The aim of this master’s thesis is to analyze ways of propagation light in optical fiber with two polarization planes. Theoretical part is focused to to the issue of polarized light, its mathematical description using Stokes and Jones vectors, the state of polarization on Poincare sphere and polarization multiplex. In the practicle part are first measured several laboratory measurements to verify the theoretical assumptions from the previous chapters. At the end of the thesis was constructed a functional polarization multiplex for wavelength 1550 nm.

En bakteriell ledinfektion kallas för septisk artrit och utan snabb behandling kan tillståndet leda till irreversibla ledskador och flera allvarliga komplikationer. Snabb diagnostik är viktigt för en god prognos och med nuvarande metod för odling av ledvätska tar det upp till fem dygn för detektion av mikroorganismer. Metoden inkluderar odling på fasta substrat och i blododlingsflaskor. Med en ny metod som använder multiplex PCR (polymerase chain reaction) skulle analystiden kunna minskas till en timme. BioFire Joint Infection (JI) Panel är ett test som kan detektera flera mikroorganismer samtidigt genom amplifiering av DNA. Mikroorganismer detekteras och identifieras genom cellysering, DNA-rening, PCR-reaktioner i två steg och smältanalys. Syftet med arbetet var att utvärdera BioFire JI Panel för detektion av mikroorganismer i ledvätska och jämföra metodens känslighet med standardiserad odling respektive odling i buljong, och svara på frågeställningen hur känslig JI-panelen är jämfört med standardiserad odlingsmetod. Prov konstruerades genom att suspendera målbakterierna Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, Neisseria gonorrhoeae, Haemophilus influenzae, Kingella kingae och Finegoldia magna i natriumklorid. Suspensionerna seriespäddes och odlades ut på fasta substrat, inokulerades i blododlingsflaskor och buljonger samt analyserades med JI-panelen för att identifiera metodernas detektionsgränser. Fasta substrat, blododlingsflaskor och buljonger visade likvärdig känslighet för majoriteten av bakterieisolaten medan JI-panelen generellt visade en sämre eller likvärdig känslighet i jämförelse med standardmetod. Panelen visade potential för detektion av svårodlade bakterier och skulle vara ett bra komplement till nuvarande odlingsmetod. Ytterligare studier hade behövts men den snabba analystiden som möjliggör snabbare behandling framhävde metodens kliniska potential. / Bacterial joint infections are called septic arthritis and without rapid diagnosis and treatment the condition could lead to irreversible joint damage and other serious complications. With the current method for synovial fluid cultivation, which includes both solid substrates and culture vials, detection of microorganisms takes up to five days. With a new method utilizing multiplex polymerase chain reaction (PCR), the time to detection could decrease to an hour. BioFire Joint Infection (JI) Panel can detect multiple microorganisms simultaneously through amplification of DNA. Microorganisms are detected and identified by cell lysis, DNA purification and two consecutive PCR reactions with subsequent melting curve analysis. The aim of the study was to evaluate the BioFire JI Panel for detection of microorganisms in synovial fluid and compare the method’s sensitivity to standard and broth cultivation. Samples were constructed by suspending target bacteria, Staphylococcus aureus, Escherichia coli, Streptococcus pyogenes, Neisseria gonorrhoeae, Haemophilus influenzae, Kingella kingae and Finegoldia magna, in sodium chloride. The suspensions were serially diluted and cultivated on solid substrates, in culture vials, broth and analyzed with the JI panel to find the detection limits for each method. Solid substrates, culture vials and broth exhibited similar sensitivity while the JI panel generally had a lower or similar sensitivity compared to standard methods. The panel showed potential detecting difficult to grow bacteria and would be a good complement to current methods. Further studies are required but the quick analysis enabling faster treatment highlighted the clinical potential.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Ying Fang / The Porcine Disease Complex (PDC) results in major economic problems for swine producers. PDC outbreaks result in increased mortality, decreased feed efficiency, higher cull rates, prolonged days to market and increased treatment costs. This disease involves the interaction and participation of many multifactorial etiologies including both bacterial and viral organisms playing a role in disease initiation and progression. The most common viral pathogens associated with the PDC include porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus (PCV2) and swine influenza virus (swIV). The recent outbreak of porcine epidemic diarrhea virus (PEDV) in the US swine herd has made the PDC even more complicated. In aid of the prevention and control of the PDC, veterinarians and producers require fast and efficient diagnostic tests for controlling the disease. In this study, we have generated recombinant nucleocapsid antigens to these viruses for use in a Luminex™ technology-based fluorescent microsphere immunoassay (FMIA). Utilizing these recombinant nucleocapsid antigens, the FMIA was developed to simultaneously detect antibodies in serum from animals infected with PEDV, PRRSV, SwIV and PCV2. The FMIA was developed based on testing experimentally derived standard positive and negative control sera, and the diagnostic specificity and sensitivity were compared to that generated from the classical enzyme-linked immunosorbent assay (ELISA) or hemagglutination inhibition (HI) test. Based on an evaluation of 4147 serum samples with known serostatus, the multiplex FMIAs reached greater than 97.5% sensitivity and 92.3 % specificity. Results showed that multiplexing did not affect the diagnostic sensitivity or specificity of each individual assay. This work provides a platform for the development of multiplex assays for detecting various swine pathogens simultaneously and aids in preventing and controlling the PDC.

Malgré des années de recherche, la mammite est encore la maladie qui engendre le plus de pertes pour les producteurs agricoles du secteur laitier. Il est possible que ce fait puisse s'expliquer en partie par l'effort important investi au niveau de l'amélioration génétique des vaches laitières de manière à favoriser des phénotypes permettant une production supérieure. Cette pression génétique s'est effectuée au détriment de la santé de celles-ci. Afin de corriger cette erreur, des technologies plus récentes sont donc appelées à être mises à contribution. L'identification et la caractérisation des différents microorganismes responsables de cette maladie sont des bonnes étapes vers la possibilité de mieux traiter les animaux atteints et de diminuer l'incidence des infections. L'utilisation de méthodes de détection moléculaires de ces pathogènes amène d'ores et déjà une capacité de diagnostic et un débit d'obtention de résultats difficilement atteignable à l'aide des méthodes d'identification microbiologiques seules, et ce, dans plusieurs domaines. Le secteur agroalimentaire n'y échappe pas et des méthodes de détection moléculaires des pathogènes causant des infections intramammaires menant à la mammite sont disponibles. Toutefois, il est important d'analyser les forces et les faiblesses des systèmes proposés actuellement de manière à évaluer leur utilité dans un cadre pratique pour l'industrie laitière. Le but du travail présenté est de proposer une méthode de détection des pathogènes causant des infections intramammaires en utilisant une approche moléculaire. De plus, celle-ci devait respecter plusieurs critères pré-établis qui sont, selon notre avis, nécessaires pour mettre au point un système qui puisse être vraiment utilisé dans un cadre appliqué. Ainsi, l'approche proposée permet d'identifier tous les pathogènes majeurs responsables de cas de mammite au Canada tout en ayant un coût d'opération raisonnable, une capacité de travail à haut débit automatisé et une sensibilité suffisamment élevée pour être utilisée avec des échantillons de lait. La méthode ainsi développée pour respecter ces critères a permis l'analyse de 273 échantillons de lait provenant de cas de mammite en utilisant une amplification PCR avec amorces fluorescentes suivie d'une électrophorèse capillaire et d'une analyse de fragments assistée par laser. Afin d'évaluer sa validité, les mêmes échantillons ont été analysés préalablement par les méthodes de microbiologie préconisées par le « National Mastitis Council », et ce, dans deux laboratoires indépendants. La méthode fut également validée à l'aide d'échantillons de lait contaminés artificiellement avec une quantité connue de chacun des microorganismes ciblés dans cette étude. Les résultats ont permis de déterminer les paramètres de sensibilité et de spécificité en comparant les résultats moléculaires avec ceux obtenus par les méthodes microbiologiques. Ces paramètres, bien qu'étant essentiels pour juger de la performance d'un outil de diagnostic moléculaire, sont trop souvent ignorés dans les études présentant de telles méthodes. Les travaux présentés sont donc à la fois prometteurs pour le diagnostic rapide de la mammite que pour la possibilité de mettre au point des marqueurs génétiques de résistance à la mammite dans le futur.

Volume Holographic Data Storage Systems (HDSS) has been of interest for almost seven decades, and are now considered as a viable option for Write Once Read Many (WORM) cold data storage applications. Thanks to the Bragg selectivity of thick volume holograms, HDSS stores several hundreds of holograms on top of each other, called multiplexed data pages, by which data recording density can be substantially increased compared to surface recordings. On the other hand, signal intensity upon reconstruction of such multiplexed data pages inversely scales with number of multiplexing squared. Therefore, longer detection time and/or a high power laser along with a large dynamic range material is needed to make HDSS a truly viable "fast and high density" option for WORM applications. Historically, the trade-off between data density and data rate is well recognized. The challenge has been partially solved by continuous efforts such as improvement of materials, optical architectures, opto-mechanical systems and signal processing [1,2]. In this dissertation, we provide an additional pathway for HDSS to further increase both data density and transfer rates which is Cavities Enhancement Techniques for HDSS, to overcome the fundamental tradeoff. Key ideas are: recycling light with cavity to enhance data rate, and increasing number of multiplexing by combining cavity-eigenmode multiplexing, a subset of orthogonal phasecode multiplexing, with angular multiplexing. Based on this idea, we design and demonstrate Cavity-enhanced HDSS in such a way that we increase data rate and/or data density by at least factor of 2 while taking advantage of previous improvements as they are, or only with the minimum amount of modifications. In Section 1, we review history of HDSS and summarize the latest research results of HDSS and requirements on modern optical data storage systems as they relate to our solutions. In Section 2, theory of volume holography is reviewed by emphasizing understanding of angular and orthogonal phase code multiplexing. In Section 3 the theory of cavity enhanced reference arms is presented. We discuss how cavities provide a coherent boost to the beam power, which can be used in recording to alleviate source power requirements and/or increase the data recording rate and demonstrate the enhancement experimentally. Beyond basic enhancement, cavities also enable orthogonal phase code multiplexing via cavity eigenmodes. In Section 4, we experimentally demonstrate angular and orthogonal phase code hybrid multiplexing to overcome the limitation of the maximum number of multiplexing imposed by the geometrical constraints of angular multiplexing. In Section 5, novel aspects of the research are discussed in conjunction with the application of the technology for commercial use. Conclusions and future research direction are addressed in Section 6.

Dried blood spots is an alternative method to collect blood samples from research subjects. However, little is known about how hemoglobin and hematocrit affect bead-based multiplex assay performance. The purpose of this study was to determine how bead-based multiplex assays perform when analyzing dried blood spot samples. A series of four experiments outline the study each with a specific purpose. A total of 167 subject samples were collected and 92 different biomarkers were measured. Median fluorescence intensity results show a positive correlation between filtered and non-filtered samples. Utilizing a smaller quantity of sample results in a positive correlation to a larger sample. Removal of hemoglobin from the dried blood spot sample does not increase detection or concentration of biomarkers. Of the 92 different biomarkers measured 56 were detectable in 100-75% of the attempted samples. We conclude that blood biomarkers can be detected using bead-based multiplex assays. In addition, it is possible to utilize a smaller quantity of sample while avoiding the use of the entire sample, and maintaining a correlation to the total sample. While our method of hemoglobin was efficient it also removed the biomarkers we wished to analyze. Thus, an alternative method is necessary to determine if removing hemoglobin increases concentration of biomarkers. More research is necessary to determine if the biomarkers measured in this study can be measured over time or within an experimental model.

When forensic biological samples yield low quality/low quantity DNA, thecurrent STR analysis methods do not generate acceptable profiles. Whole genomeamplification can be used to pre-amplify the entire genome for downstream analyses. A commercially available kit for DOP-PCR, a form of WGA, is currently being used in the clinical for downstream single locus targets. Forensic analyses utilize a multiplex amplification. This study determined that the "home brew" created by our lab performs the same as the commercially available kit. Future optimization studies of DOP-PCR can utilize this "home brew". Additionally, this research determined that a 10 second increase in electrokinetic injection time onto the Capillary Electrophoresis (CE) in combination with a post-STR amplification purification and elution into formamide produces a slightly higher percent STR allele success over the standard protocol. After future optimization studies, this may be a useful method to obtain more accurate and complete STR profiles from low quality/low quantity biological samples.

>Magister Scientiae - MSc / The objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / A doenÃa de Chagas à causada pelo parasito intracelular Trypanosoma cruzi e afeta em torno de 17 milhÃes de pessoas na AmÃrica Latina. A fase crÃnica da doenÃa caracteriza-se por baixo nÃvel de parasitemia e alto nÃvel de anticorpos Anti-T.cruzi e o diagnÃstico à feito preferencialmente utilizando-se mÃtodos sorolÃgicos, incluindo imunofluorescÃncia indireta (IFI), hemaglutinaÃÃo indireta (HAI) e imunoensaio enzimÃtico (ELISA). SÃo testes que apresentam alta sensibilidade, mas sofrem de baixa especificidade, e um variÃvel nÃmero de indivÃduos apresenta testes sorolÃgicos inconclusivos. Nos Ãltimos anos, muitos estudos tÃm usado a tecnologia da ReaÃÃo em Cadeia da Polimerase (PCR) para detectar sequÃncias de DNA de T. cruzi em sangue de pacientes chagÃsicos crÃnicos. A alta especificidade da PCR tem apontado para sua aplicaÃÃo como mÃtodo confirmatÃrio no diagnÃstico de pacientes com provas sorolÃgicas inconclusivas. O objetivo desse trabalho foi realizar uma anÃlise comparativa entre a PCR e os mÃtodos convencionais mais utilizados para o diagnÃstico - sorologia e xenodiagnÃstico, na detecÃÃo da infecÃÃo por T. cruzi, em indivÃduos com doenÃa de Chagas crÃnica. Sangue de 67 pacientes chagÃsicos crÃnicos, ambos os sexos, provenientes do ambulatÃrio de doenÃa de Chagas do Hospital UniversitÃrio Walter CantÃdio, da Universidade Federal do CearÃ, foram avaliados para T. cruzi, utilizando testes sorolÃgicos (IFI, HAI e ELISA), PCR-Multiplex e xenodiagnÃstico. AlÃm disso, foi avaliado tambÃm a PCR-Multiplex em dejetos de triatomÃneos provenientes do xenodiagnÃstico destes pacientes. De acordo com o resultado dos testes sorolÃgicos, os pacientes foram classificados em 3 grupos: 1Â. Sorologia positiva (quando 2 ou 3 resultados dentre os 3 testes sorolÃgicos foram positivos), 2Â. Sorologia inconclusiva ou indeterminado (apenas 2 resultados foram negativos) e 3Â. Sorologia negativa (quando os 3 resultados foram negativos). Dentre as 59 amostras com sorologia positiva foram obtidos 18 (30,5%) resultados positivos de PCR-Multiplex em sangue perifÃrico e 41 (69,5%) resultados negativos. Nos PCR-Multiplex em dejetos de triatomÃneos foram obtidos 20 (33,9%) resultados positivos e 39 (66,1%) resultados negativos. As 2 amostras com sorologia inconclusiva foram negativas na PCR-Multiplex, tanto em sangue perifÃrico como em dejetos de triatomÃneos. As amostras com sorologia negativa (n=6) apresentaram 2 resultados positivos (33,3%) na PCR-Multiplex em sangue e 4 resultados negativos (66,7%). Nos dejetos de triatomÃneos, as 6 amostras com sorologia negativa foram negativas na PCR-Multiplex. Estes resultados mostraram que o ensaio da PCR-Multiplex em amostras de sangue perifÃrico e em dejetos de triatomÃneos, empregando iniciadores para regiÃes diferentes, no caso, DNA nuclear e DNA do cinetoplasto, podem ser aplicados para o diagnÃstico da doenÃa de Chagas crÃnica (mesmo com diferentes metodologias de extraÃÃo de DNA total). Os dados mostraram tambÃm que a PCR-Multiplex em dejetos de triatomÃneos à mais sensÃvel que o ensaio do xenodiagnÃstico.

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No. of bitstreams: 2 Dissertação - Anniely Carvalho Rebouças Oliveira - 2016.pdf: 1878998 bytes, checksum: 1afecfd5948d2abb2a5374cbeb9ff005 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-04-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The acute respiratory infections (ARIs) are an important cause of morbidity and mortality worldwide. Viruses have an important role in the etiology of the respiratory infections. Children less than five years of age have about four to six ARIs per year and this is a common cause of hospitalization, mainly in developing countries. In Brazil, particularly in the West-Central region, studies that evaluated the circulation of respiratory viruses in the pediatric population are scarce. Thus, this study aimed to investigate the occurrence of respiratory viruses in the pediatric population, from Goiânia-Goiás. Between May/2014 and May/2015, samples of nasal swabs were collected from children from zero to six years of age presenting or not respiratory symptoms, attended at the Materno Infantil hospital in Goiânia. For the molecular screening, 16 viruses were investiged using three Multiplex Nested-PCR protocols. It two hundred and fifty one samples were collected, of wich 90 (35,9%) were positive for at least one respiratory agent, being rhinovirus (31%), respiratory syncytial virus (27,4%) and parainfluenza virus (13,3%) the most prevalent. Children under two years of age had greater overall positive, although not statistically significant. Similar detection rate was observed among the groups symptomatic (37%) and asymptomatic (34,5%). For the symptomatic group, children whose samples were collected during the dry period (p<0,05) and those with up to two years of age were most affected (p<0,05). In the asymptomatic group no statistical difference was observed. It was observed rate of co-detection of 6,4% (16/251), mostly in samples of symptomatic patients (13/16) (p<0,05). The evaluation of the temporal profile showed a higher detection rate during the dry season, less rainfall, relative humidity and cooler temperatures. The results reinforce the importance of respiratory viruses in children and contribute to further understanding of the epidemiological and temporal factors associated with different pathogens in our region. Thus, they open the way to new studies in the state of Goiás, also providing information to assist in the construction of control measures and more effective prevention of these infections. / As infecções do trato respiratório (ITRs) representam importante causa de morbimortalidade em todo o mundo, sendo os vírus importantes agentes etiológicos. Estima-se que grande parte das crianças menores de cinco anos de idade tenha a cada ano de quatro a seis ITRs, sendo esta causa comum de hospitalização, especialmente em países em desenvolvimento. No Brasil, em particular na Região Centro-Oeste, estudos que avaliem a circulação de vírus respiratórios em população pediátrica são escassos. Neste contexto, o presente estudo teve como objetivo investigar a ocorrência de diferentes vírus respiratórios em população pediátrica de Goiânia-Goiás, apresentando ou não quadro sintomático respiratório. Durante o período de maio/2014 a maio/2015, foram coletadas amostras de swab respiratório de crianças entre zero e seis anos de idade, apresentando quadro de infecção respiratória ou assintomáticas para a mesma, atendidas no Hospital Materno Infantil, em Goiânia. As amostras foram submetidas à triagem molecular para 16 vírus respiratórios por meio de três protocolos de multiplex nested-PCR. No total, 251 amostras foram coletadas, das quais 90 (35,9%) apresentaram positividade para pelo menos um agente respiratório, sendo rinovírus (31%), vírus sincicial respiratório (27,4%) e parainfluenza (13,3%) os mais frequentes. Crianças com até dois anos de idade apresentaram maior positividade global, contudo sem diferença estatística. Índice de detecção semelhante foi observado entre os grupos sintomático (37%) e assintomático (34,5%). Para as crianças do grupo sintomático, foram mais afetadas aquelas cujas amostras foram coletadas durante período de seca (p<0,05) e aquelas com até dois anos de idade (p<0,05). No grupo assintomático, nenhuma diferença estatística foi observada. Foi observado índice de co-detecção de 6,4% (16/251), sendo a maioria de indivíduos sintomáticos (13/16) (p<0,05). A avaliação do perfil temporal demonstrou maior índice de detecção nos meses de seca, com menor precipitação pluviométrica, umidade relativa do ar e temperaturas mais frias. Os resultados obtidos no presente estudo reforçam a importância dos vírus respiratórios na população pediátrica, auxiliando na compreensão dos fatores epidemiológicos e temporais associados aos diferentes patógenos em nossa região. Desta forma, abrem caminhos para novos estudos no estado de Goiás, fornecendo ainda informações que auxiliem na construção de medidas de controle e prevenção mais eficazes contra estas infecções.