Biochemical characterization of an intermediate membrane subfraction in cyanobacteria involved in an assembly network for photosystem II

Oxygenic photosynthesis converts light energy into chemical energy and is responsible for generating most of our atmosphere’s oxygen and biomass on earth. Several multimeric protein complexes are involved in the underlying photosynthetic electron transfer chain with photosystem II (PSII) representing the initial complex mediating the extraction of electrons from water molecules, thus generating molecular oxygen as a by-product. During recent years, the structural details and components of the PSII complex, including its inorganic and organic cofactors, have been elucidated in great detail. However, little is known about the assembly pathway of this at least 20 protein subunits containing machinery. Previous work indicated that PSII assembly occurs in a step-wise fashion and requires a number of facilitating factors, which interact transiently with nascent PSII complexes. Earlier studies of one of those assembly factors, the cyanobacterial PratA protein, suggested that PSII biogenesis does not only underlie a temporal order but is also organized at the spatial level, as PratA was shown to mark a special intermediate membrane subfraction (PDMs), hypothesized to represent regions for initial steps of PSII biogenesis. The presented work focused on a more detailed characterization of PDMs, clearly supporting their significance not only with regard to early protein assembly, but also concerning pigment synthesis and integration into the PSII precomplexes. The PDMs could further be allocated to special membrane regions, named biogenesis centers, at sites where thylakoid membranes converge to the plasma membrane, thus demonstrating the spatial organization of PSII assembly at the cellular level. Moreover, a novel function of PratA in preloading of PSII with Mn2+ ions, necessary for construction of the water-splitting complex, was discovered. Concomitantly with progression of the assembly, the nascent PSII complexes are transported from the PDMs to the thylakoid membrane system, where Sll0933 – a novel PSII assembly factor identified in this thesis – mediates the integration of the PSII inner antenna proteins followed by completion of the assembly process. Additionally, it could be shown that many of the so far identified facilitating factors interact with each other and, thus, form a complex network for PSII assembly. Especially the interaction between the two assembly factors YCF48 and Sll0933 was characterized in more detail, revealing a successive mode of action with YCF48 operating upstream of Sll0933. Taken together, the presented results enable the development of an extended and elaborated model of PSII assembly, which is a concerted process connecting protein and cofactor synthesis/integration in a spatiotemporal manner, and thus contribute to a more profound understanding of photosynthesis itself.Oxygene Photosynthese ermöglicht die Umwandlung von Lichtenergie in chemische Energie. Aufgrund des bei den zugrunde liegenden Reaktionen gebildeten molekularen Sauerstoffs (O2) stellt sie die Basis für den in unserer Atmosphäre angesammelten Sauerstoff und somit die Grundlage für höheres Leben auf der Erde dar. Die Entstehung von O2 wird von Photosystem II (PSII) katalysiert, dem ersten Komplex in einer Reihe von aus verschiedenen Bestandteilen aufgebauten Proteinkomplexen, welche den Transport von Elektronen zur Energiegewinnung vermitteln. Während die Zusammensetzung von PSII aus mindestens 20 verschiedenen Protein-Untereinheiten und diversen Co-Faktoren in den letzten Jahren weitgehend aufgeklärt wurde, ist das Wissen über den zugrunde liegenden Assemblierungsweg verhältnismäßig beschränkt. Bislang konnte gezeigt werden, dass dieser Ablauf schrittweise erfolgt und eine Reihe von Assemblierungsfaktoren erfordert. Dass die Biogenese von PSII neben ihrer zeitlichen Abfolge auch auf räumlicher Ebene organisiert ist, lässt sich aus früheren Studien eines dieser Assemblierungsfaktoren, des cyanobakteriellen PratA-Proteins schließen, welches eine spezielle Membranfraktion (PDMs) kennzeichnet, die offenbar an den frühen Schritten der PSII-Biogenese beteiligt ist. Die hier gezeigten Ergebnisse einer detaillierteren Charakterisierung der PDMs unterstreichen ihre Bedeutung nicht nur im Hinblick auf die frühen Schritte der Assemblierung der Proteinuntereinheiten, sondern auch auf die Pigmentsynthese und deren Integration in PSII-Präkomplexe. Strukturell konnten PDMs sogenannten Biogenesezentren zugeordnet werden, welche sich an Stellen, an denen sich die Thylakoidmembranen der Plasmamembran annähern, befinden. Somit wurde die Frage der subzellulären Lokalisierung der PSII-Assemblierung beantwortet. Außerdem konnte eine Beteiligung von PratA an der Beladung von PSII mit Mn2+-Ionen zum Aufbau des Wasserspaltungsapparates gezeigt werden. Weitere Daten weisen darauf hin, dass mit zunehmender Assemblierung ein Transport der entstehenden PSII-Komplexe von den PDMs in Richtung der Thylakoidmembranen erfolgt. Unmittelbar nach Erreichen der Thylakoide scheint dann Sll0933 – ein neuer, in dieser Arbeit identifizierter PSII-Assemblierungsfaktor – die Integration der inneren PSII-Antennenproteine zu vermitteln. Der Abschluss der Assemblierung von PSII erfolgt ebenfalls im Thylakoidmembransystem. Es wurden außerdem Hinweise dafür erhalten, dass sich viele der bisher identifizierten Assemblierungsfaktoren gegenseitig beeinflussen und somit ein komplexes Netzwerk bilden. Insbesondere die Interaktion zwischen den beiden Assemblierungsfaktoren YCF48 und Sll0933 wurde detaillierter untersucht und führte zur Schlussfolgerung, dass YCF48 an früheren Schritten als Sll0933 an der PSII-Assemblierung beteiligt ist. Alles in allem handelt es sich bei der Assemblierung von PSII also um einen Prozess mit einer auf räumlicher und zeitlicher Ebene organisierten Verknüpfung von Integration der Protein-Untereinheiten und Synthese/Insertion der Co-Faktoren. Die dargestellten Ergebnisse ermöglichen eine deutliche Ausweitung des bisher geltenden Modells der PSII-Assemblierung und tragen somit zu einem besseren Gesamtverständnis des Prozesses der oxygenen Photosynthese an sich bei

Removal of acrylic coatings from works of art by means of nanofluids: understanding the mechanism at the nanoscale

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Nodular lymphocyte predominant hodgkin lymphoma and T cell/histiocyte rich large B cell lymphoma : endpoints of a spectrum of one disease?

In contrast to the commonly indolent clinical behavior of nodular lymphocyte predominant Hodgkin lymphoma (NLPHL), T cell/histiocyte rich large B cell lymphoma (THRLBCL) is frequently diagnosed in advanced clinical stages and has a poor prognosis. Besides the different clinical presentations of these lymphoma entities, there are variants of NLPHL with considerable histopathologic overlap compared to THRLBCL. Especially THRLBCL-like NLPHL, a diffuse form of NLPHL, often presents a histopathologic pattern similar to THRLBCL, suggesting a close relationship between both lymphoma entities. To corroborate this hypothesis, we performed gene expression profiling of microdissected tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. In unsupervised analyses, the lymphomas did not cluster according to their entity. Moreover, even in supervised analyses, very few consistently differentially expressed transcripts were found, and for these genes the extent of differential expression was only moderate. Hence, there are no clear and consistent differences in the gene expression of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. Based on the gene expression studies, we identified BAT3/BAG6, HIGD1A, and FAT10/UBD as immunohistochemical markers expressed in the tumor cells of all three lymphomas. Characterization of the tumor microenvironment for infiltrating T cells and histiocytes revealed significant differences in the cellular composition between typical NLPHL and THRLBCL cases. However, THRLBCL-like NLPHL presented a histopathologic pattern more related to THRLBCL than NLPHL. In conclusion, NLPHL and THRLBCL may represent a spectrum of the same disease. The different clinical behavior of these lymphomas may be strongly influenced by differences in the lymphoma microenvironment, possibly related to the immune status of the patient at the timepoint of diagnosis

mRNA localization, reaction centre biogenesis and thylakoid membrane targeting in cyanobacteria

The thylakoid membranes of cyanobacteria form a complex intracellular membrane system with a distinctive proteome. The sites of biogenesis of thylakoid proteins remain uncertain, as do the signals that direct thylakoid membrane-integral proteins to the thylakoids rather than to the plasma membrane. Here, we address these questions by using fluorescence in situ hybridization to probe the subcellular location of messenger RNA molecules encoding core subunits of the photosystems in two cyanobacterial species. These mRNAs cluster at thylakoid surfaces mainly adjacent to the central cytoplasm and the nucleoid, in contrast to mRNAs encoding proteins with other locations. Ribosome association influences the distribution of the photosynthetic mRNAs on the thylakoid surface, but thylakoid affinity is retained in the absence of ribosome association. However, thylakoid association is disrupted in a mutant lacking two mRNA-binding proteins, which probably play roles in targeting photosynthetic proteins to the thylakoid membrane

Label-Free Optical Single-Molecule Micro- and Nanosensors

This is the author accepted manuscript. The final version is available from Wiley via the DOI in this recordLabel-free optical sensor systems have emerged that exhibit extraordinary sensitivity for detecting physical, chemical, and biological entities at the micro/nanoscale. Particularly exciting is the detection and analysis of molecules, on miniature optical devices that have many possible applications in health, environment, and security. These micro- and nanosensors have now reached a sensitivity level that allows for the detection and analysis of even single molecules. Their small size enables an exceedingly high sensitivity, and the application of quantum optical measurement techniques can allow the classical limits of detection to be approached or surpassed. The new class of label-free micro- and nanosensors allows dynamic processes at the single-molecule level to be observed directly with light. By virtue of their small interaction length, these micro- and nanosensors probe light–matter interactions over a dynamic range often inaccessible by other optical techniques. For researchers entering this rapidly advancing field of single-molecule micro- and nanosensors, there is an urgent need for a timely review that covers the most recent developments and that identifies the most exciting opportunities. The focus here is to provide a summary of the recent techniques that have either demonstrated label-free single-molecule detection or claim single-molecule sensitivity.Living Systems Institute, University of Exete

Choline as a cation for the design of low-toxic and biocompatible ionic liquids, surfactants, and deep eutectic solvents

Choline is an essential nutrient for humans which has important key functions in the body, e.g. precursor for acetylcholine or phospholipids. Further it is a quaternary ammonium cation of biological origin. However, the most important characteristic is its bulky, unsymmetrical structure. Therefore, in this thesis it is shown as a very efficient counterion to lower the Krafft temperature of surfactants and the melting or freezing point of ionic liquids and deep eutectic solvents. These substances were investigated for eventual applications as extracting agent, in formulations or for washing processes. For this purposes a low cytotoxicity is important. It was confirmed by performing MTT-assays of these substances with HeLa and SK-MEL-28 cells. The first part of this thesis is focused on the synthesis of choline carboxylate based ionic liquids. The design of the ionic liquids is divided into two approaches. Firstly, the melting point could be further lowered, beside the use of a bulky, unsymmetrical cation, by decreasing the chain length of the carboxylate anion (ChCm with m = 2, 4, 6, 8, 10) and secondly, by inserting a double bond into the alkyl chain (choline oleate). Therefore, choline carboxylates ChCm with chain lengths of m = 2, 4, 6, 8 and 10 and choline oleate were synthesized and the focus is set on the physico-chemical characterization and the investigation of the influence of the alkyl chain length and the double bond in the alkyl chain on the aqueous phase behavior and the temperature dependent behavior of the neat choline carboxylates. Several experimental techniques like wide and small angle X-ray scattering, polarizing optical microscopy, NMR spectroscopy and differential scanning calorimetry were used for the characterization. The second main part of this thesis is focused on the investigation of new promising, biocompatible and biodegradable surfactants for a possible application as laundry detergents. From other studies it is known that choline dodecylsulfate is a less salt sensitive surfactant and possesses a more acidic headgroup compared to the choline carboxylates and its Krafft temperature is lower compared to the one of sodium dodecylsulfate. A better washability at low temperatures is normally obtained by increasing the chain length, while the Krafft temperature of the surfactant remains below room temperature. Thus, choline hexadecylsulfate was synthesized as a better washing surfactant. The temperature dependent binary phase diagrams of choline dodecylsulfate and choline hexadecylsulfate with water were investigated with small angle X-ray scattering, polarizing optical microscopy and visual observations and compared to the binary phase diagrams of the sodium analogs and to choline carboxylates. In addition, the thermotropic phase behavior was studied and compared to the one of choline carboxylates ChCm with m = 8, 10 and oleate. Last but not least, the washability of choline alkylsulfate surfactants and mixtures of choline alkylsulfates with different chain length or with a common laundry detergent was evaluated with the help of washing, foamability and foam stability tests. Furthermore, the oil solubilization capacity of the pure surfactants was evaluated. These tests show a better washing ability for mixtures of choline alkylsulfates with different chain length or of choline hexadecylsulfate with a common laundry detergent compared to the single surfactants. As third part of the thesis a strategy to design room temperature liquid and fully dissociated deep eutectic solvents composed of choline bicarboxylate and choline chloride (and urea) was introduced. Therefore at room temperature liquid and fully dissociated choline glutarate based deep eutectic solvents were synthesized. The mixtures were characterized according to their solvent properties with density, viscosity and conductivity measurements

Clustering genetischer Daten auf der Basis eines konsistenten Softwareframeworks zur Datenverwaltung in klinischen Studien

Diese Arbeit beschäftigt sich mit dem modellfreien Clustering von genetischen Mutationsdaten in Form von SNPs und der Qualität von Clusteringergebnissen bzw. deren Verbesserung mit Hilfe eines Softwareframeworks zur Datenverwaltung in klinischen Studien

The Role of Slr0151, a Tetratricopeptide Repeat Protein from Synechocystis sp PCC 6803, during Photosystem II Assembly and Repair

The assembly and repair of photosystem II (PSII) is facilitated by a variety of assembly factors. Among those, the tetratricopeptide repeat (TPR) protein Slr0151 from Synechocystis sp. PCC 6803 (hereafter Synechocystis) has previously been assigned a repair function under high light conditions (Yang et al., 7014). Here, we show that inactivation of slr0151 affects thylakoid membrane ultrastructure even under normal light conditions. Moreover, the level and localization of Slr0151 are affected in a variety of PSII-related mutants. In particular, the data suggest a close functional relationship between Slr0151 and SI10933, which interacts with Ycf48 during PSII assembly and is homologous to PAM68 in Arabidopsis thaliana. Immunofluorescence analysis revealed a punctate distribution of Slr0151 within several different membrane types in Synechocystis cells