Herein, different polymer libraries were examined to determine the effect polymer structure has on intracellular events. The effect of different polyamine lengths in copolymers on cellular uptake, the effect of modifying end groups of trehalose-containing polymers on transfection efficiency, and the effect of different linker lengths between galactose and a hepatocyte-targeted polymer on transfection efficiency were studied. Furthermore, it was demonstrated that polymers with terbium chelated in their repeat units could potentially be used for Förster resonance energy transfer (FRET) studies to monitor pDNA release from the polymer. Much of the work in this dissertation focuses on elucidating the intracellular mechanisms of linear poly(ethylenimine) (PEI) and how it compares to poly(L-tartaramidopentaethylenetetramine) (T4) and poly(galactaramidopentaethylenetetramine) (G4), two poly(glycoamidoamine)s synthesized by our group. The long-term goal of this project is to develop structure-function relationships between polymers and pDNA delivery efficacy that will result in the rational design of safe, efficient vehicles for therapeutic nucleic acid delivery. Many polymers used as DNA delivery vehicles display high cytotoxicity. Often, the polymers with the highest transfection efficiency are the most toxic, as demonstrated herein by PEI and T4 with varying polymer lengths. Therefore, it was of interest to study how polymer structure influences mechanisms of cytotoxicity. To this end, studies on several mechanisms of cytotoxicity, including nuclear envelope permeabilization, were conducted. Longer polymers induced more cytotoxic responses than shorter ones, and it appears that hydroxyl groups in the repeat unit of polymers play a role in polyplex formation. This research has also led us to a potential link between transfection efficiency and cytotoxicity; the polymers with the highest transfection efficiency were also the most toxic, and were also able to induce the most nuclear envelope permeability. It is possible that these polymers' ability to permeabilize the nuclear envelope is what causes their high transfection efficiency and high toxicity. In addition, flow cytometry and confocal microscopy studies revealed that polymer structure plays a role in nuclear trafficking; poly(glycoamidoamine)s G4 and T4 more dependent on intracellular machinery than PEI. This research demonstrates the impact that changes in polymer structure have on intracellular mechanisms. / Ph. D.

No presente trabalho, desenvolvemos uma estratégia vacinal baseada em vacinas de DNA que codificam proteínas do HPV-16, o tipo viral de maior relevância epidemiológica, fusionadas à glicoproteína D (gD) do vírus herpes simplex tipo 1 (HSV-1). A vacina que contêm o gene da E7 de HPV-16 fusionada à gD de HSV-1 (pgDE7), administrada em regime vacinal de quatro doses, foi capaz de gerar significativa ativação de células T CD8+ E7-específicas e apresentar 40% de efeito protetor anti-tumoral terapêutico em camundongos desafiados com células transformadas que expressam as proteínas E6 e E7 do HPV-16 (células TC-1). A partir das evidências geradas, desenvolvemos um novo vetor vacinal que codifica as proteínas E7, E6 e E5 do HPV-16 (pgD-E7E6E5). Em ensaios em modelo murino, apenas uma dose da vacina foi capaz de gerar ativação de células T CD8+ específicas e 70% dos camundongos previamente desafiados com células TC-1 e inoculados com 3 doses da vacina mantiveram-se livres de tumores. Como tentativa de potencializar o efeito protetor terapêutico encontrado, adotamos duas medidas: a co-administração de plasmídeos que codificam citocinas e a otimização de códons da sequência gênica que codifica a proteína quimérica. A combinação das vacinas pgDE7 ou pgD-E7E6E5 com plasmídeos que carregam os genes das citocinas IL-2, IL-12 ou GM-CSF foi capaz de aumentar a proteção terapêutica para 100% em regime vacinal de dose única. A adequação da sequência antigênica ao sistema de expressão humano, aumentou em cerca de 5 vezes o potencial terapêutico do vetor vacinal pgDE7. Em conjunto, os dados apresentados nesta tese demonstram a evolução do desenvolvimento de uma estratégia vacinal contra tumores induzidos por HPV-16 e encorajam seu potencial para uso em futuros ensaios clínicos. / The development of immunotherapeutic strategies against human papillomavirus (HPV) is a priority for the control of HPV-induced lesions and cervical cancer. In this study, we developed DNA vaccines encoding HPV-16 proteins fused to glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) as an approach to control HPV-16 induced tumors. The vaccine encoding HPV-16 E7 fused to HSV-1 gD (pgDE7), when administered in a four doses vaccine regimen, was able to generate significant activation of E7-specific CD8+ T cells and showed 40% of therapeutic anti-tumor effect in mice previously challenged with tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells). Following these evidences, we developed a new vaccine vector encoding HPV-16 E7, E6, E5 proteins fused to HSV-1 gD (pgD-E7E6E5). Only one vaccine dose generated antigen-specific CD8+ T cell responses and three doses conferred 70% protection to mice previously challenged with TC-1 cells. As an attempt to enhance the observed therapeutic anti-tumor effects, we tested two approaches: co-administration of cytokine-expressing plasmids and codon optimization of the gene encoding the chimeric protein. The combination of the vaccines pgDE7 or pgD-E7E6E5 with plasmids encoding the cytokines IL-2, IL-12 or GM-CSF increased the therapeutic protection to 100% of the vaccinated animals following a single dose. The gene sequence adaptation increased by a factor of 5 the therapeutic potential of the pgDE7 vaccine. In summary, the data presented in this thesis demonstrated the development of a vaccine strategy against HPV-16 induced tumors and reinforces its potential use in future clinical trials.

O Vírus Respiratório Sincicial Humano (HRSV) é um dos patógenos mais importantes do trato respiratório. Analisamos as interações das proteínas virais nucleoproteína (N), fosfoproteína (P) e matriz (M) em células HEK-293T. N interage com as proteínas celulares Hsp70, PRMT5 e WDR77; P com Hsp70 e Tropomiosina; e M com Nucleofosmina e Tropomiosina. Cada gene celular foi co-expresso em bactérias com um gene viral possibilitando a co-precipitação das proteínas. Analisamos a interação entre Hsp70 e N ou P, confirmando sua ocorrência em bactérias. Com um conjunto de proteínas mutantes, definimos que as interações são através dos amino terminais de N e P, ou do carboxi terminal de P, e do domínio amino terminal de Hsp70. Superexpressão de Hsp70 por transfecção provocou efeito de estímulo sobre a replicação de HRSV. Imunizações em camundongos com vacinas de DNA para N e P mostraram a indução de resposta celular e humoral. Ensaios de desafio resultaram em redução da carga viral após imunização com N, indicando potencial para sua aplicação em formulação vacinal. / Human respiratory syncytial virus (HRSV) is one of the most important pathogens of the respiratory tract. We analyzed the interactions of viral nucleoprotein (N), phosphoprotein (P) and matrix (M) in HEK-293T. N interacts with the cellular proteins Hsp70, PRMT5 and WDR77; P interacts with Hsp70 and Tropomyosin; and M with Nucleophosmin and Tropomyosin. Each cellular gene was co-expressed with a viral gene in bacteria allowing co-precipitation of proteins. Hsp70 co-expression with N or P proteins confirmed that these interactions also occur in bacteria. Using a set of mutants we found that the N and P amino terminus, P carboxy terminus, and Hsp70 amino terminal domain participate in the interactions. The overexpression of Hsp70 by transfection resulted in stimulation of HRSV replication. Mice immunization with N and P showed that DNA vaccines were capable of inducing humoral and cellular response. In challenge assays it was possible to detect significant virus titer reduction in animals immunized with N, indicating its potential for a vaccine formulation.

Dez cavalos adultos clinicamente saudáveis foram inoculados por via intranasal com a estirpe A4/72 do herpesvírus eqüino tipo 1 (HVE-1). Com o intuito de estudar o efeito da dose infectante na severidade da rinopneumonite, os animais foram distribuídos em dois grupos experimentais: (a) grupo I (106,6 DICT50) e (b) grupo II (5×106,6 DICT50). Nos primeiros dez dias após a inoculação viral, todos os cavalos apresentaram manifestações de infecção respiratória leve e restrita às vias aéreas anteriores. Poucos animais desenvolveram leucopenia sangüínea envolvendo linfócitos (n=4) e neutrófilos (n=2). Somente em um houve aumento na contagem dos neutrófilos no lavado broncoalveolar (LBA). Apesar de possuírem elevados títulos de anticorpos neutralizantes antes da inoculação, alguns cavalos apresentaram soroconversão após o desafio viral. Esse padrão de resposta humoral foi determinado pelo aumento da dose infectante. O HVE-1 não foi isolado a partir das secreções nasais de nenhum animal. Entretanto, o DNA viral foi detectado pela reação em cadeia pela polimerase (PCR) nas células mononucleares sangüíneas entre o terceiro e o oitavo dias pós-inoculação (d.p.i.) em todos os animais, indicando a ocorrência de viremia. Além disso, a prova de PCR detectou o vírus nas amostras de LBA a partir do nono d.p.i. no grupo II, demonstrando que a disseminação do HVE-1 pelo trato respiratório após o desafio viral foi dose-dependente. Com base nos resultados obtidos, foi possível concluir que a PCR é uma técnica com alta sensibilidade para o diagnóstico do HVE-1, capaz de detectar a presença do DNA viral mesmo quando não ocorre a constatação do agente pelos métodos tradicionais / Ten clinically healthy adult horses were inoculated intranasally with the A4/72 strain of equine herpesvirus 1 (EHV-1). The animals were divided into two experimental groups in order to study the influence of the infective dose in the severity of rhinopneumonitis: (a) group I (106,6 TCID50) and (b) group II (5×106,6 DICT50). In the first ten days after the inoculation, they showed signs of a mild, self-limiting upper respiratory tract infection. Very few animals developed transient blood leukopenia, involving lymphocytes (n=4) as well as neutrophils (n=2). Only one horse had an increase in the neutrophils count of the bronchoalveolar lavage (BAL) samples. In spite of the presence of neutralizing antibodies before the trial, seroconversion was observed in some horses. The pattern of antibody response was determined by the increase of the challenge exposure. The virus was not isolated from nasal swabs of any horse. However, the EHV-1 was detected through the polymerase chain reaction (PCR) from peripheral blood mononuclear cells (PBMC) of all horses in the experiment within the third to the eighth day after the inoculation that illustrated the viremia. In addition, the PCR assay also detected the virus in BAL samples starting on the ninth day after the experimental infection of group II. For that reason, the dissemination of the EHV-1 throughout the respiratory tract after virus exposure was dose-dependent. Based on the results obtained from this study, it can be affirmed that the PCR is a highly effective technique in detecting the EHV-1. It may be used in circumstances where traditional methods are not efficient due to the fact that it provides an enhanced diagnostic procedure for underdiagnosed diseases

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2016-03-15T12:49:24Z No. of bitstreams: 2 Dissertação - Danilo Rezende e Silva - 2014.pdf: 795804 bytes, checksum: 70571f234568a148006646c85383db05 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-16T11:26:10Z (GMT) No. of bitstreams: 2 Dissertação - Danilo Rezende e Silva - 2014.pdf: 795804 bytes, checksum: 70571f234568a148006646c85383db05 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-16T11:26:10Z (GMT). No. of bitstreams: 2 Dissertação - Danilo Rezende e Silva - 2014.pdf: 795804 bytes, checksum: 70571f234568a148006646c85383db05 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2014-09-04 / Identification of herpesvirus in specimes fixed in formalin and paraffin embedded has been used in experimental analysis. Several molecular techniques have been used in research and in diagnosing of herpesviral meningoencephalitis. 80 cases of routine in veterinary pathology were reviewed on the Department of Animal Pathology of Veterinary and Science Animal School Medicine of Federal University of Goias, Goiania, GO (EVZ/UFG) performed between January 2007 to December 2012. Were used 18 cases with a total of 44 paraffin blocks (Group I - 14 blocks, Group II - 17 blocks and Group III - 13 blocks), which were grouped according to histopathological diagnosis, in Group I - samples without histological changes and no clinical history of neurological disease, Group II - samples with nonspecific encephalitis, Group III - samples with rabies encephalitis. Were extracted DNA fragments, from cuts blocks, using a commercial extraction kit of formalin and paraffin embedded tissue. Afterwards the technique standardization of conventional PCR for bovine herpesvirus-1 and bovine herpersvírus type-5 was performed. Were positive for BoHV-5 28% (5/18) of cases, with 11% (2/18) of the group with nonspecific encephalitis and 17% (3/18) of the group with encephalitis and positive for the rabies virus. In the test for checking of the bands for the BoHV-1, all samples, 100% (18/18) were negative. These results demonstrate that the conventional PCR technique is helpful in aiding the definitive diagnosis of encephalitis bovine herpesvirus type-5. / A extração do DNA de herpesvírus em amostras fixadas em formol, assim como amostras emblocadas em parafina, tem sido utilizada em análises experimentais, permitindo o emprego de técnicas moleculares em pesquisas e no diagnóstico das meningonencefalites por herpesvírus. Foram revisados 80 casos da rotina anatomopatológica do Setor de Patologia Animal da Escola de Veterinária e Zootecnia da Universidade Federal de Goiás, Goiânia, GO (EVZ/UFG) realizados entre janeiro de 2007 à dezembro de 2012. Foram utilizados 18 casos com um total de 44 blocos (Grupo I – 14 blocos, Grupo II – 17 blocos e Grupo III – 13 blocos), os quais foram agrupados, de acordo com diagnóstico histopatológico, em Grupo I – amostras sem alterações histológicas e sem histórico clínico de doença neurológica, Grupo II – amostras com encefalite inespecífica, Grupo III – amostras com encefalite por Raiva. DNA dos fragmentos foram obtidos a partir dos cortes dos blocos, foi extraído com kit comercial de extração de material parafinizado. Posteriormente foi realizada a padronização da técnica de PCR convencional para herpesvírus bovino tipo-1 e herpersvírus bovino tipo-5. Foram positivos para BoHV-5 28% (5/18) dos casos, sendo 11% (2/18) do grupo com encefalite inespecífica e 17% (3/18) do grupo com encefalite e positivos para o vírus da raiva. No teste para verificação das bandas para o BoHV-1, todas as amostras, 100% (18/18), foram negativas. Esses resultados comprovam que a técnica de PCR convencional é útil no auxílio do diagnóstico definitivo de encefalite por herpesvírus bovino tipo-5.

Dez cavalos adultos clinicamente saudáveis foram inoculados por via intranasal com a estirpe A4/72 do herpesvírus eqüino tipo 1 (HVE-1). Com o intuito de estudar o efeito da dose infectante na severidade da rinopneumonite, os animais foram distribuídos em dois grupos experimentais: (a) grupo I (106,6 DICT50) e (b) grupo II (5×106,6 DICT50). Nos primeiros dez dias após a inoculação viral, todos os cavalos apresentaram manifestações de infecção respiratória leve e restrita às vias aéreas anteriores. Poucos animais desenvolveram leucopenia sangüínea envolvendo linfócitos (n=4) e neutrófilos (n=2). Somente em um houve aumento na contagem dos neutrófilos no lavado broncoalveolar (LBA). Apesar de possuírem elevados títulos de anticorpos neutralizantes antes da inoculação, alguns cavalos apresentaram soroconversão após o desafio viral. Esse padrão de resposta humoral foi determinado pelo aumento da dose infectante. O HVE-1 não foi isolado a partir das secreções nasais de nenhum animal. Entretanto, o DNA viral foi detectado pela reação em cadeia pela polimerase (PCR) nas células mononucleares sangüíneas entre o terceiro e o oitavo dias pós-inoculação (d.p.i.) em todos os animais, indicando a ocorrência de viremia. Além disso, a prova de PCR detectou o vírus nas amostras de LBA a partir do nono d.p.i. no grupo II, demonstrando que a disseminação do HVE-1 pelo trato respiratório após o desafio viral foi dose-dependente. Com base nos resultados obtidos, foi possível concluir que a PCR é uma técnica com alta sensibilidade para o diagnóstico do HVE-1, capaz de detectar a presença do DNA viral mesmo quando não ocorre a constatação do agente pelos métodos tradicionais / Ten clinically healthy adult horses were inoculated intranasally with the A4/72 strain of equine herpesvirus 1 (EHV-1). The animals were divided into two experimental groups in order to study the influence of the infective dose in the severity of rhinopneumonitis: (a) group I (106,6 TCID50) and (b) group II (5×106,6 DICT50). In the first ten days after the inoculation, they showed signs of a mild, self-limiting upper respiratory tract infection. Very few animals developed transient blood leukopenia, involving lymphocytes (n=4) as well as neutrophils (n=2). Only one horse had an increase in the neutrophils count of the bronchoalveolar lavage (BAL) samples. In spite of the presence of neutralizing antibodies before the trial, seroconversion was observed in some horses. The pattern of antibody response was determined by the increase of the challenge exposure. The virus was not isolated from nasal swabs of any horse. However, the EHV-1 was detected through the polymerase chain reaction (PCR) from peripheral blood mononuclear cells (PBMC) of all horses in the experiment within the third to the eighth day after the inoculation that illustrated the viremia. In addition, the PCR assay also detected the virus in BAL samples starting on the ninth day after the experimental infection of group II. For that reason, the dissemination of the EHV-1 throughout the respiratory tract after virus exposure was dose-dependent. Based on the results obtained from this study, it can be affirmed that the PCR is a highly effective technique in detecting the EHV-1. It may be used in circumstances where traditional methods are not efficient due to the fact that it provides an enhanced diagnostic procedure for underdiagnosed diseases

No presente trabalho, desenvolvemos uma estratégia vacinal baseada em vacinas de DNA que codificam proteínas do HPV-16, o tipo viral de maior relevância epidemiológica, fusionadas à glicoproteína D (gD) do vírus herpes simplex tipo 1 (HSV-1). A vacina que contêm o gene da E7 de HPV-16 fusionada à gD de HSV-1 (pgDE7), administrada em regime vacinal de quatro doses, foi capaz de gerar significativa ativação de células T CD8+ E7-específicas e apresentar 40% de efeito protetor anti-tumoral terapêutico em camundongos desafiados com células transformadas que expressam as proteínas E6 e E7 do HPV-16 (células TC-1). A partir das evidências geradas, desenvolvemos um novo vetor vacinal que codifica as proteínas E7, E6 e E5 do HPV-16 (pgD-E7E6E5). Em ensaios em modelo murino, apenas uma dose da vacina foi capaz de gerar ativação de células T CD8+ específicas e 70% dos camundongos previamente desafiados com células TC-1 e inoculados com 3 doses da vacina mantiveram-se livres de tumores. Como tentativa de potencializar o efeito protetor terapêutico encontrado, adotamos duas medidas: a co-administração de plasmídeos que codificam citocinas e a otimização de códons da sequência gênica que codifica a proteína quimérica. A combinação das vacinas pgDE7 ou pgD-E7E6E5 com plasmídeos que carregam os genes das citocinas IL-2, IL-12 ou GM-CSF foi capaz de aumentar a proteção terapêutica para 100% em regime vacinal de dose única. A adequação da sequência antigênica ao sistema de expressão humano, aumentou em cerca de 5 vezes o potencial terapêutico do vetor vacinal pgDE7. Em conjunto, os dados apresentados nesta tese demonstram a evolução do desenvolvimento de uma estratégia vacinal contra tumores induzidos por HPV-16 e encorajam seu potencial para uso em futuros ensaios clínicos. / The development of immunotherapeutic strategies against human papillomavirus (HPV) is a priority for the control of HPV-induced lesions and cervical cancer. In this study, we developed DNA vaccines encoding HPV-16 proteins fused to glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) as an approach to control HPV-16 induced tumors. The vaccine encoding HPV-16 E7 fused to HSV-1 gD (pgDE7), when administered in a four doses vaccine regimen, was able to generate significant activation of E7-specific CD8+ T cells and showed 40% of therapeutic anti-tumor effect in mice previously challenged with tumor cells expressing HPV-16 E6 and E7 proteins (TC-1 cells). Following these evidences, we developed a new vaccine vector encoding HPV-16 E7, E6, E5 proteins fused to HSV-1 gD (pgD-E7E6E5). Only one vaccine dose generated antigen-specific CD8+ T cell responses and three doses conferred 70% protection to mice previously challenged with TC-1 cells. As an attempt to enhance the observed therapeutic anti-tumor effects, we tested two approaches: co-administration of cytokine-expressing plasmids and codon optimization of the gene encoding the chimeric protein. The combination of the vaccines pgDE7 or pgD-E7E6E5 with plasmids encoding the cytokines IL-2, IL-12 or GM-CSF increased the therapeutic protection to 100% of the vaccinated animals following a single dose. The gene sequence adaptation increased by a factor of 5 the therapeutic potential of the pgDE7 vaccine. In summary, the data presented in this thesis demonstrated the development of a vaccine strategy against HPV-16 induced tumors and reinforces its potential use in future clinical trials.

O Vírus Respiratório Sincicial Humano (HRSV) é um dos patógenos mais importantes do trato respiratório. Analisamos as interações das proteínas virais nucleoproteína (N), fosfoproteína (P) e matriz (M) em células HEK-293T. N interage com as proteínas celulares Hsp70, PRMT5 e WDR77; P com Hsp70 e Tropomiosina; e M com Nucleofosmina e Tropomiosina. Cada gene celular foi co-expresso em bactérias com um gene viral possibilitando a co-precipitação das proteínas. Analisamos a interação entre Hsp70 e N ou P, confirmando sua ocorrência em bactérias. Com um conjunto de proteínas mutantes, definimos que as interações são através dos amino terminais de N e P, ou do carboxi terminal de P, e do domínio amino terminal de Hsp70. Superexpressão de Hsp70 por transfecção provocou efeito de estímulo sobre a replicação de HRSV. Imunizações em camundongos com vacinas de DNA para N e P mostraram a indução de resposta celular e humoral. Ensaios de desafio resultaram em redução da carga viral após imunização com N, indicando potencial para sua aplicação em formulação vacinal. / Human respiratory syncytial virus (HRSV) is one of the most important pathogens of the respiratory tract. We analyzed the interactions of viral nucleoprotein (N), phosphoprotein (P) and matrix (M) in HEK-293T. N interacts with the cellular proteins Hsp70, PRMT5 and WDR77; P interacts with Hsp70 and Tropomyosin; and M with Nucleophosmin and Tropomyosin. Each cellular gene was co-expressed with a viral gene in bacteria allowing co-precipitation of proteins. Hsp70 co-expression with N or P proteins confirmed that these interactions also occur in bacteria. Using a set of mutants we found that the N and P amino terminus, P carboxy terminus, and Hsp70 amino terminal domain participate in the interactions. The overexpression of Hsp70 by transfection resulted in stimulation of HRSV replication. Mice immunization with N and P showed that DNA vaccines were capable of inducing humoral and cellular response. In challenge assays it was possible to detect significant virus titer reduction in animals immunized with N, indicating its potential for a vaccine formulation.

Le virus d’Epstein-Barr (EBV) est un Herpesvirus humain appartenant à la sous-famille des γ-Herpesvirinae. L’expression des gènes d’EBV est régulée très finement, de manière séquentielle et débute par l’expression des gènes immédiats précoces dont les produits contrôlent l’expression des gènes précoces. Les produits de ces derniers permettent la réplication de l’ADN viral et la synthèse des gènes tardifs. Les mécanismes contrôlant l’expression des gènes immédiats précoces et précoces sont assez bien compris, à contrario de ceux qui contrôlent l’expression des gènes tardifs. En effet, il est connu que ces derniers ne sont exprimés qu’après réplication de l’ADN viral. Par ailleurs, les promoteurs des gènes tardifs présentent une structure différente des promoteurs de gènes immédiats précoces, précoces ou des gènes cellulaires. Ils sont essentiellement caractérisés par la présence d’une boîte TATT à la place de la TATA-box canonique. EBV a la particularité de coder pour une TBP-like, la protéine BcRF1 qui est essentielle mais pas suffisante pour l’expression des gènes viraux tardifs et qui se fixe spécifiquement sur la séquence TATT des promoteurs viraux tardifs. Notre objectif était principalement d’identifier l’ensemble des protéines virales nécessaires à l’expression des gènes tardifs d’EBV. Nous avons ainsi démontré qu’en plus de la protéine BcRF1, EBV code pour cinq autres protéines nécessaires à l’expression des gènes viraux tardifs : BFRF2, BGLF3, BVLF1, BDLF4 et BDLF3.5. Ces six protéines virales forment un complexe qui permet le recrutement de l’ARN polymérase II sur les promoteurs des gènes viraux tardifs. Nous avons nommé ce complexe vPIC (viral Pre-Initiation Complex), par analogie avec le complexe d’initiation de la transcription cellulaire formé autour de la TATA-Binding Protein (TBP). Le vPIC est conservé chez les herpesvirus des sous familles β et γ, mais est absent chez les herpesvirus de la sous famille des α. Par ailleurs, nos résultats suggèrent que le complexe vPIC interagit avec le complexe de réplication viral ce qui peut expliquer la liaison entre réplication de l’ADN viral et transcription des gènes viraux tardifs. Ces résultats permettent ainsi de mieux comprendre les mécanismes de régulation de l’expression des gènes tardifs d’EBV et serviront de base pour la recherche de nouveaux antiviraux. / The Epstein-Barr virus (EBV) is a human Herpersvirus belonging to the subfamily of γ-Herpesvirinae. EBV genes expression is tightly regulated, in a sequential manner and begins with the expression of immediate early genes whose products control the expression of early genes. Products of these allow the viral DNA synthesis and late genes expression. Mechanisms controlling immediate early and early genes expression are well documented, in contrast to those controlling late genes expression. Indeed, it is known that late genes are expressed after viral DNA replication. Furthermore, late genes promoters have a different structure of those of immediate early, early and cellular genes. They are essentially characterized by the presence of a TATT box instead of the canonical TATA-box. EBV has the feature to encode for a TBP-like, BcRF1 protein that is essential but not sufficient for late viral genes expression and that binds specifically to the TATT sequence of the late viral promoters. Our goal was primarily to identify the set of viral proteins necessary for late EBV genes expression. Thus, we have demonstrated that in addition to the BcRF1 protein, EBV encode for five other proteins necessary for late viral genes expression: BFRF2, BGLF3, BVLF1, BDLF4 and BDLF3.5. These six viral proteins form a complex enables to recruit the RNA polymerase II on the late viral promoters. We have named this complex vPIC (viral Pre-Initiation Complex) by analogy with the cellular complex of transcription initiation, formed around the TATA-Binding Protein (TBP). The vPIC is conserved in β- and γ-Herpesvirus subfamilies, but not in the α-Herpesvirus subfamily. Furthermore, our results suggest that vPIC interacts with the viral DNA replication complex, which can explain the link between viral DNA replication and late viral genes transcription. These results bring new insights to the mechanisms regulating late EBV genes expression and provide a basis for the search of new antiviral drugs.

In order to achieve efficient therapeutic effect, it is important to understand the structure of biomaterials that are used in the therapeutic delivery system. This dissertation is dedicated towards understanding the hydrolysis pattern of plasmid DNA (pDNA) delivery vehicles comprised of poly(glycoamidoamine)s (PGAAs) under physiological conditions and effects of subtle changes in the chemical structure of the PGAAs on its biological performance. The unusual hydrolysis of the tartarate and galactarate based PGAAs was investigated by studying the hydrolysis of small model molecules which mimic the repeat unit of the respective polymers. In the case of galactarate and tartarate based molecules with terminal amines showed faster hydrolysis of the amide bonds. In addition for the tartarate based compounds, it was also found that it is necessary to have terminal amine functionality for the intramolecular hydrolysis to occur. The model compounds consists of two amide bonds and were designed symmetric, however amide bond on only one side of the tartarate moiety show underwent hydrolysis. Further studies show that one side of the amine assists the hydrolysis of the amide bond on the other side of the tartarate moiety. The degradation of poly(L-tartaramidopentaethylenetetramine) (<strong>T4</strong>) was also used to study the sustained release of pDNA from the layer-by-layer constructs of <strong>T4</strong>/pDNA. The thickness of the constructs was characterized by ellipsometry while the UV-visible spectroscopy was used to characterize the loading capacity of the constructs for pDNA. The indirect sustained release of pDNA under the physiological conditions with respect to time was characterized by the cellular uptake studies in HeLa cells. The increase in the uptake of the Cy5 labeled pDNA was seen at extended period of eleven days. The integrity of the sustained released pDNA for the transgene expression was characterized with an assay to see the expression of the green fluorescent protein (GFP) from the <strong>T4</strong>/GFP-pDNA layer-by-layer constructs. PGAAs show a very efficient delivery of the pDNA in a non-toxic manner. The chemical structure of the polymer can dictate the binding with pDNA and also the release of the pDNA form the polymer-pDNA complexes. In order to better understand the fundamentals of the nucleic acid delivery and to better design the nucleic acid delivery vehicles, subtle changes in the chemical structure of the PGAAs were designed and studied for the biological activity. The effect of charge type was investigated by designing and synthesizing guanidine based polymer series analogues to galactarate and tartarate based PGAAs (<strong>G1</strong> and <strong>T1</strong>) which incorporate secondary amines as the charge type on the polymer backbone. The guanidine based polymer series, poly(glycoamidoguanidine)s (PGAGs), show very non toxic behavior in HeLa cells at all the different polymer to pDNA ratio (<i>N/P</i> ratio) studied. Interestingly PGAGs are the only non-toxic guanidine containing polymers which are reported in the literature to the date. The cellular uptake of pDNA assisted from the PGAGs is a little higher than PGAAs compared although both the series of polymers show similar transgene expression. The transgene expression in case of PGAGs also imply the release of the polymer-pDNA complexes from the endosome. In another study of structure-bioactivity relationship based on the degree of polymerization (DP) of poly(galactaramidopentaethylenetetramine) (<strong>G4</strong>), it was found that the increase in the DP of <strong>G4</strong> increases the toxicity of the polymers in the HeLa cells. / Ph. D.