NON-CODING RNAS IN HIGH-GRADE SEROUS EPITHELIAL OVARIAN CANCER

Abstract Introduction: High-grade serous ovarian carcinoma (HGSOC) is the most lethal gynecologic malignancy, mainly because the disease is frequently diagnosed at an advanced stage and is characterized by the early onset of chemoresistant recurrences. The lack of reliable diagnostic and prognostic markers, together with the lack of effective therapies, are the major obstacles to the clinical management of patients with HGSOC. A new class of non-coding RNAs (ncRNAs), such as microRNA (miRNAs) and long non-coding RNAs (LncRNAs), with a function of gene expression regulation, have been discovered to play an important role in human cancers. Increasing evidences suggest that ncRNAs are involved in cancer progression and development of chemoresistance, and support their role as potential diagnostic, predictive and prognostic biomarkers. The hypoxic condition within the tumor microenvironment, improving the tumor neovascularization, represents an essential event contributing to the development of a more aggressive HGSOC phenotype. Recently, a group of miRNAs, termed hypoxia regulated-miRNAs (HRMs), have been identified as key elements in response to hypoxia, regulating important mechanisms involved in tumor progression. The complexity of hypoxia molecular mechanisms has not been fully elucidated yet in HGSOC, therefore there is an urgent need to discover novel biomarkers clinically useful to select patients with hypoxic tumor, that may benefit of tailored treatments. Aims of the study: My PhD project aims at elucidating transcriptional and post-transcriptional signatures characterizing HGSOC, both at the serum and tissue levels. In detail, the research effort includes: i) the investigation of circulating miRNAs as novel potential biomarkers for HGSOC detection; ii) the analysis of mRNA, miRNA and lncRNA expression profiles of HGSOC and normal tissues; iii) the evaluation of hypoxia-regulated miRNA expression in HGSOC and normal tissues. Methods: Sera from 168 HGSOC stage III-IV patients and 65 healthy donors were gathered together from two independent collections and stratified into a training set, for miRNA marker identification, and a validation set, for data validation. Nine synthetic viral/C.Elegans spike-in oligos were added to serum samples before RNA extraction, to allow accurate normalization. miRNA expression profiles were obtained using Agilent Microarray Technologies\uae. An innovative statistical approach for microarray data normalization, based on the contribute of spike-in oligos and the most invariant miRNAs, was developed to identify, in the training set, differentially expressed miRNAs. Signature validation in both the training and validation sets was performed by Real Time quantitative PCR (RT-qPCR) and confirmed by droplet digital PCR (ddPCR). A total of 99 tumor biopsies were collected from HGSOC stage III-IV patients, partially matched with the serum sample cohort (n=76). Thirty normal tissues were obtained from normal ovary (HOSE) and luminal fallopian tube surface epithelia, both representing the normal counterpart for HGSOC, whose histogenesis is still a matter of debate. Gene and miRNA expression profiles were obtained using Agilent Microarray Technologies\uae. miRNA expression levels were correlated with patient outcomes, as overall survival (OS) and progression-free survival (PFS). Additionally, a subgroup of 14 chemo-resistant and 14 chemo-sensitive HGSOC patients, together with 10 normal tissues were deep sequenced for the discovery of novel HGSOC specific coding and non-coding transcripts. Results: A panel of 97 miRNAs emerged significantly differentially expressed (92 up-regulated and five down-regulated) between sera of HGSOC patients and healthy donors by microarray analysis. Among them, the following miRNAs, i.e., miR-1246, miR-595, miR-574-5p, miR-483-3p, miR-4290, miR-2278, miR-32, miR-4281, and miR-3148, exhibiting both the highest average expression and log fold change measured in patients compared to healthy donors, were selected for further validation. miR-1246, miR-595 and miR-2278 were confirmed as significantly over-expressed in serum of HGSOC patients compared to controls by RT-qPCR (all p-values<0.03), in both the training and validation sets. Receiver Operating Characteristic (ROC) curve analysis revealed miR-1246 as the best diagnostic biomarker, with a sensitivity of 87%, a specificity of 77% and an accuracy of 84%. The absolute quantification of circulating miR-1246 by ddPCR confirmed its potential as diagnostic biomarker in HGSOC. Microarray analysis of tissue miRNA profiling revealed a total of 265 miRNAs significantly dysregulated (123 up-regulated and 142 down-regulated) in HGSOC compared to normal tissues. A group of nine miRNAs (i.e., miR-199b-5p, miR-423-5p miR-455-3p, miR-22-3p, miR-199a-3p, miR-15b-5p, miR-140-5p, miR-1246, and miR-320c) were associated with platinum response and prognosis. In particular, among tumor samples, miR-1246 up-regulation was consistently associated with platinum-resistance, poor OS and poor PFS (p-values<0.05). Kaplan-Meier survival curves, according to miR-1246 expression levels obtained by RT-qPCR, showed that OS and PFS decreased in patients with high miR-1246 expression compared to those with low miR-1246 expression (p-value<0.001, HR=2.57; p-value=0.024, HR=1.68; respectively). In addition, multivariate analysis revealed miR-1246 over-expression as an independent prognostic factor for poor OS and PFS (p-value=0.002, HR=2.31; p-value<0.05, HR=1.59; respectively). Interestingly, compared to normal tissues, both with microarray and RT-qPCR techniques, miR-1246 showed a down-regulation compared to HOSEs (p-value<0.0001), but we did not detect a significantly differential expression compared to fallopian tubes. This result mirrors the global miRNA expression trend revealed by principal component analysis (PCA) on microarray data. Subsequently, we focused our analysis on a group of 16 miRNAs belonging to the group of hypoxia-regulated miRNAs (HRMs) emerged from literature as relevant in other solid tumors. Among them, we confirmed miR-210 and miR-27a-3p/23a-3p/24-3p cluster as significantly up-regulated in HGSOC vs normal tissues by RT-qPCR (all p-values 640.002). More interestingly, we validated the significant over-expression of miR-23a-3p in the group of patients resistant to platinum-based chemotherapy compared to platinum-sensitive patients (p-value=0.03). In addition, in univariate survival analysis miR-23a-3p over-expression showed a significant correlation with decreased progression-free survival (p-value=0.009, HR=1.8), but not with overall survival variable. Importantly, miR-23a-3p over-expression has emerged as an independent prognostic marker for shortened progression-free survival in multivariate Cox regression analysis (p-value=0.01, HR=1.78). Finally, the preliminary analysis of the transcriptome sequencing allowed us to identify 1371 transcripts differentially expressed between platinum-resistant and platinum-sensitive samples. Among them, 125 transcripts showed a complete match of intron chain with known transcripts, 686 were potentially novel isoforms or showed a generic overlap with known transcripts. The remaining 560 sequences, if validated, could be novel intergenic transcripts or transcripts with an exonic overlap with reference ones. Conclusions: This study demonstrates, for the first time, miR-1246 as a potential diagnostic serum biomarker in HGSOC, as assessed by three independent technologies (microarray, RT-qPCR and ddPCR) and validated in two independent cohorts of patients. Moreover, high-throughput analysis reveals most of the gene and miRNA dysregulated in HGSOC biopsies compared to the normal counterpart. In particular, our findings indicate, for the first time, that miR-1246 over-expression correlates with a platinum-resistant HGSOC phenotype and may constitute a novel prognostic factor for HGSOC patients. Furthermore, our results regarding HRMs suggest an important role of miRNAs in response to hypoxic conditions within HGSOC. Particularly, the miR-23a-3p over-expression in the group of platinum-resistance patients may contribute to explain the importance of hypoxia in HGSOC mechanism of drug resistance and could represent an independent prognostic marker for HGSOC patients. Lastly, preliminary data emerged from transcriptome analyses, suggesting a prominent non-coding role in HGSOC platinum resistance, will be integrated with gene and miRNA expression profiles previously obtained, with the aim to identify tumor circuits associated with response to treatment and prognosis, as well as to better elucidate the molecular mechanisms characterizing HGSOC progression and adaptation to hypoxic tumor microenvironment

Improving Knowledge of Risk in Dangerous Goods Transport

In order to increase safety as far as dangerous goods transport is concerned, the DESTINATION project has been developed since 2010 in the framework of the Italy/Switzerland Operational Program for Transfrontier Co-operation 2007-2013. The project was born to satisfy the increasing needs of public bodies to share data on hazardous material land transportation and to develop instruments and methodologies to ensure territorial and environmental protection. The project aims to reach this purpose through the increased knowledge of the vulnerable subjects, people and environment, and of the transport activity itself, by using and defining an architecture of data acquisition based on “On Ground Units” (OGU) and “On Board Units” (OBU). These data will be used as an input for a new information system called GIIS (Global Integrated Information System), which manages a risk analysis model of the land transportation of hazardous materials to assess human and environmental vulnerabilities. The GIIS will provide a more effective management of land planning by providing authorities with the possibility of implementing a rational restriction to vehicles transporting dangerous goods within specific areas

A new dataset of global irrigation areas from 2001 to 2015

About 40% of global crop production takes place on irrigated land, which accounts for approximately 20% of the global farmland. The great majority of freshwater consumption by human societies is associated with irrigation, which contributes to a major modification of the global water cycle by enhancing evapotranspiration and reducing surface and groundwater runoff. In many regions of the world irrigation contributes to streamflow and groundwater depletion, soil salinization, cooler microclimate conditions, and altered land-atmosphere interactions. Despite the important role played by irrigation in food security, water cycle, soil productivity, and near-surface atmospheric conditions, its global extent remains poorly quantified. To date global maps of irrigated land are often based on estimates from circa year 2000. Here we apply artificial intelligence methods based on machine learning algorithms to satellite remote sensing and monthly climate data to map the spatial extent of irrigated areas between 2001 and 2015. We provide global annual maps of irrigated land at ≈9km resolution for the 2001-2015 and we make this dataset available online

Arbuscular mycorrhizal fungi as a tool to ameliorate the phytoremediation potential of poplar: biochemical and molecular aspects

Poplar is a suitable species for phytoremediation, able to tolerate high concentrations of heavy metals (HMs). Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with the roots of most land plants; they improve nutrient uptake and enhance phytoextraction of HMs while alleviating stress in the host plant. This review summarizes previous results from field and greenhouse studies conducted by us and dealing with this topic. In a field trial on a highly Zn- and Cu-contaminated site, differences in plant survival and growth were observed among 168 clones originating from natural populations of Populus alba L. and Populus nigra L. from northern Italy. After two and a half years from planting, the density, activity and metabolic versatility of the culturable fraction of the soil bacteria in the HM-polluted field was higher in the soil close to where larger poplar plants were growing, in spite of comparable HM concentrations recorded in these soils. One well-performing clone of P. alba (AL35), which accumulated a higher concentration of both metals and had high foliar polyamine (PA) levels, was used for further investigation. In a greenhouse study, AL35 cuttings pre-inoculated with AMF (Glomus mosseae or Glomus intraradices) and then transferred to pots containing soil, collected from the HM-polluted site, displayed growth comparable to that of controls grown on unpolluted soil, in spite of higher Cu and Zn accumulation. Such plants also showed an overall up-regulation of metallothionein (MT) and PA biosynthetic genes, together with increased PA levels. A genome-wide transcriptomic (cDNA-AFLP) analysis allowed the identification of a number of genes, mostly belonging to stress-related functional categories of defense and secondary metabolism, that were differentially regulated in mycorrhizal vs. non mycorrhizal plants. A proteomic analysis revealed that, depending on sampling time, changes in protein profiles were differentially affected by AMF and/or HMs. It is concluded that soil-borne microorganisms affect plant performance on HM-polluted soil. In particular, mycorrhizal plants exhibited increased capacity for phytostabilization of HMs, together with improved growth. Their greater stress tolerance may derive from the protective role of PAs, and from the strong modulation in the expression profiles of stress-related genes and proteins

Process Control Parameters Evaluation Using Discrete Event Simulation for Business Process Optimization

The quest for manufacturing process improvement and higher levels of customer satisfaction mandates that organizations must be equipped with advanced tools and techniques in order to respond towards ever changing internal and external customer demands by maintaining the optimal process performance, lower cost and higher profit levels. A manufacturing process can be defined as a collection of activities designed to produce a specific output for a particular customer or market. To achieve internal and external objectives, significant process parameters must be identified and evaluated to optimize the process performance. This even becomes more important to deal with fierce competition and ever changing customer demands. This paper illustrates an integrated approach using design of experiments techniques and discrete event simulation (Simul8) to understand and optimize the system dynamic based on operational control parameter evaluation and their boundary conditions. Further, the proposed model is validated using a real world manufacturing process case study to optimize the manufacturing process performance. Discrete event simulation tool is used to mimic the real world scenario, which provides a flexible and powerful way to comprehensively understand the manufacturing process variations and allows controlled 'What-If´ analysis based on design of experiments approach. Finally, this paper discusses the potential applications of the proposed methodology in the cable industry in order to optimize the cable manufacturing process by regulating the operational control parameters such as dealing with various product configurations with different equipment settings, different product flows and work in process (WIP) space limitations

Impact of Phosphatic Nutrition on Growth Parameters and Artemisinin Production in Artemisia annua Plants Inoculated or Not with Funneliformis mosseae

Artemisia annua L. is a medicinal plant appreciated for the production of artemisinin, a molecule used for malaria treatment. However, the natural concentration of artemisinin in planta is low. Plant nutrition, in particular phosphorus, and arbuscular mycorrhizal (AM) fungi can affect both plant biomass and secondary metabolite production. In this work, A. annua plants were inoculated or not with the AM fungus Funneliformis mosseae BEG12 and cultivated for 2 months in controlled conditions at three different phosphatic (P) concentrations (32, 96, and 288 µM). Plant growth parameters, leaf photosynthetic pigment concentrations, artemisinin production, and mineral uptake were evaluated. The different P levels significantly affected the plant shoot growth, AM fungal colonization, and mineral acquisition. High P levels negatively influenced mycorrhizal colonization. The artemisinin concentration was inversely correlated to the P level in the substrate. The fungus mainly affected root growth and nutrient uptake and significantly lowered leaf artemisinin concentration. In conclusion, P nutrition can influence plant biomass production and the lowest phosphate level led to the highest artemisinin concentration, irrespective of the plant mineral uptake. Plant responses to AM fungi can be modulated by cost–benefit ratios of the mutualistic exchange between the partners and soil nutrient availability