DEVELOPMENT OF AN INTEGRATED IN SILICO STRATEGY FOR THE RISK ASSESSMENT OF CHEMICALS AND THEIR MIXTURES ON DIFFERENT TOXICOLOGICAL OUTCOMES.

Daily, we are exposed to a mixture of multiple chemicals via food intake, inhalation and dermal contact. The risk for health that may result from this depends on how the effects of different chemicals in the mixture combine, and whether there is any synergism or antagonism between them. The number of different combinations of chemicals in mixtures is infinite and an efficient test strategy for mixtures is lacking. Furthermore, there is social pressure to reduce animal testing, which is the current practice in safety testing of chemicals. In this context, computational biochemistry and, more in general, bioinformatics meets all the requirements, and provides the foundation for further in vitro or in vivo studies. Aim of this PhD thesis is the development of an in silico workflow able to prioritize and discriminate chemicals that act as endocrine active substances (EAS), interfere with the retinoic acid pathway during embryo development and/or may cause liver toxicity. From the observation of the molecular initiating event to the description of the adverse outcome pathway, both ligand- and structure-based approaches were integrated with systems biology. Within this framework, (Q)SAR and molecular docking results were mixed into a majority consensus score to rank chemicals and low-mode molecular dynamic simulations were used to study their intrinsic activity, with respect to a specific nuclear receptor. Moreover, a computational approach based on both the transition state and the density functional theories was used to try discriminating a subset of chemicals as inhibitors or substrates of particular enzymes involved in the retinoic acid pathway, computing also their binding free energy values. This information was also included both in the pharmaco-dynamics (PD) and in the physiological based pharmaco-kinetics (PBPK) models. This in silico pipeline, besides being faster, has economic and ethical advantages, reducing both the research costs and the number of involved animals, in agreement with the \u201c3R\u201d principles (Reduction, Refinement and Replacement)

Literature search – Exploring in silico protein toxicity prediction methods to support the food and feed risk assessment

This report is the outcome of an EFSA procurement (NP/EFSA/GMO/2018/01) reviewing relevant scientific information on in silico prediction methods for protein toxicity, that could support the food and feed risk assessment. Several proteins are associated with adverse (toxic) effects in humans and animals, by a variety of mechanisms. These are produced by plants, animals and bacteria to prevail in hostile environments. In the present report, we present an integrated pipeline to perform a comprehensive literature and database search applied to proteins with toxic effects. \u201cToxin activity\u201d and \u201ctoxin-antitoxin system\u201d strings were used as inputs for this pipeline. UniProtKB was considered as the reference database, and only the UniProtKB curator-reviewed proteins were considered in the pipeline. Experimentally- determined structures and homology-based in silico 3D models were retrieved from protein structures repositories; family-, domain-, motif- and other molecular signature-related information was also obtained from specific databases which are part of the InterPro consortium. Protein aggregation associated with adverse effects was also investigated using different search strategies. This work can serve as the basis for further exploring novel risk assessment strategies for new proteins using in silico predictive methods

The effects of aging on thyroxine and cortisol responses to low ambient temperatures and on circadian rhythm of cortisol in the dog

Call number: LD2668 .T4 1985 P34Master of Scienc

Reducing Smooth Sumac Dominance in Native Tallgrass Prairie

Smooth sumac (Rhus glabra L.) is a resprouting shrub native to the tallgrass prairie region that increases in density without an active disturbance regime. Our objective was to use prescribed fire and herbicides to decrease smooth sumac density as a strategy to improve a degraded tall grass prairie remnant. In two separate experiments repeated in space and time, we used prescribed fire in combination with herbicides at various rates and two application methods to develop an effective management scheme for reducing smooth sumac. We used a randomized complete block design with 13 herbicide treatments and a control with three replicates in burned and non-burned areas. Results were similar in both experiments in which herbicide treatment and burning were the significant main effects. All herbicide treatments reduced smooth sumac stem density compared to the control, but no distinct advantage was detected regarding specific herbicide, application rate, or whether the herbicide was applied as a broadcast spray or with a hand-held wick. We expected burning to make the plant more susceptible to herbicides, but burning increased stem density. In this tallgrass prairie remnant, we determined that herbicides were the most effective management tool in reducing smooth sumac stem density

Gene Expression and the Diversity of Identified Neurons

Nervous systems consist of diverse populations of neurons that are anatomically and functionally distinct. The diversity of neurons and the precision with which they are interconnected suggest that specific genes or sets of genes are activated in some neurons but not expressed in others. Experimentally, this problem may be considered at two levels. First, what is the total number of genes expressed in the brain, and how are they distributed among the different populations of neurons? Second, can we identify specific genes expressed in individual neurons and relate the expression of these genes to the unique functional properties of these neurons

Novel insights into the transport mechanism of the human amino acid transporter LAT1 (SLC7A5) : probing critical residues for substrate translocation

BACKGROUND: LAT1 (SLC7A5) is the transport competent unit of the heterodimer formed with the glycoprotein CD98 (SLC3A2). It catalyzes antiport of His and some neutral amino acids such as Ile, Leu, Val, Cys, Met, Gln and Phe thus being involved in amino acid metabolism. Interestingly, LAT1 is over-expressed in many human cancers that are characterized by increased demand of amino acids. Therefore LAT1 was recently acknowledged as a novel target for cancer therapy. However, knowledge on molecular mechanism of LAT1 transport is still scarce. METHODS: Combined approaches of bioinformatics, site-directed mutagenesis, chemical modification, and transport assay in proteoliposomes, have been adopted to unravel dark sides of human LAT1 structure/function relationships. RESULTS: It has been demonstrated that residues F252, S342, C335 are crucial for substrate recognition and C407 plays a minor role. C335 and C407 cannot be targeted by SH reagents. The transporter has a preferential dimeric structure and catalyzes an antiport reaction which follows a simultaneous random mechanism. CONCLUSIONS: Critical residues of the substrate binding site of LAT1 have been probed. This site is not freely accessible by molecules other than substrate. Similarly to LeuT, K+ has some regulatory properties on LAT1. GENERAL SIGNIFICANCE: The collected data represent a solid basis for deciphering molecular mechanism underlying LAT1 function

Modified Atmosphere Packaging and low temperature storage extend marketability of cherimoya (Annona cherimola Mill.)

Cherimoya is a subtropical fruit characterized by a delicious, sweet flavor and beneficial health properties, which found suitable growing conditions in the South of Italy. However, the marketing of this product is halted by its high perishability, which limits the shelf-life of the fresh fruit to few days after harvest and does not allow for commercialization beyond local markets. Studies have shown that storage of this fruit in controlled atmosphere, using Modified Atmosphere Packaging technologies, extended the post-harvest life of Cherimoya, but little is still known about the evolution of its sensory, nutraceutical and microbiological characteristics during such storage period. In this paper, we studied the effect of a 4-days long active-MAP (30% CO2 – 10% O2 – 60% N2) storage period, associated with cold temperatures, on the physico-chemical, sensory, nutraceutical and microbiological quality traits of Italian-grown cherimoya fruits, compared with passive-MAP (Air composition, 21% O2 + 1% CO2 + 78% N2) and simple cold storage. Active-MAP proved effective in delaying the reaching of the optimal consumption point until 10 days from harvest, besides showing absence of microbial growth until after 7 days from harvest. Both active- and passive-MAP treatments maintained better nutraceutical values than control until the end of the trial period, and sensory analysis confirmed that active-MAP treated fruits were at the optimal commercial stage after 10 days from harvest

Metabolic responses of microbial community in soil amended with fresh leaves and leaf extracts from eucalyptus spp.

Field observations reveal that often large areas of soil surface beneath Eucalyptus occidentalis Endl. are completely bare or with scant vegetation. Moreover, previous studies have showed that other species of Eucalyptus, such as Eucalyptus camaldulensis Dehn, may be effective in suppressing seed germination and weed growth. Such effects have been ascribed to the large number of secondary metabolites within Eucalyptus leaves. Due to their inhibition activity against weeds, leaf extracts might be used for an integrated weed management context, in accordance with the Directive 2009/128/EC. Several studies exist about the effects of Eucalyptus leaf extracts on weeds, whereas they are lacking on soil microorganisms. Therefore, the aim of this study was to assess the effects of Eucalyptus leaf extracts on soil microbial biomass and activity, as well as on the relative abundance of main microbial groups. The extracts were obtained by hydrodistillation [essential oils (EOs) and hydrolates] and by water cold extraction (aqueous extracts) from leaves of both E. camaldulensis and E. occidentalis. Also, fresh leaves were tested. The soil was spread with EOs at doses of 2.64 or 3.52 \uf06dl g-1. They were applied by adding a proper water solution containing the given amount of EOs to 350 g of dry soil, so reaching 50% of its water holding capacity. The above solution held the emulsifier Fitoil at 0.5 mL L-1. Fresh leaves, dried at 40\ub0C for 48 hours, were chopped and applied at doses of 6.6 and 5.0 mg g-1 of dry soil for E. camaldulensis and E. occidentalis, respectively. Such litter application doses were those found, at field conditions (780 and 575 g m-2, respectively). Two controls were also prepared: one with water and another with water and Fitoil. After the addition of all the treatments, soils were incubated at room temperature (20-23\ub0C) in the dark for 35 days. At days 7 and 35, soils were analyzed for soil microbial biomass C and N. At the same days, the main microbial groups were investigated through the ester linked fatty acids (FAMEs). Moreover, 20 g of soil were incubated as above in 200 mL jars to determine the soil respiration rate (CO2 emission) during 36 days of incubation. Preliminary results showed, that EOs from the two species and at both doses exerted a significant biocidal action on soil microorganisms, while hydrolates, aqueous extracts and fresh leaves stimulated both microbial biomass and activity