Submitted by Pedro Silva Filho (pfsilva@ipen.br) on 2017-10-09T18:49:04Z No. of bitstreams: 0 / Made available in DSpace on 2017-10-09T18:49:04Z (GMT). No. of bitstreams: 0 / A Leishmaniose Visceral (LV) é uma doença infecciosa de carater sistêmico. Estima-se que 350 milhões de pessoas no mundo estão expostas ao risco de infecção, com uma prevalência de 12 milhões de infectados e letalidade mundial de 59.000 casos por ano (OMS), sendo que 90%dos casos de LV ocorrem em países onde existe grande parte da população em situação de pobreza.Na América Latina a maioria dos casos ocorre no Brasil (96%) com média de 3.500 casos/ano. As áreas de maior endemia encontram-se nas regiões mais carentes do Norte e Nordeste.A donça afeta animais e o homem, podendo levar ao óbito em 100% dos casos, tendo como seu principal vetor a Lutzomyia longipalpis e como agente etiológico, a Leishmania Infantum. O Estado do Tocantins apresenta elevado número de casos autóctones, e é considerada área endêmica pelo Minisério da Saúde devido a doença estar presente na maioria dos seus municípios. A cidade de Araguaína, com 55,8% dos casos do Tocantins, é classificada pelo Ministério da Saúde como área de transmissão intensa.O objetivo geral deste trabalho foi analisar casos confirmados de Leishmaniose visceral grave CID-B55 internados no Hospital de Doenças Tropicais do Tocantins (HDT), na cidade de Araguaína - TO que evoluíram para óbito no periodo de janeiro de 2002 a dezembro de 2015. Pretendeu-se identificar e descrever dados epidemiológicos, clínicos e laboratoriais, e variáveis relacionadas ao tratamento. Foi feito um estudo epidemiológico, observacional, retrospectivo, descritivo, utilizando dados secundários de prontuários médicos de pacientes internados, no Hospital de Doenças Tropicais do Estado do Tocantins, na cidade de Araguaína - TO. Resultados: sexo masculino, 60,8 e 39,2% sexo feminino. 49,2% idade entre 0 e 10 anos; 20% maiores de 51 anos. Local de procedência em relação ao município 40,9% são de Araguaína; e ao Estado a 89% são do Tocantins. Presença de febre em 70,8%; esplenomegalia e hepatomegalia 76,7%. Hemoglobina < 7g/dl 50%, plaquetopenia 76,7%;leucopenia 50% e hipoalbuminemia 84,9%.Tempo de febre até a internação > que 30 dias 30,8%; Tempo de diagnóstico 5,1 dias; tempo de diagnóstico ao óbito 11,6 dias; RIFI 1/80 em 53,3%; Teste Rápido positivo 88,2% droga de escolha para tratmento foi Glucantime 43% seguida por anfotericina 32% e anfotericina lipossomal 25%. A principal causa do óbito na DO(Declaração de aÓbito) foi kalazar 33,3% e também Infecções respiratorias 31,7%.Conclui-se que a maior incidencia ocorre em individuo do sexo masculino menores de 10 anos, residentes no Estado do Tocantins, apresentando quadro febril prolongado, anemia severa e hipoalbuminemia grave, podendo este quadro ter influenciado na evolução ao óbito. / Dissertação (Mestrado em Tecnologia Nuclear) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

O Urso-de-óculos (Tremarctos ornatus), único urso endêmico da América do Sul, é uma espécie severamente ameaçada devido à perda do seu hábitat e à caça. É importante do ponto de vista da conservação pela função que cumpre no ecossistema onde habita e pela sua relação com a cultura Andina. Por estas razões, torna-se necessária a aplicação de alternativas de conservação a espécie. Uma ferramenta alternativa de conservação é a reprodução assistida, porém, existe pouco conhecimento da reprodução desta espécie. A pouca informação sobre os padrões reprodutivos diminui a possibilidade de se estabelecer programas de reprodução bem sucedidos e torna-se mais difícil a criação de planos de conservação. O objetivo deste estudo foi caracterizar o ciclo ovariano na fêmea do Urso-de-óculos. Para tanto foi realizada a monitoração endócrina por meio da extração e dosagem de metabólitos fecais dos esteróides sexuais: estradiol e progesterona. O estudo foi realizado com amostras colhidas de seis fêmeas de T. ornatus mantidas em cativeiro em dois zoológicos da cidade de Lima, Peru, pelo período de treze meses, desde Fevereiro de 2010 até Abril de 2011. As extrações dos metabólitos fecais foram feitas no Laboratorio de Reproducción Animal, FMVUNMSM, em Lima, Peru; e foram quantificados por enzimaimunoensaio (EIE), no Instituto de Bioquímica Médica, Veterinärmedizinische Universität Wien-Áustria. Os resultados indicam que é possível descrever e monitorar a endocrinologia do ciclo ovariano em T. ornatus de forma não-invasiva através do uso de metabólitos fecais de esteroides sexuais; demonstrando que o Urso-de-óculos em cativeiro é uma espécie que conta com uma reprodução não sazonal, com fase folicular e lútea durando em média 08 e 22 dias, respectivamente. Nestas condições, apresenta em média, de três a quatro fases de atividade ovarianas por ano. Os resultados encontrados ajudarão a compreender o ciclo ovariano nos Ursos-de-óculos e facilitar o desenvolvimento de programas de reprodução assistida na espécie. / Spectacled bear (Tremarctos ornatus) is the only bear species that inhabits South America. It is an endangered species due to habitat loss and hunting. For the conservation point of view it is an important species for the ecosystem maintenance and also for their relationship with the Andean culture. For these reasons it is necessary to apply conservation alternatives for this species. Assisted reproduction is an alternative conservation tool; however, there is a lack of knowledge of the Spectacled bear reproduction. Little information about the reproductive parameters diminishes the possibility to establish a successful breeding program and make difficult the implementation of conservation plans for the species. The aim of this study was to characterize the ovarian cycle in the female Spectacled bear with noninvasive techniques for monitoring fecal metabolites of reproductive hormones (estradiol and progesterone). The study was carried out with samples collected by a 13-month period (Feb/10 to April/11), from six captive females T. ornatus kept in two zoological institutions in Lima, Peru. The metabolites extraction was carried out at Laboratory of Animal Reproduction, FMV-UNMSM, in Lima-Peru, and the hormonal analysis was performed in the Institute of Medical Biochemistry, Veterinärmedizinische Universität Wien-Austria. The results show us that it is possible to describe and realize a non-invasive monitoring of ovarian cycles in T. ornatus, with the use of fecal metabolites of sex steroids; and demonstrates that in captive conditions Spectacled bear is a non-seasonal reproduction species, with follicular and luteal phases lasting on average 8 and 22 days, respectively, whereas in captivity has on average three to four stages of ovarian activity per year. These results will help to understand the ovarian cycle in Spectacled Bear and facilitate the development of programs of assisted reproduction in the species.

Sete suínos com 2 meses de idade foram infectados experimentalmente com cerca de 200.000 ovos de T. solium administrados por via oral. Ao término do experimento (140 dias) os animais não apresentaram aspectos clínicos de cisticercose pelo exame de inspeção da língua; portanto eles poderiam ser classificados como animais saudáveis pelos exames usuais de inspeção utilizados em matadouros. Todos os animais foram sacrificados e a musculatura, cérebro e órgãos viscerais foram seccionados em finos pedaços para a procura de cistos. O número de cisticercos encontrados em cada animal variou de 1 a 85 (média 33,7 e desvio padrão 31,3), caracterizando-os como animais com infecção branda. Amostras de sangue dos animais infectados experimentalmente foram submetidas a imunoensaios para a pesquisa de anticorpos (ELISA Indireto, utilizando como antígeno o líquido vesicular de Taenia crassiceps; imunoblot 18/14, oriundo de antígeno de Taenia crassiceps purificado com anticorpos monoclonais e imunoblot LLGP, utilizando glicoproteínas de T. solium purificadas com lentil lectina) e para pesquisa de antígeno (ELISA Sanduíche e ELISA Direto, ambos utilizando o anticorpo monoclonal anti-ES-Tcra). O teste ELISA Direto não apresentou reatividade em qualquer amostra de soro dos diferentes animais infectados no momento do abate (t140). Foi encontrada positividade de 71% e 57%, respectivamente, para os testes ELISA Indireto para a pesquisa de anticorpos e ELISA Sanduíche para a pesquisa de antígenos nestes tempos. Pelo imunoblot (IB) 18/14 e IB LLGP, respectivamente 5 (71 %) e 6 (86%) dos animais infectados experimentalmente foram positivos no t140. O uso de ensaios imunoenzimáticos para a detecção de anticorpos específicos (IB) somados ao ELISA Sanduíche para pesquisa de antígeno permitiu que todos os animais com infecção branda fossem detectados. Dessa forma, o uso de ensaios imunoenzimáticos para detecção de anticorpos e/ou antígenos mostrou-se mais útil que o exame clínico e/ou post-mortem para a triagem de cisticercose suína em animais com infecção branda. / Seven 2-month-old swine were orally experimentally infected with approximately 200,000 eggs of Taenia solium. At the end of the experiment (140 days) the animals did not show clinical aspects of cysticercosis or parasites in tongue inspection; therefore they could be mistaken as healthy animals by usual inspection exams used in slaughterhouses. All of them were slaughtered and their whole muscles, brain and visceral organs were cut into thin slices searching for cysts. The number of cysts found in each animal varies from 1 to 85 (mean value 33.7 and standard deviation 31.3) characterizing them as slightly infected animals. Blood samples of experimentally infected animals were submitted to immunoassays for antibody detection (Indirect ELISA, using vesicular fluid antigen from Taenia crassiceps; immunoblot 18/14, from T.crassiceps antigen purified by monoclonal antibodies and immunoblot LLGP, using lentil lectin purified glycoproteins of T. solium) and for antigen detection (Sandwich ELISA and Direct ELISA, both using monoclonal antibodies anti-ES-Tcra). The Direct ELISA assay did not show any reactivity with all serum samples from the different animals at the slaughtered moment (t140). The positivity was 71% and 57% by the Indirect ELISA for searching antibodies and by the Sandwich ELISA for searching antigens, respectively. By immunoblot (IB) 18/14 and IB LLGP 5 (71 %) and 6 (86%) experimentally infected animals were positive at t140, respectively. The use of immunoassays for detecting specific antibodies (IB) together with Sandwich ELISA for antigen detection allow the detection of all slightly infected animals. Thus, the use of immunoassays for antibodies and/or antigen detection showed they are more useful than usual clinical examination and/or tongue palpation for screening cysticercosis in slightly infected pigs.

Abstract The purpose of this study was to develop sensitive and specific laboratory diagnostic methods for the demonstration of pneumococcal surface antigens or pneumococcus-specific antibodies in clinical samples. The work took account of epidemiological aspects of both pneumococcal disease and nasopharyngeal carriage of pneumococcus. We first compared the sensitivity of pneumococcal culture and antigen detection methods in nasopharyngeal samples in a developing country setting and then investigated the possibility of improving the sensitivity of the antigen detection by introducing an enrichment step in the procedure. — Further investigations were designed to determine the validity of pneumolysin-specific immune complex bound antibody assay as a tool for diagnosing pneumococcal ALRI in a developing country setting. Finally, we developed an enzyme immunoassay for the detection of pneumococcal capsular polysaccharide antigens, using type-specific antibodies produced in-house in rabbits through immunisation with an in-house-produced pneumococcal whole cell vaccine. The method was tested in nasopharyngeal and middle ear fluid samples. The first results indicated that antigen detection might be more sensitive than culture in demonstrating pneumococci in URT, particularly in children with prior antimicrobial therapy. Antigen detection is a feasible method for studies on pneumococci in developing countries. For type-specific demonstration of S. pneumoniae, detection of pneumococcal antigen after an enrichment step proved a sensitive method that can be applied for epidemiologic study purposes, e.g., in vaccine trials, in areas without ready access to a good microbiology laboratory. Determination of IC-bound pneumolysin IgG antibodies appears to be a useful method for species-specific diagnosis of pneumococcal infections. The results indicating pneumococcal aetiology in ALRI patients in this study compare well with the best results obtained by the use of lung aspirates. Increasing the number of serial samples improves the sensitivity of the assay, but even two samples provide more positive findings than other methods currently in routine use. Criteria of positivity need to be confirmed in subsequent larger studies with both healthy controls and patients with confirmed pneumococcal disease. It is also important to control the findings in patients with pneumonia of non-pneumococcal origin. The novel enzyme immunoassay was shown to work well with enrichment culture samples, with an almost 100% sensitivity compared with the culture. Middle ear fluid samples were too diluted for the enzyme immunoassay method used, and only 74% sensitivity compared with culture was achieved. Provided that adequate samples can be obtained, the method will be a useful complement to the current laboratory methods used to diagnose pneumococcal disease. With the existence of a broad spectrum of microbiological and immunological methods, it is imperative to seek international consensus for standard methods to demonstrate pneumococcus. Otherwise it is very difficult to compare results from different clinical studies. A WHO Working Group recently proposed a standard method for detecting upper respiratory carriage of pneumococcus, but a lot of work remains to be done in other areas of research on pneumococcal infections.

Cette thèse vise à développer de nouveaux matériaux hybrides pour les applications en électrochimiluminescence. Les propriétés électrochimiluminescentes de nouveaux complexes de Pt(II) et d’Ir(III) ont été explorés comme alternative aux marqueurs existants. En plus, la combinaison de complexes et de carbon nanodots portant des groupes primaires ou tertiaires à la surface comme espèces coréactives a abouti à une stratégie intéressante pour éliminer la TPrA. Les carbon nanodots dans un systéme lié par liaison covantent avec complexes métalliques sont non seulement un support innocent pour les espèces actives d’ECL, mais agissent également comme coréactif, se révélant être une plateforme auto-améliorante en ECL. Enfin, un véritable immunoessai pour la détection des marqueurs cardiaques a été mis au point avec une sensibilité et une stabilité accrues pour les applications de détection biologique et biomédicale. La même technologie peut alors être appliquée à une variété d’autres analytes, ouvrant ainsi le site à d’autres dosages. / This doctoral dissertation aim to develop new hybrid materials for ECL applications. In the field of metal complexes, the electrochemiluminescent properties of new Pt(II) and Ir(III) complexes were investigated as alternative of existing complexes. Passing to nanomaterials, the combination of labels and NCNDs bearing primary or tertiary groups on the surface as alternative co-reactant species resulted an interesting strategy to eliminate the toxic TPrA. In particular, NCNDs in covalently linked system with metal complexes is not only an innocent carrier for ECL active species, but act also as co-reactant in the ECL process, revealing itself an ECL self-enhancing platform. Finally, a real immunoassay for cardiac marker detection has been built with enhanced sensitivity and stability, which is of fundamental importance for biological and bio-medical detection applications. The same technology can be applied to a variety of other analytes opening the venue to other assays.

Pregnancy tests and related immunoassays are heavily dependent on specific and non-specific protein adsorption. These interfacial processes are affected by many factors that influence the in situ conformations of interfacially immobilised antibodies. This thesis examines a number of representative features with dual polarisation interferometry (DPI) and neutron reflection (NR), thus combining real-time dynamic monitoring with high interfacial structural resolution. Bovine serum albumin (BSA) was initially used as a model system to compare the surface coverage and thickness measurements of DPI and NR. The results show that DPI and NR provided similar surface coverage data but the measured thicknesses differed at BSA concentrations above 0.1 mg/ml. This discrepancy arose from the adoption of the uniform-layer model used by DPI for data analysis and the greater thickness sensitivity of NR. A model pregnancy immunoassay was built in steps on a silica surface so that the adsorption of each protein could be accurately monitored. Both DPI and NR provided evidence of BSA insertion into the gaps on the surface between the antibody molecules. This suggests that BSA adsorption is an excellent method to block the non-specific adsorption of target antigens to the immunoassay test surface. A magnetic tweezer system was designed and built in order to measure the specific antibody/antigen binding force. The antibodies and antigens were used to immuno-link magnetic beads to the experimental surface before the immuno-links were broken by increasing the attractive force between the magnetic tweezers and beads. The force per antibody/antigen immuno-link was estimated to lie between the values of 13.6 pN and 43.8 pN.Immuno-link detachment as a function of time was investigated. It was found that the immuno-link comprised both a strong and a weak interaction. The dissociation constant of the strong antibody/antigen interaction was found to equal 3E-4 /s and had an interaction length of 0.06 nm. The low population of beads bound by the second, weaker interaction meant that it was not possible to obtain accurate values of the dissociation constant and bond length of the second weaker interaction.

Free analyte is measured to be able to understand the pharmacological effects of a drug in the body, the binding to its ligand, and the effective drug level among other things. Thereby, it is important with correct measurements of free analyte, although it is not that easy to achieve since overestimations can occur. In this project, several immunoassays were developed for the bioanalytical methods Gyrolab and ELISA to measure free analyte, where the biotherapeutics Avastin® and Lucentis®, and the ligand VEGF were used as analytes. One difference between the methods is the short contact time of just a few seconds for Gyrolab compared to the sample incubation time of a couple of hours for ELISA. One difference between the antibodies is that Lucentis is an affinity-matured Fab region, and therefore, has a stronger affinity to VEGF compared to Avastin. When free Avastin was measured, the difference was significant, with ELISA estimating higher concentrations compared to Gyrolab. However, this was not the case for all assays. In some cases, this was probably due to differences between the methods that could not be seen. Otherwise, the results with no difference between the methods, when measuring free analyte with Lucentis as the drug, were expected due to the stronger affinity and longer halftime of dissociation. However, the difference with the longer sample incubation time for ELISA compared to the short contact time for Gyrolab seems to influence the measurement of free analyte, depending on the affinity of the components being measured.

Introduction: Pulmonary Surfactant reduces surface tension in the terminal airways thus facilitating breathing and contributes to host''s innate immunity. Surfactant Proteins (SP) A, B, C and D were recently identified as inherent proteins of the CNS. Aim of the study was to investigate cerebrospinal fluid (CSF) SP levels in hydrocephalus patients compared to normal subjects. Patients and methods: CSF SP A-D levels were quantified using commercially available ELISA kits in 126 patients (0±84 years, mean 39 years). 60 patients without CNS pathologies served as a control group. Hydrocephalus patients were separated in aqueductal stenosis (AQS, n = 24), acute hydrocephalus without aqueductal stenosis (acute HC w/o AQS, n = 16) and idiopathic normal pressure hydrocephalus (NPH, n = 20). Furthermore, six patients with pseudotumor cerebri were investigated. Results: SP AÐD are present under physiological conditions in human CSF. SP-A is elevated in diseases accompanied by ventricular enlargement (AQS, acute HC w/o AQS) in a significant manner (0.67, 1.21 vs 0.38 ng/ml in control, p<0.001). SP-C is also elevated in hydrocephalic conditions (AQS, acute HC w/o AQS; 0.87, 1.71 vs. 0.48 ng/ml in controls, p<0.001) and in Pseudotumor cerebri (1.26 vs. 0.48 ng/ml in controls, p<0.01). SP-B and SP-D did not show significant alterations. Conclusion: The present study confirms the presence of SPs in human CSF. There are significant changes of SP-A and SP-C levels in diseases affecting brain water circulation and elevation of intracranial pressure. Cause of the alterations, underlying regulatory mechanisms, as well as diagnostic and therapeutic consequences of cerebral SP''s requires further thorough investigations.

This doctoral thesis investigates the application of laser-induced breakdown spectroscopy (LIBS) for detection of labeled biomolecules on nitrocellulose paper. Nitrocellulose paper is a material often used for assays involving the concentration and labeling of a target analyte, followed by label detection. Among paper-based diagnostics are lateral-flow immuno-assays (LFIAs). Research efforts have made LFIAs into accessible, portable,and low-cost tools for detecting targets such as allergens, toxins,and microbes in food and water.Gold (Au) nanoparticles are standard biomolecular labels among LFIAs, typically detected via colorimetric means.Other labels, such as quantum dots, are also often metallic, and research is ongoing to expand the number of portable instrumentations applied to their detection. A wide diversity of lanthanide-complexed polymers (LCPs) are used as immunoassay labels but have been inapt for portable paper-based assays owing to lab-bound detection instrumentation, until now. LIBS is a multi-element characterization technique which has recently developed from a bench-top to a portable/hand-held analytical tool. This is among the first studies to show that LCPs can be considered as options for biomolecule labels in paper-based assays using bench-based and hand-held LIBS as label detection modalities.<div>Chapter one reviews the importance of rapid, multiplexed detection of chemical and biological contaminants, the application of current biosensors, and the role of LIBS as an emerging biosensor. Paper-based bioassays were identified as a promising approach for contaminant detection whose capabilities could be enhanced by LIBS. The next chapter dives into LIBS system designs to address which LIBS parameters were appropriate for label detection on paper assay material. A balance of LIBS parameters was found to be important for successful analyte detection. Chaptert hree optimizes a LIBS design for sensitive detection of 17 metals and establishes limit of detection values for 7 metals. Optimal detection parameters depended on the metal being detected and were applied to the objective of the final chapter: LIBS detection of labeled antigen immobilized on a paper-based assay. Both antibody and bacteria detection assays were successfully performed and analyzed using bench top and portable LIBS,suggesting an exciting future for the use of LIBS as a biosensor.The prospect of using LIBS for multiplexed, rapid and sensitive detection of biomolecules in assays is explored, laying grounds for future work in the ever-relevant field of biological and chemical hazard detection.<br></div>

<div>Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.</div><div><br></div><div>Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use.</div>