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Fatty acids are an essential component of the phospholipids of the inner and outer membranes of Escherichia coli. The synthesis of both fatty acids and phospholipids is regulated. Synthesis increases when growth rate increases, is inhibited when starvation occurs, and the fatty acid composition of the membrane changes with growth temperature. Several genes encoding enzymes involved in membrane synthesis are located in the rpmF-plsX-fab operon. In this operon, a gene encoding a phospholipid synthetic gene of unknown function, plsX, lies just downstream of the ribosomal protein gene rpmF and upstream of five fatty acid biosynthetic genes, fabH, fabD, fabG, acpP, and fabF. The operon is also complex; transcription is initiated from at least eight promoters. In addition, some transcripts produced by the operon are cleaved by RNases while others terminate at one of three specific points at the 5' end of plsX. This work demonstrates that a weak transcriptional terminator (an attenuator) lies at the 5' end of plsX. The attenuator was localized to a 200 bp segment. Analysis of the secondary structure of the attenuator mRNA has lead to a model which includes four stem-loop structures. In this model, the plsX start codon lies within the loop of the second stem. Two tandem stems are located directly upstream of the mapped 3' endpoints. Mutational analysis shows that all four stem-loops play a role in attenuator activity. Regulation of the attenuator and the attenuator's mechanism of controlling downstream gene expression were investigated. Ribosome binding to attenuator mRNA, the PlsX protein, ppGpp concentration, and rate of lipid synthesis all appear to have no effect on attenuator activity. Interestingly, growth temperature appears to have an effect on both attenuator activity and the activity of one or more of the promoters upstream of rpmF, P₁, P₂, and P₃. Activity of the three promoters is 4.5-fold higher at 28°C as compared with 42°C. The attenuator also appears to increase expression of downstream genes 2-fold as temperature decreases. Though the attenuator region terminates transcription, growth temperature-regulation of attenuator activity is apparently mediated by a change in stability of the mRNA. These data demonstrate that transcriptional expression of plsX is 9-fold higher at 28°C as compared with 42°C. The striking dependence on temperature of plsX expression suggests a role for PlsX in the temperature modulation of fatty acid incorporation into the membrane phospholipids. / Ph. D.

Phospholipid bilayers constitute the primary structural element of biological membranes, and as such, they play a central role in biochemical and biophysical processes at the cellular level, including cell protection, intercellular interactions, trans-membrane transport, cell morphology, and protein function, to name a few. The properties of phospholipid bilayers are thus of great interest from both experimental and theoretical standpoints. Although experiments have provided much of the macroscopic functions and properties of biological membranes, insight into specific mechanisms at the molecular level are seldom accessible by conventional methods. To obtain a better understanding of biochemical and biophysical processes at the molecular level involving phospholipid bilayers, we apply molecular simulation methods to investigate the complexity of the membrane matrix using atomistic models. Here, we discuss three specific biological processes that are associated with biological membranes: 1) membrane stabilization, 2) membrane phase behavior, and 3) fatty acid-induced toxicity in cell membranes. For membrane stabilization, molecular dynamics studies were performed for mixed phospholipid bilayers containing two of the most prevalent phospholipids (phosphatidylcholine and phosphatidylethanolamime) in biological membranes. We presented structural and dynamics properties of these systems, as well as the effect of stabilizing agents, such as trehalose, on their properties. Furthermore, we performed a comprehensive analysis of the phase transition of lipid bilayers and investigated the interactions of stabilizing agents (glucose or trehalose) with lipid bilayers under dehydrated conditions to understand the mechanisms for preservation of cellular systems. For membrane phase behavior, a comprehensive study of the structural properties of saturated and monounsaturated lipid bilayers near the main phase transition were investigated using molecular dynamics simulations. In this study, we demonstrated that atomistic simulations are capable of capturing the phase transformation process of lipid bilayers, providing a valuable set of molecular and structural information at and near its transition state. Lastly, the third study investigated the mechanism for fatty acid-induced toxicity by integrating in vitro and in silico experiments to reveal the biophysical interactions of saturated fatty acid (palmitate) with the cellular membranes and the role of trehalose and unsaturated fatty acids (oleate and linoleate) in preventing changes to the membrane structure. Knowledge gained from this study is essential in the prevention and treatment of obesity-associated cirrhosis diseases. / Ph. D.

The proteins required for catabolism of glycerol 3- phosphate are encoded by the genes of the <i>glp</i> regulon of Escherichia coli and are under negative transcriptional regulation by the <i>glpR</i>-encoded repressor. The <i>glpR</i> gene is adjacent to, and is transcribed divergently from, <i>glpD</i>. <i>GlpR</i> and <i>glpD</i> are separated by two open reading frames, designated <i>glpE</i> and <i>glpG</i>, encoding proteins of unknown function. The <i>glpD</i>-encoded aerobic sn-glycerol 3-phosphate dehydrogenase is a cytosplasmic membrane-associated respiratory enzyme. The nucleotide sequence of <i>glpD</i> was determined. An open reading frame of 501 codons was preceded by a consensus Shine-Dalgarno sequence. The proposed translational start and reading frame of glpD were confirmed by determining the nucleotide sequence across the fusion joint of a <i>glpD-lacZ</i>- translational fusion. / Ph. D.

All of the glp genes of Escherichia coli are subject to negative regulation by the glpR-encoded repressor (GlpR). Comparison of the repressor binding affinity for consensus and altered consensus operator regions showed that positions 3, 4, 5, 7 and 8 bp removed from the center of operator symmetry are most important for repressor binding. Cooperative binding of repressors to tandem operators was demonstrated. Cooperativity was maximal when two 20-base pair operators were directly repeated and decreased with the deletion of 2 base pairs or the addition of 4 base pairs between the operators. Cooperative binding was eliminated by a 6 base pair insertion between tandem operators. / Master of Science

Phospholipids are commonly found in biological membranes. They have a polar head group and two ester linked fatty acids tails. Different methods such as thin layer chromatography and high performance liquid chromatography coupled with ultraviolet, refractive index, flame ionization detector, and mass spectrometry (MS) detection have long been used in the study of phospholipids. These methods were time-consuming and lacked quantitative accuracy. In this work, phosphatidylcholine, phosphatidyl-ethanolamine, phosphatidylinositol and phosphatidylserine have been studied by supercritical fluid chromatography (SFC) coupled with evaporative light scattering detection (ELSD) and mass spectrometry (MS). Four different silica-based stationary phases were studied: 2-ethylpyridine, 4-ethylpyridine, diol and conventional cyanopropyl. The influence of different mobile phase additives on the elution of phospholipids has been studied. The results have shown that isopropylamine is a better additive compared with ammonium acetate, tetrabutyl-ammonium acetate, and trifluoroacetic acid for the elution of phospholipids. All phospholipids have been eluted with baseline separation in less than 15 minutes although there is some partial overlap on the pyridine columns. The second goal for this work was fractionation of phospholipids from lecithin (a by-product from soybean) by using supercritical fluid extraction (SFE) with methanol-modified CO2. Neutral lipids were first removed from the crude sample using pure CO2. Partrial fractionation of PE and nearly pure fractionated PC were obtained by varying the modifier concentration in the extraction fluid at 460 atm and 40oC with silica gel inside the extraction vessel. A total of six components were isolated from crude soybean lecithin. / Master of Science

No / The structural and functional diversity of lipids accounts for their involvement into a wide range of homeostatic processes and disease states, including lifestyle-related diseases as well as genetic conditions. Challenges presented by this diversity have been addressed to a great extent by the development of lipidomics, a platform that makes possible the detailed profiling and characterisation of lipid species present in any cell, organelle, tissue or body fluid, and allows for a wider appreciation of the biological role of lipid networks. Progress in the field of lipidomics has been greatly facilitated by recent advances in MS and includes a range of analytical platforms supporting applications spanning from qualitative and quantitative assessment of multiple species to lipid imaging. Here we review these MS techniques currently in routine use in lipidomics, alongside with new ones that have started making an impact in the field. Recent applications in health, disease and nutrition-related questions will also be discussed with a view to convey the importance of lipidomics contributions to biosciences and food technology.

The ability to extrapolate nutritional intervention data from experimental rodent models to humans requires standardization of dietary design. The inability to translate the level of nutrients from animal models to humans has contributed to contradictory findings between species. It is hypothesized that dietary linoleic acid (LA) promotes chronic and acute diseases by enriching tissues with arachidonic acid (AA), its downstream metabolite. However, levels of LA in rodent diets are notoriously erratic making interspecies comparisons unreliable. Therefore, the ability to extrapolate the biological effects of dietary LA from experimental rodents to humans necessitates an allometric scaling model that is rooted within a human equivalent context. To determine the physiological effect of dietary LA on tissue AA, a mathematical model for extrapolating nutrients based on energy was designed to mimic human equivalent doses. C57BL/6J mice were divided into 9 groups fed a background diet equivalent to that of the US diet (including LA, ALA, AA, EPA, DHA) with supplemental doses of LA (up to 2.3x) or AA (up to 5x). Changes in the phospholipid fatty acid compositions were monitored in plasma and erythrocytes and compared to data from humans supplemented with equivalent doses of LA or AA. Increasing dietary LA had little effect on tissue AA, while supplementing diets with AA significantly increased tissue AA levels, recapitulating results from human trials. Thus, interspecies comparisons for dietary LA between rodents and humans can be achieved when rodents are provided human equivalent doses based on differences in metabolic activity as defined by energy consumption.

Ce travail étudie l’obtention de phospholipides structurés enrichis en DHA et en acide caprylique (PC DHA-C8) par voie enzymatique. Deux voies de synthèse sont étudiées, l’acidolyse et l’estérification. Suite à un criblage enzymatique, la lipase retenue pour les deux voies de synthèse est TL-IM. Une optimisation des paramètres de la réaction d’acidolyse a été réalisée entre l’acide caprylique (C8:0) et la phosphatidylcholine de tournesol (PC) par le biais d’un plan d’expériences. Les conditions optimales déterminées sont une température de 38°C, une activité de l’eau de 0,7, une quantité d’enzyme de 15% de la masse en substrat ainsi qu’un rapport molaire C8:0/PC de 18. Ces conditions ont ensuite été utilisées pour l’acidolyse de phospholipides microalgaux riches en DHA issus de la microalgue Tisochrysis lutea afin d’obtenir de la PC DHA-C8. Les résultats n’ont pas été concluants. L’autre voie de synthèse étudiée est l’estérification par des lipases de la GPC, de l’acide caprylique et du DHA en milieu fondu. Cette réaction a été optimisée par la technique du pas par pas. Les paramètres étudiés sont la température, la quantité d’enzyme, le rapport molaire GPC/C8:0/DHA et l’application d’un vide. Pour l’obtention de PC DHA-C8, il faut fixer chacun de ces paramètres respectivement de la sorte : 45°C, 20% d’enzyme, un rapport molaire de 1/3/15 et un vide de 100 mbar. La production de PC DHA-C8, bien qu’optimisée ne dépasse pas 2% de rendement. Cependant, durant cette expérience, il a été constaté une forte production de LPC DHA, atteignant 16% sans optimisation des paramètres de synthèse. / The enzymatic synthesis of structured phsopholipids enriched in DHA and caprylic acid (PC DHA-C8) is studied. Two different ways are studied, acidolysis and esterification. An enzymatic screening led to the choice of the immobilized lipase from Thermomyces lanuginosa (TL-IM) for the 2 reactions. Parameters of the acidolysis reaction between carpylic acid (C8:0) and sunflower phosphatidylcholine (PC) were optimized by means of an experimental design. The optimum conditions determined are a temperature of 38°C, an aw of 0.7, an amount of enzyme of 15% of the mass of substrate and a molar ratio of C8:0/PC of 18. These conditions were applied to the acidolysis of microalgal phospholipids from T. lutea, rich in DHA, in order to produce PC DHA-C8. The other studied reaction is the lipase catalyzed esterification of GPC with C8:0 and DHA in a solvent-free medium This reaction has been optimized by studying each factor independently. The parameters studied are the temperature, the amount of lipase, the molar ratio GPC/C8:0/DHA and the use of reduced pressure. In order to obtain PC DHA-C8, each of theses parameters are respectively set at: 45°C, 20% of enzyme, a molar ratio of 1/3/15 and a pressure of 100 mbar. The production of PC DHA-C8, although optimized, does not exceed a yield of 2%. However, during this experiment, a high production of LPC DHA is observed, up to 16% without optimization of the synthesis parameter.

<p>\ / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished