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Anxiety is an affective state characterized by a sustained, long-lasting defensive response, induced by unpredictable, diffuse threat. In comparison, fear is a phasic response to predictable threat. Fear can be experimentally modeled with the help of cue conditioning. Context conditioning, in which the context serves as the best predictor of a threat due to the absence of any conditioned cues, is seen as an operationalization of sustained anxiety.
This thesis used a differential context conditioning paradigm to examine sustained attention processes in a threat context compared to a safety context for the first time. In three studies, the attention mechanisms during the processing of contextual anxiety were examined by measuring heart rate responses and steady-state-visually evoked potentials (ssVEPs). An additional focus was set on the processing of social cues (i.e. faces) and the influence of contextual information on these cues. In a last step, the correlates of sustained anxiety were compared to evoked responses by phasic fear, which was realized in a previously established paradigm combining predictable and unpredictable threat.
In the first study, a contextual stimulus was associated with an aversive loud noise, while a second context remained unpaired. This conditioning paradigm created an anxiety context (CTX+) and a safety context (CTX-). After acquisition, a social agent vs. an object was presented as a distractor in both contexts. Heart rate and cortical responses, with ssVEPs by using frequency tagging, to the contexts and the distractors were assessed. Results revealed enhanced ssVEP amplitudes for the CTX+ compared to the CTX− during acquisition and during presentation of distractor stimuli. Additionally, the heart rate was accelerated in the acquisition phase, followed by a heart rate deceleration as a psychophysiological marker of contextual anxiety.
Study 2 used the same context conditioning paradigm as Study 1. In contrast to the first study, persons with different emotional facial expressions were presented in the anxiety and safety contexts in order to compare the differential processing of these cues within periods of threat and safety. A similar anxiety response was found in the second study, although only participants who
Abstract
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were aware of the contingency between contexts and aversive event showed a sensory amplification of the threat context, indicated by heart rate response and ssVEP activation. All faces irrespective of their emotional expression received increased attentional resources when presented within the anxiety context, which suggests a general hypervigilance in anxiety contexts.
In the third study, the differentiation of predictable and unpredictable threat as an operationalization of fear and anxiety was examined on a cortical and physiological level. In the predictable condition, a social cue was paired with an aversive event, while in the unpredictable condition the aversive event remained unpaired with the respective cue. A fear response to the predictable cue was found, indicated by increased oscillatory response and accelerated heart rate. Both predictable and unpredictable threat yielded increased ssVEP amplitudes evoked by the context stimuli, while the response in the unpredictable context showed longer-lasting ssVEP activation to the threat context.
To sum up, all three studies endorsed anxiety as a long-lasting defensive response. Due to the unpredictability of the aversive events, the individuals reacted with hypervigilance in the anxiety context, reflected in a facilitated processing of sensory information and an orienting response. This hypervigilance had an impact on the processing of novel cues, which appeared in the anxiety context. Considering the compared stimuli categories, the stimuli perceived in a state of anxiety received increased attentional resources, irrespective of the emotional arousal conveyed by the facial expression. Both predictable and unpredictable threat elicited sensory amplification of the contexts, while the response in the unpredictable context showed longer-lasting sensory facilitation of the threat context.
Pulsars (in short for Pulsating Stars) are magnetized, fast rotating neutron stars. The basic picture of a pulsar describes it as a neutron star which has a rotation axis that is not aligned with its magnetic field axis. The emission is assumed to be generated near the magnetic poles of the neutron star and emitted along the open magnetic field lines. Consequently, the corresponding beam of photons is emitted along the magnetic field line axis. The non-alignment of both, the rotation and the magnetic field axis, results in the effect that the emission of the pulsar is only seen if its beam points towards the observer.
The emission from a pulsar is therefore perceived as being pulsed although its generation is not. This rather simple geometrical model is commonly referred to as Lighthouse Model and has been widely accepted. However, it does not deliver an explanation of the precise mechanisms behind the emission from pulsars (see below for more details).
Nowadays more than 2000 pulsars are known. They are observed at various wavelengths. Multiwavelength studies have shown that some pulsars are visible only at certain wavelengths while the emission from others can be observed throughout large parts of the electromagnetic spectrum. An example of the latter case is the Crab pulsar which is also the main object of interest in this thesis. Originating from a supernova explosion observed in 1054 A.D. and discovered in 1968, the Crab pulsar has been the central subject of numerous studies. Its pulsed emission is visible throughout the whole electromagnetic spectrum which makes it a key figure in understanding the possible mechanisms of multiwavelength emission from pulsars.
The Crab pulsar is also well known for its radio emission strongly varying on long as well as on short time scales. While long time scale behaviour from a pulsar is usually examined through the use of its average profile (a profile resulting from averaging of a large number of individual pulses resulting from single rotations), short time scale behaviour is examined via its single pulses. The short time scale anomalous behaviour of its radio emission is commonly referred to as Giant Pulses and represents the central topic of this thesis.
While current theoretical approaches place the origin of the radio emission from a pulsar like the Crab near its magnetic poles (Polar Cap Model) as already indicated by the Lighthouse model, its emission at higher frequencies, especially its gamma-ray emission, is assumed to originate further away in the geometrical region surrounding a pulsar which is commonly referred to as a pulsar magnetosphere (Outer Gap Model). Consequently, the respective emission regions are usually assumed not to be connected. However, past observational results from the Crab pulsar represent a contradiction to this assumption.
Radio giant pulses from the Crab pulsar have been observed to emit large amounts of energy on very short time scales implying small emission regions on the surface of the pulsar. Such energetic events might also leave a trace in the gamma-ray emission of the Crab pulsar.
The aim of this thesis is to search for this connection in the form of a correlation study between radio giant pulses and gamma-photons from the Crab pulsar.
To make such a study possible, a multiwavelength observational campaign was organized for which radio observations were independently applied for, coordinated and carried out with the Effelsberg radio telescope and the Westerbork Synthesis Radio Telescope and gamma-ray observations with the Major Atmospheric Imaging Cherenkov telescopes. The corresponding radio and gamma-ray data sets were reduced and the correlation analysis thereafter consisted of three different approaches:
1) The search for a clustering in the differences of the times of arrival of radio giant pulses and gamma-photons;
2) The search for a linear correlation between radio giant pulses and gamma-photons using the Pearson correlation approach;
3) A search for an increase of the gamma-ray flux around occurring radio giant pulses.
In the last part of the correlation study an increase of the number of gamma-photons centered on a radio giant pulse by about 17% (in contrast with the number of gamma-photons when no radio giant pulse occurs in the same time window) was discovered. This finding suggests that a new theoretical approach for the emission of young pulsars like the Crab pulsar, is necessary.
The active place avoidance task is a dry-arena task used to assess spatial navigation and memory in rodents. In this task, a subject is put on a rotating circular arena and avoids an invisible sector that is stable in relation to the room. Rotation of the arena means that the subject's avoidancemust be active, otherwise the subject will be moved in the to-be-avoided sector by the rotation of the arena and a slight electric shock will be administered. The present experiment explored the effect of variable arena rotation speed on the ability to avoid the to-be-avoided sector. Subjects in a group with variable arena rotation speed learned to avoid the sector with the same speed and attained the same avoidance ability as rats in a group with a stable arena rotation speed. Only a slight difference in preferred position within the room was found between the two groups. No difference was found between the two groups in the dark phase, where subjects could not use orientation cues in the room. Only one rat was able to learn the avoidance of the to-be-avoided sector in this phase. The results of the experiment suggest that idiothetic orientation and interval timing are not crucial for learning avoidance of the to-be-avoided sector. However, idiothetic orientation might be sufficient for avoiding the sector in the dark.
One of the major health consequences of the Chernobyl Nuclear Power Plant accident in 1986 was a dramatic increase in incidence of thyroid cancer among those who were aged less than 18 years at the time of the accident. This increase has been directly linked in several analytic epidemiological studies to iodine-131 (I-131) thyroid doses received from the accident. However, there remains limited understanding of factors that modify the I-131-related risk. Focusing on post-Chernobyl pediatric thyroid cancer in Belarus, we reviewed evidence of the effects of radiation, thyroid screening, and iodine deficiency on regional differences in incidence rates of thyroid cancer. We also reviewed current evidence on content of nitrate in groundwater and thyroid cancer risk drawing attention to high levels of nitrates in open well water in several contaminated regions of Belarus, i.e. Gomel and Brest, related to the usage of nitrogen fertilizers. In this hypothesis generating study, based on ecological data and biological plausibility, we suggest that nitrate pollution may modify the radiation-related risk of thyroid cancer contributing to regional differences in rates of pediatric thyroid cancer in Belarus. Analytic epidemiological studies designed to evaluate joint effect of nitrate content in groundwater and radiation present a promising avenue of research and may provide useful insights into etiology of thyroid cancer.
Background: Sub-Saharan Africa has a high prevalence of hepatitis B virus (HBV) infections. Health care workers (HCWs) are at high risk of contracting HBV infection through their occupation. Vaccination of HCWs against HBV is standard practice in many countries, but is often not implemented in resource-poor settings. We aimed with this cross-sectional study to determine HBV prevalence, HCW vaccination status, and the risk factors for HCWs contracting HBV infection in Tanzania.
Methods: We enrolled 600 HCWs from a tertiary Tanzanian hospital. Their demographics, medical histories, HBV vaccination details and risk factors for contracting blood-borne infections were collected using a standardized questionnaire. Serum samples were tested for HBV and hepatitis C virus (HCV) markers by ELISA techniques, PCR and an anti-HBs rapid test. HCWs were divided in two subgroups: those at risk of contracting HBV (rHCW 79.2 %) via exposure to potentially infectious materials, and those considered not at risk of contracting HBV (nrHCW, 20.8 %). Results: The overall prevalence of chronic HBV infection (HBsAg+, anti-HBc+, anti-HBs-) was 7.0 % (42/598). Chronic HBV infection was found in 7.4 % of rHCW versus 5.6 % of nrHCW(p-value = 0.484). HCWs susceptible to HBV (HBsAg-, anti-HBc-, anti-HBs-) comprised 31.3 %. HBV immunity achieved either by healed HBV infection (HBsAg-, anti-HBc+, anti-HBs+) or by vaccination (HBsAg-, anti-HBc-, anti-HBs+) comprised 36.5 % and 20.2 %, respectively. 4.8 % of participants had indeterminate results (HBsAg-, anti-HBc+, anti-HBc-IgM-, anti-HBs-). Only 77.1 % of HCWs who received a full vaccination course had an anti-HBs titer > 10 ml/U. An anti-HBs point-of-care test was 80.7 % sensitive and 96.9 % specific. There was a significantly higher risk for contracting HBV (anti-HBc+) among those HCW at occupational risk (rHCW) of older age (odds ratios (OR) in rHCW 3.297, p < 0.0001 vs. nrHCW 1.385, p = 0.606) and among those HCW being employed more than 11 years (OR 2.51, p < 0.0001***). HCV prevalence was low (HCV antibodies 1.2 % and HCV-RNA 0.3 %).
Conclusions: Chronic HBV infection is common among Tanzanian HCWs. One third of HCWs were susceptible to HBV infection, highlighting the need for vaccination. Due to high prevalence of naturally acquired immunity against HBV pre-testing might be a useful tool to identify susceptible individuals.
Tree invasions have substantial impacts on biodiversity and ecosystem functioning, and trees that are dispersed by animals are more likely to become invasive. In addition, hybridisation between plants is well documented as a source of new weeds, as hybrids gain new characteristics that allow them to become invasive. Corymbia torelliana is an invasive tree with an unusual animal dispersal mechanism: seed dispersal by stingless bees, that hybridizes readily with other species. We examined hybrids between C. torelliana and C. citriodora subsp. citriodora to determine whether hybrids have inherited the seed dispersal characteristics of C. torelliana that allow bee dispersal. Some hybrid fruits displayed the characteristic hollowness, resin production and resin chemistry associated with seed dispersal by bees. However, we did not observe bees foraging on any hybrid fruits until they had been damaged. We conclude that C. torelliana and C. citriodora subsp. citriodora hybrids can inherit some fruit characters that are associated with dispersal by bees, but we did not find a hybrid with the complete set of characters that would enable bee dispersal. However, around 20,000 hybrids have been planted in Australia, and ongoing monitoring is necessary to identify any hybrids that may become invasive.
Background: In order to influence every day clinical practice professional organisations issue management guidelines. Cross-sectional surveys are used to evaluate the implementation of such guidelines. The present survey investigated screening for glucose perturbations in people with coronary artery disease and compared patients with known and newly detected type 2 diabetes with those without diabetes in terms of their life-style and pharmacological risk factor management in relation to contemporary European guidelines.
Methods: A total of 6187 patients (18-80 years) with coronary artery disease and known glycaemic status based on a self reported history of diabetes (previously known diabetes) or the results of an oral glucose tolerance test and HbA1c (no diabetes or newly diagnosed diabetes) were investigated in EUROASPIRE IV including patients in 24 European countries 2012-2013. The patients were interviewed and investigated in order to enable a comparison between their actual risk factor control with that recommended in current European management guidelines and the outcome in previously conducted surveys. Results: A total of 2846 (46 %) patients had no diabetes, 1158 (19 %) newly diagnosed diabetes and 2183 (35 %) previously known diabetes. The combined use of all four cardioprotective drugs in these groups was 53, 55 and 60 %, respectively. A blood pressure target of <140/90 mmHg was achieved in 68, 61, 54 % and a LDL-cholesterol target of <1.8 mmol/L in 16, 18 and 28 %. Patients with newly diagnosed and previously known diabetes reached an HbA1c <7.0 % (53 mmol/mol) in 95 and 53 % and 11 % of those with previously known diabetes had an HbA1c >9.0 % (>75 mmol/mol). Of the patients with diabetes 69 % reported on low physical activity. The proportion of patients participating in cardiac rehabilitation programmes was low (approximate to 40 %) and only 27 % of those with diabetes had attended diabetes schools. Compared with data from previous surveys the use of cardioprotective drugs had increased and more patients were achieving the risk factor treatment targets.
Conclusions: Despite advances in patient management there is further potential to improve both the detection and management of patients with diabetes and coronary artery disease.
Biodiversity loss can affect the viability of ecosystems by decreasing the ability of communities to respond to environmental change and disturbances. Agricultural intensification is a major driver of biodiversity loss and has multiple components operating at different spatial scales: from in-field management intensity to landscape-scale simplification. Here we show that landscape-level effects dominate functional community composition and can even buffer the effects of in-field management intensification on functional homogenization, and that animal communities in real-world managed landscapes show a unified response (across orders and guilds) to both landscape-scale simplification and in-field intensification. Adults and larvae with specialized feeding habits, species with shorter activity periods and relatively small body sizes are selected against in simplified landscapes with intense in-field management. Our results demonstrate that the diversity of land cover types at the landscape scale is critical for maintaining communities, which are functionally diverse, even in landscapes where in-field management intensity is high.
Patients with refractory or relapsed and refractory multiple myeloma who no longer receive benefit from novel agents have limited treatment options and short expected survival. del(17p) and t(4;14) are correlated with shortened survival. The phase 3 MM-003 trial demonstrated significant progression-free and overall survival benefits from treatment with pomalidomide plus low-dose dexamethasone compared to high-dose dexamethasone among patients in whom bortezomib and lenalidomide treatment had failed. At an updated median follow-up of 15.4 months, the progression-free survival was 4.0 versus 1.9 months (HR, 0.50; P<0.001), and median overall survival was 13.1 versus 8.1 months (HR, 0.72; P=0.009). Pomalidomide plus low-dose dexamethasone, compared with high-dose dexamethasone, improved progression-free survival in patients with del(17p) (4.6 versus 1.1 months; HR, 0.34; P < 0.001), t(4;14) (2.8 versus 1.9 months; HR, 0.49; P=0.028), and in standard-risk patients (4.2 versus 2.3 months; HR, 0.55; P<0.001). Although the majority of patients treated with high-dose dexamethasone took pomalidomide after discontinuation, the overall survival of patients treated with pomalidomide plus low-dose dexamethasone or highdose dexamethasone was 12.6 versus 7.7 months (HR, 0.45; P=0.008) in patients with del(17p), 7.5 versus 4.9 months (HR, 1.12; P=0.761) in those with t(4;14), and 14.0 versus 9.0 months (HR, 0.85; P=0.380) in standard-risk subjects. The overall response rate was higher in patients treated with pomalidomide plus low-dose dexamethasone than in those treated with high-dose dexamethasone both among standard-risk patients (35.2% versus 9.7%) and those with del(17p) (31.8% versus 4.3%), whereas it was similar in patients with t(4; 14) (15.9% versus 13.3%). The safety of pomalidomide plus low-dose dexamethasone was consistent with initial reports. In conclusion, pomalidomide plus low-dose dexamethasone is efficacious in patients with relapsed/refractory multiple myeloma and del(17p) and/or t(4;14).
European Myeloma Network Guidelines for the Management of Multiple Myeloma-related Complications
(2015)
The European Myeloma Network provides recommendations for the management of the most common complications of multiple myeloma. Whole body low-dose computed tomography is more sensitive than conventional radiography in depicting osteolytic disease and thus we recommend it as the novel standard for the detection of lytic lesions in myeloma (grade 1A). Myeloma patients with adequate renal function and bone disease at diagnosis should be treated with zoledronic acid or pamidronate (grade 1A). Symptomatic patients without lytic lesions on conventional radiography can be treated with zoledronic acid (grade 1B), but its advantage is not clear for patients with no bone involvement on computed tomography or magnetic resonance imaging. In asymptomatic myeloma, bisphosphonates are not recommended (grade 1A). Zoledronic acid should be given continuously, but it is not clear if patients who achieve at least a very good partial response benefit from its continuous use (grade 1B). Treatment with erythropoietic-stimulating agents may be initiated in patients with persistent symptomatic anemia (hemoglobin < 10g/dL) in whom other causes of anemia have been excluded (grade 1B). Erythropoietic agents should be stopped after 6-8 weeks if no adequate hemoglobin response is achieved. For renal impairment, bortezomib-based regimens are the current standard of care (grade 1A). For the management of treatment-induced peripheral neuropathy, drug modification is needed (grade 1C). Vaccination against influenza is recommended; vaccination against streptococcus pneumonia and hemophilus influenza is appropriate, but efficacy is not guaranteed due to suboptimal immune response (grade 1C). Prophylactic aciclovir (or valacyclovir) is recommended for patients receiving proteasome inhibitors, autologous or allogeneic transplantation (grade 1A).
Fulminant myocarditis is rare but a potentially life-threatening disease. Acute or mild myocarditis following acute ischemia is generally associated with a profound activation of the host’s immune system. On one hand this is mandatory to protect the host’s heart by fighting the invading agents (i.e., bacteria, viruses or other microbial agents) and/or to induce healing and repair processes in the damaged myocardium. On other hand, uncontrolled activation of the immune system may result in the generation of auto-reactive (not always beneficial) immune cells.
Myocarditis or inflammatory cardiomyopathy is characterized by focal or diffuse infiltrates, myocyte necrosis and/or apoptosis and subsequent fibrotic replacement of the heart muscle. In humans, about 30% of the myocarditis-patients develop dilated cardiomyopathy. As the clinical picture of myocarditis is multifaceted, it is difficult to diagnose the disease. Therefore, the main goal of the present work was to test and further develop novel non-invasive methods for the detection of myocardial inflammation by employing both contrast enhanced MRI techniques as well as novel nuclear tracers that are suitable for in vivo PET/ SPECT imaging.
As a part of this thesis, a pre-clinical animal model was successfully established by immunizing female Lewis rats with whole-porcine cardiac myosin (CM). Induction of Experimental Autoimmune Myocarditis (EAM) is considered successful when anti-myosin antibody titers are increased more than 100-fold over control animals and pericardial effusion develops. In addition, cardiac tissues from EAM-rats versus controls were analyzed for the expression of various pro-inflammatory and fibrosis markers. To further exploit non-invasive MRI techniques for the detection of myocarditis, our EAM-rats were injected either with (1) ultra-small Paramagnetic iron oxide particles (USPIO’s; Feraheme®), allowing for in vivo imaging , (2) micron sized paramagnetic iron oxide particles (MPIO) for ex vivo inflammatory cell-tracking by cMRI, or (3) with different radioactive nuclear tracers (67gallium citrate, 68gallium-labeled somatostatin analogue, and 68gallium-labeled cyclic RGD-peptide) which in the present work have been employed for autoradiographic imaging, but in principle are also suitable for in vivo nuclear imaging (PET/SPECT). In order to compare imaging results with histology, consecutive heart sections were stained with hematoxylin & eosin (HE, for cell infiltrates) and Masson Goldner trichrome (MGT, for fibrosis); in addition, immuno-stainings were performed with anti-CD68 (macrophages), anti-SSRT2A (somatostatin receptor type 2A), anti-CD61 (β3-integrins) and anti-CD31 (platelet endothelial cell adhesion molecule 1).
Sera from immunized rats strongly reacted with cardiac myosin. In immunized rats, echocardiography and subsequent MRI revealed huge amounts of pericardial effusion (days 18-21). Analysis of the kinetics of myocardial infiltrates revealed maximal macrophage invasion between days 14 and 28. Disappearance of macrophages resulted in replacement-fibrosis in formerly cell-infiltrated myocardial areas. This finding was confirmed by the time-dependent differential expression of corresponding cytokines in the myocardium. Immunized animals reacted either with an early or a late pattern of post-inflammation fibrosis. Areas with massive cellular infiltrates were easily detectible in autoradiograms showing a high focal uptake of 67gallium-citrate and 68gallium labeled somatostatin analogues (68Ga DOTA-TATE). Myocardium with a loss of cardiomyocytes presented a high uptake of 68gallium labeled cyclic RGD-peptide (68Ga NOTA-RGD). MRI cell tracking experiments with Feraheme® as the contrast-agent were inconclusive; however, strikingly better results were obtained when MPIOs were used as a contrast-agent: histological findings correlated well with in vivo and ex vivo MPIO-enhanced MRI images.
Imaging of myocardial inflammatory processes including the kinetics of macrophage invasion after microbial or ischemic damage is still a major challenge in, both animal models and in human patients. By applying a broad panel of biochemical, histological, molecular and imaging methods, we show here that different patterns of reactivity may occur upon induction of myocarditis using one and the same rat strain. In particular, immunized Lewis rats may react either with an early or a late pattern of macrophage invasion and subsequent post-inflammation fibrosis. Imaging results achieved in the acute inflammatory phase of the myocarditis with MPIOs, 67gallium citrate and 68gallium linked to somatostatin will stimulate further development of contrast agents and radioactive-nuclear tracers for the non-invasive detection of acute myocarditis and in the near future perhaps even in human patients.
The impact of acquired severe motor impairments is pervasive and may lead to a complete loss of communication and voluntary motor control, rendering the patient behaviourally unresponsive. In routine clinical care it may thus be unclear, whether some of these patients are even conscious. Given that finding a cure is unlikely, care focuses on providing the best possible quality of life (QoL), and knowing its predictors might contribute to that aim. Patients who still can communicate often report a high QoL, and several predictors have been identified. However, many instruments used to assess QoL require at least residual verbal and motor abilities. Thus, a method to assess QoL independent of these requirements is desirable. In addition, many instruments assume QoL to be temporarily stable, and little information is available on predictors of instantaneous QoL, i.e. QoL as it fluctuates from moment to moment throughout the day.
The correct regulation of cell growth and proliferation is essential during normal animal development. Myc proteins function as transcription factors, being involved in the con-trol of many growth- and proliferation-associated genes and deregulation of Myc is one of the main driving factors of human malignancies.
The first part of this thesis focuses on the identification of directly regulated Myc target genes in Drosophila melanogaster, by combining ChIPseq and RNAseq approaches. The analysis results in a core set of Myc target genes of less than 300 genes which are mainly involved in ribosome biogenesis. Among these genes we identify a novel class of Myc targets, the non-coding small nucleolar RNAs (snoRNAs). In vivo studies show that loss of snoRNAs not only impairs growth during normal development, but that overexpression of several snoRNAs can also enhance tumor development in a neu-ronal tumor model. Together the data show that Myc acts as a master regulator of ribo-some biogenesis and that Myc’s transforming effects in tumor development are at least partially mediated by the snoRNAs.
In the second part of the thesis, the interaction of Myc and the Zf-protein Chinmo is described. Co-immunoprecipitations of the two proteins performed under endogenous and exogenous conditions show that they interact physically and that neither the two Zf-domains nor the BTB/POZ-domain of Chinmo are important for this interaction. Fur-thermore ChIP experiments and Myc dependent luciferase assays show that Chinmo and Myc share common target genes, and that Chinmo is presumably also involved in their regulation. While the exact way of how Myc and Chinmo genetically interact with each other still has to be investigated, we show that their interaction is important in a tumor model. Overexpression of the tumor-suppressors Ras and Chinmo leads to tu-mor formation in Drosophila larvae, which is drastically impaired upon loss of Myc.
The recently discovered human DREAM complex (for DP, RB-like, E2F and MuvB complex) is a chromatin-associated pocket protein complex involved in cell cycle- dependent gene expression. DREAM consists of five core subunits and forms a complex either with the pocket protein p130 and the transcription factor E2F4 to repress gene expression or with the transcription factors B-MYB and FOXM1 to promote gene expression.
Gas2l3 was recently identified by our group as a novel DREAM target gene. Subsequent characterization in human cell lines revealed that GAS2L3 is a microtubule and F-actin cross-linking protein, expressed in G2/M, plays a role in cytokinesis, and is important for chromosomal stability.
The aim of the first part of the study was to analyze how expression of GAS2L3 is regulated by DREAM and to provide a better understanding of the function of GAS2L3 in mitosis and cytokinesis.
ChIP assays revealed that the repressive and the activating form of DREAM bind to the GAS2L3 promoter. RNA interference (RNAi) mediated GAS2L3 depletion demonstrated the requirement of GAS2L3 for proper cleavage furrow ingression in cytokinesis. Immunofluorescence-based localization studies showed a localization of GAS2L3 at the mitotic spindle in mitosis and at the midbody in cytokinesis. Additional experiments demonstrated that the GAS2L3 GAR domain, a putative microtubule- binding domain, is responsible for GAS2L3 localization to the constriction zones in cytokinesis suggesting a function for GAS2L3 in the abscission process.
DREAM is known to promote G2/M gene expression. DREAM target genes include several mitotic kinesins and mitotic microtubule-associated proteins (mitotic MAPs). However, it is not clear to what extent DREAM regulates mitotic kinesins and MAPs, so far. Furthermore, a comprehensive study of mitotic kinesin expression in cancer cell lines is still missing.
Therefore, the second major aim of the thesis was to characterize the regulation of mitotic kinesins and MAPs by DREAM, to investigate the expression of mitotic kinesins in cancer cell line panels and to evaluate them as possible anti-cancer targets.
ChIP assays together with RNAi mediated DREAM subunit depletion experiments demonstrated that DREAM is a master regulator of mitotic kinesins. Furthermore, expression analyses in a panel of breast and lung cancer cell lines revealed that mitotic kinesins are up-regulated in the majority of cancer cell lines in contrast to non-transformed controls. Finally, an inducible lentiviral-based shRNA system was developed to effectively deplete mitotic kinesins. Depletion of selected mitotic kinesins resulted in cytokinesis failures and strong anti-proliferative effects in several human cancer cell lines.
Thus, this system will provide a robust tool for future investigation of mitotic kinesin function in cancer cells.
Staphylococcus aureus is a facultative Gram-positive human pathogen which can cause different severe infections. Staphylococci are phagocytosed by professional and non-professional phagocytes; they are strongly cytotoxic against eukaryotic cells and have been proposed to play a role in immune evasion by spreading within migrating phagocytes. This study investigated the post invasive events upon S. aureus infection. Strains which are able to escape the phagosome were identified and the responsible toxins were determined. Thereby innovative insights into host pathogen interaction were obtained.
A novel class of small amphipathic peptides with strong surfactant-like properties, the phenol soluble modulins, particularly PSMα as well as the leukocidin LukAB, are involved in phagosomal escape of the clinical S. aureus strains LAC, MW2 and 6850 in non-professional and professional phagocytes. Whereas, PSMβ, δ-toxin, α-toxin, β-toxin or phosphatidyl inositol-dependent phospholipase C did not affect phagosomal escape. By blocking the bacterial DNA-dependent RNA polymerase with rifampicin phagosomal escape is determined to start approximately 2.5 hours post infection. Phagosomal escape further was required for intracellular replication of S. aureus. Strains which are not able to escape cannot replicate in the acidic vacuole, whereas, the host cytoplasm offers a rich milieu for bacterial replication. Additionally, phagosomal escape, with intracellular bacterial replication induces the subsequent host cell death. This could be confirmed by an infection assay including S. aureus knockout mutants in psmα or lukAB which were significantly less cytotoxic, compared with those infected with escape-positive wild type strains.
Further, this study showed that phagosomal escape is not only mediated by bacterial toxins. Since, the phagocyte-specific cognate receptors for both escape relevant toxins, FPR2 (PSMα receptor) and CD11b (LukAB receptor) are produced in epithelial and endothelial cells only after infection with S. aureus in a calcium dependent fashion. The knockdown of both receptors using siRNA prevents S. aureus to escape the phagosome. Furthermore, blocking intracellular calcium release with the inositol trisphosphate receptor (IP3R) inhibitor 2-APB prohibits upregulation of fpr2 and cd11b and subsequently phagosomal escape of S. aureus.
To conclude, the current study clarifies that phagosomal escape and host cell death are interplay of both, bacterial toxins and host cell factors.
Staphylococcus aureus ist ein fakultativ Gram-positives Humanpathogen, dass verschiedene schwerwiegende Infektionen verursachen kann. Staphylokokken werden von professionellen und nicht-professionellen Phagozyten (Fresszellen) zu gleich aufgenommen. Desweitern sind sie stark zytotoxisch für eukaryotische Zellen. Außerdem wird vermutet, dass sie sich mittels migrierender Phagozyten dem angeborenen Immunsystem entziehen können. In dieser Studie werden die post-invasiven Ereignisse während einer Staphylokokken Infektion untersucht. Im Detail wurden Stämme identifiziert die aus den Phagosomen entkommen können und die dafür verantwortlichen Toxine. Im Zuge dessen wurden neue Erkenntnisse der Interaktion zwischen Bakterien und Wirtszellen gewonnen.
Eine neue Klasse von kleinen amphiphatischen Peptiden mit starken grenzflächenaktiven Eigenschaften (Surfactant), die sogenannten Phenol soluble modulins (PSMs) im Besonderen PSMα sowie das Leukozidin LukAB, sind am phagosomalen Ausbruch der klinisch relevanten S. aureus Stämmen LAC, MW2 und 6850 in nicht professionellen und professionellen Phagozyten involviert. Hingegen, sind PSMβ, δ-toxin, α-toxin, β-toxin oder Phosphatidylinositol abhängige Phospholipase C nicht am phagosomalen Ausbruch beteiligt. Durch die Hemmung der bakteriellen DNA-abhängigen RNA Polymerase mit Rifampicin wurde der Zeitpunkt für den Ausbruch auf etwa 2,5 Stunden nach der Infektion eingegrenzt. Der phagosomale Ausbruch ist weiterhin für die intrazelluläre Replikation von S. aureus notwendig. Während Stämme, die nicht ausbrechen können in der angesäuerten Vakuole nicht replizieren können, bietet das Zytoplasma ein reichhaltiges Milieu für die Vermehrung. Zudem wird der Pathogen induzierte Zelltod erst nach dem phagosomalen Ausbruch und mit anschließender Vermehrung ermöglicht. Nachgewiesen wurde dies mittels psmα und lukAB defizienten Mutanten welche signifikant weniger zytotoxisch waren als der Wildtyp Stamm. Diese Studie zeigt darüber hinaus, dass der phagosomale Ausbruch nicht nur durch bakterielle Toxine vermittelt wird. Sondern, dass die Phagozyten-spezifischen Rezeptoren für beide relevanten Toxine, FPR2 (PSMα Rezeptor) und CD11b (LukAB Rezeptor), in Epithel- und Endothelzellen nach Infektion mit S. aureus calciumabhängig produziert werden und für den Ausbruch notwendig sind. Der knockdown beider Rezeptoren mittels siRNA verhindert den Ausbruch. Wird der intrazelluläre Calciumstrom mittels des Inositoltrisphosphat Rezeptor (IP3R) Inhibitor 2-APB blockiert können die Gene fpr2 und cd11b nicht hochreguliert werden und der Ausbruch wird ebenfalls verhindert.
Folglich zeigt diese Studie, dass der phagosomale Ausbruch und Pathogen induzierte Zelltod sowohl durch bakterielle Toxine als auch Wirtsfaktoren vermittelt wird.
In Enrique Gaspar y Rimbau’s 1887 science fiction novel El anacronópete, comedy presents itself in a variety of guises. One of the central comic elements of the book is the playful way in which the lower class characters, namely the maid Juana and the soldier Pendencia, engage with language. This article will compare Gaspar’s El anacronópete with two of its official translations, Leyla Rouhi’s The Anacronópete and Yolanda Molina-Gavilán and Andrea Bell’s The Time Ship: A Chrononautical Journey, in order to ascertain to what extent the Spanish author’s comic touch is preserved in the English translations of Juana’s and Pendencia’s speech. The maid’s and the soldier’s use of double meaning, the mondegreen, and code-switching will be the specific focus of our analysis. We will see that, as Salman Rushdie claims, although «[i]t is normally supposed that something always gets lost in translation […] something can also be gained» (1991: 17).
Starting in the late 1990s ionic liquids (ILs) gained momentum both in academia as well as industry. ILs are defined as organic salts with a melting point below 100 °C. Active pharmaceutical ingredients (APIs) may be transferred into ILs by creating salts with a bulky counterion with a soft electron density. ILs have demonstrated the potential to tune important pharmaceutical features such as the solubility and the dissolution rate, particularly addressing the challenge of poor water soluble drugs (PWSD). Due to the tunability of ILs, modification of physico-chemical properties of APIs may be envisioned without any modifications of the chemical structure.
In the first chapter the potential as well as the limitation of ILs are discussed. The chapter commences with an overview of preparation and characterization of API-ILs. Moreover, examples for pharmaceutical parameters are presented which may be affected by IL formation, including the dissolution rate, kinetic solubility or hygroscopicity as well as biopharmaceutical performance and toxicology. The impact of IL formation on those pharmaceutically relevant features is highlighted, resulting in a blueprint for a novel formulation concept to overcome PWSD challenges without the need for structural changes of the API.
Within the second chapter the IL concept is detailed for one specific API - counterion combination. A poorly water soluble acidic API against migraine attacks was transformed into an IL in an effort to minimize the time to maximum plasma concentration (tmax) and optimize the overall bioavailability. These studies were conducted in parallel to a prodrug of the API for comparison of the IL strategy versus a strategy involving modification of the API’s structure. A significantly longer duration of API supersaturation and a 700 fold faster dissolution rate of the IL in comparison to the free acid were obtained and the underlying mechanism was elucidated. The transepithelial absorption was determined using Caco-2 cell layers. For the IL about 3 times more substance was transported in comparison to the prodrug when substances were applied as suspensions, despite the higher permeability of the prodrug, as increased solubility of the IL exceeded this effect. Cytotoxicity of the counterion was assessed in hepatic, renal and macrophage cell lines, respectively, and IC50 values were in the upper µM / lower mM range. The outcome of the study suggested the IL approach instrumental for tuning biopharmaceutical properties, without structural changes of the API as required for preparation of prodrugs. Thus the toolbox for formulation strategies of poorly water soluble drugs could be extended by an efficient concept.
The third chapter focuses on the effect of different counterions on the physico-chemical properties of an API-IL, in particular to overcome the challenge of poor water solubility. Therefore, the same poorly water soluble acidic API against migraine attacks mentioned above was combined with 36 counterions resulting in ILs and low lattice enthalpy salts (LLES). Depending on the counterions, different dissolution rates, durations of supersaturation and hygroscopicities were obtained and release profiles could be tailored from immediate to sustained release. Besides, in vitro the cytotoxicity of the counterions was assessed in three cell lines. Using molecular descriptors such as the number of hydrophobic atoms, the graph theoretical diameter and the number of positive charges of the counterion, the dissolution rate, supersaturation and hygroscopicity as well as the cytotoxicity of counterions could be adequately modeled, rendering it possible to predict properties of new LLESs.
Within the forth chapter different poorly water soluble APIs were combined with the counterion tetrabutylphosphonium (TBP) studying the impact on the pharmaceutical and physical properties of the APIs. TBP-ILs and low lattice enthalpy salts were prepared of the acidic APIs Diclofenac, Ibuprofen, Ketoprofen, Naproxen, Sulfadiazine, Sulfamethoxazole and Tolbutamide. NMR and IR spectroscopy, DSC, XRPD, DVS and dissolution rate measurements, release profiles and saturation concentration measurements were used to characterize the free acids and TBP salts as compared to the corresponding sodium salts. The TBP salts as compared to the free acids displayed lower melting points and glass transition temperatures and up to 1000 times higher dissolution rates. The increase in the dissolution rate directly correlated with the salts’ hygroscopicity, an aspect which is critically discussed in terms of pharmaceutical translation challenges. In summary TBP ILs of solid salts were proved instrumental to approach the challenge of poor water solubility. The outcome profiled tailor-made counterions as a powerful formulation strategy to address poor water solubility, hence bioavailability and ultimately therapeutic potential of challenging APIs.
In summary, a plethora of ILs and LLESs were prepared by combination of different acidic APIs and counterions. The IL and LLESs concept was compared to conventional salt and prodrug strategies. By choice of the counterion, biopharmaceutical relevant parameters were deliberately modified and release profiles were tuned ranging from immediate to prolonged release. The impact of distinct structural counterion features controlling the dissolution, supersaturation, hygroscopicity and counterion cytotoxicity were identified, correlations were presented and predictive models were built. ILs and LLESs could be proven to be a powerful concept for the formulation of poorly water soluble acidic APIs.
The brain-derived neurotrophic factor BDNF plays a critical role in neuronal development and the induction of L-LTP at glutamatergic synapses in several brain regions. However, the cellular and molecular mechanisms underlying these BDNF effects have not been firmly established. Using in vitro cultures of cortical neurons from knockout mice for Pld1 and Rsk2, BDNF was observed to induce a rapid RSK2-dependent activation of PLD and to stimulate BDNF ERK1/2-CREB and mTor-S6K signalling pathways, but these effects were greatly reduced in Pld1\(^{-/-}\) neurons. Furthermore, phospho-CREB did not accumulate in the nucleus, whereas overexpression of PLD1 amplified the BDNF-dependent nuclear recruitment of phospho-ERK1/2 and phospho-CREB. This BDNF retrograde signalling was prevented in cells silenced for the scaffolding protein PEA15, a protein which complexes with PLD1, ERK1/2, and RSK2 after BDNF treatment. Finally PLD1, ERK1/2, and RSK2 partially colocalized on endosomal structures, suggesting that these proteins are part of the molecular module responsible for BDNF signalling in cortical neurons.
Background: Dysferlin is reduced in patients with limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment myopathy, and in certain Ethnic clusters. Methods: We evaluated clinical and genetic patient data from three different Swiss Neuromuscular Centers.
Results: Thirteen patients from 6 non-related families were included. Age of onset was 18.8 +/- 4.3 years. In all patients, diallelic disease-causing mutations were identified in the DYSF gene. Nine patients from 3 non-related families from Central Switzerland carried the identical homozygous mutation, c.3031 + 2T>C. A possible founder effect was confirmed by haplotype analysis. Three patients from two different families carried the heterozygous mutation, c.1064_1065delAA. Two novel mutations were identified (c.2869C>T (p.Gln957Stop), c.5928G>A (p.Trp1976Stop)).
Conclusions: Our study confirms the phenotypic heterogeneity associated with DYSF mutations. Two mutations (c.3031 + 2T>C, c.1064_1065delAA) appear common in Switzerland. Haplotype analysis performed on one case (c.3031 + 2T>C) suggested a possible founder effect.
This Letter presents a search at the LHC for s-channel single top-quark production in proton–proton collisions at a centre-of-mass energy of 8 TeV. The analyzed data set was recorded by the ATLAS detector and corresponds to an integrated luminosity of 20.3 fb\(^{−1}\). Selected events contain one charged lepton, large missing transverse momentum and exactly two b-tagged jets. A multivariate event classifier based on boosted decision trees is developed to discriminate s-channel single top-quark events from the main background contributions. The signal extraction is based on a binned maximum-likelihood fit of the output classifier distribution. The analysis leads to an upper limit on the s-channel single top-quark production cross-section of 14.6 pb at the 95% confidence level. The fit gives a cross-section of σs=5.0±4.3 pb, consistent with the Standard Model expectation.