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.

Aziridinazione stereoselettiva di chetoni α,β-insaturi α-sostituiti via organocatalisi

Il lavoro della presente Tesi è stato lo sviluppo della sintesi asimmetrica di aziridine chirali a partire da chetoni α,β-insaturi α-sostituiti, verificando la possibilità di applicare ammine primarie come organocatalizzatori attraverso un meccanismo tandem ione imminio-enammina. Nelle nostre prove le migliori ammine primarie si sono rivelate gli pseudoenantiomeri 9-ammino-9-deossi-epi-idrochinina e idrochinidina, e i migliori acidi per formare il sale catalitico sono stati acido trifluoroacetico (TFA) e acido salicilico. Il fattore chiave per le reazioni di aziridinazione è stata la scelta della molecola sorgente di azoto, che deve avere comportamento nucleofilico nel primo step di aza-Michael (via ione imminio), e comportamento elettrofilico nello step di chiusura del ciclo (via enammina). Le prove preliminari sono state condotte con il sale catalitico formato dalla 9-ammino-9-deossi-epi-idrochinina e TFA in toluene a 50 °C. Migliori risultati sono stati ottenuti sostituendo la sorgente di azoto utilizzata inizialmente e utilizzando il sale catalitico composto da 9-ammino-9-deossi-epi-idrochinidina e acido salicilico in toluene a 50 °C. In questo caso la resa è stata pari a 56% ed eccesso enantiomerico (ee) del 90%. Sfruttando quindi le condizioni ottimizzate inizialmente, abbiamo provato la reazione su altri due chetoni con maggiore ingombro sterico rispetto a quello utilizzato per l’ottimizzazione iniziale del processo. In entrambi i casi la reattività è stata sensibilmente inferiore a quanto atteso, con rese non superiori al 14%. Inoltre anche i valori di ee sono stati poco soddisfacenti. Ipotizziamo che questi risultati deludenti siano causati dall’ingombro sterico della catena in posizione β che impedisce l’avvicinamento del catalizzatore, il quale, non creando un intorno asimmetrico, non crea una distinzione tra le due possibili direzioni di attacco del nucleofilo. Da questi ultimi risultati sembra che la reazione di aziridinazione da noi ottimizzata sia per ora limitata al solo chetone utilizzato nella fase iniziale del lavoro. Al fine di estendere l’applicazione di queste condizioni, nel futuro saranno effettuate prove anche con altri chetoni α,β-insaturi α-sostituiti, ma che non presentino sostituzione in posizione β, dato che abbiamo osservato che essa rappresenta il maggiore limite per la reattività e selettività. Infine sarà importante determinare la configurazione assoluta del prodotto finora ottenuto, mediante spettroscopia ECD e VCD. E’ infatti importante conoscere tutte le caratteristiche chimiche e fisiche di prodotto ottenuto, in modo da avere maggiore conoscenza del processo da noi sviluppato, per poterlo migliorare ed estenderne l’applicabilità in futuro.

Reinigung der Vinorin-Synthase aus Zellkulturen von Rauvolfia serpentina

Abstrakt - DeutschThema: Reinigung der Vinorin-Synthase aus Zellkulturen von Rauvolfia serpentina Die Arbeit befaßte sich mit der Reinigung und Charakterisierung des Enzyms Vinorin-Synthase aus Zellkulturen von Rauvolfia serpentina. Dieses Acetyl-Coenzym-A-abhängige Enzym katalysiert im Biosyntheseweg des Alkaloids Ajmalin die Umwandlung von 16-epi-Vellosimin zu Vinorin. Mit Hilfe eines neu entwickelten Enzymaktivitätstests ist eine einfache und schnelle qualitative sowie quantitative Bestimmung der Aktivität der Vinorin-Synthase möglich. Das Molekulargewicht der Vinorin-Synthase wurde durch Größenausschlußchromatographie an Superdex 75 zu 43 kD ermittelt. Das entwickelte Reinigungsschema mit den vier säulenchromatographischen Reinigungsschritten Anionenaustauschchromatographie an SOURCE 30Q, Chromatographie an Hydroxyapatit, Anionenaustauschchromatographie an Mono Q und Chromatofokussierung an Mono P führte zu einer 340fachen Anreicherung der Vinorin-Synthase. Das nach der Reinigung durchgeführten gelelektrophoretischen Untersuchungen ermöglichten keine Zuordnung einer Bande zur Bande der Vinorin-Synthase. Mögliche Ursachen für die Nichtzuordnung einer Bande im SDS-Gel zur Vinorin-Synthase sind in einer möglichen Überlagerung eines Fremdprotein mit der Vinorin-Synthase, eine niedrige Expression der Vinorin-Synthase, die eine deutlich bessere Anreicherung erfordern würde oder der Aufbau des Proteins aus Untereinheiten, in die es während der Behandlung mit SDS zerfällt, gegeben.

Charakterisierung der Vinorin-Synthase aus Rauvolfia serpentina durch Reinigung, Expression, Mutation und Kristallisation

ABSTRACTDie vorliegende Arbeit befasste sich mit der Reinigung,heterologen Expression, Charakterisierung, molekularenAnalyse, Mutation und Kristallisation des EnzymsVinorin-Synthase. Das Enzym spielt eine wichtige Rolle inder Ajmalin-Biosynthese, da es in einerAcetyl-CoA-abhängigen Reaktion die Umwandlung desSarpagan-Alkaloids 16-epi-Vellosimin zu Vinorin unterBildung des Ajmalan-Grundgerüstes katalysiert. Nach der Reinigung der Vinorin-Synthase ausHybrid-Zellkulturen von Rauvolfia serpentina/Rhazya strictamit den fünf chromatographischen TrennmethodenAnionenaustauschchromatographie an SOURCE 30Q, HydrophobeInteraktionen Chromatographie an SOURCE 15PHE,Chromatographie an MacroPrep Ceramic Hydroxyapatit,Anionenaustauschchromatographie an Mono Q undGrößenausschlußchromatographie an Superdex 75 konnte dieVinorin-Synthase aus 2 kg Zellkulturgewebe 991fachangereichert werden.Das nach der Reinigung angefertigte SDS-Gel ermöglichte eineklare Zuordnung der Protein-Bande als Vinorin-Synthase.Der Verdau der Enzymbande mit der Endoproteinase LysC unddie darauffolgende Sequenzierung der Spaltpeptide führte zuvier Peptidsequenzen. Der Datenbankvergleich (SwissProt)zeigte keinerlei Homologien zu Sequenzen bekannterPflanzenenzyme. Mit degenerierten Primern, abgeleitet voneinem der erhaltenen Peptidfragmente und einer konserviertenRegion bekannter Acetyltransferasen gelang es, ein erstescDNA-Fragment der Vinorin-Synthase zu amplifizieren. Mit derMethode der RACE-PCR wurde die Nukleoidsequenzvervollständigt, was zu einem cDNA-Vollängenklon mit einerGröße von 1263 bp führte, der für ein Protein mit 421Aminosäuren (46 kDa) codiert.Das Vinorin-Synthase-Gen wurde in den pQE2-Expressionsvektorligiert, der für einen N-terminalen 6-fachen His-tagcodiert. Anschließend wurde sie erstmals erfolgreich in E.coli im mg-Maßstab exprimiert und bis zur Homogenitätgereinigt. Durch die erfolgreiche Überexpression konnte dieVinorin-Synthase eingehend charakterisiert werden. DerKM-Wert für das Substrat Gardneral wurde mit 20 µM, bzw.41.2 µM bestimmt und Vmax betrug 1 pkat, bzw. 1.71 pkat.Nach erfolgreicher Abspaltung des His-tags wurden diekinetischen Parameter erneut bestimmt (KM- Wert 7.5 µM, bzw.27.52 µM, Vmax 0.7 pkat, bzw. 1.21 pkat). Das Co-Substratzeigt einen KM- Wert von 60.5 µM (Vmax 0.6 pkat). DieVinorin-Synthase besitzt ein Temperatur-Optimum von 35 °Cund ein pH-Optimum bei 7.8.Homologievergleiche mit anderen Enzymen zeigten, dass dieVinorin-Synthase zu einer noch kleinen Familie von bisher 10Acetyltransferasen gehört. Alle Enzyme der Familie haben einHxxxD und ein DFGWG-Motiv zu 100 % konserviert. Basierendauf diesen Homologievergleichen und Inhibitorstudien wurden11 in dieser Proteinfamilie konservierte Aminosäuren gegenAlanin ausgetauscht, um so die Aminosäuren einer in derLiteratur postulierten katalytischen Triade(Ser/Cys-His-Asp) zu identifizieren.Die Mutation aller vorhandenen konservierten Serine undCysteine resultierte in keiner Mutante, die zumvollständigen Aktivitätsverlust des Enzyms führte. Nur dieMutationen H160A und D164A resultierten in einemvollständigen Aktivitätsverlust des Enzyms. Dieses Ergebniswiderlegt die Theorie einer katalytischen Triade und zeigte,dass die Aminosäuren H160A und D164A exklusiv an derkatalytischen Reaktion beteiligt sind.Zur Überprüfung dieser Ergebnisse und zur vollständigenAufklärung des Reaktionsmechanismus wurde dieVinorin-Synthase kristallisiert. Die bis jetzt erhaltenenKristalle (Kristallgröße in µm x: 150, y: 200, z: 200)gehören der Raumgruppe P212121 (orthorhombisch primitiv) anund beugen bis 3.3 Å. Da es bis jetzt keine Kristallstruktureines zur Vinorin-Synthase homologen Proteins gibt, konntedie Struktur noch nicht vollständig aufgeklärt werden. ZurLösung des Phasenproblems wird mit der Methode der multiplenanomalen Dispersion (MAD) jetzt versucht, die ersteKristallstruktur in dieser Enzymfamilie aufzuklären.

Confocal microscopy applied to the study of single entity fluorescence and light scattering

A sample scanning confocal optical microscope (SCOM) was designed and constructed in order to perform local measurements of fluorescence, light scattering and Raman scattering. This instrument allows to measure time resolved fluorescence, Raman scattering and light scattering from the same diffraction limited spot. Fluorescence from single molecules and light scattering from metallic nanoparticles can be studied. First, the electric field distribution in the focus of the SCOM was modelled. This enables the design of illumination modes for different purposes, such as the determination of the three-dimensional orientation of single chromophores. Second, a method for the calculation of the de-excitation rates of a chromophore was presented. This permits to compare different detection schemes and experimental geometries in order to optimize the collection of fluorescence photons. Both methods were combined to calculate the SCOM fluorescence signal of a chromophore in a general layered system. The fluorescence excitation and emission of single molecules through a thin gold film was investigated experimentally and modelled. It was demonstrated that, due to the mediation of surface plasmons, single molecule fluorescence near a thin gold film can be excited and detected with an epi-illumination scheme through the film. Single molecule fluorescence as close as 15nm to the gold film was studied in this manner. The fluorescence dynamics (fluorescence blinking and excited state lifetime) of single molecules was studied in the presence and in the absence of a nearby gold film in order to investigate the influence of the metal on the electronic transition rates. The trace-histogram and the autocorrelation methods for the analysis of single molecule fluorescence blinking were presented and compared via the analysis of Monte-Carlo simulated data. The nearby gold influences the total decay rate in agreement to theory. The gold presence produced no influence on the ISC rate from the excited state to the triplet but increased by a factor of 2 the transition rate from the triplet to the singlet ground state. The photoluminescence blinking of Zn0.42Cd0.58Se QDs on glass and ITO substrates was investigated experimentally as a function of the excitation power (P) and modelled via Monte-Carlo simulations. At low P, it was observed that the probability of a certain on- or off-time follows a negative power-law with exponent near to 1.6. As P increased, the on-time fraction reduced on both substrates whereas the off-times did not change. A weak residual memory effect between consecutive on-times and consecutive off-times was observed but not between an on-time and the adjacent off-time. All of this suggests the presence of two independent mechanisms governing the lifetimes of the on- and off-states. The simulated data showed Poisson-distributed off- and on-intensities, demonstrating that the observed non-Poissonian on-intensity distribution of the QDs is not a product of the underlying power-law probability and that the blinking of QDs occurs between a non-emitting off-state and a distribution of emitting on-states with different intensities. All the experimentally observed photo-induced effects could be accounted for by introducing a characteristic lifetime tPI of the on-state in the simulations. The QDs on glass presented a tPI proportional to P-1 suggesting the presence of a one-photon process. Light scattering images and spectra of colloidal and C-shaped gold nano-particles were acquired. The minimum size of a metallic scatterer detectable with the SCOM lies around 20 nm.

Reinigung und Coexpression von Enzymen der Raubasin-Biosynthese

Die vorgestellten Arbeiten bezüglich der Biosynthese von pflanzlichen Indolalkaloiden können in einen molekularbiologischen und einen proteinche-mischen Teil aufgegliedert werden. Im molekularbiologischen Abschnitt stand die Entwicklung eines Vektorsystems im Vordergrund, das die gleichzeitige Expression mehrerer Enzyme in bakteriellen Kulturen erlaubte. Hierfür konnte zunächst die cDNA der Strictosidin-Synthase aus Rauvolfia serpentina in den Expressionsvektor pQE-70 einkloniert und das Protein aktiv exprimiert werden. Bevor es zum Einbau des Strictosidin-β-D-Glucosidase-Gens –ebenfalls aus Rauvolfia serpentina– kam, musste dessen Aktivität mit einer vorgeschalteten Ribosomenbindestelle sichergestellt werden. Diese zusätzliche Binderegion wurde an das 5’-Ende der cDNA angefügt, um die ungestörte Expression des Enzyms im späteren Coexpressions-System zu gewährleisten. Im Hinblick auf weiterführende Arbeiten in Bezug auf die komplette in vitro-Synthese bereits bekannter Alkaloide, wie z.B. das antiarrhythmisch wirkende Ajmalin oder das antihypertensiv wirkende Heteroyohimbin-Alkaloid Raubasin, wurde die ursprüngliche multiple-clonig-site des verwendeten Expressionsvektors pQE-70 um 27 zusätzliche Restriktionsschnittstellen (verteilt auf 253 bp) erweitert. Nach erfolgreicher Ligation der Strictosidin-β-D-Glucosidase-cDNA mit vorgeschalteter Ribosomenbindestelle an das 3’-Ende des Strictosidin-Synthase-Gens gelang die heterologe Coexpression beider Enzyme in einer homogenen Suspensionskultur des E. coli-Expressionsstamms M15. Dafür wurde das Vektorkonstrukt pQE-70bh-STR-RBS-SG entwickelt. Das Endprodukt der anschließenden enzymatischen Umsetzung von Tryptamin und Secologanin wurde über das Zwischenrodukt Strictosidin gebildet und konnte als Cathenamin identifiziert werden. Im proteinchemischen Teil der Dissertation wurde die Reinigung einer Cathenamin-Reduktase aus Zellsuspensionskulturen von Catharanthus roseus RC mit dem Ziel der partiellen Bestimmung der Aminosäuresequenz bearbeitet. Das gesuchte Enzym wandelte in einer NADPH-abhängigen Reaktion das Edukt Cathenamin in Raubasin um. Des weiteren wurde untersucht, wie viele Enzyme insgesamt an der Umwandlung von Cathenamin zu Raubasin und den eng verwandten Produkten Tetrahydroalstonin und 19-Epi-Raubasin beteiligt waren. Unter Anwendung eines hierfür entwickelten säulenchromatographischen Protokolls gelang die Reinigung einer Raubasin-bildenden Reduktase, deren Teilsequenz jedoch noch nicht bestimmt werden konnte. Die Anzahl der beteiligten Enzyme bei der Ausbildung von Raubasin, Tetrahydroalstonin und 19-Epi-Raubasin konnte auf mindestens zwei beziffert werden.

α-ossidazione asimmetrica organocatalizzata di aldeidi ramificate con dibenzoil perossido

In this thesis, the development of an enantioselective oxidation of α-branched aldehydes using covalent organocatalysis is described. At state of the art, the asymmetric organocatalysis approach, gave often serous difficulties for these kind of substrate respect “classic” aldehydes. We have used a primary cinchona alkaloid derived amine (specially the 9-epi-NH2-CDA) to develop the reaction in combinations with additives. With benzoyl peroxide as oxidant and 2-phenylpropionaldehyde as reference substrate, we have tried to optimize this system but we not found great results about enantiomeric excess.

Sintesi di polimeri funzionali per la catalisi asimmetrica

In recent years, the asymmetric organocatalysis has been recognized as an independent area of synthetic chemistry, where the goal is the preparation of any chiral molecule in an efficient, rapid, and stereoselective manner. In this context we have synthesized macromolecular catalysts soluble in the reaction conditions and that can finally recovered by simple precipitation and subsequent filtration. In particular different active compounds (9-epi-NH2 hyidroquinine and β –isocupreidine) have been linked to the terminal group of the main chain polyethylene glycol monomethyl ether (PEG-5000). The macromolecular catalysts have been tested in different reactions and the results have been compared with those of the correspondent low molecular compounds.

Identificazione dei fenotipi clinici dell’insufficienza renale cronica che predicono la progressione della malattia. Un’applicazione della tecnica degli alberi decisionali

The primary aim of this dissertation to identify subgroups of patients with chronic kidney disease (CKD) who have a differential risk of progression of illness and the secondary aim is compare 2 equations to estimate the glomerular filtration rate (GFR). To this purpose, the PIRP (Prevention of Progressive Kidney Disease) registry was linked with the dialysis and mortality registries. The outcome of interest is the mean annual variation of GFR, estimated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. A decision tree model was used to subtype CKD patients, based on the non-parametric procedure CHAID (Chi-squared Automatic Interaction Detector). The independent variables of the model include gender, age, diabetes, hypertension, cardiac diseases, body mass index, baseline serum creatinine, haemoglobin, proteinuria, LDL cholesterol, tryglycerides, serum phoshates, glycemia, parathyroid hormone and uricemia. The decision tree model classified patients into 10 terminal nodes using 6 variables (gender, age, proteinuria, diabetes, serum phosphates and ischemic cardiac disease) that predict a differential progression of kidney disease. Specifically, age <=53 year, male gender, proteinuria, diabetes and serum phosphates >3.70 mg/dl predict a faster decrease of GFR, while ischemic cardiac disease predicts a slower decrease. The comparison between GFR estimates obtained using MDRD4 and CKD-EPI equations shows a high percentage agreement (>90%), with modest discrepancies for high and low age and serum creatinine levels. The study results underscore the need for a tight follow-up schedule in patients with age <53, and of patients aged 54 to 67 with diabetes, to try to slow down the progression of the disease. The result also emphasize the effective management of patients aged>67, in whom the estimated decrease in glomerular filtration rate corresponds with the physiological decrease observed in the absence of kidney disease, except for the subgroup of patients with proteinuria, in whom the GFR decline is more pronounced.

Regio- e stereocontrollo nell'addizione viniloga di alchilidenossindoli a nitrostireni via organocatalisi.

The topic of this work is the simultaneous activation, promoted by 9-epi-NH2-DHQA-TU, of alkylideneoxindole and nistirene derivatives, respectively via base catalysis and hydrogen-bond catalysis. The chosen substrates, of high biological interest, are used as starting materials for a vinylogous Michael addition where we wish to control the stereochemistry of the two asymmetric carbons far away from the active site, respectively in γ and δ position. Due to the particular structure of the starting oxindoles, it is hereby presented the first variant of this reaction performed at its highest level of stereochemical complexity. It is possible as a matter of fact, to generate 24 isomers of the product. Specifically, given that the nucleophilic attack can occur from various, non equivalent regions of the starting molecule, our main goal was to achieve a complete regio- and stereocontrol of the reaction. We have verified that the reported organocatalyzed vinylogous reaction represents a valid integration of the metal-catalyzed one, since it affords highly stereochemically complex products in good to high yields and excellent optical purity.

Airway Basal Cell Vascular Endothelial Growth Factor-mediated Cross-Talk Regulates Endothelial Cell Dependent Growth Support of Human Airway Basal Cells

The human airway epithelium is a pseudostratified heterogenous layer comprised of cili-ated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epi-thelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to ex-press mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothe-lium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Total internal reflection fluorescence cross-correlation spectroscopy: theory and application for studying boundary slip phenomenon

I present a new experimental method called Total Internal Reflection Fluorescence Cross-Correlation Spectroscopy (TIR-FCCS). It is a method that can probe hydrodynamic flows near solid surfaces, on length scales of tens of nanometres. Fluorescent tracers flowing with the liquid are excited by evanescent light, produced by epi-illumination through the periphery of a high NA oil-immersion objective. Due to the fast decay of the evanescent wave, fluorescence only occurs for tracers in the ~100 nm proximity of the surface, thus resulting in very high normal resolution. The time-resolved fluorescence intensity signals from two laterally shifted (in flow direction) observation volumes, created by two confocal pinholes are independently measured and recorded. The cross-correlation of these signals provides important information for the tracers’ motion and thus their flow velocity. Due to the high sensitivity of the method, fluorescent species with different size, down to single dye molecules can be used as tracers. The aim of my work was to build an experimental setup for TIR-FCCS and use it to experimentally measure the shear rate and slip length of water flowing on hydrophilic and hydrophobic surfaces. However, in order to extract these parameters from the measured correlation curves a quantitative data analysis is needed. This is not straightforward task due to the complexity of the problem, which makes the derivation of analytical expressions for the correlation functions needed to fit the experimental data, impossible. Therefore in order to process and interpret the experimental results I also describe a new numerical method of data analysis of the acquired auto- and cross-correlation curves – Brownian Dynamics techniques are used to produce simulated auto- and cross-correlation functions and to fit the corresponding experimental data. I show how to combine detailed and fairly realistic theoretical modelling of the phenomena with accurate measurements of the correlation functions, in order to establish a fully quantitative method to retrieve the flow properties from the experiments. An importance-sampling Monte Carlo procedure is employed in order to fit the experiments. This provides the optimum parameter values together with their statistical error bars. The approach is well suited for both modern desktop PC machines and massively parallel computers. The latter allows making the data analysis within short computing times. I applied this method to study flow of aqueous electrolyte solution near smooth hydrophilic and hydrophobic surfaces. Generally on hydrophilic surface slip is not expected, while on hydrophobic surface some slippage may exists. Our results show that on both hydrophilic and moderately hydrophobic (contact angle ~85°) surfaces the slip length is ~10-15nm or lower, and within the limitations of the experiments and the model, indistinguishable from zero.

Temporal variation of genetic diversity and differentiation of albacore, Thunnus alalunga, in the Mediterranean Sea

This study is on albacore (Thunnus alalunga, Bonnaterre 1788), an epi- and mesopelagic oceanic tuna species cosmopolitan in the tropical and temperate waters of all oceans including the Mediterranean Sea, extending in a broad band between 40°N and 40°S. What it’s known about albacore population structure is based on different studies that used fisheries data, RFLP, mtDNA control region and nuDNA markers, blood lectins analysis, individual tags and microsatellite. At the moment, for T. alalunga six management units are recognized: the North Pacific, South Pacific, Indian, North Atlantic, South Atlantic and Mediterranean stocks. In this study I have done a temporal and spatial comparison of genetic variability between different Mediterranean populations of Thunnus alalunga matching an historical dataset ca. from 1920s composed of 43 individuals divided in 3 populations (NADR, SPAIN and CMED) with a modern dataset composed of 254 individuals and 7 populations (BAL, CYP, LIG, TYR, TUR, ADR, ALB). The investigation was possible using a panel of 94 nuclear SNPs, built specifically for the target species at the University of Basque Country UPV/EHU. First analysis done was the Hardy-Weinberg, then the number of clusters (K) was determined using STRUCTURE and to assess the genetic variability, allele frequencies, the average number of alleles per locus, expected (He) and observed (Ho) heterozygosis, and the index of polymorphism (P) was used the software Genetix. Historical and modern samples gives different results, showing a clear loss of genetic diversity over time leading to a single cluster in modern albacore instead of the two found in historical samples. What this study reveals is very important for conservation concerns, and additional research endeavours are needed.

La risonanza magnetica per la valutazione della funzionalità renale in pazienti affetti da malattia policistica

Scopo. La risonanza magnetica (RM) in diffusione (Diffusion Weighted imaging DWI) permette di ottenere dati quantitativi sul movimento dei protoni liberi in acqua mediante la valutazione di alcuni parametri tra cui il coefficiente di diffusione apparente (Apparent Diffusion Coefficient ADC). Questo parametro potrebbe essere usato per differenziare i diversi tipi di tessuti (cisti di diverso tipo, parenchima sano, parenchima non più funzionante, …) e stabilire il danno renale. Lo scopo del presente lavoro è quello di valutare la correlazione tra il valore di ADC e il tessuto renale al fine di verificare la potenziale capacità di questo parametro di offrire informazioni funzionali e poter predire l’insorgenza di cisti e quindi l’aumento di volume del rene. Materiali e Metodi. 11 pazienti di cui 2 donne e 9 uomini (età compresa tra i 55 e i 75 anni) sono stati sottoposti a esame RM con sistema ad alto campo 1,5T. Tutti i pazienti erano affetti da malattia policistica. Il protocollo prevedeva l’utilizzo di una sequenza SE-EPI pesata in diffusione, le immagini sono state acquisite per diversi valori di b-value (500 s/mm², 700 s/mm², 900 s/mm², e 1500 s/mm²). Risultati. Osservando i valori di ADC (coefficiente di diffusione apparente) ottenuti per diversi valori di b e in due differenti regioni del rene, corrispondenti a cisti e parenchima, si sono riscontrati valori diversi . Indipendentemente dal b-value, le cisti hanno un valore di ADC minore rispetto al valore di ADC delle zone parenchimali. Al crescere del b-value, tali valori diventano più piccoli. Conclusioni. La DWI permette di ottenere valori di ADC in grado di discriminare tessuti.

Body mass index, hormone replacement therapy, and endometrial cancer risk: a meta-analysis

Background: Body mass index (BMI) is a risk factor for endometrial cancer. We quantified the risk and investigated whether the association differed by use of hormone replacement therapy (HRT), menopausal status, and histologic type. Methods: We searched MEDLINE and EMBASE (1966 to December 2009) to identify prospective studies of BMI and incident endometrial cancer. We did random-effects meta-analyses, meta-regressions, and generalized least square regressions for trend estimations assuming linear, and piecewise linear, relationships. Results: Twenty-four studies (17,710 cases) were analyzed; 9 studies contributed to analyses by HRT, menopausal status, or histologic type, all published since 2003. In the linear model, the overall risk ratio (RR) per 5 kg/m2 increase in BMI was 1.60 (95% CI, 1.52–1.68), P < 0.0001. In the piecewise model, RRs compared with a normal BMI were 1.22 (1.19–1.24), 2.09 (1.94–2.26), 4.36 (3.75–5.10), and 9.11 (7.26–11.51) for BMIs of 27, 32, 37, and 42 kg/m2, respectively. The association was stronger in never HRT users than in ever users: RRs were 1.90 (1.57–2.31) and 1.18 (95% CI, 1.06–1.31) with P for interaction ¼ 0.003. In the piecewise model, the RR in never users was 20.70 (8.28–51.84) at BMI 42 kg/m2, compared with never users at normal BMI. The association was not affected by menopausal status (P ¼ 0.34) or histologic type (P ¼ 0.26). Conclusions: HRT use modifies the BMI-endometrial cancer risk association. Impact: These findings support the hypothesis that hyperestrogenia is an important mechanism underlying the BMI-endometrial cancer association, whilst the presence of residual risk in HRT users points to the role of additional systems. Cancer Epidemiol Biomarkers Prev; 19(12); 3119–30.