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Título de Ingeniero en Biotecnología Molecular / Diversos mecanismos de edición génica se han implementado a lo largo de los años para mutar el genoma del virus herpes simple tipo 2 (VHS-2). Éstos se han basado principalmente en el uso de procesos de recombinación homóloga. Un mecanismo comúnmente utilizado consiste en la realización de recombinación homóloga con un sustrato de intercambio alélico (AES) directamente sobre células transfectadas con genoma viral. Otra aproximación comúnmente utilizada es el desarrollo de recombinación homóloga en bacterias transformadas con un cromosoma artificial bacteriano (BAC) que contiene el genoma completo del virus herpes simple. La progenie viral mutante es luego obtenida mediante transfección de células con este material genético modificado. Si bien se han desarrollado diversas cepas mutantes de VHS-2 utilizando estas metodologías, ambas metodologías son laboriosas y presentan problemas técnicos tanto previos como posteriores a la generación de la mutación en el genoma. Dadas estas dificultades, se ha buscado desarrollar otros mecanismos más convenientes los cuales faciliten este proceso. Una metodología relativamente reciente que permite realizar modificaciones genéticas de manera más simple es aquella conocida como la tecnología de CRISPR-Cas9. Dada la mayor facilidad y especificidad de esta técnica, comparada a las más antiguas, nos propusimos mutar mediante el sistema CRISPR-Cas9, los genes virales US6, US4 y UL53 del VHS-2 para interrumpir la síntesis de las proteínas virales gD, gG y gK, respectivamente. Si bien se lograron desarrrollar los reactivos para realizar estas mutaciones, lamentablemente no se obtuvieron los aislados mutantes deseados. Se discuten los eventuales problemas que no permitieron lograr el objetivo

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 80-85).

Objetivos.- Conocer la prevalencia de la seropositividad para herpes virus I y II en pacientes con linfomas no hodking y su asociación con el linaje celular (B o T). Pacientes y Métodos.- Se tomó una muestra de 60 pacientes en el Hospital Almenara de Agosto de 1999 a Diciembre del 2000 todos ellos pacientes con diagnostico establecido de linfoma no hodking nuevos o en primera recaída, el análisis se realizo mediante bioestadística descriptiva. Resultados.- La mediana de la edad fue de 59 años, 2/3 fueron varones, 65% pacientes nuevos y el primario fue extraganglionar en un 58% de los casos. El 80% de los linfomas fueron a células B y más del 90% en estadios avanzados (III y IV), ningún caso fue positivo para IgM herpes I o II y 25% tuvieron serologia positiva para herpes I o II (2/3 positivos para IgG I) de los cuales más del 90% fueron a células B. Conclusiones.- La prevalencia de seropositividad para herpes virus I y II en pacientes con linfoma no hodking es del 25%, mayormente asociado a células B, además de tener un porcentaje considerable de linfomas a celulas T (25%) y linfomas extranodales (58%); para evaluar la posibilidad de asociación entre este virus y los linfomas requerimos de un estudio caso-control. / Objetives.- We intent to know the prevalence of seropositivity for herpes virus I and II in patients with malignant non hodking lymphoma (NHL), and the association with the cell lineage (B or T). Patients and Methods.- We considered 60 new or in first recurrence patients with NHL at the Almenara Hospital from August 1999 to December 2000, we analized the data by descriptive biostatistics in the Epi-Info program. Results.- The median age was 59 years, two thirds were men, 65% new patients and the primary site was extranodular in 58% of the cases. 80% were NHL to B cells, and more than 90% in advance stage (III and IV), none of them were positive for IgM herpes virus I or II and 25% were positive for IgG I or II (2/3 positive for IgG I) and more than 90% of them were for B cell, Conclusion.- The prevalence of seropositivity for herpes I or II in patients with NHL was 25%, usually associated to B cells, on the other hand we have an elevated percentage of T cell NHL (25%) as well as extranodulas NHL (58%). We need more studies specially a case-control study to define the association of herpes virus I or II with NHL.

Thesis (doctoral)--Rijksuniversiteit te Groningen.

Made available in DSpace on 2015-12-06T23:04:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2003 / Introdução: O Herpes simples labial recorrente e um problema de Saúde publica devido a sua elevada prevalencia. Provoca absenteismo ao trabalho e a escola, podendo estigmatizar o individuo, e evoluir para formas mais graves, como a ceratite. Os tratamentos ate agora desenvolvidos baseiam-se em antivirais, que demonstram efeitos na diminuicao da sintomatologia, no entanto, nao prevenindo as recidivas. A vacina Sabin, utilizada na prevencao de poliomielite, possui a capacidade de aumentar a imunidade atraves dos anticorpos especificos por meio dos CTLs (linfocitos T citoliticos) e potencialmente poderia ser utilizada na imunoprofilaxia de herpes simples labial recorrente. Objetivos: Estabelecer a seguranca e eficacia da utilizacao da vacina Sabin na imunoprofilaxia de Herpes simples labial recorrente. Tipo de Estudo: Ensaio clinico controlado randomizado, duplo cego. Local: Ambulatorio de Dermatologia e Pronto Atendimento do Hospital São Paulo. Amostra: pacientes de ambos os sexos, maiores de 18 anos, que apresentem mais de quatro episodios ao ano, por no minimo tres anos, de herpes simples labial, excluindo gestantes e imunodeficientes. Intervencao: administracao de vacina Sabin em duas doses com intervalo de 1 mes. Tempo de seguimento: 1 ano Desfechos clinicos: 1-Diminuicao dos eventos de herpes simples labial; 2Diminuicao da intensidade das crises herpeticas. Analise de dados: O tamanho da amostra foi calculado em 100 (a,=0,05; 0=0,1) pacientes para cada grupo, considerando-se uma melhora de 10 por cento no grupo placebo e poder estatistico para detectar melhora de 30 por cento no grupo intervencao, em relacao ao placebo. A analise sera realizada por intencao de tratamento, com a utilizacao do teste de qui-quadrado e diferenca de risco absisco absovariaveis dicotomicas e teste-t para as continuas, com nivel de significancia para o erro alfa de 5 por cento, intervalo de confianca de 95 por cento / BV UNIFESP: Teses e dissertações

A HSV 2 immediate early antigen was prepared and used as an immunogen in an attempt to produce monoclonal antibodies to this set to proteins. The results of three screening assays, ELISA, immunodiffusion and radioimmunoprecipitation with cell extracts and Protein A-Sepharose beads indicated that the hybrid cell lines are nonsecretors of antiHSV 2 antibodies. Other monoclonal cell lines from Dr. Bacchetti's laboratory were characterized by radioimmunoprecipitation with Protein A-Sepharose beads and cell extracts. / Thesis / Master of Science (MS)

The role of highly conserved amino acids within the carboxy-terminal hydrophobic domain of herpes simplex virus I (HSV-I) glycoprotein gB was studied by introducing point mutations using the method of site directed mutagenesis. A segment of this hydrophobic domain of glycoprotein gB contains a nuclear envelope (NE) targeting signal and the effect of these point mutations on targeting to the nuclear envelope was determined. A complementation assay was employed to determine the effect these mutations have on HSV-I infectivity .The point mutations created within the transmembrane domain of glycoprotein gB had no effect on nuclear envelope targeting and localization. However, single point mutations introduced into the first and second hydrophobic domains of glycoprotein gB, G₇₄₃R and F₇₇₀S, affected the targeting and localization of full-length glycoprotein gB at the nuclear envelope. When the transmembrane domain ofHSV-I glycoprotein gB containing the following point mutations A₇₉₀Q, A₇₉₁S, A₇₈₆S, A₇₈₆Y and A₇₉₀S, was introduced into a chimeric protein consisting of the cytoplasmic domain and ectodomain of a plasma membrane protein, vesicular stomatitis virus glycoprotein G, NE targeting and localization were affected. These point mutations may affect the targeting of glycoprotein gB by altering the structure of the targeting signal within the protein. It can be hypothesized that the presence of the cytoplasmic domain. ectodomain domain, and the first and second transmembrane domains within full-length glycoprotein gB can compensate for the effect these point mutations have on nuclear envelope targeting. since the same point mutations had no effect on the targeting · and localization of full-length glycoprotein gB. Complementation assays showed that the glycoprotein gB mutants, A₇₈₆S, A₇₈₆Y, A₇₈₆N, A₇₉₀Q, A₇₉₁S, F₇₇₀S, or G₇₄₃R, were unable to complement a gB-null virus even though these mutant proteins are localized at the nuclear envelope. These proteins may not have been incorporated into the viral capsid due to misfolding or due to the fact that sequences required for interaction with other viral proteins were lost. Another possibility is that the mutant proteins were incorporated into the HSV virion but were not biologically active. / Thesis / Master of Science (MS)

The glycoprotein gB of HSV-1 is involved in viral entry and membrane fusion functions. It is glycosylated, forms homodimers, and is transported to both the inner nuclear membrane and plasma membrane in infected cells. The gB glycoprotein contains a potential membrane anchoring hydrophobic sequence of 69 amino acids, located near the carboxy terminus of the glycoprotein. This sequence is predicted to span the membrane bilayer three times, and can thus be divided into three distinct segments, each of which could span the bilayer. To define both the membrane anchoring sequence and the role of this 69 residue hydrophobic domain, a series of deletion mutants were constructed. These mutants have one, two or all three of the predicted membrane spanning segments deleted, in every combination possible, thereby creating a total of 7 deletion mutants. These mutant constructs were expressed in COS-1 cells using transient expression systems. All the mutant constructs were expressed and glycosylated in a manner similar to the wild type gB glycoprotein. Mutant glycoproteins lacking the third (or most carboxy terminal) predicted spanning segment of this 69 residue hydrophobic domain were found to be secreted from the cells, indicating that this segment may specify the membrane anchoring domain of the gB glycoprotein. Further, the mutant glycoproteins containing this third segment were localized in the nuclear envelope, while mutants lacking this segment were not. All the deletion mutants, except for one, were however defective in intracellular transport and processing of the N-linked oligosaccharides. The only mutant that showed any intracellular transport and processing had only the third segment deleted, but even this mutant was transported and processed much slower than the wild type glycoprotein. The mutant glycoproteins also failed to complement a gB-null virus. These results suggest that the carboxy terminus hydrophobic domain contains essential structural determinants of the gB glycoprotein. / Thesis / Master of Science (MS)

Herpes simplex Virus Type 1 expresses three different classes of genes, immediate early, early, and late, during a lytic infection. Immediate early genes are the first class of genes expressed and they are the only genes expressed independently of de novo viral protein synthesis. This unique characteristic is thought to be the result of the activation of immediate early genes by Vmw65, a protein brought into the cell as a component of the infecting virion. Vmw65 transactivates through the target sequence TAATGARAT (R= purine) which is present at least once in all immediate early transcription regulatory regions. By inserting minimal synthetic promoters, containing the TAATGARAT sequence, into the thymidine kinase locus of the herpes simplex virus type 1 genome I determined that transactivation by Vmw65 is not sufficient to confer on the linked sequences the complete immediate early pattern of gene expression. Furthermore through a transient expression assay I determined that the TAATGARAT sequence element, by itself, when linked to a TATA box is sufficient to act as a target for Vmw65 transactivation. / Thesis / Master of Science (MS)