L'étude du pouvoir moussant de la gélatine trouve son origine dans un problème industriel important, non résolu jusqu'à présent : le choix des lots de gélatine en fonction des applications (par exemple : les gelées sucrées ou salées ainsi que les gommes nécessitent pour leur fabrication des gélatines peu moussantes ; les produits foisonnés et les "lards" requièrent des gélatines à pouvoir moussant élevé). Pour ce faire, quatre objectifs ont été définis : la caractérisation du pouvoir moussant de la gélatine - la mise en évidence des paramètres influençant les propriétés moussantes - la maîtrise de la formation non contrôlée de mousse spontanée dans les solutions de gélatine - la définition du process à appliquer à la matière première pour l'obtention de gélatine de pouvoir moussant souhaité. Dans le cadre de ce travail, seuls les deux premiers objectifs ont été entièrement réalisés.

Die durch Phosphatidylserin Decarboxylase (PSD) katalysierte Decarboxylierung von Phosphatidylserin (PS) zu Phosphatidylethanolamin (PE) ist für Mitochondrien in Hefe und Mäusen von essentieller Bedeutung. Im Rahmen der vorliegenden Dissertation wurde erstmals die Rolle dieses PE-Syntheseweges in Pflanzen untersucht. Die drei in Arabidopsis identifizierten PSD Gene atPSD1, atPSD2, atPSD3 codieren für Enzyme, die in Membranen der Mitochondrien (atPSD1), der Tonoplasten (atPSD2) und des Endoplasmatischen Retikulums (atPSD3) lokalisiert sind. Der Beitrag der einzelnen PSDs zur PE-Synthese wurde anhand von psd Null-Mutanten untersucht. Dabei stellte sich atPSD3 als das Enzym mit der höchsten Aktivität heraus. Alternativ zum PSD-Weg wird in Arabidopsis PE auch mittels Aminoalkohol-phosphotransferase synthetisiert. Der Verlust der gesamten PSD-Aktivität, wie es in der erzeugten psd Dreifachmutante der Fall ist, wirkt sich ausschließlich auf die Lipidzusammensetzung in der Mitochondrienmembran aus. Demzufolge wird extramitochondriales PE hauptsächlich über die Aminoalkoholphosphotransferase synthetisiert. Die veränderte Lipidzusammensetzung der Mitochondrienmembran hatte jedoch keinen Einfluss auf die Anzahl, Größe und Ultrastruktur der Mitochondrien sowie auf das ADP/ATP-Verhältnis und die Respiration. Neben der Bereitstellung von Reduktionsäquivalenten beeinflusst die Funktionalität der Mitochondrien auch die Bildung von Blüten- und Staubblättern. Diese Blütenorgane waren in der psd Dreifachmutante stark verändert, und der Blütenphänotyp ähnelte der APETALA3-Mutante. Dieses homöotische Gen ist für die Ausbildung von Blüten- und Staubblättern verantwortlich. Für die Erzeugung der Mutanten psd2-1 und psd3-1 wurde ein T-DNA Vektor verwendet, der den Promotor des APETALA3 Gens enthielt, welcher in den Mutanten psd2-1, psd3-1 sowie psd2-1psd3-1 und der psd1psd2-1psd3-1 Dreifachmutante eine vergleichbare Co-Suppression des APETALA3 Gens hervorruft. Der Blütenphänotyp trat jedoch nur in der psd Dreifachmutante auf, da nur in ihr die Kombination von geringen Funktionstörungen der Mitochondrien, hervorgerufen durch veränderte Lipidzusammensetzung, mit der Co-Suppression von APETALA3 auftritt. / Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine (PE) catalyzed by phosphatidylserine decarboxylase (PSD) is an essential reaction for mitochondria in yeast and mice. This dissertation describes the role of this biosynthesis pathway in plants for the first time. Three PSD genes were identified in Arabidopsis, atPSD1, atPSD2, atPSD3. The gene products localize to mitochondria (atPSD1), tonoplast (atPSD2) and endoplasmatic retikulum (atPSD3). Contribution to PE-synthesis of each PSD was analyzed using T-DNA insertion mutants. Thereby, atPSD3 was found to be the most active isoform. Alternatively, PE is also synthesized by the action of aminoalcohol phosphotransferase. Complete loss of PSD activity, like in the psd triple mutant, resulted in changes in lipid composition of mitochondria membranes exclusively. In conclusion the bulk of PE is synthesized by aminoalcohol phosphotransferase. Changed lipid composition of mitochondria did not result in changes of mitochondria number, structure, ADP/ATP ratio and respiration. Mitochondria functionality was formerly shown to effect formation of petals and stamens. These flower organs were drastically morphologically changed in psd triple mutants and showed strong similarities to APETALA3 mutants. APETALA3 is a homeotic gene responsible for specifying petals and stamens. Mutants psd2-1 and psd3-1 used for crossing psd double and triple mutants contained a T-DNA vector which include the promoter for APETALA3. This promoter caused co-suppression of the endogenous APETALA3 gene in all mutants isolated from the Arabidopsis Knockout Facility, whereas changed flower morphology occurred only in the triple mutant concluding a combined effect of co-suppression and a reduced functionality of mitochondria, caused by changed lipid composition.

This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III & IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.

Steroidogenic factor (SF1) is an orphan nuclear receptor that is essential for steroid hormone-biosynthesis and endocrine development. Recent studies have demonstrated that phospholipids are ligands for SF1. In the present study our aim was to identify endogenous ligands for SF1 and characterize their functional significance in mediating cAMP-dependent transcription of human CYP17. Using mass spectrometry we show that in H295R adrenocortical cells SF1 is bound to sphingosine (SPH) under basal conditions and that cAMP stimulation decreases the amount of SPH bound to the receptor. We also show that silencing both acid and neutral ceramidases using siRNA induces CYP17 mRNA expression, suggesting that SPH acts as an inhibitory ligand. In vitro analysis of ligand binding using scintillation proximity assays show that several sphingolipids and phospholipids, including phosphatidic acid (PA), can compete with [3H]SPH for binding to SF1, suggesting that SF1 may have more than one ligand and binding specificity may change with the changes in intracellular fluxes of phospholipids. Further, phosphatidic acid (PA) induces SF1-dependent transcription of CYP17 reporter constructs. Inhibition of diacyglycerol kinase (DAGK) activity using R59949 and silencing DAGK- expression attenuates SF1-dependent CYP17 transcriptional. We propose that PA is an activating ligand for SF1 and that cAMP-stimulated activation of SF1 takes place by displacement of SPH.

Positive strand RNA viruses, inculding tombusviruses, are known to utilize cellular membranes to assemble their replicase complexes (VRCs). Two tombusviruses , Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), replicate on different organellar membranes, peroxisomes or endoplasmic reticulum (ER) for TBSV and mitochodria outer membranes in case of CIRV. I showed that both TBSV and CIRV replicase proteins could assemble VRCs and replicate viral RNA on purified microsomes (ER) and mitochondria. Different efficiencies of assembly was shown determined by multiple domains on TBSV or CIRV replication proteins. To study why VRC assembly could occur on an alternative organellar membranes, I focused on the phospholipids, key lipid components in ER or mitochondria membranes. Phospholipids directly interact with viral replicases, however, their specific roles during (+)RNA virus replication are far less understood. I used TBSV as a model (+) RNA virus, and established a cell-free TBSV replication system using artificial membranes prepared from different phospholipids. I showed that phosphatidylethanolamine (PE) is required for full cycle replication of the viral RNA.Moreover, PE is enriched at the sites of TBSV replication in plant and yeast cells, and was up-regulated during TBSV replication. Furthermore, up-regulation of total cellular PE content in yeast due to deletion of CHO2 leads to dramatically stimulated TBSV replication. Overall, I identified PE as the key lipid component of membranes required for TBSV replication, and my data highlighted that PE, an abundant phospholipid in all eukaryotic cells, not only serves as a structural component of membrane bilayers, its interaction with the viral replication proteins also stimulates (+)RNA virus replication. Further experiments indicated both early secretory pathway and endocytic pathway are involved in PE re-distribution to site of replication. In addition to lipids and subcellular membranes, certain host proteins are also involved in (+) RNA virus replication and VRC assembly. I identified Hop-like stress- inducible protein 1 (Sti1p), which interacts with heat shock protein 70, is required for the inhibition of CIRV replication. My findings indicate that Hop/Sti1 co-chaperone could act as a virus restriction factor in case of mitochondrial CIRV, but not against peroxisomal tombusvirus.

Cells use lipid membranes to organize and define their chemical environments. All cell membranes are based on a common structure: bilayers composed of phospholipids with two hydrocarbon chains. How did biology converge on this particular solution for cellular encapsulation? The first cell membranes are proposed to have assembled from simple, single-chain lipids, such as fatty acids and their derivatives, which would have been available in the prebiotic environment. Here we argue that the physical properties of fatty acid membranes would have made them well suited for a role as primitive cell membranes and predisposed their evolution to modern, phospholipid-based membranes. We first considered models for primitive membrane self-assembly, which faces significant concentration barriers due to the entropic cost of aggregation and the solubility of single-chain lipids. We therefore identified two physical mechanisms by which fatty acid membrane assembly can proceed from dilute solutions. Thermal diffusion columns, a proposed prebiotic concentration method, drive the formation of fatty acid vesicles by concentrating an initially isotropic solution past the critical concentration necessary for aggregation. Alternatively, mixtures of fatty acids with varying chain lengths, the expected products of abiotic lipid synthesis, intrinsically reduce the concentration barrier to aggregation through their polydispersity. These results motivated us to better understand the phase behavior of fatty acids in solutions. We found that the composition of fatty acid aggregates, whether vesicles or micelles, is also determined by concentration. Fatty acid vesicles feature significant amounts of coexisting micelles, whose abundance is enriched in low concentration solutions. We utilized this micelle-vesicle equilibrium to drive the growth of pre-existing fatty acid vesicles by changing amphiphile concentration. We next considered the evolution of phospholipid membranes, which was a critical and necessary step for the early evolution of cells. We found that the incorporation of even small amounts of phospholipids drives the growth of fatty acid vesicles by competition for monomers with neighboring vesicles lacking phospholipids. This competitive growth would have provided a strong selective advantage for primitive cells to evolve the catalytic machinery needed to synthesize phospholipids from their single-chain precursors. Growth is caused by any relative difference in phospholipid content, suggesting an evolutionary arms race among primitive cells for increasingly phospholipid membranes. What would have been the consequences for early cells of such a transition in membrane composition? We found that increasing phospholipid content inhibits the permeability of fatty acid membranes through changes in bilayer fluidity. For early heterotrophic cells, the emergence of increasingly phospholipid membranes would have therefore imposed new selective pressures for the evolution of membrane transport machinery and metabolism. Our model for early membrane evolution led us to develop prebiotic models for phospholipid chemistry. The assembly of phospholipids from single-chain substrates requires a single reaction: the acyltransfer of an activated fatty acid onto a glycerol monoester or lysophospholipid. We developed a synthetic model for this reaction that incorporates a copper-catalyzed azide-alkyne cycloaddition and showed that it drives de novo vesicle assembly.

Neurological and neuroendocrine disorders result from signaling dysregulation at the molecular, cellular, and multi-cellular levels. This dissertation presents the development of separation methods, using capillary zone electrophoresis (CZE) and capillary liquid chromatography (CLC), for detecting and quantifying small molecules, peptides, and proteins involved in cellular signaling. CZE is a rapid separation technique, making it ideal for monitoring cellular dynamics with high temporal resolution. An ultraviolet - light emitting diode was used for photolytic optical gating of caged fluorophore-labeled biogenic amines, common functional groups in neurotransmitters. Additionally, a novel caged fluorophore with faster reaction kinetics than commercially available dyes was used to label reduced thiols and primary amines in the presence of o-phthalaldehyde. Together this light source and novel caged dye illustrate the utility of these methods for monitoring chemical dynamics during continuous sampling. Many cellular second messengers, including inositol phosphates, are known to exist within the cell, but their dynamics and intermolecular interactions are poorly understood since they lack chromophores or electroactive functional groups making direct detection difficult. Utilizing CZE with capacitive coupled contactless conductivity detection (C4D), biological phosphates were separated and detected based on their high anionic charge, suggesting the utility of C4D in label-free detection of biological molecules. The techniques described above require higher sensitivity to monitor physiologically relevant analyte concentrations; therefore, Hadamard transform capillary electrophoresis (HTCE) was used as a multiplexing method in which multiple separations were performed simultaneously. HTCE resulted in increased sensitivity by decreasing the random background noise. Peptides and proteins propagate signals within or between cells; yet, they are difficult to separate and detect by CZE since their highly charged surfaces result in non-specific adsorption to the capillary wall. To minimize these interactions, stable hybrid phospholipid bilayers were prepared as capillary coatings for CZE separations of cationic proteins. Additionally, stabilized phospholipid bilayer coatings were formed on silica particles through redox polymerization of synthetic, polymerizable lipids. These bilayers were stable after exposure to surfactant, organic solvents, and after storage for one month, suggesting their value as lipid chromatography stationary phases for future incorporation of transmembrane proteins to analyze binding interactions with small molecules.

No description available.

This thesis investigates the development of new methodology for the identification and quantification of organic phosphorus compounds in environmental samples. Phosphorus is a vital element for primary production and one of the factors contributing to eutrophication. Eutrophication of aquatic systems leads to algal blooms, changes in ecological balance and deteriorating water quality. Difficulties in studying organic phosphorus stem from the fact that organic phosphorus is present in the environment in a variety of forms and each form may have different degradation and turnover time, having very different effects on eutrophication. New methods for the quantification of phosphorus derived from three groups of organic phosphorus compounds were developed. For the determination of phosphorus derived from DNA and phospholipids selective extraction was combined with digestion and colorimetric determination of the extracted phosphate. For quantification of inositol phosphates high performance liquid chromatography was coupled with tandem mass spectrometry using electrospray ionization.   The methods were applied to studying the distribution of these compounds in a small catchment and in the case of DNA-P and phospholipid-P, the degradation of the fractions in lake sediments. The studies showed that phosphorus bound to DNA, phospholipids and inositol phosphates constitute a sizeable part of the total phosphorus in different environmental samples. The phospholipid-P fraction was the smallest one, accounting for, on average, only a few percent of the total phosphorus in the sample. Inositol phosphates were most prevalent in the soils, with inositol hexakisphosphate accounting for over 10% of the total phosphorus content. The highest content of DNA-P was found in sediments and it was shown that DNA-P degrades more rapidly than phospholipid-P and therefore plays a more critical role in internal loading.

Changes in the plasma membrane phospholipid composition may alter the structure and/or fluidity and lead to a variety of changes in membrane functions. Dietary fats are known to influence the composition of lipids in the plasma membrane. The purpose of this investigation was to compare effects of dietary linoleic and stearic acid on the composition of the phospholipids in the plasma membranes of mammary tumors in A/St mice.Plasma membranes were isolated and lipids were extracted. Phospholipids were separated by thin-layer chromatography and identified by detection with molybdenum blue reagent. The Rf values and integration of optical densities were used to compare phospholipid composition in membranes of tumors from mice fed experimental diets. It appears that the amount of dietary fat, but not the type, affects the phospholipid distributions. The phosphatidylinositol was the phospholipid most affected, representing the smallest amount in membranes from tumors in mice fed the low fat diets. / Department of Biology