Ovarian cancer is a high mortality disease where early stage detection may have significant survival benefits. Promising next-generation non-invasive, biomarker-based screening modalities involve longitudinal monitoring of serum biomarkers and multi-marker panel detection. Here, rapid, sensitive, precise and multiplexable diagnostic platforms can facilitate biomarker validation along with early detection and screening, and this work attempts to exploit the programmable bio-nano-chip (p-BNC) immunosensor to address these specific translational needs in ovarian cancer. First, the p-BNC was adapted for Cancer Antigen 125 (CA125) quantitation, the current FDA standard, with prominent implications in novel early detection and screening modalities. Antibody pairs binding to distinct epitopes on CA125 were identified and the p-BNC operating variables (incubation times, flow rates and reagent concentrations) were attuned to deliver optimal analytical performance (inter- and intra-assay precision of 1.2% and 1.9% and Limit-of-Detection (LOD) 1.0 U/mL), competitive with current gold standards, but with a short analysis time of 43 minutes. Further validation of the system with advanced stage patient sera (n=20) demonstrated good correlation with 'gold standard' ELISA (R² = 0.97). Next, the p-BNC was adapted for concomitant analysis of CA125 and Human Epididymis Protein 4 (HE4), a novel multiplexed biomarker panel for early detection and screening. The HE4 immunoassay was developed to perform optimally with the 'rate determining' CA125 assay. Cross-reactivity analysis demonstrated high specificity multiplexing. The dose-response curves for the multiplexed CA125 and HE4 immunoassays were congruous with their singleplex counterparts with respective LODs of 0.51 U/mL and 4.18 pM and a total analysis time of 44 minutes. A small pilot scale clinical study was conducted to discriminate between surgically confirmed patient sera (n=8) and corresponding age-matched healthy controls (n=8) utilizing the multiplexed p-BNC, interpreted with a risk of ovarian malignancy algorithm. Successful discrimination was achieved between the groups with Receiver Operating Characteristic (ROC) curve AUC (Area Under the Curve) values of 1.00, 0.984 and 1.00 respectively for CA125, HE4 and the composite marker combination. Taken together, the analytical and clinical performance, multiplexing capabilities and the short turn-around times on the p-BNC offer methodological advancements over current gold standard techniques, indicating strong promise for ovarian cancer diagnostics. / text

Most human organs consist of solid tissues. Most human disorders occur in solid tissues. No two cells are equivalent in native solid tissues as single cells widely vary in their biochemistry, specialization, and location. Yet, the fundamental limitations of solid-tissue processing and analysis have made it challenging to access the richness of molecular information in single cells of animal and human solid tissues. We have eliminated a set of limitations of solid-tissue processing and analysis. In this thesis, I first describe a new method for sampling single cells in live solid tissues. This method preserves the molecules of all molecular classes and thus fulfills the precondition for multiplexing within and across molecular classes. I then describe new analytical methods and strategies for massively multiplex analysis within and across molecular classes in each sampled single cell. Standard curves, the basis of analytical methods, can be constructed in all measurements and signals can be mapped to the corresponding quantities. Proof of principle experiments are presented. These methods will enable the quantification of the molecular mechanism of each sampled single cell in solid tissues by analytically measuring tens of proteins, transcripts and/or metabolites at once. By performing these measurements in human solid-tissue biopsies, we will be able to define a new category of diagnostic tests, to personalize single-cell pharmacology and to rapidly identify mechanistic biomarkers and drug targets.

Surveillance of domestic poultry flocks for antibodies against avian influenza and Newcastle disease to detect and differentiate between these diseases is very important. The ability to determine if the detected influenza virus antibodies belong to one of the reportable H5 or H7 subtypes is imperative. These two major viruses are continually responsible for economic loss in poultry industries all over the world. Current serological methods of detection are an effective means of detecting antibody responses to these viruses, however continually investigating improved methods of surveillance is important. Development of a serological assay using Luminex technology which involves the use of recombinantly generated influenza A nucleoprotein, hemagglutinin H5, hemagglutinin H7, and Newcastle disease nucleocapsid proteins bound to Magplex beads allowed for the simultaneous detection of antibodies against these proteins that matches the efficiency of past methods while maintaining high levels of specificity and overall accuracy. Assay development took the form of two connected projects beginning with construction of an assay that operated in duplex, detecting antibodies against influenza nucleoprotein (AIV-NP) and Newcastle disease nucleocapsid protein (APMV-1-NC). Once optimized, the second half of development involved expansion of the assay to include detection of H5 (AIV-H5) and H7 (AIV-H7) subtypes, as well as the addition of internal assay quality controls to monitor assay performance over time. Assay thresholds and overall performance of both of these functional assays were evaluated using large quantities of field and experimental sera from chickens and turkeys to maximize specificity and overall accuracy.

No description available.

Os atuais sistemas de criação na avicultura, baseados na alta densidade populacional, aumentam os riscos de disseminação de enfermidades, especialmente das doenças respiratórias e daquelas cujos agentes etiológicos possuem mais de um hospedeiro. A Cólera Aviária (CA) possui estas características e geralmente apresenta-se de forma aguda e com altos índices de morbidade e de mortalidade. Apesar de representar uma das patologias aviárias que deve ser considerada para o diagnóstico diferencial de enfermidades com notificação obrigatória que cursam com morte súbita, a patogenia e os fatores de virulência envolvidos na CA ainda estão pouco elucidados. O objetivo deste trabalho foi pesquisar quinze genes associados à virulência em 25 amostras de Pasteurella multocida isoladas de casos de CA na região sul do Brasil através do desenvolvimento de protocolos de multiplex-PCR. Além disto, o presente estudo visou comparar a capacidade de tipificação capsular do método molecular com testes fenotípicos. Os protocolos de multiplex-PCR desenvolvidos foram capazes de detectar todos os genes propostos. Os genes ompH, oma87, sodC, hgbA, hgbB, exBD-tonB, nanB, hyaD-hyaC estiveram presentes em 100% das amostras (25/25). Os genes sodA e nanH em 96% (24/25), o gene ptfA em 92% (23/25) e o gene pfhA em 60% (15/25). Os genes toxA, bcbD, dcbF não foram identificados em nenhuma das amostras pesquisadas (0/25). A tipificação capsular através do teste molecular apresentou maior capacidade de detecção quando comparada aos testes fenotípicos, pois enquanto 36% (9/25) das amostras não foram identificadas pelo teste convencional, somente 8% (1/25) não foi tipificada através do multiplex-PCR. Foram obtidos seis diferentes perfis genéticos, sendo P1 (negativo para os genes toxA, dcbF e bcbD) o mais comum. Com este trabalho, concluiu-se que os protocolos de multiplex-PCR desenvolvidos tornam-se uma ferramenta bastante útil e rápida para a detecção simultânea dos genes de virulência. Apesar da alta frequência dos genes estudados e de todas as amostras pertencerem à mesma subespécie de P. multocida, foram observados seis perfis genéticos, os quais devem ser confirmados em um estudo com um maior número de amostras. / The current breeding systems in poultry, based on high population density, increases the risk of spread of pathologies, especially respiratory diseases and those whose etiologic agents have more than one host. Fowl Cholera (FC) has these features and generally is presented in an acute form and with high rates of morbidity and mortality. Even though it represents one of the several avian diseases that should be considered in the differential diagnosis of notifiable diseases that course with sudden death, pathogenesis and virulence factors involved in the FC are still poorly understood. The objective of this study was to investigate fifteen genes related with virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. In addition, this study evaluated the ability of capsular typing comparing the method molecular with phenotypic tests. The multiplex-PCR protocols developed were capable to detect all the proposed genes. The genes ompH, oma87, sodC, hgbA, hgbB, exBD-tonB, nanB, hyaD-hyaC, bcbD and dcbF were present in 100% of the samples (25/25). Gene sodA and nanH in 96% (24/25), ptfA in 92% (23/25) and pfhA in 60% (15/25). Gene toxA, bcbD and dcbF were not identified in any of the samples studied (0/25). The capsular typing by molecular test presented a higher detection capability when compared to phenotypic tests, because while 36% (9/25) of 25 of samples were not identified by conventional testing, only 8% (1/25) was not typified by multiplex–PCR. Six different genetic profiles were obtained and P1 (negative to genes toxA, dcbF and bcbD) was the most common. With this work, it was concluded that the multiplex-PCR protocols developed can be a useful tool for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and that all samples belong to the same subspecies of P. multocida, six genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

La dyslexie est le plus fréquent des troubles neurodéveloppementaux de l’enfant. A ce jour, 9 loci de susceptibilité ont été identifiés parmi lesquels DYX 9 en Xq27. Nous avons réalisé la première étude de liaison française suivie de l’analyse de gènes candidats sur l’ensemble du génome de 58 sujets issus de 12 familles multiplex pour la dyslexie, en utilisant un phénotype catégoriel. Des résultats significatifs sont apparus pour la région Xq27.3, au sein de DYX9. Le Lod score multipoint maximal obtenu était 3.884 entre rs12558359 et rs454992. Dans cette région, les séquences codantes de 7 gènes candidats ayant un rôle dans le développement cérébral (CXORF1, CXORF51, SLITRK2, FMR1, FMR2, ASFMR1, FMR1NB), ont été étudiées à la recherche de mutations. Nous avons également étudié les séquences 5’ non codantes des gènes FMR1 et FMR2. Aucune mutation n’a été retrouvée. Ces résultats de liaison de la région Xq27.3, au locus XFRA, dans une population française confirment la région DYX9 comme région d’intérêt dans la dyslexie de développement. / Dyslexia is a frequent neurodevelopmental learning disorder. To date, 9 susceptibility loci have been determined among which DYX9 located in Xq27. We performed the first French SNP linkage study followed by candidate gene investigation in dyslexia by studying 12 multiplex families (58 subjects) with at least two children affected, according to categorical restrictive criteria for phenotype definition. Significant results emerged on Xq27.3 within DYX9. The maximum multipoint LOD score reached 3,884 between rs12558359 and rs454992. Within this region, 7 candidate genes were investigated for mutations in exonic sequences (CXORF1, CXORF51, SLITRK2, FMR1, FMR2, ASFMR1, FMR1NB), all having a role during brain development. We further looked for 5’UTR trinucleotide repeats in FMR1 and FMR2 genes. No mutation or polymorphism co-segregating with dyslexia was found. This finding in French families with Dyslexia showed significant linkage on Xq27.3 enclosing FRAXA, and consequently confirmed the DYX9 region as a robust susceptibility locus. We reduced the previously described interval from 6.8 Mb (DXS1227- DXS8091) to 4Mb also disclosing a higher Lod score.

Os atuais sistemas de criação na avicultura, baseados na alta densidade populacional, aumentam os riscos de disseminação de enfermidades, especialmente das doenças respiratórias e daquelas cujos agentes etiológicos possuem mais de um hospedeiro. A Cólera Aviária (CA) possui estas características e geralmente apresenta-se de forma aguda e com altos índices de morbidade e de mortalidade. Apesar de representar uma das patologias aviárias que deve ser considerada para o diagnóstico diferencial de enfermidades com notificação obrigatória que cursam com morte súbita, a patogenia e os fatores de virulência envolvidos na CA ainda estão pouco elucidados. O objetivo deste trabalho foi pesquisar quinze genes associados à virulência em 25 amostras de Pasteurella multocida isoladas de casos de CA na região sul do Brasil através do desenvolvimento de protocolos de multiplex-PCR. Além disto, o presente estudo visou comparar a capacidade de tipificação capsular do método molecular com testes fenotípicos. Os protocolos de multiplex-PCR desenvolvidos foram capazes de detectar todos os genes propostos. Os genes ompH, oma87, sodC, hgbA, hgbB, exBD-tonB, nanB, hyaD-hyaC estiveram presentes em 100% das amostras (25/25). Os genes sodA e nanH em 96% (24/25), o gene ptfA em 92% (23/25) e o gene pfhA em 60% (15/25). Os genes toxA, bcbD, dcbF não foram identificados em nenhuma das amostras pesquisadas (0/25). A tipificação capsular através do teste molecular apresentou maior capacidade de detecção quando comparada aos testes fenotípicos, pois enquanto 36% (9/25) das amostras não foram identificadas pelo teste convencional, somente 8% (1/25) não foi tipificada através do multiplex-PCR. Foram obtidos seis diferentes perfis genéticos, sendo P1 (negativo para os genes toxA, dcbF e bcbD) o mais comum. Com este trabalho, concluiu-se que os protocolos de multiplex-PCR desenvolvidos tornam-se uma ferramenta bastante útil e rápida para a detecção simultânea dos genes de virulência. Apesar da alta frequência dos genes estudados e de todas as amostras pertencerem à mesma subespécie de P. multocida, foram observados seis perfis genéticos, os quais devem ser confirmados em um estudo com um maior número de amostras. / The current breeding systems in poultry, based on high population density, increases the risk of spread of pathologies, especially respiratory diseases and those whose etiologic agents have more than one host. Fowl Cholera (FC) has these features and generally is presented in an acute form and with high rates of morbidity and mortality. Even though it represents one of the several avian diseases that should be considered in the differential diagnosis of notifiable diseases that course with sudden death, pathogenesis and virulence factors involved in the FC are still poorly understood. The objective of this study was to investigate fifteen genes related with virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. In addition, this study evaluated the ability of capsular typing comparing the method molecular with phenotypic tests. The multiplex-PCR protocols developed were capable to detect all the proposed genes. The genes ompH, oma87, sodC, hgbA, hgbB, exBD-tonB, nanB, hyaD-hyaC, bcbD and dcbF were present in 100% of the samples (25/25). Gene sodA and nanH in 96% (24/25), ptfA in 92% (23/25) and pfhA in 60% (15/25). Gene toxA, bcbD and dcbF were not identified in any of the samples studied (0/25). The capsular typing by molecular test presented a higher detection capability when compared to phenotypic tests, because while 36% (9/25) of 25 of samples were not identified by conventional testing, only 8% (1/25) was not typified by multiplex–PCR. Six different genetic profiles were obtained and P1 (negative to genes toxA, dcbF and bcbD) was the most common. With this work, it was concluded that the multiplex-PCR protocols developed can be a useful tool for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and that all samples belong to the same subspecies of P. multocida, six genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

Os atuais sistemas de criação na avicultura, baseados na alta densidade populacional, aumentam os riscos de disseminação de enfermidades, especialmente das doenças respiratórias e daquelas cujos agentes etiológicos possuem mais de um hospedeiro. A Cólera Aviária (CA) possui estas características e geralmente apresenta-se de forma aguda e com altos índices de morbidade e de mortalidade. Apesar de representar uma das patologias aviárias que deve ser considerada para o diagnóstico diferencial de enfermidades com notificação obrigatória que cursam com morte súbita, a patogenia e os fatores de virulência envolvidos na CA ainda estão pouco elucidados. O objetivo deste trabalho foi pesquisar quinze genes associados à virulência em 25 amostras de Pasteurella multocida isoladas de casos de CA na região sul do Brasil através do desenvolvimento de protocolos de multiplex-PCR. Além disto, o presente estudo visou comparar a capacidade de tipificação capsular do método molecular com testes fenotípicos. Os protocolos de multiplex-PCR desenvolvidos foram capazes de detectar todos os genes propostos. Os genes ompH, oma87, sodC, hgbA, hgbB, exBD-tonB, nanB, hyaD-hyaC estiveram presentes em 100% das amostras (25/25). Os genes sodA e nanH em 96% (24/25), o gene ptfA em 92% (23/25) e o gene pfhA em 60% (15/25). Os genes toxA, bcbD, dcbF não foram identificados em nenhuma das amostras pesquisadas (0/25). A tipificação capsular através do teste molecular apresentou maior capacidade de detecção quando comparada aos testes fenotípicos, pois enquanto 36% (9/25) das amostras não foram identificadas pelo teste convencional, somente 8% (1/25) não foi tipificada através do multiplex-PCR. Foram obtidos seis diferentes perfis genéticos, sendo P1 (negativo para os genes toxA, dcbF e bcbD) o mais comum. Com este trabalho, concluiu-se que os protocolos de multiplex-PCR desenvolvidos tornam-se uma ferramenta bastante útil e rápida para a detecção simultânea dos genes de virulência. Apesar da alta frequência dos genes estudados e de todas as amostras pertencerem à mesma subespécie de P. multocida, foram observados seis perfis genéticos, os quais devem ser confirmados em um estudo com um maior número de amostras. / The current breeding systems in poultry, based on high population density, increases the risk of spread of pathologies, especially respiratory diseases and those whose etiologic agents have more than one host. Fowl Cholera (FC) has these features and generally is presented in an acute form and with high rates of morbidity and mortality. Even though it represents one of the several avian diseases that should be considered in the differential diagnosis of notifiable diseases that course with sudden death, pathogenesis and virulence factors involved in the FC are still poorly understood. The objective of this study was to investigate fifteen genes related with virulence in 25 samples of Pasteurella multocida isolated from FC cases in the southern region of Brazil through the development of multiplex PCR protocols. In addition, this study evaluated the ability of capsular typing comparing the method molecular with phenotypic tests. The multiplex-PCR protocols developed were capable to detect all the proposed genes. The genes ompH, oma87, sodC, hgbA, hgbB, exBD-tonB, nanB, hyaD-hyaC, bcbD and dcbF were present in 100% of the samples (25/25). Gene sodA and nanH in 96% (24/25), ptfA in 92% (23/25) and pfhA in 60% (15/25). Gene toxA, bcbD and dcbF were not identified in any of the samples studied (0/25). The capsular typing by molecular test presented a higher detection capability when compared to phenotypic tests, because while 36% (9/25) of 25 of samples were not identified by conventional testing, only 8% (1/25) was not typified by multiplex–PCR. Six different genetic profiles were obtained and P1 (negative to genes toxA, dcbF and bcbD) was the most common. With this work, it was concluded that the multiplex-PCR protocols developed can be a useful tool for rapid and simultaneous detection of virulence genes. Despite the high frequency of the analyzed genes and that all samples belong to the same subspecies of P. multocida, six genetic profiles were observed, which should be confirmed in a study with a larger number of samples.

Magister Scientiae - MSc / South Africa

Masters of Science / Genetic analysis for resistance to Woolly Apple Aphid in apple rootstock breeding populations MC Selala MSc Thesis, Department of Biotechnology, Faculty of Science, University of the WesternCape. The Woolly Apple Aphid (WAA) Eriosoma lanigerum (Hausm.) (Homoptera: Aphididae) is economically one of the most important pests in apple commercial production in the Western Cape province, South Africa. The apple cultivar Northern Spy possesses a single major gene (Er1) responsible for E. lanigerum resistance. This cultivar has been used as a commercial rootstock in apple breeding programmes. There are other genes also implicated in resistance to E. lanigerum from other cultivars. Manipulation and pyramiding of the E. lanigerum resistance genes (Er1, Er2 and Er3) might provide a necessary control for commercial apple production. The aim of this study was to construct a genetic linkage map for apple using microsatellite markers. The use of marker-assisted selection would greatly benefit local apple breeding programmes. Ninety six seedlings from a Northern Spy × Cox Orange Pippin mapping population were used for genetic linkage construction. Phenotypic data collection and analysis were performed to determine the E. lanigerum infestation patterns and the levels of resistance conferred by the Er1 gene from Northern Spy using 52 in vitro propagated seedlings in the greenhouse. Classification and quantification analysis showed association patterns between first assessments (30 days) to second assessment (60 days) in all replicate blocks. Roots and shoots data showed that it could be useful in quantitative trait loci (QTL) analysis, but may be used in different QTLs beingidentified due to the variations between roots and shoots data. A preliminary linkage map was constructed using a mapping population from Northern Spy × Cox Orange Pippin (96 seedlings).Fluorescently labelled published and predicted microsatellite markers were used in map construction. Primers were optimised using single apple cultivar and the detection of polymorphisms using nine apple cultivars. Optimised markers were multiplexed for high throughput data generation using the Polymerase Chain Reaction (PCR) technique. Multiplexed PCR products were pooled and analysed on an ABI 310 PRISM™ Genetic Analyser to determine allele fragment sizes, and the inherited segregation types in the seedlings. Computer software GenoTyper® 2.5.2 and JoinMap® 3.0 was used in data analysis from ABI 310 PRISM™Genetic Analyser and linkage map construction. Seventy two markers were used in linkage map construction, which produced nine linkage groups with some segments from the same linkage group. Twenty-one markers were aligned on the map 20 published and one predicted. Only one linkage group consisted of five markers while other linkage groups had two markers each. This study has proved that th preliminary linkage map could be used as the basis of a complete linkage map of Northern Spy × Cox Orange Pippin.