Biomarcatori per il monitoraggio biologico di soggetti esposti ad agenti cancerogeni per il polmone

Riassunto I biomarcatori o “marcatori biologici” svolgono un ruolo fondamentale nel monitoraggio biologico. In questo lavoro ci siamo soffermati sullo studio di biomarcatori di effetto e di esposizione a xenobiotici ambientali. Nel primo caso abbiamo valutato i micro RNA (miRNA) da utilizzare per la diagnosi precoce del tumore al polmone in matrici di facile accesso, quale il CAE e il plasma, utilizzando il miRNA-21, oncogeno, e il miRNA-486, oncosoppressore. I risultati evidenziano una loro capacità di distinguere correttamente i soggetti con tumore polmonare dai soggetti sani, ipotizzando un loro utilizzo a scopo diagnostico. Nella seconda parte del lavoro di tesi sono stati studiati i biomarcatori di esposizione a benzene per valutare gli effetti dell’esposizione a concentrazioni ambientali su bambini residenti in città e a diverso livello di urbanizzazione. Lo studio ha evidenziato una correlazione dose-effetto fra le concentrazioni di benzene e dei suoi metaboliti urinari e un danno ossidativo a livello degli acidi nucleici. Tuttavia, le concentrazioni di benzene urinario non sono influenzate dal grado di industrializzazione, a differenza dell’S-PMA e degli indicatori di stress ossidativo (8-oxodGuo e 8-oxoGuo) che sembrano risentire sia della residenza che del momento del campionamento. Infine abbiamo ricercato possibili biomarcatori di esposizione a vinilcicloesene (VCH), sottoprodotto industriale nella polimerizzazione del 1,3-butadiene, poiché non sono ancora stati proposti BEI di riferimento nonostante i bassi valori di TLV-TWA (0.1 ppm) proposti dall’ACGIH. Nella prima fase del lavoro abbiamo studiato i meccanismi di tossicità del VCH tramite modelli in vitro, testando varie linee cellulari. I risultati evidenziano come la dose reale di VCH sia di molto inferiore a quella nominale per effetto dell’evaporazione. Inoltre, nelle linee cellulari più sensibili si sono evidenziati effetti citostatici, con alterazioni del ciclo cellulare, a differenza dell’esposizione agli epossidi del VCH, il VCD e l’1,2-VCHME, che determinano lisi cellulare con IC50 di 3 ordini di grandezza inferiori a quelli del VCH. La quantificazione dei metaboliti di I fase e di II fase del VCH nelle linee cellulari epatiche ha evidenziato concentrazioni di circa 1000 volte inferiori a quelle del VCH confermando come la sua tossicità sia principalmente dovuta alla produzione degli intermedi epossidici. La trasformazione nei metaboliti di II fase conferma inoltre l’effetto detossificante del metabolismo. La trasferibilità dei risultati ottenuti in vitro su sistemi in vivo fornirà le basi per poter identificare possibili metaboliti da proporre per il monitoraggio biologico di lavoratori esposti a VCH. PAROLE CHIAVE: biomarcatori di effetto e di esposizione, tumore al polmone, miRNA, benzene, vinilcicloesene.

The application of artificial neural networks to the interpretation and classification of freshwater benthic invertebrate communities

This thesis presents a thorough and principled investigation into the application of artificial neural networks to the biological monitoring of freshwater. It contains original ideas on the classification and interpretation of benthic macroinvertebrates, and aims to demonstrate their superiority over the biotic systems currently used in the UK to report river water quality. The conceptual basis of a new biological classification system is described, and a full review and analysis of a number of river data sets is presented. The biological classification is compared to the common biotic systems using data from the Upper Trent catchment. This data contained 292 expertly classified invertebrate samples identified to mixed taxonomic levels. The neural network experimental work concentrates on the classification of the invertebrate samples into biological class, where only a subset of the sample is used to form the classification. Other experimentation is conducted into the identification of novel input samples, the classification of samples from different biotopes and the use of prior information in the neural network models. The biological classification is shown to provide an intuitive interpretation of a graphical representation, generated without reference to the class labels, of the Upper Trent data. The selection of key indicator taxa is considered using three different approaches; one novel, one from information theory and one from classical statistical methods. Good indicators of quality class based on these analyses are found to be in good agreement with those chosen by a domain expert. The change in information associated with different levels of identification and enumeration of taxa is quantified. The feasibility of using neural network classifiers and predictors to develop numeric criteria for the biological assessment of sediment contamination in the Great Lakes is also investigated.

Avaliação da qualidade da água do Rio Piranhas-Açu/RN utilizando a comunidade de macroinvertebrados bentônicos

The biomonitoring technique for water quality in water bodies has been incorporated increasingly in management of the natural resources, using mainly indices of its aquatic community characteristics. In Brazil the first efforts to use this technique was restricted to the South and the Midwest region of this country. Located in the northeast region, this study was conducted at Rio Grande do Norte state and had as main objective the assessment of water quality in the Piranhas-Açu river using benthic macroinvertebrates community as biological indicators. Thus, were applied two biotic indices, the BMWP (Biological Monitoring Working Party score system) and the IBF (Family Biotic Index). According the requirements of these indices, two periods of sampling were determined as basic methodology, in dry seasons of 2006 and 2007. The sampling sites were around the municipalities of Açu, Ipanguaçu, Alto do Rodrigues and Pendências. Physical-chemical measurements; a protocol of rapid assessment of diversity of habitats, and determination of the degree of tolerance of each taxon to organic pollution were also performed. The most abundant organisms found in the river were tolerant with the prevalence of Chironomidae (Diptera) and Melanoides tuberculata. This results, together with those ones got in indices application, showed a considerable organic pollution in the four sample places and the reaches were classified as modified to impacted. According to the used indices, this study suggest that this river have significant alteration in the biotic community and water quality on the organic pollution, and this fact, occurs in the reach of the river further downstream, where were expected a good water quality. Considering these results, measures aiming the conservation of this water body are needed regarding as it is inserted into a fragile ecosystem with dry season phenomenon, and the water of this river has extremely importance for the local economy

Biomonitoramento da comunidade de macroinvertebrados de um reservatório de abastecimento público no sul do Brasil

The faunal inventory of the macroinvertebrate community is important to the environmental assessment, since this biota is sensitive to human disturbance. The reservoir of Rio Verde, located on the first plateau of Paraná, is inserted into an agricultural region with several forest fragments Araucaria. The aim of this study was to evaluate the environmental integrity of the reservoir through ecological indexes of macroinvertebrate community benthic and associated with macrophytes. Five sampling points were defined in the study area, which comprise distinct microhabitats in the basin. There were four sampling campaigns, each by weather station: Spring (2014); Summer (2015); Autumn (2015) and Winter (2015). In each sample were measured abiotic various parameters in the field and be collected water samples for nutrient analysis in the laboratory. The macroinvertebrates were collected in triplicate at adapted Macan method using mesh sieve 1 mm and CPUE (catch per unit effort) for 20 minutes. In order to pellet sample was used a dredger model Petersen 2L. Still in the field, by season, samples were collected from macrophytes Myriophyllum aquaticum (Vell) Verdc. and Potamogeton montevidensis A. Benn. in triplicates in the fluvial region of the reservoir, to analyze the associated fractal dimension and macrofauna. For this we used a PVC sampler specific volume 0.025 m3. the following ecological descriptors were calculated in each case: abundance, wealth tax, wealth Margalef, Shannon-Wiener diversity, evenness evenness through the Past software. The index Biological Monitoring Working Party (BMWP) for monitoring sampling points was also calculated. Regarding the statistical analysis, we used the analysis of PERMANOVA to compare points and seasons and canonical correspondence analysis (CCoA) for variables. Regarding M. aquaticum and P. montevidensis it was not verified difference to the average associated macroinvertebrates. However there was a difference for abundance of organisms in the fractal dimension and biomass of specimens. M. aquaticum is more complex and took more macrofauna in relation to P. montevidensis. Regarding the monitoring of the reservoir, it showed up mesotrophic with moderate nutrient concentrations and within the regulatory limits. Benthic macrofauna showed statistical differences in relation to the reservoir region, sample point and temporal variation. The BMWP index showed that the river region has the highest biotic integrity (in all samples above 70 points), and the ecological descriptors of wealth and Margalef diversity of Shannon- Wiener higher. In point 4 (dam downstream) were recorded evidence of possible impacts due to lower wealth and BMWP index which resulted in a questionable quality water. New approaches are needed to focus on the aquatic community in the best understanding of this ecosystem and also with a view to environmental preservation of the Green River Basin.

Seasonal variation of acetylcholinesterase, catalase, glutathione s transferase in different size of Pinctada radiata and metal pollution (Pb, Cd, Ni) in Persian Gulf

There are various tools for monitoring the concentration of pollutants on aquatic ecosystems. Today these studies are based on biological monitoring and biomarkers. The aim of this study was to measure the concentration of the acetylcholinesterase (AChE), glutathione S-transferase and catalase as biomarkers of heavy metal contamination in pearl oyster Pinctada radiata and their mechanism in aquatic ecosystems. Heavy metals lead, cadmium and nickel were measured in soft tissue and studied stations in four seasons. Samples were collected seasonally in Lavan stations, Hendurabi and Nakhilo (in the northern Persian Gulf) from spring 2013 to winter of that year by scuba diving. Pearl oysters are divided according to their shells size; shells separated from soft tissues and were transferred to the laboratory for analysis of heavy metals and enzymes. Moopam standard method for were used for measuring the concentration of heavy metals and for analyzing tissue concentrations of glutathione S-transferase in Clam the method recommended by Habig et al in 1974 were used. For measuring acetylcholinesterase Ellman method were used. Catalase contamination in pearl oyster in the supernatant obtained from the study based on the method homogeate soft tissue of mussels (Abei, 1974) was evaluated. The results showed that the concentration of lead has significant difference in sediments station, the concentration of lead in Lavan is significantly higher than the other two stations, This could be due to the movement of tanker, boats and floating refueling and with a considerable amount of wastewater containing oil and Petroleum into the water, and also due to precipitation and industrial discharges the lead in the region is increasing, land-disposed sewage sludge, has large concentrations of lead. Compare the results of this study with standards related and other similar studies at the regional and international level showed that pollutant concentration of heavy metals in all cases significantly less than all the standards and guide values associated. And also compared to other world research results have been far less than others, Being Less of the conclusion given in this research according that nickel is one of the indicators of oil pollution in the study area and emissions have been relatively low of oil. The concentration of acetylcholinesterase at several stations, in large and small sizes and in the seasons had no significant difference. Variations of catalase, and glutathione S-transferase were almost similar to each other and parameters, station and seasons were significantly different in the concentrations of these enzymes. The effects and interaction between various parameters indicate that following parameters has impact on the concentration of catalase and glutathione S-transferase. Stations; Seasonal changes in antioxidant enzymes related to (assuming a constant in salinity and oxygen) to age, reproductive cycle, availability of food and water temperature. With increasing temperature at warm season, antioxidant enzymes were increase, with increasing temperature and abundance of food in the environment the amount of antioxidant enzymes may increase. The presence of the enzyme concentration may indicate that the higher levels of the enzyme to eliminate ROS activities to be any healthier situation. At the time of gonads maturation and spawning season catalase activity increases. This study also indicates that catalase was significantly higher in the warm season. Due to low pollutants of heavy metals in the study area, a lower level of contaminants were observed in shellfish tissue incidents of international standards and strong correlation between the amount of heavy metal contamination in pearl oyster tissue and enzymes was not observed. Therefore, we can say that the pearl oyster remains in a healthy condition and the amount of enzyme is normal.

Multiple myeloma and bone

International audience

Avaliação da qualidade da água do Rio Piranhas-Açu/RN utilizando a comunidade de macroinvertebrados bentônicos

The biomonitoring technique for water quality in water bodies has been incorporated increasingly in management of the natural resources, using mainly indices of its aquatic community characteristics. In Brazil the first efforts to use this technique was restricted to the South and the Midwest region of this country. Located in the northeast region, this study was conducted at Rio Grande do Norte state and had as main objective the assessment of water quality in the Piranhas-Açu river using benthic macroinvertebrates community as biological indicators. Thus, were applied two biotic indices, the BMWP (Biological Monitoring Working Party score system) and the IBF (Family Biotic Index). According the requirements of these indices, two periods of sampling were determined as basic methodology, in dry seasons of 2006 and 2007. The sampling sites were around the municipalities of Açu, Ipanguaçu, Alto do Rodrigues and Pendências. Physical-chemical measurements; a protocol of rapid assessment of diversity of habitats, and determination of the degree of tolerance of each taxon to organic pollution were also performed. The most abundant organisms found in the river were tolerant with the prevalence of Chironomidae (Diptera) and Melanoides tuberculata. This results, together with those ones got in indices application, showed a considerable organic pollution in the four sample places and the reaches were classified as modified to impacted. According to the used indices, this study suggest that this river have significant alteration in the biotic community and water quality on the organic pollution, and this fact, occurs in the reach of the river further downstream, where were expected a good water quality. Considering these results, measures aiming the conservation of this water body are needed regarding as it is inserted into a fragile ecosystem with dry season phenomenon, and the water of this river has extremely importance for the local economy

Monitoreo biológico poblacional de Aulacomya atra (Molina, 1782) en el litoral de Moquegua y Tacna

La pesquería del recurso choro (Aulacomya atra) en los últimos años presenta mayor interés en los puertos de Ilo (Moquegua) y Morro Sama (Tacna), incrementándose notoriamente sus desembarques, mejorando los beneficios económicos de los extractores. Sin embargo, hay consecuencias negativas en los bancos naturales, al observarse disminución de la talla media y de las poblaciones en algunas zonas de extracción. Los desembarques en el 2014 alcanzaron 1556 t en Moquegua y Tacna, registrándose 15% de incremento en relación al 2013. Las principales zonas de extracción fueron Leonas y Cuartel (Ilo) que presentan mayores densidades por presencia de individuos juveniles, mientras que las densidades de adultos disminuyen progresivamente. En la Región Tacna destacan las zonas de Lozas y Lobera en los desembarques, con mayores densidades en Lobera y Quebrada de Burros, las zonas de Pocoma, Escoria y Mesas presentaron las menores densidades. El comportamiento reproductivo del recurso indica la existencia de dos periodos importantes de maduración y desove por año, siendo el más importante el de primavera. La composición especiológica de las matrices de A. atra estuvo conformada por 91 especies variando su frecuencia de ocurrencia por zona de extracción y periodo de muestreo, lo que indica la importancia de esta comunidad como organismo bioingeniero. El índice de diversidad de Shannon varió entre 2,6 a 4,8 bits/ind. Las condiciones oceanográficas no muestran fuerte presencia en toda el área evaluada de las Aguas Costeras Frías durante los monitoreos de abril, junio y agosto.

Iowa's Water - Ambient Monitoring Program: Biological Assessment of Iowa's Streams and Rivers, February 2001

The Iowa Department of Natural Resources has produced an 4 page article about how to assess Iowa's streams and rivers. How to use ambient monitoring.

Monitoring of biological markers indicative of doping: the athlete biological passport

The athlete biological passport (ABP) was recently implemented in anti-doping work and is based on the individual and longitudinal monitoring of haematological or urine markers. These may be influenced by illicit procedures performed by some athletes with the intent to improve exercise performance. Hence the ABP is a valuable tool in the fight against doping. Actually, the passport has been defined as an individual and longitudinal observation of markers. These markers need to belong to the biological cascade influenced by the application of forbidden hormones or more generally, affected by biological manipulations which can improve the performance of the athlete. So far, the haematological and steroid profile modules of the ABP have been implemented in major sport organisations, and a further module is under development. The individual and longitudinal monitoring of some blood and urine markers are of interest, because the intraindividual variability is lower than the corresponding interindividual variability. Among the key prerequisites for the implementation of the ABP is its prospect to resist to the legal and scientific challenges. The ABP should be implemented in the most transparent way and with the necessary independence between planning, interpretation and result management of the passport. To ensure this, the Athlete Passport Management Unit (APMU) was developed and the WADA implemented different technical documents associated to the passport. This was carried out to ensure the correct implementation of a profile which can also stand the challenge of any scientific or legal criticism. This goal can be reached only by following strictly important steps in the chain of production of the results and in the management of the interpretation of the passport. Various technical documents have been then associated to the guidelines which correspond to the requirements for passport operation. The ABP has been completed very recently by the steroid profile module. As for the haematological module, individual and longitudinal monitoring have been applied and the interpretation cascade is also managed by a specific APMU in a similar way as applied in the haematological module. Thus, after exclusion of any possible pathology, specific variation from the individual norms will be then considered as a potential misuse of hormones or other modulators to enhance performance.

Multi-analyte detection for biological fluids. Towards continous monitoring of glucose, ionized calcium and pH using a viscometric affinity biosensor.

We present a viscometric affinity biosensor that can potentially allow continuous multi-analyte monitoring in biological fluids like blood or plasma. The sensing principle is based on the detection of viscosity changes of a polymeric solution which has a selective affinity for the analyte of interest. The chemico-mechanical sensor incorporates an actuating piezoelectric diaphragm, a sensing piezoelectric diaphragm and a flow-resisting microchannel for viscosity detection. A free-standing Anodic Alumina Oxide (AAO) porous nano-membrane is used as selective interface. A glucose-sensitive sensor was fabricated and extensively assessed in buffer solution. The sensor reversibility, stability and sensitivity were excellent during at least 65 hours. Results showed also a good degree of stability for a long term measurement (25 days). The sensor behaviour was furthermore tested in fetal bovine serum (FBS). The obtained results for glucose sensing are very promising, indicating that the developed sensor is a candidate for continuous monitoring in biological fluids. Sensitive solutions for ionized calcium and pH are currently under development and should allow multi-analyte sensing in the near future.

Ultra-trace monitoring of copper in environmental and biological samples by inductively coupled plasma atomic emission spectrometry after separation and preconcentration by using octadecyl silica membrane disks modified by a new schiff's base

Ultra-trace amounts of Cu(II) were separated and preconcentrated by solid phase extraction on octadecyl-bonded silica membrane disks modified with a new Schiff,s base (Bis- (2-Hydroxyacetophenone) -2,2-dimethyl-1,3-propanediimine) (SBTD) followed by elution and inductively coupled plasma atomic emission spectrometric detection. The method was applied as a separation and detection method for copper(II) in environmental and biological samples. Extraction efficiency and the influence of sample matrix, flow rate, pH, and type and minimum amount of stripping acid were investigated. The concentration factor and detection limit of the proposed method are 500 and 12.5 pg mL-1, respectively.

Development of molecular monitoring methods and assessment of the environmental fate of the biological control agent of fire blight Pseudomonas fluorescens EPS62e

Pseudomonas fluorescens EPS62e es va seleccionar com a agent de biocontrol del foc bacterià per la seva eficàcia en el control de Erwinia amylovora. En aquest treball es van desenvolupar mètodes de traçabilitat que van permetre la seva detecció específica i quantificació. Mitjançant les tècniques RAPD i U-PCR es van obtenir fragments d'amplificació diferencial per EPS62e que es van seqüenciar i caracteritzar com marcadors SCAR per dissenyar una PCR en temps real. La PCR a temps real es va utilitzar simultàniament amb mètodes microbiològics per estudiar l'adaptabilitat epifítica de EPS62e en pomera i perera. L'ús combinat de mètodes microbiològics i moleculars va permetre la identificació de tres estats fisiològics de EPS62e: la colonització activa, l'entrada en un estat de viable però no cultivable, i la mort cel·lular. Aquest treball mostra que EPS62e està ben adaptada a la colonització de flors a camp, encoratjant la seva utilització dins d'una estratègia de control biològic contra el foc bacterià.

Defining a fish bio-assessment tool to monitoring the biological condition of a cascade reservoirs system in tropical area

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Monitoring the extracellular matrix mineralization processes in biological scaffolds using bioreactors, x-ray μct technology and 3-d modeling methods (monitoraggio dei processi di mineralizzazione della matrice extracellulare in scaffolds biologici, utilizzando bioreattore, tecnologia μct a raggi x e metodi di modellizzazione 3d)

Al fine di migliorare le tecniche di coltura cellulare in vitro, sistemi a bioreattore sono sempre maggiormente utilizzati, e.g. ingegnerizzazione del tessuto osseo. Spinner Flasks, bioreattori rotanti e sistemi a perfusione di flusso sono oggi utilizzati e ogni sistema ha vantaggi e svantaggi. Questo lavoro descrive lo sviluppo di un semplice bioreattore a perfusione ed i risultati della metodologia di valutazione impiegata, basata su analisi μCT a raggi-X e tecniche di modellizzazione 3D. Un semplice bioreattore con generatore di flusso ad elica è stato progettato e costruito con l'obiettivo di migliorare la differenziazione di cellule staminali mesenchimali, provenienti da embrioni umani (HES-MP); le cellule sono state seminate su scaffold porosi di titanio che garantiscono una migliore adesione della matrice mineralizzata. Attraverso un microcontrollore e un'interfaccia grafica, il bioreattore genera tre tipi di flusso: in avanti (senso orario), indietro (senso antiorario) e una modalità a impulsi (avanti e indietro). Un semplice modello è stato realizzato per stimare la pressione generata dal flusso negli scaffolds (3•10-2 Pa). Sono stati comparati tre scaffolds in coltura statica e tre all’interno del bioreattore. Questi sono stati incubati per 21 giorni, fissati in paraformaldehyde (4% w/v) e sono stati soggetti ad acquisizione attraverso μCT a raggi-X. Le immagini ottenute sono state poi elaborate mediante un software di imaging 3D; è stato effettuato un sezionamento “virtuale” degli scaffolds, al fine di ottenere la distribuzione del gradiente dei valori di grigio di campioni estratti dalla superficie e dall’interno di essi. Tale distribuzione serve per distinguere le varie componenti presenti nelle immagini; in questo caso gli scaffolds dall’ipotetica matrice cellulare. I risultati mostrano che sia sulla superficie che internamente agli scaffolds, mantenuti nel bioreattore, è presente una maggiore densità dei gradienti dei valori di grigio ciò suggerisce un migliore deposito della matrice mineralizzata. Gli insegnamenti provenienti dalla realizzazione di questo bioreattore saranno utilizzati per progettare una nuova versione che renderà possibile l’analisi di più di 20 scaffolds contemporaneamente, permettendo un’ulteriore analisi della qualità della differenziazione usando metodologie molecolari ed istochimiche.