(Table T2) Relative signal intensity of intact polar lipids in sediments from ODP Sites 207-1257 and 207-1258

We report results from the analysis of intact polar lipids (IPLs) in sediments from Ocean Drilling Program Sites 1257 and 1258. IPLs, constituting the cell membranes of living organisms, were detected in organic-lean sediments but not in underlying organic-rich black shales. Microbial activity in organic-lean sediments is likely due to sulfate-dependent oxidation of methane whereas difficulties detecting IPLs in black shales are interpreted to result from unfavorable signal-to-noise ratios due to low cell concentrations in combination with extremely high analytical noise created by uncharacterized organic matrix. IPLs found are consistent with a low-diversity community of archaea and bacteria. The concentrations of IPLs are more than one order of magnitude lower than those in Neogene deep subsurface sediments at the Peruvian margin, suggestive of significantly lower cell concentrations in Demerara Rise. This finding is consistent with inferred low rates of subsurface microbial activity.

Carbon isotope composition of lipids from bones and host sediments of the Atlantic and Pacific Oceans

Carbon in lipids separated from organic matter of fish and marine mammal bones from bottom of the Pacific and Atlantic oceans has d13C values ranging from -21.6 to -25.8 per mil and is isotopically lighter than that in lipids and total organic matter of host sediments. During fossilization of organic phosphate carbon isotope composition of bound lipids of fish bone becomes lighter and that of bones of mammals becomes heavier, possibly as a result of metabolisms of these organisms and composition of phospholipids in them.

Concentration of archaeal intact polar lipids (IPLs) of ROV sediment core GeoB12315-9

Here we report the amount of archaeal cardiolipins analogues in a cold seep off Pakistan (Makran accretionary prism). The published data (Yoshinaga et al., 2012) describes a series of tentatively identified cardiolipin analogues (dimeric phospholipids or bisphosphatidylglycerol), which represented 0.5% to 5% of total archaeal intact polar lipids.

Fossilization and degradation of archaeal intact polar tetraether lipids in ODP Site 201-1229 sediments

Glycerol dibiphytanyl glycerol tetraether (GDGT) lipids are part of the cellular membranes of Thaumarchaeota, an archaeal phylum composed of aerobic ammonia oxidizers, and are used in the paleotemperature proxy TEX86. GDGTs in live cells possess polar head groups and are called intact polar lipids (IPL-GDGTs). Their transformation to core lipids (CL) by cleavage of the head group was assumed to proceed rapidly after cell death but it has been suggested that some of these IPL-GDGTs can, just like the CL-GDGTs, be preserved over geological timescales. Here, we examined IPL-GDGTs in deeply buried (0.2-186 mbsf, ~2.5 Myr) sediments from the Peru Margin. Direct measurements of the most abundant IPL-GDGT, IPL-crenarchaeol, specific for Thaumarchaeota, revealed depth profiles which differed per head group. Shallow sediments (<1 mbsf) contained IPL-crenarchaeol with both glycosidic- and phosphate headgroups, as also observed in thaumarchaeal enrichment cultures, marine suspended particulate matter and marine surface sediments. However, hexose, phosphohexose-crenarchaeol is not detected anymore below 6 mbsf (~7 kyr), suggesting a high lability. In contrast, IPL-crenarchaeol with glycosidic head groups is preserved over time scales of Myr. This agrees with previous analyses of deeply buried (>1 m) marine sediments, which only reported glycosidic and no phosphate-containing IPL-GDGTs. TEX86 values of CL-GDGTs did not markedly change with depth, and the TEX86 of IPL-derived GDGTs decreased only when the proportions of monohexose- to dihexose-GDGTs changed, likely due to the enhanced preservation of the monohexose GDGTs. Our results support the hypothesis that in situ GDGT production and differential IPL degradation in sediments is not substantially affecting TEX86 paleotemperature estimations based on CL GDGTs and indicate that likely only a small amount of IPL-GDGTs present in deeply buried sediments is part of cell membranes of active Archaea. The amount of archaeal biomass in the deep biosphere based on these IPLs may have been substantially overestimated.

Intact and core tetraether lipids in water column profiles of suspended particulate matter and of surface sediment samples off Cape Blanc, NW Africa

In the reconstruction of sea surface temperature (SST) from sedimentary archives, secondary sources, lateral transport and selective preservation are considered to be mainly negligible in terms of influencing the primary signal. This is also true for the archaeal glycerol dialkyl glycerol tetraethers (GDGTs) that form the basis for the TEX86 SST proxy. Our samples represent four years variability on a transect off Cape Blanc (NW Africa). We studied the subsurface production, vertical and lateral transport of intact polar lipids and core GDGTs in the water column at high vertical resolution on the basis of suspended particulate matter (SPM) samples from the photic zone, the subsurface oxygen minimum zone (OMZ), nepheloid layers (NL) and the water column between these. Furthermore we compared the water column SPM GDGT composition with that in underlying surface sediments. This is the first study that reports TEX86 values from the precursor intact polar lipids (IPLs) associated with specific head groups (IPL -specific TEX86). We show a clear deviation from the sea surface GDGT composition in the OMZ between 300 and 600 m. Since neither lateral transport nor selective degradation provides a satisfactory explanation for the observed TEX-derived temperature profiles with a bias towards higher temperatures for both core- and IPL -specific TEX86 values, we suggest that subsurface in situ production of archaea with a distinct relationship between lipid biosynthesis and temperature is the responsible mechanism. However, in the NW-African upwelling system the GDGT contribution of the OMZ to the surface sediments does not seem to affect the sedimentary TEX86 as it shows no bias and still reflects the signal of the surface waters between 0 and 60 m.

(Table 1) Composition of lipids in particulate matter and bottom sediments of the Baltic Sea

Lipid contents in the upper layer of bottom sediments in the Baltic Sea range from 0.37 to 2.66 mg/g (1.2-25.8% Corg). It is shown that the main factors determining composition of lipids in bottom precipitates are relative roles of different sources of lipids in sediments and conditions of sediment accumulation. Runoff of the Daugava River into the Gulf of Riga contributes simple low-polarity lipids. Sterols and certain bound fatty acids originate in living organic matter. Polar lipids are formed by inheritance of complex phospholipids and glycolipids from plankton and/or by formation of polycondensates.

Composition of free lipids in bottom sediments in the tropical West Pacific and South Atlantic

Contents of free lipids in the upper layers of slightly siliceous diatomaceous oozes from the South Atlantic and of calcareous foraminiferal oozes, of coral sediments and of red clays from the western tropical Pacific amount varies from 0.014 to 0.057% of dry sediment. Their content is inversely proportional to total content of organic matter. Relative content of low-polar compounds in total amount of lipids and content of hydrocarbons, fatty acids, and sterols in the composition of these compounds can serve as an index of degree of transformation of organic matter in sediment because these compounds are resistant to various degree to microbial and hydrolytic decomposition and, consequently, are selectively preserved under conditions of biodegradation of organic compounds during oxydation-reduction processes.

Damaging effects of advanced glycation end product in the murine macrophages cell line J774A.1.

The interaction of reducing sugars, such as aldose, with proteins and the subsequent molecular rearrangements, produces irreversible advanced glycation end-products (AGEs), a heterogeneous class of non-enzymatic glycated proteins or lipids. AGEs form cross-links, trap macromolecules and release reactive oxygen intermediates. AGEs are linked to aging, and increase in several related diseases. The aim of this study was to assess, in a murine macrophage cell line, J774A.1, the effects of 48 h of exposure to glycated serum containing a known amount of pentosidine, a well-known AGE found in the plasma and tissues of diabetic and uremic subjects. Fetal bovine serum was incubated with ribose (50 mm) for 7 days at 37 °C to obtain about 10 nmol/ml of pentosidine. The cytotoxic parameters studied were cell morphology and viability by neutral red uptake, lactate dehydrogenase release and tetrazolium salt test. In the medium and in the intracellular compartment, bound and free pentosidine were evaluated by HPLC, as sensitive and specific glycative markers, and thiobarbituric acid reactive substances (TBARs), as index of the extent of lipid peroxidation. Our results confirm that macrophages are able to take up pentosidine. It is conceivable that bound pentosidine is degraded and free pentosidine is released inside the cell and then into the medium. The AGE increase in the medium was combined with an increase in TBARs, meaning that an oxidative stress occurred; marked cytotoxic effects were observed, and were followed by the release of free pentosidine and TBARs into the culture medium.

Formation of positron-atom bound states in collisions between Rydberg Ps and neutral atoms

Predicted 20 years ago, positron binding to neutral atoms has not yet been observed experimentally. A scheme is proposed to detect positron-atom bound states by colliding Rydberg positronium (Ps) with neutral atoms. Estimates of the charge-transfer reaction cross section are obtained using the first Born approximation for a selection of neutral atom targets and a wide range of incident Ps energies and principal quantum numbers. We also estimate the corresponding Ps ionization cross section. The accuracy of the calculations is tested by comparison with earlier predictions for charge transfer in Ps collisions with hydrogen and antihydrogen. We describe an existing Rydberg Ps beam suitable for producing positron-atom bound states and estimate signal rates based on the calculated cross sections and realistic experimental parameters. We conclude that the proposed methodology is capable of producing such states and of testing theoretical predictions of their binding energies.

Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Development of Imaging Mass Spectrometry Analysis of Lipids in Biological and Clinically Relevant Applications

La spectrométrie de masse mesure la masse des ions selon leur rapport masse sur charge. Cette technique est employée dans plusieurs domaines et peut analyser des mélanges complexes. L’imagerie par spectrométrie de masse (Imaging Mass Spectrometry en anglais, IMS), une branche de la spectrométrie de masse, permet l’analyse des ions sur une surface, tout en conservant l’organisation spatiale des ions détectés. Jusqu’à présent, les échantillons les plus étudiés en IMS sont des sections tissulaires végétales ou animales. Parmi les molécules couramment analysées par l’IMS, les lipides ont suscité beaucoup d'intérêt. Les lipides sont impliqués dans les maladies et le fonctionnement normal des cellules; ils forment la membrane cellulaire et ont plusieurs rôles, comme celui de réguler des événements cellulaires. Considérant l’implication des lipides dans la biologie et la capacité du MALDI IMS à les analyser, nous avons développé des stratégies analytiques pour la manipulation des échantillons et l’analyse de larges ensembles de données lipidiques. La dégradation des lipides est très importante dans l’industrie alimentaire. De la même façon, les lipides des sections tissulaires risquent de se dégrader. Leurs produits de dégradation peuvent donc introduire des artefacts dans l’analyse IMS ainsi que la perte d’espèces lipidiques pouvant nuire à la précision des mesures d’abondance. Puisque les lipides oxydés sont aussi des médiateurs importants dans le développement de plusieurs maladies, leur réelle préservation devient donc critique. Dans les études multi-institutionnelles où les échantillons sont souvent transportés d’un emplacement à l’autre, des protocoles adaptés et validés, et des mesures de dégradation sont nécessaires. Nos principaux résultats sont les suivants : un accroissement en fonction du temps des phospholipides oxydés et des lysophospholipides dans des conditions ambiantes, une diminution de la présence des lipides ayant des acides gras insaturés et un effet inhibitoire sur ses phénomènes de la conservation des sections au froid sous N2. A température et atmosphère ambiantes, les phospholipides sont oxydés sur une échelle de temps typique d’une préparation IMS normale (~30 minutes). Les phospholipides sont aussi décomposés en lysophospholipides sur une échelle de temps de plusieurs jours. La validation d’une méthode de manipulation d’échantillon est d’autant plus importante lorsqu’il s’agit d’analyser un plus grand nombre d’échantillons. L’athérosclérose est une maladie cardiovasculaire induite par l’accumulation de matériel cellulaire sur la paroi artérielle. Puisque l’athérosclérose est un phénomène en trois dimension (3D), l'IMS 3D en série devient donc utile, d'une part, car elle a la capacité à localiser les molécules sur la longueur totale d’une plaque athéromateuse et, d'autre part, car elle peut identifier des mécanismes moléculaires du développement ou de la rupture des plaques. l'IMS 3D en série fait face à certains défis spécifiques, dont beaucoup se rapportent simplement à la reconstruction en 3D et à l’interprétation de la reconstruction moléculaire en temps réel. En tenant compte de ces objectifs et en utilisant l’IMS des lipides pour l’étude des plaques d’athérosclérose d’une carotide humaine et d’un modèle murin d’athérosclérose, nous avons élaboré des méthodes «open-source» pour la reconstruction des données de l’IMS en 3D. Notre méthodologie fournit un moyen d’obtenir des visualisations de haute qualité et démontre une stratégie pour l’interprétation rapide des données de l’IMS 3D par la segmentation multivariée. L’analyse d’aortes d’un modèle murin a été le point de départ pour le développement des méthodes car ce sont des échantillons mieux contrôlés. En corrélant les données acquises en mode d’ionisation positive et négative, l’IMS en 3D a permis de démontrer une accumulation des phospholipides dans les sinus aortiques. De plus, l’IMS par AgLDI a mis en évidence une localisation différentielle des acides gras libres, du cholestérol, des esters du cholestérol et des triglycérides. La segmentation multivariée des signaux lipidiques suite à l’analyse par IMS d’une carotide humaine démontre une histologie moléculaire corrélée avec le degré de sténose de l’artère. Ces recherches aident à mieux comprendre la complexité biologique de l’athérosclérose et peuvent possiblement prédire le développement de certains cas cliniques. La métastase au foie du cancer colorectal (Colorectal cancer liver metastasis en anglais, CRCLM) est la maladie métastatique du cancer colorectal primaire, un des cancers le plus fréquent au monde. L’évaluation et le pronostic des tumeurs CRCLM sont effectués avec l’histopathologie avec une marge d’erreur. Nous avons utilisé l’IMS des lipides pour identifier les compartiments histologiques du CRCLM et extraire leurs signatures lipidiques. En exploitant ces signatures moléculaires, nous avons pu déterminer un score histopathologique quantitatif et objectif et qui corrèle avec le pronostic. De plus, par la dissection des signatures lipidiques, nous avons identifié des espèces lipidiques individuelles qui sont discriminants des différentes histologies du CRCLM et qui peuvent potentiellement être utilisées comme des biomarqueurs pour la détermination de la réponse à la thérapie. Plus spécifiquement, nous avons trouvé une série de plasmalogènes et sphingolipides qui permettent de distinguer deux différents types de nécrose (infarct-like necrosis et usual necrosis en anglais, ILN et UN, respectivement). L’ILN est associé avec la réponse aux traitements chimiothérapiques, alors que l’UN est associé au fonctionnement normal de la tumeur.

Dimethyl Acetals, an Indirect Marker of the Endogenous Antioxidant Plasmalogen Level, are Reduced in Blood Lipids of Sudanese Pre-eclamptic Subjects whose Background Diet is High in Carbohydrate

In Sudanese women with (n = 60) and without (n = 65) pre-eclampsia, circulating lipids, plasma and red cell saturated and monounsaturated fatty (MUFA) acids and dimethyl acetals (DMAs) were investigated. DMAs are an indirect marker of levels of plasmalogens, endogenous antioxidants, which play a critical role in oxidative protection, and cholesterol homeostasis. The pre-eclamptics had higher C18:1n-9 (p < 0.001) and ΣMUFA (p < 0.01) in plasma free fatty acids, C16:1n-7, C18:1n-9, ΣMUFA; 16:0/16:1n-7 (p < 0.01) in erythrocyte choline phosphoglycerides (ePC) and 16:1n-7, 18:1n-7 and 16:0/16:1n-7 (p < 0.01) in erythrocyte ethanolamine phosphoglycerides (ePE). In contrast, the DMAs 18:0, 18:1 and ΣDMAs in ePE, and 16:0, 18:0 and ΣDMAs in ePC were reduced (p < 0.001) in the pre-eclamptic women. This study of pregnant women with high carbohydrate and low fat background diet suggests pre-eclampsia is associated with oxidative stress and enhanced activity of the microsomal enzyme stearyl-CoA desaturase (delta 9 desaturase), as assessed by palmitic/palmitoleic (C16:0/C16:n-1) and stearic/oleic (C18/C18:1n-9) ratios.

Effects of Different Fatty Acids and Dietary Lipids on Adiponectin Gene Expression in 3T3-L1 Cells and C57BL/6J Mice Adipose Tissue

Obesity is positively correlated to dietary lipid intake, and the type of lipid may play a causal role in the development of obesity-related pathologies. A major protein secreted by adipose tissue is adiponectin, which has antiatherogenic and antidiabetic properties. The aim of this study was to evaluate the effects of four different high-fat diets (enriched with soybean oil, fish oil, coconut oil, or lard) on adiponectin gene expression and secretion by the white adipose tissue (WAT) of mice fed on a selected diet for either 2 (acute treatment) or 60 days (chronic treatment). Additionally, 3T3-L1 adipocytes were treated for 48 h with six different fatty acids: palmitic, linoleic, eicosapentaenoic (EPA), docosahexaenoic (DHA), lauric, or oleic acid. Serum adiponectin concentration was reduced in the soybean-, coconut-, and lard-enriched diets in both groups. Adiponectin gene expression was lower in retroperitoneal WAT after acute treatment with all diets. The same reduction in levels of adiponectin gene expression was observed in epididymal adipose tissue of animals chronically fed soybean and coconut diets and in 3T3-L1 cells treated with palmitic, linoleic, EPA, and DHA acids. These results indicate that the intake of certain fatty acids may affect serum adiponectin levels in mice and adiponectin gene expression in mouse WAT and 3T3-L1 adipocytes. The effects appear to be time dependent and depot specific. It is postulated that the downregulation of adiponectin expression by dietary enrichment with soybean oil or coconut oil may contribute to the development of insulin resistance and atherosclerosis.

Towards NIR emission: 2-(1H-tetrazol-1-yl)pyridine as versatile ligand for the synthesis of two new families of cationic and neutral Ir(III) complexes.

In the last decades, cyclometalated Ir(III) complexes have drawn a large interest for their unique properties: they are excellent triplet state emitters, thus the emission is phosphorescent in nature; typically high quantum yields and good stability make them good candidates for luminescent materials. Moreover, through an opportune choice of the ligands, it is possible to tune the emission along the whole visible spectra. Thanks to these interesting features, Ir(III) complexes have found different applications in several areas of applied science, from OLEDs to bioimaging. In particular, regarding the second application, a remarkable red-shift in the emission is required, in order to minimize the problem of the tissue penetration and the possible damages for the organisms. With the aim of synthesizing a new family of NIR emitting Ir(III) complexes, we envisaged the possibility to use for the first time 2-(1H-tetrazol-1-yl)pyridine as bidentate ligand able to provide the required red-shift of the emission of the final complexes. Exploiting the versatility of the ligand, I prepared two different families of heteroleptic Ir(III) complexes. In detail, in the first case the 2-(1H-tetrazol-1-yl)pyridine was used as bis-chelating N^N ligand, leading to cationic complexes, while in the second case it was used as cyclometalating C^N ligand, giving neutral complexes. The structures of the prepared molecules have been characterised by NMR spectroscopy and mass spectrometry. Moreover, the neutral complexes’ emissive properties have been measured: emission spectra have been recorded in solution at both room temperature and 77K, as well as in PMMA matrix. DFT calculation has then been performed and the obtained results have been compared to experimental ones.

Role of membrane lipids in the regulation of erythrocytic oxygen-transport function in cardiovascular diseases

The composition and condition of membrane lipids, the morphology of erythrocytes, and hemoglobin distribution were explored with the help of laser interference microscopy (LIM) and Raman spectroscopy. It is shown that patients with cardiovascular diseases (CVD) have significant changes in the composition of their phospholipids and the fatty acids of membrane lipids. Furthermore, the microviscosity of the membranes and morphology of the erythrocytes are altered causing disordered oxygen transport by hemoglobin. Basic therapy carried out with the use of antiaggregants, statins, antianginals, beta-blockers, and calcium antagonists does not help to recover themorphofunctional properties of erythrocytes. Based on the results the authors assume that, for the relief of the ischemic crisis and further therapeutic treatment, it is necessary to include, in addition to cardiovascular disease medicines, medication that increases the ability of erythrocytes’ hemoglobin to transport oxygen to the tissues. We assume that the use of LIM and Raman spectroscopy is advisable for early diagnosis of changes in the structure and functional state of erythrocytes when cardiovascular diseases develop.