Hochsch., Diplomarbeit, --Mittweida, 2004.

Zugl.: Mittweida, Hochsch., Diplomarbeit, 2004

Thesis (Honors)--Liberty University Honors Program, 2007. / Includes bibliographical references. Also available through Liberty University's Digital Commons.

Thesis (Ph. D.)--Biomedical Engineering, Georgia Institute of Technology, 2006. / Tannenbaum, Allen, Committee Member ; Choi, Jung, Committee Member ; Borodovsky, Mark, Committee Chair ; Voit, Eberhard, Committee Member ; Lee, Eva, Committee Member.

Thesis (Ph.D.)--University of Nebraska-Lincoln, 2006. / Title from title screen (site viewed on Oct. 6, 2006). PDF text: xiv, 307 p. : ill. (some col.) ; 4.75Mb. UMI publication number: AAT 3213861. Includes bibliographical references. Also available in microfilm, microfiche and paper format.

Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-06T18:47:19Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertacao-BrenoAlbuquerque.pdf: 1989114 bytes, checksum: 513b1650c4c44605e3bda0afef185321 (MD5) / Made available in DSpace on 2017-04-06T18:47:19Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertacao-BrenoAlbuquerque.pdf: 1989114 bytes, checksum: 513b1650c4c44605e3bda0afef185321 (MD5) Previous issue date: 2012 / O Papilomavírus bovino(BPV) é o agente etiológico da papilomatosebovina. Esta apresenta lesões que normalmente são benignas e tendem a regredir, porém podem progredir a uma neoplasia. Muitas metodologias utilizadas para detecção de BPV se mostram inespecíficas e apresentam reações cruzadas com outros organismos relacionados. No entanto, a reação quantitativa em tempo real emcadeia da polimerase (qPCR) é uma ferramenta de destaque na detecção, tipificação e quantificação de nucleotídeos e vem sendo utilizada na clínica para avaliar carga viral. O objetivo do trabalho foi desenvolver um novo protocolo de detecção, tipificação e quantificação de BPV através daqPCR. Foram desenhados cinco pares de primers, que possuem como alvo uma região conservada do genoma viral (gene L1) de diferentes BPVs. A seletividade dos primers foi testada in vitroe DNA extraído de células MDBK não infectadas foram utilizados como controle negativo. A técnica de qPCR permitiu detectar, tipificar e quantificar material viral dos BPVs 1, 2, 4, 5 e 6. O limiar relativo da detecção foi de 4fg de DNA,emtorno de 30-40 cópias de DNA/μL. Dos cinco pares de primers produzidos, quatro apresentaram mesmo perfil térmico durante a qPCR (qPCRBPV2, 4, 5 e 6), permitindo em um único procedimento detectar e tipificar os quatro tipos virais. A distinção das amostrasfoi realizada através da análise de meltingque permitiu tipificá-las. Através da metodologia desenvolvida foi observado que em lesões cutâneas de bovinos infectados com BPV a carga viral não se mostrou inferior a 1000 cópias/μL, enquanto que a técnica permite quantificar até um limiar de 40 copias de DNA/μl. Este trabalho possui relevância para validação de qPCR como diagnóstico da papilomatose bovina e particular importância quando aplicado em estudos da infecção pelo BPV e no monitoramento por veterinários da eficácia das futuras vacinas. / Bovine papillomavirus (BPVs) is the etiologic agent of bovine papilomatose which is characterized by hyper proliferative lesions. Papillomas in cattle are typically benignandoften regress, but occasionally lesions can persist and progress to malignant neoplasia.The majority of current techniques for identification of BPV is unspecific andpossessescross-reactivity with closely related organisms.The Real-time quantitative polymerase chain reaction assay (qPCR) has become an exceptional tool for detection and quantification of oligonucleotides and has been utilized increasingly on viral load evaluation.Aiming to develop a new protocol for fast detection, typification and quantification of BPV in qPCR, we designed five pairs of Oligonucleotides for BPV1, 2, 4, 5 and 6 focusing on L1 gene. The qPCR primers sets were testedin vitroandMadin-Darby Bovine Kidney Cells (MDBK)DNA was also used as negative control.The Real-time qPCR assay provided an accurate detection and quantification for the BPVs 1, 2, 4, 5 and 6. The relative detection limit for the assays was 4fg or 30 to 40genome equivalents. Four primers pairs (qPCRBPV2, 4, 5 and 6) had the same annealing temperature and their products showed differences on meltingpoints analyses. Through the meltingpoint analysis, samples can be identified and discriminated as a screening and then samples can be run for viral load. In our study we tested the viral load in bovine cutaneous skin warts and observed infections with 1000 copies/μl at least. However, this assay could reach levels of 40copies/μL. In conclusion, this methodology has an important impact on the validation of qPCR as a BPV diagnosis. Its relevance is proved when applied to BPV infection studies and the monitoring of the efficacy of future BPV vaccinesby veterinarians.

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Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 85-92). Also available on the World Wide Web.

The purpose of this investigation was to examine the structure of the murine cytomegalovirus (MCMV) genome and to study its expression during productive and non-productive infections caused by the virus. The kinetic complexity of MCMV DNA was not less than its molecular weight implying the absence of major reiterations. The restriction endonuclease EcoR^ cleaved this molecule into twenty-five fragments, which were present in the digest in equimolar amounts and ranged in molecular weights from 20 to 1 million. The sum of the molecular weights of the fragments was 136 million. The genomes of the 'K 181' and 'Smith' strains of MCMV appeared to share more than 99 percent of their sequences, although the DNAs exhibited slightly different fragmentation patterns when treated with EcoR^ and Hind III endonucleases. Control was exerted on the transcription of the MCMV genome at temporal, quantitative, and processing levels. During productive infections, approximately 25 percent of the genome was represented as stable transcripts in the cell at 6 hours post infection, i.e., before the onset of viral DNA synthesis, whereas RNA transcribed from 35 to 40 percent of the DNA was present in the cells in the later stages of infection. RNA sequences corresponding to 6 h (early) transcripts would be detected in the cell throughout the infectious cycle. Both 'early' and 'late' RNA comprised two RNA classes differing about 7 to 10 fold in concentration. Viral DNA synthesis in the host cell was required for the expression of 'late' genes since in the presence of inhibitors of protein and DNA synthesis only 'early' transcription occurred. Control was also exerted on the transport of transcripts from the nucleus to the cytoplasm of infected cells. Although RNA extracted from the nuclei of infected cells arose from 25 (early) and 35 (late) percent of the viral genome, transcripts from only 11 (early) and 15 (late) percent of the DNA were detected in the cytoplasm. Cells of mouse origin (3T3.cells), arrested in the G^ phase of the cell cycle, retained the viral genome in a non-replicating state, but could be induced to enter the lytic cycle by serum activation. Transcripts from 19 percent of the genome were observed in G^ arrested, MCMV-infected cells. Viral RNA in these cells comprised only one abundance class, which was similar to the scarce class in 'early' RNA from infected exponentially growing cells. Some evidence was also obtained for the transmission of latent MCMV genomes from mother to progeny. Cells cultured from embryos of infected mice did not normally produce infectious virus. However, the presence of the virus, at least in some of these cells, could be demonstrated by immunofluorescence, and by in-situ hybridization, using iodinated MCMV DNA as probe. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate