Neste trabalho, um eletrodo compósito à base de grafite e poliuretana (EGPU) 60% (grafite, m/m) foi avaliado quanto ao seu potencial para determinação dos antibióticos da família das tetraciclinas em amostras de água natural, de urina bovina e humana, e em amostras de leite bovino e humano. Visando desenvolver métodos simples, de baixo custo e com baixa geração de resíduos, o eletrodo foi usado sem modificação, para atender a estes propósitos e considerando os níveis de concentração das tetraciclinas nas amostras biológicas. A otimização dos parâmetros em voltametria de pulso diferencial (DPV) foi realizada para a determinação dos antibióticos tetraciclina (TC), oxitetraciclina (OTC) e clortetraciclina (CTC) em tampão fosfato 0,10 mol L-1 pH 2,3, permitindo atingir limites de detecção de 2,6 µmol L-1, 1,7 µmol L-1 e 0,82 µmol L-1, respectivamente, sem a necessidade de renovação da superfície, entre as medidas sucessivas. Durante as determinações de TC e OTC em amostras de água natural e purificada, foram observadas recuperações entre 92,6% e 100%, após etapa de pré-concentração. As amostras de urina foram acidificadas, em seguida centrifugadas para a remoção de partículas sólidas e proteínas. A TC e OTC foram determinadas diretamente, na amostra de urina tratada e diluída em tampão fosfato 0,10 mol L-1 pH 2,3. Durante a determinação de TC e OTC nas amostras de urina, após o preparo da amostra, foram obtidas recuperações entre 96% e 100%. As amostras de leite bovino e humano foram intencionalmente contaminadas com TC. Após preparo das amostras, estas foram submetidas à extração, usando cartucho para extração em fase sólida (SPE). Após eluição e retomada do analito retido no cartucho, a quantidade de TC presente nas amostras foi determinada pelo método de adição de padrão, usando DPV, com EGPU 60% (grafite, m/m). Foram obtidas recuperações entre 76% e 103% para as amostras de leite bovino e recuperações entre 91% e 100 % para as amostras de leite humano, que mostram que o eletrodo proposto pode ser usado neste tipo de determinação com as características postuladas acima. / A bare graphite-polyurethane composite electrode 60% (graphite, m/m) (GPUE) was evaluated in the determination of tetracycline (TC) class antibiotics in natural water as well as, in human and bovine urine and in milk samples. Thus, a DPV procedure was optimized for the determination of tetracycline, oxytetracycline (OTC) and chlortetracycline (CTC) antibiotics in phosphate buffer 0.10 mol L-1 pH 2.3, resulting in LOD of 2.6 µmol L-1, 1.7 µmol L-1 and 0.82 µmol L-1, respectively. During the determination of TC and OTC in the water samples, recoveries between 92.6 and 100% were found, after pre-concentration stage. The urine samples were submitted to acidification followed by centrifugation to remove solids and proteins. During the determination of TC and OTC in urine samples, after pre-treatment of urine, recoveries between 96 and 100% were found. The composite electrode was also evaluated in the determination of TC in the bovine and human milk samples. During the determination of TC in milk, recoveries between 76 and 103% were found for bovine milk and recoveries between 91 and 100% were found for human milk after pre-concentration stage, showing the feasibility of the procedure.

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Neste trabalho, um eletrodo compósito à base de grafite e poliuretana (EGPU) 60% (grafite, m/m) foi avaliado quanto ao seu potencial para determinação dos antibióticos da família das tetraciclinas em amostras de água natural, de urina bovina e humana, e em amostras de leite bovino e humano. Visando desenvolver métodos simples, de baixo custo e com baixa geração de resíduos, o eletrodo foi usado sem modificação, para atender a estes propósitos e considerando os níveis de concentração das tetraciclinas nas amostras biológicas. A otimização dos parâmetros em voltametria de pulso diferencial (DPV) foi realizada para a determinação dos antibióticos tetraciclina (TC), oxitetraciclina (OTC) e clortetraciclina (CTC) em tampão fosfato 0,10 mol L-1 pH 2,3, permitindo atingir limites de detecção de 2,6 µmol L-1, 1,7 µmol L-1 e 0,82 µmol L-1, respectivamente, sem a necessidade de renovação da superfície, entre as medidas sucessivas. Durante as determinações de TC e OTC em amostras de água natural e purificada, foram observadas recuperações entre 92,6% e 100%, após etapa de pré-concentração. As amostras de urina foram acidificadas, em seguida centrifugadas para a remoção de partículas sólidas e proteínas. A TC e OTC foram determinadas diretamente, na amostra de urina tratada e diluída em tampão fosfato 0,10 mol L-1 pH 2,3. Durante a determinação de TC e OTC nas amostras de urina, após o preparo da amostra, foram obtidas recuperações entre 96% e 100%. As amostras de leite bovino e humano foram intencionalmente contaminadas com TC. Após preparo das amostras, estas foram submetidas à extração, usando cartucho para extração em fase sólida (SPE). Após eluição e retomada do analito retido no cartucho, a quantidade de TC presente nas amostras foi determinada pelo método de adição de padrão, usando DPV, com EGPU 60% (grafite, m/m). Foram obtidas recuperações entre 76% e 103% para as amostras de leite bovino e recuperações entre 91% e 100 % para as amostras de leite humano, que mostram que o eletrodo proposto pode ser usado neste tipo de determinação com as características postuladas acima. / A bare graphite-polyurethane composite electrode 60% (graphite, m/m) (GPUE) was evaluated in the determination of tetracycline (TC) class antibiotics in natural water as well as, in human and bovine urine and in milk samples. Thus, a DPV procedure was optimized for the determination of tetracycline, oxytetracycline (OTC) and chlortetracycline (CTC) antibiotics in phosphate buffer 0.10 mol L-1 pH 2.3, resulting in LOD of 2.6 µmol L-1, 1.7 µmol L-1 and 0.82 µmol L-1, respectively. During the determination of TC and OTC in the water samples, recoveries between 92.6 and 100% were found, after pre-concentration stage. The urine samples were submitted to acidification followed by centrifugation to remove solids and proteins. During the determination of TC and OTC in urine samples, after pre-treatment of urine, recoveries between 96 and 100% were found. The composite electrode was also evaluated in the determination of TC in the bovine and human milk samples. During the determination of TC in milk, recoveries between 76 and 103% were found for bovine milk and recoveries between 91 and 100% were found for human milk after pre-concentration stage, showing the feasibility of the procedure.

Abstract Platinum group elements (PGEs) have a high commercial value and variety of applications in different fields of industry. One of the well-known applications is the use of palladium, platinum and rhodium in the catalytic converters of automobiles to reduce the amount of harmful gases emitted to the environment. Advanced analytical methods are needed to deal with issues related to development of new catalyst materials, recycling of PGEs from spent materials and for monitoring PGE emissions to the environment. In the first part of this study the emphasis was on the catalyst materials. Especially, reliable determination of ruthenium content in catalyst materials required further studies. Consequently, acid digestions in closed vessels using a microwave oven or high pressure asher were compared with a previously reported fusion method. Furthermore, the recovery of PGEs from spent materials is important due to many factors, for example, the high value of these metals, environmental aspects related to their production and possible availability issues in the future. Thus, utilization of microwave-assisted leaching and cloud point extraction (CPE) for the recovery of palladium, platinum, rhodium and ruthenium from catalyst materials was investigated. The second part of this study concentrated on the PGEs in environmental samples and the analytical challenges related to PGE determinations with inductively coupled plasma mass spectrometry (ICP-MS). Due to the use of PGEs in catalytic converters of automobiles, they are emitted to the roadside environment. The use of Pleurozium schreberi, a terrestrial moss, for active biomonitoring of these emissions was evaluated. Advanced analytical methods were needed to perform interference-free determinations of palladium, platinum and rhodium in these samples. Two alternative approaches for interference elimination were studied. Firstly, the interfering elements were removed using CPE as a chemical separation method. Secondly, interferences were eliminated using ammonia as a reaction gas with the novel ICP-MS/MS (inductively coupled plasma tandem mass spectrometry) technique. / Tiivistelmä Platinaryhmän alkuaineita hyödynnetään monissa teknisissä sovelluksissa. Yksi tunnetuimmista on autoliikenteen haitallisten päästöjen vähentäminen käyttäen palladiumia, platinaa ja rodiumia autojen pakokaasukatalysaattoreissa. Luotettavia analyysimenetelmiä tarvitaan esimerkiksi kehitettäessä uusia katalyyttimateriaaleja, kierrätettäessä platinaryhmän alkuaineita käytetyistä materiaaleista tai seurattaessa platinaryhmän alkuaineiden määrää ympäristössä. Tämän tutkimuksen ensimmäinen osa liittyi katalyyttien ruteniumpitoisuuksien määrittämiseen ja platinaryhmän alkuaineiden kierrätykseen katalyyttimateriaaleista. Erityisesti näytteenhajotusvaihe aiheuttaa usein ongelmia ruteniummäärityksissä. Tämän vuoksi tutkimuksessa verrattiin keskenään mikroaaltotekniikalla tai korkeapainetuhkistimella suoritettuja happohajotuksia ja aiemmin raportoidulla sulatemenetelmällä suoritettuja hajotuksia. Mikroaaltoavusteista liuotusta sovellettiin yhdessä samepisteuuton kanssa tutkittaessa katalyyttimateriaalien palladiumin, platinan, rodiumin ja ruteniumin kierrätysmahdollisuuksia. Aihe on ajankohtainen, kun huomioidaan platinaryhmän alkuaineiden korkea hinta, niiden tuotantoon liittyvät ympäristöasiat sekä saatavuuteen liittyvät epävarmuustekijät. Tutkimuksen toisessa osassa keskityttiin ympäristönäytteisiin ja erityisesti niiden ICP-MS -analytiikan (induktiiviplasmamassaspektrometria) haasteisiin. Autojen katalysaattoreista lähtöisin olevia platinaryhmän alkuaineita päätyy ympäristöön lähinnä teiden varsille. Näitä päästöjä arvioitiin aktiivista biomonitorointia käyttäen. Spektraaliset häiriöt vaikeuttivat kerättyjen sammalnäytteiden (Pleurozium schreberi) palladium-, platina- ja rodiumpitoisuuksien määrityksiä. Tämän vuoksi mittauksissa esiintyvien häiriöiden poistossa hyödynnettiin kahta eri lähestymistapaa. Näistä ensimmäisessä häiritsevät alkuaineet poistettiin kemiallisen erotusmenetelmän, samepisteuuton, avulla. Toisessa tavassa häiriöt poistettiin uudella ICP-MS/MS -tekniikalla (induktiiviplasmatandemmassaspektrometria) käyttäen ammoniakkia reaktiokaasuna.

<p>  </p> <p>Emerging pathogenic fungi have become a topic of conservation concern due to declines seen in several host taxa. One newly emerging fungal pathogen, <em>Ophidiomyces ophiodiicola</em>, has been well documented as the causative agent of Snake Fungal Disease (SFD). SFD has been found in a variety of snake species across the United States, including the Eastern Massasauga (<em>Sistrurus catenatus</em>), a federally threatened rattlesnake species. Most work to date has involved detecting SFD for diagnosis of infection through direct sampling from snakes. Attempts to detect <em>O. ophiodiicola</em> in the environment to better understand its distribution, seasonality, and habitat associations are lacking. I collected topsoil and ground water samples from four macrohabitat types in northern Michigan at a site where SFD infection has been seen in Eastern Massasauga. I used a quantitative PCR (qPCR) assay targeting the internal transcribed spacer region (ITS) developed for diagnosis of SFD after extracting DNA from samples. <em>Ophidiomyces</em> DNA was successfully detected in topsoil, with minimal to no detection in groundwater samples. The frequency in which <em>Ophidiomyces</em> was detected in a sample did not differ between habitats, but samples grouped seasonally showed higher detection occurring during mid-summer. Investigation of the correlation of environmental parameters on <em>Ophidiomyces</em> occurrence recovered no relationships. Our data suggests that season has some effect on the presence of <em>Ophidiomyces</em>. Differences between habitats may exist but are likely more dependent on the time of sampling and currently uninvestigated soil parameters. These findings build on our understanding of <em>Ophidiomyces</em> ecology and epidemiology and inform where snakes like the Eastern Massasauga may be encountering the fungal pathogen. Furthermore, they assist with developing conservation practices aimed at reducing <em>O. ophiodiicola </em>exposure in imperiled snake species. </p>

Cette thèse s'intéresse à la conception et le développement des techniques de bioinfor- matique qui peuvent faciliter l'utilisation de l'approche metabarcoding pour mesurer la diversité d'espèces. Le metabarcoding peut être utilisé avec le séquencage haut débit pour l'identification d'espèces multiples à partir d'un seul échantillon environnemental. La véritable force du metabarcoding réside dans l'utilisation de barcode marqueurs choisi pour une étude particulière et l'identification d'espèces ou des taxons peut être réalisé avec des marqueurs soigneusement conçu. Avec l'avancement des techniques haut débit de séquençage, une énorme quantité des données de séquences est produit qui contient un nombres substantiel des mutations. Ces mutations posent un grand problème pour les estimations correctes de la biodiversité et pour le d'assignation de taxon. Les trois problèmes majeurs dans le domaine de la bioinformatique que j'ai abordés dans cette thèse sont: i) évaluer la qualité d'une barcode marker , ii) concevoir des nouveaux région barcode et iii) d'analyser les données de séquençage pour traiter les erreurs et éliminer le bruit en séquences. Pour évaluer la qualité d'un barcode marker, on a développé deux mesures quantita- tive,formelle: la couverture (Bc) et la spécificité (Bs). La couverture donne une mesure de universalité d'une pairs de primer pour amplifier un large nombre de taxa, alors que la spécificité donne une mesure de capacité à discriminer entre les différents taxons. Ces mesures sont très utiles pour le classement des barcode marker et pour sélectionner les meilleurs markers. Pour trouver des nouveaux région barcode notamment pour les applications metabarcod- ing, j'ai développé un logiciel, ecoPrimers3. Basé sur ces deux mesures de qualité et de l'information taxinomique intégré, ecoPrimers nous permet de concevoir barcode markers pour n'importe quel niveau taxonomique . En plus, avec un grand nombre de paramètres réglables il nous permet de contrôler les propriétés des amorces. Enfin, grâce a des algorithmes efficaces et programmé en langage C, ecoPrimers est suffisamment efficace pour traiter des grosses bases de données, y compris génomes bactériens entièrement séquencés. Enfin pour traiter des erreurs présentes dans les données de séquencage , nous avons analysé un ensemble simple d'échantillons de PCR obtenus à partir de l'analyse du régime alimentaire de Snow Leopard. En mesurant les corrélations entre les différents paramètres des erreurs, nous avons observé que la plupart des erreurs sont produites pendant l'amplification par PCR. Pour détecter ces erreurs, nous avons développé un algorithme utilisant les graphes, qui peuvent différencier les vrai séquences des erreurs induites par PCR. Les résultats obtenus à partir de cet algorithme a montré que les données de-bruitée a donnent une estimation réaliste de la diversité des espèces étudiées dans les Alpes françaises. / This thesis is concerned with the design and development of bioinformatics techniques that can facilitate the use of metabarcoding approach for measuring species diversity. Metabarcoding coupled with next generation sequencing techniques have a strong po- tential for multiple species identification from a single environmental sample. The real strength of metabarcoding resides in the use of barcode markers chosen for a particular study. The identification at species or higher level taxa can be achieved with carefully designed barcode markers. Moreover with the advent of high throughput sequencing techniques huge amount of sequence data is being produced that contains a substantial level of mutations. These mutations pose a problem for the correct estimates of biodi- versity and for the taxon assignation process. Thus the three major challenges that we addressed in this thesis are: evaluating the quality of a barcode region, designing new barcodes and dealing with errors occurring during different steps of an experiment. To assess the quality of a barcode region we have developed two formal quantitative mea- sures called barcode coverage (Bc) and barcode specificity (Bs). Barcode coverage is concerned with the property of a barcode to amplify a broad range of taxa, whereas barcode specificity deals with its ability to discriminate between different taxa. These measures are extremely useful especially for ranking different barcodes and selecting the best markers. To deal with the challenge of designing new barcodes for metabarcoding applications we have developed an efficient software called ecoPrimers. Based on the above two quality measures and with integrated taxonomic information, ecoPrimers1 enables us to design primers and their corresponding barcode markers for any taxonomic level. Moreover with a large number of tunable parameters it allows us to control the properties of primers. Finally, based on efficient algorithms and implemented in C language, ecoPrimers is efficient enough to deal with large data bases including fully sequenced bacterial genomes. Finally to deal with errors present in DNA sequence data, we have analyzed a simple set of PCR samples obtained from the diet analysis of snow leopard. We grouped closely related sequences and by measuring the correlation between different parameters of mutations, we have shown that most of the errors were introduced during PCR amplification. In order to deal with such errors, we have further developed an algorithm using graphs approach, that can differentiate true sequences from PCR induced errors. The results obtained from this algorithm showed that de-noised data gave a realistic estimate of species diversity studied in French Alpes. This algorithm is implemented in program obiclean.