Bochum, Universiẗat, Diss., 2004.

Thesis (Ph. D.)--Washington State University, August 2007. / Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-22Bitstream added on 2014-06-13T19:16:44Z : No. of bitstreams: 1 moura_mf_me_botfca.pdf: 702744 bytes, checksum: d94bcbccf8a2ddefe768c5c679f20208 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O pimentão (Capsicum annuum L.) está entre as dez hortaliças mais consumidas no Brasil. Dois potyvirus são verificados nesta cultura, o Potato virus Y (PVY) e o Pepper yellow mosaic virus (PepYMV). Foi avaliada entre outubro de 2007 a novembro de 2008, a ocorrência de potyvirus infectando Capsicum spp. nos municípíos de Pirajú, Pirajuí, Paranapanema, Santa Cruz do Rio Pardo, Sorocaba, São Miguel Arcanjo, Itapetininga, Reginópolis, Lins, Iacanga e Mogi-Mirim, do Estado de São Paulo. Das 408 amostras coletadas, 105 foram positivas para a presença de potyvirus utilizando-se antissoro antipotyvirus (Agdia). Em algumas amostras foi detectada a presença de infecção mista com o Cucumber mosaic virus (CMV) e begomovírus. A inoculação de cinqüenta e um isolados na série diferencial de Capsicum spp contendo os genes pvr21, pvr22 e Pvr4, dos quais dez foram provenientes da Empresa Sakata Seed Sudamérica, possibilitou a classificação de dois isolados em patótipo 0, três em patótipo 1, seis em patótipo 1.2, onze em patótipo 1.2.3 e treze em patótipo 1-3 de PVY. Dezesseis isolados não puderam ser classificados em patótipos. Não foi verificada correlação entre local de coleta e ocorrência de um patótipo específico, evidenciando grande variabilidade biológica dos isolados de potyvirus no campo. Nenhum dos isolados coletados ocasionou sintomas evidentes em plantas de pimentão Rubia R e Magali R, indicando que a resistência conferida por estes híbridos ainda é efetiva contra os isolados de potyvirus predominantes no campo. Um par de primers PepNib (5’ GWTSGYYGMMTTGGATGATG 3’) e PepUTR (5’ AGTAGTACAGGAAAAGCC 3’) foi 2 obtido para amplificação completa da região codificadora da proteína capsidial de PVY e PepYMV. Analisando-se esta região do genoma viral, pôde-se constatar predominância da espécie PepYMV. O PVY foi encontrado somente em coletas... / The sweet pepper (Capsicum annuum L.) is one of the ten most consumed vegetables in the country. In Brazil, two potyviruses are verified in this culture, the Potato virus Y (PVY) and the Pepper yellow mosaic virus (PepYMV). Between October 2007 and November 2008, the occurrence of potyvirus infecting Capsicum spp. was evaluated on plants collected from Piraju, Pirajuí, Paranapanema, Santa Cruz do Rio Pardo, Sorocaba, São Miguel Arcanjo, Itapetininga, Reginópolis, Lins, Iacanga and Mogi-Mirim, in Sao Paulo State. Among the 408 collected samples, 105 were positive for the presence of potyvirus using antipotyvirus antiserum (Agdia). The presence of mixed infection with the Cucumber mosaic virus (CMV) and begomovirus was also verified. The inoculation of fifty one isolates on the series of Capsicum spp. containing the genes pvr21, pvr22 and Pvr4, ten of them isolates from the Sakata Seed Sudamerica Company, made it possible the classification of these isolates in different pathotypes. Two isolates were classified as pathotype 0, three in pathotype 1, six in 4 pathotype 1.2, eleven in pathotype 1.2.3 and thirteen in pathotype 1.3 of PVY. Sixteen isolates were not able to be classified in pathotypes of PVY. No correlation could be made between the origin of the isolate and the presence of an specific pathotype, indicanting a greet biological variability between the potyvirus isolates. None of the isolates collected in the field caused symptoms in Rubia R and Magali R, indicating that the resistance provided by these hybrids is still effective against the predominant isolates of potyvirus. A pair of primer PepNib (5’ GWTSGYYGMMTTGGATGATG 3’) and PepUTR (5’ AGTAGTACAGGAAAAGCC 3’) were obtained for the complete amplification of the capsid protein region of PVY and PepYMV isolates. PepYMV was the prevalent species of potyvirus found infecting sweet peppers. The PVY was found only... (Complete abstract click electronic access below)

The main aim of this master thesis was the simultaneous detection of four selected plant viruses ? Apple mosaic virus, Plum pox virus, Prunus necrotic ringspot virus and Prune harf virus, by microarrays. The intermediate step in the process of the detection was optimizing of multiplex polymerase chain reaction (PCR).

Made available in DSpace on 2014-06-11T19:27:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-26Bitstream added on 2014-06-13T19:56:56Z : No. of bitstreams: 1 leda_aro_me_botfm.pdf: 436850 bytes, checksum: e631607fe6c1cbb57e01981982378a57 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A história natural de infecção pelo HIV e a progressão para a aids podem variar entre diferentes indivíduos, possivelmente devido a fatores genéticos, entre eles os polimorfismos de nucleotídeo simples (SNPs). SNPs localizados em regiões promotoras de genes que codificam citocinas podem afetar a síntese e a regulação dessas moléculas, resultando em alterações nas respostas imunitárias. O presente estudo buscou avaliar as frequências e os possíveis efeitos de SNPs nas posições -589 e -1098 da região promotora do gene da IL-4 e SNPs nas posições -238 e -862 da região promotora do gene do TNF-α em pacientes portadores do HIV. Amostras de DNA de 157 pacientes foram obtidas através de células mononucleares de sangue periférico e a genotipagem dos SNPs foi realizada pela técnica de High Resolution Melting (HRM). Foi observado que pacientes portadores de TT em SNP/pIL-4 -589 apresentaram contagem de linfócitos T CD8 + menor em relação aos portadores de CC (p=0.0104). Além disso, portadores de TT em SNP/pIL-4 -1098 apresentaram contagem de linfócitos T CD8 + maior em comparação aos portadores de GT (p=0.0053). Em relação a SNP/pTNF-α -238, as proporções de pacientes portadores de GG e GA diferiu entre os pacientes sem HAART e pacientes com HAART e sem falha terapêutica (p=0.0205). Assim, os resultados obtidos no presente estudo fortalecem a hipótese de que SNPs em genes de citocinas podem alterar a história natural da infecção pelo HIV e o curso clínico da doença, principalmente devido a alterações no balanço da produção de citocinas pro- e antiinflamatórias. / The natural history of HIV infection and its progression towards aids may vary considerably among different individuals, possibly due to genetic factors, such as single nucleotide polymorphisms (SNPs). In cytokines genes promoters, SNPs may affect protein synthesis and regulation, resulting in more or less efficient immune responses against HIV. The present study evaluated the frequencies and possible effects of SNPs in the IL-4 gene promoter at positions -589 and -1098 and in the TNF-α gene promoter at positions -238 and -862 in HIV-infected patients from Brazil. DNA samples from 157 patients were obtained from peripheral blood mononuclear cells, and SNPs genotyping was performed by High Resolution Melting analysis (HRM). Patients carrying TT at SNP/pIL-4 -589 had lower circulating T CD8 + cells compared to CC carriers (p=0.0104). Moreover, carriers of TT at SNP/pIL-4 -1098 had more circulating T CD8 + cells compared to GT carriers (p=0.0053). Regarding SNP/pTNF-α -238, GG and GA proportions were significantly different between patients without HAART and patients on HAART without therapeutic failure (p=0.0205). In conclusion, these results provide compelling evidence that the presence of SNPs in cytokine-coding genes do modify the natural history of HIV infection, mainly due to changes in the balance between pro- and anti-inflammatory cytokines.

Memoria para optar al Título Profesional de Médico Veterinario / El virus influenza A (IAV) de origen porcino posee gran importancia en salud pública y salud animal. IAV posee gran variabilidad genética y antigénica, lo cual es muy importante dentro de su patogenia. Este estudio consistió en determinar la variabilidad genética del gen de la neuraminidasa (NA) en virus aislados de planteles porcinos de producción intensiva entre los años 2013 y 2014. Para esto, se analizaron 1167 muestras desde 25 granjas mediante aislamiento viral, RT-PCR en tiempo real y secuenciación por Next Generation Sequencing, obteniéndose 41 secuencias genómicas de NA pertenecientes al 50% de las empresas muestreadas. Se determinó la presencia de los subtipos H1N1, H1N2 y H3N2 circulando en los planteles chilenos, específicamente en la Región de Valparaíso, Región Metropolitana, Región del Libertador General Bernardo O’Higgins, Región del Maule y Región del Biobío, confirmando la gran diseminación de IAV en porcinos de Chile. Del total de secuencias de NA, 17 correspondieron a N1, las que fueron agrupadas dentro del clúster N1 pandémico, evidenciando múltiples introducciones del virus a la especie porcina. Por otro lado, 24 secuencias correspondieron a N2. La mayoría de éstas se agruparon en un clúster monofilético, que fue designado como “N2 chileno”, el cual resultó ser distinto a aquellos encontrados en otros países y evidenciaban una introducción única a la población de cerdos chilenos. Se observó también la presencia de distintas variantes virales dentro de una misma empresa y la presencia de una misma variante en empresas distintas. Estos resultados permitieron conocer la realidad de IAV y específicamente la diversidad de NA. / Swine origin influenza A virus (IAV) is an important concern in both public and animal health. IAVs have shown high genetic and antigenic diversity, which is very important for the viral pathogenesis. The aim of this study was to determine the neuraminidase gene (NA) genetic variability in IAVs obtained from intensive swine farms, during 2013 to 2014. One thousand one hundred sixty-seven samples from 25 swine farms were tested by viral isolation, real time RT-PCR and sequenced by Next Generation Sequencing. A total of 41 NA sequences were obtained, representing 50% of the swine farms in study. H1N1, H1N2 and H3N2 subtypes were detected in Valparaíso, Metropolitan, Libertador General Bernardo O´Higgins, Maule and Biobío Regions, confirming the wide spreading of IAV in intensive swine farms in Chile. Seventeen viral sequences belonged to N1 subtype. All of them were grouped into the pandemic N1 cluster, and the tree topology suggests multiple introductions of the virus into swine population. The rest of the sequences belonged to N2 subtype. Most of them were grouped into a monophyletic cluster, named “Chilean N2”. This cluster was not close related with viruses previously described, suggesting a unique introduction into the swine population. At farm level, was observed the presence of more than one IAV variant and the presence of a determined variant circulating in several farms. These results could help to understand the influenza A virus dynamic in swine in Chile. / Financiamiento: Proyecto Fondef ID14I10201.

Glioblastoma is the most common primary brain tumour in adults. Despite the aggressive standard of care currently used, median patient survival following treatment is only 14 months. Innovative treatment options are needed for these patients. Recently, oncolytic viruses have emerged as promising immunotherapies for the treatment of solid tumours. Preliminary work in our lab has demonstrated that Farmington virus, a novel brain-safe oncolytic rhabdovirus, can be engineered to encode a tumour-associated antigen (TAA) to prime and boost antigen-specific adaptive immune responses. Since other rhabdoviruses share this boosting capacity, a heterologous rhabdovirus prime/boost regimen can be designed to combine two powerful oncolytics and a robust anti-TAA adaptive immune response. We evaluated Farmington’s ability to vaccinate against a self- glioblastoma antigen and two foreign glioblastoma-associated antigens. Farmington was able to vaccinate against the foreign antigens, leading to efficacy in prophylactic and therapeutic glioblastoma models. Additionally, treatment with heterologous rhabdoviruses demonstrated efficacy in an aggressive murine mammary carcinoma model. Herein, we demonstrate promising preliminary results for a novel glioblastoma therapeutic approach. Le glioblastome est la tumeur primaire la plus fréquente chez l’adulte. La survie moyenne des patients n’excède pas 14 mois malgré une prise en charge thérapeutique agressive. Par conséquent, la mise au point de traitements innovants et efficaces est une nécessité pour ces patients. Des avancées récentes ont mise en évidence l’intérêt des virus oncolytiques dans le traitement des tumeurs solides. Des travaux préliminaires réalisés au sein de notre laboratoire ont, en effet, démontré que le virus Farmington pouvait être modifié afin d’exprimer un antigène associé aux tumeurs (AAT), pour initier et potentialiser une réponse immunitaire adaptative spécifique. D’autres rhabdovirus possèdent des capacités de potentialisation immunitaire similaires et peuvent être utilisés en association avec le virus Farmington modifié pour amorcer et amplifier la réponse immunitaire oncolytique de l’hôte. Le but de ce projet était d’évaluer le potentiel du virus Farmington comme vaccin contre des antigènes tumoraux d’origine endogène ou exogène associés au glioblastome. Nos résultats ont montré que le virus Farmington a la capacité d’induire une réponse immunitaire prophylactique et thérapeutique contre les antigènes tumoraux exogènes dans des modèles de glioblastome. De plus, l’utilisation de rhabdovirus hétérologues s’est aussi révélée efficace pour le traitement de carcinome mammaire agressif chez la souris. Cette étude préliminaire apporte des résultats prometteurs pour le développement de nouvelles approches thérapeutiques efficaces dans le traitement du glioblastome.

Los virus del grupo C (GRCV) son un complejo que pertenecen al género Orthobunyavirus, de la familia Peribunyaviridae (anteriormente denominado Bunyaviridae). Estos virus están asociados con enfermedades febriles en humanos que generalmente habitan en áreas tropicales y subtropicales de América del Sur y América Central. A pesar de que muchos GRCV han sido aislados de mosquitos, animales y seres humanos, los análisis genéticos de estos virus aún son limitados. En el Perú, el centro de investigaciones de enfermedades tropicales de la marina de los Estados Unidos (NAMRU-6) viene conduciendo desde los años noventa una vigilancia pasiva de las enfermedades febriles. Durante este tiempo, se lograron recuperar e identificar mediante la prueba de inmunofluorescencia 65 aislamientos de GRCV de pacientes febriles habitantes del norte y sur de la Amazonía peruana. Para caracterizar estos aislamientos, se secuenciaron una región de 500 pb del segmento S y una región de 750 pb de los segmentos M y L. El análisis de la secuencia de los aislamientos clínicos mostró identidades de nucleótidos que oscilaban entre el 68% y el 100% y las identidades de secuencia de aminoácidos deducidas que oscilaban entre 72% y 100%. Las secuencias se compararon con las siguientes cepas referenciales: virus Caraparu (CARV), virus Murutucu (MURV), virus Oriboca (ORIV), virus Marituba (MTBV), virus Apeu (APEUV) y virus Madrid (MADV). La comparación de secuencias de los segmentos L y S de las cepas clínicas con cepas referenciales mostró dos clados; clado I formado por aislamientos con alta correlación con CARV-MADV y clado II conformado por aislamientos con alta correlación con MURV, ORIV, APEUV y MTBV. Las secuencias del segmento M contiene algunos aislamientos de GRCV diferentes filogeneticamente a los de segmento L y S y su distribución filogenética está altamente relacionada con la microneutralización serológica. Estos resultados demuestran las relaciones genéticas y serológicas de los GRCV que circula en la Amazonía peruana y la divergencia genética de los GRCV en el norte de la Amazonía. / Tesis

African horsesickness virus (AHSV) is a dsRNA virus that belongs to the Orbivirus genus within the Reoviridae family. Each of the ten viral dsRNA segments encodes one virus-specific protein. During its life cycle AHSV replicates both in an insect vector and in a mammalian host, but while it has no detrimental effect on insect cells the virus is highly pathogenic to mammalian cells. It is postulated that this relates to different viral release mechanisms. Currently the main candidate for mediating viral release in both insects and mammals is the viral nonstructural protein NS3. In bluetongue virus (BTV), the prototype virus of the Orbivirus genus, it has been shown that NS3 interacts with both the viral outer capsid protein VP2 and a cellular exocytosis protein. For AHSV, we investigated whether the same mechanism was involved in viral release. This study aimed to identify and map possible protein-protein interaction between AHSV NS3 and VP2, and AHSV NS3 and unknown insect cellular proteins. For investigating the NS3-VP2 interactions a eukaryotic expression system (yeast twohybrid), a column binding assay utilising bacterially expressed NS3 and recombinant baculovirus expressed VP2 as well as a membrane flotation assay utilising recombinant baculovirus expressed VP2 and NS3-GFP, were used. A number of problems were encountered and no conclusive results were obtained. For investigating viral-cellular protein interactions the yeast two-hybrid system was also used, utilising NS3 as bait to screen proteins expressed from a Drosophila cDNA library. Results showed an interaction between the N-terminal region of AHSV NS3 and ubiquitin, an essential protein for the trafficking and degradation of membrane proteins from the endoplasmic reticulum. It also acts as a sorting signal in both the secretory pathway and in endosomes, where it targets proteins into multivesicular bodies in the lumen of vacuoles/lysosomes. It has been shown that ubiquitin could play a role in the pinching off of budding vesicles. An AHSV infected cell could therefore potentially use ubiquitin in its vesicular budding pathway, therefore giving the opportunity for viruses to use this to release them from the cell. The Hsp70 was another protein identified that interacts with AHSV NS3. This protein plays a role in folding reactions, protein translocation across membranes of organelles and protein assembly. It has been reported in other studies done that both ubiquitin and Hsp70 play roles in regulating the bioavailability of viral proteins, which could explain the different levels of NS3, high in insect cells and low in mammalian cells, which indirectly control the viral exit pathway used, budding versus lytic release. These results lay the foundation for explaining the potential role of NS3 in the AHSV life cycle in insect cells. / Dissertation (MS)--University of Pretoria, 2007. / Genetics / unrestricted