A altura de dossel e a adubação nitrogenada da pastagem podem afetar a nutrição nitrogenada do milho, num sistema de integração lavoura pecuária?

We conducted a field experiment near Abelardo Luz, Santa Catarina, from October 2012 to April 2014, to evaluate the effect interaction of nitrogen fertilization and height of canopy over the N nutrition of corn subsequently grown to pasture. The data belonging to this thesis are related to the first two production cycles obtained in sorghum pasture (2012/2013), oat (2013) and corn crop (2013/2014). In the evaluation of forage sorghum and oat it was used the same experimental design, consisting of randomized complete block in a factorial arrangement (2 x 2) with three replications. The first factor was considered canopy height (Low and High) and the second factor was the fertilization of cover crop pasture (0 and 200 kg N.ha-1). In phase I and II, the combination of factors evaluators were prepared in the same experimental unit. For corn crop the design was a randomized complete block in a factorial design (2X2X4X6) with three replications. Factors considered in corn were: canopy height of pasture (Low and High), nitrogen application times (NG - nitrogen in the grains and NP - nitrogen in pasture), nitrogen fertilization in corn (0, 100, 200 and 300 kg N.ha-1) and time (46, 53, 60, 67, 76 and 103 days after sowing the maize). In phases I and II, in general the use of N in the pasture increased the productive potential of the pasture and animal management and canopy height has changed the dynamics of structural components and botanical pasture. In cold conditions for long periods and not acclimatized plants the adoption of high nitrogen fertilization and height high grazing pasture leave vulnerable to damage caused by the freezing of plants. The anticipation of nitrogen fertilization on pasture positively affects the corn crop by increasing the accumulated dry matter and N content in the plant. Nitrogen nutritional content of corn with the anticipation of fertilization in pasture is suitable for obtaining high crop production in integrated crop-livestock system. When used nitrogen only coverage in corn sufficiency level in the nitrogen nutrition is achieved with the use of 100 kg N.ha-1. With the use of 200 kg N ha -1 NG and NP no difference in nitrogen content and nitrogen nutrition index.

In vitro studies of drug transformations: application to forensic toxicology

The forensic toxicologist faces challenges in the detection of drugs and poisons in biological samples due to transformations which occur both during life and after death. For example, changes can result from drug metabolism during life or from the use of formalin solution for post mortem embalming purposes. The former requires the identification of drug metabolites and the latter the identification of chemical reaction products in order to know which substances had been administered. The work described in this thesis was aimed at providing ways of tackling these challenges and was divided into two parts. Part 1 investigated the use of in vitro drug metabolism by human liver microsomes (HLM) to obtain information on drug metabolites and Part 2 investigated the chemical reactions of drugs and a carbamate pesticide with formalin solution and formalin-blood. The initial aim of part I was to develop an in vitro metabolism method using HLM, based on a literature review of previous studies of this type. MDMA was chosen as a model compound to develop the HLM method because its metabolism was known and standards of its metabolites were commercially available. In addition, a sensitive and selective method was developed for the identification and quantitation of hydrophilic phase I drug metabolites using LC/MS/MS with a conventional reverse-phase (C18) column. In order to obtain suitable retention factors for polar drug metabolites on this column, acetyl derivatives were evaluated for converting the metabolites to more lipophilic compounds and an optimal separation system was developed. Acetate derivatives were found to be stable in the HPLC mobile phase and to provide good chromatographic separation of the target analytes. In vitro metabolism of MDMA and, subsequently, of other drugs involved incubation of 4 µg drug substance in pH 7.4 buffer with an NADPH generating system (NGS) at 37oC for 90 min with addition of more NGS after 30 min. The reaction was stopped at 90 min by the addition of acetonitrile before extraction of the metabolites. Acetate derivatives of MDMA metabolites were identified by LC/MS/MS using multiple reaction monitoring (MRM). Three phase I metabolites (both major and minor metabolites) of MDMA were detected in HLM samples. 3,4-dihydroxy-methamphetamine and 4-hydroxy-3-methoxymethamphetamine were found to be major metabolites of MDMA whereas 3,4-methylenedioxyamphetamine was found to be a minor metabolite. Subsequently, ten MDMA positive urines were analysed to compare the metabolite patterns with those produced by HLM. An LC/MS method for MDMA and its metabolites in urine samples was developed and validated. The method demonstrated good linearity, accuracy and precision and insignificant matrix effects, with limits of quantitation of 0.025 µg/ml. Moreover, derivatives of MDMA and its metabolites were quantified in all 10 positive human urine samples. The urine metabolite pattern was found to be similar to that from HLM. The second aim of Part 1 was to use the HLM system to study the metabolism of some new psychoactive substances, whose misuse worldwide has necessitated the development of analytical methods for these drugs in biological specimens. Methylone and butylone were selected as representative cathinones and para-methoxyamphetamine (PMA) was chosen as a representative ring-substituted amphetamine, because of the involvement of these drugs in recent drug-related deaths, because of a relative lack of information on their metabolism, and because reference standards of their metabolites were not commercially available. An LC/MS/MS method for the analysis of methylone, butylone, PMA and their metabolites was developed. Three phase I metabolites of methylone and butylone were detected in HLM samples. Ketone reduction to β-OH metabolites and demethylenation to dihydroxy-metabolites were found to be major phase I metabolic pathways of butylone and methylone whereas N-demethylation to nor-methylone and nor-butylone were found to be minor pathways. Also, demethylation to para-hydroxyamphetamine was found to be a major phase I metabolic pathway of PMA whereas β-hydroxylation to β-OH-PMA was found to be a minor pathway. Formaldehyde is used for embalming, to reduce decomposition and preserve cadavers, especially in tropical countries such as Thailand. Drugs present in the body can be exposed to formaldehyde resulting in decreasing concentrations of the original compounds and production of new substances. The aim of part II of the study was to evaluate the in vitro reactions of formaldehyde with selected drug groups including amphetamines (amphetamine, methamphetamine and MDMA), benzodiazepines (alprazolam and diazepam), opiates (morphine, hydromorphone, codeine and hydrocodone) and with a carbamate insecticide (carbosulfan). The study would identify degradation products to serve as markers for the parent compounds when these were no longer detectable. Drugs standards were spiked in 10% formalin solution and 10% formalin blood. Water and whole blood without formalin were used for controls. Samples were analysed by LC/MS/MS at different times from the start, over periods of up to 30 days. Amphetamine, methamphetamine and MDMA were found to rapidly convert to methamphetamine, DMA and MDDMA respectively, in both formalin solution and formalin blood, confirming the Eschweiler-Clarke reaction between amine-containing compounds and formaldehyde. Alprazolam was found to be unstable whereas diazepam was found to be stable in both formalin solution and water. Both were found to hydrolyse in formalin solution and to give open-ring alprazolam and open-ring diazepam. Other alprazolam conversion products attached to paraformaldehyde were detected in both formalin solution and formalin blood. Morphine and codeine were found to be more stable than hydromorphone and hydrocodone in formalin solution. Conversion products of hydromorphone and hydrocodone attached to paraformaldehyde were tentatively identified in formalin solution. Moreover, hydrocodone and hydromorphone rapidly decreased within 24 h in formalin blood and could not be detected after 7 days. Carbosulfan was found to be unstable in formalin solution and was rapidly hydrolysed within 24 h, whereas in water it was stable up to 48 h. Carbofuran was the major degradation product, plus smaller amounts of other products, 3-ketocarbofuran and 3-hydrocarbofuran. By contrast, carbosulfan slowly hydrolysed in formalin-blood and was still detected after 15 days. It was concluded that HLM provide a useful tool for human drug metabolism studies when ethical considerations preclude their controlled administration to humans. The use of chemical derivatisation for hydrophilic compounds such as polar drug metabolites for analysis by LC/MS/MS with a conventional C18 column is effective and inexpensive, and suitable for routine use in the identification and quantitation of drugs and their metabolites. The detection of parent drugs and their metabolites or conversion and decomposition products is potentially very useful for the interpretation of cases in forensic toxicology, especially when the original compounds cannot be observed.

Low-frequency and common genetic variation in ischemic stroke: The METASTROKE collaboration

Erratum in: Low-frequency and common genetic variation in ischemic stroke: The METASTROKE collaboration. [Neurology. 2016]

Pressure ulcer management in Oman: nurses’ knowledge and views

Background: Pressure ulcers (PrUs) have a significant impact on health system expenditure and patient’s quality of life. It is a global problem. Many studies were undertaken in regard to PrU prevention and management. In Oman, no studies have been conducted to investigate nurses’ knowledge on prevention and management of PrUs. The purpose of this descriptive sequential explanatory mixed-method study was to explore the nurses’ level of knowledge in relation to prevention and management of PrUs in Oman. Methods: A mixed method design was used and the study was conducted over two Phases. In Phase I, a questionnaire was developed to explore nurses’ knowledge on PrU, policy, and resources. The main section of the questionnaire was the Pieper-Zulkowski Pressure Ulcer knowledge test (PZ-PUKT) which tests the knowledge on PrU. Another two sections were developed including questions about wound policy and resources available for PrU prevention and management in Oman. The questionnaire was distributed to nurses who were working in surgical, medical, orthopaedic, CCU, and ICU wards/units in seven hospitals. In Phase II study, semi-structured qualitative interviews were conducted with 16 of the questionnaire respondents. Interviews took approximately 30 minutes, were recorded and transcribed verbatim. Qualitative data were analysed using the Knowledge, Attitudes and Practice (KAP) model as the a priori framework. Results: In Phase I, 478 questionnaires were analysed. The knowledge test results showed the overall mean percent score for correctly answered questions was 51% suggesting a low level of knowledge. There was a significant relationship between nurses’ knowledge and age (P=0.001) and between knowledge and years of experience (P=0.001) with knowledge increasing with age and years of experience. In Phase II, four themes were identified from the interviews: knowledge, attitude, and practice (framework themes) and perception of role. Findings indicated positive and negative attitudes towards the care of PrUs. Some nurses stated feeling rewarded when they see wounds improving while others said they could not work with patients independently because they lacked the knowledge and the skills needed. There was variation in the management of PrU between hospitals. Both studies indicated that the wound management policy did not include enough information to guide nurses. Conclusion: Overall the nurses’ level of knowledge on PrU was relatively low. Most nurses were not familiar with wound management policy or different PrU prevention and management strategies. Nurses are aware of the risk of PrUs and try their best to manage them with the available resources however more training is required.

Qualidade de vida, percepção de saúde, resiliência, auto-regulação e suporte social nos adolescentes portugueses com a doencça crónica

Tese de Doutoramento em Psicologia na área de especialidade Psicologia da Saúde

L1 track finding for a time multiplexed trigger

At the HL-LHC, proton bunches will cross each other every 25. ns, producing an average of 140 pp-collisions per bunch crossing. To operate in such an environment, the CMS experiment will need a L1 hardware trigger able to identify interesting events within a latency of 12.5. μs. The future L1 trigger will make use also of data coming from the silicon tracker to control the trigger rate. The architecture that will be used in future to process tracker data is still under discussion. One interesting proposal makes use of the Time Multiplexed Trigger concept, already implemented in the CMS calorimeter trigger for the Phase I trigger upgrade. The proposed track finding algorithm is based on the Hough Transform method. The algorithm has been tested using simulated pp-collision data. Results show a very good tracking efficiency. The algorithm will be demonstrated in hardware in the coming months using the MP7, which is a μTCA board with a powerful FPGA capable of handling data rates approaching 1. Tb/s.

Reproductive performance as affected by lactation status and body fat in beef cows.

Seventy-one mature Brangus cows, 38 nonlactating (NL) and 33 in late stage of lactation (L) were fed for 192 days (Phase I) a low energy diet (L). During Phase II (65 days) 19 NL and 17 L cows were fed a high energy diet (H). The other nonlactating (19) and lactating (16) cows remained on the low energy diet. Energy restriction during Phase I did not affect (P> 0.05) cyclic ovarian activity although losses in body weight and condition were substantial. Rapid changes in body weight, condition, and percent empty body lipe (EBLP) during Phase II did not substantially influencefertility, although a five-fold difference in EBLP was observed (NL0H vs. L-L). Treatment groups did not differ (P> 0.05) in conception rate, days from the beginning of the breeding season to breeding and to conception, conception at first service, and number of services per conception. Values observed for these parameters for NL-H, L-H, NL-L, and L-L groups were respectively: 68,4%, `3,.2, 23.3, 36.8% and 1.68; 82,4% 12.7, 19.5, 58.8% and 1.29; 68.4%, 10.2, 17.4, 47.4%, and 1.41; 68.8%, 12.4, 19.5, 43.7%, and 1.50.

Biologia reprodutiva da Tainha, Mugil curema valenciennes, 1836 (osteichthyes: mugilidade) nas águas costeiras do Rio Grande do Norte

The marine fish white mullet, Mugil curema Valenciennes, 1836 (Osteichthyes: Mugilidae) exhibits a wide geographical distribution, being common in the Brazilian coast and is an important component of the artisanal fisheries. The objective of this study was to investigate the reproductive biology of M. curema in the coastal waters of Rio Grande do Norte. Fish samples were captured on a monthly basis during August, 2008 to July, 2009. The fish specimens were numbered, weighed, measured, dissected and their gonads were removed, weighed, their sex and gonadal development were identified. The length-weight relationship was determined for males and females. The sex ratio, the size at first gonadal maturation and gonadosomatic index (GSI) were calculated and ovarian development was investigated using macroscopic and histological techniques. The fecundity, spawning type and the reproductive period of the species were determined. A total of 366 specimens (186 males and 180 females) were captured. The sex ratio was 1:1 and the females were heavier than males. The estimated values of the angular coefficient for both sexes suggest that the species has isometric growth. The size at which 50% of the population began the process of maturation was 25.9 cm of total length for grouped sex. The macroscopic characteristics of the ovaries showed four stages of development: immature, maturing, mature and spent. However, the microscopic characteristics of the ovaries showed five stages of development: immature, early maturing, late maturing, mature and spent. The development of oocytes indicated five phases: Chromatin-nucleolus (phase I), initial perinucleolar (phase II), final perinuclear (phase II), formation of vitelline vesicle or yolk (phase III), vitellogenic (phase IV) and complete vitellogenesis (phase V). The species has a prolonged spawning period, with two peaks coinciding with the rainy season.

Essays on Emissions Trading Markets

This dissertation is a collection of three economics essays on different aspects of carbon emission trading markets. The first essay analyzes the dynamic optimal emission control strategies of two nations. With a potential to become the largest buyer under the Kyoto Protocol, the US is assumed to be a monopsony, whereas with a large number of tradable permits on hand Russia is assumed to be a monopoly. Optimal costs of emission control programs are estimated for both the countries under four different market scenarios: non-cooperative no trade, US monopsony, Russia monopoly, and cooperative trading. The US monopsony scenario is found to be the most Pareto cost efficient. The Pareto efficient outcome, however, would require the US to make side payments to Russia, which will even out the differences in the cost savings from cooperative behavior. The second essay analyzes the price dynamics of the Chicago Climate Exchange (CCX), a voluntary emissions trading market. By examining the volatility in market returns using AR-GARCH and Markov switching models, the study associates the market price fluctuations with two different political regimes of the US government. Further, the study also identifies a high volatility in the returns few months before the market collapse. Three possible regulatory and market-based forces are identified as probable causes of market volatility and its ultimate collapse. Organizers of other voluntary markets in the US and worldwide may closely watch for these regime switching forces in order to overcome emission market crashes. The third essay compares excess skewness and kurtosis in carbon prices between CCX and EU ETS (European Union Emission Trading Scheme) Phase I and II markets, by examining the tail behavior when market expectations exceed the threshold level. Dynamic extreme value theory is used to find out the mean price exceedence of the threshold levels and estimate the risk loss. The calculated risk measures suggest that CCX and EU ETS Phase I are extremely immature markets for a risk investor, whereas EU ETS Phase II is a more stable market that could develop as a mature carbon market in future years.

Geographical literacy, attitudes, and experiences of freshman students: A qualitative study at Florida International University

The purpose of the study was to explore the geography literacy, attitudes and experiences of Florida International University (FIU) freshman students scoring at the low and high ends of a geography literacy survey. The Geography Literacy and ABC Models formed the conceptual framework. Participants were freshman students enrolled in the Finite Math course at FIU. Since it is assumed that students who perform poorly on geography assessments do not have an interest in the subject, testing and interviewing students allowed the researcher to explore the assumption. ^ In Phase I, participants completed the Geography Literacy Survey (GLS) with items taken from the 2010 NAEP Geography Subject Area Assessment. The low 35% and high 20% performers were invited for Phase II, which consisted of semi-structured interviews. A total of 187 students participated in Phase I and 12 in Phase II. ^ The primary research question asked was what are the geography attitudes and experiences of freshman students scoring at the low and high ends of a geographical literacy survey? The students had positive attitudes regardless of how they performed on the GLS. ^ The study included a quantitative sub-question regarding the performance of the students on the GLS. The students’ performance on the GLS was equivalent to the performance of 12th grade students from the NAEP Assessment. There were three qualitative sub-questions from which the following themes were identified: the students’ definition of geography is limited, students recall more out of school experiences with geography, and students find geography valuable. In addition, there were five emergent themes: there is a concern regarding a lack of geographical knowledge, rote memorization of geographical content is overemphasized, geographical concepts are related to other subjects, taking the high school level AP Human Geography course is powerful, and there is a need for real-world applications of geographical knowledge. ^ The researcher offered as suggestions for practice to reposition geography in our schools to avoid misunderstandings, highlight its interconnectedness to other fields, connect the material to real world events/daily decision-making, make research projects meaningful, partner with local geographers, and offer a mandatory geography courses at all educational levels.^

Assessment of the Occurrence and Potential Risks of Antibiotics and their Metabolites in South Florida Waters Using Liquid Chromatography Tandem Mass Spectrometry

An automated on-line SPE-LC-MS/MS method was developed for the quantitation of multiple classes of antibiotics in environmental waters. High sensitivity in the low ng/L range was accomplished by using large volume injections with 10-mL of sample. Positive confirmation of analytes was achieved using two selected reaction monitoring (SRM) transitions per antibiotic and quantitation was performed using an internal standard approach. Samples were extracted using online solid phase extraction, then using column switching technique; extracted samples were immediately passed through liquid chromatography and analyzed by tandem mass spectrometry. The total run time per each sample was 20 min. The statistically calculated method detection limits for various environmental samples were between 1.2 and 63 ng/L. Furthermore, the method was validated in terms of precision, accuracy and linearity. The developed analytical methodology was used to measure the occurrence of antibiotics in reclaimed waters (n=56), surface waters (n=53), ground waters (n=8) and drinking waters (n=54) collected from different parts of South Florida. In reclaimed waters, the most frequently detected antibiotics were nalidixic acid, erythromycin, clarithromycin, azithromycin trimethoprim, sulfamethoxazole and ofloxacin (19.3-604.9 ng/L). Detection of antibiotics in reclaimed waters indicates that they can’t be completely removed by conventional wastewater treatment process. Furthermore, the average mass loads of antibiotics released into the local environment through reclaimed water were estimated as 0.248 Kg/day. Among the surface waters samples, Miami River (reaching up to 580 ng/L) and Black Creek canal (up to 124 ng/L) showed highest concentrations of antibiotics. No traces of antibiotics were found in ground waters. On the other hand, erythromycin (monitored as anhydro erythromycin) was detected in 82% of the drinking water samples (n.d-66 ng/L). The developed approach is suitable for both research and monitoring applications. Major metabolites of antibiotics in reclaimed wates were identified and quantified using high resolution benchtop Q-Exactive orbitrap mass spectrometer. A phase I metabolite of erythromycin was tentatively identified in full scan based on accurate mass measurement. Using extracted ion chromatogram (XIC), high resolution data-dependent MS/MS spectra and metabolic profiling software the metabolite was identified as desmethyl anhydro erythromycin with molecular formula C36H63NO12 and m/z 702.4423. The molar concentration of the metabolite to erythromycin was in the order of 13 %. To my knowledge, this is the first known report on this metabolite in reclaimed water. Another compound acetyl-sulfamethoxazole, a phase II metabolite of sulfamethoxazole was also identified in reclaimed water and mole fraction of the metabolite represent 36 %, of the cumulative sulfamethoxazole concentration. The results were illustrating the importance to include metabolites also in the routine analysis to obtain a mass balance for better understanding of the occurrence, fate and distribution of antibiotics in the environment. Finally, all the antibiotics detected in reclaimed and surface waters were investigated to assess the potential risk to the aquatic organisms. The surface water antibiotic concentrations that represented the real time exposure conditions revealed that the macrolide antibiotics, erythromycin, clarithromycin and tylosin along with quinolone antibiotic, ciprofloxacin were suspected to induce high toxicity to aquatic biota. Preliminary results showing that, among the antibiotic groups tested, macrolides posed the highest ecological threat, and therefore, they may need to be further evaluated with, long-term exposure studies considering bioaccumulation factors and more number of species selected. Overall, the occurrence of antibiotics in aquatic environment is posing an ecological health concern.

Molecular mechanisms controlling physiological plasticity in marine mussels under the influence of natural and anthropogenic stress factors

Marine mussels are exceptionally well-adapted to live in transitional habitats where they are exposed to fluctuating environmental parameters and elevated levels of natural and anthropogenic stressors throughout their lifecycle. However, there is a dearth of information about the molecular mechanisms that assist in dealing with environmental changes. This project aims to investigate the molecular mechanisms governing acclimatory and stress responses of the Mediterranean mussel (Mytilus galloprovincialis) by addressing relevant life stages and environmental stressors of emerging concern. The experimental approach consisted of two phases to explore (i) the physiological processes at early life history and the consequences of plastic pollution and (ii) the adult physiology processes under natural habitats. As the first phase, I employed a plastic leachate (styrene monomer), and polystyrene microplastics to understand the modulation of cytoprotective mechanisms during the early embryo stages. Results revealed the onset of transcriptional impairments of genes involved in MXR-related transporters and other physiological processes induced by styrene and PS-MPs. In the second phase, as a preliminary analysis, microbiota profile of adult mussels at the tissue scale and its surrounding water was explored to understand microbiota structures that may reflect peculiar adaptations to the respective tissue functions. The broader experiment has been implemented to understand the variability of transcriptional profiles in the mussel digestive glands in the natural setting. All the genes employed in this study have shown possibilities to use as molecular biomarker responses throughout the year for monitoring the physiology of mussels living in a particular environment and, in turn, more properly detecting changes in the environment. As a whole, my studies provide insights into the interactions between environmental parameters, and intrinsic characters, and physiology of marine bivalves, and it could help to interpretation of responses correctly under stress conditions and climate change scenarios.

Development of novel multi-substituted apatite nanophases with advanced functionalities for bone regeneration

In the field of bone substitutes is highly researched an innovative material able to fill gaps with high mechanical performances and able to stimulate cell response, permitting the complete restoration of the bone portion. In this respect, the synthesis of new bioactive materials able to mimic the compositional, morphological and mechanical features of bone is considered as the elective approach for effective tissue regeneration. Hydroxyapatite (HA) is the main component of the inorganic part of bone. Additionally ionic substitution can be performed in the apatite lattice producing different effects, depending from the selected ions. Magnesium, in substitution of calcium, and carbonate, in substitution of phosphate, extensively present in the biological bones, are able to improve properties naturally present in the apatitic phase, (i.e. biomimicry, solubility e osteoinductive properties). Other ions can be used to give new useful properties, like antiresorptive or antimicrobial properties, to the apatitic phase. This thesis focused on the development of hydroxyapatite nanophases with multiple ionic substitutions including gallium, or zinc ions, in association with magnesium and carbonate, with the purpose to provide double synergistic functionality as osteogenic and antibacterial biomaterial. Were developed bioactive materials based on Sr-substituted hydroxyapatite in the form of sintered targets. The obtained targets were treated with Pulsed Plasma Deposition (PED) resulting in the deposition of thin film coatings able to improve the roughness and wettability of PEEK, enhancing its osteointegrability. Were investigated heterogeneous gas-solid reactions, addressed to the biomorphic transformations of natural 3D porous structures into bone scaffolds with biomimetic composition and hierarchical organization, for application in load-bearing sites. The kinetics of the different reactions of the process were optimized to achieve complete and controlled phase transformation, maintaining the original 3-D morphology. Massive porous scaffolds made of ion-substituted hydroxyapatite and bone-mimicking structure were developed and tested in 3-D cell culture models.

Predictive And Prognostic Role Of Biological Markers In Neuroendocrine Neoplasia And Evaluation Of Activity And Safety Of Second Line Treatments In Neuroendocrine Carcinoma Patients

BACKGROUND Neuroendocrine neoplasia (NEN) are divided in well differentiated G1,G2 and G3 neuroendocrine tumors (NETs) and G3 neuroendocrine carcinomas (NECs). For the latter no standard therapy in second-line is available and prognosis is poor. METHODS Primary aim was to evaluate new prognostic and predictive biomarkers (WP1-3). In WP4 we explored the activity of FOLFIRI and CAPTEM as second-line in NEC patients in a multicenter non-comparative phase II trial RESULTS In WP1-2 we found that 4 of 6 GEP-NEC patients with a negative 68Ga-PET/CT had a loss of expression of RB1. In WP3 on 47 GEP-NENs patients the presence of DLL3 in 76.9% of G3 NEC correlate with RB1-loss (p<0.001), negative 68Ga-PET/CT(p=0.001) and a poor prognosis. In the WP4 we conducted a multicenter non-comparative phase II trial to explore the activity of FOLFIRI or CAPTEM in terms of DCR, PFS and OS given as second-line in NEC patients. From 06/03/2017 to 18/01/2021 53 out of 112 patients were enrolled in 17 of 23 participating centers. Median follow-up was 10.8 (range 1.4 – 38.6) months. The 3-month DCR was 39.3% in the FOLFIRI and 32.0 % in the CAPTEM arm. The 6-months PFS rate was 34.6% ( 95%CI 17.5-52.5) in FOLFIRI and 9.6% (95%CI 1.8-25.7) in CAPTEM group. In the FOLFIRI subgroup the 6-months and 12-months OS rate were 55.4% (95%CI 32.6-73.3) and 30.3% (CI 11.1-52.2) respectively. In CAPTEM arm the 6-months and 12-months OS rate were 57.2% (95%34.9-74.3) and 29.0% (95%10.0-43.3). The miRNA analysis of 20 patients compared with 20 healthy subjects shows an overexpression of miRNAs involved in staminality , neo-angiogenesis and mitochontrial anaerobic glycolysis activation. CONCLUSION WP1-3 support the hypothesis that G3NECs carrying RB1 loss is associated with a DLL3 expression highlighting a potential therapeutic opportunity. Our study unfortunately didn’t met the primary end–point but the results are promising

Study of the urinary biomarkers of synthetic cannabinoid receptor agonists (SCRAs) for the forensic unequivocal proof of consumption: medico-legal implications for drug abuse prevention

Introduction. Synthetic cannabinoid receptor agonists (SCRAs) represent the widest group of New Psychoactive Substances (NPS) and, around 2021-2022, new compounds emerged on the market. The aims of the present research were to identify suitable urinary markers of Cumyl-CB-MEGACLONE, Cumyl-NB-MEGACLONE, Cumyl-NB-MINACA, 5F-EDMB-PICA, EDMB-PINACA and ADB-HEXINACA, to present data on their prevalence and to adapt the methodology from the University of Freiburg to the University of Bologna. Materials and methods. Human phase-I metabolites detected in 46 authentic urine samples were confirmed in vitro with pooled human liver microsomes (pHLM) assays, analyzed by liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qToF-MS). Prevalence data were obtained from urines collected for abstinence control programs. The method to study SCRAs metabolism in use at the University of Freiburg was adapted to the local facilities, tested in vitro with 5F-EDMB-PICA and applied to the study of ADB-HEXINACA metabolism. Results. Metabolites built by mono, di- and tri-hydroxylation were recommended as specific urinary biomarkers to monitor the consumption of SCRAs bearing a cumyl moiety. Monohydroxylated and defluorinated metabolites were suitable proof of 5F-EDMB-PICA consumption. Products of monohydroxylation and amide or ester hydrolysis, coupled to monohydroxylation or ketone formation, were recognized as specific markers for EDMB-PINACA and ADB-HEXINACA. The LC-qToF-MS method was successfully adapted to the University of Bologna, as tested with 5F-EDMB-PICA in vitro metabolites. Prevalence data showed that 5F-EDMB-PINACA and EDMB-PINACA were more prevalent than ADB-HEXINACA, but for a limited period. Conclusion. Due to undetectability of parent compounds in urines and to shared metabolites among structurally related compounds, the identification of specific urinary biomarkers as unequivocal proofs of SCRAs consumption remains challenging for forensic laboratories. Urinary biomarkers are necessary to monitor SCRAs abuse and prevalence data could help in establishing tailored strategies to prevent their spreading, highlighting the role for legal medicine as a service to public health.