial age in both hemispheres. These areas extend beyond the previously established face-sensitive network and are centered on the posterior inferior temporal sulcus (pITS) and the posterior angular gyrus (pANG), an evolutionarily new formation of the human brain. Using probabilistic tractography and by calculating spatial cross-correlations as well as creating minimum intersection maps between activation and connectivity patterns we demonstrate a hitherto unrecognized link between structure and function in the human brain on the basis of cognitive age processing. According to our results, implicit age processing involves the inferior temporal sulci and is, at the same time, closely tied to quantity decoding by the presumed neural systems devoted to magnitudes in the human parietal lobes. The ventral portion of Wernicke’s largely forgotten perpendicular association fasciculus is shown not only to interconnect these two areas but to relate to their activations, i.e. to transmit age-relevant information. In particular, post-hoc age-rating competence is shown to be associated with high response levels in the left angular gyrus. Cortical activation patterns related to changes of facial age differ from those previously elicited by other fixed as well as changeable face aspects such as gender (used for comparison), ethnicity and identity as well as eye gaze or facial expressions. We argue that this may be due to the fact that individual changes of facial age occur ontogenetically, unlike the instant changes of gaze direction or expressive content in faces that can be “mirrored” and require constant cognitive monitoring to follow. Discussing the ample evidence for distinct representations of quantitative age as opposed to categorical gender varied over continuous androgyny levels, we suggest that particular face-sensitive regions interact with additional object-unselective quantification modules to obtain individual estimates of facial age.
Understanding a complex network’s structure holds the key to understanding its function. The physics community has contributed a multitude of methods and analyses to this cross-disciplinary endeavor. Structural features exist on both the microscopic level, resulting from differences between single node properties, and the mesoscopic level resulting from properties shared by groups of nodes. Disentangling the determinants of network structure on these different scales has remained a major, and so far unsolved, challenge. Here we show how multiscale generative probabilistic exponential random graph models combined with efficient, distributive message-passing inference techniques can be used to achieve this separation of scales, leading to improved detection accuracy of latent classes as demonstrated on benchmark problems. It sheds new light on the statistical significance of motif-distributions in neural networks and improves the link-prediction accuracy as exemplified for gene-disease associations in the highly consequential Online Mendelian Inheritance in Man database.
Honeybee foragers frequently fly several kilometres to and from vital resources, and communicate those locations to their nest mates by a symbolic dance language. Research has shown that they achieve this feat by memorizing landmarks and the skyline panorama, using the sun and polarized skylight as compasses and by integrating their outbound flight paths. In order to investigate the capacity of the honeybees’ homing abilities, we artificially displaced foragers to novel release spots at various distances up to 13 km in the four cardinal directions. Returning bees were individually registered by a radio frequency identification (RFID) system at the hive entrance. We found that homing rate, homing speed and the maximum homing distance depend on the release direction. Bees released in the east were more likely to find their way back home, and returned faster than bees released in any other direction, due to the familiarity of global landmarks seen from the hive. Our findings suggest that such large scale homing is facilitated by global landmarks acting as beacons, and possibly the entire skyline panorama.
Control of host cell death is of paramount importance for the survival and replication of obligate intracellular bacteria. Among these, human pathogenic Chlamydia induces the inhibition of apoptosis in a variety of different host cells by directly interfering with cell death signaling. However, the evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. Here, we investigated the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway. Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.
Acellular Pertussis Booster in Adolescents Induces Th1 and Memory CD8+ T Cell Immune Response
(2011)
In a number of countries, whole cell pertussis vaccines (wcP) were replaced by acellular vaccines (aP) due to an improved reactogenicity profile. Pertussis immunization leads to specific antibody production with the help of CD4+ T cells. In earlier studies in infants and young children, wcP vaccines selectively induced a Th1 dominated immune response, whereas aP vaccines led to a Th2 biased response. To obtain data on Th1 or Th2 dominance of the immune response in adolescents receiving an aP booster immunization after a wcP or aP primary immunization, we analyzed the concentration of Th1 (IL-2, TNF-a, INF-c) and Th2 (IL-4, IL-5, IL-10) cytokines in supernatants of lymphocyte cultures specifically stimulated with pertussis antigens. We also investigated the presence of cytotoxic T cell responses against the facultative intracellular bacterium Bordetella pertussis by quantifying pertussis-specific CD8+ T cell activation following the aP booster immunization. Here we show that the adolescent aP booster vaccination predominantly leads to a Th1 immune response based on IFNgamma secretion upon stimulation with pertussis antigen, irrespective of a prior whole cell or acellular primary vaccination. The vaccination also induces an increase in peripheral CD8+CD69+ activated pertussis-specific memory T cells four weeks after vaccination. The Th1 bias of this immune response could play a role for the decreased local reactogenicity of this adolescent aP booster immunization when compared to the preceding childhood acellular pertussis booster. Pertussis-specific CD8+ memory T cells may contribute to protection against clinical pertussis.
No abstract avDendritic cells (DC) are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC) were infected with viable resting conidia of Aspergillus fumigatus (Af293) for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; .80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes.
Background: Acquisition of information about food sources is essential for animals that forage collectively like social insects. Foragers deliver two commodities to the nest, food and information, and they may favor the delivery of one at the expenses of the other. We predict that information needs should be particularly high at the beginning of foraging: the decision to return faster to the nest will motivate a grass-cutting ant worker to reduce its loading time, and so to leave the source with a partial load. Principal Findings: Field results showed that at the initial foraging phase, most grass-cutting ant foragers (Acromyrmex heyeri) returned unladen to the nest, and experienced head-on encounters with outgoing workers. Ant encounters were not simply collisions in a probabilistic sense: outgoing workers contacted in average 70% of the returning foragers at the initial foraging phase, and only 20% at the established phase. At the initial foraging phase, workers cut fragments that were shorter, narrower, lighter and tenderer than those harvested at the established one. Foragers walked at the initial phase significantly faster than expected for the observed temperatures, yet not at the established phase. Moreover, when controlling for differences in the fragment-size carried, workers still walked faster at the initial phase. Despite the higher speed, their individual transport rate of vegetable tissue was lower than that of similarly-sized workers foraging later at the same patch. Conclusions/Significance: At the initial foraging phase, workers compromised their individual transport rates of material in order to return faster to the colony. We suggest that the observed flexible cutting rules and the selection of partial loads at the beginning of foraging are driven by the need of information transfer, crucial for the establishment and maintenance of a foraging process to monopolize a discovered resource.
Background: Patterns that arise from an ecological process can be driven as much from the landscape over which the process is run as it is by some intrinsic properties of the process itself. The disentanglement of these effects is aided if it possible to run models of the process over artificial landscapes with controllable spatial properties. A number of different methods for the generation of so-called ‘neutral landscapes’ have been developed to provide just such a tool. Of these methods, a particular class that simulate fractional Brownian motion have shown particular promise. The existing methods of simulating fractional Brownian motion suffer from a number of problems however: they are often not easily generalisable to an arbitrary number of dimensions and produce outputs that can exhibit some undesirable artefacts. Methodology: We describe here an updated algorithm for the generation of neutral landscapes by fractional Brownian motion that do not display such undesirable properties. Using Monte Carlo simulation we assess the anisotropic properties of landscapes generated using the new algorithm described in this paper and compare it against a popular benchmark algorithm. Conclusion/Significance: The results show that the existing algorithm creates landscapes with values strongly correlated in the diagonal direction and that the new algorithm presented here corrects this artefact. A number of extensions of the algorithm described here are also highlighted: we describe how the algorithm can be employed to generate landscapes that display different properties in different dimensions and how they can be combined with an environmental gradient to produce landscapes that combine environmental variation at the local and macro scales.
A Candidate Approach Implicates the Secreted Salmonella Effector Protein SpvB in P-Body Disassembly
(2011)
P-bodies are dynamic aggregates of RNA and proteins involved in several post-transcriptional regulation processes. Pbodies have been shown to play important roles in regulating viral infection, whereas their interplay with bacterial pathogens, specifically intracellular bacteria that extensively manipulate host cell pathways, remains unknown. Here, we report that Salmonella infection induces P-body disassembly in a cell type-specific manner, and independently of previously characterized pathways such as inhibition of host cell RNA synthesis or microRNA-mediated gene silencing. We show that the Salmonella-induced P-body disassembly depends on the activation of the SPI-2 encoded type 3 secretion system, and that the secreted effector protein SpvB plays a major role in this process. P-body disruption is also induced by the related pathogen, Shigella flexneri, arguing that this might be a new mechanism by which intracellular bacterial pathogens subvert host cell function.
Fanconi Anemia Core Complex Gene Promoters Harbor Conserved Transcription Regulatory Elements
(2011)
The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 59 region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 39 regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-b and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.
In this thesis eight robust and reliable LC-MS/MS methods were developed and validated to analyze atorvastatin, clopidogrel, furosemide, itraconazole, loratadine, naproxen, nisoldipine and sunitinib in human plasma. The active metabolites 2-hydroxyatorvastatin, 4-hydroxyatorvastatin, hydroxyitraconazole, descarboethoxy-loratadine, 4-hydroxynisoldipine and N-desethylsunitinib were also included in the corresponding methods. Due to the different physical, chemical and pharmacokinetic properties of the analytes a wide spectrum regarding sample preparation techniques, chromatography and mass spectrometric detection was covered. Protein precipitation methods were developed for furosemide, itraconazole, naproxen, nisoldipine and sunitinib. Liquid-liquid extraction methods were developed for atorvastatin, clopidogrel and loratadine. Criteria to choose protein precipitation or liquid-liquid extraction were the final plasma concentrations of the drugs, which are mainly dependant on the dose, bioavailability and t1/2 and of course cost-effectiveness. Altogether, the methods have a concentration range from 0.001 ng/mL (LLOQ of clopidogrel) to 50000 ng/mL (highest calibration point for naproxen), covering 5 x 107 orders of magnitude. The runtime of the methods ranged from 2 to 4 minutes, facilitating a high sample throughput. All developed methods were validated according to recent guidelines as they were used to analyze sampes from clinical trials. Excellent linearity, intra-day and inter-day precision and accuracy were observed in the validated calibration ranges. Hemolyzed, lipemic and different batches of human plasma as well as sample dilution did not affect the determiantion of the analytes. Clopidogrel, loratadine, nisoldipine and sunitinib and if available their metabolites were subjected to a matrix effect test, resulting in no influence of different batches of human plasma on the analytical methods. Noteworthy is clopidogrel that shows a slight effect on one of the two used mass spectrometers. However, that effect was reproducible and did therefore not affect clopidogrel determination. No evidence of instability during chromatography, extraction and sample storage processes for all analytes except 4-hydroxyatorvastatin was found, for which a significant decrease was observed after three months. During incurred sample reanalysis of study samples 95 % of the samples were within ±15 % with respect to the first analysis. Moreover, the atorvastatin, loratadine and clopidogrel method were compared on two generations of triple quadrupole mass spectrometers, the API 3000™ and the API 5000™. The new ion source and the changes in the ion path of the API 5000™ provided higher sensitivity, the extend depending on the substance. However, the API 3000™ had very good precision in the performed system comparison. The validated methods showed excellent performance and quality data during routine sample analysis of eight clinical trials. Moreover, they are suitable for high sample throughput due to their short run times.
In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio.
Growth factor induced signaling cascades are key regulatory elements in tissue development, maintenance and regeneration. Deregulation of the cascades has severe consequences, leading to developmental disorders and neoplastic diseases. As a major function in signal transduction, activating mutations in RAF family kinases are the cause of many human cancers. In the first project described in this thesis we focused on B-RAF V600E that has been identified as the most prevalent B-RAF mutant in human cancer. In order to address the oncogenic function of B-RAF V600E, we have generated transgenic mice expressing the activated oncogene specifically in lung alveolar epithelial type II cells. Constitutive expression of B-RAF V600E caused abnormalities in alveolar epithelium formation that led to airspace enlargements. These lung lesions showed signs of tissue remodeling and were often associated with chronic inflammation and low incidence of lung tumors. Inflammatory cell infiltration did not precede the formation of emphysema-like lesions but was rather accompanied with late tumor development. These data support a model where the continuous regenerative process initiated by oncogenic B-RAF-driven alveolar disruption provides a tumor-promoting environment associated with chronic inflammation. In the second project we focused on wild type B-RAF and its role in an oncogenic-C-RAF driven mouse lung tumor model. Toward this aim we have generated compound mice in which we could conditionally deplete B-RAF in oncogenic-C-RAF driven lung tumors. Conditional elimination of B-RAF did not block lung tumor formation however led to reduced tumor growth. The diminished tumor growth was not caused by increased cell death instead was a consequence of reduced cell proliferation. Moreover, B-RAF ablation caused a reduction in the amplitude of the mitogenic signalling cascade. These data indicate that in vivo B-RAF is dispensable for the oncogenic potential of active C-RAF; however it cooperates with oncogenic C-RAF in the activation of the mitogenic cascade.
The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes.
Background: We evaluated the effect of insulin stimulation and dietary changes on myocardial, skeletal muscle and brain [18F]-fluorodeoxyglucose (FDG) kinetics and uptake in vivo in intact mice. Methods: Mice were anesthetized with isoflurane and imaged under different conditions: non-fasted (n = 7; "controls"), non-fasted with insulin (2 IU/kg body weight) injected subcutaneously immediately prior to FDG (n = 6), fasted (n = 5), and fasted with insulin injection (n = 5). A 60-min small-animal PET with serial blood sampling and kinetic modeling was performed. Results: We found comparable FDG standardized uptake values (SUVs) in myocardium in the non-fasted controls and non-fasted-insulin injected group (SUV 45-60 min, 9.58 ± 1.62 vs. 9.98 ± 2.44; p = 0.74), a lower myocardial SUV was noted in the fasted group (3.48 ± 1.73; p < 0.001). In contrast, the FDG uptake rate constant (Ki) for myocardium increased significantly by 47% in non-fasted mice by insulin (13.4 ± 3.9 ml/min/100 g vs. 19.8 ± 3.3 ml/min/100 g; p = 0.030); in fasted mice, a lower myocardial Ki as compared to controls was observed (3.3 ± 1.9 ml/min/100 g; p < 0.001). Skeletal muscle SUVs and Ki values were increased by insulin independent of dietary state, whereas in the brain, those parameters were not influenced by fasting or administration of insulin. Fasting led to a reduction in glucose metabolic rate in the myocardium (19.41 ± 5.39 vs. 3.26 ± 1.97 mg/min/100 g; p < 0.001), the skeletal muscle (1.06 ± 0.34 vs. 0.34 ± 0.08 mg/min/100 g; p = 0.001) but not the brain (3.21 ± 0.53 vs. 2.85 ± 0.25 mg/min/100 g; p = 0.19). Conclusions: Changes in organ SUVs, uptake rate constants and metabolic rates induced by fasting and insulin administration as observed in intact mice by small-animal PET imaging are consistent with those observed in isolated heart/muscle preparations and, more importantly, in vivo studies in larger animals and in humans. When assessing the effect of insulin on the myocardial glucose metabolism of non-fasted mice, it is not sufficient to just calculate the SUV - dynamic imaging with kinetic modeling is necessary.
Background: Genome wide association studies reported two single nucleotide polymorphisms in ANK3 (rs9804190 and rs10994336) as independent genetic risk factors for bipolar disorder. Another SNP in ANK3 (rs10761482) was associated with schizophrenia in a large European sample. Within the debate on common susceptibility genes for schizophrenia and bipolar disorder, we tried to investigate common findings by analyzing association of ANK3 with schizophrenia, bipolar disorder and unipolar depression. Methods: We genotyped three single nucleotide polymorphisms (SNPs) in ANK3 (rs9804190, rs10994336, and rs10761482) in a case-control sample of German descent including 920 patients with schizophrenia, 400 with bipolar affective disorder, 220 patients with unipolar depression according to ICD 10 and 480 healthy controls. Sample was further differentiated according to Leonhard’s classification featuring disease entities with specific combination of bipolar and psychotic syndromes. Results: We found no association of rs9804190 and rs10994336 with bipolar disorder, unipolar depression or schizophrenia. In contrast to previous findings rs10761482 was associated with bipolar disorder (p = 0.015) but not with schizophrenia or unipolar depression. We observed no association with disease entities according to Leonhard’s classification. Conclusion: Our results support a specific genetic contribution of ANK3 to bipolar disorder though we failed to replicate findings for schizophrenia. We cannot confirm ANK3 as a common risk factor for different diseases.
Varicella-zoster virus infections in immunocompromised patients - a single centre 6-years analysis
(2011)
Background: Infection with varicella-zoster virus (VZV) contemporaneously with malignant disease or immunosuppression represents a particular challenge and requires individualized decisions and treatment. Although the increasing use of varicella-vaccines in the general population and rapid initiation of VZVimmunoglobulins and acyclovir in case of exposure has been beneficial for some patients, immunocompromised individuals are still at risk for unfavourable courses. Methods: In this single center, 6-year analysis we review incidence, hospitalization and complication rates of VZVinfections in our center and compare them to published data. Furthermore, we report three instructive cases. Results: Hospitalization rate of referred children with VZV-infections was 45%, among these 17% with malignancies and 9% under immunosuppressive therapy. Rate of complications was not elevated in these two high-risk cohorts, but one ALL-patient died due to VZV-related complications. We report one 4-year old boy with initial diagnosis of acute lymphoblastic leukemia who showed a rapidly fatal outcome of his simultaneous varicella-infection, one 1.8-year old boy with an identical situation but a mild course of his disease, and an 8.5-year old boy with a steroiddependent nephrotic syndrome. This boy developed severe hepatic involvement during his varicella-infection but responded to immediate withdrawl of steroids and administration of acyclovir plus single-dose cidofovir after nonresponse to acyclovir after 48 h. Conclusion: Our data show that patients with malignant diseases or immunosuppressive therapy should be hospitalized and treated immediately with antiviral agents. Despite these measures the course of VZV-infections can be highly variable in these patients. We discuss aids to individual decision-making for these difficult situations.
Background: Neuropathic pain must be correctly diagnosed for optimal treatment. The questionnaire named Neuropathic Pain Symptom Inventory (NPSI) was developed in its original French version to evaluate the different symptoms of neuropathic pain. We hypothesized that the NPSI might also be used to differentiate neuropathic from non-neuropathic pain. Methods: We translated the NPSI into German using a standard forward-backward translation and administered it in a case-control design to patients with neuropathic (n = 68) and non-neuropathic pain (headache and osteoarthritis, n = 169) to validate it and to analyze its discriminant properties, its sensitivity to change, and to detect neuropathic pain subgroups with distinct profiles. Results: Using a sum score (the NPSI-G score), we found sensitivity to change (r between 0.37 and 0.5 for pain items of the graded chronic pain scale) and could distinguish between neuropathic and other pain on a group basis, but not for individual patients. Post hoc development of a discriminant score with optimized diagnostic properties to distinguish neuropathic pain from non-neuropathic pain resulted in an instrument with high sensitivity (91%) and acceptable specificity (70%). We detected six different pain profiles in the patient group with neuropathic pain; three profiles were found to be distinct. Conclusions: The NPSI-G potentially combines the properties of a diagnostic tool and an instrument to identify subtypes of neuropathic pain.
Background: Specific cell targeting is an important, yet unsolved problem in bacteria-based therapeutic applications, like tumor or gene therapy. Here, we describe the construction of a novel, internalin A and B (InlAB)-deficient Listeria monocytogenes strain (Lm-spa+), which expresses protein A of Staphylococcus aureus (SPA) and anchors SPA in the correct orientation on the bacterial cell surface. Results: This listerial strain efficiently binds antibodies allowing specific interaction of the bacterium with the target recognized by the antibody. Binding of Trastuzumab (Herceptin®) or Cetuximab (Erbitux®) to Lm-spa+, two clinically approved monoclonal antibodies directed against HER2/neu and EGFR/HER1, respectively, triggers InlABindependent internalization into non-phagocytic cancer cell lines overexpressing the respective receptors. Internalization, subsequent escape into the host cell cytosol and intracellular replication of these bacteria are as efficient as of the corresponding InlAB-positive, SPA-negative parental strain. This specific antibody/receptormediated internalization of Lm-spa+ is shown in the murine 4T1 tumor cell line, the isogenic 4T1-HER2 cell line as well as the human cancer cell lines SK-BR-3 and SK-OV-3. Importantly, this targeting approach is applicable in a xenograft mouse tumor model after crosslinking the antibody to SPA on the listerial cell surface. Conclusions: Binding of receptor-specific antibodies to SPA-expressing L. monocytogenes may represent a promising approach to target L. monocytogenes to host cells expressing specific receptors triggering internalization.
Background: In principle, the elimination of malignancies by oncolytic virotherapy could proceed by different mechanisms - e.g. tumor cell specific oncolysis, destruction of the tumor vasculature or an anti-tumoral immunological response. In this study, we analyzed the contribution of these factors to elucidate the responsible mechanism for regression of human breast tumor xenografts upon colonization with an attenuated vaccinia virus (VACV). Methods: Breast tumor xenografts were analyzed 6 weeks post VACV infection (p.i.; regression phase) by immunohistochemistry and mouse-specific expression arrays. Viral-mediated oncolysis was determined by tumor growth analysis combined with microscopic studies of intratumoral virus distribution. The tumor vasculature was morphologically characterized by diameter and density measurements and vessel functionality was analyzed by lectin perfusion and extravasation studies. Immunological aspects of viral-mediated tumor regression were studied in either immune-deficient mouse strains (T-, B-, NK-cell-deficient) or upon cyclophosphamide-induced immunosuppression (MHCII+-cell depletion) in nude mice. Results: Late stage VACV-infected breast tumors showed extensive necrosis, which was highly specific to cancer cells. The tumor vasculature in infected tumor areas remained functional and the endothelial cells were not infected. However, viral colonization triggers hyperpermeability and dilatation of the tumor vessels, which resembled the activated endothelium in wounded tissue. Moreover, we demonstrated an increased expression of genes involved in leukocyte-endothelial cell interaction in VACV-infected tumors, which orchestrate perivascular inflammatory cell infiltration. The immunohistochemical analysis of infected tumors displayed intense infiltration of MHCII-positive cells and colocalization of tumor vessels with MHCII+/CD31+ vascular leukocytes. However, GI-101A tumor growth analysis upon VACV-infection in either immunosuppressed nude mice (MHCII+-cell depleted) or in immune-deficient mouse strains (T-, B-, NK-cell-deficient) revealed that neither MHCII-positive immune cells nor T-, B-, or NK cells contributed significantly to VACV-mediated tumor regression. In contrast, tumors of immunosuppressed mice showed enhanced viral spreading and tumor necrosis. Conclusions: Taken together, these results indicate that VACV-mediated oncolysis is the primary mechanism of tumor shrinkage in the late regression phase. Neither the destruction of the tumor vasculature nor the massive VACV-mediated intratumoral inflammation was a prerequisite for tumor regression. We propose that approaches to enhance viral replication and spread within the tumor microenvironment should improve therapeutical outcome.
Characterisation of Mena Promoter Activity and Protein Expression in Wild-type and Gene-trapped Mice
(2011)
Proteins of the Ena/VASP protein family are important regulators of actin and participate in cell-cell and cell-matrix adhesions. To date, the physiological importance of Ena/VASP proteins for integrity of the cardiovascular system has remained unclear. To study cardiovascular functions of Mena and VASP, we used an established VASP knockout mouse in combination with a novel gene-trap-based model to ablate Mena function. In the mutated Mena mouse, the endogenous Mena gene is disrupted by the insertion of a β-galactosidase construct and β-galactosidase expression is under the control of the endogenous Mena promoter. X-gal staining of mouse organs revealed Mena promoter activity in smooth muscle layers of vessels, intestines and bronchioles, but also in cells of the brain, in cardiomyocytes and in the respiratory epithelium of bronchioles. In wild-type mice, Western blotting revealed differing protein expression patterns of VASP and Mena. Mena expression was observed in almost every tissue, predominantly in heart, lung, stomach, large intestine, testis, brain and eye. Additionally, the neuronalspecific Mena isoform was expressed in brain, eye, and slightly in heart and stomach. VASP protein, in contrast, was predominantly detected in spleen and thrombocytes. In gene-trapped mice, Mena expression was largely reduced in heart, lung and stomach but only slightly decreased in brain and testis. Immunofluorescence microscopy revealed colocalisation of Mena and F-actin at intercalated discs of cardiomyocytes and strong colocalisation of Mena and α- smooth-muscle-actin in vessels and bronchioles. Functional analysis of Mena/VASP-mutated and wild-type mice using electrocardiography suggested that the depletion of either Mena or VASP does not interfere with normal heart function. However, in double-deficient mice, the resting heart rate was significantly increased, probably reflecting a mechanism to compensate defects in ventricle contraction and to maintain a normal cardiac output. In agreement, cardiac catheter investigations suggested dilated cardiomyopathy in doubledeficient mice. Thus, although Western blot analysis showed differing protein expression patterns of Mena and VASP, these findings suggest that Mena and VASP mutually compensate for each other. Concerning Mena, we propose an important role of the protein in vessel walls, cardiomyocytes and bronchioles.
The Nucleotide Excision Repair (NER) pathway is able to remove a vast diversity of structurally unrelated DNA lesions and is the only repair mechanism in humans responsible for the excision of UV induced DNA damages. The NER mechanism raises two fundamental questions: 1) How is DNA damage recognition achieved discriminating damaged from non damaged DNA? 2) How is DNA incision regulated preventing endonucleases to cleave DNA non specifically but induce and ensure dual incision of damaged DNA? Thus, the aim of this work was to investigate the mechanisms leading from recognition to incision of damaged DNA. To decipher the underlying process of damage recognition in a prokaryotic model system, the intention of the first part of this work was to co crystallize the helicase UvrB form Bacillus caldotenax together with a DNA substrate comprising a fluorescein adducted thymine as an NER substrate. Incision assays were performed to address the question whether UvrB in complex with the endonuclease UvrC is able to specifically incise damaged DNA employing DNA substrates with unpaired regions at different positions with respect to the DNA lesion. The results presented here indicate that the formation of a specific pre incision complex is independent of the damage sensor UvrA. The preference for 5’ bubble substrate suggests that UvrB is able to slide along the DNA favorably in a 5’ → 3’ direction until it directly encounters a DNA damage on the translocating strand to then recruit the endonuclease UvrC. In the second part of this work, the novel endonuclease Bax1 from Thermoplasma acidophilum was characterized. Due to its close association to archaeal XPB, a potential involvement of Bax1 in archaeal NER has been postulated. Bax1 was shown to be a Mg2+ dependent, structure specific endonuclease incising 3’ overhang substrates in the single stranded region close to the ssDNA/dsDNA junction. Site directed mutagenesis of conserved amino acids was employed to identify putative active site residues of Bax1. In complex with the helicase XPB, however, incision activity of Bax1 is altered regarding substrate specificity. The presence of two distinct XPB/Bax1 complexes with different endonuclease activities indicates that XPB regulates Bax1 incision activity providing insights into the physical and functional interactions of XPB and Bax1.
It has been proposed that different features of a face provide a source of information for separate perceptual and cognitive processes. Properties of a face that remain rather stable over time, so called invariant facial features, yield information about a face’s identity, and changeable aspects of faces transmit information underlying social communication such as emotional expressions and speech movements. While processing of these different face properties was initially claimed to be independent, a growing body of evidence suggests that these sources of information can interact when people recognize faces with whom they are familiar. This is the case because the way a face moves can contain patterns that are characteristic for that specific person, so called idiosyncratic movements. As a face becomes familiar these idiosyncratic movements are learned and hence also provide information serving face identification. While an abundance of experiments has addressed the independence of invariant and variable facial features in face recognition, little is known about the exact nature of the impact idiosyncratic facial movements have on face recognition. Gaining knowledge about the way facial motion contributes to face recognition is, however, important for a deeper understanding of the way the brain processes and recognizes faces. In the following dissertation three experiments are reported that investigate the impact familiarity of changeable facial features has on processes of face recognition. Temporal aspects of the processing of familiar idiosyncratic facial motion were addressed in the first experiment via EEG by investigating the influence familiar facial movement exerts on event-related potentials associated to face processing and face recognition. After being familiarized with a face and its idiosyncratic movement, participants viewed familiar or unfamiliar faces with familiar or unfamiliar facial movement while their brain potentials were recorded. Results showed that familiarity of facial motion influenced later event-related potentials linked to memory processes involved in face recognition. The second experiment used fMRI to investigate the brain areas involved in processing familiar facial movement. Participants’ BOLD-signal was registered while they viewed familiar and unfamiliar faces with familiar or unfamiliar idiosyncratic movement. It was found that activity of brain regions, such as the fusiform gyrus, that underlie the processing of face identity, was modulated by familiar facial movement. Together these two experiments provide valuable information about the nature of the involvement of idiosyncratic facial movement in face recognition and have important implications for cognitive and neural models of face perception and recognition. The third experiment addressed the question whether idiosyncratic facial movement could increase individuation in perceiving faces from a different ethnic group and hence reduce impaired recognition of these other-race faces compared to own-race faces, a phenomenon named the own-race bias. European participants viewed European and African faces that were each animated with an idiosyncratic smile while their attention was either directed to the form or the motion of the face. Subsequently recognition memory for these faces was tested. Results showed that the own-race bias was equally present in both attention conditions indicating that idiosyncratic facial movement was not able to reduce or diminish the own-race bias. In combination the here presented experiments provide further insight into the involvement of idiosyncratic facial motion in face recognition. It is necessary to consider the dynamic component of faces when investigating face recognition because static facial images are not able to provide the full range of information that leads to recognition of a face. In order to reflect the full process of face recognition, cognitive and neural models of face perception and recognition need to integrate dynamic facial features as a source of information which contributes to the recognition of a face.
Polarity and migration are essential for T cell activation, homeostasis, recirculation and effector function. To address how T cells coordinate polarization and migration when interacting with dendritic cells (DC) during homeostatic and activating conditions, a low density collagen model was used for confocal live-cell imaging and high-resolution 3D reconstruction of fixed samples. During short-lived (5 to 15 min) and migratory homeostatic interactions, recently activated T cells simultaneously maintained their amoeboid polarization and polarized towards the DC. The resulting fully dynamic and asymmetrical interaction plane comprised all compartments of the migrating T cell: the actin-rich leading edge drove migration but displayed only moderate signaling activity; the mid-zone mediated TCR/MHC induced signals associated with homeostatic proliferation; and the rear uropod mediated predominantly MHC independent signals possibly connected to contact-dependent T cell survival. This “dynamic immunological synapse” with distinct signaling sectors enables moving T cells to serially sample antigen-presenting cells and resident tissue cells and thus to collect information along the way. In contrast to homeostatic contacts, recognition of the cognate antigen led to long-lasting T cell/DC interaction with T cell rounding, disintegration of the uropod, T cell polarization towards the DC, and the formation of a symmetrical contact plane. However, the polarity of the continuously migrating DC remained intact and T cells aggregated within the DC uropod, an interesting cellular compartment potentially involved in T cell activation and regulation of the immune response. Taken together, 3D collagen facilitates high resolution morphological studies of T cell function under realistic, in vivo-like conditions.
In this study I investigate the role of Schwann cell and axon-derived trophic signals as modifiers of axonal integrity and sprouting in motoneuron disease and diabetic neuropathy (DNP). The first part of this thesis focuses on the role of the Schwann-cell-derived ciliary neurotrophic factor (CNTF) for compensatory sprouting in a mouse model for mild spinal muscular atrophy (SMA). In the second part, the role of the insulin-like growth factor 1 (IGF-1) and its binding protein 5 (IGFBP-5) is examined in the peripheral nerves of patients with DNP and in two corresponding mouse models. Proximal SMA is caused by homozygous loss or mutation of the SMN1 gene on human chromosome 5. The different forms of SMA can be divided into four groups, depending on the levels of SMN protein produced from a second SMN gene (SMN2) and the severity of the disease. Patients with milder forms of the disease, type III and type IV SMA, normally reach adulthood and regularly show enlargement of motor units, signifying the reinnervation of denervated muscle fibers. However, the underlying mechanisms are not understood. Smn+/- mice, a model of type III/IV SMA, are phenotypically normal, but they reveal progressive loss of motor neurons and denervation of motor endplates starting at 4 weeks of age. The progressive loss of spinal motor neurons reaches 50% at 12 months but muscle strength is not reduced. The first evidence for axonal sprouting as a compensatory mechanism in these animals was the more than 2-fold increase in amplitude of single motor unit action potentials (SMUAP) in the gastrocnemius muscle. Confocal analysis confirmed pronounced sprouting of innervating motor axons. As CNTF is highly expressed in Schwann cells and known to be involved in sprouting, its role for this compensatory sprouting response and the maintenance of muscle strength in Smn+/- mice was investigated. Deletion of CNTF in this mouse model results in reduced sprouting and decline of muscle strength in Smn+/- Cntf-/- mice. These findings indicate that CNTF is necessary for a sprouting response and thus enhances the size of motor units in skeletal muscles of Smn+/- mice. DNP afflicting motor and sensory nerve fibers is a major complication in diabetes mellitus. The underlying cellular mechanisms of motor axon degeneration are poorly understood. IGFBP-5, an inhibitory binding protein for IGF-1, is highly upregulated in peripheral nerves in patients with DNP. The study investigates the pathogenic relevance of this finding in transgenic mice overexpressing IGFBP-5 in motor axons. These mice develop motor axonopathy similar to that seen in DNP. Motor axon degeneration is also observed in mice in which the IGF-1 receptor (IGF-1R) was conditionally depleted in motoneurons, indicating that reduced activity of IGF-1 on IGF-1R in motoneurons is responsible for the observed effect. These data provide evidence that elevated expression of IGFBP-5 in diabetic nerves reduces the availability of IGF-1 for IGF-1R on motor axons leading to progressive neurodegeneration, and thus offers novel treatment strategies.
Practical optimization problems often comprise several incomparable and conflicting objectives. When booking a trip using several means of transport, for instance, it should be fast and at the same time not too expensive. The first part of this thesis is concerned with the algorithmic solvability of such multiobjective optimization problems. Several solution notions are discussed and compared with respect to their difficulty. Interestingly, these solution notions are always equally difficulty for a single-objective problem and they differ considerably already for two objectives (unless P = NP). In this context, the difference between search and decision problems is also investigated in general. Furthermore, new and improved approximation algorithms for several variants of the traveling salesperson problem are presented. Using tools from discrepancy theory, a general technique is developed that helps to avoid an obstacle that is often hindering in multiobjective approximation: The problem of combining two solutions such that the new solution is balanced in all objectives and also mostly retains the structure of the original solutions. The second part of this thesis is dedicated to several aspects of systems of equations for (formal) languages. Firstly, conjunctive and Boolean grammars are studied, which are extensions of context-free grammars by explicit intersection and complementation operations, respectively. Among other results, it is shown that one can considerably restrict the union operation on conjunctive grammars without changing the generated language. Secondly, certain circuits are investigated whose gates do not compute Boolean values but sets of natural numbers. For these circuits, the equivalence problem is studied, i.\,e.\ the problem of deciding whether two given circuits compute the same set or not. It is shown that, depending on the allowed types of gates, this problem is complete for several different complexity classes and can thus be seen as a parametrized) representative for all those classes.
In recent years high-throughput experiments provided a vast amount of data from all areas of molecular biology, including genomics, transcriptomics, proteomics and metabolomics. Its analysis using bioinformatics methods has developed accordingly, towards a systematic approach to understand how genes and their resulting proteins give rise to biological form and function. They interact with each other and with other molecules in highly complex structures, which are explored in network biology. The in-depth knowledge of genes and proteins obtained from high-throughput experiments can be complemented by the architecture of molecular networks to gain a deeper understanding of biological processes. This thesis provides methods and statistical analyses for the integration of molecular data into biological networks and the identification of functional modules, as well as its application to distinct biological data. The integrated network approach is implemented as a software package, termed BioNet, for the statistical language R. The package includes the statistics for the integration of transcriptomic and functional data with biological networks, the scoring of nodes and edges of these networks as well as methods for subnetwork search and visualisation. The exact algorithm is extensively tested in a simulation study and outperforms existing heuristic methods for the calculation of this NP-hard problem in accuracy and robustness. The variability of the resulting solutions is assessed on perturbed data, mimicking random or biased factors that obscure the biological signal, generated for the integrated data and the network. An optimal, robust module can be calculated using a consensus approach, based on a resampling method. It summarizes optimally an ensemble of solutions in a robust consensus module with the estimated variability indicated by confidence values for the nodes and edges. The approach is subsequently applied to two gene expression data sets. The first application analyses gene expression data for acute lymphoblastic leukaemia (ALL) and differences between the subgroups with and without an oncogenic BCR/ABL gene fusion. In a second application gene expression and survival data from diffuse large B-cell lymphomas are examined. The identified modules include and extend already existing gene lists and signatures by further significant genes and their interactions. The most important novelty is that these genes are determined and visualised in the context of their interactions as a functional module and not as a list of independent and unrelated transcripts. In a third application the integrative network approach is used to trace changes in tardigrade metabolism to identify pathways responsible for their extreme resistance to environmental changes and endurance in an inactive tun state. For the first time a metabolic network approach is proposed to detect shifts in metabolic pathways, integrating transcriptome and metabolite data. Concluding, the presented integrated network approach is an adequate technique to unite high-throughput experimental data for single molecules and their intermolecular dependencies. It is flexible to apply on diverse data, ranging from gene expression changes over metabolite abundances to protein modifications in a combination with a suitable molecular network. The exact algorithm is accurate and robust in comparison to heuristic approaches and delivers an optimal, robust solution in form of a consensus module with confidence values. By the integration of diverse sources of information and a simultaneous inspection of a molecular event from different points of view, new and exhaustive insights into biological processes can be acquired.
Bacterial protein toxins belong to the most potent toxins which are known. They exist in many different forms and are part of our every day live. Some of them are spread by the bacteria during infections and therefore play a crucial role in pathogenicity of these strains. Others are secreted as a defense mechanism and could be uptaken with spoiled food. Concerning toxicity, some of the binary toxins of the AB7-type belong to the most potent and dangerous toxins in the world. Even very small amounts of these proteins are able to cause severe symptoms during an infection with pathogen species of the genus Clostridium or Bacillus. Apart from the thread the toxins constitute, they exhibit a unique way of intoxication. Members of the AB7-toxin family consist of a pore-forming subunit B, that acts as a molecular syringe to translocate the enzymatic moieties A into the cytosol of target cells. This complex mechanism does not only kill cells with high efficiency and therefore should be studied for treatment, but also displays a possibility to address certain cells with a specific protein cargo if used as a molecular delivery tool. Concerning both issues, binding and translocation of the channel are the crucial steps to either block or modify the system in the desired way. To gain deeper insight into the transport of binary toxins the structure of the B subunit is of great importance, but being a membrane protein, no crystal could be obtained up to now for either protective antigen (PA) of Anthrax toxin or any other AB7-type binding domain. Therefore, the method of choice in this work is an electro-physical approach using the so-called black-lipid-bilayer system for determination of biophysical constants. Additionally, diverse cell based assays serve as a proving method for the data gained during in vitro measurements. Further information was gathered with specially designed mutants of the protein channel. The first part of this thesis focuses on the translocation process and its possible use as a molecular tool to deliver protein cargo into special cell types. The task was addressed by measuring the binding of different effector proteins related and unrelated to the AB7 toxin family. These proteins were tested in titration experiments for the blockage of the ion current through a membrane saturated with toxin channels. Especially the influence of positively charged His-tags has been determined in detail for PA and C2II. As described in chapter 2, a His-tag transferred the ability of being transported by PA, but not by C2II, to different proteins like EDIN (from S. aureus) in vitro and in cell-based experiments. This process was found to change the well-known voltage-dependency of PA to a huge extend and therefore is related to membrane potentials which play a crucial role in many processes in living cells. Chapter 3 sums up findings, which depict that binding partners of PA share certain common motives. These could be detected in a broad range of substrates, ranging from simple ions in an electrolyte over small molecules to complex protein effectors. The gathered information could be further used to design blocker-substrates for treatment of Anthrax infections or tags, which render PA possible as a molecular syringe for cargo proteins. The deeper insight to homologies and differences of binary toxin components is the core of chapter 4, in which the cross-reactivity of Anthrax and C2-toxin was analyzed. The presented results lead to a better understanding of different motives involved in binding and translocation to and via the B components PA and C2II, as well as the enzymatically active A moieties edema factor (EF), lethal factor (LF) and C2I. In the second part of the thesis, the blockage of intoxication is the center of interest. Therefore, chapter 5 focuses on the analysis of specially designed blocker-substrate molecules for PA. These molecules form a plug in the pore, abolishing translocation of the enzymatic units. Especially, if multi-resistant strains of Anthrax (said to be already produced in Russia as a biological weapon) are taken into consideration, these substrates could stop intoxication and buy time, to deal with the infection. Chapter 6 describes the blockage of PA-channels by anti-His antibody from the trans-side of the porin, an effect which was not described for any other antibody before. Interestingly, even mutation of the estimated target amino acid Histidine 310 to Glycine could not interfere with this ionic strength dependent binding.
Currently, we observe a strong growth of services and applications, which use the Internet for data transport. However, the network requirements of these applications differ significantly. This makes network management difficult, since it complicated to separate network flows into application classes without inspecting application layer data. Network virtualization is a promising solution to this problem. It enables running different virtual network on the same physical substrate. Separating networks based on the service supported within allows controlling each network according to the specific needs of the application. The aim of such a network control is to optimize the user perceived quality as well as the cost efficiency of the data transport. Furthermore, network virtualization abstracts the network functionality from the underlying implementation and facilitates the split of the currently tightly integrated roles of Internet Service Provider and network owner. Additionally, network virtualization guarantees that different virtual networks run on the same physical substrate do not interfere with each other. This thesis discusses different aspects of the network virtualization topic. It is focused on how to manage and control a virtual network to guarantee the best Quality of Experience for the user. Therefore, a top-down approach is chosen. Starting with use cases of virtual networks, a possible architecture is derived and current implementation options based on hardware virtualization are explored. In the following, this thesis focuses on assessing the Quality of Experience perceived by the user and how it can be optimized on application layer. Furthermore, options for measuring and monitoring significant network parameters of virtual networks are considered.
The present work reviews the experimental literature on the acute effects of alcohol on human behaviour related to driving performance. A meta-analysis was conducted which includes studies published between 1954 and 2007 in order to provide a comprehensive knowledge of the substance alcohol. 450 studies reporting 5,300 findings were selected from over 12,000 references after applying certain in- and exclusion criteria. Thus, the present meta-analysis comprises far more studies than reviews on alcohol up to now. In the selected studies, different performance tests were conducted which were relevant for driving. The classification system used in this work assigns these tests to eight categories. The main categories consist of several sub categories classifying the tasks more precisely. The main categories were: (1) visual functions, (2) attention (including vigilance), (3) divided attention, (4) en-/decoding (including information processing and memory), (5) reaction time (including simple reaction time and choice reaction time), (6) psychomotor skills, (7) tracking and (8) driving. In addition to the performance aspect, the classification system takes into account mood and social behaviour variables related to driving safety like tiredness or aggression. Following the evaluation method of vote-counting, the number of significant findings and the number of non-significant findings were summarised per blood alcohol concentration (BAC) group. Thereby, a quantitative estimation of the effects of alcohol depending on the BAC was established, the so-called impairment function, which shows the percentage of significantly impaired findings. In order to provide a general overview of alcohol effects on driving-related performance, a global impairment function was established by aggregating all performance findings. The function is nearly linear with about 30% significant findings at a BAC of 0.05% and 50% significant findings at a BAC of 0.08%. In addition, more specific impairment functions considering only the findings of the single behavioural categories were calculated. The results revealed that impairment depends not only on the BAC, but also clearly differs between most of the performance categories. Tracking and driving performance were most affected by alcohol with impairment beginning at very low BACs of 0.02%. Also psychomotor skills were considerably affected by rather low BACs. Impairment of visual functions and information processing occurred at BACs of 0.04% and increased substantially with higher BACs. Impairment in memory tests could be found with very low BACs of 0.02%, but varied depending on the kind of memory. Performance decrements in divided attention tests could also be found with very low BACs in some studies. Attention started to be impaired at 0.04% BAC, but – as in vigilance tasks – considerable impairment only occurred at higher BACs. Choice reaction time was affected at lower BACs than simple reaction time, which was – together with the critical flicker fusion frequency – the least sensitive parameter to the effects of alcohol. To conclude, most skills which are relevant for the safe operation of a vehicle are clearly impaired by BACs of 0.05%, with motor functions being more affected than cognitive functions and complex tasks more than simple tasks. Generally, the results provided no evidence of a threshold effect for alcohol. There was no driving-related performance category for which a sudden transition from unimpaired to impaired occurred at a particular BAC level. In addition, a comparison was made between the present meta-analysis and two reviews of Moskowitz (Moskowitz & Fiorentino, 2000; Moskowitz & Robinson, 1988). Moskowitz reported much lower BACs at which performance was impaired. The reasons for this discrepancy lies in a different way to review scientific findings. On the one hand, Moskowitz focused on significant findings when selecting studies and findings for his reviews. On the other hand, the evaluation method used by Moskowitz ignored non-significant findings and counted each study once at the lowest BAC for which impairment was found. Those non-significant findings are as important as the significant ones in order to determine thresholds of impairment. Therefore, in contrast to Moskowitz, the present work describes the effects of alcohol with functions considering also the non-significant findings. The significance of the non-significant is emphasized with respect to the selection procedure as well as to the evaluation method.
There is such vast amount of visual information in our surroundings at any time that filtering out the important information for further processing is a basic requirement for any visual system. This is accomplished by deploying attention to focus on one source of sensory inputs to the exclusion of others (Luck and Mangun 2009). Attention has been studied extensively in humans and non human primates (NHPs). In Drosophila, visual attention was first demonstrated in 1980 (Wolf and Heisenberg 1980) but this field remained largely unexplored until recently. Lately, however, studies have emerged that hypothesize the role of attention in several behaviors but do not specify the characteristic properties of attention. So, the aim of this research was to characterize the phenomenon of visual attention in wild-type Drosophila, including both externally cued and covert attention using tethered flight at a torque meter. Development of systematic quantifiable behavioral tests was a key aspect for this which was not only important for analyzing the behavior of a population of wild-type flies but also for comparing the wild-type flies with mutant flies. The latter would help understand the molecular, genetic, and neuronal bases of attention. Since Drosophila provides handy genetic tools, a model of attention in Drosophila will serve to the greater questions about the neuronal circuitry and mechanisms involved which might be analogous to those in primates. Such a model might later be used in research involving disorders of attention. Attention can be guided to a certain location in the visual field by the use of external cues. Here, using visual cues the attention of the fly was directed to one or the other of the two visual half-fields. A simple yet robust paradigm was designed with which the results were easily quantifiable. This paradigm helped discover several interesting properties of the cued attention, the most substantial one being that this kind of external guidance of attention is restricted to the lower part of the fly’s visual field. The guiding cue had an after-effect, i.e. it could occur at least up to 2 seconds before the test and still bias it. The cue could also be spatially separated from the test by at least 20° and yet attract the attention although the extent of the focus of attention (FoA) was smaller than one lower visual half-field. These observations excluded the possibility of any kind of interference between the test and the cue stimuli. Another interesting observation was the essentiality of continuous visibility of the test stimulus but not the cue for effective cuing. When the contrast of the visual scene was inverted, differences in response frequencies and cuing effects were observed. Syndirectional yaw torque responses became more frequent than the antidirectional responses and cuing was no longer effective in the lower visual field with inverted contrast. Interestingly, the test stimulus with simultaneous displacement of two stripes not only effectuated a phasic yaw torque response but also a landing response. A 50 landing response was produced in more than half of the cases whenever a yaw torque response was produced. Elucidation of the neuronal correlates of the cued attention was commenced. Pilot experiments with hydroxyurea (HU) treated flies showed that mushroom bodies were not required for the kind of guidance of attention tested in this study. Dopamine mutants were also tested for the guidance of attention in the lower visual field. Surprisingly, TH-Gal4/UAS-shits1 flies flew like wild-type flies and also showed normal optomotor response during the initial calibration phase of the experiment but did not show any phasic yaw torque or landing response at 18 °C, 25 °C or 30 °C. dumb2 flies that have almost no D1 dopamine receptor dDA1 expression in the mushroom bodies and the central complex (Kim et al. 2007) were also tested and like THGal4/ UAS-shits1 flies did not show any phasic yaw torque or landing response. Since the dopamine mutants did not show the basic yaw torque response for the test the role of dopamine in attention could not be deduced. A different paradigm would be needed to test these mutants. Not only can attention be guided through external cues, it can also be shifted endogenously (covert attention). Experiments with the windows having oscillating stripes nicely demonstrated the phenomenon of covert attention due to the production of a characteristic yaw torque pattern by the flies. However, the results were not easily quantifiable and reproducible thereby calling for a more systematic approach. Experiments with simultaneous opposing displacements of two stripes provide a promising avenue as the results from these experiments showed that the flies had a higher tendency to deliver one type of response than when the responses would be produced stochastically suggesting that attention increased this tendency. Further experiments and analysis of such experiments could shed more light on the mechanisms of covert attention in flies.
In this thesis different algorithms for the solution of generalized Nash equilibrium problems with the focus on global convergence properties are developed. A globalized Newton method for the computation of normalized solutions, a nonsmooth algorithm based on an optimization reformulation of the game-theoretic problem, and a merit function approach and an interior point method for the solution of the concatenated Karush-Kuhn-Tucker-system are analyzed theoretically and numerically. The interior point method turns out to be one of the best existing methods for the solution of generalized Nash equilibrium problems.
The acquired immunodeficiency syndrome (AIDS) is currently the most infectious disease worldwide. It is caused by the human immunodeficiency virus (HIV). At the moment there are ~33.3 million people infected with HIV. Sub-Saharan Africa, with ~22.5 million people infected accounts for 68% of the global burden. In most African countries antiretroviral therapy (ART) is administered in limited-resource settings with standardised first- and second-line ART regimens. During this study I analysed the therapy-naïve population of Cape Town, South Africa and Mwanza, Tanzania for any resistance associated mutations (RAMs) against protease inhibitors, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors. My results indicate that HIV-1 subtype C accounts for ~95% of all circulating strains in Cape Town, South Africa. I could show that ~3.6% of the patient derived viruses had RAMs, despite patients being therapy-naïve. In Mwanza, Tanzania the HIV drug resistance (HIVDR) prevalence in the therapy-naïve population was 14.8% and significantly higher in the older population, >25 years. Therefore, the current WHO transmitted HIVDR (tHIVDR) survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Based on the prevalence rates of tHIVDR in the study populations it is recommended that all HIV-1 positive individuals undergo a genotyping resistance test before starting ART. I also characterized vif sequences from HIV-1 infected patients from Cape Town, South Africa as the Vif protein has been shown to counteract the antiretroviral activity of the cellular APOBEC3G/F cytidine deaminases. There is no selective pressure on the HIV-1 Vif protein from current ART regimens and vif sequences was used as an evolutionary control. As the majority of phenotypic resistance assays are still based on HIV-1 subtype B, I wanted to design an infectious HIV-1 subtype C proviral molecular clone that can be used for in vitro assays based on circulating strains in South Africa. Therefore, I characterized an early primary HIV-1 subtype C isolate from Cape Town, South Africa and created a new infectious subtype C proviral molecular clone (pZAC). The new pZAC virus has a significantly higher transient viral titer after transfection and replication rate than the previously published HIV-1 subtype C virus from Botswana. The optimized proviral molecular clone, pZAC could be used in future cell culture and phenotypic HIV resistance assays regarding HIV-1 subtype C.
Yersinia enterocolitica subsp. palearctica serobiotype O:3/4 comprises about 80-90 % of all human patient isolates in Germany and Europe and is responsible for sporadic cases worldwide. Even though this serobiotype is low pathogenic, Y. enterocolitica subsp. palearctica serobiotype O:3/4 is involved in gastroenteritis, lymphadenitis and various extraintestinal sequelae as reactive arthritis. The main animal reservoir of this serobiotype are pigs, causing a high rate of O:3/4 contaminations of raw pork in butcher shops in Germany (e.g. Bavaria 25 %) and countries in north-east Europe. As Y. enterocolitica O:3/4 is geographically and phylogenetically distinct from the so far sequenced mouse-virulent O:8/1B strain, complete genome sequencing has been performed for the European serobiotype O:3/4 DSMZ reference strain Y11, which has been isolated from a patient stool. To gain greater insight into the Y. enterocolitica subspecies palearctica group, also draft genome sequences of two other human O:3/4 isolates (strains Y8265, patient isolate, and Y5307, patient isolate associated with reactive arthritis), a closely related Y. enterocolitica palearctica serobiotype O:5,27/3 (strain Y527P), and two biotype 1A strains (a nosocomial strain of serogroup O:5 and an environmental serogroup O:36 isolate) have been performed. Those strains were compared to the high-pathogenic Y. enterocolitica subsp. enterocolitica serobiotype O:8/1B strain 8081 to address the peculiarities of the strain Y11 and the Y. enterocolitica subspecies palearctica group. The main focus was to unravel the pathogenic potential of strain Y11 and thus to identify novel putative virulence genes and fitness factors, especially those that may constitute host specificity of serobiotype O:3/4. Y. enterocolitica subspecies palearctica serobiotype O:3/4 strains lack most of the mouse-virulence-associated determinants of Y. enterocolitica subsp. enterocolitica serotype O:8, for example the HPI, Yts1 type 2 and Ysa type three secretion systems. In comparison, serobiotype O:3/4 strains obviously acquired a different set of genes and genomic islands for virulence and fitness such as the Ysp type three secretion system, an RtxA-like putative toxin, insecticidal toxins and a functional PTS system for N-acetyl-galactosamine uptake, named aga-operon. The aga-operon is able to support the growth of the Y. enterocolitica subsp. enterocolitica O:8/1B on N-acetyl-galactosamine after transformation with the aga operon. Besides these genes, also two prophages, PhiYep-2 and PhiYep-3, and a asn tRNA-associated GIYep-01 genomic island might influence the Y. enterocolitica subsp. palearctica serobiotype O:3/4 pathoadaptation. The PhiYep-3 prophage and the GIYep-01 island show recombination activity and PhiYep-3 was not found in all O:3/4 strains of a small strain collection tested. Y. enterocolitica subsp. palearctica serobiotype O:5,27/3 strain Y527P was found to be closely related to all serobiotype O:3/4 strains, whereas the biotype 1A isolates have more mosaic-segmented genomes and share putative virulence genes both with serobiotypes O:8/1B and O:3/4, which implies their common descent. Besides the pYV virulence plasmid, biotype 1A strains lack classical virulence markers as the Ail adhesin, the YstA enterotoxin, and the virulence-associated protein C. Interestingly, there are no notable differences between the known virulence factors present in nosocomial and environmental strains, except the presence of a truncated Rtx toxin-like gene cluster and remnants of a P2-like prophage in the hospital serogroup O:5 isolate.
Understanding of complex interactions and events in a nervous system, leading from the molecular level up to certain behavioural patterns calls for interdisciplinary interactions of various research areas. The goal of the presented work is to achieve such an interdisciplinary approach to study and manipulate animal behaviour and its underlying mechanisms. Optical in vivo imaging is a new constantly evolving method, allowing one to study not only the local but also wide reaching activity in the nervous system. Due to ease of its genetic accessibility Drosophila melanogaster represents an extraordinary experimental organism to utilize not only imaging but also various optogenetic techniques to study the neuronal underpinnings of behaviour. In this study four genetically encoded sensors were used to investigate the temporal dynamics of cAMP concentration changes in the horizontal lobes of the mushroom body, a brain area important for learning and memory, in response to various physiological and pharmacological stimuli. Several transgenic lines with various genomic insertion sites for the sensor constructs Epac1, Epac2, Epac2K390E and HCN2 were screened for the best signal quality, one line was selected for further experiments. The in vivo functionality of the sensor was assessed via pharmacological application of 8-bromo-cAMP as well as Forskolin, a substance stimulating cAMP producing adenylyl cyclases. This was followed by recording of the cAMP dynamics in response to the application of dopamine and octopamine, as well as to the presentation of electric shock, odorants or a simulated olfactory signal, induced by acetylcholine application to the observed brain area. In addition the interaction between the shock and the simulated olfactory signal by simultaneous presentation of both stimuli was studied. Preliminary results are supporting a coincidence detection mechanism at the level of the adenylyl cyclase as postulated by the present model for classical olfactory conditioning. In a second series of experiments an effort was made to selecticvely activate a subset of neurons via the optogenetic tool Channelrhodopsin (ChR2). This was achieved by recording the behaviour of the fly in a walking ball paradigm. A new method was developed to analyse the walking behaviour of the animal whose brain was made optically accessible via a dissection technique, as used for imaging, thus allowing one to target selected brain areas. Using the Gal4-UAS system the protocerebral bridge, a substructure of the central complex, was highlighted by expressing the ChR2 tagged by fluorescent protein EYFP. First behavioural recordings of such specially prepared animals were made. Lastly a new experimental paradigm for single animal conditioning was developed (Shock Box). Its design is based on the established Heat Box paradigm, however in addition to spatial and operant conditioning available in the Heat Box, the design of the new paradigm allows one to set up experiments to study classical and semioperant olfactory conditioning, as well as semioperant place learning and operant no idleness experiments. First experiments involving place learning were successfully performed in the new apparatus.
Indirect Search for Dark Matter in the Universe - the Multiwavelength and Multiobject Approach
(2011)
Cold dark matter constitutes a basic tenet of modern cosmology, essential for our understanding of structure formation in the Universe. Since its first discovery by means of spectroscopic observations of the dynamics of the Coma cluster some 80 years ago, mounting evidence of its gravitational pull and its impact on the geometry of space-time has build up across a wide range of scales, from galaxies to the entire Hubble flow. The apparent lack of electromagnetic coupling and independent measurements of the energy density of baryonic matter from the primordial abundances of light elements show the non-baryonic nature of dark matter, and its clustering properties prove that it is cold, i.e. that it has a temperature lower than its mass during the time of radiation-matter equality. A generic particle candidate for cold dark matter are weakly interacting massive particles at the electroweak symmetry-breaking scale, such as the neutralinos in R-parity conserving supersymmetry. Such particles would naturally freeze-out with a cosmologically relevant relic density at early times in the expanding Universe. Subsequent clustering of matter would recover annihilation interactions between the dark matter particles to some extent and thus lead to potentially observable high-energy emission from the decaying unstable secondaries produced in annihilation events. The spectra of the secondaries would permit a determination of the mass and annihilation cross section, which are crucial for the microphysical identification of the dark matter. This the central motivation for indirect dark matter searches. However, presently neither the indirect searches, nor the complementary direct searches based on the detection of elastic scattering events, nor the production of candidate particles in collider experiments, has yet provided unequivocal evidence for dark matter. This does not come as a surprise, since the dark matter particles interact only through weak interactions and therefore the corresponding secondary emission must be extremely faint. It turns out that even for the strongest mass concentrations in the Universe, the dark matter annihilation signal is expected to not exceed the level of competing astrophysical sources. Thus, the discrimination of the putative dark matter annihilation signal from the signals of the astrophysical inventory has become crucial for indirect search strategies. In this thesis, a novel search strategy will be developed and exemplified in which target selection across a wide range of masses, astrophysical background estimation, and multiwavelength signatures play the key role. It turns out that the uncertainties regarding the halo profile and the boost due to surviving substructure are bigger for halos at the lower end of the observed mass scales, i.e. in the regime of dwarf galaxies and below, while astrophysical backgrounds tend to become more severe for massive dark matter halos such as clusters of galaxies. By contrast, the uncertainties due to unknown details of particle physics are invariant under changes of the halo mass. Therefore, the different scaling behaviors can be employed to significantly cut down on the uncertainties in observations of different targets covering a major part of the involved mass scales. This strategical approach was implemented in the scientific program carried out with the MAGIC telescope system. Observations of dwarf galaxies and the Virgo- and Perseus clusters of galaxies have been carried out and, at the time of writing, result in some of the most stringent constraints on weakly interacting massive particles from indirect searches. Here, the low-threshold design of the MAGIC telescope system plays a crucial role, since the bulk of the high-energy photons, produced with a high multiplicity during the fragmentation of unstable dark matter annihilation products, are emitted at energies well below the dark matter mass scale. The upper limits severely constrain less generic, but more prolific scenarios characterized by extraordinarily high annihilation efficiencies.
During the last decades the standard model of particle physics has evolved to one of the most precise theories in physics, describing the properties and interactions of fundamental particles in various experiments with a high accuracy. However it lacks on some shortcomings from experimental as well as from theoretical point of view: There is no approved mechanism for the generation of masses of the fundamental particles, in particular also not for the light, but massive neutrinos. In addition the standard model does not provide an explanation for the observance of dark matter in the universe. Moreover the gauge couplings of the three forces in the standard model do not unify, implying that a fundamental theory combining all forces can not be formulated. Within this thesis we address supersymmetric models as answers to these various questions, but instead of focusing on the most simple supersymmetrization of the standard model, we consider basic extensions, namely the next-to-minimal supersymmetric standard model (NMSSM), which contains an additional singlet field, and R-parity violating models. R-parity is a discrete symmetry introduced to guarantee the stability of the proton. Using lepton number violating terms in the context of bilinear R-parity violation and the munuSSM we are able to explain neutrino physics intrinsically supersymmetric, since those terms induce a mixing between the neutralinos and the neutrinos. Since 2009 the Large Hadron Collider (LHC) at CERN explores the new energy regime of Tera-electronvolt, allowing the production of potentially existing heavy particles by the collision of protons. Thus the near future might provide answers to the open questions of mass generation in the standard model and show hints towards physics beyond the standard model. Therefore this thesis works out the phenomenology of the supersymmetric models under consideration and tries to point out differences to the well-known features of the simplest supersymmetric realization of the standard model. In case of the R-parity violating models the decays of the light neutralinos can result in displaced vertices. In combination with a light singlet state these displaced vertices might offer a rich phenomenology like non-standard Higgs decays into a pair of singlinos decaying with displaced vertices. Within this thesis we present some calculations at next order of perturbation theory, since one-loop corrections provide possibly large contributions to the tree-level masses and decay widths. We are using an on-shell renormalization scheme to calculate the masses of neutralinos and charginos including the neutrinos and leptons in case of the R-parity violating models at one-loop level. The discussion shows the similarities and differences to existing calculations in another renormalization scheme, namely the DRbar scheme. Moreover we consider two-body decays of the form chi_j^0 -> chi_l^\pm W^\mp involving a heavy gauge boson in the final state at one-loop level. Corrections are found to be large in case of small or vanishing tree-level decay widths and also for the R-parity violating decay of the lightest neutralino chi_1^0 -> l^\pm W^\mp. An interesting feature of the models based on bilinear R-parity violation is the correlation between the branching ratios of the lightest neutralino decays and the neutrino mixing angles. We discuss these relations at tree-level and for two-body decays chi_1^0 -> l^\pm W^\mp also at one-loop level, since only the full one-loop corrections result in the tree-level expected behavior. The appendix describes the two programs MaCoR and CNNDecays being developed for the analysis carried out in this thesis. MaCoR allows for the calculation of mass matrices and couplings in the models under consideration and CNNDecays is used for the one-loop calculations of neutralino and chargino mass matrices and the two-body decay widths.
Attention-deficit/hyperactivity disorder (ADHD) is a genetically complex childhood onset neurodevelopmental disorder which is highly persistent into adulthood. Several chromo-somal regions associated with this disorder were identified previously in genome-wide linkage scans, association (GWA) and copy number variation (CNV) studies. In this work the results of case-control and family-based association studies using a can-didate gene approach are presented. For this purpose, possible candidate genes for ADHD have been finemapped using mass array-based SNP genotyping. The genes KCNIP4, CDH13 and DIRAS2 have been found to be associated with ADHD and, in addition, with cluster B and cluster C personality disorders (PD) which are known to be related to ADHD. Most of the associations found in this work would not withstand correction for multiple testing. However, a replication in several independent populations has been achieved and in conjunction with previous evidence from linkage, GWA and CNV studies, it is assumed that there are true associations between those genes and ADHD. Further investigation of DIRAS2 by quantitative real-time PCR (qPCR) revealed expression in the hippocampus, cerebral cortex and cerebellum of the human brain and a significant increase in Diras2 expression in the mouse brain during early development. In situ hybrid-izations on murine brain slices confirmed the results gained by qPCR in the human brain. Moreover, Diras2 is expressed in the basolateral amygdala, structures of the olfactory system and several other brain regions which have been implicated in the psychopatholo-gy of ADHD. In conclusion, the results of this work provide further support to the existence of a strong genetic component in the pathophysiology of ADHD and related disorders. KCNIP4, CDH13 and DIRAS2 are promising candidates and need to be further examined to get more knowledge about the neurobiological basis of this common disease. This knowledge is essential for understanding the molecular mechanisms underlying the emergence of this disorder and for the development of new treatment strategies.
The scope of the present work encompasses the influence of experience (i.e. expertise) for feature processing in unconscious information processing. In the introduction, I describe the subliminal priming paradigm, a method to examine how stimuli, we are not aware of, nonetheless influence our actions. The activation of semantic response categories, the impact of learned stimulus-response links, and the action triggering through programmed stimulus-response links are the main three hypotheses to explain unconscious response activation. Besides, the congruence of perceptual features can also influence subliminal priming. On the basis of the features location and form, I look at evidence that exists so far for perceptual priming. The second part of the introduction reviews the literature showing perceptual superiority of experts. This is illustrated exemplarily with three domains of expertise – playing action video games, which constitutes a general form of perceptual expertise, radiology, a more natural form of expertise, and expertise in the game of chess, which is seen as the Drosophila of psychology. In the empirical section, I report nine experiments that applied a subliminal check detection task. Experiment 1 shows subliminal response priming for chess experts but not for chess novices. Thus, chess experts are able to judge unconsciously presented chess configurations as checking or nonchecking. The results of Experiment 2 suggest that acquired perceptual chunks, and not the ability to integrate perceptual features unconsciously, was responsible for unconscious check detection, because experts’ priming does not occur for simpler chess configurations which afforded an unfamiliar classification. With a more complex chess detection task, Experiment 3 indicates that chess experts are not able to process perceptual features in parallel or alternatively, that chess experts are not able to form specific expectations which are obviously necessary to elicit priming if many chess displays are applied. The aim of Experiment 4-9 was to further elaborate on unconscious processing of the single features location and form in novices. In Experiment 4 and 5, perceptual priming according the congruence of the single features location and form outperformed semantically-based response priming. Experiment 6 and 7 show that (in contrast to form priming) the observed location priming effect is rather robust and is also evident for an unexpected form or colour. In Experiment 8, location and form priming, which was additionally related to response priming, were directly compared to each other. Location priming was again stronger than form priming. Finally, Experiment 9 demonstrates that with the subliminal check detection task it is possible to induce response priming in novices when the confounding influences of location and form are absent. In the General discussion, I first summarized the findings. Second, I discuss possible underlying mechanisms of different subliminal perception in experts and novices. Third, I focus on subliminal perceptual priming in novices, especially on the impact of the features location and form. And finally, I discuss a framework, the action trigger account that integrates the different results of the present work.
At the present day the idea of cosmological inflation constitutes an important extension of Big Bang theory. Since its appearance in the early 1980’s many physical mechanisms have been worked out that put the inflationary expansion of space that proceeds the Hot Big Bang on a sound theoretical basis. Among the achievements of the theory of inflation are the explanaition of the almost Euclidean geometry of ‘visible’space, the homogeneity of the cosmic background radiation but, in particular, also the tiny inhomogeneity of a relative amplitude of 10−5. In many models of inflation the inflationary phase ends only locally. Hence, there exists the possibility that the inflationary process still goes on in regions beyond our visual horizon. This property is commonly termed ‘eternal inflation’. In the framework of a cosmological scalar fields, eternal inflation can manifest itself in a variety of ways. On the one hand fluctuations of the field, if sufficiently large, can work against the classical trajectory and therefore counteract the end of inflation. In regions where this is the case the accelerated expansion of space continues at a higher rate. In parts of this region the process may replicate itself again and in this way may continue throughout all of time. Space and field are said to reproduce themselves. On the other hand, a mechanism that can occur in addition or independent of the latter, is so called vacuum tunneling. If the potential of the scalar field has several local minima, a semi-classical calculation suggests that within a spherical region, a bubble, the field can tunnel to another state. The respective tunneling rates depend on the potential difference and the shape of the potential between the states. Generally, the tunneling rate is exponentially suppressed, which means that the inflation lasts for a long time before tunneling takes place. The ongoing inflationary process effectively reduces local curvature, anistotropy and inhomogeneity, so that this property is known as the ‘cosmic no-hair conjecture’. For this reason cosmological considerations of the evolution of bubbles thus far almost entirely involved vacuum (de Sitter) backgrounds. However, new insights in the framework of string theory suggest high tunneling rates which allow for the possibility of bubble nucleation in non-vacuum dominated backgrounds. In this case the evolution of the bubble depends on the properties of the background spacetime. A deeper introduction in chapter 4 is followed by the presentation of the Lemaître-Tolman spacetime in chapter 5 which constitutes the background spacetime in the study of the effect of matter and inhomogeneity on the evolution of vacuum bubbles. In chapter 6 we explicitly describe the application of the ‘thin-shell’ formalism and the resulting system of equations. This is succeeded in chapter 7 by the detailed analysis of bubble evolution in various limits of the Lemaître-Tolman spacetime and a Robertson-Walker spacetime with a rapid phase transition. The central observations are that the presence of dust, at a fixed surface energy density, goes along with a smaller nucleation volume and possibly leads to a a collapse of the bubble. In an expanding background, the radially inhomogeneous dust profile is efficiently diluted so that there is essentially no effect on the evolution of the domain wall. This changes in a radially inhomogeneous curvature profile, positive curvature decelerates the expansion of the bubble. Moreover, we point out that the adopted approach does not allow for a treatment of a, physically expected, matter transfer so that the results are to be understood as preliminary under this caveat. In the second part of this thesis we consider potential observable consequences of bubble collisions in the cosmic microwave background radiation. The topological nature of the signal suggests the use of statistics that are well suited to quantify the morphological properties of the temperature fluctuations. In chapter 10 we present Minkowski Functionals (MFs) that exactly provide such statistics. The presented error analysis allows for a higher precision of numerical MFs in comparison to earlier methods. In chapter 12 we present the application of our algorithm to a Gaussian and a collision map. We motivate the expected MFs and extract their numerical counterparts. We find that our least-squares fitting procedure accurately reproduces an underlying signal only when a large number of realizations of maps are averaged over, while for a single WMAP and PLANCK resolution map, only when a highly prominent disk, with |δT| = 2√σG and ϑd = 40◦, we are able to recover the result. This is unfortunate, as it means that MF are intrinsically too noisy to be able to distinguish cold and hot spots in the CMB for small sizes.
Die Chlorophylle stellen in der Natur die wichtigsten Pigmente dar, weil sie verantwortlich für die Photosynthese sind und hierbei vielfältige Funktionen wahrnehmen, die sich aus ihrer Selbstassemblierung sowie den vorteilhaften optischen und Redox-Eigenschaften ergeben. Die in dieser Arbeit untersuchten semisynthetischen Zinkchlorine stellen Modellverbindungen des natürlichen Bacteriochlorophylls c (BChl c) der Lichtsammelsysteme (light-harvesting: LH) in Chlorosomen von Bakterien, jedoch ohne Proteingerüst, dar. Die entscheidenden Vorteile dieser Zinkchlorine (ZnChl) gegenüber den natürlichen BChls bestehen im einfachen semisynthetischen Zugang ausgehend von Chlorophyll a (Chl a), ihrer gesteigerten chemischen Stabilität sowie der Möglichkeit ihre Selbstassemblierung durch gezielte chemische Modifizierung der Seitenketten in der Peripherie zu steuern. Während bereits mehrfach über die vielversprechenden Redox- und excitonischen Eigenschaften von Aggregaten von ZnChl und natürlichem BChl c und den damit verbundene Voraussetzungen für Excitontransport über große Distanzen berichtet wurde, sind die Ladungstransporteigenschaften von Aggregaten der biomimetischen ZnChl bis heute unerforscht. Die vorliegende Arbeit beschäftigt sich mit der Aufklärung der Struktur von Aggregaten einer Vielzahl von semisynthetischen Zinkchlorophyllderivaten im Feststoff, in Lösung und auf Oberflächen durch die Kombination verschiedenster spektroskopischer, kristallographischer und mikroskopischer Techniken an die sich Untersuchungen zum Ladungstransport in den Aggregaten anschließen. Schema 1 zeigt die verschiedenen, in dieser Arbeit synthetisierten ZnChls, die entweder mit einer Hydroxy- oder Methoxygruppe in der 31-Position funktionalisiert sind sowie Substituenten unterschiedlicher Art, Länge und Verzweigung an der Benzylestergruppe in 172-Position tragen.Die Packung dieser Farbstoffe hängt entscheidend von ihrer chemischen Struktur ab. Während die ZnChls 1a, 2a, 3 mit 31-Hydroxygruppe und Alkylseitenketten (Dodecyl bzw. Oligoethylenglykol) gut lösliche stabförmige Aggregate bilden, lagern sich die analogen Verbindungen mit 31-Methoxygruppe (1b, 2b) zu Stapeln in Lösung und auf Oberflächen zusammen. Diese supramolekularen Polymere wurden im Detail in Kapitel 3 mit Hilfe von UV/Vis- und CD-Spektroskopie (circular dichroism: CD) sowie dynamische Lichtstreuung (dynamic light scattering: DLS) untersucht. Darüber hinaus lieferten temperaturabhängige UV/Vis- in Kombination mit DLS-Messungen wertvolle Informationen über die Aggregationsprozess dieser beiden Sorten von Aggregaten. Während sich die ZnChl 1a mit 31 Hydroxygruppe entsprechend dem isodesmischen Modell zu röhrenförmigen Aggregaten zusammenlagern, bilden sich die stapelförmigen Aggregate von 1b nach einem kooperativen Keimbildungs-Wachstums-Mechanismus (nucleation-elongation mechanism). Detaillierte elektronenmikroskopische Studien lieferten erstmals überzeugende Beweise für röhrenförmige Nanostrukturen der Aggregate des wasserlöslichen 31-Hydroxy Zinkchlorin 3. Die gemessenen Durchmesser der Röhren von ~ 5-6 nm dieser Aggregate liegen in hervorragender Übereinstimmung mit den Elektronenmikroskopie-Daten von BChl c Stabaggregaten in Chlorosomen (Chloroflexus aurantiacus, Durchmesser ~ 5-6 nm) und entsprechen damit dem von Holzwarth und Schaffner postulierten röhrenförmigen Modell... Im Einklang mit ihren hoch geordneten, robusten Strukturen, die sich eindimensional in einer Größenordnung von Mikrometeren erstrecken, sowie ihrer Fähigkeit zum effizienten Ladungs-trägertransport stellen diese selbstassemblierten Nanoröhren von ZnChls vielversprechende Ausgangsmaterialien für die Fertigung supramolekularer elektronischer Bauteile dar. Wissenschaftliche Bemühungen einige dieser Moleküle und ihre entsprechenden supramolekularen Polymere für die Fertigung von (opto-)elektronischen Bauteilen wie organischen Feldeffekttransistoren zu benutzten, stellen lohnende Aufgaben für die Zukunft dar...