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The trace gases CO2 and CH4 pertain to the most relevant greenhouse gases and are important exchange fluxes of the global carbon (C) cycle. Their atmospheric quantity increased significantly as a result of the intensification of anthropogenic activities, such as especially land-use and land-use change, since the mid of the 18th century. To mitigate global climate change and ensure food security, land-use systems need to be developed, which favor reduced trace gas emissions and a sustainable soil carbon management. This requires the accurate and precise quantification of the influence of land-use and land-use change on CO2 and CH4 emissions. A common method to determine the trace gas dynamics and C sink or source function of a particular ecosystem is the closed chamber method. This method is often used assuming that accuracy and precision are high enough to determine differences in C gas emissions for e.g., treatment comparisons or different ecosystem components.
However, the broad range of different chamber designs, related operational procedures and data-processing strategies which are described in the scientific literature contribute to the overall uncertainty of closed chamber-based emission estimates. Hence, the outcomes of meta-analyses are limited, since these methodical differences hamper the comparability between studies. Thus, a standardization of closed chamber data acquisition and processing is much-needed.
Within this thesis, a set of case studies were performed to: (I) develop standardized routines for an unbiased data acquisition and processing, with the aim of providing traceable, reproducible and comparable closed chamber based C emission estimates; (II) validate those routines by comparing C emissions derived using closed chambers with independent C emission estimates; and (III) reveal processes driving the spatio-temporal dynamics of C emissions by developing (data processing based) flux separation approaches.
The case studies showed: (I) the importance to test chamber designs under field conditions for an appropriate sealing integrity and to ensure an unbiased flux measurement. Compared to the sealing integrity, the use of a pressure vent and fan was of minor importance, affecting mainly measurement precision; (II) that the developed standardized data processing routines proved to be a powerful and flexible tool to estimate C gas emissions and that this tool can be successfully applied on a broad range of flux data sets from very different ecosystem; (III) that automatic chamber measurements display temporal dynamics of CO2 and CH4 fluxes very well and most importantly, that they accurately detect small-scale spatial differences in the development of soil C when validated against repeated soil inventories; and (IV) that a simple algorithm to separate CH4 fluxes into ebullition and diffusion improves the identification of environmental drivers, which allows for an accurate gap-filling of measured CH4 fluxes.
Overall, the proposed standardized data acquisition and processing routines strongly improved the detection accuracy and precision of source/sink patterns of gaseous C emissions. Hence, future studies, which consider the recommended improvements, will deliver valuable new data and insights to broaden our understanding of spatio-temporal C gas dynamics, their particular environmental drivers and underlying processes.
Over the last years there is an increasing awareness that historical land cover changes and associated land use legacies may be important drivers for present-day species richness and biodiversity due to time-delayed extinctions or colonizations in response to historical environmental changes. Historically altered habitat patches may therefore exhibit an extinction debt or colonization credit and can be expected to lose or gain species in the future. However, extinction debts and colonization credits are difficult to detect and their actual magnitudes or payments have rarely been quantified because species richness patterns and dynamics are also shaped by recent environmental conditions and recent environmental changes.
In this thesis we aimed to determine patterns of herb-layer species richness and recent species richness dynamics of forest herb layer plants and link those patterns and dynamics to historical land cover changes and associated land use legacies. The study was conducted in the Prignitz, NE-Germany, where the forest distribution remained stable for the last ca. 100 years but where a) the deciduous forest area had declined by more than 90 per cent (leaving only remnants of "ancient forests"), b) small new forests had been established on former agricultural land ("post-agricultural forests"). Here, we analyzed the relative importance of land use history and associated historical land cover changes for herb layer species richness compared to recent environmental factors and determined magnitudes of extinction debt and colonization credit and their payment in ancient and post-agricultural forests, respectively.
We showed that present-day species richness patterns were still shaped by historical land cover changes that ranged back to more than a century. Although recent environmental conditions were largely comparable we found significantly more forest specialists, species with short-distance dispersal capabilities and clonals in ancient forests than in post-agricultural forests. Those species richness differences were largely contingent to a colonization credit in post-agricultural forests that ranged up to 9 species (average 4.7), while the extinction debt in ancient forests had almost completely been paid. Environmental legacies from historical agricultural land use played a minor role for species richness differences. Instead, patch connectivity was most important. Species richness in ancient forests was still dependent on historical connectivity, indicating a last glimpse of an extinction debt, and the colonization credit was highest in isolated post-agricultural forests. In post-agricultural forests that were better connected or directly adjacent to ancient forest patches the colonization credit was way smaller and we were able to verify a gradual payment of the colonization credit from 2.7 species to 1.5 species over the last six decades.
Most of the matter in the universe consists of hydrogen. The hydrogen in the intergalactic medium (IGM), the matter between the galaxies, underwent a change of its ionisation state at the epoch of reionisation, at a redshift roughly between 6>z>10, or ~10^8 years after the Big Bang. At this time, the mostly neutral hydrogen in the IGM was ionised but the source of the responsible hydrogen ionising emission remains unclear. In this thesis I discuss the most likely candidates for the emission of this ionising radiation, which are a type of galaxy called Lyman alpha emitters (LAEs). As implied by their name, they emit Lyman alpha radiation, produced after a hydrogen atom has been ionised and recombines with a free electron. The ionising radiation itself (also called Lyman continuum emission) which is needed for this process inside the LAEs could also be responsible for ionising the IGM around those galaxies at the epoch of reionisation, given that enough Lyman continuum escapes. Through this mechanism, Lyman alpha and Lyman continuum radiation are closely linked and are both studied to better understand the properties of high redshift galaxies and the reionisation state of the universe.
Before I can analyse their Lyman alpha emission lines and the escape of Lyman continuum emission from them, the first step is the detection and correct classification of LAEs in integral field spectroscopic data, specifically taken with the Multi-Unit Spectroscopic Explorer (MUSE). After detecting emission line objects in the MUSE data, the task of classifying them and determining their redshift is performed with the graphical user interface QtClassify, which I developed during the work on this thesis. It uses the strength of the combination of spectroscopic and photometric information that integral field spectroscopy offers to enable the user to quickly identify the nature of the detected emission lines. The reliable classification of LAEs and determination of their redshifts is a crucial first step towards an analysis of their properties.
Through radiative transfer processes, the properties of the neutral hydrogen clouds in and around LAEs are imprinted on the shape of the Lyman alpha line. Thus after identifying the LAEs in the MUSE data, I analyse the properties of the Lyman alpha emission line, such as the equivalent width (EW) distribution, the asymmetry and width of the line as well as the double peak fraction. I challenge the common method of displaying EW distributions as histograms without taking the limits of the survey into account and construct a more independent EW distribution function that better reflects the properties of the underlying population of galaxies. I illustrate this by comparing the fraction of high EW objects between the two surveys MUSE-Wide and MUSE-Deep, both consisting of MUSE pointings (each with the size of one square arcminute) of different depths. In the 60 MUSE-Wide fields of one hour exposure time I find a fraction of objects with extreme EWs above EW_0>240A of ~20%, while in the MUSE-Deep fields (9 fields with an exposure time of 10 hours and one with an exposure time of 31 hours) I find a fraction of only ~1%, which is due to the differences in the limiting line flux of the surveys. The highest EW I measure is EW_0 = 600.63 +- 110A, which hints at an unusual underlying stellar population, possibly with a very low metallicity.
With the knowledge of the redshifts and positions of the LAEs detected in the MUSE-Wide survey, I also look for Lyman continuum emission coming from these galaxies and analyse the connection between Lyman continuum emission and Lyman alpha emission. I use ancillary Hubble Space Telescope (HST) broadband photometry in the bands that contain the Lyman continuum and find six Lyman continuum leaker candidates. To test whether the Lyman continuum emission of LAEs is coming only from those individual objects or the whole population, I select LAEs that are most promising for the detection of Lyman continuum emission, based on their rest-frame UV continuum and Lyman alpha line shape properties. After this selection, I stack the broadband data of the resulting sample and detect a signal in Lyman continuum with a significance of S/N = 5.5, pointing towards a Lyman continuum escape fraction of ~80%. If the signal is reliable, it strongly favours LAEs as the providers of the hydrogen ionising emission at the epoch of reionisation and beyond.
Thermoresponsive Zellkultursubstrate für zeitlich-räumlich gesteuertes Auswachsen neuronaler Zellen
(2019)
Ein wichtiges Ziel der Neurowissenschaften ist das Verständnis der komplexen und zugleich faszinierenden, hochgeordneten Vernetzung der Neurone im Gehirn, welche neuronalen Prozessen, wie zum Beispiel dem Wahrnehmen oder Lernen wie auch Neuropathologien zu Grunde liegt. Für verbesserte neuronale Zellkulturmodelle zur detaillierten Untersuchung dieser Prozesse ist daher die Rekonstruktion von geordneten neuronalen Verbindungen dringend erforderlich. Mit Oberflächenstrukturen aus zellattraktiven und zellabweisenden Beschichtungen können neuronale Zellen und ihre Neuriten in vitro strukturiert werden. Zur Kontrolle der neuronalen Verbindungsrichtung muss das Auswachsen der Axone zu benachbarten Zellen dynamisch gesteuert werden, zum Beispiel über eine veränderliche Zugänglichkeit der Oberfläche.
In dieser Arbeit wurde untersucht, ob mit thermoresponsiven Polymeren (TRP) beschichtete Zellkultursubstrate für eine dynamische Kontrolle des Auswachsens neuronaler Zellen geeignet sind. TRP können über die Temperatur von einem zellabweisenden in einen zellattraktiven Zustand geschaltet werden, womit die Zugänglichkeit der Oberfläche für Zellen dynamisch gesteuert werden kann. Die TRP-Beschichtung wurde mikrostrukturiert, um einzelne oder wenige neuronale Zellen zunächst auf der Oberfläche anzuordnen und das Auswachsen der Zellen und Neuriten über definierte TRP-Bereiche in Abhängigkeit der Temperatur zeitlich und räumlich zu kontrollieren. Das Protokoll wurde mit der neuronalen Zelllinie SH-SY5Y etabliert und auf humane induzierte Neurone übertragen. Die Anordnung der Zellen konnte bei Kultivierung im zellabweisenden Zustand des TRPs für bis zu 7 Tage aufrecht erhalten werden. Durch Schalten des TRPs in den zellattraktiven Zustand konnte das Auswachsen der Neuriten und Zellen zeitlich und räumlich induziert werden. Immunozytochemische Färbungen und Patch-Clamp-Ableitungen der Neurone demonstrierten die einfache Anwendbarkeit und Zellkompatibilität der TRP-Substrate.
Eine präzisere räumliche Kontrolle des Auswachsens der Zellen sollte durch lokales Schalten der TRP-Beschichtung erreicht werden. Dafür wurden Mikroheizchips mit Mikroelektroden zur lokalen Jouleschen Erwärmung der Substratoberfläche entwickelt. Zur Evaluierung der generierten Temperaturprofile wurde eine Temperaturmessmethode entwickelt und die erhobenen Messwerte mit numerisch simulierten Werten abgeglichen. Die Temperaturmessmethode basiert auf einfach zu applizierenden Sol-Gel-Schichten, die den temperatursensitiven Fluoreszenzfarbstoff Rhodamin B enthalten. Sie ermöglicht oberflächennahe Temperaturmessungen in trockener und wässriger Umgebung mit hoher Orts- und Temperaturauflösung. Numerische Simulationen der Temperaturprofile korrelierten gut mit den experimentellen Daten. Auf dieser Basis konnten Geometrie und Material der Mikroelektroden hinsichtlich einer lokal stark begrenzten Temperierung optimiert werden. Ferner wurden für die Kultvierung der Zellen auf den Mikroheizchips eine Zellkulturkammer und Kontaktboard für die elektrische Kontaktierung der Mikroelektroden geschaffen.
Die vorgestellten Ergebnisse demonstrieren erstmalig das enorme Potential thermoresponsiver Zellkultursubstrate für die zeitlich und räumlich gesteuerte Formation geordneter neuronaler Verbindungen in vitro. Zukünftig könnte dies detaillierte Studien zur neuronalen Informationsverarbeitung oder zu Neuropathologien an relevanten, humanen Zellmodellen ermöglichen.
Hyperspectral remote sensing of the spatial and temporal heterogeneity of low Arctic vegetation
(2019)
Arctic tundra ecosystems are experiencing warming twice the global average and Arctic vegetation is responding in complex and heterogeneous ways. Shifting productivity, growth, species composition, and phenology at local and regional scales have implications for ecosystem functioning as well as the global carbon and energy balance. Optical remote sensing is an effective tool for monitoring ecosystem functioning in this remote biome. However, limited field-based spectral characterization of the spatial and temporal heterogeneity limits the accuracy of quantitative optical remote sensing at landscape scales. To address this research gap and support current and future satellite missions, three central research questions were posed:
• Does canopy-level spectral variability differ between dominant low Arctic vegetation communities and does this variability change between major phenological phases?
• How does canopy-level vegetation colour images recorded with high and low spectral resolution devices relate to phenological changes in leaf-level photosynthetic pigment concentrations?
• How does spatial aggregation of high spectral resolution data from the ground to satellite scale influence low Arctic tundra vegetation signatures and thereby what is the potential of upcoming hyperspectral spaceborne systems for low Arctic vegetation characterization?
To answer these questions a unique and detailed database was assembled. Field-based canopy-level spectral reflectance measurements, nadir digital photographs, and photosynthetic pigment concentrations of dominant low Arctic vegetation communities were acquired at three major phenological phases representing early, peak and late season. Data were collected in 2015 and 2016 in the Toolik Lake Research Natural Area located in north central Alaska on the North Slope of the Brooks Range. In addition to field data an aerial AISA hyperspectral image was acquired in the late season of 2016. Simulations of broadband Sentinel-2 and hyperspectral Environmental and Mapping Analysis Program (EnMAP) satellite reflectance spectra from ground-based reflectance spectra as well as simulations of EnMAP imagery from aerial hyperspectral imagery were also obtained.
Results showed that canopy-level spectral variability within and between vegetation communities differed by phenological phase. The late season was identified as the most discriminative for identifying many dominant vegetation communities using both ground-based and simulated hyperspectral reflectance spectra. This was due to an overall reduction in spectral variability and comparable or greater differences in spectral reflectance between vegetation communities in the visible near infrared spectrum.
Red, green, and blue (RGB) indices extracted from nadir digital photographs and pigment-driven vegetation indices extracted from ground-based spectral measurements showed strong significant relationships. RGB indices also showed moderate relationships with chlorophyll and carotenoid pigment concentrations. The observed relationships with the broadband RGB channels of the digital camera indicate that vegetation colour strongly influences the response of pigment-driven spectral indices and digital cameras can track the seasonal development and degradation of photosynthetic pigments.
Spatial aggregation of hyperspectral data from the ground to airborne, to simulated satel-lite scale was influenced by non-photosynthetic components as demonstrated by the distinct shift of the red edge to shorter wavelengths. Correspondence between spectral reflectance at the three scales was highest in the red spectrum and lowest in the near infra-red. By artificially mixing litter spectra at different proportions to ground-based spectra, correspondence with aerial and satellite spectra increased. Greater proportions of litter were required to achieve correspondence at the satellite scale.
Overall this thesis found that integrating multiple temporal, spectral, and spatial data is necessary to monitor the complexity and heterogeneity of Arctic tundra ecosystems. The identification of spectrally similar vegetation communities can be optimized using non-peak season hyperspectral data leading to more detailed identification of vegetation communities. The results also highlight the power of vegetation colour to link ground-based and satellite data. Finally, a detailed characterization non-photosynthetic ecosystem components is crucial for accurate interpretation of vegetation signals at landscape scales.
The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid+ chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation
Topologische Datenanalyse
(2019)
Bei der Analyse von höherdimensionalen Daten kann deren Gestalt wichtige Informationen über den Datensatz liefern. Bei einer gegebenen Punktwolke, die aus einem unbekannten topologischen Raum ausgewählt wurde, versucht die Topologische Datenanalyse (TDA) den ursprünglichen Raum zu rekonstruieren. Dieser Beitrag soll eine Einführung in die Topologische Datenanalyse geben und konzentriert sich dabei auf zwei wichtige Aspekte: die Persistente Homologie und den Mapper. Dabei werden zuerst die notwendigen theoretischen Grundlagen vorgestellt und anschließend wird die Methodik bei der Visualisierung von Daten eingesetzt.
Die Persistente Homologie ist eines der Standardwerkzeuge in der TDA. Sie findet ihre Anwendung beispielsweise in den Bereichen Formerkennung und -beschreibung. Der Mapper als zweites wichtiges Konzept der TDA wandelt umfangreiche, höherdimensionale Datensätze in Simplizialkomplexe um und kann dadurch geometrische und topologische Eigenschaften der Daten bestimmen. Des Weiteren ist die Mapper-Methode ein brauchbares Werkzeug zur Visualisierungen von mehrdimensionalen Daten, woran statistische Verfahren scheitern.
Permettez à l’inventeur du manuscrit dont vous avez l’édition diplomatique sous les yeux d’exposer en exergue les principales étapes qui ont conduit en 2012 à la détermination de la langue et à l’identification de l’auteur.
Étant inscrit à la conférence de linguistique missionnaire à l’université de Brème en mars 2012 et conscient que maintes bibliothèques et archives renferment des trésors non identifiés ou mal catalogués, je suivis le conseil d’un collègue de la bibliothèque de l’université de Trèves, Hans-Ulrich Seiffert, d’inspecter le manuscrit 1136 / 2048 4° de la bibliothèque municipale de Trèves. Cette bibliothèque est riche de plus de 2.500 manuscrits et incunables provenant pour la plupart des saisies révolutionnaires françaises dans les bibliothèques des ci-devant couvents trévirois. Parmi ces trésors un manuscrit portugais des missions qui était rentré dans les fonds de la bibliothèque de Trèves dès 1799 et avait été exposé en 1991 dans le cadre d’une évocation des activités missionnaires globales des Jésuites. Michael Embach, alors directeur de la bibliothèque du séminaire épiscopal de Trèves, l’avait brièvement décrit dans une notice qui prétendait qu’il était rédigé en espagnol et en portugais et avait peut-être été composé par un auteur « Meirin » dans le cadre de ses préparations pour partir dans les missions… Dans ma copie du catalogue de 1991 se trouve inscrit « la langue reste à déterminer ».
J’allais résoudre le mystère en cette mémorable journée du jeudi 23 février 2012 quand je me résolus à l’autopsier dans la salle désormais historique de la Bibliothèque municipale de Trèves, dirigée depuis peu par le collègue Embach. Il parut à l’évidence que le manuscrit ne contenait aucun mot d’espagnol mais que dans la première partie, les entrées en portugais, strictement numérotées et arrangées dans l’ordre alphabétique, étaient suivies par leurs équivalences dans une langue inconnue qui, cependant, avait pour un linguiste une forte odeur de l’Amérique amazonienne. La partie postérieure du manuscrit présentait l’arrangement inverse. Les entrées dans la langue inconnue étaient suivies de leurs équivalences portugaises. Ce n’est que plus tard que nous avons compris le mystérieux ordre des lemmata dans cette partie, basée qu’elle est sur la rime finale des mots.
Deux facteurs ont grandement facilité l’identification de la langue inconnue : 1. la permission du directeur Embach de pouvoir prendre des photos de travail de tous les folios du manuscrit et 2. l’entrée dans le premier catalogue de la Bibliothèque de Trèves de 1802 : “Codex maxime memorabiblis est, cum nondum grammatica praeter Lusitani Anchieta nota sit, et nullum vocabularium huius linguae existet. Sine dubio scriptum est a quodam Missionario Jesuita”.
Rentrant de Trèves chez moi, je fis une brève halte dans mon bureau d’alors pour consulter Internet. L’entrée “Anchieta” dans l’encyclopédie électronique Wikipedia m’informait que ce jésuite portugais avait composé une grammaire du Vieux Tupi, publiée en 1595. Sous le vocable “Old Tupi” ensuite, j’appris que l’auto-désignation pour cette langue est ñeengatú (la bonne langue, le parler correct). De là à retrouver au folio 25 de mes photos la traduction du portugais linguagem par nheénga ne prit que quelques minutes. Dès ce premier soir donc, le mystère de la langue inconnue était résolu!
Un projet de recherche international, voire transcontinental allait se mettre peu à peu en place après. À la conférence de Brème, début mars 2012, je fis la connaissance du linguiste et romaniste, le professeur Wolf Dietrich de l’université de Münster en Westphalie, un des meilleurs connaisseurs des parlers amazoniens. Il rentrait avec une copie de mes photos de travail et confirma bientôt l’importance du manuscrit de Trèves pour la connaissance de la Lingua Geral, le parler qui s’était développé à partir du Vieux Tupi moribond.
Je communiquai également ma découverte au frère Karl-Heinz Arenz de la congrégation des pères de Steyl, originaire de l’Eifel allemande et enseignant d’histoire aux universités brésiliennes de Belém et de Santarém dans l’État du Pará. Arenz est l’auteur d’une étude sur le jésuite luxembourgeois Jean-Philippe Bettendorf, actif au Maranhão dans la seconde moitié du XVIIe siècle. Nous avions collaboré en 2007 à une exposition didactique consacrée à ce personnage. Arenz ne tarda pas à passer l’information sur le manuscrit de Trèves aux deux linguistes brésiliennes Cândida Barros et Ruth Monserrat, qui à leur tour présentaient un projet de recherche qui fut accepté par les autorités brésiliennes. Les reals de ce subside furent bien investis en effet : l’étudiant doctoral Gabriel Prudente réalisa une transcription intégrale du manuscrit de Trèves qui fit apparaître des couches dialectales et sociolectales dans le texte, et même quelques mots allemands. La bibliothèque municipale de Trèves, quant à elle, contribua « en nature » les images digitales qui sont consultables sur les pages de gauche de cette édition électronique.
Début avril 2014, nous fîmes tous connaissance personnellement dans le cadre d’un colloque à l’université de Belém où fut décidée la présente édition digitale et où furent prononcées la plupart des contributions scientifiques que vous pouvez lire en guise d’introduction. Restait à percer l’énigme du véritable auteur du manuscrit. Cândida Barros avait assez tôt proposé les noms de trois Jésuites allemands, actifs dans les années 1750 dans la région du fleuve Xingu et chassés par les mesures anti-jésuites du pouvoir portugais en 1756. L’étude paléographique des Quattuor vota des trois candidats, aux archives centrales des Jésuites à Rome, me permit début septembre 2015 d’éliminer les pères Eckart et Kaulen et de ne retenir que Antonius Meisterburg, originaire de Bernkastel sur la Moselle, comme le scribe du manuscrit de Trèves. Qu’il me soit permis d’exprimer ma GRATITUDE à toutes celles et à tous ceux qui ont contribué à cette belle aventure de découverte intellectuelle et scientifique et à rendre au Brésil une petite pierre de son histoire.
Dermal Delivery of the High-Molecular-Weight Drug Tacrolimus by Means of Polyglycerol-Based Nanogels
(2019)
Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions.
The natural abundance of Coiled Coil (CC) motifs in cytoskeleton and extracellular matrix proteins suggests that CCs play an important role as passive (structural) and active (regulatory) mechanical building blocks. CCs are self-assembled superhelical structures consisting of 2-7 α-helices. Self-assembly is driven by hydrophobic and ionic interactions, while the helix propensity of the individual helices contributes additional stability to the structure. As a direct result of this simple sequence-structure relationship, CCs serve as templates for protein design and sequences with a pre-defined thermodynamic stability have been synthesized de novo. Despite this quickly increasing knowledge and the vast number of possible CC applications, the mechanical function of CCs has been largely overlooked and little is known about how different CC design parameters determine the mechanical stability of CCs. Once available, this knowledge will open up new applications for CCs as nanomechanical building blocks, e.g. in biomaterials and nanobiotechnology.
With the goal of shedding light on the sequence-structure-mechanics relationship of CCs, a well-characterized heterodimeric CC was utilized as a model system. The sequence of this model system was systematically modified to investigate how different design parameters affect the CC response when the force is applied to opposing termini in a shear geometry or separated in a zipper-like fashion from the same termini (unzip geometry). The force was applied using an atomic force microscope set-up and dynamic single-molecule force spectroscopy was performed to determine the rupture forces and energy landscape properties of the CC heterodimers under study. Using force as a denaturant, CC chain separation is initiated by helix uncoiling from the force application points. In the shear geometry, this allows uncoiling-assisted sliding parallel to the force vector or dissociation perpendicular to the force vector. Both competing processes involve the opening of stabilizing hydrophobic (and ionic) interactions. Also in the unzip geometry, helix uncoiling precedes the rupture of hydrophobic contacts.
In a first series of experiments, the focus was placed on canonical modifications in the hydrophobic core and the helix propensity. Using the shear geometry, it was shown that both a reduced core packing and helix propensity lower the thermodynamic and mechanical stability of the CC; however, with different effects on the energy landscape of the system. A less tightly packed hydrophobic core increases the distance to the transition state, with only a small effect on the barrier height. This originates from a more dynamic and less tightly packed core, which provides more degrees of freedom to respond to the applied force in the direction of the force vector. In contrast, a reduced helix propensity decreases both the distance to the transition state and the barrier height. The helices are ‘easier’ to unfold and the remaining structure is less thermodynamically stable so that dissociation perpendicular to the force axis can occur at smaller deformations.
Having elucidated how canonical sequence modifications influence CC mechanics, the pulling geometry was investigated in the next step. Using one and the same sequence, the force application points were exchanged and two different shear and one unzipping geometry were compared. It was shown that the pulling geometry determines the mechanical stability of the CC. Different rupture forces were observed in the different shear as well as in the unzipping geometries, suggesting that chain separation follows different pathways on the energy landscape. Whereas the difference between CC shearing and unzipping was anticipated and has also been observed for other biological structures, the observed difference for the two shear geometries was less expected. It can be explained with the structural asymmetry of the CC heterodimer. It is proposed that the direction of the α-helices, the different local helix propensities and the position of a polar asparagine in the hydrophobic core are responsible for the observed difference in the chain separation pathways. In combination, these factors are considered to influence the interplay between processes parallel and perpendicular to the force axis.
To obtain more detailed insights into the role of helix stability, helical turns were reinforced locally using artificial constraints in the form of covalent and dynamic ‘staples’. A covalent staple bridges to adjacent helical turns, thus protecting them against uncoiling. The staple was inserted directly at the point of force application in one helix or in the same terminus of the other helix, which did not experience the force directly. It was shown that preventing helix uncoiling at the point of force application reduces the distance to the transition state while slightly increasing the barrier height. This confirms that helix uncoiling is critically important for CC chain separation. When inserted into the second helix, this stabilizing effect is transferred across the hydrophobic core and protects the force-loaded turns against uncoiling. If both helices were stapled, no additional increase in mechanical stability was observed. When replacing the covalent staple with a dynamic metal-coordination bond, a smaller decrease in the distance to the transition was observed, suggesting that the staple opens up while the CC is under load.
Using fluorinated amino acids as another type of non-natural modification, it was investigated how the enhanced hydrophobicity and the altered packing at the interface influences CC mechanics. The fluorinated amino acid was inserted into one central heptad of one or both α-helices. It was shown that this substitution destabilized the CC thermodynamically and mechanically. Specifically, the barrier height was decreased and the distance to the transition state increased. This suggests that a possible stabilizing effect of the increased hydrophobicity is overruled by a disturbed packing, which originates from a bad fit of the fluorinated amino acid into the local environment. This in turn increases the flexibility at the interface, as also observed for the hydrophobic core substitution described above. In combination, this confirms that the arrangement of the hydrophobic side chains is an additional crucial factor determining the mechanical stability of CCs.
In conclusion, this work shows that knowledge of the thermodynamic stability alone is not sufficient to predict the mechanical stability of CCs. It is the interplay between helix propensity and hydrophobic core packing that defines the sequence-structure-mechanics relationship. In combination, both parameters determine the relative contribution of processes parallel and perpendicular to the force axis, i.e. helix uncoiling and uncoiling-assisted sliding as well as dissociation. This new mechanistic knowledge provides insight into the mechanical function of CCs in tissues and opens up the road for designing CCs with pre-defined mechanical properties. The library of mechanically characterized CCs developed in this work is a powerful starting point for a wide spectrum of applications, ranging from molecular force sensors to mechanosensitive crosslinks in protein nanostructures and synthetic extracellular matrix mimics.