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In this thesis, the influence of an environment on molecules and, in particular, on the quantum control of such systems is investigated. Different approaches to describe system-bath dynamics are implemented and applied. The inclusion of a dissipation term in the system Hamiltonian leads to energy loss and relaxation to the ground state. As a first application, the isomerisation reaction in an aromatic complex is treated. It is shown that this simple model is able to reproduce results of time-resolved spectroscopic measurements. Next, the influence of noise is investigated. The incorporation of fluctuations reveals that energy is not conserved and coherences are destroyed. As an example, the quantum control of a population transfer in Na2 is examined. The efficiency of control processes is studied in dependence on the strength of the noise and different system-bath couplings. Starting with the unperturbed system, Local Control Theory is applied to construct a field which selectively transfers population into a single excited electronic state. The coupling to the bath is then switched on to monitor the dependence of the coupling strength on the transfer efficiency. The perturbation of the bath effects the Na2 molecule in such a way that potential energy curves and transition dipole moments are distorted. An important result is that already elastic collisions lead to a substantial loss of control efficiency. The most promising approach used in this thesis is the stochastic Schrödinger equation. It is equivalent to the commonly employed descriptions of system-bath dynamics within the reduced density matrix formalism. It includes decoherences and dissipation caused by elastic and inelastic collisions. Our contribution is the incorporation of laser excitation into the kinetic Monte-Carlo scheme. Thus we are able to apply this stochastic approach to the quantum control of population transfer in the sodium dimer. Because within our description it is possible to separate pure dephasing, inelastic transitions, and coherent time-evolution, we can identify the relative influence of these processes on the control efficiency. This leads to a far more physical picture of the basic processes underlying the perturbations of an environment then what a reduced density matrix description can provide. In utilising the stochastic wave function approach instead of the density matrix formalism, the computations are quite efficient. The stochastic Schrödinger equation is realised by N independent runs, where, in our case, an ensemble size of N = 1000 gives converged results. The efficiency of the laser control process is studied as a function of temperature and collision rates. A rise in temperature (or collision rate) reeffects a stronger fluctuation and thus results in a less efficient transfer by the control field. Though the Gaussian fluctuations used here do not strictly represent 'white'- noise, since a deterministic machine is not able to produce uncorrelated random numbers, an acceptable distribution is achieved by simple procedures. An improvement of the here applied algorithms would, for instance, include a more sophisticated sampling of the dephasing rates. Only one example of a control process is studied here and an application of the developed approach to other problems of quantum control is to be performed. This thesis established a systematic approach to understand quantum control in the presence of an environment.
In the context of this dissertation very long ranged exciton diffusion lengths (LD) were simulated for perylene-based materials under ideal conditions. This leads to the conclusion that the short LD values in existing materials result from an extrinsic and intrinsic immobilization. The latter, which is a specific material property, is based on a relaxation of the exciton into self-trapping states. An in-depth understanding of the atomistic processes defining self-trapping is essential to developing materials with long LD in the future, in which intrinsic immobilization is prevented. For the development of such a mechanistic understanding it is crucial that a clear relationship between molecular structure and LD is available. This is given by single crystals of diindeno perylene (DIP) and α-perylene tetracarboxylic anhydride (α-PTCDA). An extraordinary large LD of 90 nm was measured for the first one, while the latter possesses only 22 nm. Part of this thesis was to deliver reasons for this discrepancy. Only self-trapping comes into question to explain the different LD values. One reason for the different self-trapping in DIP and α-PTCDA could lie in the electronic structure. However, it was possible to demonstrate that a wide range of perylene-based materials possess no significant differences in their electronic structures. Consequently, such differences can be neglected for the explanation of immobilization mechanisms for the exciton. A further possible explanation could be polarization effects in the crystal, which influences the electronic structure of perylene based materials differently. Especially their influence on charge transfer (CT) states, which are located above the optically bright Frenkel state, was in question because such states could be stabilized by a polarizable surrounding. A significant influence of polarization effects on all considered states were excluded by using a polarizable continuum model. Hence, the small LD values in α-PTCDA are an evidence for self-trapping, which produces a crystal structure built up by π-stacks, while the one of DIP is of herringbone type. Since polarization effects can be neglected, is the dimer only via steric restrictions influenced by the crystal. Hence, a method describing self-trapping has to consider such effects, so that a mechanical embedding QM/MM approach is sufficient. Now, potential energy surfaces were calculated, on which wave packet dynamics were subsequently performed. In this way, atomistic mechanisms for the immobilization of excitons were described for the first time in organic materials. Self-trapping was studied in crystals of α-PTCDA by potential energy surfaces, which map an intermolecular shift motion of the dimer in the crystal. An immobilization of excitons occurs within 500 fs, which results from an irreversible energy loss together with a local deformation of the crystal lattice. This prevents a further transport of the exciton. In the case of DIP, this immobilization does not proceed due to high barriers. These barriers result from the herringbone type packing motif in the DIP crystal. This discrepancy in the dynamics explains the different LD values in DIP and α-PTCDA. In a further example, an exciton immobilization was found in helical π-aggregates of perylene tetracarboxylic bisimide (PBI) molecules. Self-trapping is caused by a relaxation mechanism, in which the exciton is transferred by asymmetric vibrations of the aggregate from the bright to a dark Frenkel state within 200 fs, whereby the transition is mediated by a CT state. However, the CT state is almost non-populated during the whole mechanism so that its participation could not yet be proven experimentally. This entire procedure is solely possible in helical aggregates, because only for such structures is there a CT state located next to the bright Frenkel state. At the final Frenkel state a torsional motion around the π-stacking axis is possible so that the loss in energy and the local rearrangement of the aggregate structure occurs, which means a self-trapping of the exciton. This mechanism is in perfect agreement with all available experimental data. These insights allow the conclusion that in future materials for organic solar cells an irreversible and ultrafast deformation of aggregates after photo-absorption must be avoided. Only in this way long LD values can be achieved and exciton self-trapping can be prevented. However, small LD values are always predicted in helical aggregates of perylene-based materials, because exciton immobilization occurs already due to small molecular motions. For this reason such aggregates are inappropriate for the use in organic solar cells. Long LD values are expected for aggregate structures with long intermolecular shifts or molecules with bulky substituents.
The inheritance of the histone H3 variant CENP-A in nucleosomes at centromeres following DNA replication is mediated by an epigenetic mechanism. To understand the process of epigenetic inheritance, or propagation of histones and histone variants, as nucleosomes are disassembled and reassembled in living eukaryotic cells, we have explored the feasibility of exploiting photo-activated localization microscopy (PALM). PALM of single molecules in living cells has the potential to reveal new concepts in cell biology, providing insights into stochastic variation in cellular states. However, thus far, its use has been limited to studies in bacteria or to processes occurring near the surface of eukaryotic cells. With PALM, one literally observes and 'counts' individual molecules in cells one-by-one and this allows the recording of images with a resolution higher than that determined by the diffraction of light (the so-called super-resolution microscopy). Here, we investigate the use of different fluorophores and develop procedures to count the centromere-specific histone H3 variant CENP-A\(^{Cnp1}\) with single-molecule sensitivity in fission yeast (Schizosaccharomyces pombe). The results obtained are validated by and compared with ChIP-seq analyses. Using this approach, CENP-A\(^{Cnp1}\) levels at fission yeast (S. pombe) centromeres were followed as they change during the cell cycle. Our measurements show that CENP-A(Cnp1) is deposited solely during the G2 phase of the cell cycle.
Background: The European Centres of Reference Network for Cystic Fibrosis (ECORN-CF) established an Internet forum which provides the opportunity for CF patients and other interested people to ask experts questions about CF in their mother language. The objectives of this study were to: 1) develop a detailed quality assessment tool to analyze quality of expert answers, 2) evaluate the intra- and inter-rater agreement of this tool, and 3) explore changes in the quality of expert answers over the time frame of the project. Methods: The quality assessment tool was developed by an expert panel. Five experts within the ECORN-CF project used the quality assessment tool to analyze the quality of 108 expert answers published on ECORN-CF from six language zones. 25 expert answers were scored at two time points, one year apart. Quality of answers was also assessed at an early and later period of the project. Individual rater scores and group mean scores were analyzed for each expert answer. Results: A scoring system and training manual were developed analyzing two quality categories of answers: content and formal quality. For content quality, the grades based on group mean scores for all raters showed substantial agreement between two time points, however this was not the case for the grades based on individual rater scores. For formal quality the grades based on group mean scores showed only slight agreement between two time points and there was also poor agreement between time points for the individual grades. The inter-rater agreement for content quality was fair (mean kappa value 0.232 ± 0.036, p < 0.001) while only slight agreement was observed for the grades of the formal quality (mean kappa value 0.105 ± 0.024, p < 0.001). The quality of expert answers was rated high (four language zones) or satisfactory (two language zones) and did not change over time. Conclusions: The quality assessment tool described in this study was feasible and reliable when content quality was assessed by a group of raters. Within ECORN-CF, the tool will help ensure that CF patients all over Europe have equal possibility of access to high quality expert advice on their illness.
We present the complete threshold enhanced predictions in QCD for the total cross section of gluino pair production at hadron colliders at next-to-next-to-leading order. Thanks to the computation of the required one-loop hard matching coefficients our results are accurate to the next-to-next-to-leading logarithm. In a brief phenomenological study we provide predictions for the total hadronic cross sections at the LHC and we discuss the uncertainties arising from scale variations and the parton distribution functions.
For many practical purposes, it is convenient to formulate unbroken non-abelian gauge theories like QCD in a color-flow basis. We present a new derivation of SU(N) interactions in the color-flow basis by extending the gauge group to U(N) × U(1)′ in such a way that the two U(1) factors cancel each other. We use the quantum action principles to show the equivalence to the usual basis to all orders in perturbation theory. We extend the known Feynman rules to exotic color representations (e.g. sextets) and discuss practical applications as they occur in automatic computation programs.
PTPN22 encodes the lymphoid tyrosine phosphatase Lyp that can dephosphorylate Lck, ZAP-70 and Fyn to attenuate TCR signaling. A single-nucleotide polymorphism (C1858T) causes a substitution from arginine (R) to tryptophan (W) at 620 residue (R620W). Lyp-620W has been confirmed as a susceptible allele in multiple autoimmune diseases, including type 1 diabetes (T1D). Several independent studies proposed that the disease-associated allele is a gain-of-function variant. However, a recent report found that in human cells and a knockin mouse containing the R620W homolog that Ptpn22 protein degradation is accelerated, indicating Lyp-620W is a loss-of-function variant. Whether Lyp R620W is a gain- or loss-of-function variant remains controversial. To resolve this issue, we generated two lines (P2 and P4) of nonobese diabetic (NOD) mice in which Ptpn22 can be inducibly silenced by RNAi. We found long term silencing of Ptpn22 increased spleen cellularity and regulatory T (Treg) cell numbers, replicating the effect of gene deletion reported in the knockout (KO) B6 mice. Notably, Ptpn22 silencing also increased the reactivity and apoptotic behavior of B lymphocytes, which is consistent with the reduced reactivity and apoptosis of human B cells carrying the alleged gain-of-function PTPN22 allele. Furthermore, loss of Ptpn22 protected P2 KD mice from spontaneous and Cyclophosphamide (CY) induced diabetes. Our data support the notion that Lyp-620W is a gain-of-function variant. Moreover, Lyp may be a valuable target for the treatment of autoimmune diseases.
Protection of healthy tissues from infection with systemically administered vaccinia virus strains
(2012)
Oncolytic virotherapy using recombinant vaccinia virus strains is a promising approach for the treatment of cancer. To further improve the safety of oncolytic vaccinia viruses, the cellular microRNA machinery can be applied as the host’s own security mechanism to avoid unwanted viral replication in healthy tissues. MicroRNAs are a class of small single-stranded RNAs which due to their ability to mediate post-transcriptional gene-silencing, play a crucial role in almost every regulatory process in cellular metabolism. Different cancers display unique microRNA expression patterns, showing significant up- or downregulation of endogenously expressed microRNAs. Furthermore, the behavior of cancer cells can be altered by either adding microRNAs known to inhibit cancer cell spread and proliferation or suppressing cancer promoting microRNAs (oncomirs) making microRNAs promising targets for cancer gene therapy. The cell’s own RNAi machinery can also be utilized to control viral replication due to the virus dependence on the host cell replication machinery, a process controlled by microRNAs. GLV-1h68 is a replication-competent recombinant oncolytic vaccinia virus constructed and generated by Genelux Corp., San Diego, CA, USA which carries insertions of three reporter gene cassettes for detection and attenuation purposes and is currently being evaluated for cancer treatment in clinical trials. Though there are hardly any side effects found in GLV-1h68 mediated oncolytic therapy an increased tropism for replication exclusively in cancer cells is desirable. Therefore it was investigated whether or not further cancer cell specificity of a recombinant vaccinia virus strain could be obtained without compromising its oncolytic activity using microRNA interference. Let-7a is a well characterized microRNA known to be expressed in high levels in healthy tissues and strongly downregulated in most cancers. To control vaccinia virus replication rates, four copies of the mature human microRNA let-7a target sequence were cloned behind the stop codon in the 3’end of the vaccinia virus D4R gene, using a GLV-1h68 derivative, GLV-1h190, as parental strain yielding the new recombinant virus strain GLV-1h250. The D4R gene belongs to the group of early transcribed vaccinia genes and encodes an essential enzyme, uracil DNA glycosylase, which catalyzes the removal of uracil residues from double-stranded DNA. A defect in D4R prevents vaccinia virus from entering into the intermediate and late phase of replication, leading to an aborted virus replication. After expression of the microRNA target sequence from the vaccinia virus genome, the endogenously expressed microRNA-let-7a should recognize its target structure within the viral mRNA transcript, thereby binding and degrading the viral mRNA which should lead to a strong inhibition of the virus replication in healthy cells. GLV-1h250 replication rates in cancerous A549 lung adenocarcinoma cells, which show a strong down-regulation of microRNA let-7a, was comparable to the replication rates of its parental strain GLV-1h190 and the control strain GLV-1h68. In contrast, GLV-1h250 displayed a 10-fold decrease in viral replication in non-cancerous ERC cells when compared to GLV-1h190 and GLV-1h68. In A549 tumor bearing nude mice GLV-1h250 replicated exclusively in the tumorous tissue and resulted in efficient tumor regression without adverse effects leading to the conclusion that GLV-1h250 replicates preferentially in cancerous cells and tissues, which display low endogenous let-7a expression levels.
Background: Recently published results of quality of life (QoL) studies indicated different outcomes of palliative radiotherapy for brain metastases. This prospective multi-center QoL study of patients with brain metastases was designed to investigate which QoL domains improve or worsen after palliative radiotherapy and which might provide prognostic information.
Methods: From 01/2007-01/2009, n=151 patients with previously untreated brain metastases were recruited at 14 centers in Germany and Austria. Most patients (82 %) received whole-brain radiotherapy. QoL was measured with the EORTC-QLQ-C15-PAL and brain module BN20 before the start of radiotherapy and after 3 months.
Results: At 3 months, 88/142 (62 %) survived. Nine patients were not able to be followed up. 62 patients (70.5 % of 3-month survivors) completed the second set of questionnaires. Three months after the start of radiotherapy QoL deteriorated significantly in the areas of global QoL, physical function, fatigue, nausea, pain, appetite loss, hair loss, drowsiness, motor dysfunction, communication deficit and weakness of legs. Although the use of corticosteroid at 3 months could be reduced compared to pre-treatment (63 % vs. 37 %), the score for headaches remained stable. Initial QoL at the start of treatment was better in those alive than in those deceased at 3 months, significantly for physical function, motor dysfunction and the symptom scales fatigue, pain, appetite loss and weakness of legs. In a multivariate model, lower Karnofsky performance score, higher age and higher pain ratings before radiotherapy were prognostic of 3-month survival.
Conclusions: Moderate deterioration in several QoL domains was predominantly observed three months after start of palliative radiotherapy for brain metastases. Future studies will need to address the individual subjective benefit or burden from such treatment. Baseline QoL scores before palliative radiotherapy for brain metastases may contain prognostic information.
Basing on the assumption that frontotemporal lobar degeneration (FTLD), schizophrenia and bipolar disorder (BPD) might share common aetiological mechanisms, we analyzed genetic variation in the FTLD risk gene progranulin (GRN) in a German population of patients with schizophrenia (n=271) or BPD (n=237) as compared with 574 age-, gender-and ethnicity-matched controls. Furthermore, we measured plasma progranulin levels in 26 German BPD patients as well as in 61 Italian BPD patients and 29 matched controls. A significantly decreased allelic frequency of the minor versus the wild-type allele was observed for rs2879096 (23.2 versus 34.2%, P<0.001, OR: 0.63, 95% CI: 0.49-0.80), rs4792938 (30.7 versus 39.7%, P=0.005, OR: 0.70, 95% CI: 0.55-0.89) and rs5848 (30.3 versus 36.8, P=0.007, OR: 0.71, 95% CI: 0.56-0.91). Mean +/- SEM progranulin plasma levels were significantly decreased in BPD patients, either Germans or Italians, as compared with controls (89.69 +/- 3.97 and 116.14 +/- 5.80 ng/ml, respectively, versus 180.81 +/- 18.39 ng/ml P<0.001) and were not correlated with age. In conclusion, GRN variability decreases the risk to develop BPD and schizophrenia, and progranulin plasma levels are significantly lower in BPD patients than in controls. Nevertheless, a larger replication analysis would be needed to confirm these preliminary results.
The objective of this thesis focuses on the development of strategies for precise control of perylene bisimide (PBI) self-assembly and the in-depth elucidation of structural and optical features of discrete PBI aggregates by means of NMR and UV/Vis spectroscopy. The strategy for discrete dimer formation of PBIs is based on delicate steric control that distinguishes the two facets of the central perylene surface. The strategy applied in this thesis for accessing discrete PBI quadruple and further oligomeric stacks relies on backbone-directed PBI self-assembly. For this purpose, two tweezer-like PBI dyads bearing the respective rigid backbones, diphenylacetylene (DPA) and diphenylbutydiyne (DPB), were synthesized. The distinct aggregation behavior of these structurally similar PBI dyads can be ascribed to the intramolecular distance between the two PBI chromophores imparted by the DPA and DPB spacers.
Background
Empirical studies investigating the prevalence of mental disorders and psychological distress in cancer patients have gained increasing importance during recent years, particularly with the objective to develop and implement psychosocial interventions within the cancer care system. Primary purpose of this epidemiological cross-sectional multi-center study is to detect the 4-week-, 12-month-, and lifetime prevalence rates of comorbid mental disorders and to further assess psychological distress and psychosocial support needs in cancer patients across all major tumor entities within the in- and outpatient oncological health care and rehabilitation settings in Germany.
Methods/Design
In this multicenter, epidemiological cross-sectional study, cancer patients across all major tumor entities will be enrolled from acute care hospitals, outpatient cancer care facilities, and rehabilitation centers in five major study centers in Germany: Freiburg, Hamburg, Heidelberg, Leipzig and Würzburg. A proportional stratified random sample based on the nationwide incidence of all cancer diagnoses in Germany is used. Patients are consecutively recruited in all centers. On the basis of a depression screener (PHQ-9) 50% of the participants that score below the cutoff point of 9 and all patients scoring above are assessed using the Composite International Diagnostic Interview for Oncology (CIDI-O). In addition, all patients complete validated questionnaires measuring emotional distress, information and psychosocial support needs as well as quality of life.
Discussion
Epidemiological data on the prevalence of mental disorders and distress provide detailed and valid information for the estimation of the demands for the type and extent of psychosocial support interventions. The data will provide information about specific demographic, functional, cancer- and treatment-related risk factors for mental comorbidity and psychosocial distress, specific supportive care needs and use of psychosocial support offers.
A new flavanone-chromone biflavonoid, preussianone (1), has been isolated from the leaves of Garcinia preussii, along with four known biflavonoids. The absolute stereostructures were elucidated by chemical, spectroscopic, and chiroptical methods. The biological properties of the new biflavonoid against several bacterial strains were evaluated.
Aims/hypothesis
Several glucose-sensing pathways have been implicated in glucose-triggered secretion of glucagon-like peptide-1 (GLP-1) from intestinal L cells. One involves glucose metabolism and closure of ATP-sensitive K\(^+\) channels, and another exploits the electrogenic nature of Na\(^+\)-coupled glucose transporters (SGLTs). This study aimed to elucidate the role of these distinct mechanisms in glucose-stimulated GLP-1 secretion.
Methods
Glucose uptake into L cells (either GLUTag cells or cells in primary cultures, using a new transgenic mouse model combining proglucagon promoter-driven Cre recombinase with a ROSA26tdRFP reporter) was monitored with the FLII\(_{12}\)Pglu-700μδ6 glucose sensor. Effects of pharmacological and genetic interference with SGLT1 or facilitative glucose transport (GLUT) on intracellular glucose accumulation and metabolism (measured by NAD(P)H autofluorescence), cytosolic Ca\(^{2+}\) (monitored with Fura2) and GLP-1 secretion (assayed by ELISA) were assessed.
Results
L cell glucose uptake was dominated by GLUT-mediated transport, being abolished by phloretin but not phloridzin. NAD(P)H autofluorescence was glucose dependent and enhanced by a glucokinase activator. In GLUTag cells, but not primary L cells, phloretin partially impaired glucose-dependent secretion, and suppressed an amplifying effect of glucose under depolarising high K\(^+\) conditions. The key importance of SGLT1 in GLUTag and primary cells was evident from the impairment of secretion by phloridzin or Sglt1 knockdown and failure of glucose to trigger cytosolic Ca\(^{2+}\) elevation in primary L cells from Sglt1 knockout mice.
Conclusions/interpretation
SGLT1 acts as the luminal glucose sensor in L cells, but intracellular glucose concentrations are largely determined by GLUT activity. Although L cell glucose metabolism depends partially on glucokinase activity, this plays only a minor role in glucose-stimulated GLP-1 secretion.
Introduction: Although there has been a worldwide emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA), little is known about the molecular epidemiology of MRSA in Tanzania.
Methodology: In this study, we characterized MRSA strains isolated from clinical specimens at the Bugando Medical Centre, Tanzania, between January and December 2008. Of 160 S. aureus isolates from 600 clinical specimens, 24 (15%) were found to be MRSA. Besides molecular screening for the Panton Valentine leukocidin (PVL) genes by PCR, MRSA strains were further characterized by Multi-Locus Sequence Typing (MLST) and spa typing.
Results: Despite considerable genetic diversity, the spa types t690 (29.1%) and t7231 (41.6%), as well as the sequence types (ST) 88 (54.2%) and 1797 (29.1%), were dominant among clinical isolates. The PVL genes were detected in 4 isolates; of these, 3 were found in ST 88 and one in ST1820. Resistance to erythromycin, clindamicin, gentamicin, tetracycline and co-trimoxazole was found in 45.8%, 62.5%, 41.6%, 45.8% and 50% of the strains, respectively.
Conclusion: We present the first thorough typing of MRSA at a Tanzanian hospital. Despite considerable genetic diversity, ST88 was dominant among clinical isolates at the Bugando Medical Centre. Active and standardized surveillance of nosocomial MRSA infection should be conducted in the future to analyse the infection and transmission rates and implement effective control measures.
Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS.
We present polarimetry, i.e. the detection of optical rotation of light polarization, in a configuration suitable for femtosecond spectroscopy. The polarimeter is based on common-path optical heterodyne interferometry and provides fast and highly sensitive detection of rotatory power. Femtosecond pump and polarimeter probe beams are integrated into a recently developed accumulative technique that further enhances sensitivity with respect to single-pulse methods. The high speed of the polarimeter affords optical rotation detection during the pump-pulse illumination period of a few seconds. We illustrate the concept on the photodissociation of the enantiomers of methyl p-tolyl sulfoxide. The sensitivity of rotatory detection, i.e. the minimum rotation angle that can be measured, is determined experimentally including all noise sources to be 0.10 milli-degrees for a measurement time of only one second and an interaction length of 250 μm. The suitability of the presented setup for femtosecond studies is demonstrated in a non-resonant two-photon photodissociation experiment.
Current changes of biodiversity result almost exclusively from human activities. This anthropogenic conversion of natural ecosystems during the last decades has led to the so-called ‘biodiversity crisis’, which comprises the loss of species as well as changes in the global distribution patterns of organisms. Species richness is unevenly distributed worldwide. Altogether, 17 so-called ‘megadiverse’ nations cover less than 10% of the earth’s land surface but support nearly 70% of global species richness. Mexico, the study area of this thesis, is one of those countries. However, due to Mexico’s large extent and geographical complexity, it is impossible to conduct reliable and spatially explicit assessments of species distribution ranges based on these collection data and field work alone. In the last two decades, Species distribution models (SDMs) have been established as important tools for extrapolating such in situ observations. SDMs analyze empirical correlations between geo-referenced species occurrence data and environmental variables to obtain spatially explicit surfaces indicating the probability of species occurrence. Remote sensing can provide such variables which describe biophysical land surface characteristics with high effective spatial resolutions. Especially during the last three to five years, the number of studies making use of remote sensing data for modeling species distributions has therefore multiplied. Due to the novelty of this field of research, the published literature consists mostly of selective case studies. A systematic framework for modeling species distributions by means of remote sensing is still missing. This research gap was taken up by this thesis and specific studies were designed which addressed the combination of climate and remote sensing data in SDMs, the suitability of continuous remote sensing variables in comparison with categorical land cover classification data, the criteria for selecting appropriate remote sensing data depending on species characteristics, and the effects of inter-annual variability in remotely sensed time series on the performance of species distribution models. The corresponding novel analyses were conducted with the Maximum Entropy algorithm developed by Phillips et al. (2004). In this thesis, a more comprehensive set of remote sensing predictors than in the existing literature was utilized for species distribution modeling. The products were selected based on their ecological relevance for characterizing species distributions. Two 1 km Terra-MODIS Land 16-day composite standard products including the Enhanced Vegetation Index (EVI), Reflectance Data, and Land Surface Temperature (LST) were assembled into enhanced time series for the time period of 2001 to 2009. These high-dimensional time series data were then transformed into 18 phenological and 35 statistical metrics that were selected based on an extensive literature review. Spatial distributions of twelve tree species were modeled in a hierarchical framework which integrated climate (WorldClim) and MODIS remote sensing data. The species are representative of the major Mexican forest types and cover a variety of ecological traits, such as range size and biotope specificity. Trees were selected because they have a high probability of detection in the field and since mapping vegetation has a long tradition in remote sensing. The result of this thesis showed that the integration of remote sensing data into species distribution models has a significant potential for improving and both spatial detail and accuracy of the model predictions.
Background: Clearance of apoptotic neutrophils in the lung is an essential process to limit inflammation, since they could become a pro-inflammatory stimulus themselves. The clearance is partially mediated by alveolar macrophages, which phagocytose these apoptotic cells. The phagocytosis of apoptotic immune cells by monocytes in vitro has been shown to be augmented by several constituents of pulmonary surfactant, e. g. phospholipids and hydrophobic surfactant proteins. In this study, we assessed the influence of exogenous poractant alfa (Curosurf (R)) instillation on the in vivo phagocytosis of apoptotic neutrophils by alveolar macrophages.
Methods: Poractant alfa (200 mg/kg) was instilled intratracheally in the lungs of three months old adult male C57/Black 6 mice, followed by apoptotic neutrophil instillation. Bronchoalveloar lavage was performed and alveolar macrophages and neutrophils were counted. Phagocytosis of apoptotic neutrophils was quantified by determining the number of apoptotic neutrophils per alveolar macrophages.
Results: Exogenous surfactant increased the number of alveolar macrophages engulfing apoptotic neutrophils 2.6 fold. The phagocytosis of apoptotic neutrophils was increased in the presence of exogenous surfactant by a 4.7 fold increase in phagocytosed apoptotic neutrophils per alveolar macrophage.
Conclusions: We conclude that the anti-inflammatory properties of surfactant therapy may be mediated in part by increased numbers of alveolar macrophages and increased phagocytosis of apoptotic neutrophils by alveolar macrophages.
Acute bacterial meningitis is a life-threatening disease in humans. Discussed as entry sites for pathogens into the brain are the blood-brain and the blood-cerebrospinal fluid barrier (BCSFB). Although human brain microvascular endothelial cells (HBMEC) constitute a well established human in vitro model for the blood-brain barrier, until now no reliable human system presenting the BCSFB has been developed. Here, we describe for the first time a functional human BCSFB model based on human choroid plexus papilloma cells (HIBCPP), which display typical hallmarks of a BCSFB as the expression of junctional proteins and formation of tight junctions, a high electrical resistance and minimal levels of macromolecular flux when grown on transwell filters. Importantly, when challenged with the zoonotic pathogen Streptococcus suis or the human pathogenic bacterium Neisseria meningitidis the HIBCPP show polar bacterial invasion only from the physiologically relevant basolateral side. Meningococcal invasion is attenuated by the presence of a capsule and translocated N. meningitidis form microcolonies on the apical side of HIBCPP opposite of sites of entry. As a functionally relevant human model of the BCSFB the HIBCPP offer a wide range of options for analysis of disease-related mechanisms at the choroid plexus epithelium, especially involving human pathogens.
Pneumolysin, a protein toxin, represents one of the major virulence factors of Streptococcus pneumoniae. This pathogen causes bacterial meningitis with especially high disease rates in young children, elderly people and immunosuppressed patients. The protein toxin belongs to the family of cholesterol-dependent cytolysins, which require membrane cholesterol in order to bind and to be activated. Upon activation, monomers assemble in a circle and undergo conformational change. This conformational change leads to the formation of a pore, which eventually leads to cell lysis. This knowledge was obtained by studies that used a higher concentration compared to the concentration of pneumolysin found in the cerebrospinal fluid of meningitis patients. Thus, a much lower concentration of pneumolysin was used in this work in order to investigate effects of this toxin on primary mouse astrocytes. Previously, a small GTPase activation, possibly leading to cytoskeletal changes, was found in a human neuroblastoma cell line. This led to the hypothesis that pneumolysin can lead to similar cytoskeletal changes in primary cells. The aim of this work was to investigate and characterise the effects of pneumolysin on primary mouse astrocytes in terms of a possible pore formation, cellular trafficking and immunological responses. Firstly, the importance of pore-formation on cytoskeletal changes was to be investigated. In order to tackle this question, wild-type pneumolysin and two mutant variants were used. One variant was generated by exchanging one amino acid in the cholesterol recognising region, the second variant was generated by deleting two amino acids in a protein domain that is essential for oligomerisation. These variants should be incapable of forming a pore and were compared to the wild-type in terms of lytic capacities, membrane binding, membrane depolarisation, pore-formation in artificial membranes (planar lipid bilayer) and effects on the cytoskeleton. These investigations resulted in the finding that the pore-formation is required for inducing cell lysis, membrane depolarisation and cytoskeletal changes in astrocytes. The variants were not able to form a pore in planar lipid bilayer and did not cause cell lysis and membrane depolarisation. However, they bound to the cell membrane to the same extent as the wild-type toxin. Thus, the pore-formation, but not the membrane binding was the cause for these changes. Secondly, the effect of pneumolysin on cellular trafficking was investigated. Here, the variants showed no effect, but the wild-type led to an increase in overall endocytotic events and was itself internalised into the cell. In order to characterise a possible mechanism for internalisation, a GFP-tagged version of pneumolysin was used. Several fluorescence-labelled markers for different endocytotic pathways were used in a co-staining approach with pneumolysin. Furthermore, inhibitors for two key-players in classical endocytotic pathways, dynamin and myosin II, were used in order to investigate classical endocytotic pathways and their possible involvement in toxin internalisation. The second finding of this work is that pneumolysin is taken up into the cell via dynamin- and caveolin-independent pinocytosis, which could transfer the toxin to caveosomes. From there, the fate of the toxin remains unknown. Additionally, pneumolysin leads to an overall increase in endocytotic events. This observation led to the third aim of this work. If the toxin increases the overall rate of endocytosis, the question arises whether toxin internalisation favours bacterial tissue penetration of the host or whether it serves as a defence mechanism of the cell in order to degrade the protein. Thus, several proinflammatory cytokines were investigated, as previous studies describe an effect of pneumolysin on cytokine production. Surprisingly, only interleukin 6-production was increased after toxin-treatment and no effect of endocytotic inhibitors on the interleukin 6-production was observed. The conclusion from this finding is that pneumolysin leads to an increase of interleukin 6, which would not depend on the endocytotic uptake of pneumolysin. The production of interleukin 6 would enhance the production of acute phase proteins, T-cell activation, growth and differentiation. On the one hand, this activation could serve pathogen clearance from infected tissue. On the other hand, the production of interleukin 6 could promote a further penetration of pathogen into host tissue. This question should be further investigated.
Plant-derived natural products and their analogs continue to play an important role in the discovery of new drugs for the treatment of human diseases. Potentially promising representatives of secondary metabolites are the naphthylisoquinoline alkaloids, which show a broad range of activities against protozoan pathogens, such as plasmodia, leishmania, and trypanosoma. Due to the increasing resistance of those pathogens against current therapies, highly potent novel agents are still urgently needed. Thus, it is worthy to discover new naphthylisoquinoline alkaloids hopefully with pronounced bioactivities by isolation from plants or by synthesis. The naphthylisoquinoline alkaloids are biosynthetically related to another class of plant-derived products, the naphthoquinones, some of which have been recently found to display excellent anti-multiple myeloma activities without showing any cytotoxicities on normal blood cells. Multiple myeloma still remains incurable, although remissions may be induced with co-opted therapeutic treatments. Therefore, more potent naphthoquinones are urgently required, and can be obtained by isolation from plants or by synthesis. In detail, the results in this thesis are listed as follows: 1) Isolation and characterization of naphthylisoquinoline alkaloids from the stems of a Chinese Ancistrocladus tectorius species. Nine new naphthylisoquinoline alkaloids, named ancistectorine A1 (60), N-methylancistectorine A1 (61), ancistectorine A2 (62a), 5-epi-ancistectorine A2 (62b), 4'-O-demethylancistectorine A2 (63), ancistectorine A3 (64), ancistectorine B1 (65), ancistectorine C1 (66), and 5-epi-ancistrolikokine D (67) were isolated from the Chinese A. tectorius and fully characterized by chemical, spectroscopic, and chiroptical methods. Furthermore, the in vitro anti-infectious activities of 60-62 and 63-66 have been tested. Three of the metabolites, 61, 62a, and 62b, exhibited strong antiplasmodial activities against the strain K1 of P. falciparum without showing significant cytotoxicities. With IC50 values of 0.08, 0.07, and 0.03 μM, respectively, they were 37 times more active than the standard chloroquine (IC50 = 0.26 μM). Moreover, these three compounds displayed high antiplasmodial selectivity indexes ranging from 100 to 3300. According to the TDR/WHO guidelines, they could be considered as lead compounds. In addition, seven alkaloids, 69-74 (structures not shown here), were isolated from A. tectorius that were known, but new to the plant, together with another fourteen known compounds (of these, only the structures of the three main alkaloids, 5a, 5b, and 78 are shown here), which had been previously found in the plant. The three metabolites ancistrocladine (5a), hamatine (5b), and (+)-ancistrocline (78) were found to show no or moderate activities against the MM cell lines. 2) Isolation and characterization of naphthylisoquinoline alkaloids from the root bark of a new, botanically yet undescribed Congolese Ancistrocladus species. An unprecedented dimeric Dioncophyllaceae-type naphthylisoquinoline alkaloid, jozimine A2 (84), as first recognized by G. Bauckmann from an as yet undescribed Ancistrocladus species, was purified and characterized as part of this thesis. Its full structural assignment was achieved by spectroscopic and chiroptical methods, and further confirmed by an X-ray diffraction analysis, which had never succeeded for any other dimeric naphthylisoquinoline alkaloids before. Structurally, the dimer is composed of two identical 4'-O-demethyldioncophylline A halves, coupled through a sterically hindered central axis at C-3',3'' of the two naphthalene moieties. Pharmacologically, jozimine A2 (84) showed an extraordinary antiplasmodial activity (IC50 = 1.4 nM) against the strain NF54 of P. falciparum. Beside jozimine A2 (85), another new alkaloid, 6-O-demethylancistrobrevine C (84), and four known ones, ancistrocladine (5a), hamatine (5b), ancistrobrevine C (86), and dioncophylline A (6) were isolated from the Ancistrocladus species, the latter in a large quantity (~500 mg), showing that the plant produces Ancistrocladaceae-type, mixed-Ancistrocladaceae/Dioncophyllaceae-type, and Dioncophyllaceae-type naphthyl- isoquinoline alkaloids. Remarkably, it is one of the very few plants, like A. abbreviatus, and A. barteri, that simultaneously contain typical representatives of all the above three classes of alkaloids. 3) Semi-synthesis of jozimine A2 (85), 3'-epi-85, jozimine A3 (93) and other alkaloids from dioncophylline A (6). The dimeric naphthylisoquinoline alkaloids, jozimine A2 (85) and 3'-epi-85, constitute rewarding synthetic targets for a comparative analysis of their antiplasmodial activities and for a further confirmation of the assigned absolute configurations of the isolated natural product of 85. They were semi-synthesized in a four-step reaction sequence from dioncophylline A (6) in cooperation with T. Büttner. The key step was a biomimetic phenol-oxidative dimerization at C-3' of the N,O-dibenzylated derivative of 89 by utilizing Pb(OAc)4. This is the first time that the synthesis of such an extremely sterically hindered (four ortho-substituents) naphthylisoquinoline alkaloid – with three consecutive biaryl axes! – has been successfully achieved. A novel dimeric naphthylisoquinoline, jozimine A3 (93), bearing a 6',6''-central biaryl axis, was semi-synthesized from 5'-O-demethyldioncophylline A (90) by a similar biomimetic phenol-oxidative coupling reaction as a key step, by employing Ag2O. HPLC analysis with synthetic reference material of 3'-epi-85 and 93 for co-elution revealed that these two alkaloids clearly are not present in the crude extract of the Ancistrocladus species from which jozimine A2 (85) was isolated. This evidences that jozimine A2 (85) is very specifically biosynthesized by the plant with a high regio- and stereoslectivity. Remarkably, the two synthetic novel dimeric naphthylisoquinoline alkaloids 3'-epi-85 and 93 were found to display very good antiplasmodial activities, albeit weaker than that of the natural and semi-synthetic product 85. Additionally, the two compounds 3'-epi-85 and 93 possessed high or moderate selectivity indexes, which were much lower than that of 85. However, they can still be considered as new lead structures. Two unprecedented oxidative products of dioncophylline A, the diastereomeric dioncotetralones A (94a) and B (94b), were synthesized from dioncophylline A (6) in a one-step reaction. Remarkably, the aromatic properties in the “naphthalene” and the “isoquinoline” rings of 94a and 94b are partially lost and the “biaryl” axis has become a C,C-double bond, so that the two halves are nearly co-planar to each other, which has never been found among any natural or synthetic naphthylisoquinoline alkaloid. Their full structural characterization was accomplished by spectroscopic methods and quantum-chemical CD calculations (done by Y. Hemberger). The presumed reaction mechanism was proposed in this thesis. In addition, one of the two compounds, 94a, exhibited a highly antiplasmodial activity (IC50 = 0.09 μM) with low cytotoxicity, and thus, can be considered as a new promising lead structure. Its 2'-epi-isomer, 94b, was inactive, evidencing a significant effect of chirality on the bioactivity. Of a number of naphthylisoquinoline alkaloids tested against the multiple-myeloma cell lines, the three compounds, dioncophylline A (6), 4'-O-demethyldioncophylline A (89), and 5'-O-demethyldioncophylline A (90) showed excellent activities, even much stronger than dioncoquinones B (10), C (102), the epoxide 175, or the standard drug melphalan. 4) Isolation and characterization of bioactive naphthoquinones from cell cultures of Triphyophyllum peltatum. Three new naphthoquinones, dioncoquinones C (102), D (103), and E (104), the known 8-hydroxydroserone (105), which is new to this plant, and one new naphthol dimer, triphoquinol A (107), were isolated from cell cultures of T. peltatum in cooperation with A. Irmer. Dioncoquinone C (102) showed an excellent activity against the MM cells, very similar to that of the previously found dioncoquinone B (10), without showing any inhibitory effect on normal cells. The other three naphthoquinones, 103105, were inactive or only weakly active. 5) Establishment of a new strategy for a synthetic access to dioncoquinones B (10) and C (102) on a large scale for in vivo experiments and for the synthesis of their analogs for first SAR studies. Before the synthesis of dioncoquinone B (10) described in this thesis, two synthetic pathways had previously been established in our group. The third approach described here involved the preparation of the joint synthetic intermediate 42 with the previous two routes. The tertiary benzamide 135 was ortho-deprotonated by using s-BuLi/TMEDA, followed by transmetallation with MgBr2▪2Et2O, and reaction with 2-methylallyl bromide (139). It resulted in the formation of ortho-allyl benzamide 140, which was cyclized by using methyl lithium to afford the naphthol 42. This strategy proved to be the best among the established three approaches with regard to its very low number of steps and high yields. By starting with 136, this third strategy yielded the related bioactive natural product, dioncoquinone C (102), which was accessed by total synthesis for the first time. To identify the pharmacophore of the antitumoral naphthoquinones, a library of dioncoquinone B (10) and C (102) analogs were synthesized for in vitro testing. Among the numerous naphthoquinones tested, the synthetic 7-O-demethyldioncoquinone C (or 7-O-hydroxyldioncoquinone B) (145), constitutes another promising basic structure to develop a new anti-MM agent. Furthermore, preliminary SAR results evidence that the three hydroxy functions at C-3, C-5, and C-6 are essential for the biological properties as exemplarily shown through the compounds 10, 102, and 145. All other mixed OH/OMe- or completely OMe-substituted structures were entirely inactive. By a serendipity the expoxide 175 was found to display the best anti-MM activity of all the tested isolated metabolites from T. peltatum, the synthesized naphthoquinones, and their synthetic intermediates. Toxic effects of 175 on normal cells were not observed, in contrast to the high toxicities of all other epoxides. Thus, the anti-MM activity of 175 is of high selectivity. The preliminary SAR studies revealed that the 6-OMe group in 175 is required, thus differed with the above described naphthoquinones (where 6-OH is a requisite in 10, 102, and 145), which evidenced potentially different modes of action for these two classes of compounds. 6) The first attempted total synthesis of the new naturally occurring triphoquinone (187a), which was recently isolated from the root cultures of T. peltatum in our group. A novel naphthoquinone-naphthalene dimer, 187a (structure shown in Chapter 10), was isolated in small quantities from the root cultures of T. peltatum. Thus, its total synthesis was attempted for obtaining sufficient amounts for selected biotestings. The key step was planned to prepare the extremely sterically hindered (four ortho-substituents) binaphthalene 188 by a coupling reaction between the two 2-methylnaphthalene derivatives. Test reactions involving a system of two simplified 2-methylnaphthylboron species and 2-methylnaphthyl bromide proved the Buchwald ligand as most promising. The optimized conditions were then applied to the two true - highly oxygenated - coupling substrates, between the 2-methylnaphthylboron derivatives 210, 211, 213, or 214 and the 2-methylnaphthyl iodides (or bromides) 215 (206), 215 (206), 212 (205), or 212 (205), respectively. Unfortunately, this crucial step failed although various bases and solvent systems were tested. This could be due to the high electron density of the two coupling substrates, both bearing strongly OMOM/OMe-donating function groups. Therefore, a more powerful catalyst system or an alternative synthetic strategy must be explored for the total synthesis of 187a. 7) Phytochemical investigation of the Streptomyces strain RV-15 derived from a marine sponge. Cyclodysidins A-D (216-219), four new cyclic lipopeptides with a- and ß-amino acids, were isolated from the Streptomyces strain RV15 derived from a marine sponge by Dr. U. Abdelmohsen. Their structures were established as cyclo-(ß-AFA-Ser-Gln-Asn-Tyr-Asn-Ser-Thr) by spectroscopic analysis using 2D NMR techniques and CID-MS/MS in the course of this thesis. In conclusion, the present work contributes to the discovery of novel antiplasmodial naphthylisoquinoline alkaloids and antitumoral naphthoquinones, which will pave the way for future studies on these two classes of compounds.
Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1
(2012)
Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.
The analysis presented in this paper applies to experimental situations where observers or objects to be studied (both stationary, with respect to each other) are located in environments the optical thickness of which is strongly different. By their large optical thickness, non-transparent media are clearly distinguished from their transparent counterparts. Non-transparent media comprise thin metallic films, packed or fluidised beds, the Earth’s crust, and even dark clouds and other cosmological objects. As a representative example, a non-transparent slab is subjected to transient disturbances, and a rigorous analysis is presented whether physical time reasonably could be constructed under such condition. The analysis incorporates mapping functions that correlate physical events, e, in non-transparent media, with their images, f(e), tentatively located on a standard physical time scale. The analysis demonstrates, however, that physical time, in its rigorous sense, does not exist under non-transparency conditions. A proof of this conclusion is attempted in three steps: i) the theorem “there is no time without space and events” is accepted, (ii) images f[e(s,t)] do not constitute a dense, uncountably infinite set, and (iii) sets of images that are not uncountably infinite do not create physical time but only time-like sequences. As a consequence, mapping f[e(s,t)] in non-transparent space does not create physical analogues to the mathematical structure of the ordered, dense half-set R+ of real numbers, and reverse mapping, f-1f[e(s,t)] would not allow unique identification and reconstruction of original events from their images. In these cases, causality and determinism, as well as invariance of physical processes under time reversal, might be violated. Existence of time holes could be possible, as follows from the sequence of images, f[e(s,t)], that is not uncountably infinite, in contrast to R+. Practical impacts are expected for understanding physical diffusion-like, radiative transfer processes, stability models to protect superconductors against quenchs or for description of their transient local pair density and critical currents. Impacts would be expected also in mathematical formulations (differential equations) of classical physics, in relativity and perhaps in quantum mechanics, all as far as transient processes in non-transparent space would be concerned. An interesting problem is whether temporal cloaking (a time hole) in a transparent medium, as very recently reported in the literature, can be explained by the present analysis. The analysis is not restricted to objects of laboratory dimensions: Because of obviously existing radiation transfer analogues, it is tempting to discuss consequences also for much larger structures in particular if an origin of time is postulated.
A quite new approach to low-cost mass production of flexible solar cells are organic photovoltaics. Even though the device efficiencies increased rapidly during the last years, further imporvements are essential for a successful market launch. One important factor influencing the device efficiency is the photocurrent of a solar cell, which is defined as the difference between the current under illumination and in the dark. In case of organic bulk heterojunction (BHJ) solar cells it is — in contrast to inorganic devices — dependent on the applied bias voltage. The voltage dependence results in a reduced fill factor and thus an even more pronounced influence of the photocurrent on the device efficiency. It is therefore crucial to understand the underlying processes determining the photocurrent in order to be able to further improve the solar cell performance. In a first step the photocurrent of P3HT:PC61BM devices was investigated by a pulsed measurement technique in order to prevent disturbing influences due to device heating under continous illumination. The resulting photocurrent was hyperbolic tangent like and featured a point symmetry, whose origin and meaning were discussed. In addition, the photocurrent was described by a combined model of Braun–Onsager and Sokel–Hughes theory for field dependent polaron pair dissociation and charge extraction, respectively. After this macroscopic view on the photocurrent, the focus of this work moves to the more basic processes determining the photocurrent: charge photogeneration and recombination. In a comparative study the field-dependence of these was investigated by time-delayed collection field (TDCF) measurements for two well-known reference systems, namely P3HT:PC61BM and MDMO-PPV:PC61BM. It was possible to identify two different dominating scenarios for the generation of free charge carriers. The first one — via a thermalized charge transfer state (CTS) — is clearly influenced by geminate recombination and therefore less efficient. In the second scenario, the free charge carriers are either generated directly or via an excited, “hot” CTS. In addition, clear differences in the nongeminate recombination dynamics of both material systems were found. Similar studies were also be presented with two modern low bandgap polymers which only differ by the bridging atom in the cyclopentadithiophene (PCPDTBT:PC71BM vs. Si-PCPDTBT:PC71BM). Such small changes in the chemical structure were already sufficient to affect the charge photogeneration as well as the morphology of the blend. These findings were set into relation to current–voltage characteristics in order to discuss the origin of the clear differences in the solar cell performance of both materials. Another crucial parameter limiting the solar cell efficiency is the builtin potential of a device. Within the range of semiconducting pn-junctions, Mott–Schottky analysis is an established method to determine the built-in potential. As it was originally derived for abrupt pn-junctions, its validity for organic BHJ solar cells — a bipolar, effective medium — was discussed. Experimental findings as well as the contradictions to Mott–Schottky theory indicated, that a direct transfer of this method to organic photovoltaics is not appropriate. Finally, the results obtained in the framework of the MOPS-project (Massengedruckte Organische Papier-Solarzellen) will be presented, in which the first completely roll-to-roll printed paper solar cells were realized.
Plant communities in the European Alps are assumed to be highly affected by climate change since temperature rise in this region is above the global average. It is predicted that higher temperatures will lead to advanced snowmelt dates and that the number of extreme weather events will increase. The aims of this study were to determine the impacts of extreme climatic events on flower phenology and to assess whether those impacts differed between lower and higher altitudes. In 2010 an experiment simulating advanced and delayed snowmelt as well as drought event was conducted along an altitudinal transect ca. every 250m (600-2000 m a.s.l.) in the Berchtesgaden National Park, Germany. The study showed that flower phenology is strongly affected by altitude; however there were few effects of the manipulative treatments on flowering. The effects of advanced snowmelt were significantly greater at higher than at lower sites, but no significant difference was found between both altitudinal bands for the other treatments. The response of flower phenology to temperature declined through the season and the length of flowering duration was not significantly influenced by treatments. The stronger effect of advanced snowmelt at higher altitudes might be a response to differences in treatment intensity across the gradient. Consequently, shifts in the date of snowmelt due to global warming may affect species more at higher than at lower altitudes since changes may be more pronounced at higher altitudes. Our data indicate a rather low risk of drought events on flowering phenology in the Bavarian Alps.
Proton–proton collisions at √s=7 TeV and heavy ion collisions at \(\sqrt{sNN}\)=2.76 TeV were produced by the LHC and recorded using the ATLAS experiment’s trigger system in 2010. The LHC is designed with a maximum bunch crossing rate of 40 MHz and the ATLAS trigger system is designed to record approximately 200 of these per second. The trigger system selects events by rapidly identifying signatures of muon, electron, photon, tau lepton, jet, and B meson candidates, as well as using global event signatures, such as missing transverse energy. An overview of the ATLAS trigger system, the evolution of the system during 2010 and the performance of the trigger system components and selections based on the 2010 collision data are shown. A brief outline of plans for the trigger system in 2011 is presented.
The measurement of missing transverse momentum in the ATLAS detector, described in this paper, makes use of the full event reconstruction and a calibration based on reconstructed physics objects. The performance of the missing transverse momentum reconstruction is evaluated using data collected in pp collisions at a centre-of-mass energy of 7 TeV in 2010. Minimum bias events and events with jets of hadrons are used from data samples corresponding to an integrated luminosity of about 0.3 nb\(^{−1}\) and 600 nb\(^{−1}\) respectively, together with events containing a Z boson decaying to two leptons (electrons or muons) or a W boson decaying to a lepton (electron or muon) and a neutrino, from a data sample corresponding to an integrated luminosity of about 36 pb\(^{−1}\). An estimate of the systematic uncertainty on the missing transverse momentum scale is presented
Internet applications are becoming more and more flexible to support diverge user demands and network conditions. This is reflected by technical concepts, which provide new adaptation mechanisms to allow fine grained adjustment of the application quality and the corresponding bandwidth requirements. For the case of video streaming, the scalable video codec H.264/SVC allows the flexible adaptation of frame rate, video resolution and image quality with respect to the available network resources. In order to guarantee a good user-perceived quality (Quality of Experience, QoE) it is necessary to adjust and optimize the video quality accurately. But not only have the applications of the current Internet changed. Within network and transport, new technologies evolved during the last years providing a more flexible and efficient usage of data transport and network resources. One of the most promising technologies is Network Virtualization (NV) which is seen as an enabler to overcome the ossification of the Internet stack. It provides means to simultaneously operate multiple logical networks which allow for example application-specific addressing, naming and routing, or their individual resource management. New transport mechanisms like multipath transmission on the network and transport layer aim at an efficient usage of available transport resources. However, the simultaneous transmission of data via heterogeneous transport paths and communication technologies inevitably introduces packet reordering. Additional mechanisms and buffers are required to restore the correct packet order and thus to prevent a disturbance of the data transport. A proper buffer dimensioning as well as the classification of the impact of varying path characteristics like bandwidth and delay require appropriate evaluation methods. Additionally, for a path selection mechanism real time evaluation mechanisms are needed. A better application-network interaction and the corresponding exchange of information enable an efficient adaptation of the application to the network conditions and vice versa. This PhD thesis analyzes a video streaming architecture utilizing multipath transmission and scalable video coding and develops the following optimization possibilities and results: Analysis and dimensioning methods for multipath transmission, quantification of the adaptation possibilities to the current network conditions with respect to the QoE for H.264/SVC, and evaluation and optimization of a future video streaming architecture, which allows a better interaction of application and network.
The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.
Susac syndrome, a rare but probably underdiagnosed combination of encephalopathy, hearing loss, and visual deficits due to branch retinal artery occlusion of unknown aetiology has to be considered as differential diagnosis in various conditions. Particularly, differentiation from multiple sclerosis is often challenging since both clinical presentation and diagnostic findings may overlap. Optical coherence tomography is a powerful and easy to perform diagnostic tool to analyse the morphological integrity of retinal structures and is increasingly established to depict characteristic patterns of retinal pathology in multiple sclerosis. Against this background we hypothesised that differential patterns of retinal pathology facilitate a reliable differentiation between Susac syndrome and multiple sclerosis. In this multicenter cross-sectional observational study optical coherence tomography was performed in nine patients with a definite diagnosis of Susac syndrome. Data were compared with age-, sex-, and disease duration-matched relapsing remitting multiple sclerosis patients with and without a history of optic neuritis, and with healthy controls. Using generalised estimating equation models, Susac patients showed a significant reduction in either or both retinal nerve fibre layer thickness and total macular volume in comparison to both healthy controls and relapsing remitting multiple sclerosis patients. However, in contrast to the multiple sclerosis patients this reduction was not distributed over the entire scanning area but showed a distinct sectorial loss especially in the macular measurements. We therefore conclude that patients with Susac syndrome show distinct abnormalities in optical coherence tomography in comparison to multiple sclerosis patients. These findings recommend optical coherence tomography as a promising tool for differentiating Susac syndrome from MS.
Pars plana vitrectomy for malignant glaucoma in non-glaucomatous and in filtered glaucomatous eyes
(2012)
Purpose: To assess the outcomes of pars plana vitrectomy for the treatment of malignant glaucoma in patients with and without previous filtration surgery. Patients and methods: Data of 15 patients developing malignant glaucoma after trabeculectomy (60%) or following ophthalmic interventions other than filtration surgery (40%) were recorded retrospectively. Pars plana vitrectomy was performed in case of failed medical or laser treatment recreating the normal pathway of aqueous humor. The main outcome measures were the postoperative intraocular pressure (IOP), the frequency of complications, and success rate based on the following criteria: IOP reduction by $20% and to #21 mmHg (definition one) or an IOP , 18 mmHg (definition two) with (qualified success) and without (complete success) glaucoma medication. Results: Vitrectomy reduced IOP from baseline in eyes with and without previous trabeculectomy during a median follow-up of 16.4 months (range 7 days to 58 months); although the majority of patients required glaucoma medication to reach desired IOP. The complete success rates were 11% (both definitions) for patients with filtering blebs and none of the patients without previous trabeculectomy had complete success at the 12-month visit. Complications were few and included transient shallowing of the anterior chamber, choroidal detachment, corneal decompensation, filtering bleb failure, and need for further IOP-lowering procedures. Conclusion: Pars plana vitrectomy is equally effective for malignant glaucoma caused by trabeculectomy or interventions other than filtration surgery, although IOP-lowering medication is necessary in nearly all cases to maintain target IOP.
The photoluminescence of annealed Cd\(_x\)Zn\(_{1-x}\)Se quantum dots (QDs) under the influence of an external magnetic field has been studied in this paper. Post-growth annealing was performed for different annealing times. Above a critical annealing time, the QD luminescence shows a pronounced red-shift of the Zeeman split magnetic subcomponents. This observation is in contrast to the blue-shift caused by the diamagnetic behavior that is usually observed in non-magnetic QDs. We attribute our finding to the paramagnetism caused by the mixing of heavy and light hole states. Hence, post-growth thermal annealing treatment might be employed to render undoped epitaxial QDs intrinsically magnetic in a controlled manner. Two theoretical models were developed: a few-particle model to account for excitonic complex effects and a multiband calculation that describes the valence band hybridization. Contrasting the two models allowed us to unambiguously elucidate the nature of such an effect.
Background: Current surgical approaches to the distal radius include dorsal and palmar plate fixation. While palmar plates have gained widespread popularity, few reports have provided data on long term clinical outcomes in comparison. This paper reports the result of a randomised clinical study comparing dorsal Pi plates and palmar, angle-stable plates for treatment of comminuted, intraarticular fractures of the distal radius over the course of twelve months.
Methods: 42 patients with unilateral, intraarticular fractures of the distal radius were included and randomised to 2 groups, 22 were treated with a palmar plate, 20 received a dorsal Pi-plate. Results were evaluated after 6 weeks, 3, 6 and 12 months postoperatively focussing on functional recovery as well as radiological results.
Results: The palmar plate group demonstrated significantly better results regarding range of motion and grip strength over the course of 12 months. While a comparable increase in function was observed in both groups, the better results from the early postoperative period in the palmar plate group prevailed over the whole course. Radiological results showed a significantly increased palmar tilt and carpal sag in dorsal plates, with other radiological parameters being comparable. Pain levels were decreased in dorsal plates after hardware removal and failed to show significant differences after 12 months. However, complications such as tendon ruptures were more frequent in the dorsal plate group.
Conclusions: Functional advantage of palmar plates gained within the first 6 weeks prevails over the course of a year. Both groups demonstrate further gradual increase of function after 6 months, although dorsal plates did not catch up completely. Improved early postoperative function seems to be the cornerstone for the best possible results. Patients with dorsal plates benefit from hardware removal more than palmar plates in terms of reduction of pain levels. The advantage of palmar plates is a faster functional recovery with lower complication rates. This is especially important in the elderly population. Radiological results did not show a superiority of palmar plates over dorsal plates.
Two brothers in their fifties presented with a medical history of suspected fungal allergy, allergic bronchopulmonary aspergillosis, alveolitis, and invasive aspergillosis and pulmonary fistula, respectively. Eventually, after a delay of 50 years, chronic granulomatous disease (CGD) was diagnosed in the index patient. We found a new splice mutation in the NCF2 (p67-phox) gene, c.1000+2T -> G, that led to several splice products one of which lacked exons 11 and 12. This deletion was in frame and allowed for remarkable residual NADPH oxidase activity as determined by transduction experiments using a retroviral vector. We conclude that p67-phox which lacks the 34 amino acids encoded by the two exons can still exert considerable functional activity. This activity can partially explain the long-term survival of the patients without adequate diagnosis and treatment, but could not prevent progressing lung damage.
A brain-computer interface (BCI) enables communication without movement based on brain signals measured with electroencephalography (EEG). BCIs usually rely on one of three types of signals: the P300 and other components of the event-related potential (ERP), steady state visual evoked potential (SSVEP), or event related desynchronization (ERD). Although P300 BCIs were introduced over twenty years ago, the past few years have seen a strong increase in P300 BCI research. This closed-loop BCI approach relies on the P300 and other components of the ERP, based on an oddball paradigm presented to the subject. In this paper, we overview the current status of P300 BCI technology, and then discuss new directions: paradigms for eliciting P300s; signal processing methods; applications; and hybrid BCIs. We conclude that P300 BCIs are quite promising, as several emerging directions have not yet been fully explored and could lead to improvements in bit rate, reliability, usability, and flexibility.
In summary, structure-activity relationships in peptide and dendrimer carriers modified with different organometal complexes were studied on a human breast cancer cell line. Variation of the organometal cargo and carrier can significantly influence their biological properties and might open the way to new approaches in chemotherapy. Furthermore, the incorporation of complexes with different C≡O vibrational signatures in a model peptide was explored to examine information encoding in biomolecules in a barcoding strategy for potential imaging applications. In particular for the latter, additional stable metal-carbonyl markers need to be prepared in future work to expand the pool of vibrational labels available.
Due to their negative water budget most recent semi-/arid regions are characterized by vast evaporates (salt lakes and salty soils). We recently identified those hyper-saline environments as additional sources for a multitude of volatile halogenated organohalogens (VOX). These compounds can affect the ozone layer of the stratosphere and play a key role in the production of aerosols. A remote sensing based analysis was performed in the Southern Aral Sea basin, providing information of major soil types as well as their extent and spatial and temporal evolution. VOX production has been determined in dry and moist soil samples after 24 h. Several C1- and C2 organohalogens have been found in hyper-saline topsoil profiles, including CH3Cl, CH3Br, CHBr3 and CHCl3. The range of organohalogens also includes trans-1,2-dichloroethene (DCE), which is reported here to be produced naturally for the first time. Using MODIS time series and supervised image classification a daily production rate for DCE has been calculated for the 15 000 km\(^2\) ranging research area in the southern Aralkum. The applied laboratory setup simulates a short-term change in climatic conditions, starting from dried-out saline soil that is instantly humidified during rain events or flooding. It describes the general VOX production potential, but allows only for a rough estimation of resulting emission loads. VOX emissions are expected to increase in the future since the area of salt affected soils is expanding due to the regressing Aral Sea. Opportunities, limits and requirements of satellite based rapid change detection and salt classification are discussed.
Optimal open-loop control, i.e. the application of an analytically derived control rule, is demonstrated for nanooptical excitations using polarization-shaped laser pulses. Optimal spatial near-field localization in gold nanoprisms and excitation switching is realized by applying a shift to the relative phase of the two polarization components. The achieved near-field switching confirms theoretical predictions, proves the applicability of predefined control rules in nanooptical light–matter interaction and reveals local mode interference to be an important control mechanism.
Neisseria meningitidis employs polysaccharides and outer membrane proteins to cope with human serum complement attack. To screen for factors influencing serum resistance, an assay was developed based on a colorimetric serum bactericidal assay. The screening used a genetically modified sequence type (ST)-41/44 clonal complex (cc) strain lacking LPS sialylation, polysaccharide capsule, the factor H binding protein (fHbp) and MutS, a protein of the DNA repair mechanism. After killing of >99.9% of the bacterial cells by serum treatment, the colorimetric assay was used to screen 1000 colonies, of which 35 showed enhanced serum resistance. Three mutant classes were identified. In the first class of mutants, enhanced expression of Opc was identified. Opc expression was associated with vitronectin binding and reduced membrane attack complex deposition confirming recent observations. Lipopolysaccharide (LPS) immunotype switch from immunotype L3 to L8/L1 by lgtA and lgtC phase variation represented the second class. Isogenic mutant analysis demonstrated that in ST-41/44 cc strains the L8/L1 immunotype was more serum resistant than the L3 immunotype. Consecutive analysis revealed that the immunotypes L8 and L1 were frequently observed in ST-41/44 cc isolates from both carriage and disease. Immunotype switch to L8/L1 is therefore suggested to contribute to the adaptive capacity of this meningococcal lineage. The third mutant class displayed a pilE allelic exchange associated with enhanced autoaggregation. The mutation of the C terminal hypervariable region D of PilE included a residue previously associated with increased pilus bundle formation. We suggest that autoaggregation reduced the surface area accessible to serum complement and protected from killing. The study highlights the ability of meningococci to adapt to environmental stress by phase variation and intrachromosomal recombination affecting subcapsular antigens.
The well-known Ugi reaction of aldehydes with amines, carboxylic acids and isocyanides leads to the formation of acyclic alpha-acylaminocarboxamides. Replacement of the carboxylic acid derivatives with beta-acyl substituted acrylic acids gives access to highly substituted 2,5-diketopiperazines in one single reaction-step without additives or complex reaction procedures. The obtained diketopiperazines show anti-proliferative effects on activated T cells and represent therefore potential candidates for targeting unwanted T cell-mediated immune responses.
Self-organized nanowires at semiconductor surfaces offer the unique opportunity to study electrons in reduced dimensions. Notably the dimensionality of the system determines it’s electronic properties, beyond the quasiparticle description. In the quasi-one-dimensional (1D) regime with weak lateral coupling between the chains, a Peierls instability can be realized. A nesting condition in the Fermi surface leads to a backfolding of the 1D electron band and thus to an insulating state. It is accompanied by a charge density wave (CDW) in real space that corresponds to the nesting vector. This effect has been claimed to occur in many surface-defined nanowire systems, such as the In chains on Si(111) or the Au reconstructions on the terraced Si(553) and Si(557) surfaces. Therefore a weak coupling between the nanowires in these systems has to be concluded. However theory proposes another state in the perfect 1D limit, which is completely destroyed upon slight coupling to higher dimensions. In this so-called Tomonaga-Luttinger liquid (TLL) state, the quasiparticle description of the Fermi liquid breaks down. Since the interaction between the electrons is enhanced due to the strong confinement, only collective excitations are allowed. This leads to novel effects like spin charge separation, where spin and charge degrees of freedom are decoupled and allowed to travel independently along the 1D-chain. Such rare state has not been realized at a surface until today. This thesis uses a novel approach to realize nanowires with improved confinement by studying the Au reconstructed Ge(001) surface. A new cleaning procedure using piranha solution is presented, in order to prepare a clean and long-range ordered substrate. To ensure optimal growth of the Au nanowires the phase diagram is extensively studied by scanning tunneling microscopy (STM) and low energy electron diffraction (LEED). The structural elements of the chains are revealed and described in high detail. Remarkably a structural phase transition of the delicate wire structure is found to occur above room temperature. Due to the lack of energy gaps a Peierls transition can be excluded as its origin. The transition is rather determined as 3D Ising type and therefore includes the substrate as well. Two hallmark properties of a TLL are found in the Au/Ge(001) wires by spectroscopic studies: Power-law suppression of the density of states (DOS) and universal scaling. This impressively proves the existence of a TLL in these chains and opens up a gateway to an atomic playground. Local studies and manipulations of a TLL state become possible for the first time. These comprise (i) doping by alkaline atoms, (ii) studies on chain ends and (iii) tunable coupling between the chains by additional Au atoms. Most importantly these manipulations offer input and test for theoretical models and predictions, and are thereby ultimately advancing the field of correlated electrons.
Oncolytic viruses refer to those that are able to eliminate malignancies by direct targeting and lysis of cancer cells, leaving non-cancerous tissues unharmed. Several oncolytic viruses including adenovirus strains, canine distemper virus and vaccinia virus strains have been used for canine cancer therapy in preclinical studies. However, in contrast to human studies, clinical trials with oncolytic viruses for canine cancer patients have not been reported. An ‘ideal’ virus has yet to be identified. This review is focused on the prospective use of oncolytic viruses in the treatment of canine tumors - a knowledge that will undoubtedly contribute to the development of oncolytic viral agents for canine cancer therapy in the future.
Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy.
Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models.
Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells.
Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors.
Melanoma is the most aggressive skin cancer with very limited treatment options. Upon appearance of metastases chemotherapeutics are used to either kill or slow down the growth of cancer cells by inducing apoptosis or senescence, respectively. With melanomas originating from melanocytes, it is vital to elucidate the mechanisms that distinguish senescence induction from proliferation and tumourigenicity. Xmrk (Xiphophorus melanoma receptor kinase), the fish orthologue of the human epidermal growth factor receptor (EGFR), causes highly aggressive melanoma in fish. Using an inducible variant, HERmrk, I showed that high receptor levels result in melanocyte senescence, whereas low and medium expression allows for cell proliferation and tumourigenicity. Mechanistically, HERmrk leads to increased reactive oxygen species (ROS) levels, which trigger a DNA damage response. Consequently, multinucleated, senescent cells develop by both endomitosis and fusion. Furthermore, oncogenic N‐RAS (N-‐RAS61K) induces a similar multinucleated phenotype in melanocytes. In addition, I found that both overexpression of C‐MYC and the knockdown of miz‐1 (Myc‐interacting zinc finger protein 1) diminished HERmrk‐induced senescence entry. C‐MYC prevent ROS induction, DNA damage and senescence, while acting synergistically with HERmrk in conveying tumourigenic features to melanocytes. Further analyses identified cystathionase (CTH) as a novel target gene of Myc and Miz-1 crucial for senescence prevention. CTH encodes an enzyme involved in the synthesis of cysteine from methionine, thereby allowing for increased ROS detoxification. Even though senescence was thought to be irreversible and hence tumour protective, I demonstrated that prolonged expression of the melanoma oncogene N‐RAS61K in pigment cells overcomes initial OIS by triggering the emergence of tumour‐initiating, mononucleated stem‐like cells from multinucleated senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis‐resistant and induces fast‐growing, metastatic tumours upon transplantation into nude mice. Our data demonstrate that induction of OIS is not only a cellular failsafe mechanism, but also carries the potential to provide a source for highly aggressive, tumour‐initiating cells.
This thesis is devoted to numerical verification of optimality conditions for non-convex optimal control problems. In the first part, we are concerned with a-posteriori verification of sufficient optimality conditions. It is a common knowledge that verification of such conditions for general non-convex PDE-constrained optimization problems is very challenging. We propose a method to verify second-order sufficient conditions for a general class of optimal control problem. If the proposed verification method confirms the fulfillment of the sufficient condition then a-posteriori error estimates can be computed. A special ingredient of our method is an error analysis for the Hessian of the underlying optimization problem. We derive conditions under which positive definiteness of the Hessian of the discrete problem implies positive definiteness of the Hessian of the continuous problem. The results are complemented with numerical experiments. In the second part, we investigate adaptive methods for optimal control problems with finitely many control parameters. We analyze a-posteriori error estimates based on verification of second-order sufficient optimality conditions using the method developed in the first part. Reliability and efficiency of the error estimator are shown. We illustrate through numerical experiments, the use of the estimator in guiding adaptive mesh refinement.
This thesis presents results covering several topics in correlated many fermion systems. A Monte Carlo technique (CT-INT) that has been implemented, used and extended by the author is discussed in great detail in chapter 3. The following chapter discusses how CT-INT can be used to calculate the two particle Green’s function and explains how exact frequency summations can be obtained. A benchmark against exact diagonalization is presented. The link to the dynamical cluster approximation is made in the end of chapter 4, where these techniques are of immense importance. In chapter 5 an extensive CT-INT study of a strongly correlated Josephson junction is shown. In particular, the signature of the first order quantum phase transition between a Kondo and a local moment regime in the Josephson current is discussed. The connection to an experimental system is made with great care by developing a parameter extraction strategy. As a final result, we show that it is possible to reproduce experimental data from a numerically exact CT-INT model-calculation. The last topic is a study of graphene edge magnetism. We introduce a general effective model for the edge states, incorporating a complicated interaction Hamiltonian and perform an exact diagonalization study for different parameter regimes. This yields a strong argument for the importance of forbidden umklapp processes and of the strongly momentum dependent interaction vertex for the formation of edge magnetism. Additional fragments concerning the use of a Legendre polynomial basis for the representation of the two particle Green’s function, the analytic continuation of the self energy for the Anderson Kane Mele Model, as well as the generation of test data with a given covariance matrix are documented in the appendix. A final appendix provides some very important matrix identities that are used for the discussion of technical details of CT-INT.
Parasitic helminths share a large degree of common genetic heritage with their various hosts. This includes cell-cell-communication mechanisms mediated by small peptide cytokines and lipophilic/steroid hormones. These cytokines are candidate molecules for host-parasite cross-communication in helminth diseases. In this work the function of two evolutionary conserved signaling pathways in the model cestode Echinococcus multilocularis has been studied. First, signaling mechanisms mediated through fibroblast growth factors (FGF) and their cognate receptors (FGFR) which influence a multitude of biological functions, like homeostasis and differentiation, were studied. I herein investigated the role of EmFR which is the only FGFR homolog in E. multilocularis. Functional analyses using the Xenopus oocyte expression system clearly indicate that EmFR can sense both acidic and basic FGF of human origin, resulting in an activation of the EmFR tyrosine kinase domain. In vitro experiments demonstrate that mammalian FGF significantly stimulates proliferation and development of E. multilocularis metacestode vesicles and primary cells. Furthermore, DNA synthesis and the parasite’s Erk-like MAPK cascade module was stimulated in the presence of exogenously added mammalian FGF. By using the FGFR inhibitor BIBF1120 the activity of EmFR in the Xenopus oocyte system was effectively blocked. Addition of BIBF1120 to in vitro cultivated Echinococcus larval material led to detrimental effects concerning the generation of metacestode vesicles from parasite stem cells, the proliferation and survival of metacestode vesicles, and the dedifferentiation of protoscoleces towards the metacestode. In conclusion, these data demonstrate the presence of a functional EmFR-mediated signaling pathway in E. multilocularis that is able to interact with host-derived cytokines and that plays an important role in larval parasite development. Secondly, the role of nuclear hormone receptor (NHR) signaling was addressed. Lipophilic and steroid hormone signaling contributes to the regulation of metazoan development. By means of in silico analyses I demonstrate that E. multilocularis expresses a set of 17 NHRs that broadly overlaps with that of the related flatworms Schistosoma mansoni and S. japonicum, but also contains several NHR encoding genes that are unique to this parasite. One of these, EmNHR1, is homolog to the DAF-12/HR-96 subfamily of NHRs which regulate cholesterol homeostasis in metazoans. Modified yeast-two hybrid analyses revealed that host serum contains a ligand which induces homodimerization of the EmNHR1 ligand-binding domain. Also, a HNF4-like homolog, EmHNF4, was characterized. Human HNF4 plays an important role in liver development. RT-PCR experiments showed that both isoforms of the EmHNF4 encoding gene are expressed stage-dependently suggesting distinct functions of the two isoforms in the parasite. Moreover, specific regulatory mechanisms on the convergence of NHR signaling and TGF-β/BMP signaling pathways in E. multilocularis have been identified. On the one hand, EmNHR1 directly interacted with the EmSmadC and on the other hand EmHNF4b interacted with EmSmadD, EmSmadE which are all downstream signaling components of the TGF-β/BMP signaling pathway. This suggests cross-communication in order to regulate target gene expression. With these results, further studies on the role of NHR signaling in the cestode will be facilitated. Also, the first serum-free in vitro cultivation system for E. multilocularis was established using PanserinTM401 as medium. Serum-free co-cultivation with RH-feeder cells and an axenic cultivation method have been established. With the help of this serum-free cultivation system investigations on the role of specific peptide hormones, like FGFs, or lipophilic/steroid hormones, like cholesterol, for the development of helminths will be much easier.
The bloodstream developmental forms of pathogenic African trypanosomes are uniquely susceptible to killing by small hydrophobic peptides. Trypanocidal activity is conferred by peptide hydrophobicity and charge distribution and results from increased rigidity of the plasma membrane. Structural analysis of lipid-associated peptide suggests a mechanism of phospholipid clamping in which an internal hydrophobic bulge anchors the peptide in the membrane and positively charged moieties at the termini coordinate phosphates of the polar lipid headgroups. This mechanism reveals a necessary phenotype in bloodstream form African trypanosomes, high membrane fluidity, and we suggest that targeting the plasma membrane lipid bilayer as a whole may be a novel strategy for the development of new pharmaceutical agents. Additionally, the peptides we have described may be valuable tools for probing the biosynthetic machinery responsible for the unique composition and characteristics of African trypanosome plasma membranes.
We study the symmetrised rank-one convex hull of monoclinic-I martensite (a twelve-variant material) in the context of geometrically-linear elasticity. We construct sets of T3s, which are (non-trivial) symmetrised rank-one convex hulls of 3-tuples of pairwise incompatible strains. Moreover we construct a five-dimensional continuum of T3s and show that its intersection with the boundary of the symmetrised rank-one convex hull is four-dimensional. We also show that there is another kind of monoclinic-I martensite with qualitatively different semi-convex hulls which, so far as we know, has not been experimentally observed. Our strategy is to combine understanding of the algebraic structure of symmetrised rank-one convex cones with knowledge of the faceting structure of the convex polytope formed by the strains.
Background: Multiple myeloma (MM) is a B-cell malignancy, where malignant plasma cells clonally expand in the bone marrow of older people, causing significant morbidity and mortality. Typical clinical symptoms include increased serum calcium levels, renal insufficiency, anemia, and bone lesions. With standard therapies, MM remains incurable; therefore, the development of new drugs or immune cell-based therapies is desirable. To advance the goal of finding a more effective treatment for MM, we aimed to develop a reliable preclinical MM mouse model applying sensitive and reproducible methods for monitoring of tumor growth and metastasis in response to therapy. Material and Methods: A mouse model was created by intravenously injecting bone marrow-homing mouse myeloma cells (MOPC-315.BM) that expressed luciferase into BALB/c wild type mice. The luciferase in the myeloma cells allowed in vivo tracking before and after melphalan treatment with bioluminescence imaging (BLI). Homing of MOPC-315.BM luciferase+ myeloma cells to specific tissues was examined by flow cytometry. Idiotype-specific myeloma protein serum levels were measured by ELISA. In vivo measurements were validated with histopathology. Results: Strong bone marrow tropism and subsequent dissemination of MOPC-315.BM luciferase+ cells in vivo closely mimicked the human disease. In vivo BLI and later histopathological analysis revealed that 12 days of melphalan treatment slowed tumor progression and reduced MM dissemination compared to untreated controls. MOPC-315.BM luciferase+ cells expressed CXCR4 and high levels of CD44 and a4b1 in vitro which could explain the strong bone marrow tropism. The results showed that MOPC-315.BM cells dynamically regulated homing receptor expression and depended on interactions with surrounding cells. Conclusions: This study described a novel MM mouse model that facilitated convenient, reliable, and sensitive tracking of myeloma cells with whole body BLI in living animals. This model is highly suitable for monitoring the effects of different treatment regimens.
Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy
(2012)
Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.
In effector T and B cells immune receptor signals induce within minutes a rise of intracellular Ca++, the activation of the phosphatase calcineurin and the translocation of NFAT transcription factors from cytosol to nucleus. In addition to this first wave of NFAT activation, in a second step the occurrence of NFATc1/αA, a short isoform of NFATc1, is strongly induced. Upon primary stimulation of lymphocytes the induction of NFATc1/αA takes place during the G1 phase of cell cycle. Due to an auto-regulatory feedback circuit high levels of NFATc1/αA are kept constant during persistent immune receptor stimulation. Contrary to NFATc2 and further NFATc proteins which dampen lymphocyte proliferation, induce anergy and enhance activation induced cell death (AICD), NFATc1/αA supports antigenmediated proliferation and protects lymphocytes against rapid AICD. Whereas high concentrations of NFATc1/αA can also lead to apoptosis, in collaboration with NF-κB-inducing co-stimulatory signals they support the survival of mature lymphocytes in late phases after their activation. However, if dysregulated, NFATc1/αA appears to contribute to lymphoma genesis and – as we assume – to further disorders of the lymphoid system. While the molecular details of NFATc1/αA action and its contribution to lymphoid disorders have to be investigated, NFATc1/αA differs in its generation and function markedly from all the other NFAT proteins which are expressed in lymphoid cells. Therefore, it represents a prime target for causal therapies of immune disorders in future.
The present antithrombotic drugs used to treat or prevent ischemic stroke have significant limitations: either they show only moderate efficacy (platelet inhibitors), or they significantly increase the risk for hemorrhages (thrombolytics, anticoagulants). Although most strokes are caused by thrombotic or embolic vessel occlusions, the pathophysiological role of platelets and coagulation is largely unclear. The introduction of novel transgenic mouse models and specific coagulation inhibitors facilitated a detailed analysis of molecular pathways mediating thrombus formation in models of acute ischemic stroke. Prevention of early platelet adhesion to the damaged vessel wall by blocking platelet surface receptors glycoprotein Ib alpha (GPIbα) or glycoprotein VI (GPVI) protects from stroke without provoking bleeding complications. In addition, downstream signaling of GPIbα and GPVI has a key role in platelet calcium homeostasis and activation. Finally, the intrinsic coagulation cascade, activated by coagulation factor XII (FXII), has only recently been identified as another important mediator of thrombosis in cerebrovascular disease, thereby disproving established concepts. This review summarizes the latest insights into the pathophysiology of thrombus formation in the ischemic brain. Potential clinical merits of novel platelet inhibitors and anticoagulants as powerful and safe tools to combat ischemic stroke are discussed.
Consider the situation where two or more images are taken from the same object. After taking the first image, the object is moved or rotated so that the second recording depicts it in a different manner. Additionally, take heed of the possibility that the imaging techniques may have also been changed. One of the main problems in image processing is to determine the spatial relation between such images. The corresponding process of finding the spatial alignment is called “registration”. In this work, we study the optimization problem which corresponds to the registration task. Especially, we exploit the Lie group structure of the set of transformations to construct efficient, intrinsic algorithms. We also apply the algorithms to medical registration tasks. However, the methods developed are not restricted to the field of medical image processing. We also have a closer look at more general forms of optimization problems and show connections to related tasks.
The visualization of energy functions is based on the possibility of separating different degrees of freedom. The most important one is the Born-Oppenheimer-approximation, which separates nucleus and electron movements. This allows the illustration of the potential energy as a function of the nuclei coordinates. Minima of the surface correspond to stable points like isomers or conformers. They are important for predicting the stability or thermodynamical of a system. Stationary points of first order correspond to transition points. They describe phase transitions, chemical reaction, or conformational changes. Furthermore, the partition function connects the potential hypersurface to the free energy of the system. The aim of the present work is the development and application of new approaches for the efficient exploration of multidimensional hypersurfaces. Initially, the Conformational Analysis and Search Tool (CAST) program was developed to create a basis for the new methods and algorithms. The development of CAST in object oriented C++ included, among other things, the implementation of a force field, different interfaces to external programs, analysis tools, and optimization libraries. Descriptions of an energy landscape require knowledge about the most stable minima. The Gradient Only Tabu Search (GOTS) has been shown to be very efficient in the optimization of mathematical test functions. Therefore, GOTS was taken as a starting point. Tabu-Search is based on the steepest descent - modest ascent strategy. The steepest descent is used for finding local minima, while the modest ascent is taken for leaving a minimum quickly. Furthermore, Tabu-Search is combined with an adaptive memory design to avoid cycling or returning. The highly accurate exploration of the phase space by Tabu-Search is often too expensive for complex optimization problems. Therefore, an algorithm for diversification of the search is required. After exploration of the proximity of the search space, the algorithm would guide the search to new and hopefully promising parts of the phase space. First application of GOTS to conformational search revealed weaknesses in the diversification search and the modest ascent part. On the one hand, the original methodology for diversification is insufficiently diverse. The algorithm is considerably improved by combining the more local GOTS with the wider searching Basin Hopping (BH) approach. The second weak point is a too inaccurate and inefficient modest ascent strategy. Analysis of common transition state search algorithms lead to the adaption of the Dimer-method to the Tabu-Search approach. The Dimer-method only requires the first derivatives for locating the closest transition state. For conformational search, dihedral angles are usually the most flexible degrees of freedom. Therefore, only those are used in the Dimer-method for leaving a local minimum. Furthermore, the exact localization of the reaction pathway and the transition state is not necessary as the local minimum position should only be departed as fast as possible. This allows for larger step sizes during the Dimer-search. In the following optimization step, all coordinates are relaxed to remove possible strains in the system. The new Tabu-Search method with Dimer-search delivers more and improved minima. Furthermore, the approach is faster for larger systems. For a system with approximately 1200 atoms, an acceleration of 40 was measured. The new approach was compared to Molecular Dynamics with optimization (MD), Simulated Annealing (SA), and BH with the help of conformational search problems of bio-organic systems. In all cases, a better performance was found. A comparison to the Monte Carlo Multiple Minima/Low Mode Sampling (MCMM/LM) method proved the outstanding performance of the new Tabu-Search approach. The solvation of the chignolin protein further revealed the possibility of uncovering discrepancies between the employed theoretical model and the experimental starting structure. Ligand optimization for improvement of x-ray structures was one further new application field. Besides the global optimization, the search for transition states and reaction pathways is also of paramount importance. These points describe different transitions of stable states. Therefore, a new approach for the exploration of such cases was developed. The new approach is based on a global minimization of a hyperplane being perpendicular to the reaction coordinate. Minima of this reduced phase space belong to traces of transition states between reactant and product states on the unchanged hypersurface. Optimization to the closest transition state using the Dimer-method delivers paths lying between the initial and the final state. An iterative approach finally yields complex reaction pathways with many intermediate local minima. The PathOpt algorithm was tested by means of rearrangements of argon clusters showing very promising results.
Background: Melatonin (MLT) has many health implications, therefore it is of valuable importance to develop specific analytical methods for determination of MLT in the presence of its main contaminant, N-{2-[1-({3-[2(acetylamino)ethyl]-5-methoxy-1H-indol-2-yl}methyl)-5-methoxy-1H-indol-3-yl]ethyl}acetamide (10). For development of these analytical methods, compound 10 had to be prepared in an adequate amount.
Results: Compound 10 was synthesized in six steps starting from 5-methoxyindole-2-carboxylic acid (1). Analytical performance of the proposed spectrofluorimetric methods was statistically validated with respect to linearity, accuracy, precision and specificity. The proposed methods were successfully applied for the assay of MLT in laboratory prepared mixtures containing up to 60 % of compound 10 and in commercial MLT tablets with recoveries not less than 99.00 %. No interference was observed from common pharmaceutical additives and the results were favorably compared with those obtained by a reference method.
Conclusions: This work describes simple, sensitive, and reliable second derivative spectrofluorimetric method in addition to two multivariate calibration methods, principal component regression (PCR) and partial least square (PLS), for the determination of MLT in the presence of compound 10.
Background: Melatonin (MLT) has many health implications, therefore it is of valuable importance to develop specific analytical methods for determination of MLT in the presence of its main contaminant, N-{2-[1-({3-[2-(acetylamino)ethyl]-5-methoxy-1H-indol-2-yl}methyl)-5-methoxy-1H-indol-3-yl]ethyl}acetamide (10). For development of these analytical methods, compound 10 had to be prepared in an adequate amount. Results: Compound 10 was synthesized in six steps starting from 5-methoxyindole-2-carboxylic acid (1). Analytical performance of the proposed spectrofluorimetric methods was statistically validated with respect to linearity, accuracy, precision and specificity. The proposed methods were successfully applied for the assay of MLT in laboratory prepared mixtures containing up to 60 % of compound 10 and in commercial MLT tablets with recoveries not less than 99.00 %. No interference was observed from common pharmaceutical additives and the results were favorably compared with those obtained by a reference method. Conclusions: This work describes simple, sensitive, and reliable second derivative spectrofluorimetric method in addition to two multivariate calibration methods, principal component regression (PCR) and partial least square (PLS), for the determination of MLT in the presence of compound 10.
The high failure rate of new drug candidates in preclinical or clinical studies due to hepatotoxicity represents a considerable problem in the drug development. Hence, there is an urgent need to develop new approaches for early and reliable prediction of drug-induced hepatotoxicity that enables a better identification of drug candidates with high potential for toxicity at early stages of drug development. Therefore, the aim of this work was to improve the prediction of drug-induced liver injury in preclinical studies through evaluation of more reliable and sensitive biomarkers of hepatotoxicity and a better understanding of the underlying mechanistic basis for drug-induced toxicity. First, the ability of a set of potential markers (NGAL, thiostatin, clusterin, PON1) to detect early signs of liver injury was assessed in rats treated with drug candidates that were dropped from further development, in part due to toxic adverse effects in the liver. In summary, PON1 and clusterin were not consistently altered in response to liver injury and thus provide no additive information to the traditional liver enzymes in detecting drug-induced hepatotoxicity. In contrast, thiostatin and NGAL were increased in serum and urine of treated animals in a time- and dose-dependent manner. These changes correlated well with mRNA expression in the target organ and generally reflected the onset and degree of drug-induced liver injury. Receiver-operating characteristics analyses supported serum thiostatin, but not NGAL, as a better indicator of drug-induced hepatobiliary injury than conventional clinical chemistry parameters, such as ALP, ALT and AST. Although thiostatin, an acute phase protein expressed in a range of tissues, may not be specific for liver injury, our results indicate that thiostatin may serve as a sensitive, minimally-invasive diagnostic marker of inflammation and tissue damage in preclinical safety assessment. In the second part of this work, combined application of genomics profiling technology and RNAi to inhibit the pharmacological target of a drug candidate BAY16, a glucagon receptor (GCGR) antagonist, was used to determine if interference with the pharmacological target plays a role in the toxic response to BAY16, and to narrow down those molecular changes that are associated with toxicity, and not the pharmacological action of BAY16. In contrast to Bay 16, which was found to be cytotoxic at concentrations of 75 µM, silencing of the glucagon receptor did not affect cell viability in primary rat hepatocytes. Thus, it can be concluded that hepatotoxicity of Bay 16 was not related to the drugs inhibitory effect on the glucagon receptor in vitro and in vivo. These findings were supported by the fact that most of BAY16-induced changes in gene expression occurred independently of the pharmacological modulation of GCGR. These off-target effects include altered xenobiotic metabolism, oxidative stress, increased fatty acid synthesis, and alterations in cholesterol and bile acid metabolic processes. Although it was not possible to draw a final conclusion about the mechanism of BAY16 hepatotoxicity, changes in these molecular mechanisms appear contribute to progression of hepatic injury. With regard to drug safety assessment in preclinical studies, the utilization of siRNA technology in vitro represents a new approach to improve mechanistic understanding of the nature of drug’s toxicity, being either chemically mediated or due to primary or secondary pharmacological mode of action.
We review the particle physics ingredients affecting the normalization, shape, and flavor composition of astrophysical neutrinos fluxes, such as different production modes, magnetic field effects on the secondaries muons, pions, and kaons, and flavor mixing, where we focus on p? interactions. We also discuss the interplay with neutrino propagation and detection, including the possibility to detect flavor and its application in particle physics, and the use of the Glashow resonance to discriminate p? from pp interactions in the source. We illustrate the implications on fluxes and flavor composition with two different models: 1 the target photon spectrum is dominated by synchrotron emission of coaccelerated electrons and 2 the target photon spectrum follows the observed photon spectrum of gamma-ray bursts. In the latter case, the multimessenger extrapolation from the gamma-ray fluence to the expected neutrino flux is highlighted.
The 7th International Symposium on Neuroprotection and Neurorepair was held from May 2nd to May 5th, 2012 in Potsdam, Germany. The symposium, which directly continues the successful Magdeburg meeting series, attracted over 330 colleagues from 29 countries to discuss recent findings and advances in the field. The focus of the 2012 symposium was widened from stroke and traumatic brain injury to neurodegenerative diseases, notably dementia, and more generally the ageing brain. Thereby, emphasis was given on neurovascular aspects of neurodegeneration and stroke including the blood–brain barrier, recent findings regarding the pathomechanism of Alzheimer’s disease, and brain imaging approaches. In addition, neurobiochemical aspects of neuroprotection, the role of astrogliosis, the clinical progress of cell-based approaches as well as translational hurdles and opportunities were discussed in-depth. This review summarizes some of the most stimulating discussions and reports from the meeting.
Neuroprotection aims to prevent salvageable neurons from dying. Despite showing efficacy in experimental stroke studies, the concept of neuroprotection has failed in clinical trials. Reasons for the translational difficulties include a lack of methodological agreement between preclinical and clinical studies and the heterogeneity of stroke in humans compared to homogeneous strokes in animal models. Even when the international recommendations for preclinical stroke research, the Stroke Academic Industry Roundtable (STAIR) criteria, were followed, we have still seen limited success in the clinic, examples being NXY-059 and haematopoietic growth factors which fulfilled nearly all the STAIR criteria. However, there are a number of neuroprotective treatments under investigation in clinical trials such as hypothermia and ebselen. Moreover, promising neuroprotective treatments based on a deeper understanding of the complex pathophysiology of ischemic stroke such as inhibitors of NADPH oxidases and PSD-95 are currently evaluated in preclinical studies. Further concepts to improve translation include the investigation of neuroprotectants in multicenter preclinical Phase III-type studies, improved animal models, and close alignment between clinical trial and preclinical methodologies. Future successful translation will require both new concepts for preclinical testing and innovative approaches based on mechanistic insights into the ischemic cascade.
Mice overexpressing proteolipid protein (PLP) develop a leukodystrophy-like disease involving cytotoxic, CD8+ T-lymphocytes. Here we show that these cytotoxic T-lymphocytes perturb retrograde axonal transport. Using fluorogold stereotactically injected into the colliculus superior, we found that PLP overexpression in oligodendrocytes led to significantly reduced retrograde axonal transport in retina ganglion cell axons. We also observed an accumulation of mitochondria in the juxtaparanodal axonal swellings, indicative for a disturbed axonal transport. PLP overexpression in the absence of T-lymphocytes rescued retrograde axonal transport defects and abolished axonal swellings. Bone marrow transfer from wildtype mice, but not from perforin- or granzyme B-deficient mutants, into lymphocyte-deficient PLP mutant mice led again to impaired axonal transport and the formation of axonal swellings, which are predominantly located at the juxtaparanodal region. This demonstrates that the adaptive immune system, including cytotoxic T-lymphocytes which release perforin and granzyme B, are necessary to perturb axonal integrity in the PLP-transgenic disease model. Based on our observations, so far not attended molecular and cellular players belonging to the immune system should be considered to understand pathogenesis in inherited myelin disorders with progressive axonal damage.
Imprinted genes play important roles in brain development. As the neural developmental capabilities of human parthenogenetic embryonic stem cells (hpESCs) with only a maternal genome were not assessed in great detail, hence here the potential of hpESCs to differentiate into various neural subtypes was determined. In addition DNA methylation and expression of imprinted genes upon neural differentiation was also investigated. The results demonstrated that hpESC-derived neural stem cells (hpNSCs) showed expression of NSC markers Sox1, Nestin, Pax6, and Musashi1 (MS1), the silencing of pluripotency genes (Oct4, Nanog) and the absence of activation of neural crest (Snai2, FoxD3) and mesodermal (Acta1) markers. Moreover, confocal images of hpNSC cultures exhibited ubiquitous expression of NSC markers Nestin, Sox1, Sox2 and Vimentin. Differentiating hpNSCs for 28 days generated neural subtypes with neural cell type-specific morphology and expression of neuronal and glial markers, including Tuj1, NeuN, Map2, GFAP, O4, Tau, Synapsin1 and GABA. hpNSCs also responded to region-specific differentiation signals and differentiated into regional phenotypes such as midbrain dopaminergic- and motoneuron-type cells. hpESC-derived neurons showed typical neuronal Na+/K+ currents in voltage clamp mode, elicited multiple action potentials with a maximum frequency of 30 Hz. Cell depicted a typical neuron-like current pattern that responded to selective pharmacological blockers of sodium (tetrodotoxin) and potassium (tetraethylammonium) channels. Furthermore, in hpESCs and hpNSCs the majority of CpGs of the differentially methylated regions (DMRs) KvDMR1 were methylated whereas DMR1 (H19/Igf2 locus) showed partial or complete absence of CpG methylation, which is consistent with a parthenogenetic (PG) origin. Upon differentiation parent-of-origin-specific gene expression was maintained in hpESCs and hpNSCs as demonstrated by imprinted gene expression analyses. Together this shows that despite the lack of a paternal genome, hpNSCs are proficient in differentiating into glial- and neuron-type cells, which exhibit electrical activity similar to newly formed neurons. Moreover, maternal-specific gene expression and imprinting-specific DNA-methylation are largely maintained upon neural differentiation. hpESCs are a means to generate histocompatible and disease allele-free ESCs. Additionally, hpESCs are a unique model to study the influence of imprinting on neurogenesis.
An important feature of addiction is the high drug craving that may promote the continuation of consumption. Environmental stimuli classically conditioned to drug-intake have a strong motivational power for addicts and can elicit craving. However, addicts differ in the attitudes towards their own consumption behavior: some are content with drug taking (consonant users) whereas others are discontent (dissonant users). Such differences may be important for clinical practice because the experience of dissonance might enhance the likelihood to consider treatment. This fMRI study investigated in smokers whether these different attitudes influence subjective and neural responses to smoking stimuli. Based on self-characterization, smokers were divided into consonant and dissonant smokers. These two groups were presented smoking stimuli and neutral stimuli. Former studies have suggested differences in the impact of smoking stimuli depending on the temporal stage of the smoking ritual they are associated with. Therefore, we used stimuli associated with the beginning (BEGIN-smoking-stimuli) and stimuli associated with the terminal stage (END-smoking-stimuli) of the smoking ritual as distinct stimulus categories. Stimulus ratings did not differ between both groups. Brain data showed that BEGIN-smoking-stimuli led to enhanced mesolimbic responses (amygdala, hippocampus, insula) in dissonant compared to consonant smokers. In response to END-smoking-stimuli, dissonant smokers showed reduced mesocortical responses (orbitofrontal cortex, subcallosal cortex) compared to consonant smokers. These results suggest that smoking stimuli with a high incentive value (BEGIN-smoking-stimuli) are more appetitive for dissonant than consonant smokers at least on the neural level. To the contrary, smoking stimuli with low incentive value (END-smoking-stimuli) seem to be less appetitive for dissonant smokers than consonant smokers. These differences might be one reason why dissonant smokers experience difficulties in translating their attitudes into an actual behavior change.
Background: Ascorbic acid demonstrates a cytotoxic effect by generating hydrogen peroxide, a reactive oxygen species (ROS) involved in oxidative cell stress. A panel of eleven human cancer cell lines, glioblastoma and carcinoma, were exposed to serial dilutions of ascorbic acid (5-100 mmol/L). The purpose of this study was to analyse the impact of catalase, an important hydrogen peroxide-detoxifying enzyme, on the resistance of cancer cells to ascorbic acid mediated oxidative stress. Methods: Effective concentration (EC50) values, which indicate the concentration of ascorbic acid that reduced the number of viable cells by 50%, were detected with the crystal violet assay. The level of intracellular catalase protein and enzyme activity was determined. Expression of catalase was silenced by catalase-specific short hairpin RNA (sh-RNA) in BT-20 breast carcinoma cells. Oxidative cell stress induced apoptosis was measured by a caspase luminescent assay. Results: The tested human cancer cell lines demonstrated obvious differences in their resistance to ascorbic acid mediated oxidative cell stress. Forty-five percent of the cell lines had an EC50>20 mmol/L and fifty-five percent had an EC50<20 mmol/L. With an EC50 of 2.6–5.5 mmol/L, glioblastoma cells were the most susceptible cancer cell lines analysed in this study. A correlation between catalase activity and the susceptibility to ascorbic acid was observed. To study the possible protective role of catalase on the resistance of cancer cells to oxidative cell stress, the expression of catalase in the breast carcinoma cell line BT-20, which cells were highly resistant to the exposure to ascorbic acid (EC50: 94,9 mmol/L), was silenced with specific sh-RNA. The effect was that catalase-silenced BT-20 cells (BT-20 KD-CAT) became more susceptible to high concentrations of ascorbic acid (50 and 100 mmol/L). Conclusions: Fifty-five percent of the human cancer cell lines tested were unable to protect themselves against oxidative stress mediated by ascorbic acid induced hydrogen peroxide production. The antioxidative enzyme catalase is important to protect cancer cells against cytotoxic hydrogen peroxide. Silenced catalase expression increased the susceptibility of the formerly resistant cancer cell line BT-20 to oxidative stress.
The nothotaxa of the genus Sedum series Rupestria Berger (Crassulaceae) are reviewed, three new hybrids (S. ×affomarcoi, S. ×henkii and S. ×pascalianum) are described and Sedum brevierei Chass. is typified. The identity of the western (French) populations of S. rupestre L. subsp. erectum ‘t Hart is also clarified, and a table with all known combinations is given.
Natalizumab is a recombinant monoclonal antibody raised against integrin alpha-4 (CD49d). It is approved for the treatment of patients with multiple sclerosis (MS), a chronic inflammatory autoimmune disease of the CNS. While having shown high therapeutic efficacy, treatment by natalizumab has been linked to progressive multifocal leukoencephalopathy (PML) as a serious adverse effect. Furthermore, drug cessation sometimes induces rebound disease activity of unknown etiology. Here we investigated whether binding of this adhesion-blocking antibody to T lymphocytes could modulate their phenotype by direct induction of intracellular signaling events. Primary CD4+ T lymphocytes either from healthy donors and treated with natalizumab in vitro or from MS patients receiving their very first dose of natalizumab were analyzed. Natalizumab induced a mild upregulation of IL-2, IFN-c and IL-17 expression in activated primary human CD4+ T cells propagated ex vivo from healthy donors, consistent with a pro-inflammatory costimulatory effect on lymphokine expression. Along with this, natalizumab binding triggered rapid MAPK/ERK phosphorylation. Furthermore, it decreased CD49d surface expression on effector cells within a few hours. Sustained CD49d downregulation could be attributed to integrin internalization and degradation. Importantly, also CD4+ T cells from some MS patients receiving their very first dose of natalizumab produced more IL-2, IFN-c and IL-17 already 24 h after infusion. Together these data indicate that in addition to its adhesion-blocking mode of action natalizumab possesses mild direct signaling capacities, which can support a pro-inflammatory phenotype of peripheral blood T lymphocytes. This might explain why a rebound of disease activity or IRIS is observed in some MS patients after natalizumab cessation.
Studying the function and malfunction of genes and proteins associated with inherited forms of peripheral neuropathies has provided multiple clues to our understanding of myelinated nerves in health and disease. Here, we have generated a mouse model for the peripheral neuropathy Charcot–Marie–Tooth disease type 4H by constitutively disrupting the mouse orthologue of the suspected culprit gene FGD4 that encodes the small RhoGTPase Cdc42-guanine nucleotide exchange factor Frabin. Lack of Frabin/Fgd4 causes dysmyelination in mice in early peripheral nerve development, followed by profound myelin abnormalities and demyelination at later stages. At the age of 60 weeks, this was accompanied by electrophysiological deficits. By crossing mice carrying alleles of Frabin/Fgd4 flanked by loxP sequences with animals expressing Cre recombinase in a cell type-specific manner, we show that Schwann cell-autonomous Frabin/Fgd4 function is essential for proper myelination without detectable primary contributions from neurons. Deletion of Frabin/Fgd4 in Schwann cells of fully myelinated nerve fibres revealed that this protein is not only required for correct nerve development but also for accurate myelin maintenance. Moreover, we established that correct activation of Cdc42 is dependent on Frabin/Fgd4 function in healthy peripheral nerves. Genetic disruption of Cdc42 in Schwann cells of adult myelinated nerves resulted in myelin alterations similar to those observed in Frabin/Fgd4-deficient mice, indicating that Cdc42 and the Frabin/Fgd4–Cdc42 axis are critical for myelin homeostasis. In line with known regulatory roles of Cdc42, we found that Frabin/Fgd4 regulates Schwann cell endocytosis, a process that is increasingly recognized as a relevant mechanism in peripheral nerve pathophysiology. Taken together, our results indicate that regulation of Cdc42 by Frabin/Fgd4 in Schwann cells is critical for the structure and function of the peripheral nervous system. In particular, this regulatory link is continuously required in adult fully myelinated nerve fibres. Thus, mechanisms regulated by Frabin/Fgd4–Cdc42 are promising targets that can help to identify additional regulators of myelin development and homeostasis, which may crucially contribute also to malfunctions in different types of peripheral neuropathies.
The "Large Hadron Collider" (LHC) is currently the most powerful particle accelerator. It provides particle collisions at a center of mass energy in the Tera-electronvolt range, which had never been reached in a laboratory before. Thereby a new era in high energy particle physics has began. Now it is possible to test one of the most precise theories in physics, the Standard Model of particle physics, at these high energies. The purpose is particularly served by four large experiments installed at the LHC, namely "A Toroidal LHC ApparatuS" (ATLAS), the "Compact-Muon-Solenoid" (CMS), the "Large Hadron Collider beauty" (LHCb) and "A Large Ion Collider Experiment" (ALICE). Besides exploring the high energy behavior of the well-established portions of the Standard Model, one of the main objectives is to find the Higgs boson included in the model, but not discovered by any preceding effort. It is of tremendous importance since fermions and heavy electroweak gauge bosons acquire mass because of this boson. Although the success of the Standard Model in describing nature is already undisputed, there are some flaws due to observations inexplicable within this theory only. Therefore searches for physics beyond the Standard Model are promoted at the LHC experiments as well. In order to achieve the defined goals, crucial aspects are firstly precise measurements, to verify Standard Model predictions in detail, and secondly an evaluation of as much information as accessible by the detectors, to recognize new phenomena as soon as possible for subsequent optimizations. Both challenges are only possible with a superior understanding of the detectors. An inevitable contribution to attain this knowledge is a realistic simulation, partially requiring new implementation techniques to describe the very complex instrumentation. The research presented here is performed under the patronage of the ATLAS collaboration with a special focus on measurements done with muon spectrometer. Thus a first central issue is the performance of the spectrometer in terms of physics objects that are recognized by the device, the compatibility of data and the existing simulation as well as its improvement and finally the extension of the acceptance region. Once the excellent behavior and comprehension of the muon spectrometer is demonstrated, a second part addresses one physics use case of reconstructed muons. The electroweak force is part of the Standard Model and causes the interaction of heavy electroweak gauge bosons with fermions as well as their self-interaction. In proton-proton collisions such gauge bosons are produced. However, they decay immediately into a pair of fermions. In case of the Z boson, which is one of the gauge bosons, oppositely charged fermions of the same generation, including muons, emerge. The various decay modes are determined precisely at particle accelerators other than the LHC. However, the associated production of two Z bosons is measured less exactly at those facilities because of a very low cross section. The corresponding results acquired with the ATLAS experiment exceed all previous measurements in terms of statistics and accuracy. They are reported in this thesis as obtained from the observation of events with four charged leptons. The enhancement of the signal yield based on the extension of the muon spectrometer acceptance is especially emphasized as well as alternative methods to estimate background events. Furthermore, the impact on the probing of couplings of three Z bosons and intersection with the search for the Standard Model Higgs boson are pointed out.
Resolving the physiological mechanisms by which rhizobacteria enhance plant growth is difficult, since many such bacteria contain multiple plant growth-promoting properties. To understand further how the 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCd)-containing rhizobacterium Variovorax paradoxus 5C-2 affects plant growth, the flows and partitioning of mineral nutrients and abscisic acid (ABA) and ABA metabolism were studied in pea (Pisum sativum) plants following rhizosphere bacterial inoculation. Although root architecture was not affected, inoculation increased root and shoot biomass, and stomatal conductance, by 20, 15, and 24%, respectively, and increased N, P, K, Ca, and Mg uptake by 16, 81, 50, 46, and 58%, respectively. P deposition in inoculated plant roots was 4.9 times higher than that in uninoculated controls. Rhizobacterial inoculation increased root to shoot xylem flows and shoot to root phloem flows of K by 1.8- and 2.1-fold, respectively. In control plants, major sinks for K deposition were the roots and upper shoot (43% and 49% of total uptake, respectively), while rhizobacterial inoculation increased K distribution to the lower shoot at the expense of other compartments (xylem, phloem, and upper shoot). Despite being unable to metabolize ABA in vitro, V. paradoxus 5C-2 decreased root ABA concentrations and accumulation by 40–60%. Although inoculation decreased xylem ABA flows, phloem ABA flows increased. Whether bacterial ACCd attenuates root to shoot ABA signalling requires further investigation, since ABA is critical to maintain growth of droughted plants, and ACCd-containing organisms have been advocated as a means of minimizing growth inhibition of plants in drying soil.
The present cumulative dissertation comprises three neuroimaging studies using different techniques, functional tasks and experimental variables of diverse nature to investigate human prefrontal cortex (PFC) (dys)function as well as methodological aspects of functional near-infrared spectroscopy (fNIRS). (1) Both dopamine (DA) availability (“inverted U-model”) and excitatory versus inhibitory DA receptor stimulation (“dual-state theory”) have been linked to PFC processing and cognitive control function. Electroencephalography (EEG) was recorded during a Go/NoGo response inhibition task in 114 healthy controls and 181 adult patients with attention-deficit/hyperactivity disorder (ADHD). As a neural measure of prefrontal cognitive response control the anteriorization of the P300 centroid in NoGo- relative to Go-trials (NoGo anteriorization, NGA) was investigated for the impact of genetic polymorphisms modulating catechol-O-methyltransferase efficiency (COMT, Val158Met) in degrading prefrontal DA and inhibitory DA receptor D4 sensitivity (DRD4, 48bp VNTR). Single genes and ADHD diagnosis showed no significant impact on the NGA or behavioral measures. However, a significant COMT×DRD4 interaction was revealed as subjects with relatively increased D4-receptor function (DRD4: no 7R-alleles) displayed an “inverted U”-relationship between the NGA and increasing COMT-dependent DA levels, whereas subjects with decreased D4-sensitivity (7R) showed a U-relationship. This interaction was supported by 7R-allele dose-effects and also reflected by an impact on task behavior, i.e. intraindividual reaction time variability. Combining previous theories of PFC DA function, neural stability at intermediate DA levels may be accompanied by the risk of overly decreased neural flexibility if inhibitory DA receptor function is additionally decreased. The findings of COMT×DRD4 epistasis might help to disentangle the genetic basis of dopaminergic mechanisms underlying prefrontal (dys)function. (2) While progressive neurocognitive impairments are associated with aging and Alzheimer's disease (AD), cortical reorganization might delay difficulties in effortful word retrieval, which is one of the earliest cognitive signs of AD. Therefore, cortical hemodynamic responses were measured with fNIRS during phonological and semantic verbal fluency, and investigated in 325 non-demented, healthy subjects (age: 51-82 years). The predictive value of age, sex, verbal fluency performance and years of education for the cortical hemodynamics was assessed using multiple regression analyses. Age predicted bilaterally reduced inferior frontal junction (IFJ) and increased middle frontal and supramarginal gyri activity in both task conditions. Years of education as well as sex (IFJ activation in females > males) partly predicted opposite effects on activation compared to age, while task performance was not a significant predictor. All predictors showed small effect sizes (-.24 < β < .22). Middle frontal and supramarginal gyri activity may compensate for an aging-related decrease in IFJ recruitment during verbal fluency. The findings of aging-related (compensatory) cortical reorganization of verbal fluency processing might, in combination with other (risk) factors and using longitudinal observations, help to identify neurodegenerative processes of Alzheimer's disease, while individuals are still cognitively healthy. (3) Individual anatomical or systemic physiological sources of variance may hamper the interpretation of fNIRS signals as neural correlates of cortical functions and their association with individual personality traits. Using simultaneous fNIRS and functional magnetic resonance imaging (fMRI) of hemodynamic responses elicited by an intertemporal choice task in 20 healthy subjects, variability in crossmodal correlations and divergence in associations of the activation with trait "sensitivity to reward" (SR) was investigated. Moreover, an impact of interindividual anatomy and scalp fMRI signal fluctuations on fNIRS signals and activation-trait associations was studied. Both methods consistently detected activation within right inferior/middle frontal gyrus, while fNIRS-fMRI correlations showed wide variability between subjects. Up to 41% of fNIRS channel activation variance was explained by gray matter volume (simulated to be) traversed by near-infrared light, and up to 20% by scalp-cortex distance. Extracranial fMRI and fNIRS time series showed significant temporal correlations at the temple. Trait SR was negatively correlated with fMRI but not fNIRS activation elicited by immediate rewards of choice within right inferior/middle frontal gyrus. Higher trait SR increased the correlation between extracranial fMRI signal fluctuations and fNIRS signals, suggesting that task-evoked systemic arousal-effects might be trait-dependent. Task-related fNIRS signals might be impacted by regionally and individually weighted sources of anatomical and systemic physiological error variance. Traitactivation correlations might be affected or biased by systemic physiological arousal-effects, which should be accounted for in future fNIRS studies of interindividual differences.
High background noise is an impediment to signal detection and perception. We report the use of multiple solutions to improve signal perception in the acoustic and visual modality by the Bornean rock frog, Staurois parvus. We discovered that vocal communication was not impaired by continuous abiotic background noise characterised by fast-flowing water. Males modified amplitude, pitch, repetition rate and duration of notes within their advertisement call. The difference in sound pressure between advertisement calls and background noise at the call dominant frequency of 5578 Hz was 8 dB, a difference sufficient for receiver detection. In addition, males used several visual signals to communicate with conspecifics with foot flagging and foot flashing being the most common and conspicuous visual displays, followed by arm waving, upright posture, crouching, and an open-mouth display. We used acoustic playback experiments to test the efficacy-based alerting signal hypothesis of multimodal communication. In support of the alerting hypothesis, we found that acoustic signals and foot flagging are functionally linked with advertisement calling preceding foot flagging. We conclude that S. parvus has solved the problem of continuous broadband low-frequency noise by both modifying its advertisement call in multiple ways and by using numerous visual signals. This is the first example of a frog using multiple acoustic and visual solutions to communicate in an environment characterised by continuous noise.
Multi-Wavelength Observations of the high-peaked BL Lacertae objects 1ES 1011+496 and 1ES 2344+514
(2012)
BL Lacertae objects belong to the most luminous sources in the Universe. They represent a subclass of active galactic nuclei with a spectrum that is dominated by non-thermal emission, extending from radio wavelengths to tera electronvolt (TeV) energies. The emission is strongly variable on time scales of years down to minutes, and arises from relativistic jets pointing at small angles to the line of sight of the observer, which is the reason for naming them “blazars”. Blazars are the dominant extragalactic source class in the radio, microwave and gamma-ray regime, are prime candidates for the origin of the Cosmic Rays and excellent laboratories to study black hole and jet physics as well as relativistic effects. Despite more than 20 years of observational efforts, the physical mechanisms driving their emission are not yet fully understood. So far, studies of their broad-band continuum emission were mostly concentrated on bright, flaring states. However, for a better understanding of the central engine powering the jets, the bias from flux-limited observations of the past must be overcome and their long-term average continuum spectral energy distributions (SEDs) must be determined. This work presents the first simultaneous multi-wavelength campaigns from the radio to the TeV regime of two high-frequency peaked BL Lacertae objects known to emit at TeV energies. The first source, 1ES 1011+496, was observed between February and May 2008, the second one, 1ES 2344+514, between September 2008 and February 2009. The extensive observational campaigns were organised independently from an external trigger for the presence of a flaring state. Since the duty cycle of major flux outbursts is known to be rather low, the campaigns were expected to yield SEDs representative of the long-term average emission. Central for this thesis is the analysis of data obtained with the MAGIC Cherenkov telescope, measuring energy spectra and light curves from ~0.1 to ~10 TeV. For the remaining instruments, observation time was proposed and additional data was organised by collaboration with the instrument teams by the author of this work. Such data was obtained mostly in a fully reduced state. Individual light curves are investigated as well as combined in a search for inter-band correlations. The data of both sources reveal a notable lack of a correlation between the emission at radio and optical wavelengths, indicating that the radio and short-wavelength emission arise in different regions of the jet. Quasi-simultaneous SEDs of two different flux states are observationally determined and described by a one-zone as well as a self-consistent two-zone synchrotron self-Compton model. First approaches to model the SEDs by means of a Chi2 minimisation technique are briefly discussed. The SEDs and the resulting model parameters, characterising the physical conditions in the emission regions, are compared to archival data. Though the models can describe the data well, for 1ES 1011+496 the model parameters indicate that in addition to the synchrotron and inverse-Compton emission of relativistic electrons, emission due to accelerated protons seems to be required. The SEDs of 1ES 2344+514 reveal one of the lowest activity states ever detected from the source. Despite that, the model parameters are not indicative of a distinct quiescent state, which may be caused by the degeneracy of the different parameters in one-zone models. Moreover, indications accumulate that the radiation can not be attributed to a single emission region. The results disfavour some of the current blazar classification schemes and the so-called “blazar sequence”, emphasising the need for a more realistic explanation of the systematics of the blazar SEDs in terms of fundamental parameters.
Magnetic resonance imaging can be used for preprocedural assessment of complex anatomy for radiofrequency (RF) ablations, e.g., in a univentricular heart. This case report features the treatment of a young patient with a functionally univentricular heart who suffered from persistent sudden onset tachycardia with wide complexes that required RF ablation as treatment.
Background
MRI of the lung is recommended in a number of clinical indications. Having a non-radiation alternative is particularly attractive in children and young subjects, or pregnant women.
Methods
Provided there is sufficient expertise, magnetic resonance imaging (MRI) may be considered as the preferential modality in specific clinical conditions such as cystic fibrosis and acute pulmonary embolism, since additional functional information on respiratory mechanics and regional lung perfusion is provided. In other cases, such as tumours and pneumonia in children, lung MRI may be considered an alternative or adjunct to other modalities with at least similar diagnostic value.
Results
In interstitial lung disease, the clinical utility of MRI remains to be proven, but it could provide additional information that will be beneficial in research, or at some stage in clinical practice. Customised protocols for chest imaging combine fast breath-hold acquisitions from a “buffet” of sequences. Having introduced details of imaging protocols in previous articles, the aim of this manuscript is to discuss the advantages and limitations of lung MRI in current clinical practice.
Conclusion
New developments and future perspectives such as motion-compensated imaging with self-navigated sequences or fast Fourier decomposition MRI for non-contrast enhanced ventilation- and perfusion-weighted imaging of the lung are discussed.
Main Messages
• MRI evolves as a third lung imaging modality, combining morphological and functional information.
• It may be considered first choice in cystic fibrosis and pulmonary embolism of young and pregnant patients.
• In other cases (tumours, pneumonia in children), it is an alternative or adjunct to X-ray and CT.
• In interstitial lung disease, it serves for research, but the clinical value remains to be proven.
• New users are advised to make themselves familiar with the particular advantages and limitations.
Background
Among the modalities for lung imaging, proton magnetic resonance imaging (MRI) has been the latest to be introduced into clinical practice. Its value to replace X-ray and computed tomography (CT) when radiation exposure or iodinated contrast material is contra-indicated is well acknowledged: i.e. for paediatric patients and pregnant women or for scientific use. One of the reasons why MRI of the lung is still rarely used, except in a few centres, is the lack of consistent protocols customised to clinical needs.
Methods
This article makes non-vendor-specific protocol suggestions for general use with state-of-the-art MRI scanners, based on the available literature and a consensus discussion within a panel of experts experienced in lung MRI.
Results
Various sequences have been successfully tested within scientific or clinical environments. MRI of the lung with appropriate combinations of these sequences comprises morphological and functional imaging aspects in a single examination. It serves in difficult clinical problems encountered in daily routine, such as assessment of the mediastinum and chest wall, and even might challenge molecular imaging techniques in the near future.
Conclusion
This article helps new users to implement appropriate protocols on their own MRI platforms.
Main Messages
• MRI of the lung can be readily performed on state-of-the-art 1.5-T MRI scanners.
• Protocol suggestions based on the available literature facilitate its use for routine
• MRI offers solutions for complicated thoracic masses with atelectasis and chest wall invasion.
• MRI is an option for paediatrics and science when CT is contra-indicated
Proton magnetic resonance imaging (MRI) has recently emerged as a clinical tool to image the lungs. This paper outlines the current technical aspects of MRI pulse sequences, radiofrequency (RF) coils and MRI system requirements needed for imaging the pulmonary parenchyma and vasculature. Lung MRI techniques are presented as a “technical toolkit”, from which MR protocols will be composed in the subsequent papers for comprehensive imaging of lung disease and function (parts 2 and 3). This paper is pitched at MR scientists, technicians and radiologists who are interested in understanding and establishing lung MRI methods. Images from a 1.5 T scanner are used for illustration of the sequences and methods that are highlighted.
Main Messages
• Outline of the hardware and pulse sequence requirements for proton lung MRI
• Overview of pulse sequences for lung parenchyma, vascular and functional imaging with protons
• Demonstration of the pulse-sequence building blocks for clinical lung MRI protocols
The actin cytoskeleton is essential for many cellular functions, such as the regulation of cell morphology, cell migration and vesicle transport processes. The functional diversity of actin structures is reflected in a variety of distinct molecular mechanisms regulating the polymerization of actin filaments. The spontaneous polymerization of actin however is inhibited, by both the instability of small actin oligomers and by actin monomer binding proteins, which prevent the formation of such oligomers. Actin nucleation factors help to overcome this kinetic barrier of filament initiation and are essential for the generation of novel actin filaments at specified subcellular compartments. Spir proteins are the founding members of the novel class of WH2 domain containing actin nucleation factors. They initiate actin polymerization by binding of actin monomers to four WH2 domains in the central part of the protein. Despite their ability to nucleate actin polymerization in vitro by themselves, Spir proteins form a regulatory complex with the distinct actin nucleators of the formin subgroup of formins. Spir functions in the regulation of vesicular originated filamentous actin structures, vesicle transport processes and the assembly of the cleavage furrow during asymmetric meiotic cell divisions. The mammalian genome encodes two spir genes, spir-1 and spir-2. The corresponding proteins have an identical structural array and share a high degree of homology. In order to elucidate the Spir function in developing and adult mouse tissues, the yet unknown expression of the mouse spir-2 gene was addressed. Real-time PCR analysis revealed highest expression of spir-2 in oocytes, the brain, throughout the gastrointestinal tract, testis and kidney of adult mice. In situ hybridizations were performed to substantiate the cellular nature of spir gene expression. During embryogenesis in situ hybridizations show spir-2 to be expressed in the developing nervous system and intestine. In adult mouse tissues highest expression of spir-2 was detected in the epithelial cells of the digestive tract, in neuronal cells of the nervous system and in spermatocytes. In contrast to the more restricted expression of the mouse spir-1 gene, which is mainly found in the nervous system, oocytes and testis, the data presented here show a distinct and broader expression pattern of the spir-2 gene and by this support a more general cell biological function of the novel actin nucleators. In order to address the function of Spir proteins in the developing and adult nervous system, Spir-1 deficient mice were generated by a gene trap method. Spir-1 deficient mice are viable and provide a perfect tool to address the neurobiological function of the Spir-1 protein. Analyses of primary cortical neurons from Spir-1 deficient mice revealed a specific reduction of dendritic branchpoints and are the first description of a neuronal Spir-1 function. Further, a transgenic mouse line (thy1-GFP-M) was employed that expresses the green fluorescent protein (GFP) under the control of neuron specific elements from the thy1 promoter. GFP is thereby expressed in only a subset of neurons and labels the neurons in their entirety. Spir-1 deficient mice carrying the GFP transgene were generated and analyzed. It was found that Spir-1 deficient mice exhibit a reduced number of dendritic spines in the entorhinal cortex compared to wildtype littermates. All together this study gives novel information about the cell biological function of Spir and provides insights how cytoskeletal functions structure the mammalian neuronal network.
We describe monozygotic twins discordant for childhood leukemia and secondary thyroid carcinoma. We used bisulfite pyrosequencing to compare the constitutive promoter methylation of BRCA1 and several other tumor suppressor genes in primary fibroblasts. The affected twin displayed an increased BRCA1 methylation (12%), compared with her sister (3%). Subsequent bisulfite plasmid sequencing demonstrated that 13% (6 of 47) BRCA1 alleles were fully methylated in the affected twin, whereas her sister displayed only single CpG errors without functional implications. This between-twin methylation difference was also found in irradiated fibroblasts and untreated saliva cells. The BRCA1 epimutation may have originated by an early somatic event in the affected twin: approximately 25% of her body cells derived from different embryonic cell lineages carry one epigenetically inactivated BRCA1 allele. This epimutation was associated with reduced basal protein levels and a higher induction of BRCA1 after DNA damage. In addition, we performed a genome-wide microarray analysis of both sisters and found several copy number variations, i.e., heterozygous deletion and reduced expression of the RSPO3 gene in the affected twin. This monozygotic twin pair represents an impressive example of epigenetic somatic mosaicism, suggesting a role for constitutive epimutations, maybe along with de novo genetic alterations in recurrent tumor development.
Early-life stress has been shown to influence the development of the brain and to increase the risk for psychiatric disorders later in life. Furthermore, variation in the human serotonin transporter (5-HTT, SLC6A4) gene is suggested to exert a modulating effect on the association between early-life stress and the risk for depression. At the basis of these gene x environment (G x E) interactions, epigenetic mechanisms, such as DNA-methylation, seem to represent the primary biological processes mediating early-life programming for stress susceptibility or resilience, respectively. The exact molecular mechanisms however remain to be elucidated, though. In the present study, we used two different stress paradigms to assess the molecular mechanisms mediating the relationship between early-life stress and disorders of emotion regulation later in life. First, a 5-Htt x prenatal stress (PS) paradigm was applied to investigate whether the effects of PS are dependent on the 5-Htt genotype. For this purpose, the effects of PS on cognition and anxiety- / depression-related behavior were examined using a maternal restraint stress paradigm of PS in C57BL/6 wild-type (WT) and heterozygous 5-Htt deficient (5-Htt+/-) mice. Additionally, in female offspring, a genome-wide hippocampal gene expression and DNA methylation profiling was performed using the Affymetrix GeneChip® Mouse Genome 430 2.0 Array and the AffymetrixGeneChip® Mouse Promoter 1.0R Array. Some of the resulting candidate genes were validated by quantitative real-time PCR. Further, the gene expression of these genes was measured in other brain regions of the PS animals as well as in the hippocampus of offspring of another, 5-Htt x perinatal stress (PeS) paradigm, in which pregnant and lactating females were stressed by an olfactory cue indicating infanticide. To assess resilience to PS and PeS, correlation studies between gene expression and behaviour were performed based on an initial performance-based LIMMA analysis of the gene expression microarray. 5-Htt+/- offspring of the PS paradigm showed enhanced memory performance and signs of reduced anxiety as compared to WT offspring. In contrast, exposure of 5-Htt+/- mice to PS was associated with increased depression-like behavior, an effect that tended to be more pronounced in female offspring. Further, 5-Htt genotype, PS and their interaction differentially affected the expression and DNA methylation of numerous genes and related pathways within the female hippocampus. Specifically, MAPK and neurotrophin signaling were regulated by both the 5-Htt+/- genotype and PS exposure, whereas cytokine and Wnt signaling were affected in a 5-Htt genotype x PS manner, indicating a gene x environment interaction at the molecular level. The candidate genes of the expression array could be validated and their expression patterns were partly consistent in the prefrontal cortex and striatum. Furthermore, the genotype effect of XIAP associated factor 1 (Xaf1) was also detected in the mice of the PeS paradigm. Concerning resilience, we found that the expression of growth hormone (Gh), prolactin (Prl) and fos-induced growth factor (Figf) were downregulated in WTPS mice that performed well in the forced swim test (FST). At the same time, the results indicated that Gh and Prl expression correlated positively with adrenal weight, whereas Figf expression correlated positively with basal corticosteron concentration, indicating an intricate relationship between depression-like behavior, hippocampal gene expression and the hypothalamo-pituitary-adrenal (HPA) axis activity. Correlation studies in the PeS animals revealed a link between Gh / Prl expression and anxiety-like behavior. In conclusion, our data suggest that although the 5-Htt+/- genotype shows clear adaptive capacity, 5-Htt+/- mice, particularly females, appear to be more vulnerable to developmental stress exposure when compared to WT offspring. Moreover, hippocampal gene expression and DNA methylation profiles suggest that distinct epigenetic mechanisms at the molecular level mediate the behavioral effects of the 5-Htt genotype, PS exposure, and their interaction. Further, resilience to early-life stress might be conferred by genes whose expression is linked to HPA axis function.
The sexual phase of Plasmodium falciparum begins with the differentiation of intraerythrocytic sexual stages, termed gametocytes, in the human host. Mature gametocytes circulate in the peripheral blood and are taken up by the mosquito during the blood meal. These stages are essential for the spread of the malaria disease and form gametes in the mosquito midgut within minutes. A highly conserved family of six secreted proteins has been identified in Plasmodium falciparum. They comprise multiple adhesive domains and are termed PfCCp1 through PfCCp5, and PfFNPA. It was revealed in this work that PfCCp multi-domain adhesion proteins form protein complexes in gametocytes and on the surface of newly emerged macrogametes by adhesion domain-mediated binding. Co-Immunoprecipitation assays with activated gametocyte lysates show interactions between PfCCp proteins and indicate surface association via Pfs230 and Pfs25. Pfs230 is connected with the plasma membrane of the parasite by its interaction partner Pfs48/45. This protein is linked to the plasma membrane by a GPI anchor and presumably retains the multi-protein complex on the surface of newly emerged macrogametes in the mosquito midgut. A WD40 domain containing protein was identified to be part of this protein complex. It might serve as platform for the assembly of the multi protein complex or mediate the interplay among proteins, as suggested from known functions of the WD40 domain repeats. During egress from the host erythrocyte, the emerging gametes become vulnerable to factors of the human complement, which is taken up with the blood meal. In this thesis it was found that the complement system is active for about one hour post feeding. Macrogametes defend against complement-mediated lysis by co-opting the human complement regulators Factor H and FHL-1 from the blood-meal. These serum proteins bind via its SCR domains 5-7 to the surface of macrogametes. Once bound, they trigger complement inactivation of the alternative pathway, which prevents induction of complement lysis on the surface of the malaria parasite. Antibodies against Factor H are able to impair the sexual development in vitro and are able to block transmission to the mosquito. Interaction studies on endogenous proteins and immobilized recombinant proteins revealed the PfGAP50 protein as binding partner of Factor H and FHL-1. This protein was hitherto described as a glideosome-associated protein in invasive parasite stages, but has not yet been characterized in gametes. First localization studies indicate a relocation of PfGAP50 from the inner membrane complex to the surface of macrogametes. Malaria still persists as one of the deadliest infectious diseases worldwide. Investigations on the essential transmissive stages, gametocytes and gametes of Plasmodium falciparum, stood in the background of research for a long time. This work deciphered details on protein interactions on the surface of the malaria parasite and provides first information about coactions between the parasite and the human complement in the mosquito midgut.
Upon synthesis, nascent polypeptide chains are subject to major rearrangements of their side chains to obtain an energetically more favorable conformation in a process called folding. About one third of all cellular proteins pass through the secretory pathway and undergo oxidative folding in the endoplasmic reticulum (ER). During oxidative folding, the conformational rearrangements are accompanied by the formation of disulfide bonds – covalent bonds between cysteine side chains that form upon oxidation. Protein disulfide isomerase (PDI) assists in the folding of substrates by catalyzing the oxidation of pairs of cysteine residues and the isomerization of disulfide bonds as well as by acting as chaperones. In addition to PDI itself, a family of related ER-resident proteins has formed. All PDI family members share the thioredoxin fold in at least one of their domains and exhibit a subset of the PDI activities. Despite many studies, the role of most PDI family members remains unclear. The project presented in this thesis was aimed to establish tools for the biochemical characterization of single members of the PDI family and their role in the folding process. A combination of fluorescence based assays was developed to selectively study single functions of PDI family members and relate their properties of either catalysis of oxidation or catalysis of isomerization or chaperone activity to the rest of the protein family. A binding assay using isothermal titration calorimetry (ITC) was established to complement the activity assays. Using ITC we could show for the first time that members of the PDI family can distinguish between folded and unfolded proteins selectively binding the latter. The unique information provided by this method also revealed a two-site binding of unfolded proteins by PDI itself. In addition to the functional characterization, experiments were conducted to further investigate the oligomeric state of PDI. We could show that the equilibrium between structurally different states of PDI is heavily influenced by the redox state of the protein and its environment. This new data could help to further our understanding of the interplay between oxidases like PDI and their regenerative enzymes like Ero1, which may be governed by structural changes in response to the change in redox status. Another structural approach was the screening of all investigated PDI family members for suitable crystallization conditions. As a result of this screening we could obtain protein crystals of human ERp27 and were able to solve the structure of this protein with X-ray crystallography. The structure gives insight into the mechanisms of substrate binding domains within the PDI family and helps to understand the interaction of ERp27 with the redox active ERp57. In collaboration with the group of Heike Hermanns we could further show the physiological importance of this interaction under oxidative stress. In conclusion, the project presented in this thesis provides novel tools for an extensive analysis of the activities of single PDI family members as well as a useful set of methods to characterize novel oxidoreductases and chaperones. The initial results obtained with the our novel methods are very promising. At the same time, the structural approach of this project could successfully solve the structure of a PDI family member and give information about the interplay within the PDI family.
Human adult cartilage is an aneural and avascular type of connective tissue, which consequently reflects reduced growth and repair rates. The main cell type of cartilage are chondrocytes, previously derived from human mesenchymal stem cells (hMSCs). They are responsible for the production and maintainance of the cartilaginous extracellular matrix (ECM), which consists mainly of collagen and proteoglycans. Signal transmission to or from chondrocytes, generally occurs via interaction with signalling factors connected to the cartilaginous ECM. In this context, proteins of the CCN family were identified as important matricellular and multifunctional regulators with high significance during skeletal development and fracture repair. In this thesis, main focus lies on WISP1/CCN4, which is known as a general survival factor in a variety of cell types and seems to be crucial during lineage progression of hMSCs into chondrocytes. We intend to counter the lack of knowledge about the general importance of WISP1-signalling within the musculoskeletal system and especially regarding cell death and survival by a variety of molecular and cell biology methods. First, we established a successful down-regulation of endogenous WISP1 transcripts within different cell types of the human musculoskeletal system through gene-silencing. Interestingly, WISP1 seems to be crucial to the survival of all examined cell lines and primary hMSCs, since a loss of WISP1 resulted in cell death. Bioinformatical analyses of subsequent performed microarrays (WISP1 down-regulated vs. control samples) confirmed this observation in primary hMSCs and the chondrocyte cell line Tc28a2. Distinct clusters of regulated genes, closely related to apoptosis induction, could be identified. In this context, TRAIL induced apoptosis as well as p53 mediated cell death seem to play a crucial role during the absence of WISP1 in hMSCs. By contrast, microarray analysis of WISP1 down-regulated chondrocytes indicated rather apoptosis induction via MAPK-signalling. Despite apoptosis relevant gene regulations, microarray analyses also identified clusters of differentially expressed genes of other important cellular activities, e.g. a huge cluster of interferon-inducible genes in hMSCs or gene regulations affecting cartilage homeostasis in chondrocytes. Results of this thesis emphasize the importance of regulatory mechanisms that influence cell survival of primary hMSCs and chondrocytes in the enforced absence of WISP1. Moreover, findings intensified the assumed importance for WISP1-signalling in cartilage homeostasis. Thus, this thesis generated an essential fundament for further examinations to investigate the role of WISP1-signalling in cartilage homeostasis and cell death.
Electrophysiological analyses conducted about 25 years ago detected two types of anion channels in the plasma membrane of guard cells. One type of channel responds slowly to changes in membrane voltage while the other responds quickly. Consequently, they were named SLAC, for SLow Anion Channel, and QUAC, for QUick Anion Channel. Recently, genes SLAC1 and QUAC1/ALMT12, underlying the two different anion current components, could be identified in the model plant Arabidopsis thaliana. Expression of the gene products in Xenopus oocytes confirmed the quick and slow current kinetics. In this study we provide an overview on our current knowledge on slow and quick anion channels in plants and analyze the molecular evolution of ALMT/QUAC-like and SLAC-like channels. We discovered fingerprints that allow screening databases for these channel types and were able to identify 192 (177 non-redundant) SLAC-like and 422 (402 non-redundant) ALMT/QUAC-like proteins in the fully sequenced genomes of 32 plant species. Phylogenetic analyses provided new insights into the molecular evolution of these channel types. We also combined sequence alignment and clustering with predictions of protein features, leading to the identification of known conserved phosphorylation sites in SLAC1-like channels along with potential sites that have not been yet experimentally confirmed. Using a similar strategy to analyze the hydropathicity of ALMT/QUAC-like channels, we propose a modified topology with additional transmembrane regions that integrates structure and function of these membrane proteins. Our results suggest that cross-referencing phylogenetic analyses with position-specific protein properties and functional data could be a very powerful tool for genome research approaches in general.
Alveolar echinococcosis (AE), a severe and life-threatening disease is caused by the small fox tapeworm Echinococcus multilocularis. Currently, the options of chemotherapeutic treatment are very limited and are based on benzimidazole compounds, which act merely parasitostatic in vivo and often display strong side effects. Therefore, new therapeutic drugs and targets are urgently needed. In the present work the role of two evolutionarily conserved signalling pathways in E. multilocularis, namely the insulin signalling cascade and Abl kinases, has been studied in regard to host-parasite interaction and the possible use in anti-AE chemotherapy.
Molecular characterization of antimicrobial peptide genes of the carpenter ant Camponotus floridanus
(2012)
The production of antimicrobial peptides (AMPs) is a major defense mechanism against pathogen infestation and of particular importance for insects relying exclusively on an innate immune system. Here, we report on the characterization of three AMPs from the carpenter ant Camponotus floridanus. Due to sequence similarities and amino acid composition these peptides can be classified into the cysteine-rich (e.g. defensin) and glycine-rich (e.g. hymenoptaecin) AMP groups, respectively. The gene and cDNA sequences of these AMPs were established and their expression was shown to be induced by microbial challenge. We characterized two different defensin genes. The defensin-2 gene has a single intron, whereas the defensin-1 gene has two introns. The deduced amino acid sequence of the C. floridanus defensins is very similar to other known ant defensins with the exception of a short C-terminal extension of defensin-1. The hymenoptaecin gene has a single intron and a very peculiar domain structure. The corresponding precursor protein consists of a signal- and a pro-sequence followed by a hymenoptaecin-like domain and six directly repeated hymenoptaecin domains. Each of the hymenoptaecin domains is flanked by an EAEP-spacer sequence and a RR-site known to be a proteolytic processing site. Thus, proteolytic processing of the multipeptide precursor may generate several mature AMPs leading to an amplification of the immune response. Bioinformatical analyses revealed the presence of hymenoptaecin genes with similar multipeptide precursor structure in genomes of other ant species suggesting an evolutionary conserved important role of this gene in ant immunity.
γ-Aminobutyric acid type A receptors (GABAARs) and glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the central nervous system. For proper synaptic function their precise localization and exact concentration within the neuronal surface membrane is essential. These properties are mediated by scaffolding proteins which directly contact the large intracellular loops of the receptors and tether them to cytoskeletal elements of the neuronal cells. In my thesis I deciphered the molecular details of several underlying protein-protein interactions, namely the interaction of a subset of GABAAR and GlyR subunits with the scaffolding proteins gephyrin, radixin and collybistin. I determined short linear motifs within the large intracellular loops of the receptors that directly engage in subunit specific scaffold protein interactions. My quantitative binding studies revealed that gephyrins E domain primarily recognizes the GABAAR α1 (Kd = 17 M) and α3 (Kd = 5 M) subunits, in contrast, the SH3 domain of collybistin mainly interacts with the GABAAR α2 subunit (Kd = 1 µM), while the FERM domain of radixin tightly binds to the GABAAR α5 subunit (Kd = 8 µM). My work additionally demonstrated that this simple relationship is complicated by (i) missing or (ii) overlapping binding specificities between the scaffold proteins and the receptor subunits. Moreover, this thesis addressed the possibility of (iii) posttranslational negative regulation as well as amplification generated by (iv) avidity effects as summarized below. (i) First, using biochemical methods I mapped the radixin-GABAAR α5 interaction in detail. My structural analysis and competition assays suggest that radixin mediates the receptor subunit binding via a universal binding site within the F3 subdomain of its FERM domain. This binding site is formed by an α-helix that offers a large hydrophobic pocket, which accepts a variety of different hydrophobic residues adopting different conformations, and a β-strand that readily engages in peptide backbone interactions. Not surprisingly, this binding site has been implicated in a wide variety of different scaffold interactions, thus emphasizing the importance of the essential FERM activation mechanism described earlier and suggesting additional pathways to allow tight regulation of this interaction. (ii) Next, I analyzed in detail the process of gephyrin-mediated GABAAR clustering. My X-ray crystallographic studies and binding assays revealed that gephyrin mediates binding of the GABAAR α1, α2 and α3 subunit via a universal binding site that also mediates the interactions with the GlyR β subunit. Using structure-guided mutagenesis I identified key residues within gephyrin and the receptor subunits that act as major contributors to the overall binding strength. Namely, two conserved aromatic residues within the N-terminal half of the receptor binding region engage in crucial hydrophobic interactions with gephyrin. Accordingly, J. Mukherjee from the group of our collaborator Steven J. Moss verified a substantial decrease in GABAAR cluster number and size in primary hippocampal neurons upon exchange of these residues within the GABAAR α2 subunit. Extension of my studies to collybistin (CB) revealed an overlapping but reciprocal subunit preference for this protein in comparison to gephyrin. The GABAAR α3 subunit exclusively binds gephyrin, in contrast the GABAAR α1 subunit mainly targets gephyrin (Kd = 17 µM) but additionally displays a moderate affinity (Kd ≈ 400 µM) towards the SH3 domain of CB. The GABAAR α2 subunit binds tightly to the SH3 domain of CB (Kd = 1 µM) and additionally displays a weak gephyrin affinity (Kd ≈ 500 µM). Notably, I could exclude the possibility of synergistic effects between gephyrins E domain, the SH3 domain of CB and the GABAAR α2 subunit. Instead, I found that the GABAAR α2 subunit binds gephyrin and CB in a mutually exclusive manner. These results suggest that CBs role in receptor clustering is solely determined by competing binding events of its constituting domains. Namely, the intra-molecular association between the PH/DH domain and the SH3 domain within CB competes with different inter-molecular interactions of CB: GABAAR α2 binding to the SH3 domain, PIP2 binding to the PH domain and gephyrin presumably binding to the PH and DH domain of CB. (iii) Interestingly, the receptor motifs, which have been mapped in my thesis to directly interact with the scaffold proteins, were shown in earlier studies to be posttranslationally modified in vivo. In particular, the GABAAR α1 and GlyR β subunits have been implicated as targets of the ERK/MAPK and PKC phosphorylation-pathways, respectively, while the GABAAR α5 subunit motif was shown to be ubiquitinated. In this dissertation, I analyzed Thr348, a possible ERK phosphorylation site within GABAAR α1. My binding assays verified a severe reduction of the direct gephyrin binding strength upon introduction of the respective phosphomimetic residue. The relevance of this in vitro result was highlighted by J. Mukherjee who confirmed a significant reduction in GABAAR cluster number and size upon introduction of the same mutation. The ERK/MAPK pathway is therefore a promising candidate for regulation of GABAergic transmission. (iv) In vivo, gephyrin presumably forms a multivalent scaffold, which is based on the self-association of its G (GephG) and E domains (GephE). Given the multimeric nature of gephyrin and the pentameric receptor architecture, I tested the possibility of avidity in the clustering of inhibitory neurotransmitter receptors. Cocrystallization of selected minimum peptides with GephE and their crystal structure analyses enabled me to define a receptor-derived peptide that offers a maximized gephyrin affinity. The structure of the GephE-GlyR receptor complex reveals two receptor-binding sites in close spatial vicinity (15 Å). I therefore designed bivalent peptides that enable to target both GephE sites at the same time and, as expected, a variety of biophysical methods verified an avidity-potentiated and unmatched high gephyrin affinity for these bidentate compounds. Notably, I could extend the dimerization approach to low affinity gephyrin ligands, namely short GABAAR-derived peptides that could not be studied using conventional monomeric ligands. Additionally, I verified that this compound specifically targets GephEs receptor binding site, and that it thereby inhibits its receptor binding activity. Further development of this molecule may offer the possibility to specifically analyze the effect of uncoupling the gephyrin-receptor interaction in cell culture-based assays, without altering protein function or expression level that accompanies conventional methods such as protein knock-out, RNA interference or the usage of antibodies.
Streptococcus pneumoniae is one of the major causes of bacterial meningitis, which mainly affects young infants in the developing countries of Africa, Asia (esp. India) and South America, and which has case fatality rates up to 50% in those regions. Bacterial meningitis comprises an infection of the meninges and the sub-meningeal cortex tissue of the brain, whereat the presence of pneumolysin (PLY), a major virulence factor of the pneumococcus, is prerequisite for the development of a severe outcome of the infection and associated tissue damage (e. g. apoptosis, brain edema, and ischemia). Pneumolysin belongs to the family of pore forming, cholesterol-dependent cytolysins (CDCs), bacterial protein toxins, which basically use membrane-cholesterol as receptor and oligomerize to big aggregates, which induce cell lysis and cell death by disturbance of membrane integrity. Multiple recent studies, including this work, have revealed a new picture of pneumolysin, whose cell-related properties go far beyond membrane binding, pore formation and the induction of cell death and inflammatory responses. For a long time, it has been known that bacteria harm the tissues of their hosts in order to promote their own survival and proliferation. Many bacterial toxins aim to rather hijack cells than to kill them, by interacting with cellular components, such as the cytoskeleton or other endogenous proteins. This study was able to uncover a novel capacity of pneumolysin to interact with components of the actin machinery and to promote rapid, actin-dependent cell shape changes in primary astrocytes. The toxin was applied in disease-relevant concentrations, which were verified to be sub-lytic. These amounts of toxin induced a rapid actin cortex collapse in horizontal direction towards the cell core, whereat membrane integrity was preserved, indicating an actin severing function of pneumolysin, and being consistent with cell shrinkage, displacement, and blebbing observed in live cell imaging experiments. In contrast to neuroblastoma cells, in which pneumolysin led to cytoskeleton remodeling and simultaneously to activation of Rac1 and RhoA, in primary astrocytes the cell shape changes were seen to be primarily independent of small GTPases. The level of activated Rac1 and RhoA did not increase at the early time points after toxin application, when the initial shape changes have been observed, but at later time points when the actin-dependent displacement of cells was slower and less severe, probably presenting the cell’s attempt to re-establish proper cytoskeleton function. A GUV (giant unilamellar vesicle) approach provided insight into the effects of pneumolysin in a biomimetic system, an environment, which is strictly biochemical, but still comprises cellular components, limited to the factors of interest (actin, Arp2/3, ATP, and Mg2+ on one side, and PLY on the other side). This approach was able to show that the wildtype-toxin, but not the Δ6 mutant (mutated in the unfolding domain, and thus non-porous), had the capacity to exhibit its functions through a membrane bilayer, meaning it was able to aggregate actin, which was located on the other side of the membrane, either via direct interaction with actin or in an Arp2/3 activating manner. Taking a closer look at these two factors with the help of several different imaging and biochemical approaches, this work unveiled the capacity of pneumolysin to bind and interact both with actin and Arp2 of the Arp2/3 complex. Pneumolysin was capable to slightly stabilize actin in an actin-pyrene polymerization assay. The same experimental setup was applied to show that the toxin had the capacity to lead to actin polymerization through activation of the Arp2/3 complex. This effect was additionally confirmed with the help of fluorescent microscopy of rhodamine (TRITC)-tagged actin. Strongest Arp2/3 activation, and actin nucleation/polymerization is achieved by the VCA domain of the WASP family proteins. However, addition of PLY to the Arp2/3–VCA system led to an enhanced actin nucleation, suggesting a synergistic activation function of pneumolysin. Hence, two different effects of pneumolysin on the actin cytoskeleton were observed. On the one hand an actin severing property, and on the other hand an actin stabilization property, both of which do not necessarily exclude each other. Actin remodeling is a common feature of bacterial virulence strategies. This is the first time, however, that these properties were assigned to a toxin of the CDC family. Cytoskeletal dysfunction in astrocytes leads to dysfunction and unregulated movement of these cells, which, in context of bacterial meningitis, can favor bacterial penetration and spreading in the brain tissue, and thus comprises an additional role of pneumolysin as a virulence factor of Streptococcus pneumonia in the context of brain infection.
Late-stage age-related macular degeneration (AMD) is a common sight-threatening disease of the central retina affecting approximately 1 in 30 Caucasians. Besides age and smoking, genetic variants from several gene loci have reproducibly been associated with this condition and likely explain a large proportion of disease. Here, we developed a genetic risk score (GRS) for AMD based on 13 risk variants from eight gene loci. The model exhibited good discriminative accuracy, area-under-curve (AUC) of the receiver-operating characteristic of 0.820, which was confirmed in a cross-validation approach. Noteworthy, younger AMD patients aged below 75 had a significantly higher mean GRS (1.87, 95% CI: 1.69-2.05) than patients aged 75 and above (1.45, 95% CI: 1.36-1.54). Based on five equally sized GRS intervals, we present a risk classification with a relative AMD risk of 64.0 (95% CI: 14.11-1131.96) for individuals in the highest category (GRS 3.44-5.18, 0.5% of the general population) compared to subjects with the most common genetic background (GRS -0.05-1.70, 40.2% of general population). The highest GRS category identifies AMD patients with a sensitivity of 7.9% and a specificity of 99.9% when compared to the four lower categories. Modeling a general population around 85 years of age, 87.4% of individuals in the highest GRS category would be expected to develop AMD by that age. In contrast, only 2.2% of individuals in the two lowest GRS categories which represent almost 50% of the general population are expected to manifest AMD. Our findings underscore the large proportion of AMD cases explained by genetics particularly for younger AMD patients. The five-category risk classification could be useful for therapeutic stratification or for diagnostic testing purposes once preventive treatment is available.
Sensory limitation plays an important role in the evolution of animal behaviour. Animals have to find objects of interest (e.g. food, shelters, predators). When sensory abilities are strongly limited, animals adjust their behaviour to maximize chances for success. Bats are nocturnal, live in complex environments, are capable of flight and must confront numerous perceptual challenges (e.g. limited sensory range, interfering clutter echoes). This makes them an excellent model for studying the role of compensating behaviours to decrease costs of finding resources. Cavity roosting bats are especially interesting because the availability of tree cavities is often limited, and their quality is vital for bats during the breeding season. From a bat's sensory point of view, cavities are difficult to detect and finding them requires time and energy. However, tree cavities are also long lasting, allowing information transfer among conspecifics. Here, we use a simple simulation model to explore the benefits of tree selection, memory and eavesdropping (compensation behaviours) to searches for tree cavities by bats with short and long perception range. Our model suggests that memory and correct discrimination of tree suitability are the basic strategies decreasing the cost of roost finding, whereas perceptual range plays a minor role in this process. Additionally, eavesdropping constitutes a buffer that reduces the costs of finding new resources (such as roosts), especially when they occur in low density. We conclude that natural selection may promote different strategies of roost finding in relation to habitat conditions and cognitive skills of animals.
The transcription factor Miz1 forms repressive DNA-binding complexes with the Myc, Gfi-1 and Bcl-6 oncoproteins. Known target genes of these complexes encode the cyclin-dependent kinase inhibitors (CKIs) cdkn2b (p15\(^{Ink4}\)), cdkn1a (p21\(^{Cip1}\)), and cdkn1c (p57\(^{Kip2}\)). Whether Miz1-mediated repression is important for control of cell proliferation in vivo and for tumor formation is unknown. Here we show that deletion of the Miz1 POZ domain, which is critical for Miz1 function, restrains the development of skin tumors in a model of chemically-induced, Ras-dependent tumorigenesis. While the stem cell compartment appears unaffected, interfollicular keratinocytes lacking functional Miz1 exhibit a reduced proliferation and an accelerated differentiation of the epidermis in response to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Tumorigenesis, proliferation and normal differentiation are restored in animals lacking cdkn1a, but not in those lacking cdkn2b. Our data demonstrate that Miz1-mediated attenuation of cell cycle arrest pathways via repression of cdkn1a has a critical role during tumorigenesis in the skin.
Nephrogenic Systemic Fibrosis is a rare condition appearing only in patients with severe renal impairment or failure and presents with dermal lesions and involvement of internal organs. Although many cases are mild, an estimated 5 % have a progressive debilitating course. To date, there is no known effective treatment thus stressing the necessity of ample prevention measures. An association with the use of Gadolinium based contrast agents (GBCA) makes Nephrogenic Systemic Fibrosis a potential side effect of contrast enhanced magnetic resonance imaging and offers the opportunity for prevention by limiting use of gadolinium based contrast agents in renal failure patients. In itself toxic, Gadolinium is embedded into chelates that allow its safe use as a contrast agent. One NSF theory is that Gadolinium chelates distribute into the extracellular fluid compartment and set Gadolinium ions free, depending on multiple factors among which the duration of chelates exposure is directly related to the renal function. Major medical societies both in Europe and in North America have developed guidelines for the usage of GBCA. Since the establishment of these guidelines and the increased general awareness of this condition, the occurrence of NSF has been nearly eliminated. Giving an overview over the current knowledge of NSF pathobiochemistry, pathogenesis and treatment options this review focuses on the guidelines of the European Medicines Agency, the European Society of Urogenital Radiology, the FDA and the American College of Radiology from 2008 up to 2011 and the transfer of this knowledge into every day practice.
When a key press causes a stimulus, the key press is perceived later and the stimulus earlier than key presses and stimuli presented independently. This bias in time perception has been linked to the intention to produce the effect and thus been called intentional binding (IB). In recent studies it has been shown that the IB effect is stronger when participants believed that they caused the effect stimulus compared to when they believed that another person caused the effect (Desantis et al., 2011). In this experiment we ask whether causal beliefs influence the perceived time of an effect when the putative effect occurs temporally close to another stimulus that is also an effect. In our study two participants performed the same task on connected computers with separate screens. Each trial started synchro- nously on both computers. When a participant pressed a key, a red and a yellow stimulus appeared as action effects simultaneously or with a slight delay of up to 50 ms. The partic- ipants’ task was to judge the temporal order of these two effect stimuli. Participants were either told that one participant caused one of the two stimuli while the other participant seated at the other computer caused the other stimulus, or each participant was told that he/she caused both stimuli. The different causal beliefs changed the perceived time of the effects’ appearance relative to each other. When participants believed they each caused one effect, their “own” effect was perceived earlier than the other participant’s effect. When the participants believed each caused both effects, no difference in the perceived temporal order of the red and yellow effect was found. These results confirm that higher order causal beliefs change the perceived time of an action effect even in a setting in which the occurrence of the putative effect can be directly compared to a reference stimulus.
MicroRNAs (miRs) are small non- coding RNA molecules controlling a plethora of biological processes such as development, cellular survival and senescence. We here determined miRs differentially regulated during cardiac postnatal development and aging. Cardiac function, morphology and miR expression profiles were determined in neonatal, 4 weeks, 6 months and 19 months old normotensive male healthy C57/Bl6N mice. MiR-22 was most prominently upregulated during cardiac aging. Cardiac expression of its bioinformatically predicted target mimecan (osteoglycin, OGN) was gradually decreased with advanced age. Luciferase reporter assays validated mimecan as a bona fide miR-22 target. Both, miR-22 and its target mimecan were co- expressed in cardiac fibroblasts and smooth muscle cells. Functionally, miR-22 overexpression induced cellular senescence and promoted migratory activity of cardiac fibroblasts. Small interference RNA-mediated silencing of mimecan in cardiac fibroblasts mimicked the miR-22-mediated effects. Rescue experiments revealed that the effects of miR-22 on cardiac fibroblasts were only partially mediated by mimecan. In conclusion, miR-22 upregulation in the aging heart contributed at least partly to accelerated cardiac fibroblast senescence and increased migratory activity. Our results suggest an involvement of miR-22 in age-associated cardiac changes, such as cardiac fibrosis.
Within the scope of this thesis two main topics have been investigated: the examination of micromagnetic sensors and transport of massive and massless Dirac fermions in HgTe quantum wells. For the investigation of localized, inhomogeneous magnetic fields, the fabrication and characterization of two different non-invasive and ultra sensitive sensors has been established at the chair ”Experimentelle Physik” of the University of Würzburg. The first sensor is based on the young technique named micro-Hall magnetometry. The necessary semiconductor devices (Hall cross structures) were fabricated by high-resolution electron beam lithography based on two different two dimensional electron gases (2DEGs), namely InAs/(Al,Ga)Sb- and HgTe/(Hg,Cd)Te- heterostructures. The characteristics have been examined in two different ways. Measurements in homogeneous magnetic fields served for characterization of the sensors, whereas the investigation of artificially produced sub-µm magnets substantiates the suitability of the devices for the study of novel nanoscale magnetic materials (e.g. nanowires). Systematic experiments with various magnets are in accordance with the theory of single-domain particles and anisotropic behavior due to shapes with high aspect ratio. The highest sensitivity for strongly localized fields was obtained at T = 4.2 K for a (200x200) nm^2 Hall cross - made from shallow, high mobility HgTe 2DEG. Although the field resolution was merely δB ≈ 100 µT, the nanoscale sensor size yields an outstanding flux resolution of δΦ = 2 10^(−3) Φ0, where Φ0 = h/2e is the flux quantum. Translating this result in terms of magnetic moment, the sensitivity allows for the detection of magnetization changes of a particle centered on top of the sensor as low as δM ≈ 10^2 µB, with the magnetic moment of a single electron µB, the Bohr magneton. The further examination of a permalloy nanomagnet with a cross-section of (100x20) nm^2 confirms the expected resolution ability, extracted from the noise of the sensor. The observed high signal-to-noise ratio validates the detection limit of this sensor in terms of geometry. This would be reached for a magnet (same material) with quadratic cross-section for an edge length of 3.3 nm. Moreover, the feasibility of this sensor for operation in a wide temperature range (T = mK... > 200 K) and high magnetic fields has been confirmed. The second micromagnetic sensor is the micro-SQUID (micro-Superconducting-QUantum-Interference-Device) based on niobium. The typical sensor area of the devices built in this work was (1.0x1.0) µm^2, with constrictions of about 20 nm. The characterization of this device demonstrates an amazing field sensitivity (regarding its size) of δB < 1 µT. Even though the sensor was 25 times larger than the best micro-Hall sensor, it provided an excellent flux resolution in the order of δΦ ≈ 5 10^(−4) Φ0 and a similar magnetic moment resolution of δM ≈ 10^2 µB. Furthermore, the introduction of an ellipsoidal permalloy magnet (axes: 200 nm and 400 nm, thickness 30 nm) substantiates the suitability for the detection of minuscule, localized magnetic fields. The second part of the thesis deals with the peculiar transport properties of HgTe quantum wells. These rely on the linear contribution to the band structure inherent to the heterostructure. Therefore the system can be described by an effective Dirac Hamiltonian, whose Dirac mass is tunable by the variation of the quantum well thickness. By fabrication and characterization of a systematical series of substrates, a system with vanishing Dirac mass (zero energy gap) has been confirmed. This heterostructure therefore resembles graphene (a monolayer of graphite), with the difference of exhibiting only one valley in the energy dispersion of the Brillouin zone. Thus parasitical intervalley scattering cannot occur. The existence of this system has been proven by the agreement of theoretical predictions, based on widely accepted band structure calculations with the experiment (Landau level dispersion, conductivity). Furthermore, another particularity of the band structure - the transition from linear to parabolic character - has been illustrated by the widths of the plateaus in the quantum Hall effect. Finally, the transport of ”massive” Dirac fermions (with finite Dirac mass) is investigated. In particular the describing Dirac Hamiltonian induces weak localization effects depending on the Dirac mass. This mechanism has not been observed to date, and survives in higher temperatures compared to typical localization mechanisms.
Time-resolved spectroscopic studies of energy transfer between molecules in solution form a basis for both, our understanding of fundamental natural processes like photosynthesis as well as directed synthetic approaches to optimize organic opto-electronic devices. Here, coherent two-dimensional (2D) spectroscopy opens up new possibilities, as it reveals the correlation between absorption and emission frequency and hence the full cause-and-effect chain. In this thesis two optical setups were developed and implemented, permitting the recording of electronic 2D spectra in the visible and in the hitherto unexplored ultraviolet spectral range. Both designs rely on the exclusive manipulation of beam pairs, which reduces the signal modulation to the difference between the transition frequency of the system and the laser frequency. Thus - as has been shown experimentally and theoretically - the timing precision as well as mechanical stability requirements are greatly reduced, from fractions of the oscillation period of the exciting light wave to fractions of the pulse duration. Two-dimensional spectroscopy and femtosecond transient absorption (TA) as well as different theoretical approaches and simulation models were then applied to coupled multichromophore systems of increasing complexity. Perylene bisimide-perylene monoimide dyads were investigated in cooperation with Prof. Dr. Frank Würthner and Prof. Dr. Bernd Engels at the University of Würzburg. In these simplest systems studied, global analysis of six different TA experiments unequivocally revealed an ultrafast interchromophoric energy transfer in the 100 fs range. Comparison between the obtained transfer rates and the predictions of Förster theory suggest a breakdown of this point-transition-dipole-based picture at the donor-acceptor distances realized in our compounds. Furthermore, a model including conformational changes and an interchromophoric charge transfer has been derived to consistently describe the observed pico- to nanosecond dynamics and fluorescence quantum yields. A second collaboration with Prof. Dr. Gregory Scholes (University of Toronto, Canada) and Prof. Dr. Paul Burn (University of Queensland, Australia) addressed the photophysics of a series of uorene-carbazole dendrimers. Here, a combination of 2D-UV spectroscopy and femtosecond ansiotropy decay experiments revealed the initial delocalization of the excited state wave function that saturates with the second generation. In room temperature solution, disorder-induced localization takes place on the time scales comparable to our instrument response, i.e. 100 fs, followed by energy transfer via incoherent hopping processes. Lastly, in tubular zinc chlorin aggregates, semi-synthetic analogues of natural lightharvesting antennae that had again been synthesized in the group of Prof. Dr. Frank Würthner, the interchromophoric coupling is so strong that coherently coupled domains prevail even at room temperature. From an analysis of intensity-dependent TA measurements the dimensions of these domains, the exciton delocalization length, could be determined to span 5-20 monomers. In addition, 2D spectra uncovered efficient energy transfer between neighboring domains, i.e. ultrafast exciton diffusion.
In this study, a double-donor concept is used to improve the performance of thermally evaporated merocyanine(s)/C60 bulk heterojunction (BHJ) solar cells. It is shown that the co-evaporation of two merocyanine dyes with absorption bands at ~ 500 nm (SW dye) and ~ 650 nm (LW dye), respectively, together with C60 fullerene results in an improvement of open-circuit voltage (VOC), short-circuit current (JSC) as well as total power conversion efficiency (PCE) compared to the best single-donor cell. The enhancement of JSC is attributed to a higher photon harvesting efficiency of the mixed-donor devices due to a better spectral coverage.
Chlamydiales are obligate intracellular gram-negative bacteria that have gained high medical relevance. These important human pathogens cause diverse diseases including trachoma and wide spread sexually transmitted diseases. Chlamydia establishes membrane bound inclusions in the host cell and loots the host for nutritional requirements. Infections are usually recognized by the host immune system and eliminated systematically, by triggering apoptosis. However, the pathogen Chlamydia has evolved various strategies to prevent the detection as well as protect the invaded cell against apoptosis or any other form of cell death. 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. The present study was aimed at investigating 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. Caspases were differentially regulated upon Sn infection. Caspase-3 and -9 were not activated upon Sn infection and apoptosis induction; whereas caspases-8 was activated in Sn infected cells even without apoptosis induction. This indicates that, Sn utilizes death receptor association independent caspase activation for thriving in the host environment. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Proteins (IAP-1/2 and XIAP) and the Akt/PI3K pathway. Sn infection also 20 activated the pro-survival transcription factor NF-кB. Blocking any of these survival pathways sensitized the infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. The NF-кB mutant cells also showed reduced infectivity of Sn, which indicated an essential role of NF-кB in Sn infection. It was interesting to observe that, Acanthamoeba castellanii, a natural host of Sn, survived maintaining its trophozoite forms after infection with Sn upon starvation. The metacaspases, responsible for encystment could be regulated by Sn upon infection. This suggests an early level of gene regulation indicating how the pathogen evolved its ability to inhibit apoptosis in higher organisms. The resistance to apoptosis pathways subverted in Sn-infected cells was similar but not identical to those modulated by Chlamydia. Together, the data supports the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.
SUMMARY Mast cell activation in allergic and inflammatory disease causes increased vascular permeability and edema. This thesis identifies a paracrine mechanism, by which heparin released from intracellular granules, is involved in mast cell-evoked alteration of endothelial barrier function in vivo. Negatively charged heparin initiated factor XII-driven contact activation. Activated factor XII triggered the formation of the inflammatory mediator bradykinin in plasma. Congenital deficiency and pharmacological targeting of factor XII and kinin B2 receptor provided protection from mast cell-heparin-induced leukocyte-endothelial adhesion and hypotension in rats and mice. Intravital laser scanning microscopy and tracer measurements showed that heparin increased leakage with fluid extravasation in skin microvessels in mice. Deficiency in factor XII or kinin B2 receptor conferred resistance to heparin-induced skin edema and largely protected mice from endothelial barrier dysfunction, caused by allergen-induced mast cell activation and anaphylactic reactions. In contrast, heparin and mast cell activation caused excessive edema formation in mice, deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Hereditary angioedema patients, lacking C1 esterase inhibitor, suffered from allergeninduced edema. The data indicate that mast cell-heparin-initiated bradykinin formation plays a fundamental role in defective barrier function of pathological mast cell-mediated inflammation, hypotension and edema formation.