Stuttgart, Univ., Diss., 2003.

Techn. University, Diss., 2003--Berlin.

Techn. University, Diss., 2004--Berlin. / Parallelt.: Untersuchung zur Zusammensetzung und der funktionalen Eigenschaften der Lipide einiger exotischer Ölsaaten.

Phaeodactylum et Nannochloropsis sont des espèces photosynthétiques modèles pour le métabolisme des glycérolipides, se distinguant par un enrichissement en acides gras polyinsaturés à très longues chaînes (VLC-PUFA) et de grandes quantités en triacylglycérol (TAG). Les proportions des différents lipides sont influencées par des facteurs environnementaux. Nous avons caractérisé le remodelage lipidique chez Phaeodactylum en réponse à la carence en azote et en phosphate. Ces limitations en nutriments induisent une accumulation de TAG, exploitable comme biocarburant. Nous avons identifié de nouveaux composés induisant l'accumulation de TAG et étudié le rôle potentiel du monoxyde d’azote (NO•) dans la régulation du métabolisme lipidique. Nous avons montré qu’en fonction du site de production, le NO• était un signal émis lorsque les conditions de vie étaient critiques, déclenchant l'accumulation de TAG.Les VLC-PUFAs sont produits par des élongases et des désaturases localisées dans le RE. Nous avons identifié une nouvelle classe d’élongases d’acides gras saturés, agissant sur le 16:0, et appelées Δ0-ELO. Le knock out de Δ0-ELO1 de Nannochloropsis réduit le niveau du monogalactosyldiacylglycérol (MGDG), principal lipide des chloroplastes. Ce phénotype met en évidence le rôle de Δ0-ELO1 dans la «voie oméga» qui contrôle le trafic des VLC-PUFAs. Nous avons débuté une dissection de la «voie oméga» par des approches de génétique et des analyses du remodelage lipidique à basse température chez Nannochloropsis. Le diacylglycéryl hydroxyméthyltriméthyl-β-sérine (DGTS) apparaît comme le précurseur de base pour importer des VLC-PUFAs vers le chloroplaste, suivant une voie très régulée du DGTS au MGDG. De plus nous avons montré des fonctions possibles du MGDG et des VLC-PUFAs dans la photoprotection et la régulation de la fluidité membranaire latérale. / Phaeodactylum and Nannochloropsis are photosynthetic model species for glycerolipid metabolism, standing out by an enrichment of very-long-chain polyunsaturated fatty acids (VLC-PUFAs) and high contents of neutral lipids such as triacylglycerol (TAG). Lipid profiles are influenced by environmental factors. We characterized the lipid remodelling occurring in Phaeodactylum in response to nitrogen and phosphate starvation. Nutrient limitations induce neutral lipid accumulation, which may be exploited as biofuels. We identified new triggers of TAG accumulation and investigated a potential role of nitric oxide (NO•) as second messenger in the regulation of neutral lipid levels. We conclude that in dependence of the production site, NO• serves as a signalling molecule for critical life conditions and thereby triggers TAG accumulation.VLC-PUFAs are produced by ER-located elongases and desaturases. We identified a novel class of elongases, called Δ0-ELOs, acting on saturated fatty acids, most importantly 16:0. Knock out of Δ0-ELO1 in Nannochloropsis resulted in reduced monogalactosyldiacylglycerol (MGDG) levels. MGDG is the major chloroplast lipid. This indicated a role of this initial elongase in fatty acid fate determination and thus in the elusive “omega pathway” for VLC-PUFA trafficking. We have started to investigate the “omega pathway” by reverse genetic approaches and analyses of low-temperature induced lipid remodelling in Nannochloropsis. Diacylglyceryl hydroxymethyltrimethyl-β-serine (DGTS) appears most likely at the base for the chloroplast import of VLC-PUFA, following a dynamically regulated DGTS-to-MGDG pathway. Additionally, we gave insights into possible functions of MGDG and VLC-PUFA in photoprotection and regulation of membrane fluidity.

Les décharges électriques (plasmas) sont capables de produire simultanément de grandes concentrations d'espèces chargées (électrons, ions), d'espèces neutres réactives (atomes, états métastables) et des états radiatifs émetteurs de rayonnement dans une large gamme spectrale allant de l'UV à l'infra-rouge. Certaines de ces décharges sont susceptibles de fonctionner à une température proche de la température ambiante, ce qui leur confère un fort potentiel d'interaction avec la matière vivante. La plupart des travaux actuels dans ce domaine sont focalisés sur les effets induits sur le vivant par les ROS (reactive oxygen species) mais une récente étude a établi que les atomes d'azote produits par une post-décharge en flux d'azote pur fonctionnant à pression réduite (1-20 Torr, 1 Torr = 133.3 Pa) étaient vraisemblablement l'agent principal de l'interaction avec le vivant. L'objectif de la présente thèse est de poursuivre ce travail initial en cherchant à confirmer cette hypothèse et en améliorant la compréhension générale des phénomènes physico-biochimiques mis en jeu au cours de l'interaction plasma à base d'azote / matière vivante. Pour ce faire, deux types de décharges ont été utilisés : l'une est une post-décharge en flux micro-onde fonctionnant à pression réduite dans l'azote pur ou dans un mélange Argon/Azote, l'autre est un jet de décharge couronne (corona) opérant dans l'air ambiant ou sous atmosphère contrôlée d'azote à pression atmosphérique. La première partie de l'étude est consacrée à la caractérisation physique des deux décharges par spectroscope d'émission : détermination de leur température, identification des espèces chimiques produites. Dans le cas des post-décharges à base d'azote, il a été possible de quantifier la concentration en atomes d'azote et de maximiser leur production en fonction des paramètres opératoires. Dans la deuxième partie de l'étude, l'interaction entre les espèces plasma et le vivant est quantifiée à travers la réduction logarithmique d'une population initiale de bactéries Gram - (Escherichia coli, E. coli) en fonction de la durée d'exposition aux décharges. Cette réduction est mise en corrélation avec des modifications morphologiques observées par microscopie électronique à balayage (MEB) sur les bactéries traitées et par analyse de la viabilité cellulaire obtenue via les tests MTT et DAPI. En parallèle, la capacité des deux décharges à éliminer ou à dénaturer le lipide A, sous composant pyrogène et hydrophobe d'une endotoxine (Lipopolysaccharide ou LPS) présente dans la membrane de la plupart des bactéries Gram - est établie. Au vu de ces différents éléments, un scénario de l'inactivation bactérienne par les atomes d'azote contenus dans les post-décharges à base d'azote est proposé. / Electrical discharges (plasmas) are able to simultaneously produce large concentrations of charged species (electrons, ions), of neutral reactive species (atoms, metastable states) and of radiative states emitting radiation from infrared to ultraviolet. Some of them can operate at a temperature close to the room temperature, which confer them a strong interaction potential with the living matter. Most of the existing works in this field focus on the effects induced by the ROS (reactive oxygen species) but a study recently established that the nitrogen atoms produced in a pure nitrogen flowing late afterglow operating at reduced pressure (1-20 Torr, 1 Torr = 133.3 Pa) were probably the main agent of the interaction with the living. The goal of the present thesis is to pursue this initial work by seeking to confirm this hypothesis and by improving the general comprehension of the physical and bio-chemical processes involved in the interaction between nitrogen plasmas and living matter. To do such, two types of discharges were used : one is a flowing microwave afterglow operating at reduced pressure in pure nitrogen or in mixtures of argon and nitrogen, the other is a corona discharge jet working at atmospheric pressure in ambient air or in a controlled nitrogen atmosphere. The first part of the study is devoted to the physical characterization of both discharges by emission spectroscopy, temperature determination, and identification of the produced chemical species. For the nitrogen-based afterglows, it was possible to quantify the nitrogen atoms absolute concentration and to maximize their production through the operating parameters. In the second part of the study, the interaction between plasma species and living matter is quantified by the use of the logarithmic reduction of an initial Gram - bacteria population (Escherichia coli, E. coli) vs. the discharge exposure time. This reduction is correlated with morphologic changes observed by scanning electron microscopy (SEM) on treated bacteria and by an analysis of the cell viability obtained via MTT tests and DAPI. In parallel, the ability of the two discharges to remove or alter lipid A, a pyrogenic hydrophobic sub-component of an endotoxin (Lipopolysaccharide or LPS) present in the membrane of most of the Gram - bacteria is established. Considering these elements, a scenario of bacterial inactivation by the nitrogen atoms of the late nitrogen afterglow is proposed.

The development of cellular life on earth is coupled to the formation of lipid-based biological membranes. Although many tools to analyze their biophysical properties already exist, their variety and number is still relatively small compared to the field of protein studies. One reason for this, is their small size and complex assembly into an asymmetric tightly packed lipid bilayer showing characteristics of a two-dimensional heterogenous fluid. Since membranes are capable to form dynamic, nanoscopic domains, enriched in sphingolipids and cholesterol, their detailed investigation is limited to techniques which access information below the diffraction limit of light. In this work, I aimed to extend, optimize and compare three different labeling approaches for sphingolipids and their subsequent analysis by the single-molecule localization microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM). First, I applied classical immunofluorescence by immunoglobulin G (IgG) antibody labeling to detect and quantify sphingolipid nanodomains in the plasma membrane of eukaryotic cells. I was able to identify and characterize ceramide-rich platforms (CRPs) with a size of ~ 75nm on the basal and apical membrane of different cell lines. Next, I used click-chemistry to characterize sphingolipid analogs in living and fixed cells. By using a combination of fluorescence microscopy and anisotropy experiments, I analyzed their accessibility and configuration in the plasma membrane, respectively. Azide-modified, short fatty acid side chains, were accessible to membrane impermeable dyes and localized outside the hydrophobic membrane core. In contrast, azide moieties at the end of longer fatty acid side chains were less accessible and conjugated dyes localized deeper within the plasma membrane. By introducing photo-crosslinkable diazirine groups or chemically addressable amine groups, I developed methods to improve their immobilization required for dSTORM. Finally, I harnessed the specific binding characteristics of non-toxic shiga toxin B subunits (STxBs) and cholera toxin B subunits (CTxBs) to label and quantify glycosphingolipid nanodomains in the context of Neisseria meningitidis infection. Under pyhsiological conditions, these glycosphingolipids were distributed homogenously in the plasma membrane but upon bacterial infection CTxB detectable gangliosides accumulated around invasive Neisseria meningitidis. I was able to highlight the importance of cell cycle dependent glycosphingolipid expression for the invasion process. Blocking membrane accessible sugar headgroups by pretreatment with CTxB significantly reduced the number of invasive bacteria which confirmed the importance of gangliosides for bacterial uptake into cells. Based on my results, it can be concluded that labeling of sphingolipids should be carefully optimized depending on the research question and applied microscopy technique. In particular, I was able to develop new tools and protocols which enable the characterization of sphingolipid nanodomains by dSTORM for all three labeling approaches. / Die Entwicklung von zellulären Lebensformen auf der Erde basiert auf der Entstehung biologischer Lipid-Membranen. Obwohl viele Techniken zur Verfügung stehen, welche es erlauben deren biophysikalische Eigenschaften zu untersuchen, sind die Möglichkeiten, verglichen mit der Analyse von Proteinen, eher eingeschränkt. Ein Grund hierfür, ist die geringe Größe von Lipiden und deren komplexe Zusammenlagerung in eine asymmetrische dicht gepackte Lipiddoppelschicht, welche sich wie eine heterogene zweidimensionale Flüssigkeit verhält. Durch die lokale Anreicherung von Sphingolipiden und Cholesterol sind Membranen in der Lage dynamische, nanoskopische Domänen auszubilden, welche lediglich mit Techniken, welche die optische Auflösungsgrenze umgehen, detailliert untersucht werden können. Ein wesentliches Ziel meiner Arbeit war es, drei Färbeverfahren für Sphingolipide zu vergleichen, erweitern und optimieren, um eine anschliessende Untersuchung mit Hilfe der einzelmolekülsensitiven Technik dSTORM (direct stochastic optical reconstruction microscopy) zu ermöglichen. Zunächst verwendete ich das klassische Färbeverfahren der Immunfluoreszenz, um Sphingolipid-Nanodomänen auf eukaryotischen Zellen mit Hilfe von Farbstoff-gekoppelten Antikörpern zu detektieren und quantifizieren. Dieses Vorgehen ermöglichte es mir, Ceramid-angereicherte Plattformen mit einer Größe von ~ 75nm auf der basalen und apikalen Membran verschiedener Zell-Linien zu identifizieren und charakterisieren. Als nächstes Verfahren verwendete ich die Klick-Chemie, um Sphingolipid-Analoge in lebenden und fixierten Zellen zu untersuchen. Eine Kombination aus Fluoreszenz-Mikroskopie und Anisotropie-Messungen erlaubte es mir Rückschlüsse über deren Zugänglichkeit und Konfiguration innerhalb der Plasmamembran zu ziehen. Hierbei lokalisierten Azid-Gruppen am Ende kurzkettiger Fettsäurereste außerhalb des hydrophoben Membrankerns, wodurch sie mittels membran-undurchlässige Farbstoffe angeklickt werden konnten. Im Gegensatz dazu, waren Azide an längeren Fettsäureresten weniger zugänglich und konjugierte Farbstoffe tauchten tiefer in die Plasmamembran ein. Durch die Einführung photoreaktiver Diazirin-Gruppen oder chemisch modifzierbarer Amin-Gruppen wurden Wege geschaffen, welche eine Immobilisierung und anschließende Analyse mit Hilfe von dSTORM ermöglichen. Schließlich nutzte ich das spezifische Bindeverhalten der nicht toxischen B Untereinheiten von Shiga- (STxB) und Cholera-Toxin (CTxB) aus, um Glycosphingolipid Nanodomänen im Kontext einer Neisseria meningitidis Infektion zu untersuchen. Unter physiologischen Bedingungen waren diese homogen in der Plasmamembran verteilt, jedoch reicherten sich CTxB-detektierbare Ganglioside um eindringende Bakterien an. Darüber hinaus konnte ich einen Zusammenhang zwischen der zellzyklusabhängigen Expression von Glycosphingolipiden und dem Eindringen der Bakterien herstellen. Eine Absättigung der Zucker an der äußeren Membran durch CTxB-Vorbehandlung reduzierte die Anzahl von invasiven Bakterien signifikant und bestätigte die Schlüsselrolle von Gangliosiden bei der Aufnahme von Bakterien. Meine Ergebnisse legen Nahe, dass das Färbeverfahren für Sphingolipide an die jeweilige Fragestellung und Mikroskopietechnik angepasst werden sollte. Im Rahmen dieser Arbeit konnten neue Werkzeuge und Protokolle geschaffen werden, die die Charakterisierung von Sphingolipid-Nanodomänen mittels dSTORM für alle drei Färbeverfahren ermöglichen.

Cells, whether bacterial, fungal or mammalian, are all equipped with metabolic pathways capable of producing an assortment of structurally and functionally distinct lipid species. Despite the structural diversity of lipids being recognized and correlated to specific cellular phenomena and disease states, the molecular mechanisms that underpin this structural diversity remain poorly understood. In part, this is due to the lack of adequate analytical techniques capable of measuring the structural details of lipid species in a direct, comprehensive and quantitative manner. The aim of my thesis study was to establish methodology for automated and quantitative analysis of molecular lipid species based on mass spectrometry. From this work a novel high-throughput methodology for lipidome analysis emerged. The main assets of the methodology were the structure-specific mass analysis by powerful hybrid mass spectrometers with high mass resolution, automated and sensitive infusion of total lipid extracts by a nanoelectrospray robot, and automated spectral deconvolution by dedicated Lipid Profiler software. The comprehensive characterization and quantification of molecular lipid species was achieved by spiking total lipid extracts with unique lipid standards, utilizing selective ionization conditions for sample infusion, and performing structure-specific mass analysis by hybrid quadrupole time-of-flight and ion trap mass spectrometry. The analytical routine allowed the comprehensive characterization and quantification of molecular glycerophospholipid species, molecular diacylglycerol species, molecular sphingolipid species including ceramides, glycosphingolipids and inositol-containing sphingolipids, and sterol lipids including cholesterol. The performance of the methodology was validated by comparing its dynamic quantification range to that of established methodology based on triple quandrupole mass spectrometry. Furthermore, its efficacy for lipidomics projects was demonstrated by the successful quantitative deciphering of the lipid composition of T cell receptor signaling domains, mammalian tissues including heart, brain and red blood cells, and the yeast Saccharomyces cerevisiae.

Compartmentalization of cell membranes, in particular of the apical membrane of columnar epithelia, is the topic of this thesis. The first part characterizes the apical membrane and its specialized organization and morphology, whereas the second part focuses on the formation of this unique plasma membrane domain during epithelial polarization. The apical membrane of columnar epithelia is enriched in glycosphingolipids, a class of lipids that are known to interact with cholesterol to form liquid ordered domains, also termed "rafts", in cell membranes. Imaging the apical surface of untreated and raft lipid depleted MDCK cells with atomic force microscopy revealed that raft lipids are involved in the formation and/or maintenance of microvilli, actin based protrusions of the apical plasma membrane, indicating a regulatory link between membrane domains and the cytoskeleton. Furthermore, antibody patching and photobleaching experiments performed during the work of this thesis suggest that the organization into raft and non-raft domains is very different in the apical membrane of MDCK cells compared to the plasma membrane of a fibroblast. In fact, the data support the hypothesis that the apical membrane could be a percolating raft membrane in which rafts constitute the major phase and non-raft domains exist as isolated entities. The second part of this thesis analyses the segregation of apical and basolateral membrane domains during epithelial polarization. This segregation can either be achieved by generating scaffolded domains prior to junction formation or by polarized secretory and endocytic membrane traffic after the establishment of cell junctions. While most apical and basolateral marker proteins in MDCK cells follow the latter mechanism, this thesis reports that the apical marker gp135 is confined to the dorsal face already in single attached cells. The unknown antigen was purified and identified as podocalyxin. Analysis of a series of domain mutants revealed that the C-terminal PDZ-binding motif of podocalyxin is mainly responsible for its special localization, which it shares with the PDZ protein NHERF-2. Knocking down podocalyxin by RNA interference resulted in retardation of cell growth and epithelial polarization. Taken together, the data suggest that podocalyxin and NHERF-2 could be part of an early apical polarity scaffolding system based on PDZ-binding and PDZ-containing proteins.

Ayant une structure particulière avec une tête hydrophile et une queue hydrophobe, des molécules amphiphile ont de nombreuses applications importantes, comme par exemple, la fabrication des détergents, la protection et la fonctionnalisation de surfaces, etc. Des agrégats de diverses formes, micelles, véhicules, membranes etc., peuvent se former à partir des amphiphiles. La complexité de ces agrégats moléculaires rend l’étude théorique de ce type de systèmes extrêmement difficile. Jusqu’à présent, notre connaissance sur l’interaction entre des nanoparticules et des agrégats des amphiphiles reste encore incomplète. A l’aide de certaines méthodes de simulations moléculaire et une approche théorique, nous avons entrepris une série d’études pour mieux comprendre les questions fondamentales suivantes :1. Comment la présence de nanoparticules, notamment la courbure de ses surfaces, affecte l’agrégation de molécules amphiphile ?2. Comment une bicouche de lipide, une forme d’agrégat particulier des amphiphile, peut induire l’assemblage auto-organisé de nanoparticules hydrophobes ?3. Est-ce que la présence des nanoparticules peut provoquer des transitions morphologiques d’un nanotube membranaire ? / Amphiphile molecules, endowed with a particular structure containing a hydrophilic head and a hydrophobic tail, have many important applications, e.g., fabrication of detergents, surface coating or surface functionalization, etc. Molecular aggregates of various forms, micelles, vehicle, membranes, etc. can be formed from amphiphile molecules. The complexity of these molecular aggregates involving a large number of atoms make the theoretical study of these system very challenging. Up to now, our understanding of the interaction between nanoparticles and aggregates of amphiphiles remains quite incomplete. Using a variety of molecular simulation methods and some theoretical approaches (Helfrich theory and perturbation theory), we have studied the following issues in the present thesis: 1. How the presence of nanoparticles, especially due to their highly curved surfaces, affects the aggregation of the amphiphiles? 2. How a lipid bilayer, a particular amphiphile aggregate, induces the self-assembly of hydrophobic nanoparticles.3. How the morphology transition of a membrane nanotube can be induced by nanoparticles?

Compartmentalization of cell membranes, in particular of the apical membrane of columnar epithelia, is the topic of this thesis. The first part characterizes the apical membrane and its specialized organization and morphology, whereas the second part focuses on the formation of this unique plasma membrane domain during epithelial polarization. The apical membrane of columnar epithelia is enriched in glycosphingolipids, a class of lipids that are known to interact with cholesterol to form liquid ordered domains, also termed "rafts", in cell membranes. Imaging the apical surface of untreated and raft lipid depleted MDCK cells with atomic force microscopy revealed that raft lipids are involved in the formation and/or maintenance of microvilli, actin based protrusions of the apical plasma membrane, indicating a regulatory link between membrane domains and the cytoskeleton. Furthermore, antibody patching and photobleaching experiments performed during the work of this thesis suggest that the organization into raft and non-raft domains is very different in the apical membrane of MDCK cells compared to the plasma membrane of a fibroblast. In fact, the data support the hypothesis that the apical membrane could be a percolating raft membrane in which rafts constitute the major phase and non-raft domains exist as isolated entities. The second part of this thesis analyses the segregation of apical and basolateral membrane domains during epithelial polarization. This segregation can either be achieved by generating scaffolded domains prior to junction formation or by polarized secretory and endocytic membrane traffic after the establishment of cell junctions. While most apical and basolateral marker proteins in MDCK cells follow the latter mechanism, this thesis reports that the apical marker gp135 is confined to the dorsal face already in single attached cells. The unknown antigen was purified and identified as podocalyxin. Analysis of a series of domain mutants revealed that the C-terminal PDZ-binding motif of podocalyxin is mainly responsible for its special localization, which it shares with the PDZ protein NHERF-2. Knocking down podocalyxin by RNA interference resulted in retardation of cell growth and epithelial polarization. Taken together, the data suggest that podocalyxin and NHERF-2 could be part of an early apical polarity scaffolding system based on PDZ-binding and PDZ-containing proteins.