Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T17:48:59Z No. of bitstreams: 2 Dissertacao_LarissaDaianeWillrichDeMelo.pdf: 1512214 bytes, checksum: b264a462161b24ec3ce2aacf05afea14 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-16T17:48:59Z (GMT). No. of bitstreams: 2 Dissertacao_LarissaDaianeWillrichDeMelo.pdf: 1512214 bytes, checksum: b264a462161b24ec3ce2aacf05afea14 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2013-03-25 / Efforts to develop an effective vaccine against Herpes Simplex Virus-1 (HSV-1) has been intense enough, however, there is no effective vaccine yet. The using of nanocapsules (Ncs) for production of viral vaccines may contribute to the enhancement and effectiveness of the already developed vaccines models. The main properties of the nanostructured system, which may contribute to the improvement of the immune response generated by vaccine is the slow and gradual release of the encapsulated content, protection of the encapsulated molecule or protein and adjuvanticity, inherent in every Ncs. The objective of this study was to produce and characterize nanocapsules with aqueous core to encapsulate the SSIEFARL peptide from HSV-1. This peptide has hydrophilic characteristics which impossibility the encapsulation in conventional nanocapsules with oil core. For the standardization of the tunnel several adaptations where modified in the system previously developed by Lambert et al. Among these, the stirring of the suspension, temperature, amount of surfactant, amount of peptide and settling time were adapted. All protocols two aqueous suspensions were tested, one with the organic solvent ethanol in the aqueous phase or only water. The results showed that the production with magnetic stirring in the presence of ethanol in the aqueous phase lead to Ncs with better physicochemical characteristics. Empty Ncs presented an average of particle size of 285 nm (+ 19,4) and zeta potential of -40.1 mV (+ 1,7) and the Ncs containing SSIEFARL peptide presented the size of 315 nm (+ 20,7) and zeta potential of -35,5 mV (+ 2,4). Other condition of production tested was the water temperature added during the production. When water was used at 40 °C the physico-chemical parameters were better than when room temperature water was used. Additionally, we tested the stability of the suspensions after storage at room temperature or 4 °C. The Ncs were stable for longer periods of time under refrigeration. For the characterization of morphostructure, Ncs were analyzed by transmission electron microscopy (TEM). This analysis revealed that Ncs present as spherical structures, homogeneous and uniform as standards in nanostructure for biological application. The analysis of Ncs by confocal microscopy was applied to investigate the encapsulation. Three amounts of SSIEFARL-6 FAM peptide were used (20, 30 or 50 μL), and the 20 μL showed better encapsulation. The analysis of the release profile of the peptide from Ncs showed that of the suspension produced with 20 μL was more linear, demonstrating slow release for at least 24 h. These data indicate that the method of production under magnetic stirring with ethanol and 20 μL of peptide in the aqueous phase is more suitable for production of aqueous core Ncs. Furthermore, empty Ncs at low concentrations showed no cytotoxicity to lymphoid cells from BALB-c in cell culture. Thus, we suggest that this approach has promising potential for developing a vaccine against HSV-1. / Os esforços para o desenvolvimento de uma vacina eficaz contra o Herpes Simplex Vírus-1 (HSV-1) tem sido bastante intensos, entretanto, ainda não existe uma vacina eficaz. A utilização de nanocápsulas (Ncs) para a produção de vacinas anti-virais pode contribuir para o aprimoramento e eficácia dos modelos vacinas já desenvolvidos. As principais propriedades desse sistema nanoestruturado, que podem contribuir para a melhora da resposta imune gerada por uma vacina são liberação lenta e gradual do conteúdo encapsulado, a proteção da molécula ou proteína encapsulada e a adjuvanticidade, inerente a cada Ncs. Assim, o objetivo deste estudo foi produzir e caracterizar nanocápsulas de núcleo aquoso, capazes de encapsular o peptídeo SSIEFARL do HSV-1. Este peptídeo possui características hidrofílicas sendo impossível a encapsulação em nanocápsulas convencionais de núcleo oleoso. Para a padronização do encapsulamento, foram combinadas diversas adaptações ao sistema previamente desenvolvido por Lambert e colaboradores. Dentre essas, foram adaptados o modo de agitação das suspensões, temperatura, quantidade de tensoativo, quantidade de peptídeo e tempo de sedimentação. Em todos os métodos foram testados duas alterações na fase aquosa das suspensões, foram produzidas Ncs com solvente etanol, na fase aquosa e/ou sem. Como resultados foram observados que a produção com agitação magnética em presença de etanol na fase aquosa foi a que produziu NCs com melhores características físico-químicas. Foram obtidas Ncs brancas com tamanho de partícula médio de 285 nm (+ 19,4) e potencial zeta de -40,1 mV (+ 1,7) e Ncs contendo o peptídeo SSIEFARL com tamanho de 315 nm (+ 20,7) e potencial zeta de -35,5 mV (+2,4). Outra condição alterada foi a temperatura da água adicionada durante os processos de produção. Quando foi utilizada água na temperatura de 40 oC os parâmetros físico-químicos foram melhores do que quando foi utilizada água na temperatura ambiente (T.A). Além disso, foi testada a estabilidade das suspensões após armazenamento em temperatura ambiente e geladeira (4oC). As nanocápsulas se mantiveram estáveis por mais tempo em sob refrigeração. Para a caracterização morfoestrutural das Ncs produzidas, foi utilizada a microscopia eletrônica de transmissão (MET). Essa análise revelou que as Ncs apresentam-se como estruturas esféricas, homogêneas e uniformes, conforme padrões estabelecidos para esse tipo de nanoestrutura. A análise das Ncs por microscopia confocal foi utilizada para comprovação do encapsulamento do peptídeo SSIEFARL-6-Fam, assim como a melhor quantidade. Foram utilizadas três quantidades de peptídeo (20, 30 ou 50 L), sendo que a quantidade de 20 L foi a que apresentou melhores resultados. O gráfico do perfil de liberação da suspensão produzida com 20 μL mostrou-se mais linear, demonstrando liberação lenta por pelo menos 24 h. Estes dados apontam que a metodologia de produção sob agitação magnética com etanol e 20 μL de peptídeo na fase aquosa apresentaram os melhores resultados para produção de nanocápsulas de núcleo aquoso. Além disso, as nanocápsulas brancas em baixas concentrações não apresentaram citotoxicidade para células de órgãos linfóides de camundongos BALB-c em cultura de células. Com isso, podemos sugerir que esta metodologia possui potencial promissor para desenvolvimento de vacina contra o HSV-1.

of thesis Diagnostics of neuroinfection caused by human herpesviruses using nucleic acid amplification methods author: MUDr. Klára Labská supervisor: doc. MUDr. Vilma Marešová, CSc. In recent years, the diagnosis of neuroinfections has undergone a shift towards molecular biology methods. Our research focused on the predictive value of the capture of herpesvirus (HV) DNA in cerebrospinal fluid. In the first study, we examined the presence of DNA neurotropic herpes viruses (HSV1, HSV2, VZV and HHV6) in cerebrospinal fluid in immunocompetent patients with laboratory-confirmed tick-borne meningoencephalitis and enterovirus meningitis and meningoencephalitis. The control group consisted of patients with proven absence of an inflammation in the cerebrospinal fluid. Patients were followed for 6 months. The course of the disease and its consequences, including laboratory tests, were compared between groups of patients with and without the presence of HV DNA. In the second study, we tried to demonstrate the presence of HSV1 DNA in cerebrospinal fluid during its symptomatic reactivation in patients with purulent meningitis. In our group of immunocompetent patients with non-purulent inflammation in the cerebrospinal fluid, the proportion of HV DNA positive patients reached 7.5% (13 out of 173), we also...

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Purpose of Review: Recent technological developments have vastly improved our ability to study the host microbiome and its role in many disease states. Numerous other reviews have contributed to our understanding of single viruses and gut microbiota or immunological outcomes. Here, we report, in aggregate, the newest data on genital microbiota interactions with the three most common viral STIs. Recent Findings: Four themes emerge: (1) the repeatability of specific community state types corresponding with infection risk, (2) a role for the microbiota as both therapeutic target and major player in treatment efficacy, (3) a need for models in which to study the mechanisms at play in microbiota/virus interactions, and (4) the impact of microbiota populating external genitalia on viral transmission. Summary: The studies reviewed herein suggest a convoluted interplay between host microbiota and viral STIs. More mechanistic studies are needed in order to leverage these interactions to improve prevention and treatment strategies.

The purpose of this investigation was to observe changes induced by types 1 and 2 herpes simplex viruses (HSY 1 and HSY 2) when these viruses were cultivated in fish 2 cells. The possibility of attempting to use human strains of herpesviruses in order to transform normal fish tissue culture preparations was considered. Three different fish cell cultures were tested: CHSE-214 cell lines were derived from embryonic chinook salmon (Oncorhynohus tshawytscha), and STE-137 cell lines were obtained from embryonic steelhead trout (Salmo gairdneri). Cells isolated from immature ovaries of rainbow trout (Salmo gairdneri) were propagated by the investigator in these studies. Both HSV 1 and HSV 2 demonstrated cytopathic effects on the 3 different cell types described above when these cells were observed during the exponential growth phase. If virus was added before cells were dividing actively, or when the cells were maintained in a stationary phase of growth, no effect was observed. Several methods were employed in an attempt to trans- form fish cells grown in tissue culture. Different quantities of live virus, preparations of ultraviolet light in- activated virus and purified viral DNA were used in these studies but malignant conversion by known viral components was not achieved. In these experiments, the 2B strain of cells obtained originally from immature ovaries of rainbow trout appeared to undergo spontaneous transformation thereby becoming established as a permanent cell line. To date tissue cultures designated 2B have undergone 25 transfers in this laboratory.

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The classical description of the innate antiviral response involves the production of type I interferon (IFN) and the subsequent expression of hundreds of interferon stimulated genes (ISGs), which cooperatively repress viral replication and spread. More recently, an IFN-independent antiviral response has also been described, in which the entry of an enveloped virus induces a subset of ISGs without requiring the production of IFN, although the details of this response remain unclear. In this work, multiple approaches were used to further characterize antiviral signaling pathways. Initially, the potential involvement in the IFN-independent response of the small GTPase Rac1, which has been implicated in both viral entry and antiviral signaling, was investigated. Here, Rac1 was shown to have a possible function in the negative regulation of ISG expression, although technical complications prevented definitive conclusions. As an alternative strategy to identify novel aspects of antiviral signaling, the mechanism of action of ICP0, a herpes simplex virus (HSV) protein involved in innate immune evasion, was investigated. Although ICP0 is generally thought to perform its actions in the nucleus, by tagging proteins for proteasome-mediated degradation via the E3 ubiquitin ligase activity of its RING finger domain, here it was shown that not only does cytoplasmic ICP0 have a RING-dependent but proteasome-independent ability to block antiviral signaling, but also that ICP0 has a previously unknown RING-independent function in the promotion of viral replication in the cytoplasm. To further investigate the cytoplasmic activities of ICP0, proteins interacting with ICP0 in the cytoplasm were identified using quantitative mass spectrometry. This revealed several intriguing binding partners for ICP0, including WDR11, a poorly-characterized cellular protein which was shown to undergo a dramatic relocation during HSV infection, although it was not required for viral replication in cultured cells. Therefore, this study has uncovered several new and unexpected insights into ICP0 behavior. / Thesis / Doctor of Philosophy (PhD)

When presented with certain unfavorable environmental conditions, C. trachomatis reticulate bodies (RBs) enter into a viable, yet noncultivable state called persistence. Two hallmarks of persistent chlamydiae are swollen, aberrantly shaped RBs, as viewed by transmission electron microscopy and a decrease in infectious progeny. Several models of chlamydial persistence have been described, including interferon-γ (IFN-γ), IFN-α, IFN-β, and tumor necrosis factor-α-exposure and nutrient deprivation. Previously, we established an in vitro co-infection model of two of the most common sexually transmitted pathogens in the United States, C. trachomatis and Herpes Simplex Virus-2 (HSV). Data from this tissue culture model indicate that: i) viral co-infection stimulates the formation of persistent chlamydiae and ii) productive HSV replication is not required for persistence induction. Further studies indicate that, co-infection-induced persistence is not mediated by: i) any known anti-chlamydial cytokine; ii) activation of inducible nitric oxide synthase or indoleamine 2, 3-dioxygenase; iii) inhibition of vesicular trafficking or sphingomyelin transport to the inclusion or; iv) amino acid, iron or glucose deprivation. These data demonstrate that co-infection-induced persistence is mediated by a previously undescribed, novel mechanism. During long-term co-infection with UV-inactivated HSV-2, chlamydiae recover following an initial suppression of chlamydial infectivity. These data indicate that HSV-induced persistence, like other persistence models, is reversible. Co-incubation of fixed, HSV-2-infected inducer cells with viable, C. trachomatis infected responder cells suppresses production of infectious chlamydial progeny and stimulates the formation of swollen, aberrantly shaped RBs. Antibody neutralization of HSV glycoprotein D (gD), which prevents viral attachment to one of four known HSV co-receptors on the host cell surface, also prevents co-infection-induced persistence, suggesting that HSV gD interaction with host cell surface receptors can provide the necessary stimulus to alter C. trachomatis development. Finally, exposure of C. trachomatis infected cells to soluble, recombinant HSV-2 gD:Fc fusion proteins decreases production of infectious EBs to a similar degree observed in co-infected cultures. Thus, we hypothesize that interaction of HSV gD with the host cell surface triggers a novel host anti-chlamydial pathway that restricts chlamydial development.

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