Made available in DSpace on 2014-10-09T12:52:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:55Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Orientador: Wilter Ricardo Russiano Vicente / Coorientador: Frederico Ozanan Barros Monteiro / Coorientador: Marcus Antonio Rossi Feliciano / Banca: Priscila Viau Furtado / Banca: Rodrigo del Rio do Valle / Banca: Maria Emília Franco Oliveira / Banca: Lindsay Unno Gimenes / Resumo: Macacos-da-noite são tidos como excelentes modelos experimentais, que podem contribuir com o desenvolvimento de biotécnicas da reprodução em primatas. O monitoramento do ciclo é um procedimento básico, porém complexo em animais selvagens. Tem-se desenvolvido métodos não invasivos para avaliar o perfil hormonal reprodutivo desses animais expandindo o conhecimento sobre a fisiologia reprodutiva de primatas em cativeiro e vida livre. O presente estudo visa monitorar a atividade folicular e as concentrações fecais de metabólitos de progesterona para ampliar os conhecimentos sobre a fisiologia reprodutiva dessa espécie. Foram utilizadas 12 fêmeas adultas, pertencentes à colônia de reprodução de macacos-da-noite do CENP. O estudo foi realizado a partir do exame ultrassonográfico do ovário e colheita de fezes para monitoramento dos níveis de metabólitos de progesterona dos animais por enzimaimunoensaio. Por meio da dosagem de metabólitos de progesterona foi possível apenas sgerir o ciclo da espécie para o período estudado. Porém, os níveis basais (2,1 ± 0,8 ng/g de fezes secas), a médio do pico (36,6 ± 8,6 ng/g fezes secas), os níveis médios (4,7 ± 1,8 ng/g de fezes secas) e os valores mínimo (0,4 ng/g de fezes secas) e máximo (49,9 ng/g de fezes secas) de metabólitos de progesterona foram determinados para espécie em cativeiro sob estas condições. A determinação do pico e concentrações basais de metabólitos de progesterona, conjuntamente a avaliação ultrassonográfica são ferramentas não invasivas e factíveis na avaliação do ciclo estral de macacos-da-noite / Abstract: Owl Monkeys are considered excellent experimental models and can contribute to the development of biotechnologies of reproduction in primates. Monitoring the reproductive cycle is a basic procedure, however complex in wild animals. Noninvasive methods has been developed to assess the reproductive hormonal profile of these animals expanding knowledge on reproductive physiology of primates in captivity and the wild. This study aims to monitor follicular activity and fecal progesterone levels to increase knowledge about reproductive physiology of this species. 12 adult females belonging to the breeding colony of owl monkeys of CENP were used. The study was performed using the ultrasound examination of the ovaries and feces collection for monitoring the levels of metabolites of progesterone by enzymeimuneassay. By progesterone assay we may suggest the cycle of the species for the period studied. However, the baseline levels (2.1 ± 0.8 ng/g), mean peak (36.6 ± 8.6 ng/g), mean levels (4.7 ± 1.8 ng/g) and the minimum (0.4 ng/g) and maximum (49.9 ng/g) metabolites of progesterone were determined for the specie in captivity. The determination of the peak and basal levels of progesterone together to the sonographic evaluation are noninvasive and feasible in the evaluation of the estrous cycle in owl monkeys / Doutor

Made available in DSpace on 2014-10-09T12:52:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T13:58:55Z (GMT). No. of bitstreams: 0 / A Leishmaniose Visceral Humana (LVH) é uma protozoonose sistêmica grave que tem apresentado mudança no comportamento epidemiológico, com uma tendência crescente no Brasil. Na Região Norte, com a criação do Estado do Tocantins em 1989, houve aumento da incidência de LV em decorrência das modificações ecoepidemiológicas. Foi realizado um estudo descritivo de corte transversal para avaliar o quadro da LVH no município de Araguaína (TO) no período de janeiro de 2007 a agosto de 2010. Adicionalmente, foram realizados ensaios para comparar as abordagens diagnósticas sorológicas para o diagnóstico da doença. Na distribuição dos casos de LVH nos municípios do norte do estado do Tocantins, Araguaína representa 79,8% no total dos casos no período de 2.007 a agosto de 2.010. Os resultados deste estudo revelaram que, nesse período, 769 casos foram confirmados, sendo que 98,7% foram de casos novos. Houve um predomínio do sexo masculino de 57,8% e a faixa etária mais acometida foi a de 0 a 14 anos, com 71,3% dos casos notificados. Na distribuição por zona de residência, foram notificados 98,5% dos casos na zona urbana, mostrando a urbanização da doença. Nos ensaios, foram utilizadas uma abordagem usual da rede do SUS: um ensaio de imunofluorescência indireta (IFI), um ELISA convencional, somados a um ELISA dissociativo. Os ensaios de ELISA apresentaram alta reprodutibilidade. Os pacientes positivos à IFI apresentaram uma frequência de 4% de falso-positivos quando testados em ambos os ELISA. Entre os pacientes suspeitos, houve uma alta frequência de positivos ao ELISA, maior no ELISA dissociativo do que no ELISA convencional. Na população assintomática, houve um achado de amostras positivas ao ELISA dissociativo. A presença de imunocomplexos em pacientes assintomáticos no início da doença ou sua presença em pacientes com resposta humoral menos intensa, como crianças, pode explicar estes achados de maior positividade no ELISA dissociativo. A confirmação destes resultados depende da pesquisa parasitológica em pacientes positivos ao ELISA dissociativo. Estes resultados mostram a importância de novas abordagens sorológicas no diagnóstico da LVH que viabilizariam uma triagem de paciente para diagnóstico parasitológico invasivo. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Vários autores já enfatizaram a importância do monitoramento do ciclo estral em cadelas e citaram exemplos de como ele pode ser feito. O objetivo deste estudo foi avaliar a técnica de dosagem de progesterona salivar para monitorar o ciclo estral da espécie. Para composição do grupo experimental, foram utilizadas 13 cadelas. As amostras de sangue e saliva foram colhidas paralelamente em todos os animais, a partir dos primeiros sinais de proestro. As amostras salivares foram obtidas com o uso de dispositivo específico para coleta Salivette®, método que se mostrou eficaz, visto que foi possível obter volume suficiente para dosagem de progesterona na grande maioria das amostras. As concentrações de progesterona no soro foram determinadas pela técnica de RIE e na saliva por EIE. Embora haja uma relação linear crescente e positiva entre a progesterona sérica e salivar (r=0,704; p<0,0001), não é possível utilizar o parâmetro salivar para determinar o momento da ovulação. / Several authors have already emphasized the importance of monitoring estrous cycle in bitches and mentioned examples of how it can be done. The aim of this study was to evaluate the salivary progesterone quantification technique in order to monitor the estrous cycle in this species. To compound the experimental group, 13 bitches were used. Blood and saliva samples were collected simultaneously in all animals, starting about the first day of proestrus signs. Salivary samples were collected with a specific device: Salivette®. This method was effective, since it was possible to obtain enough volume in almost all samples to quantify progesterone. Serum progesterone was quantified by radioimmunoassay and salivary progesterone by enzyme immunoassay. Although there is an increasing, linear and positive correlation between salivary and serum progesterone (r=0,704; p<0,0001), it is not possible to use the salivary parameter to set the moment of ovulation.

Abstract Natriuretic peptides have emerged as important candidates for the development of diagnostic tools in cardiovascular disease. Their increased concentrations have been found to be useful for ruling out disease of cardiac origin, as prognostic indicators, and in the follow-up of patients with heart failure. In order for natriuretic peptides to be efficient biomarkers, analytical problems in assay specificity and calibration need to be resolved. The aim of the present study was to elucidate circulating molecular components of N-terminal fragments of A- and B-type natriuretic peptides (NT-proANP and NT-proBNP) in human blood, and to develop reliable and novel assays for their measurement with clinical application. Reliable immunoassays for NT-proANP and NT-proBNP were set up based on recombinant calibrators and antisera against different epitopes. A novel immunoassay for detecting the activation of A- and/or B-type natriuretic peptide systems, referred to as NT-proXNP, was also developed. The chromatographic results of human plasma and serum samples indicated that NT-proANP and especially NT-proBNP are heterogeneous in human circulation. They are truncated at both termini, causing a serious risk of preanalytical errors. Further studies with recombinant peptides confirmed that the central parts of NT-proANP and NT-proBNP are stable in plasma and serum even at harsh storage conditions. Thus the most reliable assays are directed at the central portions of the molecule only. All developed assays were applicable to clinical samples of cardiac patients. NT-proXNP showed a diagnostic efficiency equal to or slightly better compared to individual NT-proANP and NT-proBNP assays. Furthermore, the prognostic value of NT-proANP and NT-proBNP was investigated in a population-based sample of men. Both peptides were strong predictors of mortality and its co-morbidities, adding to the prognostic value of conventional risk factors.

This thesis aims to address the challenges of the development of cost-effective and rapid assays for the accurate counting of CD4+ T cells and quantification of HIV-1 viral load for resource-constrained settings. The lack of such assays has severely affected people living in disease prevalent areas. CD4+ T cells count information plays a vital role in the effective management of HIV-1 disease. Here, we present a flow-free magnetic actuation platform that uses antibody-coated magnetic beads to efficiently capture CD4+ T cells from a 30 μL drop of whole blood. On-chip cell lysate electrical impedance spectroscopy has been utilized to quantify the isolated CD4 cells. The developed assay has a limit of detection of 25 cells per μL and provides accurate CD4 counts in the range of 25–800 cells per μL. The whole immunoassay along with the enumeration process is very rapid and provides CD4 quantification results within 5 min time frame. The assay does not require off-chip sample preparation steps and minimizes human involvement to a greater extent. The developed impedance-based immunoassay has the potential to significantly improve the CD4 enumeration process especially for POC settings. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2020. / FAU Electronic Theses and Dissertations Collection

As forensic scientists, we are required to accurately test for certain substances. This may necessitate the use of presumptive tests such as the One Step Multi-Drug Screen Test Card with the Integrated iCup®/iCup®A.D . There are many circumstances where these tests are applicable, such as job-related drug testing, custody and parole cases. An immunoassay, or presumptive test, is designed to give the analyst, even a non-scientific analyst, a general idea of what substance(s) are present in the individuals system, so that he or she is able to more accurately confirm what substances, if any, the individual may have used or consumed. The goal of the validation study of the One Step Multi-Drug Screen Test Card with the Integrated iCup®/iCup®A.D was to determine the sensitivity of various THC containing compounds (delta-9-THC, 11-nor-9-carboxy-delta9-THC, and 11-hydroxy-delta-9-THC) as well as different solutions containing Synthetic Cannabinoids at various concentrations and stored at different temperatures. Each of the drugs were tested below, at and above the cut-off of the drug stated by the manufacturer. The cut-off of 11-nor-9-carboxy-delta9-THC, given by the manufacturer, was 50 ng/mL. For every trial that was conducted, the drug could be detected in the iCup® at this limit of detection of 50 ng/mL, except when the drug had been stored in the freezer for approximately two months prior to use. Delta-9-THC was given a cut-off of 15,000 ng/mL, which is a high concentration, especially when these assays are used in custodial cases and job-related drug tests, where living individuals are providing a fresh specimen. The concentrations of delta-9-THC and 11-hydroxy-delta-9-THC were higher than the cut-off for a positive result of 11-nor-9-carboxy-delta9-THC, but it was tested below the 15,000 ng/mL cut-off for delta-9-THC, established by the manufacturer. After these adjustments were made, both delta-9-THC and 11-hydroxy-delta-9-THC could be detected in a range between 1,500 ng/mL and 5,000 ng/mL. While 1,500 ng/mL is still high for a living specimen, it is substantially lower than 15,000 ng/mL. Analyzing the higher concentration of the synthetic cannabinoid working stock solution of 10,000 ng/mL, positive results were detected at 3,500 ng/mL and 5,000 ng/mL. There were eight cannabinoids, metabolites, and synthetic cannabinoids found in the working stock solution: (1) THC, (2) 11-Hyroxy-delta-9-THC, (3) 11-nor-9-Carboxy-delta-9-THC, (4) AB-FUBINACA, (5) AB-FUBINACA Met. 3, (6) AB-FUBINACA Met. 2a, (7) AB-PINACA-blood, and (8) AB-PINACA Pentatonic Acid metabolite. As the concentrations decreased, a positive result was not produced. Ultimately, the final conclusions of all the testing was that the One Step Multi-Drug Screen Test Card with the Integrated iCup®/iCup®A.D is not as sensitive when it comes to the synthetic cannabinoids, the primary compound present in marijuana (delta-9-THC), and the active metabolite of marijuana (11-hydroxy-delta-9-THC). In order to gain more accurate results using this presumptive test, the sensitivity of the iCup® for a detection of delta-9-THC and 11-hydroxy-delta-9-THC at a lower concentration should be done. By, doing this, an analyst can be more confident when deciding what confirmatory test to use based on what substances are present in a given sample.

Epithelial ovarian cancer is the fifth leading cause of cancer-related deaths in the United States. However, the mortality rate is relatively high, due to in part to the cancer being in an advance stage at diagnosis, since it is often asymptomatic at the early-stage with a ~94% of five-year survival rate if it is diagnosed at a localized stage (stage 1). Early detection of cancer would likely improve the survival rate. Scientists are searching for novel promising methods to detect ovarian cancer at an asymptomatic early stage; also, the method is cheap and user-friendly despite there are various techniques for ovarian cancer detection. Cancer antigen 125 (CA125), a type of serum biomarker that elevates ~50% of women with early-stage and ~80% of women with advanced-stage, is used mostly for screening epithelial ovarian cancer. However, the lack of sensitivity and specificity are known to be the main drawback of CA125. Finding new potential biomarkers that diagnose cancer at a localized stage will significantly reduce the mortality rate. Human epididymis protein 4 (HE4) is such a biomarker that has a higher sensitivity and specificity compared to all other known biomarkers, and recently it has been approved by food and drug administration (FDA) for clinical applications.In this project, we developed sandwich-type micro particles immunoassay for sensitive detection of HE4 biomarker in plasma. Here, we cross-link elemental particles to a specific functional group of the targeted biomolecules based on a covalent and non-covalent linking chemistry to improve the sensitivity and the selectivity of biomarker detection in which Fe3O4 and SiO2 microparticles were used to conjugate and purify the antibody-antigen in a media. The purified assay with the microparticles was analyzed with laser-induced breakdown spectroscopy (LIBS) for detection and quantization analysis of the HE4 biomarker. Furthermore, along with LIBS, Raman, Fourier transform infrared (FT-IR), and UV- Vis spectroscopic techniques were utilized to understand the conjugation dynamic and confirm the conjugation process.

While 3D printing technologies first appeared in the 1980s, prohibitive costs, limited materials, and the relatively small number of commercially available printers confined applications mainly to prototyping for manufacturing purposes. As technologies, printer cost, materials, and accessibility continue to improve, 3D printing has found widespread implementation in research and development in many disciplines due to ease-of-use and relatively fast design-to-object workflow. Several 3D printing techniques have been used to prepare devices such as milli- and microfluidic flow cells for analyses of cells and biomolecules as well as interfaces that enable bioanalytical measurements using cellphones. This review focuses on preparation and applications of 3D-printed bioanalytical devices.

Les béta-lactamines sont les antibiotiques préférentiellement utilisés contre les bactéries Gram négatif responsables d’infections. La dissémination mondiale d’organismes produisant des béta-lactamases à spectre élargi (BLSE) ou des carbapénémases est une préoccupation générale ainsi qu’une menace économique.Parmi ces organismes, les Entérobactéries jouent un rôle important dans les infections nosocomiales (ainsi que les infections communautaires pour E. coli). L’émergence et la dissémination d’Entérobactéries productrices de BLSE (E-BLSE), exprimant principalement des béta-lactamases de la famille des CTX-Ms, et dans une mesure plus inquiétante de carbapénémases (EPC), principalement les enzymes NDM, KPC, IMP, VIM et OXA-48, sont sans le moindre doute un problème de santé publique majeur.Les CTX-Ms hydrolysent les céphalosporines à large spectre et sont les BLSEs principalement rencontrées chez les Entérobactéries lors d’infections urinaires communautaires, mais aussi les bactériémies à E. coli qui peuvent en découler. Ces infections sévères sont traitées avec des carbapénèmes, considérés comme les antibiotiques de dernier recours. Malheureusement, leur utilisation croissante à soumis les entérobactéries à une pression de sélection conduisant à de plus en plus de souches montrant une sensibilité réduite aux carbapénèmes pouvant aboutir à un échec thérapeutique.Si l’on considère les possibilités de traitement limitées pour les E-BLSEs, que les EPCs sont souvent résistantes à plusieurs si ce n’est toutes les classes d’antibiotiques, et que pour certaines peu ou pas de traitements antibiotiques sont disponibles, leur rapide détection et identification sont essentielles. Des tests fiables sont nécessaires pour aider les cliniciens à, rapidement mettre en place des mesures de contrôle de ces infections, adapter les traitements antibiotiques et optimiser les stratégies de soins et leur issue favorable.Lors de la détection des E-BLSEs et des EPCs, il est aussi crucial d’identifier la béta-lactamase impliquée pour la mise en place d’une thérapie adaptée. Les méthodes basées sur la spécificité des anticorps sont sans aucun doute parmi les plus appropriées pour atteindre cet objectif.Pour répondre aux besoins actuels, les méthodes de détection des résistances ont antibiotiques doivent être peu coûteuses (coûts réduits des consommables et des équipements) et facile à mettre en place (technicité faible) pour l’utilisateur. C’est pourquoi nous avons décidé de développer des tests immunochromatographiques qui répondent parfaitement à ce cahier des charges. Pour atteindre cet objectif, nous avons produit des anticorps monoclonaux dirigés contre les CTX-Ms, et les familles de carbapénémases NDM, KPC, et OXA-48. Les tests immuno-chromatographiques correspondants ont été développés et validés. Nos tests sont robustes, facilement transférables dans une version commerciale et stables pour 24 mois sans réfrigération. Ils sont conviviaux, performants en termes de spécificité et sensibilité, et peu couteux, de 7€ (pour un mono-test) à moins de 15€ (pour un multiplex). De plus les résultats sont obtenus dans un court délai sans la nécessité d’un équipement particulier pour la lecture. Nous avons validé un mono-test pour la détection des CTX-Ms du groupe 1, et évalué la détection des groupes 1, 2, 8 et 9 directement dans des échantillons cliniques comme les hémocultures ou l’urine. Des mono-tests pour la détection des NDMs, KPCs et des OXA-48 et un multiplex pour la détection simultanée des cinq principales carbapénémases ont également été validés. Pour ce faire, nous avons utilisés 180 souches isolées sur boites, provenant du Centre National de Référence pour la résistance aux carbapénémases chez les Entérobactéries, dont le contenu en béta-lactamase est caractérisé. / Beta-lactams are antibiotics preferentially used against gram-negative bacilli infections. The worldwide spread of extended spectrum beta-lactamases (ESBL) or carbapenemase-producing organisms is a global concern and also an economic threat.Within those organisms, Enterobacteriaceae have a major role as causes of nosocomial infections (and, for E. coli, also of community-acquired infections). The emergence and dissemination of ESBL-producing Enterobacteriaceae (ESBL-E), mainly expressing beta-lactamases from the CTX-M family, and in a worrier aspect of carbapenemase-producing Enterobacteriaceae (CPE), mainly NDM, KPC, IMP, VIM and OXA-48 like enzymes, are undoubtedly a matter of great public health concern.CTX-Ms hydrolyze broad-spectrum cephalosporins and are the most encountered BLSE in Enterobacteriaceae, and CTX-Ms producers have been reported as the most prevalent ESBL producers in community-onset urinary tract infections (UTIs). Moreover, CTX-M-producing E.coli are a major cause of bloodstream infections that are often secondary to UTIs. These severe infections are treated with carbapenems, considered as last resort antibiotics. Unfortunately, their increasing use put a selective pressure on Enterobacteriaceae, leading to more and more strains showing decreased susceptibility to carbapenems and potentially leading to therapeutic failure.Considering the limited treatment options for ESBL-E and that CPE are often resistant to several if not all classes of antibiotics, and for which very few (or no) antibiotic options remain available, their rapid detection and identification are essential. Reliable tests are needed to help physicians, to quickly provide appropriate infection control measures, to adapt rapidly antibiotic treatment and optimize care strategies and outcomes.While detecting ESBL-Es or EPCs, it is also crucial to identify the implicated beta-lactamase for accurate therapy implementation. To do so, the antibody-specificity based methods are undoubtedly appropriate. To respond to the current needs, antimicrobial drug resistance detection methods must be cheap (reduced costs of consumables and equipment) and easy to use (reduced technical complexity) for the end user, and LFIAs respond to this requirements. Our objective was to develop such tests, and this led us to produce monoclonal antibodies against CTX-Ms, NDM, KPC, IMP, VIM and OXA-48 carbapenemase families and to develop and validate the corresponding LFIAs. Our tests are robust assays, easily transferable in a commercialized version, stable for more than 24 months without refrigeration, user-friendly (no requirement of trained staff), high performance (sensitive and specific), low cost, from 7€ (monotest) to less than 15€ (multiplex). Moreover, the detection results are obtained in short delay without the need for highly technical equipment for the readout.Here, we validated a LFIA for the detection of CTX-Ms (from group 1) and to a wider extent evaluated the direct detection of CTX-Ms from groups 1, 2, 8 and 9 in clinical samples such as blood culture and urine. Mono-tests to detect NDMs and OXA-48-like, and a multiplex for the simultaneous detection of the five main carbapenemases were also validated. These validations were conducted using 180 well characterized isolates in terms of their -lactamase content from the French National Reference Centre for carbapenem-resistant Enterobacteriaceae.