Analysis of the membrane binding mechanism of Remorins and their role in beneficial endosymbioses

The plasma membrane is highly organized and within the plasma membrane proteins cluster into so-called membrane domains. Remorins are well-established membrane domain marker proteins. However, the general plasma membrane anchoring mechanism of these proteins was so far unknown. Biochemical approaches and localization studies investigating different remorins from Medicago truncatula and Arabidopsis thaliana enabled us to demonstrate that S-acylation (palmitoylation) within a C-terminal plasma membrane anchoring motif mediates tight plasma membrane attachment of these proteins. However, we could show that S-acylation is not the sole driving force for remorin immobilization in membrane nanodomains. The focus of the second part of this thesis was on the beneficial interaction between plants and symbionts. More than 80% of today´s land plants can undergo an interaction with endosymbiotic fungi that is known as Arbuscular Mycorrhiza (AM). In addition, legume plants have gained the ability to establish a second type of endosymbiosis by interacting with nitrogen-fixing rhizobia: the Root Nodule Symbiosis (RNS). Both interactions are partly controlled by the same pathway, the so-called Common Symbiosis Pathway (CSP) that has evolved through recruitment of signaling components from the evolutionary older AM to the more recently evolved RNS signaling pathway. Depending on the recognition of either fungi or rhizobia downstream of this pathway two morphologically different symbiotic structures are formed within the inner root cortex, either arbuscules or root nodules, respectively. In parallel to the evolution of RNS a local negative regulatory circuit must have evolved to suppress root nodule organogenesis when both interacting symbionts are present and arbuscule formation takes place. In this study first evidence for such a postulated regulatory pathway is presented based on the characterization of the legume-specific remorin MYCREM, which co-evolved with RNS. Phenotypic studies of mutant plants revealed that in the presence of both symbionts MYCREM functions as a negative regulator with respect to root nodule organogenesis events in a CSP-dependent manner. Analyzing the effect of overexpression of auto-active CSP-signaling components, which are known to spontaneously induce root nodule organogenesis, demonstrated a negative regulatory function of MCYREM as well. In summary, this work could serve as basis for further studies to understand the tripartite interaction of legume plants, fungi and rhizobia, as it is found in nature

Using personalised cardiovascular models to identify new diagnostic predictors for pre-eclampsia

Haemodynamic adaptations play a crucial role in uteroplacental perfusion during pregnancy. In particular, modifications of the utero-ovarian arterial network cause a significant increase in blood volume distributed to the placenta and foetus. Failure to make these cardiovascular modifications results in complicated pregnancies caused by different disorders such as hypertension, pre-eclampsia, intrauterine growth restriction (IUGR), and placental insufficiency. In pre-eclampsia, the modifications of the utero-ovarian arterial network are unsuccessful and cause less blood volume to be distributed to the placenta and foetus. Pre-eclampsia is a hypertensive disorder that is still not fully understood, and clinicians still fail at identifying pre-eclamptic women during controls, especially at differentiating between hypertensive women and pre-eclamptic women. One reason for this is that clinicians rely heavily on blood pressure when diagnosing pre-eclampsia, and this biomarker has similar readings for both pre-eclampsia and hypertension. As part of the diagnosis of pre-eclampsia, proteinuria is used. In order to improve the diagnosis of pre-eclampsia, other biomarkers are being researched. A dataset of 21 patients was used to find novel biomarkers that can classify pre-eclampsia. The dataset is divided into two groups: uncomplicated pregnancies with hypertensive women and complicated pregnancies with pre-eclampsia. A computational model of the cardiovascular system is used to simulate blood and pressure solutions based on patient-specific observations in order to develop a new biomarker. The model employs 1D modelling which incorporates a wave intensity analysis that models forward and backward waves to provide more precise predictions of wave propagation across the artery system, particularly in the utero-ovarian system. The proposed biomarkers will include dimensionless terms formed by global maternal parameters such as systolic blood pressure, stroke volume, pulse wave velocity, etc., or local uterine parameters such as pressure and velocity in specific vessels of the uterine system. Afterwards, their ability as a classifier of pre-eclampsia will be investigated. Besides this, a case study of the prone position in pregnancy and its effects on cardiovascular changes will be carried out. To do this, the computational model will be used to study what happens when a pregnant woman is positioned in the prone position and how vital metrics like blood pressure and cardiac output are altered. It was found that the biomarkers based on the radial and arcuate arteries have a better classification ability for pre-eclampsia, even higher than the Doppler-measured Resistance Index (RI) and Pulsatility Index (PI). The novelty of this work is the introduction of new biomarkers through the use of a computational model, as well as the demonstration of the dependability and use of 1D modelling in pregnancy. The model demonstrated how biomarkers that could not be measured clinically may be easily calculated using 1D modelling and provide critical information about the utero-ovarian circulation. Future work should concentrate on changing the existing solver into a much faster and simpler solver, as well as validating the biomarkers in a larger dataset

Die Rolle der Epstein-Barr Virus nukleären Antigene 3A und 3C in der B-Zellimmortalisierung

Das Epstein-Barr Virus (EBV) infiziert ruhende primäre humane B-Zellen und indu-ziert deren unbegrenzte Proliferation. Dieser Prozess der Wachstumstransformation stellt ein Modellsystem dar, das die pathogenen Mechanismen in der Tumorentsteh-ung widerspiegelt. Die Epstein-Barr Virus nukleären Antigene 3A und 3C (EBNA-3A und EBNA-3C) werden in Publikationen aus dem Zeitraum von 1993 bis 1996 als essentiell für den Prozess der B-Zellimmortalisierung eingestuft. In dieser Arbeit wurde mit einer neuen Technologie, der Maxi-EBV Methode, die Rolle der EBNA-3A und -3C Proteine erneut untersucht. Sowohl mit EBNA-3A negativen als auch mit EBNA-3C negativen Viren konnten erstmals Kulturen von infizierten B-Zellen etabliert werden. Während sich aus EBNA-3A negativen B-Zellkulturen Langzeitkulturen etablieren ließen, starben EBNA-3C negative B-Zellkulturen in der Regel nach 40-70 Tagen ab. Die Effizienz der B-Zellimmortalisierung von EBNA-3A negativen Viren war im Vergleich zur Wildtyp infizierten B-Zellen 24-fach, die der EBNA-3C negativen Viren 140-fach erniedrigt. Sowohl EBNA-3A negative, als auch EBNA-3C negative LCLs sind in ihrer Viabilität eingeschränkt, weisen jedoch unveränderte Zellteilungsraten auf. Die weitere Charakterisierung der EBNA-3A negativen LCLs ergab, dass diese eine Variante des viralen LMP1-Proteins exprimieren. Offen blieb, ob diese Variante das Auswachsen der EBNA-3A negativen B-Zellkulturen begünstigt hat. In der Folge wurden die EBNA-3A negativen LCLs zur Identifizierung von EBNA-3A-Zielgenen eingesetzt und zahlreiche aktivierte und reprimierte Kandidatengene identifiziert. Eines dieser Kandidatengene, Matrix-Metalloproteinase 7 (MMP-7), das durch EBNA-3A induziert wird, wurde im Rahmen dieser Arbeit validiert. Auch mit EBNA-3C negative Viren konnten wider Erwarten LCLs erzeugt werden, die für einen begrenzten Zeitraum in Kultur gehalten werden können. Aus dem Material eines Spenders war es auch möglich, EBNA-3C negative Langzeitkulturen zu etablieren. Die Mehrzahl der EBNA-3C negativen infizierten B-Zellkulturen durchlaufen jedoch zwischen Tag 40 und 70 eine Krise und sterben. Mit der Generierung eines konditionalen EBNA-3C Systems, durch Transfektion eines Tetrazyklin-regulierbaren EBNA-3C Expressionsvektors in frisch isolierte primäre B-Zellen und anschließender Infektion mit EBNA-3C negativen Viren, wurde ein neuer Weg geschaffen, um EBNA-3C-Funktionen zu untersuchen. Dieses 2-Schrittsystem kann nun im Prinzip für jede Virusmutante eingesetzt werden

Analysis of the membrane binding mechanism of Remorins and their role in beneficial endosymbioses

The plasma membrane is highly organized and within the plasma membrane proteins cluster into so-called membrane domains. Remorins are well-established membrane domain marker proteins. However, the general plasma membrane anchoring mechanism of these proteins was so far unknown. Biochemical approaches and localization studies investigating different remorins from Medicago truncatula and Arabidopsis thaliana enabled us to demonstrate that S-acylation (palmitoylation) within a C-terminal plasma membrane anchoring motif mediates tight plasma membrane attachment of these proteins. However, we could show that S-acylation is not the sole driving force for remorin immobilization in membrane nanodomains. The focus of the second part of this thesis was on the beneficial interaction between plants and symbionts. More than 80% of today´s land plants can undergo an interaction with endosymbiotic fungi that is known as Arbuscular Mycorrhiza (AM). In addition, legume plants have gained the ability to establish a second type of endosymbiosis by interacting with nitrogen-fixing rhizobia: the Root Nodule Symbiosis (RNS). Both interactions are partly controlled by the same pathway, the so-called Common Symbiosis Pathway (CSP) that has evolved through recruitment of signaling components from the evolutionary older AM to the more recently evolved RNS signaling pathway. Depending on the recognition of either fungi or rhizobia downstream of this pathway two morphologically different symbiotic structures are formed within the inner root cortex, either arbuscules or root nodules, respectively. In parallel to the evolution of RNS a local negative regulatory circuit must have evolved to suppress root nodule organogenesis when both interacting symbionts are present and arbuscule formation takes place. In this study first evidence for such a postulated regulatory pathway is presented based on the characterization of the legume-specific remorin MYCREM, which co-evolved with RNS. Phenotypic studies of mutant plants revealed that in the presence of both symbionts MYCREM functions as a negative regulator with respect to root nodule organogenesis events in a CSP-dependent manner. Analyzing the effect of overexpression of auto-active CSP-signaling components, which are known to spontaneously induce root nodule organogenesis, demonstrated a negative regulatory function of MCYREM as well. In summary, this work could serve as basis for further studies to understand the tripartite interaction of legume plants, fungi and rhizobia, as it is found in nature

A Modular Plasmid Assembly Kit for Multigene Expression, Gene Silencing and Silencing Rescue in Plants

The Golden Gate (GG) modular assembly approach offers a standardized, inexpensive and reliable way to ligate multiple DNA fragments in a pre-defined order in a single-tube reaction. We developed a GG based toolkit for the flexible construction of binary plasmids for transgene expression in plants. Starting from a common set of modules, such as promoters, protein tags and transcribed regions of interest, synthetic genes are assembled, which can be further combined to multigene constructs. As an example, we created T-DNA constructs encoding multiple fluorescent proteins targeted to distinct cellular compartments (nucleus, cytosol, plastids) and demonstrated simultaneous expression of all genes in Nicotiana benthamiana, Lotus japonicus and Arabidopsis thaliana. We assembled an RNA interference (RNAi) module for the construction of intron-spliced hairpin RNA constructs and demonstrated silencing of GFP in N. benthamiana. By combination of the silencing construct together with a codon adapted rescue construct into one vector, our system facilitates genetic complementation and thus confirmation of the causative gene responsible for a given RNAi phenotype. As proof of principle, we silenced a destabilized GFP gene (dGFP) and restored GFP fluorescence by expression of a recoded version of dGFP, which was not targeted by the silencing construct

Structural change in coal regions as a process of economic and social-ecological transition : lessons learnt from structural change processes in Germany

Effective policies to mitigate climate change need to be accompanied by a socially just transition. Based on experiences of past and ongoing transition policies in coal regions in Europe and with indications to the specificity of framework conditions and challenges and to the potential effectiveness and transferability of approaches, this paper presents lessons learnt which can be inspirational for similar transitions in other coal regions and for transitions in other sectors

Revealing the Chemical Composition of Birch Pollen Grains by Raman Spectroscopic Imaging

The investigation of the biochemical composition of pollen grains is of the utmost interest for several environmental aspects, such as their allergenic potential and their changes in growth conditions due to climatic factors. In order to fully understand the composition of pollen grains, not only is an in-depth analysis of their molecular components necessary but also spatial information of, e.g., the thickness of the outer shell, should be recorded. However, there is a lack of studies using molecular imaging methods for a spatially resolved biochemical composition on a single-grain level. In this study, Raman spectroscopy was implemented as an analytical tool to investigate birch pollen by imaging single pollen grains and analyzing their spectral profiles. The imaging modality allowed us to reveal the layered structure of pollen grains based on the biochemical information of the recorded Raman spectra. Seven different birch pollen species collected at two different locations in Germany were investigated and compared. Using chemometric algorithms such as hierarchical cluster analysis and multiple-curve resolution, several components of the grain wall, such as sporopollenin, as well as the inner core presenting high starch concentrations, were identified and quantified. Differences in the concentrations of, e.g., sporopollenin, lipids and proteins in the pollen species at the two different collection sites were found, and are discussed in connection with germination and other growth processes

Small phytoplankton drive high summertime carbon and nutrient export in the Gulf of California and Eastern Tropical North Pacific

Author Posting. © American Geophysical Union, 2015. This article is posted here by permission of American Geophysical Union for personal use, not for redistribution. The definitive version was published in Global Biogeochemical Cycles 29 (2015): 1309–1332, doi:10.1002/2015GB005134.Summertime carbon, nitrogen, and biogenic silica export was examined using 234Th:238U disequilibria combined with free floating sediment traps and fine scale water column sampling with in situ pumps (ISP) within the Eastern Tropical North Pacific and the Gulf of California. Fine scale ISP sampling provides evidence that in this system, particulate carbon (PC) and particulate nitrogen (PN) concentrations were more rapidly attenuated relative to 234Th activities in small particles compared to large particles, converging to 1–5 µmol dpm−1 by 100 m. Comparison of elemental particle composition, coupled with particle size distribution analysis, suggests that small particles are major contributors to particle flux. While absolute PC and PN export rates were dependent on the method used to obtain the element/234Th ratio, regional trends were consistent across measurement techniques. The highest C fixation rates were associated with diatom-dominated surface waters. Yet, the highest export efficiencies occurred in picoplankton-dominated surface waters, where relative concentrations of diazotrophs were also elevated. Our results add to the increasing body of literature that picoplankton- and diazotroph-dominated food webs in subtropical regions can be characterized by enhanced export efficiencies relative to food webs dominated by larger phytoplankton, e.g., diatoms, in low productivity pico/nanoplankton-dominated regions, where small particles are major contributors to particle export. Findings from this region are compared globally and provide insights into the efficiency of downward particle transport of carbon and associated nutrients in a warmer ocean where picoplankton and diazotrophs may dominate. Therefore, we argue the necessity of collecting multiple particle sizes used to convert 234Th fluxes into carbon or other elemental fluxes, including <50 µm, since they can play an important role in vertical fluxes, especially in oligotrophic environments. Our results further underscore the necessity of using multiple techniques to quantify particle flux given the uncertainties associated with each collection method.NSF Grant Numbers: OCE-0726290, OCF-0962362, OCE-0726543, OCE-0726422; EU Grant Number: FP7-MC-IIF-220485; MEC Grant Number: CTM2007-31241-E/MAR; ICREA Academia; MERS Grant Number: 2014 SGR – 1356; Spain's Ministerio de Educación y Ciencia Grant Numbers: AP-2009-4733, BES-2004-3348; NASA New Investigator Award Grant Number: NNX10AQ81G; Sloan Research Fellowship2016-02-2

Automated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands

A Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinhei