Schilddrüsenhormone (TH) regulieren Metabolismus und Energiestoffwechsel. Der TH‐Metabolit (THM) 3,5‐T2 (3,5‐Diiod‐L‐Thyronin) aktiviert Fett‐Oxidation und mitochondriale Atmung. Der THM 3‐Iodothyronamin (3‐T1AM) beeinflusst zusätzlich glukoregulatorische Prozesse. THM können zur Reduktion von Körperfett beitragen. Um 3,5‐T2 im humanen Serum nachzuweisen sollte ein Immunoassay aufgebaut, validiert und angewendet werden. In intakten hepatozellulären (HepG2) sowie pankreatischen ß‐Zellen (MIN6) sollte untersucht werden ob THM durch Modulation der mitochondrialen Aktivität die zelluläre Substratverstoffwechslung (3,5‐T2) und Insulinsekretion (3‐T1AM) regulieren können. Der Immunoassay ist sensitiv, spezifisch und misst zuverlässig 3,5‐T2 im humanen Serum. Hyper‐ und Hypothyreose zeigen vergleichbare 3,5‐T2 Konzentrationen, jedoch akkumuliert 3,5‐T2 bei sekundären Erkrankungen der Schilddrüse und athyreoten Patienten unter Thyroxin‐Supplementation. In HepG2‐Zellen konnte die Aktivierung der mitochondrialen Atmung durch 3,3‘,5‐Triiod‐L‐Thyronin (T3), jedoch nicht durch 3,5‐T2 stimuliert werden. Die Expression von TH‐transporters (THT) war gering verglichen mit Maus‐Hepatozyten. MIN6 exprimiert THT vergleichbar mit Langerhansschen Inselzellen der Maus. 3‐T1AM wird in die Zelle aufgenommen, zu 3‐Iodothyroessigsäure (TA1) metabolisiert, und wieder exportiert. Nach 3‐T1AM Gabe ist die mitochondriale ATP‐Produktion sowie die Glukose‐stimulierte Insulinsekretion (GSIS) vermindert. 3,5‐T2 zirkuliert in euthyreoten Individuen, ist nicht an der zentralen Regulation der TH‐Achse beteiligt, wird extrathyroidal gebildet und niedrige T3‐Werte können durch erhöhtes 3,5‐T2 erklärt werden. HepG2 erwies sich als ungeeignetes Zellmodell, da wenige THT vorhanden sind, 3,5‐T2 die Plasmamembran wahrscheinlich nicht passieren kann und damit die Aktivierung der Mitochondrien aus bleibt. In MIN6 wurde gezeigt, dass die GSIS nicht ausschließlich an der Plasmamembran durch 3‐T1AM reguliert wird. / Thyroid hormones (TH) regulate metabolism and energy metabolism. The TH‐metabolite (THM) 3,5‐T2 (3,5‐diiodo‐L‐thyronine) activates fat oxidation and mitochondrial respiration. The THM 3‐T1AM (3‐iodothyronamine) influences in addition glucoregulatory processes. THM may support reduction in body fat mass. It was the idea to establish, validate and apply an immunoassay to determine 3,5‐T2 in human serum. Using intact hepatocellular (HepG2) as well as pancreatic ß‐cells (MIN6) it should be tested if THM can modulate mitochondrial activity, resulting in increased cellular substrate usage (3,5‐T2) as well as decreased insulin secreation (3‐T1AM). The established immunoassay is sensitive, specific and detects precisely 3,5‐T2 in human serum. Hyper‐ and hypothyroidism shows similar 3,5‐T2 concentrations, although 3,5‐T2 accumulates in secondary thyroidal illness as well as in athyreotic patients under thyroxine‐supplementation. Using HepG2 cells, mitochondrial respiration was stimulated by 3,3‘,5‐triiodo‐L‐thyronine (T3), but 3,5‐T2 had no effect. Expression of TH‐transporters (THT) was low compared to murine hepatocytes. In contrast, MIN6 express THT comparable to murine Langerhans islets. 3‐T1AM is taken up by the cell, metabolized to 3‐iodothyroacetic acid (TA1) and following export. After 3‐T1AM application mitochondrial ATP‐production as well as glucose‐stimulated insulin secretion (GSIS) was reduced. 3,5‐T2 circulates in euthyroid individuals, is not involved in central regulation of TH‐axis, is produced extrathyroidally and low T3 values can be explained by increased 3,5‐T2. HepG2 was shown to be an inappropriate cellmodel, because THT are merely expressed, suggesting that 3,5‐T2 is not able to pass the plasma membrane, thereby preventing mitochondrial activation. In addition, it was shown in MIN6 cells, that GSIS is not exclusively regulated at the plasma membrane level via 3‐T1AM.

Thyronamine (TAM) sind eine neue Molekülklasse, die endokrinologische und metabolische Prozesse miteinander vereinen. Der biologisch aktive Metabolit 3-Iod-L-Thyronamin (3-T1AM) wird durch eine kombinierte Deiodierung und Decarboxylierung von Schilddrüsenhormonen (TH) gebildet. Existierende Methoden zum Nachweis und zur Quantifizierung von 3-T1AM im menschlichen Serum sind immer noch umstritten. Auch die an der Biosynthese vermutlich beteiligte TH-Decarboxylase konnte noch nicht identifiziert werden. Für die Identifizierung und Quantifizierung von TH und TAM Profilen wurde die Flüssigchromatographie-Tandem-Massenspektrometrie (LC-MS/MS) verwendet. In der bisherigen präanalytischen Aufarbeitung liefern weder Flüssig-Flüssig- noch Festphasenextraktionen reproduzierbare Ergebnisse des 3-T1AM-Gehalts im Serum. Mit der Entwicklung eines spezifischen Extraktionsverfahrens und nachfolgender Detektion mittels LC-MS/MS gelang der gleichzeitige Nachweis der häufigsten TH im humanen Serum. Parallel dazu wurden monoklonale Antikörper gegen 3-T1AM entwickelt, auf deren Basis ein quantitativer 3-T1AM Chemilumineszenz-Immunoassay entstand. Ergebnisse aus klinischen Kollektiven zeigen, dass 3-T1AM im Serum im nM Konzentrationsbereich vorkommt und dass 3-T1AM bei Patienten außerhalb der Schilddrüse produziert wird. Viele Forscher gehen davon aus, dass die aromatische L-Aminosäure Decarboxylase (AADC) die Synthese von TAM über Decarboxylierung von TH katalysiert. Diese Hypothese wurde durch Inkubation von rekombinanter humaner AADC mit TH getestet. In keinem der Experimente konnte AADC die Decarboxylierung von TH katalysieren. Zusammenfassend ist die Bestimmung von 3-T1AM im Serum mittels LC-MS/MS aufgrund der nicht reproduzierbaren präanalytischen Probenaufbereitung problematisch. In dieser Arbeit wird der erste MAb-basierte 3-T1AM assay vorgestellt, der 3-T1AM zuverlässig in humanem Serum quantifiziert. Die AADC ist wahrscheinlich nicht an der Biosynthese von TAM beteiligt. / Thyronamines (TAM) are a new class of molecules linking endocrinology and metabolism. Combined deiodination and decarboxylation of thyroid hormones (TH) generates a biologically active ‘cooling’ metabolite, 3-iodo-L-thyronamine (3-T1AM).. It remains controversial, which methods are able or not to reliably detect 3-T1AM in human serum, and the presumed TH decarboxylase is still elusive. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for the simultane-ous identification and quantification of TH and TAM profiles in biological samples. Several preanalytical methods were tested for complete extraction of 3-T1AM in human serum. Thus far, neither liquid-liquid nor solid-phase extraction methods allowed reproducible extraction of 3-T1AM from human serum samples in the preanalytical sample workup. Nevertheless, a rapid and sensitive extraction procedure was developed for detection of the major TH by LC-MS/MS in a single human serum sample. In parallel, monoclonal antibodies (MAb) targeting 3-T1AM were developed and characterized, and a highly specific quantitative 3-T1AM MAb-based chemiluminescence immunoassay was developed. Studies in clinical cohorts provide evidence that 3-T1AM is present in human serum in the nM concentration range and that 3-T1AM is produced extrathyroidally. Many researchers have reasoned that the aromatic L-amino acid decarboxylase (AADC) mediates TAM synthesis via decarboxylation of TH. This hypothesis was tested by incubating recombinant human AADC with several TH. In all tested conditions, AADC failed to catalyze the decarboxylation of TH. These in vitro observations are supported by the finding that 3-T1AM is also present in plasma samples of patients with AADC deficiency. In summary, 3-T1AM detection in serum using LC-MS/MS encounters preanalytical problems. The first MAb-based 3-T1AM CLIA is presented, which reliably quantifies 3-T1AM in human serum. AADC is likely not involved in TAM biosynthesis.

Escherichia coli enteroagregativa (EAEC), destaca-se como um importante patógeno emergente causador de diarréia persistente em países em desenvolvimento e de diarréia aguda em países desenvolvidos. A grande heterogeneidade dos fatores de virulência caracteriza esta categoria, porém não foi estabelecido um marcador genético comum a todas as amostras de EAEC. O padrão de adesão agregativa (AA) em células HEp-2 e HeLa é a forma de caracterização e diagnóstico mais precisos desta categoria. Uma das toxinas envolvidas na patogênese é Plasmid-encoded toxin (Pet) pertencente à classe das proteínas autotransportadoras com características de uma serino protease denominada SPATEs. Iniciou-se este estudo com a determinação do padrão de adesão de 164 amostras EAEC, previamente caracterizadas como sonda pCVD432 ou onda AA positiva. Assim, 141 (86%) amostras, que apresentaram padrão de adesão agregativo, foram caracterizadas como EAEC. Face aos resultados obtidos, confirmou-se a baixa especificidade da sonda AA. A pesquisa do gene pet, por meio de ensaio de PCR, resultou na positividade de 12 (8,5%) amostras. Prosseguiu-se esse estudo com a padronização da reação de immuno-dot. Utilizando-se 300 µL do sobrenadante bacteriano, soro policlonal anti-Pet e o conjugado nas diluições 1/50 e 1/2.500, respectivamente, resultados bastante reprodutíveis foram obtidos. O método foi mais sensível que a detecção do gene por PCR. Por esse ensaio, detectou-se a toxina Pet em 16 (11,3%) das 141 amostras EAEC. Nenhuma das amostras controle negativo foi reconhecida pelo soro anti-Pet, assim como as amostras de E. coli produtoras das mais diversas toxinas. Apesar da baixa prevalência de amostras de EAEC produtoras da toxina Pet, neste estudo padronizou-se um método rápido, sensível, específico e de baixo custo para pesquisa desta toxina mostrando o potencial diagnóstico deste ensaio para uso em inquéritos epidemiológicos, o que poderá permitir determinar o papel da Pet no desenvolvimento de diarréia aquosa. / Enteroaggregative Escherichia coli (EAggEC) is an emerging diarrheal pathogen, whose pathogenesis is thought to comprise colonization of the intestinal mucosa with the release of secretogenic toxins. One of the toxin involved is the plasmid-encoded toxin (Pet), which is secreted by the autotransporter mechanism and belongs to a growing class of Enterobacteriaceae autotransporter proteins. Since the characteristic aggregative adherence pattern of EAggEC is associated with the presence of a large plasmid called pCVD432, DNA probes and PCR primers derived from this plasmid have been recommended as a screening method for EAggEC in the clinicai laboratory. In this study 164 E. coli isolates positive for the pCVD432 probe were tested for adherence to HEp-2 cells in which 141 isolates showed aggregative pattern, 12 isolates from them amplify a 1037-bp DNA fragment corresponding to pet gene by PCR. Using this samples we standardized an immuno-dot assay for EAggEC detection through Pet toxin as target antigen. 300 µl of bacterial supernatant were applied in a PVDF membrane, and using a rabbit polyclonal sera anti-Pet the expression of the toxin by immuno-dot was in the same isolates in which the gene was detected. Besides no negative controls reacted with Pet antisera, in which we included 40 isolates with no virulence markers for diarrheagenic E. coli and E. coli expressing toxins other than Pet. This method proves to be rapid, sensitive, specific and low cost, demonstrating this potential as diagnosis for Pet expression and its association with diarrhea.

L’étude proposée est une étude préliminaire en vue d’utiliser des microrésonateurs en silicium fonctionnalisé en vue de la détection électromécanique d’espèces biologiques présentes à l’état de trace. Deux aspects ont été principalement étudiés : la capture ultra-sensible mais aussi la capture ultra-spécifique d’espèces biologiques comme des marqueurs pour la maladie d’Alzheimer. Elle a été effectuée dans le cadre d’une collaboration entre le département MINASYS (Micro et Nano-Système) de l’Institut d’Electronique Fondamental (IEF) et le Laboratoire Protéines et Nanotechnologies en Sciences Séparatives de la Faculté de Pharmacie de Châtenay-Malabry.Il s’est ainsi s’agit de développer un micro-résonateur en silicium intégrant des canaux enterrés permettant la circulation d’un fluide biologique à l’intérieur de la poutre et non à l’extérieur comme ils le sont plus couramment, et ce, dans le but de limiter au maximum l’amortissement du résonateur. En ce qui concerne les aspects en biologie, nous nous sommes plus particulièrement interessé à la détection d’un bio-marqueur de la maladie d’Alzheimer, le peptide amyloïde Aβ1-42. Jusqu’à présent, le dépistage de la maladie nécessite une concentration suffisamment importante de bio-marqueurs et le recours à une ponction du liquide céphalorachidien après l’apparition des symptômes de la maladie est donc nécessaire. Le microsystème présenté ici permettra de nous approcher d’un système capable de détecter des traces de biomarqueurs présentes dans le sang ou l’urine par exemple. Ainsi, dans un premier temps, nous avons mis au point un mode de détection précoce du peptide Aβ1-42 couplant un canal micro-fluidique et la microscopie à fluorescence. La surface des canaux en silicium doit être fonctionnalisée afin de permettre le greffage spécifique des antigènes. Pour cela, nous avons mis au point une technique s’appuyant sur la reconnaissance spécifique anticorps-antigène, celle-ci nécessitant une étape préalable de fonctionnalisation chimique de surface.Le manuscrit présenté ici s’articule en quatre parties principales. Dans un premier temps, une étude bibliographique permet de faire l’état de l’art sur le principe de fonctionnement de différents types bio-capteurs couramment utilisés et leurs performances. Une seconde partie décrit la fonctionnalisation de surface du silicium et plus spécifiquement de la réaction de silanisation en phase liquide réalisée sur des surfaces planes et dans des canaux fluidique. Nous abordons ensuite le domaine de la reconnaissance spécifique d’entités biologiques et détaillons les étapes de greffage des protéines réalisées sur les surfaces ainsi que la conception d’immuno-sandwich entreprise dans des canaux fluidiques. Enfin la dernière partie du manuscrit rassemble des différents résultats préliminaires obtenus en vue de l’élaboration du micro-capteur de type poutre résonante à canaux enterrés. / This study is a preliminary study to use a fonctionnalized microsensor based on electromechanical detection of biological species present at trace levels. Two aspects were involved in this work: capture high-sensitivity but also the capture of ultra-specific biological species as markers for Alzheimer's disease. It was conducted as part of collaboration between the department MINASYS (Micro and Nanobio and Microsystems) of the Institute of Fundamental Electronics (IEF) and the Laboratory Proteins and Nanotechnologies in Separative Sciences, at the Faculty of Pharmacy of Chatenay-Malabry.The sensor includes a micro-resonator, placed in vaccum, incorporating silicon buried channels for the circulation of a biological fluid and not outside as they are more commonly, and in order to minimize the damping of the resonator. As regards the biological aspects, we primily focus on the detection of a biomarker of Alzheimer's disease, amyloidal peptide Aβ1-42. Until now, screening requires the use of a puncture and cerebrospinal fluid after onset of symptoms, therefore a sufficiently high concentration of biomarkers is required. Initially, we developed a method of early detection of Aβ1-42 peptide coupling a microfluidic system and fluorescence microscopy. Therefore, the micro-resonator that are present here allow to detect these biomarkers that are present at very low concentrations in other biological fluids in the first stage of the disease, for example, in the blood or urine. Thus, in a first step, we have developed a method of early detection of Aβ1-42 peptide coupling channel microfluidic and fluorescence microscopy. The surface of the channels in silicon must be functionalized to allow the grafting of specific antigens. For this, we developed a technique based on the specific antibody-antigen recognition, the latter requires a preliminary step of chemical functionalization of surfaces.This manuscript consists of four main parts. Initially, we present a literature review of the state of the art on the principle of operation of various types commonly used bio-sensors and the performance obtained. A second chapter describes the functionalization of silicon area and more specifically of the silanization reaction in liquid phase performed on flat surfaces and in fluidic channels. In the following chapter, we discuss the field of specific recognition of biological entities and detail the steps of grafting performed on protein surfaces and design immunoassay sandwich in fluidic channels. The last chapter of the manuscript brings together various preliminary results for the development of micro-sensor type resonant beam.

Le but de cette étude est de concevoir, fabriquer et caractériser une puce microfluidique afin de mettre en oeuve la capture de nanoparticules magnétiques fonctionnalisées en vue de la reconnaissance d’anticorps spécifiques (couplage d’une très grande spécificité et sensibilité). Après avoir modélisé et simulé les performances de la microbobine intégrée dans le canal de la puce microfluidique en prenant soin de limiter la température du fluide à 37°C, la capture devant être effective, le microsystème est fabriqué en salle blanche en utilisant des procédés de fabrication collective. La fabrication du microdispositif en PDMS a aussi donné lieu à l’optimisation de procédés de modification de surface afin d’assurer la ré-utilisation du microdispositif (packaging réversible) et la limitation de l’adsorption non spécifique. L’immobilisation des anticorps su les billes (300 nm) a été menée à l’intérieur du canal en utilisant un protocole de type ELISA éprouvé. Le procédé a montré qu’il était également efficient pour cet environnement puisque nous avons pu mettre ne évidence la capture de nanoparticules / In this study, a concept of microfluidic chip with embedded planar coils is designed and fabricated for the aim of trapping effectively functionalized magnetic nanobeads and immobilizing antibody (IgG type). The planar coils as a heart of microfluidic chip is designed with criterion parameters which are optimized from simulation parameters of the maximum magnetic field, low power consumption and high power efficiency by FE method. The characterization of microcoils such as effectively nanobeads (300 nm) at low temperature (<37oC) is performed and confirmed. The channel network in PDMS material is designed for matching with entire process (including mixing and trapping beads) in microfluidic chip. A process of PDMS’s surface modification is also carried out in the assemble step of chip in order to limit the non-specific adsorption of many bio substances on PDMS surface. The microfluidic chip assemble is performed by using some developed techniques of reversible packaging PDMS microfluidic chip (such as stamping technique, using non-adhesive layer, oxygen plasma combining with solvent treatment). These packaging methods are important to reused microchip (specially the bottom substrate) in many times. The immobilization of antibody IgG-type is performed inside microfluidic chip following the standard protocol of bead-based ELISA in micro test tube. The result showed that IgG antibodies are well grafted on the surface of carboxyl-beads (comparing to result of standard protocol); these grafted antibodies are confirmed by coupling them with labeled second antibody (Fab-FITC conjugation).

Ce travail de thèse a porté sur le développement de réseaux de micro- et nanocapteurs opto-électrochimiques pour la bioanalyse. Ils répondent à la demande grandissante dans le domaine de la recherche et du diagnostic pour des outils permettant de réaliser de multiples analyses simultanément avec des échantillons de faibles volumes. Ces nouvelles biopuces de haute densité sont fabriquées à partir de faisceaux cohérents de fibres optiques. Une des deux faces est micro- ou nanostructurée par une attaque chimique, puis fonctionnalisée avec une sonde biologique. La première biopuce est un réseau de nanocapteurs fluorescents à ADN où les sondes ont été immobilisées grâce aux propriétés d’électropolymérisation du pyrrole. La lecture est réalisée à distance au travers du faisceau d’imagerie. En combinant la technique d’immobilisation avec des microleviers électrochimiques, plusieurs sondes différentes ont pu être adressées sur le même réseau nanostructuré. La seconde biopuce permet d’effectuer des immunodosages multiplexés en utilisant l’imagerie électrochimiluminescente résolue à l’échelle d’une microsphère. Le développement de cette technique permet de combiner les avantages de l’électrochimiluminescence avec des immunodosages multiplexés. L’élaboration de ces réseaux allie différentes techniques physico-chimiques, notamment électrochimiques, pour obtenir des biopuces avec un fort potentiel, grâce à une densité et un degré de multiplexage importants. / This work presents the development of optoelectrochemical micro- and nanosensor arrays for bioanalytical applications. These platforms respond to the growing need in research and diagnostic for tools allowing multiple and simultaneous analysis in small-volume samples. These new high density biochips are made from coherent optical fiber bundles: one face is micro- or nanostructured by chemical etching and then functionnalized with biological probes. The first biochip is a fluorescent DNA nanosensor array where probes have been immobilized by electrodeposition of a polypyrrole thin film. The detection of the hybridization is remotely performed through the imaging fiber. Different probes were succesfully addressed onto the same nanostructured array thanks to electrochemical cantilevers. The second biochip allows multiplexed sandwich immunoassays using electrochimiluminescent imaging resolved at the single bead level. In particular, the development of this new readout mechanism allows extending electrochemiluminescent detection for multiplexed immunoassays. Design and implementations of both platforms take advantages of different physical and chemical techniques, especially electrochemical, to obtain biochips with a great potential through high density and high multiplexing level.

Escherichia coli enteroagregativa (EAEC), destaca-se como um importante patógeno emergente causador de diarréia persistente em países em desenvolvimento e de diarréia aguda em países desenvolvidos. A grande heterogeneidade dos fatores de virulência caracteriza esta categoria, porém não foi estabelecido um marcador genético comum a todas as amostras de EAEC. O padrão de adesão agregativa (AA) em células HEp-2 e HeLa é a forma de caracterização e diagnóstico mais precisos desta categoria. Uma das toxinas envolvidas na patogênese é Plasmid-encoded toxin (Pet) pertencente à classe das proteínas autotransportadoras com características de uma serino protease denominada SPATEs. Iniciou-se este estudo com a determinação do padrão de adesão de 164 amostras EAEC, previamente caracterizadas como sonda pCVD432 ou onda AA positiva. Assim, 141 (86%) amostras, que apresentaram padrão de adesão agregativo, foram caracterizadas como EAEC. Face aos resultados obtidos, confirmou-se a baixa especificidade da sonda AA. A pesquisa do gene pet, por meio de ensaio de PCR, resultou na positividade de 12 (8,5%) amostras. Prosseguiu-se esse estudo com a padronização da reação de immuno-dot. Utilizando-se 300 &#181;L do sobrenadante bacteriano, soro policlonal anti-Pet e o conjugado nas diluições 1/50 e 1/2.500, respectivamente, resultados bastante reprodutíveis foram obtidos. O método foi mais sensível que a detecção do gene por PCR. Por esse ensaio, detectou-se a toxina Pet em 16 (11,3%) das 141 amostras EAEC. Nenhuma das amostras controle negativo foi reconhecida pelo soro anti-Pet, assim como as amostras de E. coli produtoras das mais diversas toxinas. Apesar da baixa prevalência de amostras de EAEC produtoras da toxina Pet, neste estudo padronizou-se um método rápido, sensível, específico e de baixo custo para pesquisa desta toxina mostrando o potencial diagnóstico deste ensaio para uso em inquéritos epidemiológicos, o que poderá permitir determinar o papel da Pet no desenvolvimento de diarréia aquosa. / Enteroaggregative Escherichia coli (EAggEC) is an emerging diarrheal pathogen, whose pathogenesis is thought to comprise colonization of the intestinal mucosa with the release of secretogenic toxins. One of the toxin involved is the plasmid-encoded toxin (Pet), which is secreted by the autotransporter mechanism and belongs to a growing class of Enterobacteriaceae autotransporter proteins. Since the characteristic aggregative adherence pattern of EAggEC is associated with the presence of a large plasmid called pCVD432, DNA probes and PCR primers derived from this plasmid have been recommended as a screening method for EAggEC in the clinicai laboratory. In this study 164 E. coli isolates positive for the pCVD432 probe were tested for adherence to HEp-2 cells in which 141 isolates showed aggregative pattern, 12 isolates from them amplify a 1037-bp DNA fragment corresponding to pet gene by PCR. Using this samples we standardized an immuno-dot assay for EAggEC detection through Pet toxin as target antigen. 300 &#181;l of bacterial supernatant were applied in a PVDF membrane, and using a rabbit polyclonal sera anti-Pet the expression of the toxin by immuno-dot was in the same isolates in which the gene was detected. Besides no negative controls reacted with Pet antisera, in which we included 40 isolates with no virulence markers for diarrheagenic E. coli and E. coli expressing toxins other than Pet. This method proves to be rapid, sensitive, specific and low cost, demonstrating this potential as diagnosis for Pet expression and its association with diarrhea.

A growing body of evidence supports the observation that marine disease outbreaks, especially those caused by opportunistic pathogens, are increasing in frequency and severity. One genus of such pathogens, Labyrinthula, has been identified as the causative agent of seagrass wasting disease, an epidemic that has historically plagued seagrass beds around the world. It is suspected that pathogenicity is intimately linked to the ability of the host to initiate defense responses, but a lack of compelling evidence prevents any meaningful application of preliminary observations. This body of work investigated the roles of host genotype, host immune status, and environmental stressors in dictating the susceptibility of Thalassia testudinum (turtlegrass) to seagrass wasting disease, through two investigational studies. The first, a lab-based study, addressed the deficit in empirical methods through the development of techniques that measured: 1) Labyrinthula loading in host tissue through a novel qPCR-based assay and 2) immune status in the seagrass host via four immune biomarker assays, measuring peroxidase (POX), exochitinase (EXOC), polyphenol oxidase (PPO), and lysozyme (LYS) activity. These methods were used to analyze turtlegrass individuals exposed to 1) abiotic stressors alone or 2) abiotic stressors followed by pathogen-challenge, in a controlled laboratory setting. The qPCR assay successfully quantified pathogen loading in seagrass tissue with high specificity. All four biomarkers were constitutively active in host tissue, but expression was largely unaffected by the chosen abiotic stressors. There were significant positive relationships between pathogen loading and two of the four biomarkers (POX and EXOC), regardless of abiotic stress treatment. Finally, despite the widely variable response among individuals, regardless of treatment, we identify a potential trade-off mediated immune response in T. testudinum, when faced with pathogen invasion. The second investigation was a field study conducted in Florida Bay, a shallow, subtropical estuary characterized by many spatiotemporally unique basins, where T. testudinum dominates. Samples collected from 15 representative sites were analyzed using the methods developed in the first study as well as historical monitoring databases, in an effort to identify ecologically significant trends that existed in patterns between: 1) pathogen loading and immune status; 2) pathogen loading and geographic site; 3) pathogen loading and morphometric characteristics; 4) pathogen loading and water quality data; 5) immune status and geographic site; 6) immune status and morphometric characteristics; and 7) immune status and water quality data. The results revealed that both pathogen loading and immune status varied as a function of location in Florida Bay. Furthermore, based on the trends observed among and between sites with regards to pathogen loading, immune status, leaf morphology and water quality, a mechanism in which all four of these parameter sets interact is proposed as a potential explanation for the differences observed in Labyrinthula prevalence and severity within the bay. The results of both investigations address whether wasting disease susceptibility is driven primarily by variability in the environment or in the host species, and provide valuable insight regarding the extent to which seagrasses possess the capacity for resilience against marine pathogens.

Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods. Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated. Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale.

Semiconductor quantum dots (QDs) have emerged as a new class of fluorescent probes and labeling agents for biological samples. QDs are bright, highly photostable and allow simultaneous excitation of multiple emissions. Owing to these properties, QDs hold exceptional promise in enabling intracellular biochemical studies and diagnosis with unprecedented sensitivity and accuracy. However, use of QD probes inside living cells remains a challenge due to difficulties in delivery of nanoparticles without causing aggregation and imaging single nanoparticles inside living cells. In this dissertation, a systematic approach to deliver, image and locate single QDs inside living cells is presented and the properties of molecular motor protein driven QD transport are studied. First, spectroscopic and imaging methods capable of differentiating single nanoparticles from the aggregates were developed. These technologies were validated by differentiating surface protein expression on viral particles and by enabling rapid counting of single biomolecules. Second, controlled delivery of single QDs into living cells is demonstrated. A surprising finding is that single QDs associate non-specifically with the dynein motor protein complex and are transported to the microtubule organizing center. Accurate localization and tracking of QDs inside cell cytoplasm revealed multiple dynein motor protein attachment resulting in increased velocity of the QDs. Further, spectrin molecule which is known to recruit dynein motor protein complex to phospholipid micelles was found to associate with the QDs. These results may serve as a benchmark for developing new QD surface coatings suitable for intracellular applications. Since, nanoparticles are similar in size to viral pathogens; better understanding of nanoparticle-cell interactions should also help engineer nanoparticle models to study virus-host cell interactions. (Contains AVI format multimedia files)