Le diabète de type 1 est une maladie auto-immune caractérisée par la destruction des cellules bêta des îlots de Langerhans du pancréas. Au cours de ce processus auto-immun, des auto-anticorps sont produits contre plusieurs antigènes des cellules bêta, par exemple l'insuline, l'acide glutamique décarboxylase (GAD65), la protéine tyrosine phosphatase (IA-2) et le transporteur de zinc (ZnT8). Au moins un auto-anticorps contre l'un de ces antigènes est présent dans> 95% des personnes atteintes de diabète de type 1 lors de la détection de l'hyperglycémie. Ces auto-anticorps peuvent servir de marqueurs précoces de diabète de type 1, car ils peuvent être présents des années avant l'apparition de la maladie, ce qui permet un diagnostic précoce avant les manifestations cliniques. Dans le cadre de cette thèse, nous avons développé, en partenariat avec une équipe de recherche clinique, une série de tests diagnostiques originaux, basée sur la détection précoce des différents auto-anticorps d’îlots de Langerhans à partir d'échantillons de sérum humain. Ces tests de diagnostic comprennent des tests bridging ELISA pour la détection d'auto-anticorps contre l'insuline, IA-2 et GAD65, qui sont rapides, facile à utiliser et n’utilisent pas de radioactivité. De plus, un test immunochromatographique sur bandelette pour la détection des auto-anticorps contre IA-2 a été développé. Le principal avantage des tests bandelettes est sa convivialité : les résultats peuvent être obtenus en 45 min en utilisant de très petits volumes de sérums et sans l'utilisation d’appareils spécialisés. Tous ces tests développés en interne ont été validés avec des échantillons de sérum de patients atteints de diabète de type 1 et de témoins sains et leurs performances ont été comparées avec celles de tests disponibles sur le marché. En outre, nous avons développé un test multiplex pour la détection simultanée de plusieurs auto-anticorps associés au diabète de type 1, ce qui permet de gagner du temps et d’augmenter la valeur diagnostic et prédictive du test par rapport à la détection d’un seul autoanticorps. Ce test multiplex a été validé pour la détection de deux autoanticorps (IA-2A et GADA) et comparé à nos tests ELISA de IA-2A et GADA. / Type 1 diabetes is an autoimmune disease characterized by the destruction of pancreatic beta cells within the islets of Langerhans. In the course of this autoimmune process, autoantibodies are generated against several beta-cell antigens, e.g. insulin, glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (IA-2) and zinc transporter 8 (ZnT8). At least one autoantibody against one of these antigens is present in >95% of individuals with type 1 diabetes upon hyperglycemia detection. These autoantibodies can serve as early markers of type 1 diabetes, since they can be present years before disease onset, allowing for an early diagnosis before clinical manifestations. In the course of this thesis we have developed, in partnership with a clinical research team, a series of original diagnostic tests, based on the early detection of the different anti-Langerhans islet autoantibodies from human serum samples. These diagnostic tests include bridging ELISAs for the detection of autoantibodies to insulin, IA-2 and GAD65, which are rapid, non-radioactive and easy-to-use. Moreover, a lateral flow immunoassay (dipstick) for detection of autoantibodies to IA-2 was developed. The key advantage of lateral flow immunoassay is its user-friendly format: results can be obtained within 45 min using very small volumes of sera and without the use of any specialized apparatus. All these in-house assays were validated with diabetic and healthy human serum samples and the assay performances were compared to commercially available tests on the market. In addition, we have developed a multiplex assay for simultaneous detection of multiple diabetes-associated autoantibodies, which is time-effective and increases the diagnostic and predictive values of the assay, comparing to single autoantibody detection. This multiplex assay was validated for detection of two autoantibodies i.e. IA-2A and GADA and compared to in-house IA-2A and GADA bridging ELISAs.

La recherche de nouvelles réactions est un des enjeux fondamentaux de la chimie organique. En dehors de l’approche classique basée sur la conception d’une réaction en s’appuyant sur les propriétés chimiques des substrats, une nouvelle approche utilisant le criblage systématique de combinaisons aléatoires de fonctions réactives a été récemment adoptée par plusieurs groupes. Cette stratégie nécessite un outil analytique permettant de cribler un très grand nombre de réactions par jour et d’identifier les meilleures combinaisons conduisant à la formation de produits intéressants. Les travaux de thèse présentés dans ce mémoire s’inscrivent dans le contexte de l’utilisation des techniques de dosages immuno-enzymatiques (ELISA) comme outil de criblage pour la recherche de nouvelles réactions de couplage. Dans un premier temps le criblage de 2688 combinaisons de fonctions réactives et de catalyseurs choisies au hasard a été effectué. Ce criblage a permit de mettre en évidence deux nouveaux couplages en présence de sels de cuivre : une réaction entre les thiourées et les phénols conduisant à la formation des isourées et une réaction entre les N-hydroxythiourées et les alcynes conduisant à la formation des thiazole-2-imines. Dans un second temps le criblage de 2816 combinaisons de fonctions sélectionnées, cette fois-ci, de façon rationnelle a été effectué. Ce criblage a visé la découverte de nouvelles cycloadditions [3+2] répondant aux critères de la chimie « click ». Ainsi l’utilisation de dosage immuno-enzymatique a été étendue à l’optimisation des nouvelles réactions découvertes ainsi qu’à l’évaluation de leurs cinétique, chimiosélectivité et biocompatibilité. Près de 3000 tests complémentaires effectuées sur les « hits » issus du criblage primaire ont ainsi permit de mettre en évidence 4 nouvelles réactions de couplage dont une nouvelle réaction « click » : la cycloaddition sydnone-alcyne catalysée au cuivre (CuSAC). Dans la dernière partie de ce manuscrit les études plus détaillées sur la réaction CuSAC ont été effectuées, notamment l’identification de la structure du produit de couplage et l’étendue du champ d’application de cette réaction. Enfin, l’aspect « click » de la réaction CuSAC a été illustré par l’application de cette réaction au marquage d’une protéine. / Discovery of new reactions is one of the fundamental goals in organic chemistry. In addition to the traditional approach to reaction discovery, consisting in designing a reaction on the basis of known chemical properties of reagents, new approaches based on the screening of random combinations of reactive functions and catalysts have been recently developed. The main prerequisite of this strategy is an analytical tool allowing screening of a big number of reactions per day and identifying combinations leading to the formation of unanticipated products. In the work presented herein a high-throughput immunoassay screening has been used for the discovery of new coupling reactions. In the first part of this work a screening of 2688 combinations of randomly chosen reactive functions and catalysts was carried out. This screening led to the discovery of two copper-promoted coupling reactions: a reaction between thioureas and phenols leading to the formation of isoureas through desulfurization; and a reaction between N-hydroxythioureas and alkynes leading to the formation of thiazole-2-imines. In the second part of the work a screening of 2816 combinations of rationally designed chemical functions and catalysts was carried out. This screening was focused on the discovery of catalytic [3+2] cycloadditions that comply with the standards of “click” chemistry. In this study, the use of immunoassay screening was extended to optimize new reactions and to evaluate their kinetics, chemoselectivity and biocompatibility. Therefore, around 3000 complementary tests were carried out on the hits, identified in the primary screening. This allowed the discovery of 3 new coupling reactions and one new “click” reaction: a copper-catalyzed sydnone-alkyne cycloaddition (CuSAC). The last part of the work was focused on detailed studies of the CuSAC reaction. Identification of the structure of the coupling product and substrate scope of this reaction was carried out. Finally, the applicability of the CuSAC reaction for bioconjugation was demonstrated by an example of protein labeling.

Foram testadas, com o teste não treponêmico VDRL e com o teste treponêmico imunoenzimático de competição, 23.531 amostras de soros, coletados em todas as regiões do Brasil, com o objetivo de verificar o comportamento do teste imunoenzimático treponêmico na triagem de amostras. A prevalência obtida foi de 0,63% com o VDRL e de 0,84% para o teste imunoenzimático. A análise dos dados foi feita comparando-se os resultados dos dois testes com os resultados do teste treponêmico de imunofluorescência indireta (FTA-ABS), considerado como teste de referência. No total, 1120 amostras foram submetidas ao teste FTA-ABS, incluindo todas as que foram reagentes em qualquer um dos testes de triagem e 872 amostras negativas. Amostras com resultados discordantes entre os testes foram submetidas a um teste imunoenzimático do tipo Western blot. Nas amostras por nós estudadas, o teste imunoenzimático apresentou sensibilidade de 89,95% e especificidade de 99,78%, muito superior aos 55,11% de sensibilidade e 97,43% de especificidade que encontramos para o VDRL. Os resultados dos testes detectaram positividade em amostras diferentes portanto, recomendamos utilizar a associação dos dois testes, como método de triagem, quando se trata de populações de baixa prevalência. Resultados preliminares do Western blot sugerem a participação doas proteínas de 43 kD, 17 kD e 15,5 kD na reação de ELISA treponêmico competitivo. / The VDRL, a non treponemal test, and a treponemal competitive ELISA were used to test 23,531 serum samples, collected from conscript men throughout Brazil, with the objective of assessing the performance of the competitive ELISA on the screening of serum samples. The VDRL showed a prevalence of 0.63% contrasting with a 0.84% prevalence showed by the competitive ELISA. The results obtained with the two tests were then compared to those obtained by fluorescent treponemal antibody absorption (FTA-ABS) test which is considered the gold standard method for detection of antibodies for syphilis. A total number of 1,120 samples, which included all that were reagent in at least one of the screening test plus 872 that were negative in both tests, were submitted to the FTA_ABS test. In addition, some of the samples that presented discrepant results between the two tests studied were also submitted to the Western blot test. The results of the screening tests showed an 89.95% sensitivity and a 99.78% specificity for the competitive ELISA, which are much higher than the 55.11% sensitivity and 97.43% specificity presented by the VDRL. Also, the tests detected positivity in different samples. In conclusion, we recommended the use in tandem of both tests as screening for syphilis antibodies in low prevalence populations. In addition, the results of the Western blot seemed to suggest the positivity of the ELISA becoming non reactive after treatment of the patient and that the 43 kD, 17 kD and 15 kD proteins are the main proteins involved in the ELISA competitive reaction.

Le transfert d'énergie par résonance de type Förster (FRET) est un transfert d'énergie non radiatif d'un donneur à un accepteur à proximité. En raison de sa dépendance de la distance extrêmement sensible entre env. 1 et 20 nm, FRET joue un rôle important dans la nanobiotechnologie. Ainsi FRET peut être utilisé comme système de transduction du signal, mais aussi pour l'estimation de la distance entre le donneur et l'accepteur.Les accepteurs de FRET utilisés dans ce travail étaient des nanocristaux semi-conducteurs (quantum dots, QD). Ce type de luminophore est bien connu pour ses propriétés photophysiques supérieures. Leur absorption forte et spectralement large, et leur photoluminescence (PL) brillante et spectralement fine et de l'accordabilité spectrale de la PL sont idéalement adaptés aux applications de FRET. La combinaison des QDs comme accepteurs de FRET avec des complexes luminescents de terbium (CLT) comme donneurs permet des grandes distances de FRET (> 10 nm). La distance de FRET est caractéristique d'une paire FRET et décrit la distance à laquelle l'efficacité de FRET est égale à 50%. CLT sont idéal comme donneurs de FRET parce qu'ils fournissent des longues durées de vie des états excités à l'ordre de la milliseconde. Cette longue période de décroissance de PL permet de mesurer en temps décalé pour une répression d’autofluorescence et la PL des QDs directement excités, ce qui augmente fortement la sensibilité de détection. Les bandes d'émission de PL structurés de CLT et la PL accordable de QDs sont idéales pour l'application dans le diagnostic multiplexé.La thèse se compose de deux parties. Dans la première le couple FRET CLT-QD a été utilisé dans des immunodosages de FRET homogènes pour la détection de marqueurs biologiques antigène prostatique spécifique (TPSA), énolase specifique des neurones (NSE), antigène carcino-embryonnaire (CEA), et le récepteur du facteur de croissance épidermique (EGFR). La sensibilité du dosage immunologique a été optimisé en utilisant des différents types d'anticorps IgG, F (ab')2, F (ab), et pour EGFR un anticorps de chaîne lourde unique. Des limites de détection picomolaires étaient réalisées en utilisant des échantillons de sérum de petit volume et des mesures sur des lecteurs de microplaques cliniques. Une étude détaillée des différents systèmes de FRET en utilisant la spectroscopie résolue en temps a été réalisée pour étudier l'influence des différents anticorps sur la distance, la fonctionnalité, et la sensibilité des immunodosages. L'étude a été complétée par la mesure de NSE et CEA dans un format duplexé et des échantillons réels de patients.Dans la deuxième partie le FRET pour des mesures de distance nanométriques (réglette moléculaire ou spectroscopique) étaient étudiés. FRET en résolution temporelle a permis de calculer la distance entre le donneur et l’accepteur. Par conséquent, deux stratégies de liaisons différentes ont été étudiées pour établir une proximité entre le CLT et le QD : la reconnaissance biotine-streptavidine et l’auto-assemblage médié par polyhistidine. Une étude en résolution temporelle détaillée a été effectuée avec des QDs de différentes tailles, formes et revêtements de surface combiné avec des CLT liés à trois différentes biomolécules. L'analyse des courbes de décroissance multiexponentielle des donneurs et accepteurs permettait à obtenir des informations sur la taille, la forme et la biofonctionnalité des bioconjugués CLT-QD. Les résultats étaient en accord avec d'autres méthodes d'analyse de structure, telles que la microscopie électronique à transmission (MET) ou la diffusion de lumière dynamique (DLS), mais avec l'avantage d'une mesure homogène à la résolution 3-dimensionelle (impossible pour le MET), sans l'inclusion d'une couche d'hydratation (l’inconvénient de DLS) et en faible concentration dans le même environnement que celui utilisé pour l'application biologique. / Förster resonance energy transfer (FRET) is a non-radiative energy transfer from a donor to an acceptor in close proximity. Due to its extremely sensitive distance dependence in the 1 – 20 nm range, FRET plays an important role in nanobiotechnology. Thereby FRET can be used as signal transduction system but also for the distance estimation between donor and acceptor. The selected FRET acceptors in this work were semiconductor nanocrystals (quantum dots, QDs). This type of luminophore is well known for its superior photophysical properties. Their strong and broad absorption and their bright, narrow-band, and size-tunable photoluminescence (PL) emission make QDs ideally suited for FRET application. Combing QDs as FRET acceptors with luminescent terbium complexes (LTC) as FRET donors offers exceptionally large Förster distances of more than 10 nm. The Förster distance is characteristic of a FRET pair and is the distance at which the FRET efficiency equals 50 %. A large Förster distance is desirable as it offers the detection of biological interactions over large distances. LTC are suitable FRET donors for QDs because they provide long excited-state lifetimes in the millisecond range. This long PL decay time enables time-gated measurements for the suppression of autofluorescence and PL of directly excited QDs, which strongly increases the detection sensitivity. Additionally, the structured PL emission bands of LTCs together with the size-tunable PL emission bands of QDs make this FRET pair ideal for the application in multiplexed diagnostics, which is the measurement of multiple biomarkers in a single sample.The PhD thesis consists of two parts. In the first part the LTC-QD FRET pair was used within homogeneous FRET immunoassays for the detection of the biomarkers prostate specific antigen (TPSA), neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), and epidermal growth factor receptor (EGFR). The immunoassay sensitivity was optimized using different types of antibodies IgG, F(ab’)2,F(ab), and for EGFR single heavy chain antibodies, which differ largely in their size. The use of small-volume serum samples and measurements on clinical as well customized fluorescence plate readers result in picomolar detection limits for all measured biomarkers. In addition to these QD-based in vitro diagnostic tests, a detailed study of the different FRET-systems using time-resolved spectroscopy was performed. The investigation revealed the influence of the different antibodies on distance, functionality, and sensitivity of the FRET immunoassays. The study was completed by the measurement of NSE and CEA in a duplexed format and real patient samples were investigated.The second part was to use FRET for nanometric distance measurements as molecular or spectroscopic ruler. Time-resolved FRET measurements enabled the calculation of the distance between donor and acceptor. Therefore two different binding strategies were investigated to establish a close proximity between the LTC-donor to the QD-acceptor, namely biotin-streptavidin recognition and polyhistidine mediated self-assembly. A detailed time-resolved study was performed of QDs with different sizes, shapes, and surface coatings in combination with LTC bound to three different host biomolecules, which also possessed different sizes, shapes, orientations, and binding conditions. The analysis of the multi-exponential decay curves of donor and acceptor allowed to obtain information about the size, shape, and biofunctionality of the investigated QD bioconjugates. The results were in agreement with other structural analysis methods, such as transmission electron microscopy (TEM) or dynamic light scattering (DLS), but with the advantage of a homogeneous measurement with three-dimensional resolution (not possible for TEM), without the inclusion of a hydration shell (drawback for DLS), and at low concentration in the same environment as used for the biological application.

A Doença de Chagas é um dos maiores problemas de saúde pública na América do Sul causando um elevado prejuízo à população. A despeito dos inúmeros esforços para o seu controle, a doença não tem cura e apresenta problemas científicos ainda não esclarecidos. Considerando-se que vários pesquisadores têm usado a radiação ionizante para modificar protozoários ou propriedades imunológicas de biomoléculas, neste trabalho foram estudados aspectos da resposta imunológica induzida em camundongos, resistentes e suscetíveis ao T. cruzi, utilizando formas irradiadas deste parasita. Doses baixas de radiação preservaram a capacidade reprodutiva e de invasão celular. Animais resistentes e suscetíveis, imunizados com os parasitas tratados por radiação, produziram anticorpos específicos. Após o desafio, os animais apresentaram baixa parasitemia, com exceção dos grupos imunizados com parasitas que receberam apenas altas doses de radiação. A seleção de formas tripomastigotas foi obtida irradiando-se os parasitas com baixas doses, o que promoveu aprimoramento da qualidade da resposta imune, a exemplo do que se observa quando da utilização de complemento. Estes dados evidenciam a importância da seleção das formas tripomastigotas para a imunização contra o T. cruzi e apontam a radiação ionizante como alternativa para este fim, uma vez que quando a seleção é feita utilizando-se complemento, depara-se com a dificuldade de sua remoção, colocando em risco o processo de imunização por introduzir substancias estranhas ao organismo. / Chagas\'s disease is one of the major public health problems in South America, promoting high prejudice to the local population. Despite the massive efforts to control it, this disease has no cure and presents puzzling unsolved questions. Considering that many researchers have used ionizing radiation to modify protozoans or biomolecules, we investigated the immunological response aspects of susceptible and resistant mice using irradiated parasites. Low radiation doses preserved the reproductive and invasive capacities of the parasite. Both susceptible and resistant animals, after immunization with irradiated parasites produced specific antibodies. After a challenge, the animals presented low parasitaemia, excepting those immunized with the antigen irradiated with higher doses. Using low radiation doses, we were able to selectively isolate trypomastigotes, leading to an improvement in the quality of the immune response, as previously reported when performing complement system assays. These data highlight the importance of selecting trypomastigote forms for immunization against T. cruz; and point towards ionizing radiation as an alternative to achieve this selection, since when this procedure is performed using complement, the subsequent steps are impaired by the difficulties to remove this component from the system.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T18:55:13Z No. of bitstreams: 1 117110032.pdf: 700015 bytes, checksum: 794f9c0fa96f18e2200227f043531c61 (MD5) / Made available in DSpace on 2015-03-08T18:55:13Z (GMT). No. of bitstreams: 1 117110032.pdf: 700015 bytes, checksum: 794f9c0fa96f18e2200227f043531c61 (MD5) Previous issue date: 2013-08-02 / A maturação in vitro (MIV) é um dos pontos críticos da produção in vitro de embriões bovinos, sendo que vários fatores podem interferir na MIV, como a tensão de oxigênio e a densidade de oócitos por volume de meio. O objetivo deste estudo foi avaliar o efeito da tensão de oxigênio associada a diferentes densidades de oócitos durante a MIV. Para tanto, três experimentos foram conduzidos com oócitos bovinos obtidos de ovários de abatedouro. O experimento I consistiu na avaliação da maturação citoplasmática e nuclear, o experimento II na avaliação da produção de espécies reativas de oxigênio (ROS) e atividade antioxidante, e o experimento III na avaliação das taxas de fecundação in vitro. Após a seleção, os oócitos foram submetidos a MIV distribuídos aleatoriamente em 4 tratamentos: Tratamento 1:10/5%: 1 oócito em 10μl de meio de MIV em 5% de O 2 ; Tratamento 1:10/20%: 1 oócito em 10μl de meio em 20% de O 2 ; Tratamento 1:20/5%: 1 oócito em 20μl em 5% de O 2 e Tratamento 1:20/20%: 1 oócito em 20μl de meio em 20% de O 2 . A MIV foi conduzida em grupos de 15 oócitos em meio TCM 199 modificado, acrescido de FSH, LH, EGF, soro de égua em estro (SEE) e piruvato por 24h. Decorrido o período de MIV foi conduzida a fecundação in vitro em gotas de 300μl de meio Fert-TALP, sendo realizada pelo co-cultivo de oócitos e espermatozóides (2x10 6 sptz/mL) selecionados por gradientes de mini-Percoll por 18h. No experimento I, as taxas de maturação nuclear (69,66%) e maturação citoplasmática (71,55%) foram similares entre os tratamentos (P>0,05). No experimento II, a produção de ROS foi avaliada nos oócitos e no meio de MIV, assim como a atividade antioxidante foi avaliada após 24 h de MIV. A produção de ROS pelos oócitos foi superior nos tratamentos com baixa tensão de oxigênio (5%; 13,3UF) em relação a alta tensão de oxigênio (20%; 7,0UF) independentemente da densidade de oócitos (P<0,05). Os níveis de ROS detectados no meio de MIV foram superiores nos tratamentos com alta densidade de oócitos (1:10) independentemente da tensão de oxigênio (P<0,05). A atividade da SOD (21,3UI) e os níveis de GSH (6,95 nmol GSH/ml) mensurados nos oócitos foram similares entre os tratamentos (P>0,05). As taxas de fecundação e penetração foram superiores nos tratamentos com 20% de O 2 e com alta densidade de oócitos (1:10; 48,8%) em relação aos tratamentos 1:10/5% (29,5%) e 1:20/20% (29,1%; P<0,05). Adicionalmente a taxa de polispermia foi maior no tratamento com alta tensão de oxigênio e baixa densidade de oócitos. (1:20/20%; 27.8%) em relação ao tratamento 1:10/20% (13,41%; P<0,05). Os resultados deste estudo mostram interação entre a tensão de oxigênio e a densidade de oócitos aumentando a produção de ROS em determinadas associações e influenciando posteriormente as taxas de fecundação in vitro de oócitos bovinos. / The in vitro maturation is one of the critical points on in vitro production of bovine embryos so many factors can do an interference on IVM, like oxygen tension and oocyte density by volume of medium. The aim of this study was evaluate the effects of association of oxygen tension with different oocyte density during IVM Three experiments were performed with bovine oocytes obtained from abattoir ovaries, on experiment I was performed the nuclear and cytoplasmic evaluation, on the experiment III the biochemical assay of ROS production and antioxidant activity and on experiment III was realized the evaluation of in vitro fertilization. After selection, the oocytes were randomly distributed in 4 treatments: Treatment 1:10/5%: 1:10µl in 5% of O2; Treatment 1:10/20%: 1:10µl in 20% of O2; Treatment 1:20/5%: 1:20µl in 5% of O2; Treatment 1:20/20%: 1:20µl in 20% of O2. The IVM was performed in droplets (150µl or 300µl) of TCM 199 plus FSH, LH, EGF, EMS and pyruvate. The IVF were performed in droplets (300µl) of Fert-TALP. Was realized IVF with oocytes and spermatozoa (2x106 sptz/mL) selected by Percoll density gradients for 18h. On experiment I, the nuclear maturation rates (69.66%) and reorganization mitochondrial (71.55%) rates were similar among treatments (P>0.05). In Experiment II, the ROS production in oocytes, IVM medium and antioxidant activity were evaluated after 24 h of IVM. ROS production in oocytes was higher on treatments with low tension (5%; 13.3 UF) than 20% oxygen tension (7.0 UF) independently of oocyte density (P<0.05). ROS levels on IVM medium was higher on treatments with high oocyte density (1:10) independently of oxygen tension (P<0.05). The GSH levels (6.95 nmol GSH/ml) and SOD activity (21.3UI) were similar among treatments (P>0.05). The rates of normal fertilization and normal penetration were higher in treatments with 20% of O2 with high oocyte density (1:10;48.8%) than treatments 1:10/5% (29.5%) and 1:20/20% (29.1%; P<0.05). In addiction the polysperm rates were higher on treatment with high oxygen tension and low oocyte density (1:20/20%; 27.8%) than treatment 1:10/20% (13.4%; P<0.05). The results of this study show an interaction between oxygen tension and oocyte density, that increase ROS production on certain associations and subsequently affects the IVF rates. / The viruses are significant important pathogenic agents of several animal species, including cattle. In Brazil, several viral agents causing infections have been described in cattle and they produce significant economic losses. The identification of animals infected by a virus can be performed in different ways; however, definitive confirmation requires demonstration of the agent or immune response. For this purpose, various methods with the capacity to detect the viral particle, biological activity, genome, viral antigens, or specific immune response have been developed. Immunoassays are widely used in laboratory routine for detection of viral antigens in clinical or research. These assays exhibit good sensitivity, specificity and easy for implantation. The immunoassay methodologies are based on the employment of monoclonal or polyclonal antibodies specific to the viral antigens. Therefore, the aim of this study was to produce polyclonal antibodies for some bovine virus, and evaluate their reactivity in immunofluorescence, immunoperoxidase and slot blot tests. For this purpose, strains and/or isolates of bovine herpesvirus type 1 (BoHV-1), bovine herpesvirus type 2 (BoHV-2), bovine herpesvirus type 5 (BoHV-5), bovine herpesvirus type 5 gE deleted (BoHV-5 gEΔ), bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bluetongue virus (BTV), and vaccinia virus (VACV) were amplified in cell culture and the supernatant were used to immunize rabbits. The animals were immunized five times by the subcutaneous route, and five days after the last boost the blood was collected. The serum was obtained by centrifugation. The serum was diluted (1:100 a 1:204.800) and used as primary antibodies in the immunofluorescence, immunoperoxidase and slot blot assays. The working dilution was selected among those produced specific reaction with infected cells and absent or weak background in control cells. The antiserum showed higher reactivity in immunoperoxidase technique than the immunofluorescence and slot blot. The antiserum of the BoHV-1, BoHV-5, BVDV and BRSV presented the reactivity when tested with heterologous isolates in immunofluorescence, immunoperoxidase assays. In summary, that the polyclonal antibodies raised in rabbits have high concentrations of specific antibodies, which were demonstrated by the reactivity in immunofluorescence, immunoperoxidase and slot blot assays. Additionally, these reagents can be considered an important tool for the detection and characterization of various bovine viruses in diagnostic and research routine.

La détection et quantification d’agent biologique occupe une place prépondérante dans la prévention et la détection des dangers possibles pour la santé publique (épidémie ou pandémie), l’environnement ainsi que d’autres risques contextuelles (bioterrorisme, armes biologique ou chimiques…etc.). Par conséquent, le développement d’un système portable et à moindre coût permettant de détecter ces dangers constitue l’axe de recherche pluridisciplinaire de la collaboration entre différents laboratoires de l’UPMC (Paris 6) et « RWTH university » à Aachen en Allemagne. Dans ce projet, nous avons étudié les aspects pluridisciplinaires d’un microsystème (LoC) électromagnétique de détection immunologique basé sur l’utilisation de nanoparticules magnétiques (MNP). En raison de leur extractabilité et de leur triabilité, les MNP sont adaptées à l'examen d'échantillons biologiques, servant de marqueurs pour des réactions biochimiques. La plupart des techniques classiques de détection existantes sont basées sur des méthodes colorimétrique, fluorescence ou électrochimique qui souffrent en majorité de problème de temps d’analyse et de sensibilité. A cet égard, Les méthodes d’immuno-détection magnétiques constituent une alternative prometteuse. Cette détection est effectuée à l’aide des MNP qui sont spécifiquement bio-fonctionnalisés en surface afin d’être liée à la cible (virus, anticorps…etc). La nouvelle méthode magnétique de mélange de fréquence permet la détection et la quantification de ces MNP avec une grande dynamique. Dans cette thèse, l’effort est dirigé vers la miniaturisation de ce système. Pour ce faire, nous avons développé un ensemble d’outils analytiques et de simulations multiphysiques afin d’optimiser les dimensions des parties électromagnétique (bobines planaires) et microfluidiques. Par la suite, des prototypes de cette structure de détection à partir de bobines en circuits imprimés et de réservoirs microfluidiques en PDMS sont dimensionnés et réalisés. Les performances de ces prototypes ont été évaluées en termes de limite de détection de MNP, linéarité et plage dynamique. En outre, ces prototypes ont permis de valider les outils de dimensionnement réalisés. Une limite de détection de nanoparticules magnétiques de 15ng/mL a été mesurée avec un volume d'échantillon de 14 μL correspondant à une goutte de sang. Finalement, la validation du système quant à l’immuno-détection est abordée avec un état de l’art et le développement d’une procédure de fonctionnalisation biochimique de surface ainsi que des premiers tests pour sa validation. / The detection and quantification of a biological agent or entity has become paramount to anticipate a possible health threat (epidemic or pandemic), environmental threat or to combat other contextual threats (bioterrorism, chemical and biological weapons, drugs). Consequently, developing a portable cost effective device that could detect and quantify such threats is the research focus of the joint multidisciplinary project between UPMC (Paris 6) laboratories and RWTH university in Aachen, Germany. In the framework of this project, we have studied the multidisciplinary aspects of an electromagnetic microsystem for immunologic detection based on magnetic nanoparticles (MNP) in a microfluidic lab-on-chip (LoC). Because of their extractability and sortability, magnetic nanoparticles are adapted for examination of biological samples, serving as markers for biochemical reactions. So far, the final detection step is mostly achieved by well-known immunochemical or fluorescence-based techniques which are time consuming and have limited sensitivity. Therefore, magnetic immunoassays detecting the analyte by means of magnetic markers constitute a promising alternative. MNP covered with biocompatible surface coating can be specifically bound to analytes, cells, viruses or bacteria. They can also be used for separation and concentration enhancement. The novel frequency mixing magnetic detection method allows quantifying magnetic nanoparticles with a very large dynamic measurement range. In this thesis, emphasis is put on the miniaturized implementation of this detection scheme. Following the development of analytical and multiphysics simulations tools for optimization of both excitation frequencies and detection planar coils, first multilayered printed circuit board prototypes integrating all three different coils along with an adapted microfluidic chip has been designed and realized. These prototypes have been tested and characterized with respect to their performance for limit of detection (LOD) of MNP, linear response and validation of theoretical concepts. Using the frequency mixing magnetic detection technique, a LOD of 15ng/mL for 20 nm core sized MNP has been achieved with a sample volume of 14 μL corresponding to a drop of blood. Preliminary works for biosensing have also been achieved with a state of the art of surface functionalization and a developed proposed biochemical immobilization procedure and preliminary tests of its validation.

Les travaux présentés dans cette thèse concernent le développement d'un outil d'analyse multiparamétrique pour la quantification de biomarqueurs urinaires du cancer de la vessie.La première partie des travaux de recherche a pour objectif la sélection de marqueurs pour le diagnostic et la récidive des tumeurs urothéliales. Une première étude a permis l'évaluation de la sélectivité de marqueurs candidats dans des échantillons urinaires de patients atteints de cancer de la vessie. Cinq des vingt marqueurs initiaux ont été sélectionnés pour leur performance diagnostique, définissant le Panel 1 : VEGF, MMP9, IL8, PTGS2 et EN2. Une seconde étude a été réalisée afin d'évaluer le potentiel de marqueurs et de paramètres cliniques pour le diagnostic de la récidive des tumeurs urothéliales. Les échantillons urinaires évalués provenaient donc de patients présentant une récidive du cancer de la vessie et de patients ne présentant pas de récidive. Le Panel 2 a ainsi été défini, basé sur le modèle de régression multiple le plus performant. Il comprend les paramètres cliniques et moléculaires suivants : nombre de récidives antérieures, nombre de thérapies par BCG, stade de la tumeur au moment du diagnostic, CDH1, IL8, ErbB2, IL6, EN2 et VEGF.La seconde partie concerne le développement d'un test multiparamétrique pour la quantification des marqueurs sélectionnés. Il s'agit d'une plateforme automatisée, à haut-débit et sous un format de plaque de microtitration 96-puits. La méthode de quantification choisie est un immunoessai de type sandwich sous la forme de puce à protéines. Le développement de la plateforme a débuté avec le Panel 1 dont trois des cinq marqueurs (VEGF, MMP9 et IL8) ont été intégrés avec succès. Suite à la seconde étude de sélection de marqueurs, le développement de l'immunoessai multiparamétrique a été orienté vers le Panel 2. À l'exception du marqueur EN2, nécessitant une configuration d'immunoessai différente, tous les marqueurs du Panel 2 ont pu être intégrés à la plateforme / The work reported in this thesis focuses on the development of a multiplex analytical tool for the quantification of selected bladder cancer urinary biomarkers.The aim of the first part of this work is the selection of urinary biomarkers for the diagnosis and recurrence of urothelial tumors. A first study evaluated the selectivity of candidate markers in urine samples of bladder cancer patients. Five of the twenty initial markers were selected for their diagnostic performance. They define Panel 1: VEGF, MMP9, IL8, PTGS2 and EN2. A second study was then conducted to assess the potential of urinary markers and clinical parameters for the diagnosis of bladder cancer recurrence. Two types of urine samples were thus evaluated: samples from recurrent bladder cancer patients and samples from bladder cancer patients without recurrence. Panel 2 was then defined based on the best performing multivariate regression model. It includes the following clinical and molecular parameters: number of past recurrences, number of BCG therapies, tumor stage at diagnosis, CDH1, IL8, ErbB2, IL6, EN2 and VEGF.The second part involves the development of a multiplex test for the quantification of the selected markers. It is a high-throughput automated platform in a 96-well microtiter plate format. It was designed as a multiplex sandwich immunoassay based on a protein microarray. The platform development began with Panel 1 for which three of the five markers (VEGF, MMP9 and IL8) were successful integrated into a multiplex immunoassay. The end of the second marker selection study marked the development transition from Panel 1 to Panel 2. With the exception of EN2, requiring a different immunoassay configuration, all the Panel 2 markers were integrated into the platform

The number of monoclonal antibodies approved for therapeutic use has increased rapidlyover the last decade. As a consequence, precise and robust kinetic characterization techniquesare crucial in order to select the best suitable candidates. A kinetic characterization methodwas developed in Gyrolab with automated sample transfers. The characterization wasperformed in solution in a mixing CD, containing an integrated nanoliter mixing chamberwith affinity binding columns. Association rate constants were determined for four anti-TSHantibodies with values ranging from 3x105 M-1s-1 to 10x105 M-1s-1. The antibodies wereranked according to kass. Reproducibility

In der vorliegenden Arbeit wird eine integrierbare Pumpentechnologie für polymerbasierte mikrofluidische Systeme entwickelt. Ausgehend von den Anforderungen für die Durchführung molekulardiagnostischer Nachweise kommen dabei Fertigungsverfahren zum Einsatz, die sich auch für Einweg-Anwendungen eignen. Das genutzte Aktorprinzip für die integrierten Mikropumpen basiert auf der Elektrolyse von Wasser. Zur besseren technologischen Integrierbarkeit wird das Wasser in Form eines Hydrogels appliziert. Der Elektrolyt wird dabei mit einer Polymermembran mit geringer Wasserdampfdurchlässigkeit verschlossen. Die Membran wird in ihrem plastischen Verformbereich genutzt. Zur Dimensionierung der Mikropumpen und des mikrofluidischen Systems werden analytische und numerische Modelle entwickelt, die eine gute Übereinstimmung mit den Messwerten zeigen. Die Funktionsfähigkeit wird anhand zweier vollständig integriert ablaufender Immunoassays demonstriert. Dabei kommt ein polymerbasierter, optischer Biosensor zum Einsatz.