Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The general problem of alteration in viral susceptibility by the irradiation of monolayers of tissue cells in culture was examined in this study; specifically an increased susceptibility of HeLa (an established cell line which was derived from an epidermaid carcinoma of the cervix) to destruction by herpes simplex virus (the virus commonly associated with cold sores or fever blisters). The experimental procedures included the study of the radiosensitivity of the cell line, survival curve analyses expressed as the efficiency of plating, that is the per cent of viable cells capable of forming colonies visible to the unaided eye within twelve days, and finally infectivity studies. Tissue culture has proved to be a very useful tool in the study of radiation effects on tissues of higher animals since the effects of radiation can apparently be explained on the cellular level. Many types of cells have been studied but in all cases the most striking characteristic in irradiated populations is the increased cell size. Ionizing radiation effects both the reproductive and synthesizing capacity of cells with the former being the more sensitive. Some irradiated cells never divide while others divide several times before reproduction stops. After the cells stop dividing, they continue to grow in size forming giants because synthesis of the cellular constituents continues. Giant cells resulting from x-irradiation are more readily destroyed by the action of viruses than are non-irradiated cells. PUCK & MARCUS reported that NDV when plated on a mixture of giant and normal cells, destroyed more of the giants than normal cells. An enhancement of cell susceptibility following irradiation was also demonstrated for two enteroviruses by HSIUNG. The increased susceptibility of x-ray-induced giant cells to CPE of virus and the earlier release of virus by such cells was also demonstrated by LEVINE in studies with the Leon strain of type 3 polio. Many tissue culture-virus systems have been used to demonstrate alterations in susceptibility induced by x-irradiation, but few investigators have used a HeLa-HSV system to study this altered susceptibility using low levels of irradiation (50 roentgens to 500 roentgens) [TRUNCATED] / 2999-01-01

Herpes simplex virus 1 (HSV-1) is a human pathogen that modulates many aspects of RNA metabolism and chromatin structure. We have previously reported that HSV-1 infection leads to disruption of transcription termination (DoTT) by cellular RNA Polymerase II (Pol II) of many cellular genes. By employing various mutant viruses, we observed that the HSV-1 immediate early protein ICP27 is a major contributor to DoTT. This termination defect contributes to host cell shut-off as affected read-through transcripts are retained in the nucleus and thus cannot be translated. HSV-1-induced DoTT also leads to a massive increase in transposon-accessible chromatin in downstream gene regions mediated by the viral immediate-early protein ICP22. The interplay of ICP22 with cellular and viral proteins contributes to various processes in host nuclear systems ranging from alterations of Pol II to chaperone relocalization. To better understand the role of ICP22 in open chromatin formation, a deep understanding of its interaction partners and the involved protein domains is required. Here, we report on a detailed list of ICP22 interaction partners and potential sites of interaction by Co-IP mass spectrometry (MS) analysis of both full-length as well as partial ICP22 deletion mutants. We found that many of the identified ICP22 interactors are functionally linked to the phosphorylation of the Pol II C-terminal domain, transcription regulation, and histone modifications. Additionally, we created a set of ICP22 mutants comprising small deletions of highly conserved regions or point mutations in the core region. Analysis of these mutants using ATAC-seq suggests that the ICP22's conserved core region is crucial for the induction of dOCR. Although some mutants were significantly attenuated compared to WT, a correlation between fitness and induction of open chromatin downstream of genes was not observed. Additional work is required to completely understand the molecular mechanism and biological significance of this process, as well as all the cellular factors involved. This research makes important advancements in our understanding of the ICP22 protein, its interactions with cellular factors, the importance of its domains and associated changes, as well as the consequences of those changes. Additionally, the datasets generated in this study will be important to support subsequent research on ICP22 and its role in HSV-1 infection. / Das Herpes-simplex-Virus 1 (HSV-1) ist ein menschlicher Krankheitserreger, der viele Aspekte des RNA-Stoffwechsels und der Chromatinstruktur moduliert. Wir haben in früheren Arbeiten beschrieben, dass eine HSV-1-Infektion zu einer Unterbrechung der Transkriptionstermination (DoTT) durch die zelluläre RNA-Polymerase II (Pol II) bei vielen zellulären Genen führt. Durch den Einsatz verschiedener mutierter Viren konnten wir zeigen, dass das HSV-1-"immediate early protein" ICP27 einen wesentlichen Beitrag zur DoTT leistet. Dieser Terminierungsdefekt trägt zur Abschaltung der Wirtszelle bei, da die betroffenen ReadThrough-Transkripte im Zellkern verbleiben und somit nicht in Proteine übersetzt werden können. HSV-1-induzierte DoTT führt zudem zur Bildung von transposon-zugänglichem Chromatin in downstream gelegenen Genregionen, was durch das Immediate-Early-Protein ICP22 vermittelt wird. Das Zusammenspiel von ICP22 mit zellulären und viralen Proteinen trägt zu verschiedenen Prozessen in Wirtskernsystemen bei, die von Veränderungen von Pol II bis zur Relokalisierung von Chaperonen reichen. Um die Rolle von ICP22 bei der Bildung von offenem Chromatin zu verstehen, ist ein tiefes Verständnis seiner Interaktionspartner und der beteiligten Proteindomänen erforderlich. Daher haben wir mittels Co-IP-Massenspektrometrie (MS)-Analyse eine detaillierte Liste von ICP22-Interaktionspartnern und potenziellen Interaktionsstellen sowohl von Volllängen- als auch von partiellen ICP22-Deletionsmutanten erstellt. Im Folgenden konnten wir zeigen, dass viele der identifizierten ICP22-Interaktoren funktionell mit der Phosphorylierung der C-terminalen Domäne von Pol II, der Transkriptionsregulation und istonmodifikationen verbunden sind. Darüber hinaus haben wir eine Reihe von ICP22 Mutanten mit Deletionen in hochkonservierten Regionen oder Punktmutationen in der Kernregion erstellt. Die Analyse dieser Mutanten mittels ATAC-seq legt nahe, dass die konservierte Kernregion von ICP22 für die Induktion von „downstream open chromatin“ (dOCR) entscheidend ist. Obwohl einige Mutanten im Vergleich zu WT signifikant geschwächt waren, wurde keine Korrelation zwischen der Fitness und der Induktion von offenem Chromatin in den downstream Genen beobachtet. Weitere Arbeiten sind erforderlich, um den molekularen Mechanismus und die biologische Bedeutung dieses Prozesses sowie alle beteiligten zellulären Faktoren umfassend zu verstehen. Diese Forschung bringt wichtige Fortschritte in unserem Verständnis des ICP22-Proteins, seiner Interaktionen mit zellulären Faktoren, der Bedeutung seiner Domänen und ihrer Veränderungen sowie der Folgen dieser Veränderungen für die Infektion der Zelle. Darüber hinaus werden die in dieser Studie erzeugten Datensätze für die weitere Forschung zu ICP22 und seiner Rolle bei der HSV-1 Infektion von Bedeutung sein.

In CBA/NJ mice, splenic natural killer (NK) cell activity varies with stages of estrous. Susceptibility of ICR mice to intravaginal inoculation of herpes simplex virus type 2 (HSV-2) decreases with age. Susceptibility of female ICR and CBA/NJ mice to HSV-2 inoculated intravaginally and intraperitoneally was examined during the estrous cycle. In cycling ICR mice, greatest susceptibility to intravaginal inoculation was observed during diestrous and the least during metestrous. CBA/NJ mice were most susceptible to intravaginal inoculation of HSV-2 during diestrous. ICR mice were ovariectomized to mimic diestrous and found to be highly susceptible to intravaginal inoculation at all virus doses. No difference in susceptibility among phases of the estrous cycle was seen following intraperitoneal inoculation.

Thesis (Ph. D.)--University of Sydney, 2005. / Title from title screen (viewed 20 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Faculty of Medicine. Degree awarded 2005; thesis originally submitted 2004, corrected version submitted April 2005. Includes bibliographical references. Also available in print form.

Ocorrências comuns de infecções virais graves, aparecimento de cepas resistentes e um limitado número de quimioterápicos antivirais disponíveis mostram a necessidade da busca por novas substâncias ativas como antivirais. Muitas substâncias derivadas de plantas são candidatas ao estudo do seu potencial na terapia sistêmica e/ou profilaxia de herpes simplex virus 1 (HSV-1). Dentre as plantas com atividade antiviral, destacam-se as do gênero Baccharis. O objetivo geral do trabalho foi o estudo da composição química e avaliação da atividade antiviral das folhas de Baccharis anomala D.C. através de fracionamento bioguiado. Utilizando precipitação com etanol e fracionamento por permeação molecular foi possível separar os constituintes químicos ativos contra o vírus HSV-1 presentes no extrato aquoso de Baccharis anomala. Os testes de prospecção de constituintes químicos indicaram a presença de taninos, catequinas e saponinas no extrato aquoso da espécie. Através da análise cromatográfica foi possível detectar a presença de compostos fenólicos, utilizando-se coloração com cloreto férrico e reagente natural. Em relação à atividade antiviral, a fração ativa denominada AQ PPT FR 4-5 apresentou pronunciada atividade antiviral, inibindo a replicação viral em 100 % nas concentrações de 1,25, 0,625, 0,312 e 0,156 mg/mL, contra as cepas ATCC-VR733 e Aciclovir-resistente 29-R do HSV-1. Em relação ao mecanismo de ação, observou-se atividade virucida da fração AQ PPT FR 4-5. Estes resultados são muito importantes, pois, de acordo com a literatura, ainda não foram relatados compostos com atividade virucida úteis clinicamente para o tratamento de infecções por HSV-1. Conclui-se, portanto, que Baccharis anomala possui potente atividade antiviral contra o vírus HSV-1 e é promissora para estudos posteriores que visem ao isolamento, identificação e estudo do mecanismo de ação antiviral de compostos ativos da espécie, considerando a emergência de cepas resistentes e a necessidade de compostos com novos mecanismos de ação. / Common occurrences of severe viral infections, emergence of resistant strains and a limited number of available antiviral chemotherapeutics show the need to search for new active substances as antiviral. Many compounds derived from plants are candidates for the study of their potential in systemic therapy and/or prophylaxis of herpes simplex virus type 1 (HSV-1). Among the plants with antiviral activity, those from the genus Baccharis are remarkable. The main objective of the work was the study of the chemical composition and evaluation of the antiviral activity of extracts from Baccharis anomala D.C., using bioactivity guided fractionation. Through precipitation with ethanol and fractionation by molecular permeation it was achieved the separation of the active chemical constituents against HSV-1 virus in the aqueous extract of Baccharis anomala. Phytochemical tests indicated the presence of tannins, catechins and saponins in the aqueous extract. By thin layer chromatography it was detected the presence of phenolic compounds using ferric chloride and natural reagent. Concerning the antiviral activity, the active fraction named AQ PPT FR 4-5 showed pronounced antiviral activity, represented by 100 % inhibition of viral replication at concentrations of 1.25, 0.625, 0.312 and 0.156 mg/mL, against the strains ATCC-VR733 and Acyclovir-resistant 29-R of HSV-1 virus. Regarding the mechanism of action, virucidal activity on the fraction AQ PPT FR 4-5 was detected, which is also very important because, as far as we know, compounds with virucidal activity for clinical use in the treatment of HSV-1 infections were not reported yet. In conclusion, Baccharis anomala displayed pronounced antiviral activity against HSV-1 virus and it is promising for further studies aimed to the isolation, identification and mechanism of action of antiviral active compounds of the species, considering the emergence of resistant strains and the need for compounds with new mechanisms of action.

Ocorrências comuns de infecções virais graves, aparecimento de cepas resistentes e um limitado número de quimioterápicos antivirais disponíveis mostram a necessidade da busca por novas substâncias ativas como antivirais. Muitas substâncias derivadas de plantas são candidatas ao estudo do seu potencial na terapia sistêmica e/ou profilaxia de herpes simplex virus 1 (HSV-1). Dentre as plantas com atividade antiviral, destacam-se as do gênero Baccharis. O objetivo geral do trabalho foi o estudo da composição química e avaliação da atividade antiviral das folhas de Baccharis anomala D.C. através de fracionamento bioguiado. Utilizando precipitação com etanol e fracionamento por permeação molecular foi possível separar os constituintes químicos ativos contra o vírus HSV-1 presentes no extrato aquoso de Baccharis anomala. Os testes de prospecção de constituintes químicos indicaram a presença de taninos, catequinas e saponinas no extrato aquoso da espécie. Através da análise cromatográfica foi possível detectar a presença de compostos fenólicos, utilizando-se coloração com cloreto férrico e reagente natural. Em relação à atividade antiviral, a fração ativa denominada AQ PPT FR 4-5 apresentou pronunciada atividade antiviral, inibindo a replicação viral em 100 % nas concentrações de 1,25, 0,625, 0,312 e 0,156 mg/mL, contra as cepas ATCC-VR733 e Aciclovir-resistente 29-R do HSV-1. Em relação ao mecanismo de ação, observou-se atividade virucida da fração AQ PPT FR 4-5. Estes resultados são muito importantes, pois, de acordo com a literatura, ainda não foram relatados compostos com atividade virucida úteis clinicamente para o tratamento de infecções por HSV-1. Conclui-se, portanto, que Baccharis anomala possui potente atividade antiviral contra o vírus HSV-1 e é promissora para estudos posteriores que visem ao isolamento, identificação e estudo do mecanismo de ação antiviral de compostos ativos da espécie, considerando a emergência de cepas resistentes e a necessidade de compostos com novos mecanismos de ação. / Common occurrences of severe viral infections, emergence of resistant strains and a limited number of available antiviral chemotherapeutics show the need to search for new active substances as antiviral. Many compounds derived from plants are candidates for the study of their potential in systemic therapy and/or prophylaxis of herpes simplex virus type 1 (HSV-1). Among the plants with antiviral activity, those from the genus Baccharis are remarkable. The main objective of the work was the study of the chemical composition and evaluation of the antiviral activity of extracts from Baccharis anomala D.C., using bioactivity guided fractionation. Through precipitation with ethanol and fractionation by molecular permeation it was achieved the separation of the active chemical constituents against HSV-1 virus in the aqueous extract of Baccharis anomala. Phytochemical tests indicated the presence of tannins, catechins and saponins in the aqueous extract. By thin layer chromatography it was detected the presence of phenolic compounds using ferric chloride and natural reagent. Concerning the antiviral activity, the active fraction named AQ PPT FR 4-5 showed pronounced antiviral activity, represented by 100 % inhibition of viral replication at concentrations of 1.25, 0.625, 0.312 and 0.156 mg/mL, against the strains ATCC-VR733 and Acyclovir-resistant 29-R of HSV-1 virus. Regarding the mechanism of action, virucidal activity on the fraction AQ PPT FR 4-5 was detected, which is also very important because, as far as we know, compounds with virucidal activity for clinical use in the treatment of HSV-1 infections were not reported yet. In conclusion, Baccharis anomala displayed pronounced antiviral activity against HSV-1 virus and it is promising for further studies aimed to the isolation, identification and mechanism of action of antiviral active compounds of the species, considering the emergence of resistant strains and the need for compounds with new mechanisms of action.

Ocorrências comuns de infecções virais graves, aparecimento de cepas resistentes e um limitado número de quimioterápicos antivirais disponíveis mostram a necessidade da busca por novas substâncias ativas como antivirais. Muitas substâncias derivadas de plantas são candidatas ao estudo do seu potencial na terapia sistêmica e/ou profilaxia de herpes simplex virus 1 (HSV-1). Dentre as plantas com atividade antiviral, destacam-se as do gênero Baccharis. O objetivo geral do trabalho foi o estudo da composição química e avaliação da atividade antiviral das folhas de Baccharis anomala D.C. através de fracionamento bioguiado. Utilizando precipitação com etanol e fracionamento por permeação molecular foi possível separar os constituintes químicos ativos contra o vírus HSV-1 presentes no extrato aquoso de Baccharis anomala. Os testes de prospecção de constituintes químicos indicaram a presença de taninos, catequinas e saponinas no extrato aquoso da espécie. Através da análise cromatográfica foi possível detectar a presença de compostos fenólicos, utilizando-se coloração com cloreto férrico e reagente natural. Em relação à atividade antiviral, a fração ativa denominada AQ PPT FR 4-5 apresentou pronunciada atividade antiviral, inibindo a replicação viral em 100 % nas concentrações de 1,25, 0,625, 0,312 e 0,156 mg/mL, contra as cepas ATCC-VR733 e Aciclovir-resistente 29-R do HSV-1. Em relação ao mecanismo de ação, observou-se atividade virucida da fração AQ PPT FR 4-5. Estes resultados são muito importantes, pois, de acordo com a literatura, ainda não foram relatados compostos com atividade virucida úteis clinicamente para o tratamento de infecções por HSV-1. Conclui-se, portanto, que Baccharis anomala possui potente atividade antiviral contra o vírus HSV-1 e é promissora para estudos posteriores que visem ao isolamento, identificação e estudo do mecanismo de ação antiviral de compostos ativos da espécie, considerando a emergência de cepas resistentes e a necessidade de compostos com novos mecanismos de ação. / Common occurrences of severe viral infections, emergence of resistant strains and a limited number of available antiviral chemotherapeutics show the need to search for new active substances as antiviral. Many compounds derived from plants are candidates for the study of their potential in systemic therapy and/or prophylaxis of herpes simplex virus type 1 (HSV-1). Among the plants with antiviral activity, those from the genus Baccharis are remarkable. The main objective of the work was the study of the chemical composition and evaluation of the antiviral activity of extracts from Baccharis anomala D.C., using bioactivity guided fractionation. Through precipitation with ethanol and fractionation by molecular permeation it was achieved the separation of the active chemical constituents against HSV-1 virus in the aqueous extract of Baccharis anomala. Phytochemical tests indicated the presence of tannins, catechins and saponins in the aqueous extract. By thin layer chromatography it was detected the presence of phenolic compounds using ferric chloride and natural reagent. Concerning the antiviral activity, the active fraction named AQ PPT FR 4-5 showed pronounced antiviral activity, represented by 100 % inhibition of viral replication at concentrations of 1.25, 0.625, 0.312 and 0.156 mg/mL, against the strains ATCC-VR733 and Acyclovir-resistant 29-R of HSV-1 virus. Regarding the mechanism of action, virucidal activity on the fraction AQ PPT FR 4-5 was detected, which is also very important because, as far as we know, compounds with virucidal activity for clinical use in the treatment of HSV-1 infections were not reported yet. In conclusion, Baccharis anomala displayed pronounced antiviral activity against HSV-1 virus and it is promising for further studies aimed to the isolation, identification and mechanism of action of antiviral active compounds of the species, considering the emergence of resistant strains and the need for compounds with new mechanisms of action.

Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2018-01-11T13:56:55Z No. of bitstreams: 1 FIORELLA ZIBETTI.pdf: 1848554 bytes, checksum: a4058071f9ee8b5dc5ab5c3abb07a729 (MD5) / Made available in DSpace on 2018-01-11T13:56:55Z (GMT). No. of bitstreams: 1 FIORELLA ZIBETTI.pdf: 1848554 bytes, checksum: a4058071f9ee8b5dc5ab5c3abb07a729 (MD5) / O óleo essencial de Rosmarinus officinalis (OERo) possui atividade anti- herpética in vitro. Entretanto, os constituintes químicos estão sujeitos à degradação por oxidação ou volatilização. Desta forma, surge a necessidade de desenvolvimento de sistemas carreadores para este ativo vegetal. O presente trabalho teve como objetivo desenvolver nanoemulsões contendo OERo, comparando dois métodos de baixo aporte energético, titulação à frio e inversão de fases, quanto à estabilidade, teor do marcador químico, perfil de liberação in vitro, permeação e retenção cutânea in vitro e atividade anti-herpética in vitro. O OERo, utilizado nesse estudo, apresentou como constituinte majoritário o eucaliptol (27,9%), descrito na literatura por inibir o vírus Herpes simplex. As nanoemulsões e seus respectivos hidrogéis foram desenvolvidos e conservados em temperatura ambiente por 60 dias. Todas as formulações mantiveram-se estáveis durante o período estudado, apresentando tamanho de gotícula inferior a 200nm. Nenhum dos parâmetros avaliados apresentou diferença significativa entre os métodos utilizados, com exceção do teor, que foi menor em T0 nas nanoemulsões produzidas com aquecimento. Aas formulações apresentaram cerca de 60% de liberação do óleo em 150 minutos em ensaio in vitro. No estudo de permeação/retenção cutânea in vitro foi possível detectar o eucaliptol, por Cromatografia Líquida de Alta Eficiência, no meio de lavagem (~32%), estrato córneo (~12%) e meio receptor (~56%), não sendo observado na epiderme e derme. Para avaliação da atividade anti-herpética, as nanoemulsões apresentaram índice de inibição viral superior a 98,2% (HSV-1) e 99,5% (HSV- 2), apresentando maior especificidade pelo sorotipo 2. As diferentes metodologias não impactaram significativamente na atividade anti-herpética. Dessa forma, conclui-se que ambos os métodos são capazes de preparar nanoemulsões com características semelhantes e estudos in vivo serão necessários para melhor entendimento da atividade do óleo / The essential oil of Rosmarinus officinalis (OERo) has anti-herpetic activity in vitro. However, the chemical constituents are subject to degradation by oxidation or volatilization. Thus, indicating a need to develop carriers systems for this plant active.The aim of the present work was to develop nanoemulsions containing OERo, comparing two low energy methods, cold titration and phase inversion, in terms of stability, chemical marker content, in vitro release profile, in vitro skin permeation and retention, and anti-herpetic activity in vitro. The OERo used in this study presented as main constituent eucalyptol (27.9%), which was described in the literature for inhibiting the Herpes simplex virus. Nanoemulsions and their respective hydrogels were developed by two different methodologies and stored at room temperature for 60 days. All formulations remained stable during the study period, with particle size smaller than 200nm. None of the parameters evaluated showed a significant difference between the methods used, except for the chemical marker content, which was lower in T0 for the nanoemulsions prepared with heating. The in vitro release profile of the formulations showed 60% oil release in 150 minutes. In the in vitro skin permeation / retention study, it was possible to detect eucalyptol, by High Performance Liquid Chromatography, in washing liquid (~32%), stratum corneum (~12%) and receiving liquid (~56%), but none was detected in epidermis and dermis. In order to evaluate the anti-herpetic activity, the nanoemulsions showed a viral inhibition index higher than 98.2% (HSV-1) and 99.5% (HSV-2), presenting a better specificity for serotype 2. Despite the loss of volatile constituents of the oil by heating, the different methodologies did not significantly impact the antiherpetic activity. Thus, it could be concluded that both low-energy methods are able to prepare nanoemulsions with similar characteristics and it is necessary further studies to better understand the oil activity

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