Biochemische Analyse und Charakterisierung der White Collar Proteinkomplexe: Aufklärung des molekularen Mechanismus des negativen Feedbacks von FRQ auf den WCC in der circadianen Uhr von Neurospora crassa

Fast alle Lebewesen haben ihren eigenen Lebensrhythmus an den täglichen Tag- Nacht- Rhythmus angepasst. Der Rhythmus wird gesteuert von der sogenannten inneren Uhr. Das sind molekulare Oszillatoren mit einer endogenen Periode von circa einem Tag. Daher der Name circadiane Uhr (lateinisch: „circa“ bedeutet ungefähr und „dies“ bedeutet Tag). Für das Verständnis der inneren Uhr ist es essentiell, die Interaktion der verschiedenen bekannten Uhrenproteine auf molekularem Niveau zu analysieren. Der Schleimpilz Neurospora crassa besitzt mindestens 3 Proteine, die für das Funktionieren der inneren Uhr notwendig sind. Das zentrale Uhrengen von Neurospora crassa heißt frequency (frq) und wird rhythmisch synthetisiert. Diese Rhythmik lässt sich sowohl auf RNA Ebene wie auch auf Proteinebene erkennen, wobei Protein-Peak und RNA-Peak jeweils 4-6 h phasenversetzt zu detektieren sind. Von grundlegender Bedeutung für das Funktionieren der inneren Uhr ist eine negative Rückkopplungsschleife, die durch das Uhrenprotein FRQ ausgeführt wird. Die Expression von FRQ führt zunächst zu einer Assemblierung von FRQ in einem hochmolekularen Komplex unbekannter Stöchiometrie und Zusammensetzung, der dann phosphoryliert und auf einem bisher nicht genauer untersuchten Weg in den Kern transportiert wird. Dort bewirkt der FRQ- Komplex eine Abschaltung seiner eigenen Biosynthese (transkriptionelle Regulation) wie auch das Abschalten verschiedener "Output"- Gene (ebenfalls auf transkriptioneller Ebene). Der genaue Mechanismus dieser Abschaltung ist unbekannt. Dieser ist Gegenstand der vorliegenden Arbeit und läuft wahrscheinlich zum großen Teil durch eine von FRQ induzierte Phosphorylierung des White Collar Proteinkomplexes (WCC). Dabei kommt es zu einer direkten Interaktion von FRQ mit dem WCC, der von den beiden anderen essentiellen Uhrenproteinen White- Collar 1 und White- Collar 2 gebildet wird. Bei diesen beiden Proteinen handelt es sich um Transkriptionsfaktoren, die über eine PAS- Domäne miteinander interagieren können und über einen GATA-Typ Zn-Finger die Transkription sowohl von frq als auch von anderen für das Output wichtigen Genen aktivieren. Ich habe mich mit der molekularen Analyse der verschiedenen Komplexe beschäftigt, d.h. mit der Anzahl, dem stöchiometrischen Aufbau und der Regulierung. Die zentralen Fragen sind, wie viele verschiedene Komplexe existieren und wie ihre genaue Stöchiometrie ist, wo diese Komplexe zu welchem circadianen Zeitpunkt lokalisiert sind und was ihre genaue Funktion in dem jeweiligen Kompartiment ist. Außerdem stellt sich die Frage, ob diese Funktion durch weitere Ereignisse wie z.B. Phosporylierung weiter reguliert wird. Im Rahmen der vorliegenden Arbeit gelang es mir, die Natur des negativen Feedback des FRQ Proteins auf den WCC auf eine FRQ abhängige Phosphorylierung des WCC zurückzuführen. Ich habe die Natur dieser Phosphorylierung eingehend untersucht und charakterisiert. Gleichzeitig habe ich die Phosphorylierungsstellen eingegrenzt und entsprechende Punktmutanten hergestellt und ebenfalls untersucht. Darüber hinaus gelang es mir WC-2 als TAP-Tag zu klonieren und nativ aus Neurospora aufzureinigen. Außerdem habe ich versucht, über verschiedene Ansätze neue, potentielle Interaktionspartner und deren Funktion zu identifizieren

Amphiphilic nanogels: influence of surface hydrophobicity on protein corona, biocompatibility and cellular uptake

Background and purpose: Nanogels (NGs) are promising drug delivery tools but are typically limited to hydrophilic drugs. Many potential new drugs are hydrophobic. Our study systematically investigates amphiphilic NGs with varying hydrophobicity, but similar colloidal features to ensure comparability. The amphiphilic NGs used in this experiment consist of a hydrophilic polymer network with randomly distributed hydrophobic groups. For the synthesis we used a new synthetic platform approach. Their amphiphilic character allows the encapsulation of hydrophobic drugs. Importantly, the hydrophilic/hydrophobic balance determines drug loading and biological interactions. In particular, protein adsorption to NG surfaces is dependent on hydrophobicity and critically determines circulation time. Our study investigates how network hydrophobicity influences protein binding, biocompatibility and cellular uptake. Methods: Biocompatibility of the NGs was examined by WST-1 assay in monocytic-like THP-1 cells. Serum protein corona formation was investigated using dynamic light scattering and two-dimensional gel electrophoresis. Proteins were identified by liquid chromatography-tandem mass spectrometry. In addition, cellular uptake was analyzed via flow cytometry. Results: All NGs were highly biocompatible. The protein binding patterns for the two most hydrophobic NGs were very similar to each other but clearly different from the hydrophilic ones. Overall, protein binding was increased with increasing hydrophobicity, resulting in increased cellular uptake. Conclusion: Our study supports the establishment of structure–property relationships and contributes to the accurate balance between maximum loading capacity with low protein binding, optimal biological half-life and good biocompatibility. This is an important step to derive design principles of amphiphilic NGs to be applied as drug delivery vehicles

Controllable Non-Markovianity for a Spin Qubit in Diamond

We present a flexible scheme to realize non-artificial non-Markovian dynamics of an electronic spin qubit, using a nitrogen-vacancy center in diamond where the inherent nitrogen spin serves as a regulator of the dynamics. By changing the population of the nitrogen spin, we show that we can smoothly tune the non-Markovianity of the electron spin's dynamic. Furthermore, we examine the decoherence dynamics induced by the spin bath to exclude other sources of non-Markovianity. The amount of collected measurement data is kept at a minimum by employing Bayesian data analysis. This allows for a precise quantification of the parameters involved in the description of the dynamics and a prediction of so far unobserved data points.Comment: 12 pages, 9 figure, including supplemental materia

Precision Limits in Quantum Metrology with Open Quantum Systems

The laws of quantum mechanics allow to perform measurements whose precision supersedes results predicted by classical parameter estimation theory. That is, the precision bound imposed by the central limit theorem in the estimation of a broad class of parameters, like atomic frequencies in spectroscopy or external magnetic field in magnetometry, can be overcome when using quantum probes. Environmental noise, however, generally alters the ultimate precision that can be achieved in the estimation of an unknown parameter. This tutorial reviews recent theoretical work aimed at obtaining general precision bounds in the presence of an environment. We adopt a complementary approach, where we first analyze the problem within the general framework of describing the quantum systems in terms of quantum dynamical maps and then relate this abstract formalism to a microscopic description of the system's dissipative time evolution. We will show that although some forms of noise do render quantum systems standard quantum limited, precision beyond classical bounds is still possible in the presence of different forms of local environmental fluctuations.Comment: slightly modified versio

The Contact Allergen NiSO4 Triggers a Distinct Molecular Response in Primary Human Dendritic Cells Compared to Bacterial LPS

Dendritic cells (DC) play a central role in the pathogenesis of allergic contact dermatitis (ACD), the most prevalent form of immunotoxicity in humans. However, knowledge on allergy-induced DC maturation is still limited and proteomic studies, allowing to unravel molecular effects of allergens, remain scarce. Therefore, we conducted a global proteomic analysis of human monocyte-derived dendritic cells (MoDC) treated with NiSO4, the most prominent cause of ACD and compared proteomic alterations induced by NiSO4 to the bacterial trigger lipopolysaccharide (LPS). Both substances possess a similar toll-like receptor (TLR) 4 binding capacity, allowing to identify allergy-specific effects compared to bacterial activation. MoDCs treated for 24 h with 2.5 mu g/ml LPS displayed a robust immunological response, characterized by upregulation of DC activation markers, secretion of pro-inflammatory cytokines and stimulation of T cell proliferation. Similar immunological reactions were observed after treatment with 400 mu M NiSO4 but less pronounced. Both substances triggered TLR4 and triggering receptor expressed on myeloid cells (TREM) 1 signaling. However, NiSO4 also activated hypoxic and apoptotic pathways, which might have overshadowed initial signaling. Moreover, our proteomic data support the importance of nuclear factor erythroid 2-related factor 2 (Nrf2) as a key player in sensitization since many Nrf2 targets genes were strongly upregulated on protein and gene level selectively after treatment with NiSO4. Strikingly, NiSO4 stimulation induced cellular cholesterol depletion which was counteracted by the induction of genes and proteins relevant for cholesterol biosynthesis. Our proteomic study allowed for the first time to better characterize some of the fundamental differences between NiSO4 and LPS-triggered activation of MoDCs, providing an essential contribution to the molecular understanding of contact allergy

A comparative proteomics analysis of four contact allergens in THP-1 cells shows distinct alterations in key metabolic pathways

Allergic contact dermatitis (ACD) is the predominant form of immunotoxicity in humans. The sensitizing potential of chemicals can be assessed in vitro. However, a better mechanistic understanding could improve the current OECD-validated test battery. The aim of this study was to get insights into toxicity mechanisms of four contact allergens, p-benzoquinone (BQ), 2,4-dinitrochlorobenzene (DNCB), p-nitrobenzyl bromide (NBB) and NiSO4, by analyzing differential proteome alterations in THP-1 cells using two common proteomics workflows, stable isotope labeling by amino acids in cell culture (SILAC) and label-free quantification (LFQ). Here, SILAC was found to deliver more robust results. Overall, the four allergens induced similar responses in THP-1 cells, which underwent profound metabolic reprogramming, including a striking upregulation of the TCA cycle accompanied by pronounced induction of the Nrf2 oxidative stress response pathway. The magnitude of induction varied between the allergens with DNCB and NBB being most potent. A considerable overlap between transcriptome-based signatures of the GARD assay and the proteins identified in our study was found. When comparing the results of this study to a previous proteomics study in human primary monocyte-derived dendritic cells, we found a rather low share in regulated proteins. However, on pathway level, the overlap was high, indicating that affected pathways rather than single proteins are more eligible to investigate proteomic changes induced by contact allergens. Overall, this study confirms the potential of proteomics to obtain a profound mechanistic understanding, which may help improving existing in vitro assays for skin sensitization

Assessing streams in Germany with benthic invertebrates: development of a practical standardised protocol for macroinvertebrate sampling and sorting

AbstractIn the past, no single standardised method for sampling and sorting benthic macroinvertebrates has been implemented in Germany. Therefore, we tested the suitability of two common sorting protocols, RIVPACS and AQEM/STAR, by taking samples with each protocol at 44 sampling sites. Our results reveal that different methods deliver slightly different assessment results. Moreover these two methods differ in costs. Although the AQEM/STAR protocol takes longer than the RIVPACS protocol, we favoured the AQEM/STAR protocol because of its higher level of standardisation. In order to limit costs to an acceptable level, a modification of the AQEM/STAR protocol (MAS method) is developed. This method is highly standardised, gives stable assessment results and is relatively inexpensive (€ 224.00 for processing of an average sample). A detailed protocol of the newly developed method is given

High-intensity interval training for overweight adolescents: program acceptance of a media supported intervention and changes in body composition

High-intensity interval training (HIIT) consists of short intervals of exercise at high intensity intermitted by intervals of lower intensity and is associated with improvement of body composition and metabolic health in adults. Studies in overweight adolescents are scarce. We conducted a randomized controlled trial in overweight adolescents to compare acceptance and attendance of HIIT with or without weekly motivational encouragement through text messages and access to a study website. HIIT was offered for six months (including summer vacation) twice a week (60 min/session). Participation rates were continuously assessed and acceptance was measured. Clinical parameters were assessed at baseline and after six months. Twenty-eight adolescents participated in this study (age 15.5 +/- 1.4; 54% female). The standard deviation score for body mass index over all participants was 2.33 at baseline and decreased by 0.026 (95% CI - 0.048 to 0.10) units, p = 0.49. Waist to height ratio was 0.596 at baseline and decreased by 0.013 (95% CI 0.0025 to 0.024), p = 0.023. Participation within the first two months ranged from 65% to 75%, but fell to 15% within the last three months. Attendance in the intervention group was 14% (95% CI - 8 to 37), p = 0.18, higher than the control group. Overall program content was rated as \"good\" by participants, although high drop-out rates were observed. Summer months constitute a serious problem regarding attendance. The use of media support has to be assessed further in appropriately powered trials