372 research outputs found

    Metabolomica e certificazione chimica

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    Allo scopo di affrontare le principali difficoltà incontrate nello sviluppo e nell’applicazione di tecnologie chimico-analitiche per l’ottenimento di nuovi prodotti diagnostici per il controllo della salute umana e animale, o prodotti/processi necessari per la certificazione chimica di prodotti agro-alimentari, il progetto metterà a disposizione delle imprese interessate le piattaforme tecnologiche di seguito descritte: Piattaforma di Risonanza Magnetica e Chimica Analitica; Piattaforma di Proteomica; Piattaforma di Tecnologie Alimentari e Biotecnologie Microbiche. L'obiettivo principale è quello di promuovere un network di imprese impegnate nello sviluppo di processi, servizi, prodotti analitici e diagnostico-molecolari per la salute, per la tutela ambientale, per la tracciabilità e la qualità dei prodotti agroalimentari in Sardegna.Finanziamenti::Fondo Consortile - Sardegna Ricerch

    Polimorfismi genetici dell'interleuchina 1 e dell'interleuchina 6 nella popolazione sarda con particolare riferimento alle fasce di etĂ  avanzata

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    Introduction. A unique approach to the science of longevity is the natural model of centenarians. Studies of this kind are particularly interesting because they offer people the opportunity to know the critical factors that lead to aging successfully. It is indeed a population that is homogeneous for longevity, this term a long life regardless of its quality. The design and subsequent implementation of a genetic study is not without obstacles and barriers, both from an organizational perspective (research of centenarians and their validation by official records) and because we live in a multiethnic society. The data reported in the literature indicate that human longevity could be related to the optimal functioning of the immune system. So one of the genetic determinants of longevity might reside in those polymorphisms that regulate immune response. Centenarians are exceptional individuals, who through a mechanism not yet clarified, have developed a level of biological homeostasis that is compatible with the maintenance of life for an exceptional period of time. The project AKeA studied for over ten years, the phenomenon of longevity in Sardinia, a region in which there are constantly 22 centenarians per 100,000 inhabitants. The AKeA project is based on the methodology of certification: one study only individuals in possession of official documents and people in life who sign the informed consent and accept the researchers 'home visit' s University of Sassari. The work done in recent years has focused on the study of some cluster polymorphisms of interleukin 1 and interleukin 6. The study of interleukin 1 genes in the cluster, which expresses three different proteins, two agonists, interleukin-1 alpha and interleukin 1 beta and an antagonist, interleukin-1 Ra Materials and methods 1038 were examined DNA samples from Sardinian residents aged 60 years, and centenarians centenaries AKeA part of the project. The DNA was extracted, after drawing blood from white blood cells with the standard method of phenol-chloroform We studied three polymorphisms in the promoter of the gene for interleukin-6, -174 G / C, -597 G / A and -574 G / C. We studied two olymorphisms in the promoter of the gene for interleukin-1, -511 C / C and -889 C / T Furthermore, we studied interleukin-1 RA polymorphism that is linked to the presence of different alleles that differ in sequence repea t (VNTR) of 86 bp present in intron 2. 1038 samples were tested for DNA testing of which 849 were valid. The genotype distribution and allele frequency was estimated by dividing the individuals in the following age groups: centenarians, nova ntacinquenni, nonagenarians, octogenarians and sixties. In addition, a comparison was made between the sexes. Results. The distribution of genotypes of polymorphisms -174 G / C and -597 G / A for interleukin 6 follows the distribution expected under the Hardy-Weinberg equilibrium and there were no significant differences between the boomers and centenarians. The results for interleukin 1 did not show a meaningful if not for the distribution of genotypes among women nonagenarians, who do not follow the Hardy-Weinberg equilibrium. This figure is still the subject of extensive studies for his confirmation through the analysis of a larger sample

    Contenuto di Piombo, Cadmio e Selenio nei cibi sardi e comparazione con i livelli plasmatici degli stessi in soggetti longevi

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    Lifestyle, genetic factors and dietary habits may affect the process of healthy aging. Sardinia is known for its high incidence of centenarians. Some foods consumed by old Sardinian people (wine, cheese, fruit, vegetables, etc.) have nutraceutical and antioxidants properties that are able to maintain the integrity and physiological stability, suggesting a diet-gene interaction. By contrast, some foods may have substances with harmful properties deleterious to the body such as heavy and transition metals that can cause damages. The plasma samples analyzed came from a research project, called Akea, funded by sardianan region (LR 7/2007 and L.R. 2/2007). Wines and cheeses are all of own production while the fruit is native. From the analysis of the plasma, there was a positive correlation between Cd and Se (rs = 0.21, p <0.05) and a negative correlation between Se and drinkers (rs = -0.15, p <0.05). There were no significant differences between the levels of Cd and Se in the blood in non-smokers and smokers (p> 0.05). The plasma values of Mg, Fe, Cu, Zn and Ca in plasma are located inside the reference values, also the activity of GPX3 is normal. In food, the concentrations of Cd, Se and Pb did not exceed the maximum limits permitted by law and they appeared reassuring for the risk of contamination of the soil, water and air

    La salvaguardia delle specie ittiche di allevamento attraverso la conoscenza del loro benessere

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    Il benessere animale in acquacoltura è divenuto una tematica di grande interesse a livello nazionale, europeo e mondiale. La crescente richiesta di prodotto ittico, in considerazione sia dell’aumento della popolazione mondiale, sia del sempre maggiore apprezzamento per le qualità del prodotto e per le sue proprietà nutrizionali, ha posto nuovi interrogativi in termini di sicurezza e di benessere. Le ricerche condotte sul campo stimolate anche dai rispettivi governi a tutela del prodotto, hanno permesso di accrescere le conoscenze sull’anatomia, la fisiologia e sul comportamento dei pesci. La capacità da parte del pesce di percepire l’ambiente circostante, così come il suo comportamento in relazione ai conspecifici, ha permesso di chiarire molti aspetti legati alle sue attività e, al contempo, di riconoscere per queste specie il concetto di essere senziente. Sicuramente le conoscenze fino ad oggi acquisite in relazione a più moderne tecnologie di biologia e di genetica molecolare hanno contribuito ad approfondire le suddette tematiche con interessanti risultati

    La metabolomica: nuovo approccio alla medicina predittiva per il bambino e il neonato

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    BACKGROUND Metabolomics is an emerging science, with promising application in medical practice. The metabolomic approach is based on the global identification, not driven by a priori hypothesis, of a high number of metabolites in a biological fluid; this allows the characterization of the metabolic profile typical of a certain condition, permitting to identify which metabolites or pattern of metabolites could be useful in discriminating different groups of subjects. By analysis of spectroscopic data, multivariate statistical analysis tools allow you to extract data relevant metabolic characterization of specific physiological and patho-physiological. Furthermore, the analysis needs metabolomics low amounts of biological sample, a feature that makes it applicable to multiple arrays but which especially makes it easy to use in the paediatric field. The impact of this science in the field of maternal and child studies is very important, to investigate whether there are metabolites useful to diagnose some of the most widespread diseases in childhood and to investigate whether there are correlations between the maternal condition and the development of diseases in the newborn. In the specific case we applied the metabolomic approach to some of the most common diseases of the scope for Childcare: food allergy, the preterm delivery, the development of BPD and defects of the β-oxidation of fatty acids. OBJECTIVES The aim of my PhD was to apply metabolomics analysis and mass spectrometry to investigate some of the most common diseases of children and maternal field (food allergy, pre-term birth, BPD development and defects of the β-oxidation of fatty acids) using different biological matrices: urine, amniotic fluid, plasma and blood spot. Two different approaches were applied: a target approach, very specific, that focuses on the qualitative and quantitative analysis of a single analyte, such as a marker disease or the substrate of an enzyme reaction; an untargeted approach (fongerprinting) focused on the analysis of a group of metabolites belonging to a specific metabolic pathway or a class of compounds. METHODS The untargeted approach was applied to two studies: the first to see on a group of children with suspected milk allergy to differentiate the allergic reaction in urine samples taken before the outbreak; the second to evaluate the metabolic profiles of amniotic fluid of patients with preterm delivery and development of BPD in order to be able to predict preterm birth and development of BPD in the child. The first is a prospective study including 30 children (19 males and 11 females, average age 4 years; they were all affected by IgE mediated cow’s milk protein allergy). The patients submitted to a milk oral challenge test and, accordingly, were divided in two groups: positives (15 children) and negatives (15 children). A urine sample was collected for each patient before the milk challenge test. The second is a pilot study including 32 infants of mothers who had undergone amniocentesis between the 21st and the 28th week of gestation have been included in the study: 10 preterm infants with BPD (A), 11 healthy preterm infants without BPD (B), and 11 healthy term infants (C). The metabolomic analysis of the urine and amniotic fluid was performed by means of Q-Tof (Synapt G2; Waters) high performance mass spectrometry platform coupled with a UPLC (Ultra Performance Liquid Chromatography, Waters) system, characterized by a high chromatographic resolution and a short analytic time. The sample were analysed by means of two different types of chromatographic columns (one column, HSS T3, with a reversed phase; the other with a polar phase, ACQUITY BEH HILIC). For each column the sample were analysed in positive and negative polarity. The results of the LC-MS analysis were processed with the MarkerLynx software and submitted to multivariate statistics methods. The variables which emerged from the statistic analysis were then confronted with the variables in the databases on the internet (HMDB and Metlin). As far as the targeted approach is concerned, it was applied to the study of the metabolism of the fatty acids during β-oxidation. 157 newborn children were taken into consideration, and they were divided in four groups, according to the gestational age (SG): group 1 (22-27 +6 SG); group 2 (28-31 +6 SG) group 3 (32-36 +6 SG) group 4 (37-41 +6 SG). 48-72 hours after birth, DBS (blood spot on blotting paper) and plasma samples were collected from each subject. For the analysis, unmarked acylcarnitine donated by Dr. Piero Rinaldo (Mayo Clinic, Rochester, MN, USA) were used, together with stable isotopes marked compositions, purchased from the ChromSystems Instruments & Chemicals (Munich, Germany), which were used as internal standards. The acetonitrile, the methanol, and the n-butanol were purchased from Carlo Erba (Milan, Italy); the trifluoroacetic acid (TFA) and the acetyl chloride from the Sigma-Aldrich (Milan, Italy). All the solvents were chromatographically pure. In order to quantify the acylcarnitine, calibration curves were built for each of them by the isotopic dilution method. For the statistic analysis the STATISTICA 6.0 (Stat Soft Inc, Tulsa, USA) and SPSS for Windows (version 21.0, IBM Corp., Armonk, NY) software packs were used. RESULTS The study on the prediction of the pre-term delivery and the BPD development based on the analysis of the amniotic fluid had led to the development of two data sets. The analysis of these two sets (one for each ionization method applied during the spectroscopic analysis) has allowed us to build two robust PLS-DA (Partial Least Square regression-Discriminant Analysis) models (R2=0.78 and Q2=0.56; R2=0.80 and Q2=0.56), which are able to clearly discriminate the AF samples belonging to the three groups of infants (A,B,C). A preliminary data analysis through PCA (Principal Component Analysis) had permitted to exclude the hypothesis that this discrimination could be due to the confounding effect of some important clinical variables (mother age at amniocentesis, gestational age at amniocentesis and therapy the mother submitted to during pregnancy). Last, in the study of the acylcarnitine and the amino acids, the reported results define the normal range of concentrations of the carnitine pool components, including isomers and isobaric metabolites in plasma and DBS samples, for infants of different gestational ages. The values obtained from the analysis of the samples do not fit a Gaussian distribution, therefore medians and percentiles (10°, 25°, 75°, 90°) of the concentrations of each metabolite were calculated for each group (G1, G2, G3, G4). We have also carried out the statistical analysis of the differences between the different groups. CONCLUSION The two untargeted studies have demonstrated that the metabolomic analysis is able to predict the milk oral challenge test response, on the basis of the urine metabolomic profile, and to predict pre-term birth and BPD development through the analysis of amniotic fluid. Especially as far as food allergy is concerned, 4 variables have emerged as particularly relevant in the differentiation of the two groups. Despite the fact that the confrontation of the chemical-physical characteristics of these variable with the metabolomic databases has not allowed the identification of the exact nature of the variables, it is worth underlining that the use of 2 of the most important identified variables leads to the creation of a decisional tree which allows the correct prediction in response to the test in all the subjects but one. Nevertheless, regardless of the complete identification of the involved metabolites, the most relevant result is the existence of a “metabolic fingerprint” that individuates children with higher risk of positive response to challenge test. The development and the validation of this method will allow us to avoid submitting to the oral challenge test children who are highly at risk of developing a generalized (systemic) allergic reaction. The study on the amniotic fluids had demonstrated that the metabolic profile of the amniotic fluid collected between the 21st and the 28th pregnancy week can discriminate the pregnancies subject to a pre-term delivery from those subject to an on term delivery, and identify the newborn children who will develop a BPD. These results support the hypothesis that some prenatal metabolic alterations may have a key role in the pre-term birth and in the development of BPD in the newborn. Nevertheless, further studies are necessary in order to confirm these preliminary results and to explain the interaction between the maternal-fetal environment and the development of BPD in the newborn. The results of the analysis of the acylcarnitine and the amino acids profile confirm the necessity to define specific normal intervals for every matrix and for each group of different gestational age; moreover, the comparison with results obtained by other Authors reveals the importance that each laboratory have its own reference values, both for diagnostic and research purposes. The use of HPLC-MS/MS method has allowed to complete the carnitine analysis in pre-term and on term newborn children, allowing for the first time the simultaneous analysis of more than 40 acylcarnitines, including some isomeric and isobaric forms. These characteristics and the obtained results make this test suitable for diagnostic purposes, such as second-tier test for expanded newborn screenin

    Sviluppo di un nuovo sistema microfluidico-amperometrico per lo studio della secrezione di dopamina in cellule PC12 utilizzando un microsensore nanostrutturato

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    In this study we developed a microfluidic device for the detection of dopamine (DA) secreted by PC12 cells using microdialysis and constant potential amperometry (CPA). The PC12 cells are used as an in vitro model for the study of age-related diseases associated to an impairment of the movement consequent to a deficit of DA (i.e. Parkinson's disease). The proposed system has been optimized through the use of nanostructured microsensors based on multiwall carbon nanotubes (MWCNT) with a diameter of 15 nm and a length of about 10 µm. The nanotubes are allotropes of carbon with a cylindrical structure, are efficient thermal and electrical conductors and have huge reactive surfaces; for these characteristics they are used as transducers of electrochemical signals. The MWCNT were solubilized and deposited on top of epoxy-based graphite sensors or directly incorporated in the resin itself. The nanostructured microsensors have been calibrated with different concentrations of DA. We conducted several experiments for detecting the secretion of DA induced by drugs such as nicotine and KCl. This system is a rapid and reliable model for the study of the effects of certain drugs on the secretion of DA in vitro. The proposed model may be used for studying different cell types and the DA sensor may be easily replaced with different microsensors or biosensors that allow the in vitro detection of other important molecules, such as nitric oxide, glutamate, glucose and lactate

    Metabolomica: un nuovo approccio per lo studio della predittivitĂ  di farmacoresistenza e di complicanze cardiovascolari in epilessia

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    About 30% of epileptic subjects is “drug resistant” (DR) to mono and multiple treatments. The risk of sudden unexpected death (SUDEP) is more than 20 times higher in epileptic patients than that in the general population; SUDEP represents the main cause of death in patients with refractory epilepsy. Several mechanisms probably exist, but most research has focused on seizure related cardiac arrhythmia. No evidence-based intervention to prevent SUDEP exists. Biological basis of DR are still unclear and applying innovative diagnostic techniques for prediction of outcome in the pharmacotherapy of epilepsy is a mission with important social and economical aspects. Genomic and proteomic are not able to depict the holistic approach that is fundamental for a complete description of phenotiping in human physiology. Metabolomic responses, as integration of genomic and proteomic expressions with the environmental solicitations, can give us the possibility to investigate about complex interactions in the metabolic networks induced by pathologies or by drugs. Data presented in this paper are 1H-NMR spectroscopy measurements performed by our team in our Clinical Metabolomic Labs, University of Cagliari, on blood samples of epileptic patients, previously classified by us as drug-responders or not. Using supervised models we are extracting a metabolic characterization, higher in specificity and sensibility with increasing of number of patient, to realize a profile of drug resistance in epilepsy. In this study we present a PLS_DA model. This is a preliminary model of metabolic alterations in our cohort of DR patients, which could represent a new and non-invasive method for early detection of epileptic patients at risk of sudden death and simultaneously help to clarify the pathophysiological mechanisms of DR and SUDEP

    Collaborazioni pubblico privato e capitale umano nel rilancio dello Spazio Europeo della Ricerca

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    Il lavoro analizza le politiche europee in tema di ricerca e innovazione e commenta le problematiche relative al sistema Italia. In particolare vengono presentati: il tema delle risorse umane, il contesto relativo alla situazione delle pari opportunit\ue0 e gli aspetti dei finanziamenti alla ricerca. Seguendo gli indirizzi europei in tema di \u201cpartnership pubblico privata\u201d viene quindi introdotto il concetto di Distretto Tecnologico e le sue caratteristiche. L\u2019autore, in base alla sua esperienza personale di creazione e gestione di un Distretto Tecnologico, presenta i punti di forza di tali modelli
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