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- Theodor-Boveri-Institut für Biowissenschaften (105)
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Sonstige beteiligte Institutionen
- Clinical Trial Center (CTC) / Zentrale für Klinische Studien Würzburg (ZKSW) (3)
- Röntgen Center for Complex Material Systems (RCCM), Am Hubland, 97074 W¨urzburg, Germany (2)
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- Department of Paediatric Radiology, Institute of Diagnostic and Interventional Radiology, Josef-Schneider-Straße 2, Wuerzburg 97080, Germany (1)
ResearcherID
- I-5818-2014 (1)
The composition of stable-isotope labelled isotopologues/isotopomers in metabolic products can be measured by mass spectrometry and supports the analysis of pathways and fluxes. As a prerequisite, the original mass spectra have to be processed, managed and stored to rapidly calculate, analyse and compare isotopomer enrichments to study, for instance, bacterial metabolism in infection. For such applications, we provide here the database application ‘Isotopo’. This software package includes (i) a database to store and process isotopomer data, (ii) a parser to upload and translate different data formats for such data and (iii) an improved application to process and convert signal intensities from mass spectra of \(^{13}C\)-labelled metabolites such as tertbutyldimethylsilyl-derivatives of amino acids. Relative mass intensities and isotopomer distributions are calculated applying a partial least square method with iterative refinement for high precision data. The data output includes formats such as graphs for overall enrichments in amino acids. The package is user-friendly for easy and robust data management of multiple experiments.
A novel magnetic ground state is reported for the Hubbard Hamiltonian in strained graphene. When the chemical potential lies close to the Dirac point, the ground state exhibits locally both the Neel and ferromagnetic orders, even for weak Hubbard interaction. Whereas the Neel order parameter remains of the same sign in the entire system, the magnetization at the boundary takes the opposite sign from the bulk. The total magnetization vanishes this way, and the magnetic ground state is globally only an antiferromagnet. This peculiar ordering stems from the nature of the strain-induced single-particle zero-energy states, which have support on one sublattice of the honeycomb lattice in the bulk, and on the other sublattice near the boundary of a finite system. We support our claim with the self-consistent numerical calculation of the order parameters, as well as by the Monte Carlo simulations of the Hubbard model in both uniformly and nonuniformly strained honeycomb lattice. The present result is contrasted with the magnetic ground state of the same Hubbard model in the presence of a true magnetic field (and for vanishing Zeeman coupling), which is exclusively Neel ordered, with zero local magnetization everywhere in the system.
Mutations in the oncogenic PIK3CA gene are found in 10-20% of colorectal cancers (CRCs) and are associated with poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic TRAIL death receptor antibodies emerged as promising anti-neoplastic therapeutics, but to date failed to prove their capability in the clinical setting as especially primary tumors exhibit high rates of TRAIL resistance. In our study, we investigated the molecular mechanisms underlying TRAIL resistance in CRC cells with a mutant PIK3CA (PIK3CA-mut) gene. We show that inhibition of the constitutively active phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway only partially overcame TRAIL resistance in PIK3CA-mut-protected HCT116 cells, although synergistic effects of TRAIL plus PI3K, Akt or cyclin-dependent kinase (CDK) inhibitors could be noted. In sharp contrast, TRAIL triggered full-blown cell death induction in HCT116 PIK3CA-mut cells treated with proteasome inhibitors such as bortezomib and MG132. At the molecular level, resistance of HCT116 PIK3CA-mut cells against TRAIL was reflected by impaired caspase-3 activation and we provide evidence for a crucial involvement of the E3-ligase X-linked inhibitor of apoptosis protein (XIAP) therein. Drugs interfering with the activity and/or the expression of XIAP, such as the second mitochondria-derived activator of caspase mimetic BV6 and mithramycin-A, completely restored TRAIL sensitivity in PIK3CA-mut-protected HCT116 cells independent of a functional mitochondrial cell death pathway. Importantly, proteasome inhibitors and XIAP-targeting agents also sensitized other CRC cell lines with mutated PIK3CA for TRAIL-induced cell death. Together, our data suggest that proteasome-or XIAP-targeting drugs offer a novel therapeutic approach to overcome TRAIL resistance in PIK3CA-mutated CRC.
Words are built from smaller meaning bearing parts, called morphemes. As one word can contain multiple morphemes, one morpheme can be present in different words. The number of distinct words a morpheme can be found in is its family size. Here we used Birth-Death-Innovation Models (BDIMs) to analyze the distribution of morpheme family sizes in English and German vocabulary over the last 200 years. Rather than just fitting to a probability distribution, these mechanistic models allow for the direct interpretation of identified parameters. Despite the complexity of language change, we indeed found that a specific variant of this pure stochastic model, the second order linear balanced BDIM, significantly fitted the observed distributions. In this model, birth and death rates are increased for smaller morpheme families. This finding indicates an influence of morpheme family sizes on vocabulary changes. This could be an effect of word formation, perception or both. On a more general level, we give an example on how mechanistic models can enable the identification of statistical trends in language change usually hidden by cultural influences.
This thesis explores the influence of social and environmental cues on the nest building behavior of leaf-cutting ants. Especially, the investigations are aimed at evaluating the mechanisms of nest building and how the nest environment can spatially guide building responses that lead to an adaptive nest architecture. The emergence of nest chambers in the nest of the leaf-cutting ant Acromyrmex lundi were evaluated. Rather than excavating nest chambers in advance, at places where workers encounter suitable environmental conditions for brood and fungus rearing, these items have to be present at a site. When presented in the laboratory with a choice between two otherwise identical digging sites, offering suitable environmental conditions, but one containing brood, the workers displayed a higher excavation activity at the site where they encountered the putative content of a chamber. The shape of the excavated cavity was also more round and chamber-like. It is concluded that leaf-cutting ants respond to social cues during nest building. Excavation is a costly process and colonies have to spend a part of their energy stores on nest building, so that regulatory responses for the control of nest excavation are expected to occur. Worker density at the beginning of the digging process influenced digging activity while the presence of in-nest stores did not. Stored brood and fungus did however influence the architecture of the excavated nest, leading to the excavation of larger chambers and smaller tunnels. While self-organized mechanisms appear to be involved in the nest building process, the social cues of the ants’ environment during building clearly influence the nest architecture and lead to an adjustment of the nest size to the current space needs of the colony. Workers secondarily regulated nest size by the opportunistic refilling of unused space with excavated soil pellets. As the ants should provide suitable conditions for brood and fungus rearing, they should show a behavioral response to CO2 concentrations, as the gas is known to hinder fungus respiration. Workers of A. lundi did indeed avoid high CO2-levels for fungus rearing but actually preferred CO2-values in the range encountered close to the soil surface, where this species excavates their nests. However, different CO2-levels did not affect their excavation behavior. While fungus chambers make up part of a leaf-cutting ant nest, most leaf-cutting ants of the genus Atta also spent part of the colony’s energy on excavating large, voluminous chambers for waste disposal, rather than scattering the material aboveground. It is expected that leaf-cutting ants also show environmental preferences for waste management. In experiments Atta laevigata workers preferred deposition in a warm and dry environment and showed no preference for specific CO2-levels. The continued accumulation of waste particles in a waste chamber seems to be based on the use of volatiles. These originate from the waste itself, and seem to be used as an orientation cue by workers relocating the material. The ensuing large accumulation of waste at one site should result in the emergence of more voluminous chambers for waste disposal.
Objective: The aim of this longitudinal study was to identify predictors of instantaneous well-being in patients with amyotrophic lateral sclerosis (ALS). Based on flow theory well-being was expected to be highest when perceived demands and perceived control were in balance, and that thinking about the past would be a risk factor for rumination which would in turn reduce well-being.
Methods: Using the experience sampling method, data on current activities, associated aspects of perceived demands, control, and well-being were collected from 10 patients with ALS three times a day for two weeks.
Results: Results show that perceived control was uniformly and positively associated with well-being, but that demands were only positively associated with well-being when they were perceived as controllable. Mediation analysis confirmed thinking about the past, but not thinking about the future, to be a risk factor for rumination and reduced well-being.
Discussion: Findings extend our knowledge of factors contributing to well-being in ALS as not only perceived control but also perceived demands can contribute to well-being. They further show that a focus on present experiences might contribute to increased well-being.
Despite improved survival in the Rituximab (R) era, a considerable number of patients with diffuse large B-cell lymphoma (DLBCL) ultimately die from the disease. Functional imaging using [18F]fluorodeoxyglucose-PET is suggested for assessment of residual viable tumor very early during treatment but is compromised by non-specific tracer retention in inflammatory lesions. The PET tracer [18F]fluorodeoxythymidine (FLT) as surrogate marker of tumor proliferation may overcome this limitation. We present results of a prospective clinical study testing FLT-PET as superior and early predictor of response to chemotherapy and outcome in DLBCL. 54 patients underwent FLT-PET prior to and one week after the start of R-CHOP chemotherapy. Repetitive FLT-PET imaging was readily implemented into the diagnostic work-up. Our data demonstrate that the reduction of FLT standard uptake valuemean (SUVmean) and SUVmax one week after chemotherapy was significantly higher in patients achieving complete response (CR, n=48; non-CR, n=6; p<0.006). Martingale-residual and Cox proportional hazard analyses showed a significant monotonous decrease of mortality risk with increasing change in SUV. Consistent with these results, early FLT-PET response showed relevant discriminative ability in predicting CR. In conclusion, very early FLT-PET in the course of R-CHOP chemotherapy is feasible and enables identification of patients at risk for treatment failure.
Background and Purpose
In animal models, von Willebrand factor (VWF) is involved in thrombus formation and propagation of ischemic stroke. However, the pathophysiological relevance of this molecule in humans, and its potential use as a biomarker for the risk and severity of ischemic stroke remains unclear. This study had two aims: to identify predictors of altered VWF levels and to examine whether VWF levels differ between acute cerebrovascular events and chronic cerebrovascular disease (CCD).
Methods
A case–control study was undertaken between 2010 and 2013 at our University clinic. In total, 116 patients with acute ischemic stroke (AIS) or transitory ischemic attack (TIA), 117 patients with CCD, and 104 healthy volunteers (HV) were included. Blood was taken at days 0, 1, and 3 in patients with AIS or TIA, and once in CCD patients and HV. VWF serum levels were measured and correlated with demographic and clinical parameters by multivariate linear regression and ANOVA.
Results
Patients with CCD (158±46%) had significantly higher VWF levels than HV (113±36%, P<0.001), but lower levels than AIS/TIA patients (200±95%, P<0.001). Age, sex, and stroke severity influenced VWF levels (P<0.05).
Conclusions
VWF levels differed across disease subtypes and patient characteristics. Our study confirms increased VWF levels as a risk factor for cerebrovascular disease and, moreover, suggests that it may represent a potential biomarker for stroke severity, warranting further investigation.
The interim reporting process provides decision-useful information to investors and market participants. However the legal circumstances of external interim auditor reviews differ worldwide. A mandatory review rule in the US as opposed to a contrary decision of the German legislator raises the question of the cost-benefit-relation of auditor reviews. Using a German sample of 1,023 firm-year observations from 2007 to 2010, I extract the costs and the benefits of voluntary semi-annual reviews. The unique German legal environment makes it possible to split the cost effect of a review in the price effect (included in audit-related fees) and a possible reduction of audit fees resulting from an improved year-around audit process. I observe a significant increase of audit and audit-related fees of around 14.5% (total fee effect). Additionally, the study provides evidence on declining audit fees for reviewed firms as compared to a matched sample of non-reviewed firms. The effect of an interim review on quarterly earnings quality – using discretionary accruals as an earning management proxy – shows no significant influence.
This study investigates the effect of the error announcement risk on the demand for voluntary interim auditor reviews. Material changes in the German legal environment in 2007 introduced an enforcement system for semi-annual financial reports. The demand for voluntary semi-annual reviews increased significantly from 0.8% in 2006 to 14.6% in 2007 and increased further to 19.5% until 2010 for a sample of 1,278 firm-year observations. This study addresses the question whether the enforcement structure and the resulting error announcement risk exposure have an influence on voluntary external monitoring. After controlling for agency costs, the corporate governance structure, and selected review cost factors, results of a logistic regression analysis show a positive influence of error announcement risk on the likelihood of engaging an auditor to review the semi-annual interim report. The findings contribute to the literature by demonstrating that the quality of the enforcement system and the risk of error findings influence the review decision of the board of directors positively.
In 2004 German legislation established the Financial Reporting Enforcement Panel. In 147 cases since then, the panel has ordered the announcement of errors in previously disclosed and audited financial statements of German firms. We use this unique dataset to evaluate the consequences of increasing earnings management over time on enforcement releases and their recognition in audit fees. Ettredge et al. (2010) provide evidence on a phenomenon called ‘balance sheet bloat’ that is due to income increasing earnings management and later influences the disclosure of misstated financial statements. Thus, the evidence of earnings management recognition in audit fees (Abbott et al. 2006) and the hypothesis of future information content in fees by Stanley (2011) leads us to hypothesize that auditors recognize increasing audit risk in audit fees before the enforcement process starts. We extend related earnings management and audit fee literature by modeling the development of earnings management within the misstatement firms and systematically link it to auditor reactions. We find significant predictive power of different commonly used accrual measures for enforcement releases in the period prior and up to the misstatement period. In this period of time, we also observe an audit fee increase, e.g. the recognition of increased audit risk. We investigate an audit fee effect after the misstatement period but find no significant relation.
Background: Different parameters have been determined for prediction of treatment outcome in hepatitis c virus genotype 1 infected patients undergoing pegylated interferon, ribavirin combination therapy. Results on the importance of vitamin D levels are conflicting. In the present study, a comprehensive analysis of vitamin D levels before and during therapy together with single nucleotide polymorphisms involved in vitamin D metabolism in the context of other known treatment predictors has been performed.
Methods: In a well characterized prospective cohort of 398 genotype 1 infected patients treated with pegylated interferon-alpha and ribavirin for 24-72 weeks (INDIV-2 study) 25-OH-vitamin D levels and different single nucleotide polymorphisms were analyzed together with known biochemical parameters for a correlation with virologic treatment outcome.
Results: Fluctuations of more than 5 (10) ng/ml in 25-OH-vitamin D-levels have been observed in 66 (39) % of patients during the course of antiviral therapy and neither pretreatment nor under treatment 25-OH-vitamin D-levels were associated with treatment outcome. The DHCR7-TT-polymorphism within the 7-dehydrocholesterol-reductase showed a significant association (P = 0.031) to sustained viral response in univariate analysis. Among numerous further parameters analyzed we found that age (OR = 1.028, CI = 1.002-1.056, P = 0.035), cholesterol (OR = 0.983, CI = 0.975-0.991, P<0.001), ferritin (OR = 1.002, CI = 1.000-1.004, P = 0.033), gGT (OR = 1.467, CI = 1.073-2.006, P = 0.016) and IL28B-genotype (OR = 2.442, CI = 1.271-4.695, P = 0.007) constituted the strongest predictors of treatment response.
Conclusions: While 25-OH-vitamin D-levels levels show considerable variations during the long-lasting course of antiviral therapy they do not show any significant association to treatment outcome in genotype 1 infected patients.
Modulation of sensorimotor rhythms (SMR) was suggested as a control signal for brain-computer interfaces (BCI). Yet, there is a population of users estimated between 10 to 50% not able to achieve reliable control and only about 20% of users achieve high (80–100%) performance. Predicting performance prior to BCI use would facilitate selection of the most feasible system for an individual, thus constitute a practical benefit for the user, and increase our knowledge about the correlates of BCI control. In a recent study, we predicted SMR-BCI performance from psychological variables that were assessed prior to the BCI sessions and BCI control was supported with machine-learning techniques. We described two significant psychological predictors, namely the visuo-motor coordination ability and the ability to concentrate on the task. The purpose of the current study was to replicate these results thereby validating these predictors within a neurofeedback based SMR-BCI that involved no machine learning.Thirty-three healthy BCI novices participated in a calibration session and three further neurofeedback training sessions. Two variables were related with mean SMR-BCI performance: (1) a measure for the accuracy of fine motor skills, i.e., a trade for a person’s visuo-motor control ability; and (2) subject’s “attentional impulsivity”. In a linear regression they accounted for almost 20% in variance of SMR-BCI performance, but predictor (1) failed significance. Nevertheless, on the basis of our prior regression model for sensorimotor control ability we could predict current SMR-BCI performance with an average prediction error of M = 12.07%. In more than 50% of the participants, the prediction error was smaller than 10%. Hence, psychological variables played a moderate role in predicting SMR-BCI performance in a neurofeedback approach that involved no machine learning. Future studies are needed to further consolidate (or reject) the present predictors.
Staphylococcus aureus is a major threat to public health systems all over the globe. This second most cause of nosocomial infections is able to provoke a wide variety of different types of infection in humans and animals, ranging from superficial skin and skin structure infections to invasive disease like sepsis or pneumonia. But not enough, this pathogen is also notorious in acquiring and/or developing resistance to antimicrobial compounds, thus limiting available treatment options severely. Therefore, development of new compounds and strategies to fight S. aureus is of paramount importance. But since only 1 out of 5 compounds, which entered clinical trials, becomes a drug, the preclinical evaluation of promising compounds has to be reconsidered, too. The aim of this thesis was to address both sides of this problem: first, to improve preclinical testing by incorporating in vivo imaging technologies to the preclinical testing procedure in order to acquire additional and clearer data about efficacy of promising compounds and second, by evaluating lysostaphin, which is a promising, new option to fight S. aureus infections.
The first aim of this thesis focused on the establishment of a dual modality in vivo imaging platform, consisting of Bioluminescence Imaging (BLI) and Magnetic Resonance Imaging (MRI), to offer detailed insights into the course and gravity of S. aureus infection in the murine thigh infection model. Since luciferase-expressing S. aureus strains were generated in former studies and enabled thus bioluminescence imaging of bacterial infection, this technology should be implemented into the compound evaluation platform in order to non-invasively track the bacterial burden over time. MRI, in contrast, was only rarely used in earlier studies to visualize and measure the course of infection or efficacy of anti-bacterial therapy. Thus, the first set of experiments was performed to identify benefits and drawbacks of visualizing S. aureus infections in the mouse model by different MR methods. Native, proton-based MR imaging showed in this regard increased T2 relaxation times in the infected thigh muscles, but it was not possible to define a clear border between infected and uninfected tissue. Iron oxide nanoparticles and perfluorocarbon emulsions, two MR contrast agents or tracer, in contrast, offered this distinction. Iron oxide particles were detected in this regard by their distortion of 1H signal in proton-based MRI, while perfluorocarbon emulsion was identified by 19F MRI. Mammals do not harbor sufficient intrinsic amounts of 19F to deliver specific signal and therefore, 19F MR imaging visualizes only the signal of administered perfluorocarbon emulsion. The in vivo accumulation of perfluorocarbon emulsion can be imaged by 19F MRI and overlayed on a simultaneously acquired 1H MR image, which shows the anatomical context in clear detail. Since this is advantageous compared to contrast agent based MR methods like iron oxide particle-based MRI, further experiments were performed with perfluorocarbon emulsions and 19F MRI.
Experimental studies to elucidate the accumulation of perfluorocarbon emulsion at the site of infection showed robust 19F MR signals after administration between day 2 and at least day 8 p.i.. Perfluorocarbon emulsion accumulated in all investigated mice in the shape of a ‘hollow sphere’ at the rim of the abscess area and the signal remained stable as long as the infection prevailed. In order to identify the mechanism of accumulation, flow cytometry, cell sorting and histology studies were performed. Flow cytometry and cell sorting analysis of immune cells at the site of infection showed that neutrophils, monocytes, macrophages and dendritic cells carried contrast media at the site of infection with neutrophils accounting for the overwhelming portion of perfluorocarbon signal. In general, most of the signal was associated with immune cells, thus indicating specific immune cell dependent accumulation. Histology supported this observation since perfluorocarbon emulsion related fluorescence could only be visualized in close proximity to immune cell nuclei.
After establishing and testing of 19F MRI with perfluorocarbon emulsions as infection imaging modality, the effects of antibiotic therapy upon MR signal was investigated in order to evaluate the capability of this modality for preclinical testing procedure. Thus, the efficacy of vancomycin and linezolid, two clinically highly relevant anti - S. aureus compounds, were tested in the murine thigh infection model. Both of them showed reduction of the colony forming units and bioluminescence signal, but also of perfluorocarbon emulsion accumulation strength and volume at the site of infection, which was visualized and quantified by 19F MRI. The efficacy pattern with linezolid being more efficient in clearing bacterial infection was shown similarly by all three methods. In consequence, 19F MRI with perfluorocarbon emulsion as MR tracer proved to be capable to visualize antibacterial therapy in preclinical testing models.
The next step was consequently to evaluate a promising new compound against S. aureus infections. Thus, lysostaphin, an endo-peptidase that cleaves the cell wall of S. aureus, was tested in different concentrations alone or in combination with oxacillin for efficacy in murine thigh and catheter associated infection models. Lysostaphin only in the concentration of 5 mg/kg body weight or combined with oxacillin in the concentration of 2 mg/kg showed strong reduction of bacterial burden by colony forming unit determination and bioluminescence imaging in both models. The perfluorocarbon accumulation was investigated in the thigh infection model by 19F MRI and was strongly reduced in terms of volume and signal strength in both above-mentioned groups. In general, lysostaphin showed comparable or superior efficacy than vancomycin or oxacillin alone. Therefore, further development of lysostaphin for the treatment of S. aureus infections is recommended by these experiments. Overall, the antibiotic efficacy pattern of all applied antibiotic regimens was similar with all three applied methods, demonstrating the usefulness of MRI for antibiotic efficacy testing. Importantly, treatment with oxacillin either alone or in combination with lysostaphin resulted in stronger perfluorocarbon emulsion accumulation at the site of infection than expected compared to the results from bioluminescence imaging and colony forming unit determination. This might be an indication for immunomodulatory properties of oxacillin.
Further murine infection experiments demonstrated in this context a differential release of cytokine and chemokines in the infected thigh muscle in dependence of the applied antibacterial therapy. Especially treatment with oxacillin, but to a less degree with minocycline or linezolid, too, exhibited high levels of various cytokines and chemokines, although they reduced the bacterial burden efficiently. In consequence, possible immunomodulatory effects of antibacterial compounds have to be taken into account for future applications of imaging platforms relying on the visualization of the immune response. However, this observation opens a new field for these imaging modalities since it might be extraordinary interesting to study the immunomodulatory effects of compounds or even bacterial factors in vivo. And finally, a two modality imaging platform which combines methods to visualize on the one hand the bacterial burden and on the other hand the immune response offers an innovative, new platform to study host-pathogen interaction in vivo in a non-invasive fashion.
In summary, it could be shown that perfluorocarbon emulsions accumulate in immune cells at the site of infection in the murine S. aureus thigh infection model. The accumulation pattern shapes a ‘hollow sphere’ at the rim of the abscess area and its size and perfluorocarbon content is dependent on the severity of disease and/or efficacy of antibiotic therapy. Thus, 19F MRI with perfluorocarbon emulsions is a useful imaging modality to visualize sites and course of infection as well as to evaluate promising antibacterial drug candidates. Furthermore, since the accumulation of tracer depends on immune cells, it might be additionally interesting for studies regarding the immune response to infections, auto-immune diseases or cancer, but also to investigate the efficacy of immunomodulatory compounds and immunization.
Background: Myofibrillar myopathies (MFM) are a group of phenotypically and genetically heterogeneous neuromuscular disorders, which are characterized by protein aggregations in muscle fibres and can be associated with multisystemic involvement.
Methods: We screened a large cohort of 38 index patients with MFM for mutations in the nine thus far known causative genes using Sanger and next generation sequencing (NGS). We studied the clinical and histopathological characteristics in 38 index patients and five additional relatives (n = 43) and particularly focused on the associated multisystemic symptoms.
Results: We identified 14 heterozygous mutations (diagnostic yield of 37%), among them the novel p. Pro209Gln mutation in the BAG3 gene, which was associated with onset in adulthood, a mild phenotype and an axonal sensorimotor polyneuropathy, in the absence of giant axons at the nerve biopsy. We revealed several novel clinical phenotypes and unusual multisystemic presentations with previously described mutations: hearing impairment with a FLNC mutation, dysphonia with a mutation in DES and the first patient with a FLNC mutation presenting respiratory insufficiency as the initial symptom. Moreover, we described for the first time respiratory insufficiency occurring in a patient with the p. Gly154Ser mutation in CRYAB. Interestingly, we detected a polyneuropathy in 28% of the MFM patients, including a BAG3 and a MYOT case, and hearing impairment in 13%, including one patient with a FLNC mutation and two with mutations in the DES gene. In four index patients with a mutation in one of the MFM genes, typical histological findings were only identified at the ultrastructural level (29%).
Conclusions: We conclude that extraskeletal symptoms frequently occur in MFM, particularly cardiac and respiratory involvement, polyneuropathy and/or deafness. BAG3 mutations should be considered even in cases with a mild phenotype or an adult onset. We identified a genetic defect in one of the known genes in less than half of the MFM patients, indicating that more causative genes are still to be found. Next generation sequencing techniques should be helpful in achieving this aim.
The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.
The experimental technique predominantly employed within the scope of this Thesis constitutes one subarea of femtochemistry: the time-resolved spectroscopy of photoin-
duced chemical reactions in the liquid phase by means of molecular signatures in the mid-infrared (MIR) spectral range. Probing transient vibrational states, i.e., dynamic
changes in the vibrational motion of specic molecular subunits or functional Groups allows for a distinct separation and assignment of measured signals to emerging molecular species. For this purpose, one key building block is indispensable, which most of the investigations carried out within the eld of femtochemistry have in common: a coherent light source delivering ultrashort laser pulses with a temporal duration that matches the femtosecond time scale on which molecular motions typically occur. This instrumentation enables the observation of photoinduced chemical reactions from the
starting point|the excitation event to the appearance of intermediates to the nal formation of stable photoproducts after several pico- or nanoseconds.
This work comprises the acquisition and presentation of time-resolved spectroscopic data related to promising molecular systems upon photoexcitation as well as the im-
plementation and testing of experimental optical techniques both for the presented experiments but as well for experiments conceivable in the future. In addition, linear spectroscopy measurements and quantum-chemical simulations on the emerging chemical species have been carried out. In so doing, the primary processes and subse-
quently emerging reaction products of two compounds on a timescale of several nanoseconds after photoexcitation have been elucidated in great detail. Both compounds, the
[Mn(CO)3(tpm)]+ (tpm = tris(2-pyrazolyl)methane) CO-releasing molecule (CORM) and the 5-diazo Meldrum's acid (DMA), are of academic interest but in addition belong
to molecular classes that might be utilized in the near future as dark-stable prodrugs under physiological conditions or that are already utilized in industrial chemistry procedures, respectively. The ndings of both studies gave rise to implement and examine two techniques for prospective transient absorption experiments, namely the shaping and characterization of ultraviolet (UV) laser pulses and the recording of two-photon excitation spectra. Beyond that, since each of the depicted experiments is based on the detection of weak transient absorption signals in the MIR spectral region, two dif-
ferent detection schemes, via chirped-pulse upconversion (CPU) on the one hand and via direct multichannel MCT detection on the other hand, have been juxtaposed at the
conclusion of this work. Since both techniques are suitable in femtosecond pump-probe measurements but thereby exhibit individual strengths and weaknesses, a comparative study provides clarication of the respective pros and cons. The first study introduced within this work investigates the complex photochemistry
of DMA, a photoactive compound used in lithography and industrial chemistry. By femtosecond MIR transient absorption spectroscopy covering several nanoseconds, the
light-induced dynamics and ultrafast formation of several photoproducts from the manifold of reaction pathways have been disclosed to form a coherent picture of the overall
reaction scheme. After UV excitation of DMA dissolved in methanol to the second excited state S2, 70% of excited molecules relax back to the S0 ground state. In compet-
ing processes, they can either undergo an intramolecular Wolff rearrangement to form ketene, which reacts with a solvent molecule to an enol intermediate and further to carboxylate ester, or they rst relax to the DMA S1 state, from where they can isomerize to a diazirine. The third competing reaction channel, having the lowest quantum efficiency with respect to the rst two channels, is the formation of a singlet carbene out of the S1 state. From there an ylide can arise or, via an intersystem crossing, the triplet form of the carbene follows. Whereas the primary reaction steps occur on a picosecond timescale, the subsequently arising intermediates and stable photoproducts are formed
within a few hundreds to thousands of picoseconds. For a reliable identication of the involved compounds, density functional theory calculations on the normal modes and
Fourier-transform infrared spectroscopy of the reactant and the photoproducts in the chemical equilibrium accompany the analysis of the transient spectra. Additional experiments in ethanol and isopropanol led to slight spectral shifts as well as elongated time
constants due to steric hindrance in transient spectra connected with the ester Formation channel, further substantiating the assignment of the occurring reaction pathways and photoproducts.
The study demonstrated that the combination of linear and time-resolved spectroscopic measurements in conjunction with quantum-chemical calculations constitutes a powerful tool to unravel even highly complex photoreactions exhibiting multiple consecutive intermediate states within parallel reaction pathways. Although some of the individual reaction steps, for example the ketene formation via Wolff rearrangement, have been observed on ultrashort time scales before, this work encompassed the Observation of the whole set of appearing photoproducts of DMA in different alcohol solutions within several nanoseconds. In this sense, the ultrafast photochemistry of DMA represents a prototype example for a multisequential reaction scheme, elucidated by the capabilities of femtosecond MIR spectroscopy.
With a modified instrumentation concerning amongst others the system delivering the fundamental laser pulses or the generation of the UV pump pulses, the next ob-
jective within this work was to elucidate the primary processes upon UV Irradiation of a manganese tricarbonyl CORM in aqueous environment. The time-resolved
experiment was performed with two different pump wavelengths and furthermore supported by linear spectroscopy methods and time-dependent density functional theory (TDDFT) calculations on the excited states as well as DFT calculations on the ground
states. The measurements revealed that irradiating the compound with UV excitation pulses primarily leads to ultrafast photolysis of one CO ligand. Geminate recombination may occur within one picosecond but it remains a minor process as the photolyzed CO
group is liberated and the unoccupied coordination site is predominantly filled by an incoming solvent molecule. There was no evidence for hot CO bands, i.e., the remaining CO ligands|in the dicarbonyl photoproduct as well as in the intact CORM are not vibrationally excited through the UV excitation of the CORM. According to this, the excess energy merges into low-frequency vibrational modes associated with the molecule as a whole. Since studies on a macroscopic scale at irradiation times of several minutes prove that UV irradiation eventually leads to the release of two or even all three CO
ligands, further loss of CO most likely necessitates manganese oxidation or another interaction with light. To clarify the latter, a consecutive UV pulse was employed in order to excite the photoproducts subsequent to the initial pump interaction. However, the data obtained was not instructive enough to denitely exclude the manganese oxidation being responsible for the loss of further CO groups. Besides the exchange of a CO Group by a solvent molecule or the geminate recombination, the employment of two different excitation wavelengths in combination with ndings derived from the TDDFT calculations suggested another reaction process, namely the possibility that the excitation does not lead to any bond cleavage at all. As the CORM under investigation is tissue-selective and cytotoxic against cancer cells, knowledge of these rst photoinduced reaction steps is essential for a full understanding of its biological activity. Inspired by these two studies, experimental techniques for prospective transient absorption measurements have been implemented and tested within preparative measure-
ments. First, in the course of a UV-pump-MIR-probe experiment with specically tailored excitation pulses, one could pursue the aim of coherently controlling the outcome of a photoreaction in the liquid phase. Out of the rich photochemistry of DMA the vibrational signature of a particular molecular species might thereby serve as a feedback signal, which is a central part of a learning loop that adaptively determines the pulse shape that steers the quantum mechanical system upon photoexcitation into a desired direction. This motivated the installation and testing of devices by means of which the shaping and characterization of ultrashort laser pulses in the UV could be performed. Second, motivated by the biological applications of CORMs, one can imagine a scenario where a certain amount of CORMs is deposited inside cancerous tissue. Since the activation of CO loss by means of UV pulses is not possible due to the absorption characteristics of biological tissue, the simultaneous excitation via two photons from the visible spectral regime seems appealing. However, success or failure of such an application depends on whether the deposited compound efficiently absorbs two photons simultaneously, i.e., whether the two-photon absorption cross section is large enough. Therefore, a setup to record two-photon excitation spectra under full consideration of
the crucial laser pulse parameters like the pulse duration, energy and central wavelength was arranged and tested. The rst results were obtained with a commercially available reference system (Mn2CO10) but the setup as well as the described measurement and
data analysis procedure can easily be applied to record the two-photon absorption cross section of more promising molecular systems. Third, as the detection of probe pulses
in the MIR spectral region is part of each time-resolved measurement throughout this thesis, a comparison between the newly established technique of CPU and direct multi-
channel MCT detection is presented by means of pump{probe experiments on Mn2CO10 and Co4CO12 with a 1 kHz shot-to-shot data acquisition. It was shown that the CPU detection technique scores with its high spectral resolution and coverage of the easy-to-handle and more cost-effective CCD detectors. On the other hand, in the course of the additional nonlinear upconversion process intensity fluctuations of the chirped fundamental pulses are transferred to the probe spectrum in the visible regime. This entails a lower signal-to-noise ratio than the direct MCT detection, which can be compensated by an additional normalization procedure applied to the CPU probe pulses. As a consequence, the CPU detection scheme offers more flexibility for future investigations
employing MIR probe pulses. This is of great importance for many applications within the presented eld of femtochemistry as a huge variety of time-resolved investigations on a multitude of systems in the liquid phase is based on the detection of weak transient
absorption signals in the MIR spectral region.
The paracaspase Malt1 is a central regulator of antigen receptor signaling that is frequently mutated in human lymphoma. As a scaffold, it assembles protein complexes for NF-kappa B activation, and its proteolytic domain cleaves negative NF-kappa B regulators for signal enforcement. Still, the physiological functions of Malt1-protease are unknown. We demonstrate that targeted Malt1-paracaspase inactivation induces a lethal inflammatory syndrome with lymphocyte-dependent neurodegeneration in vivo. Paracaspase activity is essential for regulatory T cell (Treg) and innate-like B cell development, but it is largely dispensable for overcoming Malt1-dependent thresholds for lymphocyte activation. In addition to NF-kappa B inhibitors, Malt1 cleaves an entire set of mRNA stability regulators, including Roquin-1, Roquin-2, and Regnase-1, and paracaspase inactivation results in excessive interferon gamma (IFN gamma) production by effector lymphocytes that drive pathology. Together, our results reveal distinct threshold and modulatory functions of Malt1 that differentially control lymphocyte differentiation and activation pathways and demonstrate that selective paracaspase blockage skews systemic immunity toward destructive autoinflammation.
Background and purpose - Due to the relative lack of reports on the medium- to long-term clinical and radiographic results of modular femoral cementless revision, we conducted this study to evaluate the medium- to long-term results of uncemented femoral stem revisions using the modular MRP-TITAN stem with distal diaphyseal fixation in a consecutive patient series.
Patients and methods - We retrospectively analyzed 163 femoral stem revisions performed between 1993 and 2001 with a mean follow-up of 10 (5-16) years. Clinical assessment included the Harris hip score (HHS) with reference to comorbidities and femoral defect sizes classified by Charnley and Paprosky. Intraoperative and postoperative complications were analyzed and the failure rate of the MRP stem for any reason was examined.
Results - Mean HHS improved up to the last follow-up (37 (SD 24) vs. 79 (SD 19); p < 0.001). 99 cases (61%) had extensive bone defects (Paprosky IIB-III). Radiographic evaluation showed stable stem anchorage in 151 cases (93%) at the last follow-up. 10 implants (6%) failed for various reasons. Neither a breakage of a stem nor loosening of the morse taper junction was recorded. Kaplan-Meier survival analysis revealed a 10-year survival probability of 97% (95% CI: 95-100).
Interpretation - This is one of the largest medium- to longterm analyses of cementless modular revision stems with distal diaphyseal anchorage. The modular MRP-TITAN was reliable, with a Kaplan-Meier survival probability of 97% at 10 years.
It is natural to consider the possibility that the most energetic particles detected (> 10^18 eV), ultra-high-energy cosmic rays (UHECRs), are originated at the most luminous transient events observed (> 10^52 erg s^-1), gamma-ray bursts (GRBs). As a result of the interaction of highly-accelerated, magnetically-confined protons and ions with the photon field inside the burst, both neutrons and UHE neutrinos are expected to be created: the former escape the source and beta-decay into protons which propagate to Earth, where they are detected as UHECRs, while the latter, if detected, would constitute the smoking gun of hadronic acceleration in the sources.
Recently, km-scale neutrino telescopes such as IceCube have finally reached the sensitivities required to probe the neutrino predictions of some of the existing GRB models. On that account, we present here a revised, self-consistent model of joint UHE proton and neutrino production at GRBs that includes a state-of-the-art, improved numerical calculation of the neutrino flux (NeuCosmA); that uses a generalised UHECR emission model where some of the protons in the sources are able to "leak out" of their magnetic confinement before having interacted; and that takes into account the energy losses of the protons during their propagation to Earth. We use our predictions to take a close look at the cosmic ray-neutrino connection and find that the current UHECR observations by giant air shower detectors, together with the upper bounds on the flux of neutrinos from GRBs, are already sufficient to put tension on several possibilities of particle emission and propagation, and to point us towards some requirements that should be fulfilled by GRBs if they are to be the sources of the UHECRs. We further refine our analysis by studying a dynamical burst model, where we find that the different particle species originate at distinct stages of the expanding GRB, each under particular conditions. Finally, we consider a possibility of new physics: the effect of neutrino decay in the flux of UHE neutrinos from GRBs. On the whole, our results demonstrate that self-consistent models of particle production are now integral to the advancement of the field, given that the full picture of the UHE Universe will only emerge as a result of looking at the multi-messenger sky, i.e., at gamma-rays, cosmic rays, and neutrinos simultaneously.
Two-dimensional electron gases (2DEGs) at transition-metal oxide (TMO) interfaces, and boundary states in topological insulators, are being intensively investigated. The former system harbors superconductivity, large magneto-resistance, and ferromagnetism. In the latter, honeycomb-lattice geometry plus bulk spin-orbit interactions lead to topologically protected spin-polarized bands. 2DEGs in TMOs with a honeycomb-like structure could yield new states of matter, but they had not been experimentally realized, yet. We successfully created a 2DEG at the (111) surface of KTaO3, a strong insulator with large spin-orbit coupling. Its confined states form a network of weakly-dispersing electronic gutters with 6-fold symmetry, a topology novel to all known oxide-based 2DEGs. If those pertain to just one Ta-(111) bilayer, model calculations predict that it can be a topological metal. Our findings demonstrate that completely new electronic states, with symmetries not realized in the bulk, can be tailored in oxide surfaces, promising for TMO-based devices.
Obligate intracellular bacteria depend entirely on nutrients from the host cell for their reproduction. Here, we show that obligate intracellular Chlamydia downregulate the central tumor suppressor p53 in human cells. This reduction of p53 levels is mediated by the PI3K-Akt signaling pathway, activation of HDM2, and subsequent proteasomal degradation of p53. The stabilization of p53 in human cells severely impaired chlamydial development and caused the loss of infectious particle formation. DNA-damage-induced p53 interfered with chlamydial development through downregulation of the pentose phosphate pathway (PPP). Increased expression of the PPP key enzyme glucose-6-phosphate dehydrogenase rescued the inhibition of chlamydial growth induced by DNA damage or stabilized p53. Thus, downregulation of p53 is a key event in the chlamydial life cycle that reprograms the host cell to create a metabolic environment supportive of chlamydial growth.