Differential regulation of IL-12p70 and IL-23 in murine dendritic cells

Dendritic cells are the sentinels between the innate and the adaptive immunity. They are professionals that capture invading pathogens, recognize specific microbial structures and induce naïve T lymphocytes to polarize into a specific T cell subset. To initiate the T cell polarization DCs secrete cytokines which are induced upon Toll-like receptor activation by microbial structures. The recognition of these structures and the discrimination between non-self and self structures by TLRs is fine tuned, but under defined circumstances deregulation of immune responses appears. Consequently, this can result in immune disorders such as autoimmunity, chronic inflammatory diseases or cancer. In this thesis the investigations are focused on the regulation of the IL-12 family members IL-12p70 and IL-23 in DCs. The objective was to investigate three different endogenous and exogenous factors that regulate IL-12p70 or IL-23. In the first part Selenium, an essential trace element and important factor in several metabolic pathways including the cellular redox status and reactive oxygen species (ROS) dependent signaling was applied as supplement in immature Langerhans cell culture. Because Selenium also plays a role in the immune system the TLR-induced IL-23 production of the DCs upon Selenium treatment was analyzed. In the immature Langerhans cell line XS-52 the strongest inducer of IL-23 was TLR4 ligand LPS. Furthermore increased levels of TLR4-induced IL-23 in cells treated with Selenium were detected in a concentration dependent manner. Whereas the IL-23 subunit p40 was upregulated upon Selenium treatment the second subunit p19 was completely unaffected. This effect was detected on mRNA and protein level. In addition, as expected, IFN-gamma inhibited the TLR4-induced IL-23 secretion of both, Selenium treated and untreated cells. In the second part of this thesis p47phox, an organizing protein of the NADPH oxidase was analyzed regarding its potential to regulate IL-12p70 and/or IL-23 secreted by different DC subtypes. Since it was demonstrated that p47phox deficiency is associated with enhanced autoimmunity and chronic inflammation we wanted to prove whether it has a function in addition to that within the NADPH oxidase. We found some hints that p47phox may be interact with proteins of the TLR signaling pathway and thus we hypothesized that p47phox may have a function for the regulation of TLR-mediated cytokine production in DCs. In several experiments with DCs from the spleen of different p47phox deficient mice we detected an increased production of TLR9-induced IL-12p70 compared to wild type cells. In contrast TLR4 stimulation with LPS displayed no significant differences between p47phox deficient and wild type cells. In spleen cells IL-23 was not detected. Confirming the results of this new negative feedback by p47phox on IL-12p70 rats, with a single nucleotide polymorphism in the p47phox gene, were investigated. Interestingly this polymorphism is located in the phosphorylation site of IRAK4, an important kinase in the TLR pathway. In rats with a methionine residue at this position in the p47phox protein enhanced IL-12p70 level were found, compared to the rats with threonine, which can be phosphorylated by IRAK4. All analyzed mice and rats have defects in the NADPH oxidase function due to a non functional p47phox protein which results in a defective ROS production. To determine whether the observed negative feedback mechanism is connected to the lack of ROS production experiments with gp91phox deficient mice, which also have a defective NADPH oxidase function, were performed. In several experiments the enhanced IL-12p70 production in cells from p47phox deficient mice could be confirmed, but no differences between gp91phox deficient and wild type mice have been observed. In further studies was found that the inhibition of the NADPH oxidase function did not alter the negative feedback on TLR9-induced IL-12p70 secretion by p47phox. Interestingly upon treatment with the inhibitor a feedback mechanism in wild type cells also after TLR4 stimulation was observed. Hence, blocking a ROS-dependent TLR4 pathway by the inhibitor uncovered the LPS induced ROS-independent pathway of the TLR4 signaling. These findings strongly approve a NADPH oxidase/ROS-independent function of p47phox in DCs. Because splenic DCs do not secrete IL-23, in vitro differentiated DCs from the bone marrow were investigated regarding the negative feedback mechanism. In DCs from p47phox deficient mice, differentiated with GM-CSF, the upregulation of IL-12p70 was confirmed, whereas Flt3-L cultured DCs did not display the negative feedback. In contrast to IL-12p70 no difference for the IL-23 production between wild type and p47phox deficient cells has been detected. Thus, we concluded that IL-23 production is not regulated by p47phox. IL-12p70 is the major cytokine in the Th1 polarization whereas IL-23 is important for the maintenance and survival of Th17 cells. To prove whether the regulation of IL-12p70 influences the T cell response immunization experiments closely resembling the classical DTH-like protocols were performed. Groups of p47phox deficient and wild type mice received either PBS, OVA alone or mixed with TLR9 ligand CpG2216 in IFA s.c. to activate and polarize naïve T cells towards Th1 or Th17 cells. After ten days isolated lymph node cells were incubated in an ELISA spot assay with or without OVA and the frequency of IFN-gamma and IL-17 producing T cells was quantified. In vitro recall of OVA immunization of wild type and p47phox deficient mice resulted in an increased IFN-gamma and IL-17 frequency in the p47phox deficient cells. The combination with CpG2216 as adjuvant and inducer of the 3rd signal enhanced the frequency of IFN-gamma and IL-17 producing T cells in wild type mice significantly. However, in p47phox deficient cells the IFN-gamma and IL-17 response, being already detectable without in vitro OVA re-stimulation, was strongly augmented upon OVA restimulation. These findings confirmed our in vitro data for IL-12p70. Hence, the data supports our hypothesis that the p47phox dependent regulation of IL-12p70 and the consequences for the T cell response is an important mechanism to prevent uncontrolled immune responses. In the last part of this thesis the immunomodulatory property of vitamin D3 on the IL-12p70 production of DCs was examined. Since it was shown that VD3 influences the differentiation and maturation of monocytes and DCs, splenic DCs from C57BL/6 and BALB/c mice were investigated regarding their IL-12p70 production after VD3 treatment. Spleen cells, stimulated with LPS or CpG2216, exhibited a decreased IL-12p70 production when treated with VD3 before stimulation phase. In contrast treatment with VD3 only during TLR stimulation had no influence on the IL-12p70 production. Since it was demonstrated that VD3 stimulates the expression of p47phox mRNA cells from p47phox deficient mice were also treated with VD3. In initial experiments only a slight inhibition of IL-12p70 has been detected in p47phox deficient cells compared to the wild type. In summary the thesis displays three different possibilities to influence the TLR-induced cytokine secretion of DCs, although with different intensities and specificities

The Mos pathway regulates cytoplasmic polyadenylation in Xenopus oocytes

Cytoplasmic polyadenylation controls the translation of several maternal mRNAs during Xenopus oocyte maturation and requires two sequences in the 3\u27 untranslated region (UTR), the U-rich cytoplasmic polyadenylation element (CPE), and the hexanucleotide AAUAAA. c-mos mRNA is polyadenylated and translated soon after the induction of maturation, and this protein kinase is necessary for a kinase cascade culminating in cdc2 kinase (MPF) activation. Other mRNAs are polyadenylated later, around the time of cdc2 kinase activation. To determine whether there is a hierarchy in the cytoplasmic polyadenylation of maternal mRNAs, we ablated c-mos mRNA with an antisense oligonucleotide. This prevented histone B4 and cyclin A1 and B1 mRNA polyadenylation, indicating that the polyadenylation of these mRNAs is Mos dependent. To investigate a possible role of cdc2 kinase in this process, cyclin B was injected into oocytes lacking c-mos mRNA. cdc2 kinase was activated, but mitogen-activated protein kinase was not. However, polyadenylation of cyclin B1 and histone B4 mRNA was still observed. This demonstrates that cdc2 kinase can induce cytoplasmic polyadenylation in the absence of Mos. Our data further indicate that although phosphorylation of the CPE binding protein may be involved in the induction of Mos-dependent polyadenylation, it is not required for Mos-independent polyadenylation. We characterized the elements conferring Mos dependence (Mos response elements) in the histone B4 and cyclin B1 mRNAs by mutational analysis. For histone B4 mRNA, the Mos response elements were in the coding region or 5\u27 UTR. For cyclin B1 mRNA, the main Mos response element was a CPE that overlaps with the AAUAAA hexanucleotide. This indicates that the position of the CPE can have a profound influence on the timing of cytoplasmic polyadenylation

Einfluss der Qualität des ÖPNV auf die Verkehrsmittelwahl im Regionalverkehr

In dieser Arbeit wird der Zusammenhang zwischen Qualität und Verkehrsmittelwahl untersucht. Dazu werden Discrete Choice Experimente mit über 2000 Nutzern und Nicht-Nutzern des regionalen ÖPNV durchgeführt. Durch die Verwendung des Verfahrens der integrierten hierarchischen Informationsintegration (HII-I) kann eine größere Anzahl von Qualitätsattributen in den Experimenten berücksichtigt werden, da hierbei ähnliche Attribute zu Konstrukten zusammengefasst werden. Diese Konstrukte werden im Rahmen der Arbeit empirisch hergeleitet. Mit zahlreichen statistischen Tests wird der Einfluss der Qualität auf die Verkehrsmittelwahl, Unterschiede zwischen einzelnen Nutzergruppen sowie die hierarchische Struktur des eingesetzten Verfahrens überprüft.Aufbauend auf den Ergebnissen dieser Arbeit lassen sich Veränderungen des Modal Splits infolge einer Änderung der Qualität simulieren und Empfehlungen für die Praxis ableiten.<br

Ozone Production in the Atmosphere Simulation Chamber SAPHIR

Tropospheric ozone in high concentrations is harmful for mankind and the environment as a whole. As it is a greenhouse gas, its rising due to anthropogenic emissions of the precursor species contributes to global warming. By being the precursor specie for all oxidizing agents in the atmosphere, e.g. the highly reactive OH radical, and being an oxidizing agent itself, ozone is very important in atmospheric chemistry. Due to this importance, a sound understanding of the chemical ozone production processes is needed. The necessary precursors for the photochemical production are the mainly anthropogenic nitrogen oxides NO$_{x}$ and the both anthropogenic and biogenic volatile organic compounds. In principle, the processes are fairly understood. In details huge uncertainties still exist. These have to be examined further to allow for well-founded predictions of short and long term ozone concentrations, e.g. to early warn the population off injurious values to come or for the use in climate change modelling. In the atmosphere simulation chamber SAPHIR chemical processes of the troposphere can be examined nearly without physical caused changes, like transport, mixing, or unknown sources and sinks of trace constituents. Ambient conditions concerning trace gas concentrations, temperature, pressure and lighting conditions characterize the SAPHIR experiments. To understand the complex processes influencing trace gas concentrations in nature, field experiments are obligatory. For the interpretation of measured field data model calculations are needed to distinguish between chemical and physical influences. The test of these models is only feasible under the physically controlled conditions inside the SAPHIR chamber. In this thesis, three different approaches, which strongly vary concerning their needed (measured) input and the computational effort, for the prediction of the photochemical ozone production were tested against SAPHIR chamber experiments. First of all model runs on the basis of the Master ChemicalMechanism, which compiles the state of the art knowledge in atmospheric chemistry in one mechanism, were tested at ambient trace gas concentrations for the first time. These model runs only need few measured input but a high computational effort. The newly developed First Degradation Step approach in contrast needs a lot of measured input, which then is combined by fundamental arithmetic to calculate the ozone production. The third, also new approach tested is an even simpler method, which estimates the ozone production by a simple combination of measured OH concentrations and OH lifetimes. As the initial organic compound for the SAPHIR experiments isoprene and its degradation products methacrolein and methyl vinyl ketone were selected as on a global scale isoprene is the mostly emitted volatile organic compound, which dominates photochemical ozone production in many regions. In the second part of this thesis special attention was directed on the methacrolein degradation. The Master Chemical Mechanism model showed strong deviations concerning the measured NO$_{x}$ concentrations. These discrepancies could partly be explained and were traced back to errors of the Master Chemical Mechanism

Turning points in the transition to parenthood: Variability of father involvement over time

"Although fathers' involvement in care work has increased, the transition to parenthood still implies a gendered division of labour. In order to gain more knowledge of this ambivalence, we focus on the variability of father involvement at this transition. Based on an Austrian qualitative longitudinal study with couples experiencing the transition to first-time parenthood, we examined how fathers' affective, cognitive and behavioural involvement varies across the transition process. Changes in fathers' involvement culminated at particular points in time, conceptualised as turning points. Results show that the transition to fatherhood is characterised by a variety of prepregnancy, prenatal and postnatal turning points at which father involvement undergoes crucial transformations. Father involvement varies not only between fathers, but also within individual transitions. The study indicates that turning points contribute to the dynamics and fluidity of the transition process." (author's abstract)"Obwohl die väterliche Beteiligung an der Betreuungsarbeit im Steigen begriffen ist, ist der Übergang zur Elternschaft nach wie vor mit einer geschlechtsspezifischen und ungleichen Arbeitsteilung verbunden. Um diese Ambivalenz zu verstehen, konzentrieren wir uns auf die Variabilität väterlicher Beteiligung bei diesem Übergang. Anhand einer österreichischen qualitativen Längsschnittstudie mit Paaren, die das erste Mal Eltern werden, wurde untersucht, wie sich die affektive, kognitive und verhaltensmäßige Beteiligung in diesem Transitionsprozess verändert. Die Ergebnisse zeigen, dass der Übergang zur Elternschaft sich durch zahlreiche Wendepunkte (turning points) auszeichnet, an denen väterliche Beteiligung eine wesentliche Änderung erfährt. Diese Wendepunkte können vor und während der Schwangerschaft sowie nach der Geburt auftreten. Die Beteiligung variiert nicht nur zwischen Vätern, sondern auch innerhalb individueller Übergänge. Wendepunkte implizieren einen Wechsel in der Arbeitsteilung zwischen Müttern und Vätern und tragen zur Dynamik und Fluidität des Transitionsprozesses bei." (Autorenreferat

Does Your Smartphone “Know” Your Social Life? A Methodological Comparison of Day Reconstruction, Experience Sampling, and Mobile Sensing

Mobile sensing is a promising method that allows researchers to directly observe human social behavior in daily life using people’s mobile phones. To date, limited knowledge exists on how well mobile sensing can assess the quantity and quality of social interactions. We therefore examined the agreement among experience sampling, day reconstruction, and mobile sensing in the assessment of multiple aspects of daily social interactions (i.e., face-to-face interactions, calls, and text messages) and the possible unique access to social interactions that each method has. Over 2 days, 320 smartphone users (51% female, age range = 18–80, M = 39.53 years) answered up to 20 experience-sampling questionnaires about their social behavior and reconstructed their days in a daily diary. Meanwhile, face-to-face and smartphone-mediated social interactions were assessed with mobile sensing. The results showed some agreement between measurements of face-to-face interactions and high agreement between measurements of smartphone-mediated interactions. Still, a large number of social interactions were captured by only one of the methods, and the quality of social interactions is still difficult to capture with mobile sensing. We discuss limitations and the unique benefits of day reconstruction, experience sampling, and mobile sensing for assessing social behavior in daily life

Counterpropagating dipole-mode vector soliton

We experimentally observe a counterpropagating dipole-mode vector soliton in a photorefractive SBN:60Ce crystal. We investigate the transient formation dynamics and show that the formation process differs significantly from the copropagating geometry. The experimental results are compared with fully anisotropic numerical simulations which show good qualitative agreement.Comment: 4 pages, 4 figure

Frequent epigenetic inactivation of RASSF2 in thyroid cancer and functional consequences

<p>Abstract</p> <p>Background</p> <p>The Ras association domain family (RASSF) encodes for distinct tumor suppressors and several members are frequently silenced in human cancer. In our study, we analyzed the role of RASSF2, RASSF3, RASSF4, RASSF5A, RASSF5C and RASSF6 and the effectors MST1, MST2 and WW45 in thyroid carcinogenesis.</p> <p>Results</p> <p>Frequent methylation of the <it>RASSF2 </it>and <it>RASSF5A </it>CpG island promoters in thyroid tumors was observed. <it>RASSF2 </it>was methylated in 88% of thyroid cancer cell lines and in 63% of primary thyroid carcinomas. <it>RASSF2 </it>methylation was significantly increased in primary thyroid carcinoma compared to normal thyroid, goiter and follicular adenoma (0%, 17% and 0%, respectively; p < 0.05). Patients which were older than 60 years were significantly hypermethylated for <it>RASSF2 </it>in their primary thyroid tumors compared to those younger than 40 years (90% vs. 38%; p < 0.05). <it>RASSF2 </it>promoter hypermethylation correlated with its reduced expression and treatment with a DNA methylation inhibitor reactivated <it>RASSF2 </it>transcription. Over-expression of RASSF2 reduced colony formation of thyroid cancer cells. Functionally our data show that RASSF2 interacts with the proapoptotic kinases MST1 and MST2 and induces apoptosis in thyroid cancer cell lines. Deletion of the MST interaction domain of RASSF2 reduced apoptosis significantly (p < 0.05).</p> <p>Conclusion</p> <p>These results suggest that <it>RASSF2 </it>encodes a novel epigenetically inactivated candidate tumor suppressor gene in thyroid carcinogenesis.</p

Counterpropagating beams in biased photorefractive crystals: anisotropic theory

We formulate an anisotropic nonlocal theory of the space charge field induced by the coherent counterpropagating beams in biased photorefractive crystals. We establish that the competition between the drift and diffusion terms has to be taken into account when the crystal cˆ axis is tilted with respect to the propagation direction of the beams. We demonstrate that this configuration combines the features of both spatial soliton formation without energy exchange and two-wave mixing with energy exchange leading to pattern formation

Improve Performance Management in Flexible Business Processes

The performance of business processes is evaluated and mon- itored with the aim of identifying whether strategic and operational goals are being achieved. Most approaches about performance measurement have been de ned over traditional highly repetitive and well-structured processes. However, cur- rent organizational and business needs have encouraged the appearance of customizable processes to manage collections of process variants derived from a process, and loosely speci- ed processes to manage non-repeatable and unpredictable processes. However, current techniques of performance mea- surement have not evolved to the same pace that business processes, thus generating a gap between processes and the measurement of their performance. The thesis introduced in this paper, is focused on enhancing the performance mea- surement of business processes by means of the improvement of existing techniques for the de nition of process perfor- mance indicators and their applicability to different types of processes. With this purpose a set of artifacts, including a metamodel, notations, tools and methodologies will be developed. They will be validated by means of case studies based on real scenarios.Ministerio de Economía y Competitividad TIN2015-70560-RJunta de Andalucía P12-TIC-1867Junta de Andalucía P10-TIC-590