Implementation of a new reference values database for semiquantification in123I-FP-CIT brain single emission tomography

Brain dopamine transporters imaging by Single Emission Tomography (SPECT) with 123I-FP-CIT (DaTScanTM) has become an important tool in the diagnosis and evaluation of Parkinson syndromes.This diagnostic method allows the visualization of a portion of the striatum – where healthy pattern resemble two symmetric commas - allowing the evaluation of dopamine presynaptic system, in which dopamine transporters are responsible for dopamine release into the synaptic cleft, and their reabsorption into the nigrostriatal nerve terminals, in order to be stored or degraded. In daily practice for assessment of DaTScan TM, it is common to rely only on visual assessment for diagnosis. However, this process is complex and subjective as it depends on the observer’s experience and it is associated with high variability intra and inter observer. Studies have shown that semiquantification can improve the diagnosis of Parkinson syndromes. For semiquantification, analysis methods of image segmentation using regions of interest (ROI) are necessary. ROIs are drawn, in specific - striatum - and in nonspecific – background – uptake areas. Subsequently, specific binding ratios are calculated. Low adherence of semiquantification for diagnosis of Parkinson syndromes is related, not only with the associated time spent, but also with the need of an adapted database of reference values for the population concerned, as well as, the examination of each service protocol. Studies have concluded, that this process increases the reproducibility of semiquantification. The aim of this investigation was to create and validate a database of healthy controls for Dopamine transporters with DaTScanTM named DBRV. The created database has been adapted to the Nuclear Medicine Department’s protocol, and the population of Infanta Cristina’s Hospital located in Badajoz, Spain.

Fonte de Iluminação LED – Avaliação dos Efeitos Fotobiológicos no Olho e Pele

Tendo por referência a diretiva 2006/95/CE, o trabalho desenvolvido no contexto da disciplina de Dissertação/Projeto/Estágio do Mestrado de Engenharia de Instrumentação e Metrologia, decorreu nas instalações do IEP (Instituto Electrotécnico Português) e teve como objetivo principal o desenvolvimento de um procedimento de avaliação dos efeitos fotobiológicos no olho e pele provocados por fontes de emissão contínua (LED), doravante designado método alternativo ao de referência. Os dois métodos, alternativo e de referência, utilizam respectivamente um foto-radiómetro multicanal e um espetro-radiómetro. O procedimento desenvolvido (método alternativo) de acordo com a norma EN/IEC62471) consiste na aquisição dos valores de irradiância com recurso a um foto-radiómetro e posterior determinação dos valores da radiância, com os quais se faz a avaliação dos efeitos fotobiológicos, para fontes de luz LED (Light Emitting Diode) ou GLS (General Lighting Service). A consulta detalhada da norma EN/IEC62471 e a pesquisa sobre os conceitos, definições, equipamentos e metodologias relacionadas com o tema em causa, constituiu o primeiro passo deste projecto. Com recurso aos dois equipamentos, uma fonte de luz LED (módulo de 12 lâmpadas LED) é avaliada em relação aos perigos (ou riscos) actínico UV e UV-A, ao perigo da luz azul e ainda o perigo térmico na retina e térmico na pele, permitindo fazer uma análise comparativa dos resultados. O método alternativo revelou-se bastante flexível e eficaz, proporcionando bons resultados em termos da irradiância e radiância dos referidos efeitos fotobiológicos. A comparação destes resultados com os valores limites de exposição mencionados na norma EN/IEC6247 permitiu afirmar que a fonte de luz LED avaliada não representa perigo fotobiológico para a saúde humana e classifica-se no grupo de risco “isento”. Uma vez cumpridos os objectivos, entendeu-se que seria uma mais-valia para o trabalho já realizado, estudar outro caso prático. Sendo assim, fez-se a avaliação da radiação de apenas um dos LED´s que constituíam a fonte usada nos ensaios anteriores, com o espetro-radiómetro (método de referência) e com uma distância de 200 mm entre a fonte e o medidor. Neste caso verificaram-se diferenças significativas nas quantidades obtidas quando comparadas com os valores normativos. Concluiu-se que o efeito fotobiológico da luz azul insere-se no grupo de “isento”, sem perigo para a saúde. Contudo, o efeito térmico da retina apresenta um aumento considerável da quantidade de radiância, embora dentro do grupo de risco “isento”. Esta classificação de grupos de risco. Face aos resultados obtidos, pode confirmar-se que as lâmpadas LED apresentam segurança fotobiológica, atendendo aos baixos valores de irradiância e radiância dos efeitos fotobiológicos estudados. Pode ainda afirmar-se que a utilização do foto-radiómetro em alternativa ao espetro-radiómetro se revela mais eficaz do ponto de vista de metodologia prática. Este trabalho demonstra a robustez desses dois equipamentos de avaliação dos efeitos fotobiológicos, e procura estabelecer uma linha de orientação para a prevenção dos efeitos adversos na pele e olhos de todos os seres humanos sujeitos à radiação ótica artificial. Quanto às incertezas de medições, em relação ao processo de medição com foto-radiómetro, a sua estimação não se realizou, devido a não rastreabilidade entre as medições indicadas pelo fabricante, no certificado de calibração e as medidas realizadas por outras entidades. Contudo, é propõe-se a sua realização em trabalhos futuros dentro desse âmbito. As incertezas dos resultados de medições com espetro-radiómetro foram parcialmente estimadas. Atendendo às potencialidades do sistema de medição, propõe-se como trabalho futuro, a aplicação da norma IEC62478, que faz parte da aplicação da norma EN/IEC62471 na avaliação do efeito da luz azul, com base na determinação da temperatura de cor correlacionada (CCT) de lâmpadas ou sistemas de lâmpadas incluindo luminárias. Os valores de irradiância e radiância adquiridos nos processos de avaliação, tanto com foto-radiómetro como espectro-radiómetro foram gravados em ficheiro Excel para um CD e anexados a este trabalho.

Rapid automated method for on-site determination of sulfadiazine in fish farming: a stainless steel veterinary syringe coated with a selective membrane of PVC serving as a potentiometric detector in a flow-injection-analysis system

Sulfadiazine is an antibiotic of the sulfonamide group and is used as a veterinary drug in fish farming. Monitoring it in the tanks is fundamental to control the applied doses and avoid environmental dissemination. Pursuing this goal, we included a novel potentiometric design in a flow-injection assembly. The electrode body was a stainless steel needle veterinary syringe of 0.8-mm inner diameter. A selective membrane of PVC acted as a sensory surface. Its composition, the length of the electrode, and other flow variables were optimized. The best performance was obtained for sensors of 1.5-cm length and a membrane composition of 33% PVC, 66% onitrophenyloctyl ether, 1% ion exchanger, and a small amount of a cationic additive. It exhibited Nernstian slopes of 61.0 mV decade-1 down to 1.0×10-5 mol L-1, with a limit of detection of 3.1×10-6 mol L-1 in flowing media. All necessary pH/ionic strength adjustments were performed online by merging the sample plug with a buffer carrier of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 4.9. The sensor exhibited the advantages of a fast response time (less than 15 s), long operational lifetime (60 days), and good selectivity for chloride, nitrite, acetate, tartrate, citrate, and ascorbate. The flow setup was successfully applied to the analysis of aquaculture waters. The analytical results were validated against those obtained with liquid chromatography–tandem mass spectrometry procedures. The sampling rate was about 84 samples per hour and recoveries ranged from 95.9 to 106.9%.

Impacto Ambiental do Processo de Tingimento de uma Indústria de Curtumes e Avaliação do Desempenho da ETAR

Um dos objetivos desta dissertação de mestrado foi avaliar o impacto ambiental do processo de tingimento usado na indústria de curtumes, em termos de volume de efluente produzido e sua carga poluente. Pretendeu-se também encontrar alternativas ao nível do processo de tingimento que levem a uma melhoria na qualidade do efluente produzido. Outro objetivo deste trabalho foi avaliar o funcionamento da ETAR existente na empresa JR Fontes, no que diz respeito aos parâmetros em estudo (carência química de oxigénio - CQO, pH, crómio e teor de sólidos suspensos totais - SST), propondo alterações que permitam respeitar os valores de emissão exigidos na licença de descarga da empresa. Verificou-se que o processo de tingimento aplicado pela empresa JR Fontes é muito poluente, em termos de carga orgânica e matéria em suspensão. Este facto é comprovado pelos resultados obtidos para os três processos estudados: Montana, Galáctico e Navak. Todos os processos apresentam um efluente com pH ácido (aproximado a 3) e valores de CQO superiores a 3550 mg O2/L, sendo o processo Navak aquele que apresenta o valor mais alto para o banho composto, 8362 mg O2/L. Relativamente ao teor de crómio, o banho de recurtume com concentração mais elevada de crómio total é o banho 1 do processo Navak, com 2297 mg/L, sendo que a concentração destes banhos é sempre elevada, igual ou superior a 746 mg/L. No que diz respeito à matéria em suspensão, o processo Navak é novamente o mais poluente, com um valor de 3842 mg SST/L, não sendo obtidos nos outros processos, valores inferiores a 1205 mg SST/L. Na realização de um processo alternativo de tingimento verificou-se que é possível diminuir a carga orgânica do efluente originado. A aplicação de recurtumes sintéticos deu origem a efluentes menos poluentes. O melhor valor obtido para a CQO foi de 1113 mg O2/L, sendo obtidos valores não superiores a 7185 mg O2/L para processos de características semelhantes aos aplicados nesta indústria. Relativamente à ETAR, apesar das restrições ao funcionamento que esta apresenta, no geral pode ser considerada eficiente, embora não consiga atingir o objetivo pretendido de remoção para a CQO, ou seja o valor de 1100 mg O2/L. Nos restantes parâmetros é cumprido o limite de emissão (350 mg SST/L, pH entre 6 e 9 e 2 mg Cr/L para o crómio total): os SST apresentam o valor de 98 mg SST/L, o crómio total de 1,2 mg Cr/L e o pH encontra-se entre 8 e 9. São aqui feitas duas abordagens para solucionar os problemas existentes na ETAR. A primeira considera um ajuste no tratamento e equipamentos existentes, através da reconstrução do tanque de equalização, da substituição do coagulante por sulfato ferroso e da reconstrução do sedimentador, assim como a substituição das tubagens por umas de maior diâmetro, solucionando assim problemas de manutenção de toda a instalação e do incumprimento da legislação. A outra abordagem implica a substituição do sistema de afinação existente, os filtros de areia e carvão ativados, por um sistema de membranas de ultrafiltração ou por dois filtros de carvão ativado. Para tratar um efluente com um valor de CQO de 3000 mg O2/L com o carvão estudado, seriam necessários 132 kg de carvão por coluna, aproveitando-se os equipamentos existentes. No caso das membranas filtrantes, estas são eficazes, reduzindo a CQO em cerca de 70%. Ao contrário do carvão, a aplicação deste sistema implicaria a aquisição de novos equipamentos. Futuramente propõe-se a avaliação dos vários tipos de reagentes usados no tingimento de couro de forma a aplicar no tratamento aqueles que produzam as características desejadas no produto final e a que apresentem um menor resultado de CQO. Propõe-se também o estudo de viabilidade da remoção de crómio dos banhos de recurtume e consequente avaliação da aplicabilidade de um tratamento biológico em substituição ou como complemento do tratamento existente.

Application of geographic information system and modelling in health impact assessment for urban road mobility in Vietnam

Transport is an essential sector in modern societies. It connects economic sectors and industries. Next to its contribution to economic development and social interconnection, it also causes adverse impacts on the environment and results in health hazards. Transport is a major source of ground air pollution, especially in urban areas, and therefore contributing to the health problems, such as cardiovascular and respiratory diseases, cancer, and physical injuries. This thesis presents the results of a health risk assessment that quantifies the mortality and the diseases associated with particulate matter pollution resulting from urban road transport in Hai Phong City, Vietnam. The focus is on the integration of modelling and GIS approaches in the exposure analysis to increase the accuracy of the assessment and to produce timely and consistent assessment results. The modelling was done to estimate traffic conditions and concentrations of particulate matters based on geo-references data. A simplified health risk assessment was also done for Ha Noi based on monitoring data that allows a comparison of the results between the two cases. The results of the case studies show that health risk assessment based on modelling data can provide a much more detail results and allows assessing health impacts of different mobility development options at micro level. The use of modeling and GIS as a common platform for the integration of different assessments (environmental, health, socio-economic, etc.) provides various strengths, especially in capitalising on the available data stored in different units and forms and allows handling large amount of data. The use of models and GIS in a health risk assessment, from a decision making point of view, can reduce the processing/waiting time while providing a view at different scales: from micro scale (sections of a city) to a macro scale. It also helps visualising the links between air quality and health outcomes which is useful discussing different development options. However, a number of improvements can be made to further advance the integration. An improved integration programme of the data will facilitate the application of integrated models in policy-making. Data on mobility survey, environmental monitoring and measuring must be standardised and legalised. Various traffic models, together with emission and dispersion models, should be tested and more attention should be given to their uncertainty and sensitivity

Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus

Introduction Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. Methods Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. Results The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. Conclusions The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies.

Effects of bariatric surgery on the central nervous system and eating behavior in humans: a systematic review on the neuroimaging studies

INTRODUCTION: Neuroimaging studies suggest that obese people might show hyperactivity of brain areas regarding reward processing, and hypoactivity of brain areas concerning cognitive control, when exposed to food cues. Although the effects of bariatric surgery on the central nervous system and eating behavior are well known, few studies have used neuroimage techniques with the aim of investigating the central effects of bariatric surgery in humans. OBJECTIVES: This paper systematically and critically reviews studies using functional neuroimaging to investigate changes on the patterns of activation of central areas related to the regulation of eating behavior after bariatric surgery. METHOD: A search on the databases Medline, Web of Science, Lilacs and Science Direct on Line, was conducted in February 2013, using the keywords "Neuroimaging", "Positron-Emission Tomography", "Magnetic Resonance Imaging", "Gastric Bypass", "Gastroplasty", "Jejunoileal Bypass", "Bariatric Surgery". RESULTS: Seven manuscripts were included; the great majority studied the central effects of Roux en Y gastric bypass, using positron emission tomography or functional magnetic resonance. CONCLUSIONS: Bariatric surgery might normalize the activity of central areas concerned with reward and incentive salience processing, as the nucleus accumbens and mesencephalic tegmental ventral area, as well as circuitries processing behavioral inhibition, as the dorsolateral prefrontal cortex.

Instrumentation and software for the VERITAS Project and the detection of TeV Gamma-Ray Emission from the BL Lacertae Object 1ES1959+650

Transmission of Cherenkov light through the atmosphere is strongly influenced by the optical clarity of the atmosphere and the prevailing weather conditions. The performance of telescopes measuring this light is therefore dependent on atmospheric effects. This thesis presents software and hardware developed to implement a prototype sky monitoring system for use on the proposed next-generation gamma-ray telescope array, VERITAS. The system, consisting of a CCD camera and a far-infrared pyrometer, was successfully installed and tested on the ten metre atmospheric Cherenkov imaging telescope operated by the VERITAS Collaboration at the F.L. Whipple Observatory in Arizona. The thesis also presents the results of observations of the BL Lacertae object, 1ES1959+650, made with the Whipple ten metre telescope. The observations provide evidence for TeV gamma-ray emission from the BL Lacertae object, 1ES1959+650, at a level of more than 15 standard deviations above background. This represents the first unequivocal detection of this object at TeV energies, making it only the third extragalactic source seen at such levels of significance in this energy range. The flux variability of the source on a number of timescales is also investigated.

Development of a rehabilitation system for the red mud waste generated at the Aughinish Alumina Bayer Plant

Aughinish Alumina Limited (AAL) have an obligation by terms of their Integrated Pollution Control Licence (IPCL) and Planning Permission to establish vegetation on the red mud stack at their plant at Aughinish, Co. Limerick. High pH and high exchangeable sodium percentage are the main known factors limiting the establishment of vegetation on red mud. Gypsum addition has been known to assist in alleviating these problems in other countries. However, there is no experience or published information on red mud rehabilitation under Irish conditions. Red mud with organic and inorganic waste-derived ameliorants as well as selected grassland species were examined under laboratory controlled environment conditions as well as in field plot trials. Also, in order that it would be economically achievable, the research utilised locally available waste products as the organic amendments. Screening trials found that physical constraints severely limit plant germination and growth in red mud. Gypsum addition effectively lowers pH, exchangeable sodium percentage and the availability of A1 and Fe in the mud. A strong relationship between pH, ESP and A1 levels was also found. Gypsum addition increased germination percentages and plant growth for all species investigated. Greenhouse trials demonstrated that organic wastes alone did not greatly improve conditions for plant growth but when used in conjunction with gypsum plant performances for all species investigated was significantly increased. There was a high mortality rate for grasses in non-gypsum treatments. An emerging trend of preferential iron uptake and calcium deficiency in non-gypsum treatments was found at pot screening stage. Species also displayed manganese and magnesium deficiencies.

Non-classical error bounds in the central limit theorem

The classical central limit theorem states the uniform convergence of the distribution functions of the standardized sums of independent and identically distributed square integrable real-valued random variables to the standard normal distribution function. While first versions of the central limit theorem are already due to Moivre (1730) and Laplace (1812), a systematic study of this topic started at the beginning of the last century with the fundamental work of Lyapunov (1900, 1901). Meanwhile, extensions of the central limit theorem are available for a multitude of settings. This includes, e.g., Banach space valued random variables as well as substantial relaxations of the assumptions of independence and identical distributions. Furthermore, explicit error bounds are established and asymptotic expansions are employed to obtain better approximations. Classical error estimates like the famous bound of Berry and Esseen are stated in terms of absolute moments of the random summands and therefore do not reflect a potential closeness of the distributions of the single random summands to a normal distribution. Non-classical approaches take this issue into account by providing error estimates based on, e.g., pseudomoments. The latter field of investigation was initiated by work of Zolotarev in the 1960's and is still in its infancy compared to the development of the classical theory. For example, non-classical error bounds for asymptotic expansions seem not to be available up to now ...

Gas emission during laparoscopic colorectal surgery using a bipolar vessel sealing device: A pilot study on four patients.

BACKGROUND: Dissection during laparoscopic surgery produces smoke containing potentially toxic substances. The aim of the present study was to analyze smoke samples produced during laparoscopic colon surgery using a bipolar vessel sealing device (LigaSuretrade mark). METHODS: Four consecutive patients undergoing left-sided colectomy were enrolled in this pilot study. Smoke was produced by the use of LigaSuretrade mark. Samples (5,5l) were evacuated from the pneumoperitoneum in a closed system into a reservoir. Analysis was performed with CO2-laser-based photoacoustic spectroscopy and confirmed by a Fourier-transform infrared spectrum. The detected spectra were compared to the available spectra of known toxins. RESULTS: Samples from four laparoscopic sigmoid resections were analyzed. No relevant differences were noted regarding patient and operation characteristics. The gas samples were stable over time proven by congruent control measurements as late as 24 h after sampling. The absorption spectra differed considerably between the patients. One broad absorption line at 100 ppm indicating H2O and several unknown molecules were detected. With a sensitivity of alpha min ca 10-5 cm-1 no known toxic substances like phenol or indole were identified. CONCLUSION: The use of a vessel sealing device during laparoscopic surgery does not produce known toxic substances in relevant quantity. Further studies are needed to identify unknown molecules and to analyze gas emission under various conditions.

A general multiserver state-dependent queueing system

The work studies a general multiserver queue in which the service time of an arriving customer and the next interarrival period may depend on both the current waiting time and the server assigned to the arriving customer. Stability of the system is proved under general assumptions on the predetermined distributions describing the model. The proof exploits a combination of the Markov property of the workload process with a regenerative property of the process. The key idea leading to stability is a characterization of the limit behavior of the forward renewal process generated by regenerations. Extensions of the basic model are also studied.

On the use of reporter bacteria for measuring availability of organic contaminants

Natural environments are constantly challenged by the release of hydrophobic organic contaminants, which represent a threat for both the ecosystem and human health. Despite a substantial degradation by naturally occurring micro-organisms, a non negligible fraction of these pollutants tend to persist in soil and sediments due to their reduced accessibility to microbial degraders. This lack of 'bioavailability' is acknowledged as a key parameter for the natural and stimulated clean-up (bioremediation) of contaminated sites. We developed a bacterial bioreporter that responds to the presence of polyaromatic hydrocarbons (PAHs) by the production of the green fluorescent protein (GFP), based on the PAH-degrading bacterium Burkholderia sartisoli. We showed in this study that the bacterial biosensor B. sartisoli strain RP037 was faithfully reporting the degradation of naphthalene and phenanthrene (two PAHs of low molecular weight) via the production of GFP. What is more, the magnitude of GFP induction was influenced by change in the PAH flux triggered by a variety of physico-chemical parameters, such as the contact surface between the pollutant and the aqueous suspension. Further experiments permitted to test the influence of dissolved organic matter, which is an important component of natural habitats and can interact with organic pollutants. In addition, we tested the influence of two types of biosurfactants (tensio-active agents produced by living organisms) on phenanthrene's degradation by RP037. Interestingly, the surfactant's effects on the biodegradation rate appeared to depend on the type of biosurfactant and probably on the type of bacterial strain. Finally, we tagged B. sartisoli strain RP037 with a constitutively expressed mCherry fluorescent protein. The presence of mCherry allowed us to visualize the bacteria in complex samples even when GFP production was not induced. The new strain RP037-mChe embedded in a gel patch was used to detect PAH fluxes from a point source, such as a non-aqueous liquid or particles of contaminated soil. In parallel, we also developed and tested a so-called multiwell bacterial biosensor platform, which permitted the simultaneous use of four different reporter strains for the detection of major crude oil components (e.g., saturated hydrocarbons, mono- and polyaromatics) in aqueous samples. We specifically constructed the strain B. sartisoli RP007 (pPROBE-phn-luxAB) for the detection of naphthalene and phenanthrene. It was equipped with a reporter plasmid similar to the one in strain RP037, except that the gfp gene was replaced by the genes luxAB, which encoded the bacterial luciferase. The strain was implemented in the biosensor platform and detected an equivalent naphthalene concentration in oil spilled-sea water. We also cloned the gene for the transcriptional activator AlkS and the operator/promoter region of the operon alkSB1GHJ from the alkane-degrader bacterium Alcanivorax borkumensis strain SK2 in order to construct a new bacterial biosensor with higher sensitivity towards long-chain alkanes. However, the resulting strain showed no increased light emission in presence of tetradecane (C14), while it still efficiently reported low concentrations of octane (C8). RÉSUMÉ : Les écosystèmes naturels sont constamment exposés à nombre de contaminants organiques hydrophobes (COHs) d'origine industrielle, agricole ou même naturelle. Les COHs menacent à la fois l'environnement, le bien-être des espèces animales et végétales et la santé humaine, mais ils peuvent être dégradés par des micro-organismes tels que les bactéries et les champignons, qui peuvent être capables des les transformer en produits inoffensifs comme le gaz carbonique et l'eau. La biodégradation des COHs est cependant fréquemment limitée par leur pauvre disponibilité envers les organismes qui les dégradent. Ainsi, bien que la biodégradation opère partiellement, les COHs persistent dans l'environnement à de faibles concentrations qui potentiellement peuvent encore causer des effets toxiques chroniques. Puisque la plupart des COHs peuvent être métabolisés par l'activité microbienne, leur persistance a généralement pour origine des contraintes physico-chimiques plutôt que biologiques. Par exemple, leur solubilité dans l'eau très limitée réduit leur prise par des consommateurs potentiels. De plus, l'adsorption à la matière organique et la séquestration dans les micropores du sol participent à réduire leur disponibilité envers les microbes. Les processus de biodisponibilité, c'est-à-dire les processus qui gouvernent la dissolution et la prise de polluants par les organismes vivants, sont généralement perçus comme des paramètres clés pour la dépollution (bioremédiation) naturelle et stimulée des sites contaminés. Les hydrocarbures aromatiques polycycliques (HAPs) sont un modèle de COH produits par les activités aussi bien humaines que naturelles, et listés comme des contaminants chroniques de l'air, des sols et des sédiments. Ils peuvent être dégradés par un vaste nombre d'espèces bactériennes mais leur taux de biodégradation est souvent limité par les contraintes mentionnées ci-dessus. Afin de comprendre les processus de biodisponibilité pour les cellules bactériennes, nous avons décidé d'utiliser les bactéries elles-mêmes pour détecter et rapporter les flux de COH. Ceci a été réalisé par l'application d'une stratégie de conception visant à produire des bactéries `biocapteurs-rapporteurs', qui littéralement s'allument lorsqu'elles détectent un composé cible pour lequel elles ont été conçues. En premier lieu, nous nous sommes concentrés sur Burkholderia sartisoli (souche RP007), une bactérie isolée du sol et consommatrice de HAP .Cette souche a servi de base à la construction d'un circuit génétique permettant la formation de la protéine autofluorescente GFP dès que les cellules détectent le naphtalène ou le phénanthrène, deux HAP de faible masse moléculaire. En effet, nous avons pu montrer que la bactérie obtenue, la souche RP037 de B. sartisoli, produit une fluorescence GFP grandissante lors d'une exposition en culture liquide à du phénanthrène sous forme cristalline (0.5 mg par ml de milieu de culture). Nous avons découvert que pour une induction optimale il était nécessaire de fournir aux cellules une source additionnelle de carbone sous la forme d'acétate, ou sinon seul un nombre limité de cellules deviennent induites. Malgré cela, le phénanthrène a induit une réponse très hétérogène au sein de la population de cellules, avec quelques cellules pauvrement induites tandis que d'autres l'étaient très fortement. La raison de cette hétérogénéité extrême, même dans des cultures liquides mélangées, reste pour le moment incertaine. Plus important, nous avons pu montrer que l'amplitude de l'induction de GFP dépendait de paramètres physiques affectant le flux de phénanthrène aux cellules, tels que : la surface de contact entre le phénanthrène solide et la phase aqueuse ; l'ajout de surfactant ; le scellement de phénanthrène à l'intérieur de billes de polymères (Model Polymer Release System) ; la dissolution du phénanthrène dans un fluide gras immiscible à l'eau. Nous en avons conclu que la souche RP037 détecte convenablement des flux de phénantrène et nous avons proposé une relation entre le transfert de masse de phénanthrène et la production de GFP. Nous avons par la suite utilisé la souche afin d'examiner l'effet de plusieurs paramètres chimiques connus dans la littérature pour influencer la biodisponibilité des HAP. Premièrement, les acides humiques. Quelques rapports font état que la disponibilité des HAP pourrait être augmentée par la présence de matière organique dissoute. Nous avons mesuré l'induction de GFP comme fonction de l'exposition des cellules RP037 au phénanthrène ou au naphtalène en présence ou absence d'acides humiques dans la culture. Nous avons testé des concentrations d'acides humiques de 0.1 et 10 mg/L, tandis que le phénanthrène était ajouté via l'heptamethylnonane (HMN), un liquide non aqueux, ce qui au préalable avait produit le plus haut flux constant de phénanthrène aux cellules. De plus, nous avons utilisé des tests en phase gazeuse avec des concentrations d'acides humiques de 0.1, 10 et 1000 mg/L mais avec du naphtalène. Contrairement à ce que décrit la littérature, nos résultats ont indiqué que dans ces conditions l'expression de GFP en fonction de l'exposition au phénanthrène dans des cultures en croissance de la souche RP037 n'était pas modifiée par la présence d'acides humiques. D'un autre côté, le test en phase gazeuse avec du naphtalène a montré que 1000 mg/L d'acides humiques abaissent légèrement mais significativement la production de GFP dans les cellules de RP037. Nous avons conclu qu'il n'y a pas d'effet général des acides humiques sur la disponibilité des HAP pour les bactéries. Par la suite, nous nous sommes demandé si des biosurfactants modifieraient la disponibilité du phénanthrène pour les bactéries. Les surfactants sont souvent décrits dans la littérature comme des moyens d'accroître la biodisponibilité des COHs. Les surfactants sont des agents tensio-actifs qui augmentent la solubilité apparente de COH en les dissolvant à l'intérieur de micelles. Nous avons ainsi testé si des biosurfactants (des surfactants produits par des organismes vivants) peuvent être utilisé pour augmenter la biodisponibilité du phénanthrène pour la souche B. sartisoli RP037. Premièrement, nous avons tenté d'obtenir des biosurfactants produits par une autre bactérie vivant en co-culture avec les biocapteurs bactériens. Deuxièmement, nous avons utilisé des biosurfactants purifiés. La co-cultivation en présence de la bactérie productrice de lipopeptide Pseudomonas putida souche PCL1445 a augmenté l'expression de GFP induite par le phénanthrène chez B. sartisoli en comparaison des cultures simples, mais cet effet n'était pas significativement différent lorsque la souche RP037 était co-cultivée avec un mutant de P. putida ne produisant pas de lipopeptides. L'ajout de lipopeptides partiellement purifiés dans la culture de RP037 a résulté en une réduction de la tension de surface, mais n'a pas provoqué de changement dans l'expression de GFP. D'un autre côté, l'ajout d'une solution commerciale de rhamnolipides (un autre type de biosurfactants produits par Pseudomonas spp.) a facilité la dégradation du phénanthrène par la souche RP037 et induit une expression de GFP élevée dans une plus grande proportion de cellules. Nous avons ainsi conclu que les effets des biosurfactants sont mesurables à l'aide de la souche biocapteur, mais que ceux-ci sont dépendants du type de surfactant utilisé conjointement avec le phénanthrène. La question suivante que nous avons abordée était si les tests utilisant des biocapteurs peuvent être améliorés de manière à ce que les flux de HAP provenant de matériel contaminé soient détectés. Les tests en milieu liquide avec des échantillons de sol ne fournissant pas de mesures, et sachant que les concentrations de HAP dans l'eau sont en général extrêmement basses, nous avons conçu des tests de diffusion dans lesquels nous pouvons étudier l'induction par les HAPs en fonction de la distance aux cellules. Le biocapteur bactérien B. sartisoli souche RP037 a été marqué avec une seconde protéine fluorescente (mCherry), qui est constitutivement exprimée dans les cellules et leur confère une fluorescence rouge/rose. La souche résultante RP037-mChe témoigne d'une fluorescence rouge constitutive mais n'induit la fluorescence verte qu'en présence de naphtalène ou de phénanthrène. La présence d'un marqueur fluorescent constitutif nous permet de visualiser les biocapteurs bactériens plus facilement parmi des particules de sol. Un test de diffusion a été conçu en préparant un gel fait d'une suspension de cellules mélangées à 0.5 % d'agarose. Des bandes de gel de dimensions 0.5 x 2 cm x 1 mm ont été montées dans des chambres d'incubation et exposées à des sources de HAP (soit dissouts dans du HMN ou en tant que matériel solide, puis appliqués à une extrémité de la bande). En utilisant ce montage expérimental, le naphtalène ou le phénanthrène (dissouts dans du HMN à une concentration de 2.5 µg/µl) ont induit un gradient d'intensité de fluorescence GFP après 24 heures d'incubation, tandis que la fluorescence mCherry demeurait comparable. Un sol contaminé par des HAPs (provenant d'un ancien site de production de gaz) a induit la production de GFP à un niveau comparable à celui du naphtalène. Des biocapteurs bactériens individuels ont également détecté un flux de phénanthrène dans un gel contenant des particules de sol amendées avec 1 et 10 mg/g de phénanthrène. Ceci a montré que le test de diffusion peut être utilisé pour mesurer des flux de HAP provenant de matériel contaminé. D'un autre côté, la sensibilité est encore très basse pour plusieurs sols contaminés, et l'autofluorescence de certains échantillons rend difficile l'identification de la réponse de la GFP chez les cellules. Pour terminer, un des points majeurs de ce travail a été la production et la validation d'une plateforme multi-puits de biocapteurs bactériens, qui a permis l'emploi simultané de plusieurs souches différentes de biocapteurs pour la détection des constituants principaux du pétrole. Pour cela nous avons choisi les alcanes linéaires, les composés mono-aromatiques, les biphényls et les composés poly-aromatiques. De plus, nous avons utilisé un capteur pour la génotoxicité afin de détecter la `toxicité globale' dans des échantillons aqueux. Plusieurs efforts d'ingénierie ont été investis de manière à compléter ce set. En premier lieu, chaque souche a été équipée avec soit gfp, soit luxAB en tant que signal rapporteur. Deuxièmement, puisqu'aucune souche de biocapteur n'était disponible pour les HAP ou pour les alcanes à longues chaînes, nous avons spécifiquement construit deux nouveaux biocapteurs. L'un d'eux est également basé sur B. sartisoli RP007, que nous avons équipé avec le plasmide pPROBE-phn-luxAB pour la détection du naphtalène et du phénanthrène mais avec production de luciférase bactérienne. Un autre est un nouveau biocapteur bactérien pour les alcanes. Bien que nous possédions une souche Escherichia coli DHS α (pGEc74, pJAMA7) détectant les alcanes courts de manière satisfaisante, la présence des alcanes à longues chaînes n'était pas rapportée efficacement. Nous avons cloné le gène de l'activateur transcriptionnel A1kS ainsi que la région opérateur/promoteur de l'opéron alkSB1GHJ chez la bactérie dégradant les alcanes Alcanivorax borkumensis souche SK2, afin de construire un nouveau biocapteur bactérien bénéficiant d'une sensibilité accrue envers les alcanes à longues chaînes. Cependant, la souche résultante E. coli DHSα (pAlk3} n'a pas montré d'émission de lumière augmentée en présence de tétradécane (C14), tandis qu'elle rapportait toujours efficacement de basses concentrations d'octane (C8). De manière surprenante, l'utilisation de A. borkumensis en tant que souche hôte pour le nouveau plasmide rapporteur basé sur la GFP a totalement supprimé la sensibilité pour l'octane, tandis que la détection de tétradécane n'était pas accrue. Cet aspect devra être résolu dans de futurs travaux. Pour calibrer la plateforme de biocapteurs, nous avons simulé une fuite de pétrole en mer dans une bouteille en verre ouverte de 5L contenant 2L d'eau de mer contaminée avec 20 ml (1%) de pétrole brut. La phase aqueuse a été échantillonée à intervalles réguliers après la fuite durant une période allant jusqu'à une semaine tandis que les principaux contaminants pétroliers étaient mesurés via les biocapteurs. L'émission de bioluminescence a été mesurée de manière à déterminer la réponse des biocapteurs et une calibration intégrée faite avec des inducteurs types a servi à calculer des concentrations d'équivalents inducteurs dans l'échantillon. E. coli a été utilisée en tant que souche hôte pour la plupart des spécificités des biocapteurs, à l'exception de la détection du naphtalène et du phénanthrène pour lesquels nous avons utilisé B. sartisoli. Cette souche, cependant, peut être employée plus ou moins selon la même procédure. Il est intéressant de noter que le pétrole répandu a produit une apparition séquentielle de composés dissouts dans la phase aqueuse, ceux-ci .étant détectables par les biocapteurs. Ce profil contenait d'abord les alcanes à courtes chaînes et les BTEX (c'est-à dire benzène, toluène, éthylbenzène et xylènes), apparaissant entre des minutes et des heures après que le pétrole a été versé. Leurs concentrations aqueuses ont par la suite fortement décru dans l'eau échantillonnée après 24 heures, à cause de la volatilisation ou de la biodégradation. Après quelques jours d'incubation, ces composés sont devenus indétectables. Les HAPs, en revanche, sont apparus plus tard que les alcanes et les BTEX, et leur concentration a augmenté de pair avec un temps d'incubation prolongé. Aucun signal significatif n'a été mis en évidence avec le biocapteur pour le biphényl ou pour la génotoxicité. Ceci démontre l'utilité de ces biocapteurs, spécifiquement pour la détection des composés pétroliers, comprenant les alcanes à courtes chaînes, les BTEX et les HAPs légers.

Emissió ràdio a baixa freqüència en fenòmens eruptius i en fonts esteses de raigs gamma d'alta i molt alta energia

La tasca investigadora presentada en aquesta memòria s'ha centrat en les fonts galàctiques de raigs gamma de molt alta energia LS I +61 303, HESS J1708-410 i HESS J1858+020. La primera és una binària de raigs gamma molt estudiada, formada per una estrella massiva i un objecte compacte. S'ha proposat un escenari on l'objecte compacte seria un púlsar jove, i la interacció del seu vent amb el vent de l'estrella generaria els raigs gamma. De totes formes, no s'ha detectat polsos procedents d'aquest putatiu púlsar. L'investigador va realitzar observacions en fase a 1280 MHz amb el radiotelescopi GMRT, sense trobar-hi polsos, cosa que implica un estricte límit superior de 0,38 mJy a la densitat mitjana de flux polsat en un putatiu púlsar amb un període major que 2 mil•lisegons en el sistema binari LS I +61 303. Per altra banda, HESS J1708-410 i HESS J1858+020 són dues fonts esteses de raigs gamma de molt alta energia de les quals no es coneix cap contrapart a d'altres longituds d'ona. L'investigador les va observar amb el GMRT, quatre vegades HESS J1708-410 (dues a 610 MHz i dues a 1400 MHz) i dues vegades HESS J1858+020 (una a cada freqüència). En les imatges realitzades amb aquestes dades no hi ha emissió estesa coincident amb les regions d'emissió de raigs gamma. HESS J1858+020 se solapa parcialment amb una font estesa que podria ser un SNR. De confirmar-se la falta de contrapartida ràdio de HESS J1708-410, estaríem parlant d'un accelerador hadrònic extraordinàriament eficient, d'una classe desconeguda fins ara.

Assessment of coronary vasoreactivity by multidetector computed tomography: feasibility study with rubidium-82 cardiac positron emission tomography.

BACKGROUND: Positron emission tomography (PET) during the cold pressor test (CPT) has been used to assess endothelium-dependent coronary vasoreactivity, a surrogate marker of cardiovascular events. However, its use remains limited by cardiac PET availability. As multidetector computed tomography (MDCT) is more widely available, we aimed to develop a measurement of endothelium-dependent coronary vasoreactivity with MDCT and similar radiation burden as with PET. METHODS AND RESULTS: A study group of 18 participants without known cardiovascular risk factor (9F/9M; age 60±6 years) underwent cardiac PET with (82)Rb and unenhanced ECG-gated MDCT within 4h, each time at rest and during CPT. The relation between absolute myocardial blood flow (MBF) response to CPT by PET (ml·min(-1)·g(1)) and relative changes in MDCT-measured coronary artery surface were assessed using linear regression analysis and Spearman's correlation. MDCT and PET/CT were analyzed in all participants. Hemodynamic conditions during CPT at MDCT and PET were similar (P>0.3). Relative changes in coronary artery surface because of CPT (2.0-21.2%) correlated to changes in MBF (-0.10-0.52ml·min(-1)·g(1)) (ρ=0.68, P=0.02). Effective dose was 1.3±0.2mSv for MDCT and 3.1mSv for PET/CT. CONCLUSIONS: Assessment of endothelium-dependent coronary vasoreactivity using MDCT CPT appears feasible. Because of its wider availability, shorter examination time and similar radiation burden, MDCT could be attractive in clinical research for coronary status assessment.