An alternative molecular recognition approach was developed for sensing small molecule analytes using the differential binding of an allosteric transcription factor (TF, specifically TetR) to its cognate DNA as the molecular recognition element coupled with Förster resonance energy transfer (FRET) to yield an internally calibrated optical signal transduction mechanism. Sensors were evaluated comprising Cy5-modified DNA (FRET acceptor) with either a tdTomato-TetR fusion protein (FP-TF) or quantum dot-TetR conjugate (QD-TF) as the FRET donor by measuring the ratio of acceptor and donor fluorescence intensities (FA/FD) with titrations of a derivative of the antibiotic tetracycline, anhydrous tetracycline (aTc). A proof-of-concept FRET-based biosensor was successfully demonstrated through the modulation of FA/FD signal intensities based on varying analyte concentrations. Sensor design parameters affecting overall signal-to-noise ratio and sensitivity of the sensors are also identified. / 2020-06-03T00:00:00Z

In the present study DNA damage was measured in peripheral blood lymphocytes from polyposis coli and colorectal cancer patients, treated with different dietary and environmental compounds and compared with lymphocytes from healthy individuals. In addition, confounding factors such as age, gender, alcohol intake and smoking habits were taken into consideration. The assays used in this study included the Comet assay, the Micronucleus assay, the Micronucleus ¿ FISH assay and the sister chromatid exchange assay. The food mutagens, PhIP and IQ, as well as titanium dioxide nanoparticles (TiO2 NPs) induced a dose dependent increase in the DNA damage and chromosomal abnormalities in all tested groups regardless of confounding factors. Prior to experiments physicochemical characterisation of nanoparticles was conducted. In the presence of the flavonoids, quercetin and rutin that were acting in an antioxidant manner, the DNA damage resulting from the highest doses of food mutagens was significantly reduced. Thus, dietary supplementation with flavonoid-rich vegetables and fruits may prove very effective in protection against oxidative stress. The polyposis coli and colon cancer patients were more susceptible to food mutagens, PhIP and IQ, as well as TiO2 NPs, and in the majority of cases had a higher level of DNA damage in the Comet assay and higher cytogenetic damage in the Micronucleus assay. In the final project, twelve frequently encountered (NewGeneris) chemical compounds were evaluated to establish their damaging potential in lymphocytes and spermatozoa from healthy donors. The highest damage was produced by DNA reactive aldehydes, food mutagens and benzo[a]pyrene when assessed with the neutral and alkaline Comet assay with and without metabolic activation. / EU NewGeneris Programme and United Kingdom - India Education and Research Initiative (UKIERI)

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Syftet med projektet var att utveckla en multiplexanalys för att detektera antigen från SARS-CoV-2, influensa och respiratoriskt syncytial virus genom att använda en masspektrometrisk metod som involverar antigenspecifika bindare. Bindarna klonades, renades och biotinylerades innan de användes i en analys utvecklad genom en målinriktad metod som involverade antigenerna. En slutsats som drog var att det var möjligt för Avi-märkta bindare att specifikt binda antigen-härledda peptidmål i multiplexanalysen. / The purpose of project was to develop a multiplex assay capable of detecting antigens from SARSCoV-2, influenza and respiratory syncytial virus by utilizing a mass spectrometric approach involving antigen-specific binders. Binders were cloned, purified and biotinylated before being employed in an assay developed by though a targeted method involving the antigens. It was concluded to be possible for Avi-tagged binders to specifically bind antigen-derived peptide targets in the multiplex assay.

No description available.

No description available.

Polychlorinated biphenyls (PCBs) are widespread persistent organic pollutants. The metabolism of PCBs by various organisms involves many steps that can lead to the formation of a wide range of metabolites. These metabolites frequently exhibit a toxicity and biodegradability different than the parent compounds. There is currently little information available about the biological effects of PCB hydroxylated metabolites that can be generated by various organisms and potentially released into the environment. The objective of the present research is to compare the toxicity of selected PCB congeners and their corresponding mono-hydroxylated metabolites. To achieve this objective, the following specific aims were performed: (1) to determine the effect of selected PCBs and PCB hydroxylated metabolites on the growth rate of a model PCB-degrading bacterium, Burkholderia xenovorans LB 400, (2) to determine the microbial toxicity of PCBs and PCB metabolites using the bioluminescent assay Microtox®, and (3) to determine the estrogenicity of PCBs and PCB metabolites using the Yeast Estrogen Screen assay (YES). The effects of a range of PCBs (PCB-2, -3, -8, -9, -30, -35, -36, -39, -61, -68, and -79) and their mono-hydroxylated metabolites on the growth rate of the PCB degrader, Burkholderia xenovorans LB400, were recorded. The results showed that the parent PCBs (50 mg L-1) did not affect the growth rate of LB400 although their hydroxylated metabolites strongly inhibited microbial growth. Using Microtox® assay, Parent PCBs (50 mg L-1) did not exhibit observable toxicity, while their hydroxylated metabolites showed a high level of toxicity (EC50 ranges from 2 mg L-1 to 46 mg L-1). Results using the YES assay also showed that the estrogenicity of hydroxylated metabolites of PCBs (50 mg L-1) was higher than the parent PCBs. The results obtained from the present study show that mono-hydroxylated metabolites of PCBs are more toxic than the corresponding parent PCBs. Because hydroxylated PCB derivatives are produced by a range of organisms and potentially released into the environment, this work raises new concerns associated with the environmental fate of PCBs. / Civil Engineering

Yes / Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1 mM, respectively) of Dox, and fixed 1 h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1 mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells. / Higher Education Funding Council for England (HEFCE)

Les zones côtières sont soumises à des pressions anthropiques multiples notamment de nature chimique qui peuvent faire peser un risque réel pour la pérennité des espèces aquatiques. Le bassin d’Arcachon, lagune macrotidale située sur la façade atlantique, est aussi le siège d’une activité ostréicole importante. Cependant depuis plusieurs années, les exploitations ostréicoles sont confrontées à une baisse de recrutement et une forte mortalité des naissains d’huître. La contamination chimique du milieu comme facteur pouvant contribuer aux effets observés sur les huîtres n’a pour l’instant pas été vérifiée. L’étude présentée ici porte sur l’évaluation, à travers différentes approches, de la toxicité potentielle de métaux et pesticides sur les stades précoces de développement de l’huître creuse C. gigas. Les réponses embryotoxiques, génotoxiques et l’expression de gènes d’intérêt ont été étudiés. Les différents pesticides (S-métolachlore, irgarol et diuron) et métaux (cuivre et cadmium) ont tout d’abord été testés séparément pour déterminer leur spectre d’effets et leur mode d’action. Il a été montré qu’une exposition des gamètes ou des embryons d’huître aux pesticides étudiés et au cuivre conduit à une augmentation des malformations larvaires et des dommages à l’ADN, une diminution du succès de fécondation et un impact sur la qualité de la descendance à des teneurs environnementales. Le cadmium, quant à lui, ne présente pas d’effet embryotoxique et génotoxique aux concentrations présentes dans le milieu aquatique. Les métabolites du métolachlore, ESA métolachlore et OA métolachlore sont retrouvés dans le bassin d’Arcachon à de plus fortes concentrations que le composé parent, cependant rien n’est actuellement connu sur les effets toxiques de ces métabolites. Il a été montré que ces métabolites sont moins embryotoxiques et génotoxiques sur les embryons et sur les spermatozoïdes d’huître que le métolachlore. Des variations dans l’expression des gènes impliqués dans les défenses antioxydantes sont observées pour les larves d’huître exposées au métolachlore et au métolachlore ESA. La toxicité d’un mélange de pesticides représentatifs de la contamination du bassin d’Arcachon en présence ou non de cuivre a ensuite été évaluée. L’exposition des embryons d’huître à ces mélanges conduit à des défauts de développement, des dommages à l’ADN et des modifications de l’expression des gènes impliqués majoritairement dans le stress oxydant. Finalement, une cartographie de la toxicité des sédiments du bassin d’Arcachon a été réalisée au cours des 4 saisons de l’année 2011 à l’aide du test embryolarvaire huître. Les sédiments d’Arguin présentent une faible toxicité quelle que soit la saison considérée. En revanche, les sédiments du Tès montrent un effet embryotoxique plus important au printemps et en été par rapport à la saison hivernale. L’ensemble de ce travail permet d’émettre l’hypothèse d’un risque chimique accru pour le développement des premiers stades de vie de huître creuse dans le bassin d’Arcachon. / The coastal areas are subject to multiple anthropogenic pressures including chemical pollution that can pose a real risk to the sustainability of aquatic species. The Arcachon Bay, macrotidal lagoon located on the French Atlantic coast, is the important ecosystem for oyster farming. But for several years, the oyster farms face lower recruitment and high mortality of oyster spat. Chemical contamination of the environment as a factor that may contribute to the observed effects on oysters has so far not been investigated.The present thesis aimed at evaluating through different approaches, of the potential toxicity of heavy metals and pesticides representative of the Arcachon Bay contamination on the early life stages of the Pacific oyster, Crassostrea gigas. Embryotoxicity, genotoxicity and expression levels of eleven targeted genes were studied. Firstly, different pesticides (S-metolachlor, irgarol, and diuron) and metals (copper and cadmium) were separately tested to determine their spectrum of effects. It were shown that exposure of gametes and embryos of oyster to environmental concentrations of pesticides and copper increased developmental abnormalities and DNA damage, and reduced fertilization success and affected offpring quality. Cadmium, meanwhile, showed no embryotoxic and genotoxic effects at the concentrations found in the Arcachon Bay. Metabolites of metolachlor, metolachlor ESA and metolachlor OA, are found in the Arcachon Bay at higher concentrations than their parent compound. The results showed that these metabolites were less embryotoxic and genotoxic on oyster embryos and spermatozoa than metolachor. Significant changes in expression of genes involved in antioxidant defense were observed for oyster larvae exposed to metolachlor and metolachlor ESA. Toxicity of mixtures of pesticides representative of the Arcachon Bay contamination with and without copper was then evaluated. Exposures of oyster embryos to these mixtures lead to development defects, DNA damage and changes in the expression of genes involved mainly in oxidative stress responses. Finally, mapping of toxicity of sediments from the Arcachon Bay was conducted for four seasons of 2011 with the oyster embryo-larvae assay. Sediments collected from Arguin exhibited low toxicity, regardless any season. In contrast, sediments from Le Tès showed higher toxicity in spring and summer seasons compared to winter season.From this work, it can be hypothesized that chemical contamination of the Arcachon Bay represents a threat for oyster reproduction and development.

O processo de tratamento da madeira utiliza substâncias que geram compostos perigosos que podem contaminar os compartimentos ambientais. O presente estudo avaliou uma área sob influência da contaminação de solo proveniente das atividades de uma usina de tratamento de madeira desativada. A presença e o efeito de compostos mutagênicos em amostras ambientais foram utilizados como marcadores de exposição associada à avaliação de marcadores genéticos de efeito precoce em humanos, com foco em crianças como grupo sensível. Uma área 1750 m distante da usina, fora do quadrante dispersão preferencial atmosférica e em oposição à drenagem do local, foi utilizada como local de referência para a coleta de amostras e comparação. Extratos orgânicos de água de abastecimento, poeira de sótão e material particulado atmosférico fino (PM2,5) foram avaliados para mutagenicidade por meio do ensaio Salmonella/microssoma. Cobre (Cu), cromo (Cr), arsênio (As) e pentaclorofenol (PCP) foram quantificados em amostras de poeira do sótão. Os 16 Hidrocarbonetos Policíclicos Aromáticos (HPAs) prioritários foram avaliados nos extratos de PM2,5 e poeira do sótão. Crianças residentes no entorno da usina e na área de referência foram avaliadas quanto à presença de micronúcleos em amostras de sangue e mucosa oral, e danos primários no DNA, pelo ensaio cometa em linfócitos de sangue periférico. De acordo com a análise de metais, as residências perto da entrada da usina foram as mais afetadas. PCP foi identificado em amostras de poeira de sótão (0,49 mg/kg) e a concentração total de HPAs nesta matriz variou 0,40-13,31 mg/g, com maior dispersão. Todas as amostras de poeira do sótão em que a concentração total de HPAs estava acima de 2μg/g apresentaram resposta positiva para a atividade mutagênica. A contribuição dos HPAs para a mutagênese na poeira de sótão representou 10%, indicando que outros compostos podem contribuir para o efeito mutagênico. A atividade mutagênica e a concentração de HPAs nas amostras de PM2,5foram, de maneira geral, mais elevadas na área de risco, embora em alguns períodos de amostragem a área de referência atingiu valores semelhantes ou mesmo superiores. O efeito mutagênico e as concentrações de HPAs observados nas amostras de PM2,5 foram semelhantes aos valores encontrados em estudos que avaliaram áreas urbanas e com influência industrial. Extratos orgânicos de água de abastecimento não apresentaram mutagenicidade. As frequências de MN em linfócitos de sangue periférico e de células binucleadas na mucosa oral foram significativamente maiores no grupo de risco. Nos demais biomarcadores avaliados não foram observadas diferenças significativas entre os grupos. O conjunto de resultados indica a necessidade de novas avaliações utilizando grupo de referência menos suscetível às influências da área contaminada. O conjunto de dados coletados neste estudo indica a necessidade de uma avaliação mais cuidadosa dos biomarcadores individualmente e de um grupo de referência menos suscetível a influências da área contaminada. Apesar da ausência de diferenças significativas entre os grupos de risco e de referência em biomarcadores de danos no DNA avaliados em crianças, os resultados observados nas amostras de poeira de sótão e PM2,5 sugerem que a população esteve ou ainda está potencialmente exposta a substâncias capazes de causar efeitos adversos à saúde humana. / The mutagenic activity and the concentration of PAHs in PM2.5 samples were generally higher in the risk area, although in some periods the reference area has reached similar or even higher values. The mutagenic effect and the concentrations of PAHs recorded in the PM2.5 samples were similar to those found in studies that assessed areas of intense urban occupation and industrial influence. Organic extracts from supply water showed no mutagenicity. The MN frequencies in peripheral blood lymphocytes and binucleated cells of the oral mucosa were significantly higher in the risk group. No significant differences between children from the reference and risk area were observed in others genetic biomarkers assessed. The result set indicates the need for further evaluations using reference group less susceptible to the influences of the contaminated area. The set of data collected in this study indicates the need for a more cautious assessment of biomarkers individually, and a reference group less susceptible to influences from the contaminated area is necessary. Despite the absence of significant differences between the risk and reference groups in biomarkers of DNA damage assessed in children, the results in attic and PM2.5 samples suggest that the population was or is still potentially exposed to substances with strong negatives effects on human health.