Oxytocin interference in the effects induced by inhalation of 7.5% CO2 in healthy volunteers

Objective The objective of this study was to assess the acute effect of intranasally administered oxytocin (OT) on subjective states, cardiovascular, and endocrine parameters in healthy volunteers who inhaled 7.5% CO2. Methods Forty-five subjects were allocated into three matched groups of subjects who received 24?international units (IU) of OT, 2?mg of lorazepam (LZP), or placebo (PL). The challenge consisted of medical air inhalation for 20?min, 10?min of rest, and CO2 7.5% inhalation for 20?min. Subjective effects were evaluated by self-assessment scales; heart rate, blood pressure, skin conductance, and salivary cortisol were also measured. Assessments were performed at four time points: (i) baseline (-15?min); (ii) post-air inhalation (90?min); (iii) post-CO2 inhalation (120?min), and (iv) post-test (160?min). Results CO2 inhalation significantly increased the anxiety score in the PL group compared with the post-air measurement but not in the OT or LZP groups. The LZP reduced anxiety after medical air inhalation. Other parameters evaluated were not affected by OT. Conclusion OT, as well as LZP, prevented CO2-induced anxiety, suggesting that this hormone has anxiolytic properties. Copyright (C) 2012 John Wiley & Sons, Ltd.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0 min at a flow-rate of 1.5 mL min(-1) into a Phenomenex Gemini (R) C18, 5 mu m analytical column (150 x 4.6 mm id.). The calibration curve was linear over the range from 0.2 to 200 ng mL(-1) for dextromethorphan and doxylamine and 0.05 to 10 ng mL(-1) for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5 mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study. (C) 2012 Elsevier B.V. All rights reserved.

In vitro organogenesis of zucchini squash cv. Caserta

A protocol for the in vitro culture of Cucurbita pepo cv. Caserta was studied, using a cotyledon segment with an attached hypocotyl fragment as an explant. First, to determine the optimal seedling age, explants were collected from 4 to 6-day-old in vitro germinated seedlings and cultured in MS basal medium supplemented with benzylaminopurine (BAP, 4.5 mu M), under a 16-h photoperiod at 27 degrees C. Based on the results obtained, the explants collected from the 4-day-old seedlings were then cultured in MS basal medium supplemented with different concentrations of BAP (0, 1.1, 2.2, 3.3, 4.5, or 5.5 mu M) and incubated under a 16-h photoperiod at 27 degrees C. In vitro organogenesis was most efficient with explants collected from 4-day-old seedlings cultured in medium supplemented with 4.5 mu M of BAP. After 4 weeks of incubation the development of adventitious buds at the cotyledon/hypocotyl junction could be observed. These buds were transferred to elongation and rooting medium and the developed plants were acclimatized to greenhouse conditions. The morphogenic process was characterized using light and scanning electron microscopy analyses to confirm the organogenesis. The results showed that this alternate explant is efficient for in vitro culture of zucchini squash cv. Caserta. The protocol will be further examined for future use in genetic transformation experiments in this species.

FIG CLONES SELECTION OF cv. ROXO DE VALINHOS OBTAINED BY IRRADIATED BUDS

The fig tree (Ficus carica L.) is a fruit tree of great world importance and, therefore, the genetic improvement becomes an important field of research for the crop improvement, being necessary to gather information on this species, mainly regarding its genetic variability so that appropriate propagation projects and management are made. However, the fig, in Brazil, is all produced from only one cultivar, Roxo de Valinhos, which produces seedless fruit, making impossible the conventional breeding. So, the fig breeding through induced mutagenic becomes a very important research line, greatly contributing to the fig culture development. The objective of this study was to select fig plants formed by cuttings treated with gamma ray. The plants used were obtained from buds of the cv. Roxo de Valinhos. The cuttings were irradiated with gamma rays in an irradiator Gamma Cell at 10 cm from the tip of the cutting, at doses of 30 Gy with dose rate of 238 Gy/h. The experiment consisted of 450 treatments, where each formed plant was a treatment. The treatments were numbered sequentially from 1 to 450 and spaced 2.5 x 1.5 m. It was evaluated the vegetative and the fruits characteristics, and the incidence of major crop pests and diseases. The analysis data showed that there is genetic variability among treatments and that the plants under numbers 1, 5, 20, 79, 164, 189, 194, 201, 221, 214, 258, 301, 322, 392, 433 and 440 are probably genetic mutants that should be tested as commercial orchards.

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.

A Phase I clinical trial of lodenafil carbonate, a new phosphodiesterase Type 5 (PDE5) inhibitor, in healthy male volunteers

Lodenafil carbonate is a new phosphodiesterase Type 5 (PDE5) inhibitor used in treatment of erectile dysfunction. Objective: The present study was conducted to evaluate the safety, tolerability, and pharmacokinetics of lodenafil carbonate after administering ascending (1 - 100 mg) single oral doses to healthy male volunteers (n = 33). Methods: The study was an open-label, dose-escalation, Phase I clinical trial involving the administration of single oral doses of lodenafil carbonate. Lodenafil carbonate was administered sequentially, escalating in single doses of 1 mg - 100 mg with a washout period of at least 1 week between each dose. The progression to the next dose was allowed after clinical and laboratory exams, Ambulatory Monitoring of Arterial Pressure (AMAP) without relevant clinical modifications and adverse events without clinical relevancy. Blood samples were collected at pre-dose, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 14, 16, 20 and 24 h post-dosing. Plasma samples for measurement of lodenafil carbonate and lodenafil were analyzed by liquid chromatography coupled to tandem mass spectrometry. Results: No serious adverse events were observed, and none of the subjects discontinued the study due to intolerance. The AMAP measurements, clinical and laboratory exams and ECG revealed no significant changes even at higher doses. Lodenafil carbonate was not detected in any samples, indicating that it acts as a prodrug. The mean lodenafil pharmacokinetic parameters for t(max) and t(1/2) were 1.6 (+/- 0.4) h and 3.3 (+/- 1.1) h, respectively. This study demonstrated that lodenafil carbonate was well tolerated and showed a good safety profile in healthy male volunteers.

Evaluation of Inductively Coupled Plasma Mass Spectrometry for Determining Ca, Cu, Fe, Mg, Mn, Se and Zn in Bovine Semen Samples using a Simple Sample Dilution Method

A simple and fast method for the determination of Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen by quadrupole inductively coupled plasma spectrometry (q-ICP-MS) is described. Prior to analysis, samples (200 mu L) were diluted 1:50 in a solution containing 0.01% v/v Triton (R) X-100 and 0.5% v/v nitric acid and directly analyzed by ICP-MS. The limits of detection of the method are 0.3, 0.03, 0.2, 0.04, 0.04, 0.03 and 0.03 mu g L-1 for Ca-44, Cu-63, Fe-57, Mg-24, Zn-64, Se-82 and Mn-55, respectively. For purposes of comparison and method validation, four ordinary bovine semen samples were directly analyzed by ICP-MS and by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GF AAS), with no statistical difference between the techniques at the 95% level when applying the t-test. Then, the proposed method was applied in the determinations of Ca, Cu, Fe, Mg, Mn, Se and Zn in collected samples of bovine semen from different breeds, which are used in reproduction programs and artificial insemination.

Caracterização de uma floresta estacional semidecidual em Varginha, MG. E comparação com remanescentes da região.

Foi realizado o levantamento florístico e fitossociológico na porção mais extensa de mata no Parque Florestal Municipal São Francisco de Assis, na cidade de Varginha, sul de Minas Gerais. A área tem cerca de 10 ha e é cortada por um pequeno córrego. Foram lançadas 25 parcelas de 20m x 20m e medidos e cadastrados todos os indivíduos arbóreos com DAP > 5cm. Foram encontrados 1568 indivíduos que, juntos, somaram área basal de 23,65 m2, sendo registrados 102 espécies, 72 gêneros e 42 famílias no levantamento fitossociológico e 111 espécies, 77 gêneros e 43 famílias no levantamento florístico. As famílias com maior número de espécies foram Myrtaceae, Fabaceae e Melastomataceae. As espécies de maior valor de importância foram Casearia arborea, Copaifera langsdorffii, Tachigali rugosa e Myrcia splendens. Os resultados encontrados são os esperados para as Florestas Estacionais Semideciduais da região.

Citrus aurantium L. essential oil exhibits anxiolytic-like activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment

Abstract Background The current treatments for anxiety disorders and depression have multiple adverse effects in addition to a delayed onset of action, which has prompted efforts to find new substances with potential activity in these disorders. Citrus aurantium was chosen based on ethnopharmacological data because traditional medicine refers to the Citrus genus as useful in diminishing the symptoms of anxiety or insomnia, and C. aurantium has more recently been proposed as an adjuvant for antidepressants. In the present work, we investigated the biological activity underlying the anxiolytic and antidepressant effects of C. aurantium essential oil (EO), the putative mechanism of the anxiolytic-like effect, and the neurochemical changes in specific brain structures of mice after acute treatment. We also monitored the mice for possible signs of toxicity after a 14-day treatment. Methods The anxiolytic-like activity of the EO was investigated in a light/dark box, and the antidepressant activity was investigated in a forced swim test. Flumazenil, a competitive antagonist of benzodiazepine binding, and the selective 5-HT1A receptor antagonist WAY100635 were used in the experimental procedures to determine the mechanism of action of the EO. To exclude false positive results due to motor impairment, the mice were submitted to the rotarod test. Results The data suggest that the anxiolytic-like activity observed in the light/dark box procedure after acute (5 mg/kg) or 14-day repeated (1 mg/kg/day) dosing was mediated by the serotonergic system (5-HT1A receptors). Acute treatment with the EO showed no activity in the forced swim test, which is sensitive to antidepressants. A neurochemical evaluation showed no alterations in neurotransmitter levels in the cortex, the striatum, the pons, and the hypothalamus. Furthermore, no locomotor impairment or signs of toxicity or biochemical changes, except a reduction in cholesterol levels, were observed after treatment with the EO. Conclusion This work contributes to a better understanding of the biological activity of C. aurantium EO by characterizing the mechanism of action underlying its anxiolytic-like activity.

Seleção de clones de figueira cv. roxo de Valinhos formados por gemas irradiadas

A figueira (Ficus carica L.), pertencente à família das Moráceas, constitui-se numa das mais importantes frutíferas cultivadas, elevando o Brasil à condição de décimo maior produtor e exportador de figos do mundo. Porém, a ficicultura apresenta alguns problemas fitossanitários, além de, no Brasil, estar toda implantada com uma única cultivar, a Roxo de Valinhos, que produz frutos sem sementes, inviabilizando o melhoramento convencional. Nesse sentido, o melhoramento genético, com o uso de mutagênicos, passa a ser uma linha de pesquisa altamente importante, podendo contribuir enormemente para o desenvolvimento da cultura. Diante disto, o objetivo do presente trabalho foi selecionar mutantes em plantas de figueira formadas por estacas irradiadas com raios gama, a fim de aumentar sua variabilidade genética com relação ao desenvolvimento vegetativo e reprodutivo. Utilizaram-se plantas formadas por estacas originadas de gemas da cultivar Roxo de Valinhos irradiadas com raios gama, no irradiador tipo Gamma a 0,10 m do ápice, na dose de Gy com taxa de dose de 238 Gy/h. O experimento constou de 450 tratamentos, sendo cada planta formada considerada um tratamento, numerando-as sequencialmente de 1 a 450 e cultivadas em espaçamento de 2,5 x 1,5 m.. As avaliações foram realizadas a partir das características tanto das folhas quanto dos frutos, bem como da incidência das principais pragas e doenças da cultura nestas plantas. Da análise dos dados, conclui-se que há variabilidade genética entre os tratamentos e que algumas plantas são prováveis mutantes, mostrando-se assim com potencial para posteriores estudos, devendo ser testadas em plantios comerciais.

Análise sensorial, teores de nitrato e de nutrientes de alface cultivada em hidroponia sob águas salinas

O presente trabalho teve como objetivos determinar os teores de nutrientes e de nitrato e realizar análise sensorial de alface cultivada em sistema hidropônico sob água salina. O experimento foi conduzido em ambiente protegido no período de dezembro de 2007 a janeiro de 2008, em Piracicaba (SP). O delineamento experimental foi de blocos ao acaso, sendo estudados cinco níveis de salinidade da água de irrigação, utilizando-se NaCl (Condutividade elétrica da água (CEa): 0,42, 1,53, 3,52, 5,55 e 7,43 dS m-1) em duas cultivares de alface (Verônica e Pira Roxa), em esquema fatorial. Foram avaliados a análise sensorial aplicando escala hedônica e através dessa, o que os provadores mais apreciaram e o que menos gostaram de cada amostra, intenção de compra e consumo, teor de nitrato, absorção de nutrientes pela folhas e determinação dos nutrientes presentes na solução nutritiva. Para o atributo sabor a cultivar Verônica recebeu as melhores notas no nível de salinidade 1,53 e 5,55 dS m-1. Para a cultivar Pira Roxa a melhor nota foi atribuída para a alface produzida na água não salina. Para os demais atributos não houve diferença significativa nos diferentes níveis de salinidade. Com relação à intenção de compra, constatou-se que as alfaces cv. Verônica e Pira Roxa apresentaram boa aceitação de mercado. Os menores níveis de nitrato (1960 mg kg-1 e 2620 mg kg-1 de massa de matéria fresca), da Verônica e Pira Roxa, respectivamente, foram relacionados à condutividade elétrica de 0,42 dS m-1, aumentando a salinidade da água para 7,43 dS m-1, o teor foliar de nitrato aumentou para 2500 mg kg-1 e 3420 mg kg-1 para as cultivares Verônica e Pira Roxa. Como o tempo de exposição da alface à salinidade em sistema hidropônico foi curto, em torno de 25 dias, não se verificou sintomas de deficiência nutricional e toda alface foi classificada como apropriada para consumo humano.

Evaluation of inductively coupled plasma mass spectrometry for determining Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen samples using a simple sample dilution method

A simple and fast method for the determination of Ca, Cu, Fe, Mg, Mn, Se and Zn in bovine semen by quadrupole inductively coupled plasma spectrometry (q-ICP-MS) is described. Prior to analysis, samples (200 µL) were diluted 1:50 in a solution containing 0.01% v/v Triton® X-100 and 0.5% v/v nitric acid and directly analyzed by ICP-MS. The limits of detection of the method are 0.3, 0.03, 0.2, 0.04, 0.04, 0.03 and 0.03 µg L-1 for 44Ca, 63Cu, 57Fe, 24Mg, 64Zn, 82Se and 55Mn, respectively. For purposes of comparison and method validation, four ordinary bovine semen samples were directly analyzed by ICP-MS and by flame atomic absorption spectrometry (FAAS) or graphite furnace atomic absorption spectrometry (GF AAS), with no statistical difference between the techniques at the 95% level when applying the t-test. Then, the proposed method was applied in the determinations of Ca, Cu, Fe, Mg, Mn, Se and Zn in collected samples of bovine semen from different breeds, which are used in reproduction programs and artificial insemination.

Acúmulo de macronutrientes na soja influenciado pelo cultivo prévio do capim-marandu, correção e compactação do solo

O presente trabalho foi desenvolvido com o objetivo de avaliar o efeito do cultivo prévio do capim-Marandu (Brachiaria brizantha cv. Marandu), da aplicação de corretivos e da compactação do solo no acúmulo de macronutrientes pela soja cultivada em sucessão. O delineamento experimental foi inteiramente casualizado em esquema fatorial 4 x 6 x 2, com três repetições. Os fatores de estudo foram quatro densidades do solo: 1,0; 1,20; 1,40 e 1,60 Mg m-3; seis tratamentos de correção: 1) controle, sem correção; 2) calcário; 3) silicato de cálcio; 4) gesso; 5) calcário + gesso; 6) silicato de cálcio + gesso; além de dois sistemas de cultivo: com e sem cultivo prévio do capim-Marandu. As unidades experimentais foram compostas por vasos de tubos de PVC de 20 cm de diâmetro, compostos por dois anéis: o anel inferior, de 40 cm de altura, recebeu o solo sob condições naturais e densidade de 1,0 Mg m-3; o anel superior, com 20 cm de altura representando 6,28 dm³, recebeu os tratamentos de densidades, correção e gesso como descrito adiante. Em cada um foram conduzidas três plantas de soja (cv. Conquista) até o final do ciclo, quando o acúmulo de macronutrientes pela cultura foi avaliado. Os resultados mostraram que o cultivo prévio do capim-Marandu e o uso de corretivos amenizaram os efeitos negativos da compactação do solo sobre a nutrição da soja. A utilização de corretivos do solo contribuiu para o aumento no acúmulo de macronutrientes na parte aérea da soja, porém o incremento na compactação diminuiu o acúmulo de N, P, K, Ca, Mg e S. A compactação do solo persistiu parcialmente mediante o cultivo prévio com o capim marandu.

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Ground state properties of neutron-rich Mg isotopes

Studies in regions of the nuclear chart in which the model predictions of properties of nuclei fail can bring a better understanding of the strong interaction in the nuclear medium. To such regions belongs the so called "island of inversion" centered around Ne, Na and Mg isotopes with 20 neutrons in which unexpected ground-state spins, large deformations and dense low-energy spectra appear. This is a strong argument that the magic N = 20 is not a closed shell in this area. In this thesis investigations of isotope shifts of stable 24,25,26Mg, as well as spins and magnetic moments of short-lived 29,31Mg are presented. The successful studies were performed at the ISOLDE facility at CERN using collinear laser and beta-NMR spectroscopy techniques. The isotopes were investigated as single-charged ions in the 280-nm transition from the atomic ground state 2S1/2 to one of the two lowest excited states 2P1/2,3/2 using continuous wave laser beams. The isotope-shift measurements with fluorescence detection for the three stable isotopes show that it is feasible to perform the same studies on radioactive Mg isotopes up to the "island of inversion". This will allow to determine differences in the mean charge square radii and interpret them in terms of deformation. The high detection efficiency for beta particles and optical pumping close to saturation allowed to obtain very good beta-asymmetry signals for 29Mg and 31Mg with half-lives around 1 s and production yields about 10^5 ions/s. For this purpose the ions were implanted into a host crystal lattice. Such detection of the atomic resonances revealed their hyperfine structure, which gives the sign and a first estimate of the value of the magnetic moment. The nuclear magnetic resonance gave also their g-factors with the relative uncertainty smaller than 0.2 %. By combining the two techniques also the nuclear spin of both isotopes could be unambiguously determined. The measured spins and g-factors show that 29Mg with 17 neutrons lies outside the "island of inversion". On the other hand, 31Mg with 19 neutrons has an unexpected ground-state spin which can be explained only by promoting at least two neutrons across the N = 20 shell gap. This places the above nucleus inside the "island". However, modern shell-model approaches cannot predict this level as the ground state but only as one of the low-lying states, even though they reproduce very well the experimental g-factor. This indicates that modifications to the available interactions are required. Future measurements include isotope shift measurements on radioactive Mg isotopes and beta-NMR studies on 33Mg.