In vitro activity of extracts and isolated polyphenols from West African medicinal plants against Plasmodium falciparum

The aim of the study was to screen 11 selected traditional medicinal plants from West Africa for their in vitro antiplasmodial activity in order to determine the activity of single and of combination of plant extracts and to examine the activity of isolated pure compounds. Ethanolic and aqueous extracts of the 11 selected plants and pure compounds from Phyllanthus muellerianus and Anogeissus leiocarpus were tested in vitro against Plasmodium falciparum 3D7. Proliferation inhibitory effects were monitored after 48 h. Among the plants and pure compounds investigated in this study, geraniin from P. muellerianus, ellagic, gentisic, and gallic acids from A. leiocarpus, and extracts from A. leiocarpus, P. muellerianus and combination of A. leiocarpus with P. muellerianus affected the proliferation of P. falciparum most potently. Significant inhibitory activity was observed in combination of A. leiocarpus with P. muellerianus (IC50 = 10.8 mu g/ml), in combination of A. leiocarpus with Khaya senegalensis (IC50 = 12.5 mu g/ml), ellagic acid (IC50 = 2.88 mu M), and geraniin (IC50 = 11.74 mu M). In general growth inhibition was concentration-dependent revealing IC50 values ranging between 10.8 and -40.1 mu g/ml and 2.88 and 11.74 mu M for plant extracts and pure substances respectively. Comparison with literature sources of in vivo and in vitro toxicity data revealed that thresholds are up to two times higher than the determined IC50 values. Thus, the present study suggests that geraniin from P. muellerianus; ellagic acid, gallic acid, and gentisic acid from A. leiocarpus; and combination of extracts from A. leiocarpus with either P. muellerianus or K. senegalensis could be a potential option for malaria treatment.

Chemical constituents from red algae Bostrychia radicans (Rhodomelaceae): new amides and phenolic compounds

This study describes the isolation and structural determination of two amides, isolated for the first time: N,4-dihydroxy-N-(2'-hydroxyethyl)-benzamide (0.019%) and N,4-dihydroxy-N-(2'-hydroxyethyl)-benzeneacetamide (0.023%). These amides, produced by the red macroalgae Bostrychia radicans, had their structures assigned by NMR spectral data and MS analyses. In addition, this chemical study led to the isolation of cholesterol, heptadecane, squalene, trans-phytol, neophytadiene, tetradecanoic and hexadecanoic acids, methyl hexadecanoate and methyl 9-octadecenoate, 4-(methoxymethyl)-phenol, 4-hydroxybenzaldehyde, methyl 4-hydroxybenzeneacetate, methyl 2-hydroxy-3-(4-hydroxyphenyl)-propanoate, hydroquinone, methyl 4-hydroxymandelate, methyl 4-hydroxybenzoate, 4-hydroxybenzeneacetic acid and (4-hydroxyphenyl)-oxo-acetaldehyde. This is the first report concerning these compounds in B. radicans, contributing by illustrating the chemical diversity within the Rhodomelaceae family.

Antioxidant activity and chemical composition of the non polar fraction of Gracilaria domingensis (Kützing) Sonder ex Dickie and Gracilaria birdiae (Plastino & Oliveira)

Gracilaria domingensis (Kützing) Sonder ex Dickie and Gracilaria birdiae (Plastino & Oliveira) (Gracilariales, Rhodophyta) are seaweeds that occur on the Brazilian coast. Based on their economic and pharmaceutical importance, we investigated the antioxidant activity of the methanolic, ethyl acetate and hexane extracts of both species. The hexane extracts display a high antioxidant activity and comparative analyses indicated G. birdiae as the most active species. Chemical investigation of these fractions showed several carotenoids and fatty acids, as well as cholesterol and sitosterol derivatives. HPLC-DAD analysis of G. birdiae showed violaxanthin (0.04 μg.mg-1 of dry material), antheraxanthin (5.31 μg.mg-1), aloxanthin (0.09 μg.mg-1), zeaxanthin (0.45 μg.mg-1) and β-carotene (0.37 μg.mg-1) as the major carotenoids. G. domingensis showed a similar carotenoid profile, however, with much lower concentration than G. birdiae. Gas chromatography coupled to mass spectrometry was used to determine other nonpolar compounds of these seaweeds. The main compounds detected in both studied species were the fatty acids 16:0; 18:1 Δ9; 20:3 Δ6,9,12, 20:4 Δ5,8,11,14. We found no specificity of compounds in either species. However, G. birdiae, presented higher contents of carotenoids and arachidonic acid than G. domingensis.

Accuracy, precision and robustness of different methods to obtain samples from silages in fermentation studies

The objective of this study was to evaluate accuracy, precision and robustness of two methods to obtain silage samples, in comparison with extraction of liquor by manual screw-press. Wet brewery residue alone or combined with soybean hulls and citrus pulp were ensiled in laboratory silos. Liquor was extracted by a manual screw-press and a 2-mL aliquot was fixed with 0.4 mL formic acid. Two 10-g silage samples from each silo were diluted in 20 mL deionized water or 17% formic acid solution (alternative methods). Aliquots obtained by the three methods were used to determine the silage contents of fermentation end-products. The accuracy of the alternative methods was evaluated by comparing mean bias of estimates obtained by manual screw-press and by alternative methods, whereas precision was assessed by the root mean square prediction error and the residual error. Robustness was determined by studying the interaction between bias and chemical components, pH, in vitro dry matter digestibility (IVDMD) and buffer capacity. The 17% formic acid method was more accurate for estimating acetic, butyric and lactic acids, although it resulted in low overestimates of propionic acid and underestimates of ethanol. The deionized water method overestimated acetic and propionic acids and slightly underestimated ethanol. The 17% formic acid method was more precise than deionized water for estimating all organic acids and ethanol. The robustness of each method with respect to variation in the silage chemical composition, IVDMD and pH is dependent on the fermentation end-product at evaluation. The robustness of the alternative methods seems to be critical at the determination of lactic acid and ethanol contents.

Effects of microbial inoculants and amino acid production by-product on fermentation and chemical composition of sugarcane silages

The objective of this study was to evaluate the chemical composition, fermentation patterns and aerobic stability of sugarcane silages with addition of amino acid production (monosodium glutamate) by-product (APB) and microbial inoculants. Mature sugarcane was chopped and ensiled in laboratory silos (n = 4/treatment) without additives (control) and with APB (10 g/kg), Pioneer 1174® (PIO, 1.0 mg/kg, Lactobacillus plantarum + Streptoccoccus faecium, Pioneer), Lalsil Cana (2.0 mg/kg, Lactobacillus buchineri, Lallemand) or Mercosil Maís 11C33® (1.0 mg/kg, Lactobacillus buchineri + Lactobacillus plantarum + Streptoccoccus faecium, Timac Agro). Fresh silage and silage liquor samples were obtained to assess pH, chemical composition and organic acid concentrations. Silage temperature was recorded throughout seven days to evaluate aerobic stability. The addition of APB decreased lactic acid levels, increased pH and N-NH3 and did not alter ethanol, acetic and butyric acids concentrations or dry matter (DM) losses. Microbial inoculants enhanced acetic acid levels, although only Pioneer 1174® and Mercosil Maís 11C33® lowered ethanol, butyric acid and DM losses. The addition of APB increased CP content and did not modify DM, soluble carbohydrates contents or in vitro dry matter digestibility. Additives did not alter silage maximum temperature or temperature increasing rate; however, Pioneer 1174® and Mercosil Maís 11C33® increased the time elapsed to reach maximum temperature. Monosodium glutamate production by-product does not alter fermentation patterns or aerobic stability of sugarcane silages, whereas homofermentative bacteria can provide silages of good quality.

Guava pomace: a new source of anti-inflammatory and analgesic bioactives

Abstract Background Guava pomace is an example of the processing waste generated after the manufacturing process from the juice industry that could be a source of bioactives. Thus, the present investigation was carried out in order to evaluate the anti-inflammatory and antinociceptive potential and determinate the main phenolic compounds of a guava pomace extract (GPE). Methods The anti-inflammatory activity was evaluated by carrageenan, dextran, serotonin, histamine-induced paw edema and neutrophils migration in the peritoneal cavity models. Acetic acid-induced abdominal writhing and formalin test were performed to investigate the antinociceptive effects. In addition, the content of total phenolic and of individual phenolic compounds was determined by GC/MS. Results GPE showed anti-inflammatory activity by carrageenan, dextran, serotonin, histamine-induced paw edema and neutrophils migration in the peritoneal cavity models (p < 0.05). GPE also demonstrated antinociceptive activity by acetic acid-induced abdominal writhing and formalin test (p < 0.05). The total phenolic value was 3.40 ± 0.09 mg GAE/g and epicatechin, quercetin, myricetin, isovanilic and gallic acids were identified by GC/MS analysis. Conclusions The presence of bioactive phenolic compounds as well as important effects demonstrated in animal models suggest that guava pomace could be an interesting source of anti-inflammatory and analgesic substances.

Food safety in wine: optimization of analytical controls and evaluation of production technologies

This PhD thesis has been proposed to validate and then apply innovative analytical methodologies for the determination of compounds with harmful impact on human health, such as biogenic amines and ochratoxin A in wines. Therefore, the influence of production technology (pH, amino acids precursor and use of different malolactic starters) on biogenic amines content in wines was evaluated. An HPLC method for simultaneous determination of amino acids and amines with precolumnderivatization with 9-Fluorenyl-methoxycarbonyl chloride (FMOC-Cl) and UV detection was developed. Initially, the influence of pH, time of derivatization, gradient profile were studied. In order to improve the separation of amino acids and amines and reduce the time of analysis, it was decided to study the influence of different flows and the use of different columns in the chromatographic method. Firstly, a C18 Luna column was used and later two monolithic columns Chromolith in series. It appeared to be suitable for an easy, precise and accurate determination of a relatively large number of amino acids and amines in wines. This method was then applied on different wines produced in the Emilia Romagna region. The investigation permitted to discriminate between red and white wines. Amino acids content is related to the winemaking process. Biogenic amines content in these wines does not represent a possible toxicological problem for human health. The results of the study of influence of technologies and wine composition demonstrated that pH of wines and amino acids content are the most important factors. Particularly wines with pH > 3,5 show higher concentration of biogenic amines than wines with lower pH. The enrichment of wines by nutrients also influences the content of some biogenic amines that are higher in wines added with amino acids precursors. In this study, amino acids and biogenic amines are not statistically affected by strain of lactic acid bacteria inoculated as a starter for malolactic fermentation. An evaluation of different clean-up (SPE-MycoSep; IACs and LLE) and determination methods (HPLC and ELISA) of ochratoxin A was carried out. The results obtained proved that the SPE clean-up are reliable at the same level while the LLE procedures shows lowest recovery. The ELISA method gave a lower determination and a low reproducibility than HPLC method.

Determination of bioactive and potentially toxic compounds in food: raw material analysis and shelf life evaluation

"Bioactive compounds" are extranutritional constituents that typically occur in small quantities in food. They are being intensively studied to evaluate their effects on health. Bioactive compounds include both water soluble compounds, such as phenolics, and lipidic substances such as n-3 fatty acids, tocopherols and sterols. Phenolic compounds, tocopherols and sterols are present in all plants and have been studied extensively in cereals, nuts and oil. n-3 fatty acids are present in fish and all around the vegetable kingdom. The aim of the present work was the determination of bioactive and potentially toxic compounds in cereal based foods and nuts. The first section of this study was focused on the determination of bioactive compounds in cereals. Because of that the different forms of phytosterols were investigated in hexaploid and tetraploid wheats. Hexaploid cultivars were the best source of esterified sterols (40.7% and 37.3% of total sterols for Triticum aestivum and Triticum spelta, respectively). Significant amounts of free sterols (65.5% and 60.7% of total sterols for Triticum durum and Triticum dicoccon, respectively) were found in the tetraploid cultivars. Then, free and bound phenolic compounds were identified in barley flours. HPLCESI/ MSD analysis in negative and positive ion mode established that barley free flavan-3- ols and proanthocyanidins were four dimers and four trimers having (epi)catechin and/or (epi)gallocatechin (C and/or GC) subunits. Hydroxycinnamic acids and their derivatives were the main bound phenols in barley flours. The results obtained demonstrated that barley flours were rich in phenolic compounds that showed high antioxidant activity. The study also examined the relationships between phenolic compounds and lipid oxidation of bakery. To this purpose, the investigated barley flours were used in the bakery production. The formulated oven products presented an interesting content of phenolic compounds, but they were not able to contain the lipid oxidation. Furthermore, the influence of conventional packaging on lipid oxidation of pasta was evaluated in n-3 enriched spaghetti and egg spaghetti. The results proved that conventional packaging was not appropriated to preserve pasta from lipid oxidation; in fact, pasta that was exposed to light showed a high content of potentially toxic compounds derived from lipid oxidation (such as peroxide, oxidized fatty acids and COPs). In the second section, the content of sterols, phenolic compounds, n-3 fatty acids and tocopherols in walnuts were reported. Rapid analytical techniques were used to analyze the lipid fraction and to characterize phenolic compounds in walnuts. Total lipid chromatogram was used for the simultaneous determination of the profile of sterols and tocopherols. Linoleic and linolenic acids were the most representative n-6 and n-3 essential dietary fatty acids present in these nuts. Walnuts contained substantial amounts of γ- and δ-tocopherol, which explained their antioxidant properties. Sitosterol, Δ5-avenasterol and campesterol were the major free sterols found. Capillary electrophoresis coupled to DAD and microTOF was utilized to determine phenolic content of walnut. A new compound in walnut ((2E,4E)- 8-hydroxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-β-D-glucopiranosyl ester, [M−H]− 403.161m/z) with a structure similar to glansreginins was also identified. Phenolic compounds corresponded to 14–28% of total polar compounds quantified. Aglycone and glycosylated ellagic acid represented the principal components and account for 64–75% of total phenols in walnuts. However, the sum of glansreginins A, B and ((2E,4E)-8-hydroxy- 2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-β-D-glucopiranosyl ester was in the range of 72–86% of total quantified compounds.

Evaluation of fluid transport processes in dental enamel. Methods to assess the relevance of enamel permeability in caries prevention and etching treatments.

This thesis evaluated in vivo and in vitro enamel permeability in different physiological and clinical conditions by means of SEM inspection of replicas of enamel surface obtained from polyvinyl siloxane impressions subsequently later cast in polyether impression ma-terial. This technique, not invasive and risk-free, allows the evaluation of fluid outflow from enamel surface and is able to detect the presence of small quantities of fluid, visu-alized as droplets. Fluid outflow on enamel surface represents enamel permeability. This property has a paramount importance in enamel physiolgy and pathology although its ef-fective role in adhesion, caries pathogenesis and prevention today is still not fully under-stood. The aim of the studies proposed was to evaluate enamel permeability changes in differ-ent conditions and to correlate the findings with the actual knowledge about enamel physiology, caries pathogenesis, fluoride and etchinhg treatments. To obtain confirmed data the replica technique has been supported by others specific techniques such as Ra-man and IR spectroscopy and EDX analysis. The first study carried out visualized fluid movement through dental enamel in vivo con-firmed that enamel is a permeable substrate and demonstrated that age and enamel per-meability are closely related. Examined samples from subjects of different ages showed a decreasing number and size of droplets with increasing age: freshly erupted permanent teeth showed many droplets covering the entire enamel surface. Droplets in permanent teeth were prominent along enamel perikymata. These results obtained through SEM inspection of replicas allowed innovative remarks in enamel physiology. An analogous testing has been developed for evaluation of enamel permeability in primary enamel. The results of this second study showed that primary enamel revealed a substantive permeability with droplets covering the entire enamel sur-face without any specific localization accordingly with histological features, without changes during aging signs of post-eruptive maturation. These results confirmed clinical data that showed a higher caries susceptibility for primary enamel and suggested a strong relationship between this one and enamel permeability. Topical fluoride application represents the gold standard for caries prevention although the mechanism of cariostatic effect of fluoride still needs to be clarified. The effects of topical fluoride application on enamel permeability were evaluated. Particularly two dif-ferent treatments (NaF and APF), with different pH, were examined. The major product of topical fluoride application was the deposition of CaF2-like globules. Replicas inspec-tion before and after both treatments at different times intervals and after specific addi-tional clinical interventions showed that such globule formed in vivo could be removed by professional toothbrushing, sonically and chemically by KOH. The results obtained in relation to enamel permeability showed that fluoride treatments temporarily reduced enamel water permeability when CaF2-like globules were removed. The in vivo perma-nence of decreased enamel permeability after CaF2 globules removal has been demon-strated for 1 h for NaF treated teeth and for at least 7 days for APF treated teeth. Important clinical consideration moved from these results. In fact the caries-preventing action of fluoride application may be due, in part, to its ability to decrease enamel water permeability and CaF2 like-globules seem to be indirectly involved in enamel protection over time maintaining low permeability. Others results obtained by metallographic microscope and SEM/EDX analyses of or-thodontic resins fluoride releasing and not demonstrated the relevance of topical fluo-ride application in decreasing the demineralization marks and modifying the chemical composition of the enamel in the treated area. These data obtained in both the experiments confirmed the efficacy of fluoride in caries prevention and contribute to clarify its mechanism of action. Adhesive dentistry is the gold standard for caries treatment and tooth rehabilitation and is founded on important chemical and physical principles involving both enamel and dentine substrates. Particularly acid etching of dental enamel enamel has usually employed in bonding pro-cedures increasing microscopic roughness. Different acids have been tested in the litera-ture suggesting several etching procedures. The acid-induced structural transformations in enamel after different etching treatments by means of Raman and IR spectroscopy analysis were evaluated and these findings were correlated with enamel permeability. Conventional etching with 37% phosphoric acid gel (H3PO4) for 30 s and etching with 15 % HCl for 120 s were investigated. Raman and IR spectroscopy showed that the treatment with both hydrochloric and phosphoric acids induced a decrease in the carbonate content of the enamel apatite. At the same time, both acids induced the formation of HPO42- ions. After H3PO4 treatment the bands due to the organic component of enamel decreased in intensity, while in-creased after HCl treatment. Replicas of H3PO4 treated enamel showed a strongly reduced permeability while replicas of HCl 15% treated samples showed a maintained permeability. A decrease of the enamel organic component, as resulted after H3PO4 treatment, involves a decrease in enamel permeability, while the increase of the organic matter (achieved by HCl treat-ment) still maintains enamel permeability. These results suggested a correlation between the amount of the organic matter, enamel permeability and caries. The results of the different studies carried out in this thesis contributed to clarify and improve the knowledge about enamel properties with important rebounds in theoretical and clinical aspects of Dentistry.

Archaeological Baltic amber: degradation mechanisms and conservation measures

The aim of this project was to achieve a deep understanding of the mechanisms by which Baltic amber degrades, in order to develop techniques for preventive conservation of archaeological amber objects belonging to the National Museum of Denmark’s collections. To examine deterioration of Baltic amber, a starting point was to identify and monitor surface and bulk properties which are affected during degradation. The way to operate consisted of the use of accelerated ageing to initiate degradation of raw Baltic amber samples in different conditions of relative humidity, oxygen exposure or pH and, successively, of the use of non/micro-destructive techniques to identify and quantify changes in visual, chemical and structural properties. A large piece of raw Baltic amber was used to prepare several test samples for two different kinds of accelerated ageing: thermal-ageing and photo-ageing. During the ageing, amber samples were regularly examined through several analytical techniques related to different information: appearance/colour change by visual examination, photography and colorimetry; chemical change by infrared spectroscopy, Raman spectroscopy and elemental analysis; rate of oxidation by oxygen measurement; qualitative analysis of released volatiles by gas chromatography – mass spectrometry. The obtained results were analysed through both critical evaluation and statistical study. After the interpretation of the achieved data, the main relations between amber and environmental factors during the degradation process became clearer and it was possible to identify the major pathways by which amber degrades, such as hydrolysis of esters into alcohols and carboxylic acids, thermal-oxidation and photo-oxidation of terpenoid components, depolymerisation and decomposition of the chemical structure. At the end it was possible to suggest a preventive conservation strategy based on the control of climatic, atmospheric and lighting parameters in the environment where Baltic amber objects are stored and displayed.

Bioactive and toxic compounds in different foodstuffs: analytical techniques and sensory analysis

Foods that provide medical and health benefits or have a role in disease risk prevention are termed functional foods. The functionality of functional foods is derived from bioactive compounds that are extranutritional constituents present in small quantities in food. Bioactive components include a range of chemical compounds with varying structures such as carotenoids, flavonoids, plant sterols, omega-3 fatty acids (n-3), allyl and diallyl sulfides, indoles (benzopyrroles), and phenolic acids. The increasing consumer interest in natural bioactive compounds has brought about a rise in demand for these kinds of compounds and, in parallel, an increasing number of scientific studies have this type of substance as main topic. The principal aim of this PhD research project was the study of different bioactive and toxic compounds in several natural matrices. To achieve this goal, chromatographic, spectroscopic and sensorial analysis were performed. This manuscript reports the main results obtained in the six activities briefly summarized as follows: • SECTION I: the influence of conventional packaging on lipid oxidation of pasta was evaluated in egg spaghetti. • SECTION II: the effect of the storage at different temperatures of virgin olive oil was monitored by peroxide value, fatty acid activity, OSI test and sensory analysis. • SECTION III: the glucosinolate and phenolic content of 37 rocket salad accessions were evaluated, comparing Eruca sativa and Diplotaxis tenuifolia species. Sensory analysis and the influence of the phenolic and glucosinolate composition on sensory attributes of rocket salads has been also studied. • SECTION IV: ten buckwheat honeys were characterised on the basis of their pollen, physicochemical, phenolic and volatile composition. • SECTION V: the polyphenolic fraction, anthocyanins and other polar compounds, the antioxidant capacity and the anty-hyperlipemic action of the aqueous extract of Hibiscus sabdariffa were achieved. • SECTION VI: the optimization of a normal phase high pressure liquid chromatography–fluorescence detection method for the quantitation of flavanols and procyanidins in cocoa powder and chocolate samples was performed.

Adipose-derived stem cells and tissue revascularization: enhancing islet survival and performance for diabetes care.

Pancreatic islet transplantation represents a fascinating procedure that, at the moment, can be considered as alternative to standard insulin treatment or pancreas transplantation only for selected categories of patients with type 1 diabetes mellitus. Among the factors responsible for leading to poor islet engraftment, hypoxia plays an important role. Mesenchymal stem cells (MSCs) were recently used in animal models of islet transplantation not only to reduce allograft rejection, but also to promote revascularization. Currently adipose tissue represents a novel and good source of MSCs. Moreover, the capability of adipose-derived stem cells (ASCs) to improve islet graft revascularization was recently reported after hybrid transplantation in mice. Within this context, we have previously shown that hyaluronan esters of butyric and retinoic acids can significantly enhance the rescuing potential of human MSCs. Here we evaluated whether ex vivo preconditioning of human ASCs (hASCs) with a mixture of hyaluronic (HA), butyric (BU), and retinoic (RA) acids may result in optimization of graft revascularization after islet/stem cell intrahepatic cotransplantation in syngeneic diabetic rats. We demonstrated that hASCs exposed to the mixture of molecules are able to increase the secretion of vascular endothelial growth factor (VEGF), as well as the transcription of angiogenic genes, including VEGF, KDR (kinase insert domain receptor), and hepatocyte growth factor (HGF). Rats transplanted with islets cocultured with preconditioned hASCs exhibited a better glycemic control than rats transplanted with an equal volume of islets and control hASCs. Cotransplantation with preconditioned hASCs was also associated with enhanced islet revascularization in vivo, as highlighted by graft morphological analysis. The observed increase in islet graft revascularization and function suggests that our method of stem cell preconditioning may represent a novel strategy to remarkably improve the efficacy of islets-hMSCs cotransplantation.

Advances in methods to detect, isolate and quantify foodborne pathogens

Foodborne diseases impact human health and economies worldwide in terms of health care and productivity loss. Prevention is necessary and methods to detect, isolate and quantify foodborne pathogens play a fundamental role, changing continuously to face microorganisms and food production evolution. Official methods are mainly based on microorganisms growth in different media and their isolation on selective agars followed by confirmation of presumptive colonies through biochemical and serological test. A complete identification requires form 7 to 10 days. Over the last decades, new molecular techniques based on antibodies and nucleic acids allow a more accurate typing and a faster detection and quantification. The present thesis aims to apply molecular techniques to improve official methods performances regarding two pathogens: Shiga-like Toxin-producing Escherichia coli (STEC) and Listeria monocytogenes. In 2011, a new strain of STEC belonging to the serogroup O104 provoked a large outbreak. Therefore, the development of a method to detect and isolate STEC O104 is demanded. The first objective of this work is the detection, isolation and identification of STEC O104 in sprouts artificially contaminated. Multiplex PCR assays and antibodies anti-O104 incorporated in reagents for immunomagnetic separation and latex agglutination were employed. Contamination levels of less than 1 CFU/g were detected. Multiplex PCR assays permitted a rapid screening of enriched food samples and identification of isolated colonies. Immunomagnetic separation and latex agglutination allowed a high sensitivity and rapid identification of O104 antigen, respectively. The development of a rapid method to detect and quantify Listeria monocytogenes, a high-risk pathogen, is the second objective. Detection of 1 CFU/ml and quantification of 10–1,000 CFU/ml in raw milk were achieved by a sample pretreatment step and quantitative PCR in about 3h. L. monocytogenes growth in raw milk was also evaluated.

Nanoscale surface properties and interaction with fundamental biomolecules of chlorite and phlogopite

The surface properties of minerals have important implications in geology, environment, industry and biotechnology and for certain aspects in the research on the origin of life. This research project aims to widen the knowledge on the nanoscale surface properties of chlorite and phlogopite by means of advanced methodologies, and also to investigate the interaction of fundamental biomolecules, such as nucleotides, RNA, DNA and amino acid glycine with the surface of the selected phyllosilicates. Multiple advanced and complex experimental approaches based on scanning probe microscopy and spatially resolved spectroscopy were used and in some cases specifically developed. The results demonstrate that chlorite exposes at the surface atomically flat terraces with 0.5 nm steps typically generated by the fragmentation of the octahedral sheet of the interlayer (brucitic-type). This fragmentation at the nanoscale generates a high anisotropy and inhomogeneity with surface type and isomorphous cationic substitutions determining variations of the effective surface potential difference, ranging between 50-100 mV and 400-500 mV, when measured in air, between the TOT surface and the interlayer brucitic sheet. The surface potential was ascribed to be the driving force of the observed high affinity of the surface with the fundamental biomolecules, like single molecules of nucleotides, DNA, RNA and amino acids. Phlogopite was also observed to present an extended atomically flat surface, featuring negative surface potential values of some hundreds of millivolts and no significant local variations. Phlogopite surface was sometimes observed to present curvature features that may be ascribed to local substitutions of the interlayer cations or the presence of a crystal lattice mismatch or structural defects, such as stacking faults or dislocation loops. Surface chemistry was found similar to the bulk. The study of the interaction with nucleotides and glycine revealed a lower affinity with respect to the brucite-like surface of chlorite.

Exchange of nitrogen dioxide (NO 2) between plants and the atmosphere under laboratory and field conditions

Nitrogen is an essential nutrient. It is for human, animal and plants a constituent element of proteins and nucleic acids. Although the majority of the Earth’s atmosphere consists of elemental nitrogen (N2, 78 %) only a few microorganisms can use it directly. To be useful for higher plants and animals elemental nitrogen must be converted to a reactive oxidized form. This conversion happens within the nitrogen cycle by free-living microorganisms, symbiotic living Rhizobium bacteria or by lightning. Humans are able to synthesize reactive nitrogen through the Haber-Bosch process since the beginning of the 20th century. As a result food security of the world population could be improved noticeably. On the other side the increased nitrogen input results in acidification and eutrophication of ecosystems and in loss of biodiversity. Negative health effects arose for humans such as fine particulate matter and summer smog. Furthermore, reactive nitrogen plays a decisive role at atmospheric chemistry and global cycles of pollutants and nutritive substances.rnNitrogen monoxide (NO) and nitrogen dioxide (NO2) belong to the reactive trace gases and are grouped under the generic term NOx. They are important components of atmospheric oxidative processes and influence the lifetime of various less reactive greenhouse gases. NO and NO2 are generated amongst others at combustion process by oxidation of atmospheric nitrogen as well as by biological processes within soil. In atmosphere NO is converted very quickly into NO2. NO2 is than oxidized to nitrate (NO3-) and to nitric acid (HNO3), which bounds to aerosol particles. The bounded nitrate is finally washed out from atmosphere by dry and wet deposition. Catalytic reactions of NOx are an important part of atmospheric chemistry forming or decomposing tropospheric ozone (O3). In atmosphere NO, NO2 and O3 are in photosta¬tionary equilibrium, therefore it is referred as NO-NO2-O3 triad. At regions with elevated NO concentrations reactions with air pollutions can form NO2, altering equilibrium of ozone formation.rnThe essential nutrient nitrogen is taken up by plants mainly by dissolved NO3- entering the roots. Atmospheric nitrogen is oxidized to NO3- within soil via bacteria by nitrogen fixation or ammonium formation and nitrification. Additionally atmospheric NO2 uptake occurs directly by stomata. Inside the apoplast NO2 is disproportionated to nitrate and nitrite (NO2-), which can enter the plant metabolic processes. The enzymes nitrate and nitrite reductase convert nitrate and nitrite to ammonium (NH4+). NO2 gas exchange is controlled by pressure gradients inside the leaves, the stomatal aperture and leaf resistances. Plant stomatal regulation is affected by climate factors like light intensity, temperature and water vapor pressure deficit. rnThis thesis wants to contribute to the comprehension of the effects of vegetation in the atmospheric NO2 cycle and to discuss the NO2 compensation point concentration (mcomp,NO2). Therefore, NO2 exchange between the atmosphere and spruce (Picea abies) on leaf level was detected by a dynamic plant chamber system under labo¬ratory and field conditions. Measurements took place during the EGER project (June-July 2008). Additionally NO2 data collected during the ECHO project (July 2003) on oak (Quercus robur) were analyzed. The used measuring system allowed simultaneously determina¬tion of NO, NO2, O3, CO2 and H2O exchange rates. Calculations of NO, NO2 and O3 fluxes based on generally small differences (∆mi) measured between inlet and outlet of the chamber. Consequently a high accuracy and specificity of the analyzer is necessary. To achieve these requirements a highly specific NO/NO2 analyzer was used and the whole measurement system was optimized to an enduring measurement precision.rnData analysis resulted in a significant mcomp,NO2 only if statistical significance of ∆mi was detected. Consequently, significance of ∆mi was used as a data quality criterion. Photo-chemical reactions of the NO-NO2-O3 triad in the dynamic plant chamber’s volume must be considered for the determination of NO, NO2, O3 exchange rates, other¬wise deposition velocity (vdep,NO2) and mcomp,NO2 will be overestimated. No significant mcomp,NO2 for spruce could be determined under laboratory conditions, but under field conditions mcomp,NO2 could be identified between 0.17 and 0.65 ppb and vdep,NO2 between 0.07 and 0.42 mm s-1. Analyzing field data of oak, no NO2 compensation point concentration could be determined, vdep,NO2 ranged between 0.6 and 2.71 mm s-1. There is increasing indication that forests are mainly a sink for NO2 and potential NO2 emissions are low. Only when assuming high NO soil emissions, more NO2 can be formed by reaction with O3 than plants are able to take up. Under these circumstance forests can be a source for NO2.