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Study of DNA methylation dynamics in a model of differential developmental competence in the ovine species
No full textDNA methylation is an important epigenetic mark that plays a role in gene regulation by the addition of a methyl group to CpGs in the DNA. Despite being relatively stable in somatic cells, DNA methylation is subject to reprogramming during embryo development and gametogenesis. Aim of this work was to evaluate different aspects of DNA methylation in relation to oocyte quality in the ovine species. A model of differential developmental competence consisting in ovine oocytes and in vitro produced (IVP) embryos derived from prepubertal (P) and adult (A) donors was used. The methylation dynamics were analysed in terms of gene expression of a panel of genes involved in DNA methylation reprogramming (DNA methyltransferases (DNMTs), ten-eleven translocation dioxygenases (TET) and methylbinding proteins (DMB) and immunolocalization of TET3 protein, 5-methylcytosine (5-mc) and 5-hydroxymethylcitosine (5-hmc). Gene relative quantification was performed by RNA reverse transcription followed by Real-Time PCR. Pools of growing oocytes at different diameter (70/90 µm, 90/110 µm, 110/130 µm), germinal vesicles (GV), IVM metaphase II (MII) oocytes, IVP embryos at two (2C) and four cell (4C) stage and blastocysts derived from P and A donors were analysed. The analysis in growing oocytes revealed that DNMT1, DNMT3B, TET1 and TET3 transcript abundance increases in relation to oocyte diameter, suggesting that synthesis and storage occur during the final stage of oocyte growth. P-GV oocytes present lower expression level of TET1, TET2 and TET3 (ANOVA; P< 0.05). DNMT1 expression is lower in 4C embryos from prepubertal oocytes and MBD1 and MBD3 expression decreases during the transition from 2C to 4C stage in embryos derived from P-animals. No significant differences were observed in embryos at the blastocyst stage (ANOVA; P>0.05). These observations confirm different gene expression patterns in our model.
Immunolocalization was performed by direct immunofluorescence. TET3 immunostaining revealed the presence of the protein only in the cytoplasm of early embryos (2C and 4C) from both P and A animals. The analysis of global DNA methylation showed high levels in all analyzed samples. Interestingly, pronuclear (PN) stage zygotes displayed a decrease in 5-mC signal in male PN compared to maternal PN from both young and adult animals; conversely, hydroxymethylation signal was scattered evident throughout the chromatin of all analyzed samples; the analysis of zygotes revealed 5-hmc signal only in male PN. Observations demonstrate that male PN undergoes a decrease in methylation and an increase in DNA hydroxylation suggesting that also in sheep an active DNA demethylation occurs soon after fertilization. Our results are in accordance with the recent hypothesis that the acquisition of epigenetic modifications may be involved in the gaining of oocyte competence during its growth. They indicate that epigenetic mechanisms may affect oocyte quality and pave the way to better understanding methylation dynamics during sheep pre-implantation development
Application of support vector machines as support to early prediction of mastitis in Sarda dairy ewes
No full textMastitis is one of the major reasons for the reduction of both milk yield and quality with consequent strong economic losses.
The Sardinian dairy ewe industry consists of 2,400,000 ewes, which is of relevant economic importance for the Island (SRDP 2007 - 2013).
In normal standard hygienic conditions of ewe farm, the prevalence of clinical mastitis is about 5%. Unfortunately it represents the tip of the iceberg. The submerge part of the iceberg is represented by subclinical mastitis, which in some cases can reach 65% of the flock (Fthenakis and Jones, 1990).
The economic loss due to mastitis is estimated around 69 NZ$ (≈ 42 €) per infected half-udder per entire period of lactation (Cuccuru et al., 2011).
The current investigation presents an alternative approach to predict the presence of udder inflammation in Sarda dairy ewes through the application of Support Vector Machines (SVMs), a sub-discipline in the field of artificial intelligence. The milk lactose content (MLC) and milk electrical conductivity (MEC) are used as predictive variable and the milk somatic cell count (MSCC) as classifier. Data used was collected from a 10-years historical database of ARAS laboratory (Sardinian Regional Association of Farmers). The SVMs has shown a sensitivity and specificity of 62% and 75% respectively. This method could therefore to be suggested as first screening system for udder inflammation detection before carrying out expensive and time-consuming bacteriological analysis
Se Hace camino al andar: architettura in Italia e Spagna nel secondo dopoguerra
No full textAfter the Second World War, Italian and Spanish architects evolve the International Style to explore new possibilities for architecture. They rethink and renovate the topics and the fundamental elements of the project with the aim of establishing a new relationship among architecture and users, places and traditions. They do not have an explicit programme but they just have a common cultural background and references made of rationalism, vernacular architecture and scandinavian architecture.
This thesis is an investigation about the new architecture developed in the Postwar Period by Italian and Spanish architects like Gardella, Ridolfi, Albini, Moretti in Italy and Coderch, Cabrero, De la Sota, Bohigas in Spain.
The investigation studies the projects of the authors of that period and wants to define the topics and the elements of this architectural attitude. The aim is to demonstrate the common effort of Italy and Spain to explore a new architectural field
Two case studies on plant genetic diversity: comparison of two historic collections of barley landraces and characterization of a common bean segregant population for domestication traits
No full textIn this study we studied the change in the time of population of barley and in second chapter we analisys the traits of Syndrome Domestication in common bean
Sviluppo e studio di biosensori sostenibili e performanti dedicati alla determinazione di composti nutraceutici e contaminanti di interesse agroalimentare
No full textThe ability of a biosensor to respond to an analyte lies in the nature of the biological component. Based on the evidence that aflatoxin is able to inhibit the enzymatic activity of the acetyl cholinesterase enzyme (ACE) (Arduini et al., 2007), an amperometric biosensor based on enzymes ACE and choline oxidase has been developed. The biosensor was tested with different mixtures of mycotoxins (aflatoxin, patulin, type B trichothecene and ochratoxin) and reported the presence of aflatoxins and patulin in a biological matrix (Chapter 1), showing different sensitivity to the two mycotoxins.
In parallel, new chemical components from sustainable sources were synthesized and evaluated in order to improve the performance of the biosensor. To solve the problem of the interfering signals present in the matrix, such as ascorbic acid (AA) and dopamine (DA), the transducer is commonly covered with polymeric films derived from orthofenilendiammine (PPD) (Killoran and O'Neill, 2008), a monomer toxic and mutagenic (Murata et al., 2006). In a first step, the monomers chosen as candidates for obtaining new permselctive films were represented by non-toxic phenols, naturally occurring in nature, such as eugenol, isoeugenol, magnolol and dehydrodieugenol. Among these, for practical reason, dehydrodieugenol was obtained through chemical synthesis starting from eugenol. Molecules were elettropolymerized using two techniques: constant potential amperometry and cyclic voltammetry. The polymers were characterized through permselectivity studies and Scanning Electron Microscopy (SEM); the studies were repeated over time to assess the stability of obtained polymers. High electropolymerization potentials were used, in order to overoxidate polymers and enhance charge repulsion characteristics of the polymer. Through the calculation of the permselectivity parameter (S%), permselective properties of the polymer films were quantified. The poly-magnolol proved the best alternative for PPD and it was included in the construction of a biosensor based on the enzyme glucose oxidase (Chapter 2).
The next step’s study of alternative polymers from natural monomers comprised the addition of a further molecule, guaiacol. This compound presents a simpler structure compared to the other molecules studied, being constituted of one guaiacyl unit. Taking into account the significant changes in permselectivity imparted by the polymerization conditions applied, in this part of the work the different phenols were polymerized by constant potential amperometry exploring a discrete range of electropolymerization potentials. Each phenol was subject to four different potentials and the characteristics of the resulting film were observed compared to H2O2, ascorbic acid and dopamine, and accompanied by images obtained by scanning electron microscopy (SEM). The experiments showed that the permeability characteristics are strongly related to the potential applied during polymerization; also permselectivity values were improved compared to previously reported work (Chapter 3).
Several molecules, that can alter the characteristics of βCD, have been selected in order to strengthen and widen βCD use in biosensors. The introduction of a bridge, created by a covalent bond between a linker molecule and two βCD, can enhance the ability of inclusive power of macrocycle; also the nature of the linker may make new functional groups and / or charges that help the interaction with the enzyme. A good attachment site is represented by the primary OH present on C6 of βCD, where, under certain conditions, can take place an esterification reaction between linker and βCD. As linker molecules have been used the polycarboxylic acid (PCA), the glyceroldiglycidilether (GDGE) and dimethyl carbonate (DMC). The products of the reactions were examined by means of nuclear magnetic resonance (NMR). Under the experimental conditions used, the reaction between the anhydride of 1,2,3,4-butanetetracarboxylic acid (BTCAa) and the βCD shows the formation of a mixture of different complexes. The complex was tested with carvacrol, a molecule that easily forms stable complexes with the βCD. The strong interaction BTCA-βCD has prevented the formation of the βCD-carvacrol complex and esterification between BTCAa and βCD (Chapter 4).
To include the βCD during electropolymerization various procedures were developed to obtain complexes between βCD and phenols. The βCD-eugenol complex, obtained in 85% of yield, was characterized by NMR spectroscopy; the analysis in D2O show a modification of the signal of the H5 βCD and the presence of well resolved eugenol signals in solution. Also the formation of βCD-guaiacol complex was confirmed by NMR with a yield of 47%. Repeated NMR analysis have confirmed a long-term stability for at least 2 weeks. Different conditions of complexation, all sustainable, including the technology to microwaves, have not been successful in obtaining a βCD- magnolol inclusion complex (Chapter 4).
The properties of a sulfur-containing phenol (S-PhOH) derivative product from Chemiplastica Specialties SpA were evaluated. The formation of strong complexes between S-PhOH and the βCD was studied by NMR spectroscopy under different experimental conditions, such as microwave, coprecipitation, sonication and solvent evaporation under vacuum (Rotavapor). These complexes are highly soluble and stable in water. The electro-deposition of S-PhOH is able to modify a platinum electrode imparting interesting characteristics towards ascorbic acid presence (Chapter 5)
The Seagrass effect turned upside down changes the prospective of sea urchin survival and landscape implications
No full textHabitat structure plays an important mediating role in predator-prey interactions. However the effects are strongly dependent on regional predator pools, which can drive predation risk in habitats with very similar structure in opposite directions. In the Mediterranean Sea predation on juvenile sea urchins is commonly known to be regulated by seagrass structure. In this study we test whether the possibility for juvenile Paracentrotus lividus to be predated changes in relation to the fragmentation of the seagrass Posidonia oceanica (four habitat classes: continuous, low-fragmentation, high-fragmentation and rocks), and to the spatial arrangement of such habitat classes at a landscape scale. Sea urchin predation risk was measured in a 20-day field experiment on tethered individuals placed in three square areas 35×35 m2 in size. Variability of both landscape and habitat structural attributes was assessed at the sampling grain 5×5 m2. Predation risk changed among landscapes, as it was lower where more ‘rocks’, and thus less seagrass, were present. The higher risk was found in the ‘continuous’ P. oceanica rather than in the low-fragmentation, high-fragmentation and rock habitats (p-values = 0.0149, 0.00008, and 0.0001, respectively). Therefore, the expectation that juvenile P. lividus survival would have been higher in the ‘continuous’ seagrass habitat, which would have served as shelter from high fish predation pressure, was not met. Predation risk changed across habitats due to different success between attack types: benthic attacks (mostly from whelks) were overall much more effective than those due to fish activity, the former type being associated with the ‘continuous’ seagrass habitat. Fish predation on juvenile sea urchins on rocks and ‘high-fragmentation’ habitat was less likely than benthic predation in the ‘continuous’ seagrass, with the low seagrass patch complexity increasing benthic activity. Future research should be aimed at investigating, derived from the complex indirect interactions among species, how top-down control in marine reserves can modify seagrass habitat effects
Emerging pests and diseases threaten <i>Eucalyptus camaldulensis</i> plantations in Sardinia, Italy
No full textThe rapid growth and environmental adaptability of Eucalyptus species has favored their global cultivation for pulpwood production. On the island of Sardinia, Italy, eucalypt plantations were established in the 20th century primarily in areas reclaimed from marshland, but the trees are now grown all over the island as ornamentals or windbreaks, and for timber, pulp and honey production. In recent years, an unusual decline and mortality of unknown etiology has been observed in Eucalyptus camaldulensis (river red gum) plantations throughout the island. Given the ecological and economic importance of eucalypt ecosystems in Sardinia, a survey was carried out in 2013 to determine which insect pests and fungal pathogens are directly involved in these phenomena. Field surveys throughout the island revealed severe infestations with the red gum lerp psyllid (Glycaspis brimblecombei) at all 12 surveyed sites, with the greatest numbers of pre-imaginal stages and adults occurring between May and July. The adult population reached its peak in July, followed 2 months later by the peak population of its specific parasitoid, Psyllaephagus bliteus. Symptoms of leaf chlorosis, crown thinning, shoot and branch dieback, sunken cankers, epicormic shoots and exudations of kino gum were also observed at the 12 field sites. Symptomatic woody samples yielded fungal isolates representing three distinct families: Botryosphaeriaceae, Diaporthaceae and Valsaceae. Morphological and DNA sequence data revealed seven distinct fungal species, namely Diaporthe foeniculina, Neofusicoccum australe, N. luteum, N. mediterraneum, N. parvum, N. vitifusiforme and Valsa fabianae. Two putative new species of Cytospora were also identified. Neofusicoccum australe was the only species recovered from all 12 sites, with isolation frequencies of 51-95%. Pathogenicity trials revealed that all Neofusicoccum species except N. vitifusiforme are directly involved in the etiology of the observed decline in the E. camaldulensis population on Sardinia
Use of BEST Procedure to assess soil physical quality in the Baratz Lake catchment (Sardinia, Italy)
No full textConversion of Mediterranean maquis and/or natural forest into agro-pastoral lands is a cause of soil degradation in many Mediterranean areas. Indicators of soil physical quality (SPQ) quantitatively linked to soil hydraulic
properties are a valuable tool to assess the effect of land use changes. In this investigation, the Beerkan Estimation of Soil Transfer parameters (BEST) procedure for soil hydraulic characterization was used to estimate SPQ
indicators. Four areas of the Baratz Lake watershed, Sardinia, Italy, characterized by both typical natural vegetation (holm oak [Quercus ilex L.] forest and high maquis) and degraded vegetation (grassland established
after fire or clearing of the maquis) were considered. The SPQ was assessed by either independently measured soil physical attributes, like soil bulk density, organic C content, saturated hydraulic conductivity, and sorptivity, and “capacitive” and “dynamic” indicators calculated from the water retention curve estimated by the BEST procedure. Measured and estimated SPQ indicators unanimously showed that clearing of the maquis caused a severe deterioration of SPQ associated with soil compaction, organic matter loss,
and decrease of macropore volume and soil aeration capacity as well as reduced water circulation. A different and unexpected result was obtained for the fire-affected area, where the SPQ was comparable to that of the neighboring
oak forest area. We deduced that vegetation restoration after fire passage contributed to maintain a high organic matter content and to mitigate rain compaction effects. We concluded that SPQ indicators derived by applying the BEST procedure are suitable to detect land degradation in the
natural environments studied
The Novel cannabinoid antagonist SM-11 reduces hedonic aspect of food intake through a dopamine-dependent mechanism
No full textCannabinoids, endogenous and exogenously administered, are known to positively regulate food intake and energy balance. Since CB1 receptor antagonists reduce food intake and antagonize overweight, we developed a new CB1 receptor antagonist in an attempt to identify a compound with potential application in overeating disorders. The newly developed SM-11 compound dose-dependently decreases food intake in rats by 15–20%. Moreover, SM-11 reduces self-administration of palatable food in both food restricted and ad libitum fed rats, suggesting an action on the hedonic component of food intake. Thus, we next tested the effect of SM-11 on the stimulating properties of the CB1 receptor agonist WIN55,212-2 (WIN) on the electrophysiological activity of Nucleus Accumbens-projecting dopaminergic neurons of the ventral tegmental area (VTA). SM-11 fully and readily antagonized the WIN-induced increments in single spiking and burst firing of antidromically-identified dopamine neurons. When administered to naïve (no WIN-pretreated) rats, SM-11 did not alter basal neuronal activity, thereby suggesting a pure antagonistic profile. SM-11 thus appears as a promising candidate in the search of potential anti-obesity medications