A Brucella canis é a espécie de Brucella que acomete os cães, acarretando problemas reprodutivos e prejuízos econômicos aos proprietários de canis comericias, além do risco que representa para os manipuladores e os proprietários dos cães, os quais podem adquirir a infecção pelo contato com os animais infectados e/ou materiais contaminados por B. canis. Vários métodos de diagnósticos foram desenvolvidos com o intuito de diagnosticar a brucelose nos cães, no entanto cada teste apresenta um desempenho diferente em relação à sensibilidade, especificidade, rapidez e praticidade na identificação dos cães acometidos. Estudos indicam que a PCR em tempo real tende a apresentar maior sensibilidade e especificidade, além da rapidez na execução. Diante disso o objetivo deste trabalho foi desenvolver, padronizar e validar a reação em cadeia da polimerase (PCR) em tempo real para diagnóstico da brucelose canina causada por B. canis utilizando amostras de sangue total de cães. Um total de 56 cães de canis comercias e animais domiciliados, foram submetidos à hemocultura, sorodiagnóstico, PCR convencional (PCRc) e PCR em tempo real (PCRtr) e posteriormente os resultados obtidos em cada teste foram comparados por meio do coeficiente Kappa. Na padronização das reações de PCRtr foram testados três conjuntos de primers e sondas (PCRtr-A, B e C), utilizando-se diferentes temperaturas de annealing e concentrações de primers. A PCRtr-B teve melhor desempenho sob temperatura de annealing de 59° C, utilizando 900 nM de primers tendo detectado um maior número de cães positivos em relação à hemocultura e PCRc. Porém o teste não se apresentou confiável, uma vez que ocorreram reações inespecíficas em amostras provenientes de cães não infectados, sugerindo uma baixa especificidade do teste. Portanto, as técnicas de hemocultura e PCRc apresentaram melhor desempenho do que a PCRtr avaliada para o diagnóstico de B. canis. / Brucella canis is the species that affects dogs, causing reproductive problems and economic losses mainly for the owners of commercial kennels, besides the risk that it represents for the manipulators and the owners of dogs that can acquire the infection by the contact with infected animals or contaminated materials. Several diagnostic methods have been developed for the diagnosis of the infection in dogs. However, the performance of the tests vary regarding sensitivity, specificity, speed and practicality for the identification of infected dogs. Studies indicated that the real-time PCR (rtPCR) might show higher sensitivity and specificity, as well as speed of execution. Therefore, the objective of this study was to develop, standardize and validate a rtPCR assay for the diagnosis of canine brucellosis caused by B. canis using whole blood samples from dogs. A total of 56 dogs from commercial kennels and domiciled animals were tested through blood culturing, serological test, conventional PCR (cPCR) and rtPCR, and subsequently the results obtained in each test were compared using the Kappa coefficient. During the standardization of the rtPCR, three sets of primers and probes (rtPCR-A, B and C) were tested using different annealing temperatures and primer concentrations. rtPCR-B showed better performance under the annealing temperature of 59° C and using 900 nM of primers having detected a higher number of positive dogs when compared to blood culturing and PCRc. However, the test was considered not reliable to be used in canine brucellosis diagnosis, since non-specific reactions occurred in samples from uninfected dogs, suggesting a low specificity of the test. Therefore, the blood culturing and cPCR techniques showed a better performance than the developed rtPCR assay to be used in the diagnosis of B. canis infection in dogs.

A Brucella canis é a espécie de Brucella que acomete os cães, acarretando problemas reprodutivos e prejuízos econômicos aos proprietários de canis comericias, além do risco que representa para os manipuladores e os proprietários dos cães, os quais podem adquirir a infecção pelo contato com os animais infectados e/ou materiais contaminados por B. canis. Vários métodos de diagnósticos foram desenvolvidos com o intuito de diagnosticar a brucelose nos cães, no entanto cada teste apresenta um desempenho diferente em relação à sensibilidade, especificidade, rapidez e praticidade na identificação dos cães acometidos. Estudos indicam que a PCR em tempo real tende a apresentar maior sensibilidade e especificidade, além da rapidez na execução. Diante disso o objetivo deste trabalho foi desenvolver, padronizar e validar a reação em cadeia da polimerase (PCR) em tempo real para diagnóstico da brucelose canina causada por B. canis utilizando amostras de sangue total de cães. Um total de 56 cães de canis comercias e animais domiciliados, foram submetidos à hemocultura, sorodiagnóstico, PCR convencional (PCRc) e PCR em tempo real (PCRtr) e posteriormente os resultados obtidos em cada teste foram comparados por meio do coeficiente Kappa. Na padronização das reações de PCRtr foram testados três conjuntos de primers e sondas (PCRtr-A, B e C), utilizando-se diferentes temperaturas de annealing e concentrações de primers. A PCRtr-B teve melhor desempenho sob temperatura de annealing de 59° C, utilizando 900 nM de primers tendo detectado um maior número de cães positivos em relação à hemocultura e PCRc. Porém o teste não se apresentou confiável, uma vez que ocorreram reações inespecíficas em amostras provenientes de cães não infectados, sugerindo uma baixa especificidade do teste. Portanto, as técnicas de hemocultura e PCRc apresentaram melhor desempenho do que a PCRtr avaliada para o diagnóstico de B. canis. / Brucella canis is the species that affects dogs, causing reproductive problems and economic losses mainly for the owners of commercial kennels, besides the risk that it represents for the manipulators and the owners of dogs that can acquire the infection by the contact with infected animals or contaminated materials. Several diagnostic methods have been developed for the diagnosis of the infection in dogs. However, the performance of the tests vary regarding sensitivity, specificity, speed and practicality for the identification of infected dogs. Studies indicated that the real-time PCR (rtPCR) might show higher sensitivity and specificity, as well as speed of execution. Therefore, the objective of this study was to develop, standardize and validate a rtPCR assay for the diagnosis of canine brucellosis caused by B. canis using whole blood samples from dogs. A total of 56 dogs from commercial kennels and domiciled animals were tested through blood culturing, serological test, conventional PCR (cPCR) and rtPCR, and subsequently the results obtained in each test were compared using the Kappa coefficient. During the standardization of the rtPCR, three sets of primers and probes (rtPCR-A, B and C) were tested using different annealing temperatures and primer concentrations. rtPCR-B showed better performance under the annealing temperature of 59° C and using 900 nM of primers having detected a higher number of positive dogs when compared to blood culturing and PCRc. However, the test was considered not reliable to be used in canine brucellosis diagnosis, since non-specific reactions occurred in samples from uninfected dogs, suggesting a low specificity of the test. Therefore, the blood culturing and cPCR techniques showed a better performance than the developed rtPCR assay to be used in the diagnosis of B. canis infection in dogs.

Thesis (M. Sc.) Uppsala : Sveriges lantbruksuniv., 2007.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-09-04T19:13:52Z No. of bitstreams: 2 Dissertação - Thiago Menegazzo Montes - 2017.pdf: 2026174 bytes, checksum: 1098b4a6affe272e15c02c0f8f99b7b4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Rejected by Luciana Ferreira (lucgeral@gmail.com), reason: Fiquei em dúvida com relação ao título, pois não entendi o que a banca escreveu na ata, se é para substituir o título por : Estudo.... adultas NA regional.. Goiás , ou , Estudo.... adultas DA regional.. Goiás. No formulário usou:" adultas DA regional Rio das Antas, Goiás" , já folha de rosto usou: adultas NA regional ... Goiás on 2017-09-15T14:35:29Z (GMT) / Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-09-20T17:13:34Z No. of bitstreams: 2 Dissertação - Thiago Menegazzo Montes - 2017.pdf: 2026174 bytes, checksum: 1098b4a6affe272e15c02c0f8f99b7b4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-09-21T10:47:17Z (GMT) No. of bitstreams: 2 Dissertação - Thiago Menegazzo Montes - 2017.pdf: 2026174 bytes, checksum: 1098b4a6affe272e15c02c0f8f99b7b4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-09-21T10:47:17Z (GMT). No. of bitstreams: 2 Dissertação - Thiago Menegazzo Montes - 2017.pdf: 2026174 bytes, checksum: 1098b4a6affe272e15c02c0f8f99b7b4 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-07-31 / Brucellosis is an infectious-contagious and zoonotic disease caused by bacteria of the genus Brucella. It is an endemic disease in Brazil, with a variable prevalence and it mainly affects the reproductive tract of the animals, resulting in several damages in the countryside, industry and public health. This research aimed to determine the prevalence of brucellosis in adult cows in the Rio das Antas Regional (Goiás), its spatial distribution and the factors associated with this disease. A total of 95 farms were randomly selected for blood sampling in cows over 24 months of age, totalizing 992 samples. These samples were submitted to the Buffered, Acidified Plate Antigen test, and for those positive cases, to the 2-Mercaptoethanol test. An epidemiological survey was applied to evaluate the risk factors for brucellosis in each farm. There was an apparent prevalence of 13.68% [7.49 - 22.26] for farms and 2.11% [1.32 - 3.22] for animals. In bovine females, the multiple logistic model for brucellosis risk factors detected a significant effect for the B19 vaccine variable (OR = 2.85; IC [1.05 – 7.04]). For these results, it can be stated that bovine brucellosis in herds and animals is considered high in the evaluated region, with a wide spatial distribution, dispersed throughout the studied territory and in all production systems. Among the factors associated with the risk of brucellosis in adult bovine females, vaccination is associated. / A brucelose é uma enfermidade infecto-contagiosa e zoonótica, causada por bactérias do gênero Brucella. Trata-se de uma doença endêmica no Brasil, com prevalência variável e que acomete principalmente o trato reprodutivo dos animais, resultando em diversos prejuízos no campo, indústria e saúde pública. Esta pesquisa foi desenvolvida com o objetivo de determinar a prevalência da brucelose em fêmeas bovinas adultas na Regional Rio das Antas / Goiás, sua distribuição espacial e os fatores associados a esta enfermidade. Foram amostradas 95 propriedades rurais sorteadas aleatoriamente para colheita de amostras de sangue em fêmeas bovinas com idade acima de 24 meses, gerando um total de 992 amostras. Os soros colhidos foram submetidos ao teste do Antígeno Acidificado Tamponado (AAT) e para os casos positivos, ao 2-Mercaptoetanol (2-ME), realizados em série. Foi aplicado um questionário epidemiológico para avaliação dos fatores de risco para brucelose em cada propriedade. Foi verificada uma prevalência aparente de 13,68% [7,49 – 22,26] para propriedades e 2,11% [1,32 – 3,22] para animais. O modelo logístico múltiplo para os fatores de risco da brucelose em fêmeas bovinas, detectou efeito significativo somente para a variável vacina B19 (OR = 2,85; IC [1,05 – 7,04]). Por tais resultados, pode-se afirmar que a brucelose bovina em rebanhos e animais é considerada alta na região avaliada, com ampla distribuição espacial, dispersa em todo o território estudado e em todos os sistemas de produção. Dentre os fatores associados ao risco de brucelose em fêmeas bovinas adultas, a vacinação mostra-se associada.

The species of Brucella were isolated and characterized almost 120 years ago and their genomes sequenced for almost 4 years. Compared to other bacterial pathogens relatively, little is known about the factors contributing to their persistence in hosts and multiplication within phagocytic cells. Also, many aspects of the interactions between Brucella and its host remain unclear. Molecular characterization of intracellular survival processes of Brucella will provide guidance for additional prevention and control measures. One of the features that distinguishes Brucella is that they do not express classic virulence factors. Thus identification of virulence factors has been elusive and some of the identified virulence genes are putative. Disruption of putative virulence genes and studying the consequent effect on attenuation in cell lines or mouse models is a widely used method. However, in most cases it is not apparent whether the mutated genes encode virulence factors or merely affect normal metabolic or biological functions. Some mutations in Brucella can be compensated by redundancy or backup mechanisms. One method for identifying putative virulence genes involved in pathogenesis is to express these genes in a nonpathogenic host and isolate recombinants with increased virulence or survival ability either in cell culture or animal model. We hypothesize that over-expression of Brucella putative virulence genes in the non-pathogenic and close phylogenic relative Ochrobactrum anthropi should enhance its survival in infection models in vivo. O. anthropi is one of the closest Brucella relatives based on DNA, rRNA, and protein analyses but it is unable to establish chronic infection and considered as opportunistic pathogen that, under certain circumstances, may produce disease in immunocompromised humans. Therefore, we established enhanced expression system in Brucella and Ochrobactrum to identify B. suis virulence genes. We created an enhanced expression system that can be used for cloning and expression of heterologous genes in Brucella and Ochrobactrum. We studied the transcriptional activity of several promoters and created some tools to enhance the expression, detection and purification of Brucella recombinant protein in Ochrobactrum. The presumable importance of alkyl hydroperoxide reductases encoded by ahpC and ahpD genes and their contribution to intracellular survival of Brucella were studied by over-expressing them. The recombinant O. anthropi expressing B. suis ahpC and ahpD genes were able to resist in vitro killing by H2O2 and or cumene hydroperoxide and survived longer in the macrophage J774 A.1 cell line. The control O. anthropi was cleared from BALB/c mice in five days while the recombinants were recovered from spleens, livers and lungs of infected mice up to eight days post-infection. We tested the contribution of B. suis cyclic glucan synthetase gene (cgs) to virulence by over-expressing it in O. anthropi. We studied the ability of the recombinant O. anthropi to resist killing in vitro and in vivo. We generated evidence that B. suis cgs when over-expressed in O. anthropi increased the amount of cyclic glucans synthesized and accumulated in the periplasmic space. This accumulation changed the virulence of the microorganism from a soil bacterium that cleared from mice in less than five days into a pathogenic organism that could survive up to 9 days and at higher doses killed the mice. In summary, several vectors have been constructed for gene expression and protein purification in Brucella and Ochrobactrum. Novel useful tools for enhancement of heterologous gene expression were created and demonstrated to work in Brucella and Ochrobactrum. Brucella putative virulence genes were studied in Ochrobactrum using the newly constructed vectors and tools. Ochrobactrum as a gain of function model for studying putative virulence genes of intracellular pathogens in general and for Brucella in particular proved to be a very useful model. / Ph. D.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-05-05T15:46:59Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_InfeccaoBrucellaAbortus.pdf: 5798071 bytes, checksum: 784c93d38504d18d1af018a238f979ca (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-05-10T14:51:17Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_InfeccaoBrucellaAbortus.pdf: 5798071 bytes, checksum: 784c93d38504d18d1af018a238f979ca (MD5) / Made available in DSpace on 2017-05-10T14:51:17Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_InfeccaoBrucellaAbortus.pdf: 5798071 bytes, checksum: 784c93d38504d18d1af018a238f979ca (MD5) Previous issue date: 2016 / O objetivo deste trabalho foi verificar a presença de Brucella abortus e as lesões causadas por esse agente nos anexos fetais, nos fetos e linfonodos de búfalas brucélicas gestantes. Para isso, 20 búfalas nos diferentes meses de gestação, sorologicamente positivas para brucelose, foram submetidas ao abate sanitário. A idade fetal foi determinada através de exames ultrassonográficos associados à mensuração dos fetos durante a necropsia. Do útero fechado desses animais foram coletadas amostras para histopatologia e reação em cadeia da polimerase em tempo real, além de fragmentos de diversos linfonodos. A partir do segundo mês de gestação foi possível detectar a presença de DNA de B. abortus em líquido amniótico, líquido alantoide e em útero e, a partir do quinto mês, na placenta, coração, baço, rim, pulmão, intestino, fígado e linfonodos dos fetos. Os principais achados anatomopatológicos foram placentite fibrinopurulenta necrótica e endometrite supurativa crônica. A detecção de DNA de B. abortus nos linfonodos das búfalas avaliadas foi verificada a partir do quarto mês de gestação em sete búfalas e em uma búfala pós-parição. Os achados histológicos foram linfadenite aguda a crônica. A presença de DNA de B. abortus foi detectada em todos os grupos de linfonodos avaliados, sendo que os linfonodos mais acometidos foram os mamários. A transmissão intrauterina a partir do segundo mês de gestação representa uma rota importante de infecção na cadeia epidemiológica da brucelose em bubalinos. / The objective of this study was to detect Brucella abortus and injuries caused by the bacteria in fetal membranes and fetuses, and in lymph nodes of buffaloes as well as to describe the lesions caused. Twenty buffaloes serologically positive for brucellosis were used and subjected to stamping for collection of material from the closed uterus of several months gestation. Fragments of lymph nodes were collected. Fetal age was determined by ultrasound examination and the size of fetuses was measured at necropsy. The samples were subjected to histopathology and qPCR. From the second month of pregnancy on it was possible to detect the presence of B. abortus DNA in amniotic fluid, allantoic liquid and uterus, and from the fifth month on in placenta, heart, spleen, kidney, lung, intestine, liver and lymph nodes of the fetuses. The main pathological findings were fibrinous suppurative necrotic placentitis, and chronic endometritis. The detection of B. abortus DNA in the lymph nodes was checked from the fourth month of pregnancy in seven buffaloes and in a post-calving buffalo. Acute to chronic lymphadenitis was histologically diagnosed. B. abortus DNA was detected in all evaluated groups of lymph nodes; the mammary lymph nodes were the most affected.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The dissertation was divided in three studies. Our first study determined the correlation between high somatic cell counts and positive milk ring tests (MRT). The milk ring test was performed on 181 milk samples from expansion tanks located at various rural sites in the regions of Brazil. Somatic cell counts (SCC) via flow cytometry were also performed. MRT identified 11 positive samples (6%) where the majority were reactive and 5 (2.8%) were found in the 6x105 cel/mL range. The results showed that there was no statistical relationship between high somatic cell counts and positive milk ring tests. Our second study evaluated the efficiency of an enzyme linked immunosorbent assay kit (ELISA) in diagnosing brucellosis in collective milk samples. The indirect ELISA test was used on 181 samples of fresh milk and identified 42 positive, 11 suspect and 128 negative samples. These results were compared to the MR test, the only test approved for collective milk samples by the Brazilian Ministry of Agriculture, Livestock and Supply (Ministério da Agricultura Pecuária e Abastecimento). The ELISA test had sensitivity of 72.7%, specificity of 78.6% and a Kappa concordance index of 0.22. Subsequently, random samples from lactating animals in nine herds were tested individually by the rose Bengal test (RBT) and the 2-mercaptoethanol 2(ME) tests. Positive animals were found only in herds that tested positive in both ELISA and MRT. The ELISA test was neither as sensitive nor as specific as reported in other countries where the ELISA test is used and where brucellosis outbreaks have been confirmed in dairy herds. Our third study used the Polymerase Chain Reaction (PCR) technique to detect Brucella abortus in samples of fresh milk. Ninety samples were preselected for the PCR test including 45 that tested positive in ELISA and/or MRT, 11 suspect in ELISA and 34 negative in both tests. Extraction was carried out by enzymatic lysis followed by amplification using BAB and IS 711 primers, which amplify fragments of 498 base pairs. Visualization was accomplished with 1% agarose gel and an Alpha Digidoc Photo Documentation system. Brucella abortus DNA was not amplified in any of the samples, demonstrating that PCR is not the best screening technique for brucellosis in milk samples at the herd level, given that these microorganisms do not always escape into the milk. / A dissertação foi dividida em três estudos. O primeiro objetivou verificar a existência de correlação entre altas contagens de células somáticas e reações positivas no Teste do anel em leite (TAL). Foram coletadas 181 amostras de leite oriundas de tanques de expansão provenientes de diferentes propriedades rurais e submetidas ao Teste de Anel em Leite (TAL). Também foi realizada a contagem de células somáticas (CCS) através do método de citometria de fluxo. O TAL resultou em 11 amostras positivas (6%), da quais, 5 (45,4%) encontravam-se na faixa de 6x105 cel/mL. Os resultados das análises demonstraram não haver associação estatística entre altas contagens de células somáticas e reações positivas no Teste de Anel em Leite. O segundo estudo teve como objetivo avaliar a eficiência de um kit de teste imunoenzimático indireto (ELISA) para diagnóstico de brucelose em amostras coletivas de leite. Amostras de leite in natura, totalizando 181, foram submetidas ao teste ELISA indireto. O teste ELISA identificou 42 (23,2%) amostras positivas, 11 (6%) suspeitas e 128 (70,7%) negativas. Para comparação foi utilizado o TAL. O ELISA apresentou sensibilidade de 72,7%, especificidade de 78,6% e um índice de concordância Kappa de 0,22. Posteriormente 9 rebanhos foram selecionados para realização de testes individuais de Antígeno Acidificado Tamponado (AAT) e 2-Mercaptoetanol 2(ME), por meio de amostragem aleatória dos animais em lactação. Foram encontrados animais reagentes apenas em rebanhos considerados positivos no TAL e no ELISA simultaneamente. Constatou-se que o teste ELISA apresentou valores de sensibilidade e especificidade inferiores aos relatados em outros países onde o teste ELISA já é utilizado, assim como foi confirmada a existência de focos de brucelose em rebanhos leiteiros. No terceiro estudo, a Reação em Cadeia de Polimerase (PCR) foi empregada para detecção de Brucella abortus em amostras de leite in natura. Foram selecionadas 90 amostras para realização da técnica de PCR, sendo 8 positivas no TAL, 37 positivas no ELISA, 11 suspeitas no ELISA e 34 negativas em ambos os testes. A extração foi realizada por meio de lise enzimática, seguida de amplificação utilizando primers BAB e IS 711, que amplificam fragmentos de 498 pares de bases. A visualização foi realizada utilizando gel de agarose 1% em fotodocumentador Alpha Digi Doc. Não foi amplificado DNA de Brucella abortus, demonstrando a PCR não se a melhor técnica para o diagnóstico de brucelose em amostras coletivas de leite. / Mestre em Ciências Veterinárias

Ionic self-assembled multilayers (ISAMs) can be formed by alternately dipping a substrate in anionic and cationic polyelectrolytes. Each immersion deposits a monolayer via electrostatic attraction, allowing for nanometer-scale control over film thickness. Additionally, ISAM films can be applied to arbitrary substrate geometries and can easily incorporate a variety of polymers and nanoscale organic or inorganic inclusions. The ISAM technique was used to tune and functionalize a rapid, sensitive fiber optic biosensor for textit{Brucella}, a family of bacteria that are detrimental to livestock and can also infect humans. The sensor was based on a turn-around point long period fiber grating (TAP-LPG). Unlike conventional LPGs, in which the attenuation peaks shift wavelength in response to environmental changes, TAP-LPGs have a highly sensitive single wavelength peak with variable attenuation. ISAMs were applied to a TAP-LPG to tune it to maximum sensitivity and to facilitate cross-linking of receptor molecules. Biotin and streptavidin were used to attach biotinylated hybridization probes specific to distinct species of textit{Brucella}. The sensor was then exposed to lysed cell cultures and tissue samples in order to evaluate its performance. The best results were obtained when using samples from textit{Brucella} infected mice, which produced a transmission change of 6.0 ± 1.4% for positive controls and 0.5 ± 2.0% for negative controls. While the sensor was able to distinguish between positive and negative samples, the relatively short dynamic range of the available fiber limited its performance. Attempts to fabricate new TAP-LPGs using a CO2 laser were unsuccessful due to poor laser stability. A second application of the ISAM technique was as a source of second-harmonic generation (SHG). SHG is a nonlinear optical process in which light is instantaneously converted to half its wavelength in the presence of intense electric fields. Localized surface plasmons (LSPs) in metal nanoparticles produce strong electric field enhancements, especially at sharp tips and edges, that can be used to increase SHG. Colloidally grown silver nanoprisms were deposited onto nonlinear ISAM films and conversion of 1064 nm Nd:YAG radiation to its 532 nm second-harmonic was observed. Little enhancement was observed when using nanoprisms with LSP resonance near 1064 nm due to their large size and low concentration. When using shorter wavelength nanoprisms, enhancements of up to 35 times were observed when they were applied by immersion, and up to 1380 times when concentrated nanoprisms were applied via dropcasting at high enough densities to broaden their extinction peak towards the excitation wavelength. A maximum enhancement of 2368 times was obtained when concentrated silver nanoprisms with LSP resonance around 900 nm were spincast with an additional layer of PCBS. / Doctor of Philosophy / Polyelectrolytes are long molecules composed of chains of charged monomers. When a substrate with a net surface charge is dipped into an oppositely charged polyelectrolyte solution, a single layer of molecules will be electrostatically deposited onto the substrate. Because the surface charge now appears to match the charge of the solution, no further deposition occurs. However, the process can be repeated by rinsing the substrate and immersing in a solution with the opposite charge. This technique forms ionic self-assembled multilayers (ISAMs), which can be assembled with nanometer-level control over thickness. The flexibility of polymer chemistry allows ISAMs to be formed from polyelectrolytes with a wide variety of properties. Additionally, the technique can easily incorporate other nanoscale materials, such as nanoparticles, clay platelets, and biological molecules, and has been investigated for applications ranging from dye-sensitized organic solar cells to drug delivery and medical implant coatings. This dissertation presents two applications of ISAM films. In one, ISAM films were used to tune and functionalize an optical biosensor for Brucella. Brucellosis primarily infects livestock, in which it causes significant reproductive problems leading to economic losses, but can also cause flu-like symptoms and more serious complications in humans. A rapid, sensitive test for Brucella is required to monitor herds and adjacent wild carriers, such as elk and bison. Optical biosensors, which operate by detecting changes due to the interaction between light and the stimulus, could satisfy this need. Long period fiber gratings (LPGs) are periodic modulations induced in the core of an optical fiber that cause transmitted light to be scattered at a resonant wavelength, resulting in attenuation. Conventional LPGs respond to changes in strain, temperature, or external refractive index by shifting their resonant wavelength. When special conditions are met, an LPG may exhibit a turn-around point (TAP), where dual peaks coalesce into a single peak with a constant wavelength but variable attenuation depth. TAP-LPGs are more sensitive than ordinary LPGs, and could be developed into inexpensive sensors with single-wavelength light sources and detectors. In this work, ISAMs were deposited onto an LPG to tune it near its TAP. Segments of single-stranded DNA, called hybridization probes, that were specific to individual species of Brucella were attached to the ISAM film before the sensor was exposed to lysed bacterial cultures. It was found that the sensor could distinguish between Brucella and other types of bacteria, but was less successful at distinguishing between Brucella species. The project was limited by the available TAP-LPGs, which had less dynamic range than those used in prior work by this group. Attempts were made to establish a new supply of TAP-LPGs by fabrication with a CO2 laser, but these efforts were unsuccessful due to poor laser stability. The second project discussed in this dissertation investigated ISAM films as a source of second-harmonic generation (SHG), a nonlinear optical process in which light is converted to half its fundamental wavelength in the presence of intense electric fields. Nonlinear ISAMs were constructed by choosing a polyelectrolyte with a hyperpolarizable side group in which SHG can occur. The SHG efficiency was increased by factors of several hundred to several thousand by the addition of silver nanoprisms. Metal nanoparticles can produce strong electric field enhancements, especially at their tips and edges, when incident light causes resonant collective oscillations in their electrons called localized surface plasmons (LSPs). It was found that while silver nanoprisms whose LSP resonant wavelength matched the fundamental wavelength were too dilute to produce noticeable enhancement, better results could be obtained by depositing shorter wavelength nanoprisms at sufficient density to broaden their extinction peak via interparticle interactions. The best enhancement observed was for a sample where concentrated silver nanoprisms with LSP resonance around 900 nm were dropcast onto an ISAM film and coated with an additional polymer layer, resulting in 2368 times more SHG than the plain ISAM film.

Submitted by Cleide Dantas (cleidedantas@ufpa.br) on 2014-07-28T15:14:47Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoSoroepidemiologicaBrucelose.pdf: 308098 bytes, checksum: b3fd5f9507ad94e60dd1653ab755d98c (MD5) / Approved for entry into archive by Ana Rosa Silva (arosa@ufpa.br) on 2014-09-12T12:13:16Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoSoroepidemiologicaBrucelose.pdf: 308098 bytes, checksum: b3fd5f9507ad94e60dd1653ab755d98c (MD5) / Made available in DSpace on 2014-09-12T12:13:16Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_InvestigacaoSoroepidemiologicaBrucelose.pdf: 308098 bytes, checksum: b3fd5f9507ad94e60dd1653ab755d98c (MD5) Previous issue date: 2007 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O objetivo do trabalho foi a detecção de anticorpos anti - Brucella abortus e anti – Leptospira interrogans em soros de eqüídeos e seus tratadores nos bairros das cidades de Belém e Ananindeua, abrangendo os meses de abril a agosto de 2005, utilizando para este fim, 195 soros sanguíneos de eqüídeos e 70 soros sanguíneos de homens que manipulavam os animais direta ou indiretamente. Para a pesquisa de animais sororeagentes à B. abortus, foram usadas as provas do Antígeno Acidificado Tamponado (AAT) como teste de triagem e a Soro Aglutinação Lenta em Tubos (SAL) e o teste do 2-Mercaptoetanol (2-ME), como teste confirmatórios. Para a Leptospirose, foi utilizada a prova de Soroaglutinação Microscópica (SAM), sendo realizada a triagem dos soros frente à 25 sorovares de L. interrogans, considerando-se positivos aquelas amostras com titulação igual ou maior que 100. De 195 amostras de soros sanguíneos de eqüídeos, 184 (94,87%) foram positivas para todos os sorovares analisados, sendo que os mais frequentemente encontrados foram: Patoc, Automnalis, Ictehaemohragiae, Pyrogenes e Bratislava. Para as amostras sanguíneas de homens, a positividade foi de 49 (70%) de soros reagentes, com os sorovares Patoc, Ictehaemohragiae, Bratislava, Butembo, Copenhageni e Automnalis os mais detectados. Das amostras positivas de animais e seus respectivos tratadores, 47/70 (67,14%) foram semelhantes para os mesmos sorovares de Leptospira spp., sendo que 2/70 (2,86%) amostras foram negativas em ambos os grupos pesquisados, 2/70 (2,86%) foram somente positivas em homens e 19/70 (27,14%) foram exclusivamente positivas nas amostras de soros de eqüídeos. Os bairros do Coqueiro, Guamá, 40 horas, Barreiro e Bengui apresentaram a maior percentagem de casos soropositivos. Não houve diferença significativa em relação às outras variantes estudadas, como: idade (animal e homem), tempo de serviço (animal e homem), espécie do animal, escore corporal do animal e grau de instrução do homem. Tanto nos animais quanto nos homens não foram detectadas reações positivas para B. abortus. / The objective of the work was the detention of antibodies anti - Brucella abortus and anti - Leptospira interrogans in serum of equines and equine workers of the quarters of the cities of Belém and Ananindeua, enclosing the months from April to August of 2005, using for this end, 195 sanguineous serum of eqquines and 70 sanguineous serum of men that manipulated the animals direct or indirectly. For the research of reagents serum of animals to B. abortus, the tests used had been Antigen Acidified Test (AAT), as a selection test, and slow seroagglutination (SAL) and and the 2-mercaptoetanol (2-ME) as confirmatory tests. To Leptospirosis, the test used was of microscopical seroagglutination being carried through the selection of the serum front to the 25 serovars of L. interrogans, considering positive those samples with titulation equal or bigger that 100. Of 195 samples of equine sanguineous serum, 184 (94.87%) had been positive for all serovars analyzed, being that more frequent found: Patoc, Automnalis, Ictehaemohragiae, Pyrogenes and Bratislava. For the sanguineous samples of men, the positivity was of 49 (70%) of reacting serum, with the most detected serovars: Patoc, Ictehaemohragiae, Bratislava, Butembo, Copenhageni and Automnalis. Of the positive samples of equine and its respective workers, 47/70 (67.14%) were similar for same serovars of Leptospira spp., being that 2/70 (2.86%) samples had been negative in both the searched groups, 2/70 (2.86%) were only positive in humans and 19/70 (27.14%) were exclusively positive in the samples of equine serum. The quarters of Coqueiro, Guamá, 40 horas, Barreiro and Bengui had presented the biggest percentage of positive cases serum. It did not have significant difference in relation to the other studied variants, as: climate, age (animal and man), time of service (animal and man), species of the animal, props up corporal of the animal and degree of instruction of the man. As much in the animals how much in the men positive reactions for B. abortus had not been detected.

Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-05-15T11:31:56Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_RespostaImuneHumoral.PDF: 1103336 bytes, checksum: f20142b1495f0855c37bd778d9a9dda5 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-05-16T17:13:52Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_RespostaImuneHumoral.PDF: 1103336 bytes, checksum: f20142b1495f0855c37bd778d9a9dda5 (MD5) / Made available in DSpace on 2017-05-16T17:13:52Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_RespostaImuneHumoral.PDF: 1103336 bytes, checksum: f20142b1495f0855c37bd778d9a9dda5 (MD5) Previous issue date: 2013-08-31 / A brucelose está distribuída em todo território nacional, com maior prevalência para a espécie Brucella abortus que apesar de ser endêmica, apresenta-se de forma heterogênea entre as diferentes regiões do Brasil. O presente estudo tem por objetivo avaliar a resposta imune humoral de bezerras bubalinas criadas na Mesorregião do Médio Amazonas, vacinadas com a cepa B19 de B. abortus na idade preconizada pelo Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose (PNCEBT). Foram selecionadas e acompanhadas 36 fêmeas (grupo teste) e 6 machos (grupo controle) com idades entre 3-8 meses. No dia 0 foi coletado sangue de todos os animais, em seguida todas as fêmeas receberam vacina comercial com amostra B19 em dose padrão conforme orienta o PNCEBT, com posteriores coletas de sangue realizadas aos 30, 60, 90, 120, 150, 180, 210, 330, 360 e 390 dias. Os soros sanguíneos foram avaliados pelo soro aglutinação das provas de triagem: Antígeno Acidificado Tamponado - AAT e pela prova confirmatória: 2 Mercaptoetanol - 2ME. Os resultados obtidos para todas as amostras coletas no dia 0 apresentaram resultado negativo na reação. Aos 30 e 60 dias todas as fêmeas apresentaram 100% de reação na prova AAT e titulação de 1:200, iniciando o declínio da titulação aos 90 dias nas duas provas. Aos 210 dias as fêmeas ainda reagentes apresentaram resultados similares nas duas provas, tendo apenas um animal se mantido reativo nas duas provas até 360 dias e, somente aos 390 dias 100% das fêmeas obtiveram reação negativa nas duas provas, durante todo o experimento os machos foram não reativos. Assim, pela avaliação dos resultados, a vacinação mostrou-se eficaz para a imunização, sendo uma ferramenta importante na profilaxia da brucelose na espécie estudada, bem como as provas aplicadas para o diagnostico dessa enfermidade em búfalos a nível regional. / Brucellosis is spread throughout the country, with the highest prevalence for the strain Brucella abortus that despite being endemic presents unevenly among different regions of Brazil. This study aims to evaluate the humoral and immune response of buffalo calves raised in the Middle Amazon Mesoregion, Pará state, vaccinated with B19 strain of B. abortus according the age recommended by the National Program for the Control and Eradication of Brucellosis and Tuberculosis (PNCEBT). Thirty-six female (test group) and six males (control group) aged 3-8 months were selected and monitored. On day 0 of the experiment, blood was collected from all animals, then all females received commercial vaccine with standard-dose sample B19 as recommended by PNCEBT with subsequent blood samples were taken at 30, 60, 90, 120, 150, 180, 210, 330, 360 and 390 days, respectively. The blood serums were evaluated for evidence of agglutination screening through Buffered Acidified Antigen - AAT and the confirmatory test through 2 Mercaptoethanol - 2ME. The results for all samples collected on day 0 were negative in the reaction, however at 30 and 60 days all females were 100% positive through test reaction AAT and titration 1:200, starting the decline through the titration at 90 days in both events. Females at 210 days still present reagents with similar results in both tests, however only one animal remained reactive in both tests up to 360 days. After 390 days of the begin of the experiment 100% of the females had a negative reaction in both events and throughout the experiment males were negative. Thus, by the evaluation of results, vaccination was effective for immunization, as well as an important tool in the prophylaxis of brucellosis in the species studied, as well as the tests applied to the diagnosis of this disease in buffaloes species at regional level.