Made available in DSpace on 2018-06-27T18:55:57Z (GMT). No. of bitstreams: 3 Danieli Ferrari da Rosa.pdf: 3718059 bytes, checksum: bb8ef19a5af8a3faa7687e991e0d5c3d (MD5) Danieli Ferrari da Rosa.pdf.txt: 158457 bytes, checksum: b6b83ac5211b5e9ee8b9dfba9feba1d9 (MD5) Danieli Ferrari da Rosa.pdf.jpg: 3270 bytes, checksum: 1f0862e05ecb2e7b1da2056322eeeaab (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sialic acid is the generic name of carboxylated monosaccharides family with nine carbon glycoconjugated at terminal portion. These molecule family are involved in several biological processes such cell recognition processes, platelet adhesion, migration, invasion and metastatic potential, it also work as a receptor for bacteria and viruses. High concentrations of total sialic acid in the blood have been reported in different groups of patients with brain tumors, leukemia, melanoma, carcinoma and other kinds of cancers. The cleavage of sialic acid is a crucial step in virus infection influenzae, since this acid is part of the cellular receptor that the virus uses during the process of cellular internalization. The neuraminidase, an enzyme produced by the virus, cleaves the bond between sialic acid and the viral glycoproteins, allowing the entry of viruses into cells.The aim of this study was the analysis of serum sialic acid levels in murine melanoma and Herpes Simplex virus-1 (HSV-1) infection model. In the tumor model were used C57BL/6 and in the viral model BALB/c mice. Mice were injected with 2x105 B16F10 cells subcutaneously in the thigh and the tumor progression was followed each day till it became visible. The HSV-1 infection was conducted by intraperitoneally injection of with 102 PFU of virus. The sialic acid in serum samples was quantified by thiobarbituric method in spectrophotometer at 549 nm. A standard curve with commercial sialic acid was used as parameter for quantification. The results showed that in tumor model the sialic acid was increased compared with control group and have significant difference (p <0.05) in the first day after administration of cells. For the viral infection the concentration of sialic acid showed a significant difference (p <0,05) in the first day after infection when compared infected with control group. The histological analysis in thigh of mice performed 24 hours after administration of B16F10 cells were found compact groups of round or polygonal melanocytes with clear and large cytoplasm, irregular chromatin, hyperchromatic and vacuolated nuclei, eosinophilic nucleoli and atypical mitosis. / O ácido siálico é o nome genérico dado a família de monossacarídeos carboxilados com nove átomos de carbono que aparece na porção terminal de glicoconjugados. Estas moléculas estão envolvidas em vários processos biológicos, tais como, processos de reconhecimento celular, adesão plaquetária, migração, invasão, potencial metastático, sendo também um receptor para bactérias e vírus. O aumento das concentrações séricas de ácido siálico total tem sido descrito em vários grupos de pacientes que sofrem de tumores cerebrais, leucemia, melanoma, carcinoma e outros tipos de cânceres. A clivagem do ácido siálico é um passo crucial para a infecção do vírus Influenza, uma vez que este ácido é parte do receptor celular usado pelo vírus durante o processo de internalização celular. A neuraminidase, enzima produzida pelo vírus, cliva a ligação entre o ácido siálico e as glicoproteínas virais, permitindo a entrada dos vírus nas células. O objetivo desse estudo foi analisar os níveis séricos de ácido siálico em modelo de melanoma murino e modelo de infecção herpética (HSV-1). No modelo tumoral foram utilizados camundongos C57BL/6 e no modelo viral camundongos BALB/c. Os camundongos receberam 2x105 células B16F10 através da administração subcutânea na coxa e a progressão do tumor foi acompanhada todos os dias até o tumor se tornar visível. A infecção com HSV-1 foi realizada através da administração intraperitoneal de 102 PFU de vírus. O ácido siálico das amostras de soro foram quantificadas pelo método tiobarbitúrico em espectrofotômetro à 549 nm. Uma curva padrão com ácido siálico comercial foi usada como parâmetro para a quantificação. Os resultados mostraram que as concentrações de ácido siálico no modelo tumoral foram aumentadas nos animais com tumor quando comparadas ao grupo controle e houve diferença significativa (p< 0,05) no primeiro dia após a administração das células. Para o modelo de infecção viral houve diferença significativa (p< 0,05) no primeiro dia após a infecção quando comparado o grupo infectado com o controle. Na análise histológica da coxa dos camundongos realizada após 24 horas da administração de células B16F10 foram encontrados grupos compactos de melanócitos arredondados ou poligonais, com citoplasma amplo e claro, cromatina irregular, núcleos hipercromáticos e vacuolizados, nucléolos eosinofílicos e mitoses atípicas.

Herpes simplex virus type 1 (HSV-1) is a double stranded DNA virus that causes epithelial skin infections and persists through the life of the host by infecting neurons, where it can switch to a latent state to evade an immune response. In rare cases during primary infection or after reactivation, instead of undergoing lytic infection at the epithelial surface, it instead travels to the brain and causes herpes simplex virus encephalitis (HSVE) which can have a ≥70% mortality rate if untreated. As the virus takes over its host cell, it gains control of the host cell machinery and manipulates host gene expression in order to evade the immune system and to pool its resources into the replication of the virus. One aspect of the dysregulated gene expression involves microRNAs (miRNAs). MiRNAs are short, non-coding RNAs that bind to the 3' untranslated region (3'UTR) of messenger RNAs (mRNAs), leading to translational repression of the target. Dysregulated miRNAs are often down-regulated during infection as the virus takes over, but many miRNAs have also been found to be up-regulated as well1–5. The aim of this study is to observe the full cellular miRNA changes in the context of an acute viral encephalitic infection using HSV-1, and to further characterize selected up-regulated miRNAs to determine their function in the context of the disease state. Of particular note were miR-141 and miR-200c which showed anti-apoptotic effects on neuronal cell culture and did not impact cell viability during an over-expression of the miRNAs. MiR-141, miR-183 and miR-200a expression was enriched within specific areas of the brain during infection. In addition, the potential for miR-150 to bind to a bioinformatically predicted target site within the shared 3'UTR of the HSV-1 UL18, UL19 and UL20 genes was explored. Examining the changes in expression of this class of regulatory RNAs and investigating their potential functions may yield new insight into the relationship between host and virus during infection.

Abstract Gastric erosions are superficial mucosal breaks. With the exception of bleeding, they are considered harmless, but their aetiology, histopathology and long-term course have remained unknown and even the evolution of gastritis in patients with gastric erosions is unclear. The present study aimed to solve clinical significance and pathology of gastric erosions in a long-term follow-up study. Initially, 117 patients and 117 controls were studied in 1974–1981, and a follow-up study was performed in 1996. We evaluated the presence of Helicobacter pylori and Herpes simplex virus (HSV) infections, use of NSAIDs and alcohol, smoking, and assessed features of gastric histopathology. For follow-up, 52 patients and 66 controls were available. In the follow-up visit, 39% patients still had gastric erosions while 11% of the controls had developed erosions (p&#160;=&#160;0.001). In H. pylori-positive subjects, peptic ulcer or a scar was more common in patients (17%) than in controls (4%, p&#160;=&#160;0.006), but otherwise no increased morbidity or mortality was seen. High antibody titres against HSV predicted the persistence of erosions (p&#160;=&#160;0.000), but H. pylori infection, use of NSAIDs, alcohol or smoking were not associated. Initially, inflammation was more active in the region of erosions than elsewhere in the antral mucosa, and more active inflammation in the erosion was associated with HSV seropositivity, H. pylori infection and the recent use of NSAIDs. Initially, H. pylori-positive subjects with chronic or recurrent erosions had higher scores of neutrophils compared to those with non-chronic/non-recurrent erosions. In H. pylori-positive subjects, body gastritis was initially less active in the patient group. With time, antral gastritis worsened only in the patient group. In H. pylori-negative subjects, there was no evolution of gastritis. These results show that a significant proportion of gastric erosions are chronic/recurrent but mostly without serious complications. However, H. pylori-positive patients have a significant risk to develop a peptic ulcer. A significant proportion of chronic gastric erosions is related to HSV infection. Focally enhanced inflammation modified by HSV or NSAID may be important in the pathogenesis of gastric antral erosions. Active inflammation in the erosions seems to predict their chronicity/recurrency. Patients with erosions share the characteristics of gastritis of the duodenal ulcer phenotype. / Tiivistelmä Eroosiot ovat mahalaukun pinnallisia limakalvovaurioita. Niitä pidetään vaarattomina lukuun ottamatta niihin liittyvää verenvuototaipumusta. Niiden etiologiaa, histopatologiaa ja taudinkulkua ei tunneta. Ei myöskään tiedetä eroosiopotilaiden mahan limakalvon tulehduksen kulkua. Tämän tutkimuksen tavoitteena oli selvittää mahalaukun eroosioiden kliininen merkitys ja patologia pitkäkestoisena seurantatutkimuksena. Alkujaan 117 potilasta ja 117 kontrollihenkilöä tutkittiin vuosina 1974–1981, ja seurantatutkimus tehtiin vuonna 1996. Selvitimme helikobakteerin ja Herpes simplex -viruksen (HSV) aiheuttamien infektioiden, tulehduskipulääkkeiden (NSAID) ja alkoholin käytön, sekä tupakoinnin esiintymistä. Lisäksi tutkimme histopatologisesti mahalaukun limakalvoa. Lopulta oli 52 potilaan ja 66 kontrollihenkilön aineisto käytettävissä. Seurantakäynnillä 39 prosentilla potilaista oli yhä mahalaukun eroosioita, kun taas kontrolliryhmästä vain 11 prosentilla oli kehittynyt eroosioita. Helikobakteeri -infektoituneilla maha- tai pohjukaissuolen haava/arpi oli yleisempää eroosioryhmässä (17&#160;%) kuin kontrolleilla (4&#160;%), mutta muuten ei esiintynyt lisääntynyttä sairastuvuutta tai kuolleisuutta. Tulehdus oli aktiivisempaa eroosioissa kuin viereisellä limakalvolla, ja tämä tulehdus liittyi korkeisiin HSV-vasta-ainetasoihin, helikobakteeri-infektioon ja NSAID:n käyttöön. Korkeat HSV-vasta-ainetasot ennustivat eroosioiden pysyvyyttä. Ensimmäisellä käynnillä aktiivinen tulehdus eroosioissa oli voimakkaampaa niillä helikobakteeri-infektoituneilla, joilla eroosiot olivat pysyviä kuin niillä, joilla eroosiot eivät uusineet. Helikobakteeri-infektoituneilla eroosiopotilailla mahalaukun runko-osan limakalvon tulehdus oli aluksi vähemmän aktiivista kuin vastaavilla kontrolliryhmän henkilöillä, mutta ajan myötä mahalaukun corpusosan limakalvon tulehdus voimistui vain eroosioryhmällä. Limakalvotulehdus ei edennyt helikobakteeri-infektoitumattomilla henkilöillä. Tulokset osoittavat, että merkittävä osa mahalaukun eroosioista on kroonisia/toistuvia, mutta enimmäkseen ilman vakavia komplikaatioita. Kuitenkin helikobakteeri-infektoituneilla eroosiopotilailla on merkittävä riski saada maha- tai pohjakaissuolen haava. HSV- infektio liittyy merkittävään osaan kroonisia mahalaukun eroosioita. Paikallisella tulehdusaktiivisuudella, jota HSV ja NSAID:n käyttö muokkaavat, saattaa olla tärkeä rooli eroosioiden synnyssä ja niiden kroonistumisessa. Eroosiopotilailla on samanlainen mahalaukun limakalvon tulehduksen jakauma kuin pohjakaissuolihaavaa sairastavilla.

The herpes virus family consists of more than hundred members that infect organisms, of which eight, differing markedly in the biology are known to infect humans. HSV- I is the most common one, causing oral lesions and sporadic encephalitis. These infections are highly prevalent affecting at least one in three individuals in the United States.The entry of the herpes virus into the cell is a two-step process. The initial step involves the cell surface heparan sulfate and glycoproteins in the viral envelope which enables the virus to penetrate into the cell. The second step is the fusion step. Depending on the nature of interaction and size of HS chain, a single chain may bind multiple viral ligands on a virion. There is substantial evidence showing that HS plays an important role in viral binding.HS is a heterogeneous, linear sulfated oligosaccharide composed of alternating glucosamine and uronic acid residues, which could specify distinct receptor for various viral ligands. HS, present on most exposed cell surfaces, make an ideal snare for the capture of most herpes viruses and may facilitate subsequent interactions with other co-receptors required for entry. Number of viruses, including HSV- I, HSV- II, HIV- I and dengue virus use sites of HS as receptors for binding to cells. Recently 2000 Liu et.al have characterized a HS based octasaccharide that binds to HSV-I gD. The distinguished feature in the composition of the octasaccharide is the presence of 3-O-sulfate glucosamine residue, which is an uncommon structural modification in HS. Its presence in the HSV-I gD binding sequence may confer specificity of interaction and assist HSV-I entry into the cell.Numerous sulfated molecules have been explored as mimics of HS in the inhibition of HSV-1 entry into cells. To date, most of the sulfated molecules screened for anti-viral activity have been carbohydrates. So, we reasoned that it should be possible to mimic critical interactions of HS with one or more viral glycoprotein using synthetic, non-polysaccharide, sulfated compounds. Further, it may be possible to mimic specific sequence(s) in HS, which play a role in HSV infection, with small synthetic, sulfated, non-carbohydrate molecules. In a search for synthetic mimics of HS as inhibitors of HSV-I infection, we screened a small, synthetic, sulfated flavonoids to discover a potent inhibitory activity arising from sulfation of a macromolecule present as an impurity in a crude natural product.The active principle was identified through an array of biophysical and chemical analyses as lignin sulfate, a heterogeneous; polydisperse network polymer composed of substituted phenylpropanoid monomers. Further, LC-MS with APCI in negative ionization mode, which have been reported in here for the first time for analysis of lignin, has been successfully used to deduce oligomeric structures present in the precursor of the active macromolecule based on the spectrum of the depolymerized lignin. This corroborates well with the structural information obtained using other analytical techniques. We hypothesize that the structural heterogeneity and polydispersity of lignin coupled with optimal combination of sulfate charge and hydrophobicity result in high potency. Given that the native lignin is inactive, lignin sulfate discovered here provides a variety of organic scaffolds that with the critical sulfate groups in space can mimic the HSV-I gD binding sequence.

Enveloped viruses acquire their membrane coat from the plasma membrane or intracellular organelles and rely on cellular machinery to facilitate envelopment and egress of virus progeny. This thesis examines egress-related interactions between host cell factors and proteins of two different enveloped viruses: hepatitis D virus (HDV) and herpes simplex virus 1 (HSV-1). HDV is a small RNA virus causing fulminant hepatitis or severely aggravating cirrhosis and hepatocellular carcinoma. HSV-1 is a large DNA virus infecting epithelial and neuronal cells. Infection with HSV-1 not only triggers the development of recurring sores on oral or genital mucosa, but can also cause severe disease in neonates and immunocompromised patients. The interaction between the large antigen of HDV (HDAg-L) and the N-terminal domain (NTD) of clathrin, a protein crucial for endocytosis and intracellular vesicular trafficking, was examined by structural, biochemical and biophysical techniques. Co-crystal structures of NTD bound to HDAg-L peptides derived from different HDV genotypes revealed that HDV interacts with multiple binding sites on NTD promiscuously, prompting re-evaluation of the binding between cellular peptides and NTD. Surprisingly, co-crystal structures and pull-down capture assays showed that cellular peptides containing clathrin-binding motifs can also bind multiple sites on the surface of NTD simultaneously. In addition, the structures of viral and cellular peptides bound to NTD enabled the molecular characterization of the fourth peptide binding site on NTD, the “Royle box”, and led to the identification of a novel binding mode at the “arrestin box” peptide binding site on NTD. The work in this thesis therefore not only identifies the molecular basis of HDV:clathrin interactions, but also furthers our understanding of basic clathrin biology. Even though many HSV-1 proteins have been implicated in the envelopment and egress of viral particles, only few interactions between HSV-1 and cellular proteins promoting these processes have been described. Therefore, the HSV-1 proteins gE, UL21 and UL56 were selected and characterized bioinformatically and/or biochemically. Cellular proteins interacting with UL56 were identified by yeast two-hybrid screening and quantitative mass spectrometry. Co-immunoprecipitation and pull-down experiments confirmed the Golgi-trafficking protein GOPC, components of the mammalian trafficking protein particle complex, and the ubiquitin ligase NEDD4 as novel binding partners of UL56, thereby suggesting exciting new avenues for the investigation of cellular mechanisms contributing to HSV-1 envelopment and egress.

Yau, Kwok Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 64-71). / Abstracts in English and Chinese. / Chapter Chapter 1: --- Introduction --- p.1 / Trichosanthin --- p.2 / Apoptosis --- p.10 / Herpes Simplex Virus --- p.16 / Conclusion --- p.28 / Chapter Chapter 2: --- Materials and Methods --- p.29 / Cell lines and virus --- p.30 / Infectivity assay --- p.30 / Treatment of cells and virus infection --- p.32 / MTT assay for cytotoxicity --- p.34 / Preparation of cell lysate --- p.35 / Bradford assay for protein concentration --- p.36 / Western blot analysis --- p.37 / ELISA for quantification of HSV-1 antigen --- p.38 / Statistical analyses --- p.39 / Chapter Chapter 3: --- Results --- p.40 / Cytotoxicity and anti-herpetic activity of TCS and CHX --- p.41 / Selective cytotoxicity of TCS toward HSV-1 infected cells --- p.44 / Selective cytotoxicity is implicated in the antiviral activity of TCS --- p.50 / The effect of selective cytotoxicity on TI value --- p.53 / Chapter Chapter 4: --- Discussion --- p.55 / References --- p.64

Background: Genital herpes is one of the most common sexually transmitted infections in the United States. As genital herpes is incurable and contagious, individuals with genital herpes face the decision to disclose their status to potential sexual partners with each new relationship formed. Such disclosure places individuals with genital herpes in a position to face rejection, which is commonly reported as one of the most concerning aspects of having genital herpes. The present study seeks to further understand the nature of genital herpes disclosure by addressing two core aims: 1) to understand determinants of and reasons for disclosure and non-disclosure and 2) to explore the relationship between past partner reactions to a disclosure and future intentions to disclose. Methods: Data on genital herpes disclosure experiences were collected via an online questionnaire, which was distributed through a variety of online channels including social media websites and email lists. Individuals who self-identified as having genital herpes and were 18 years and older were eligible for participation. Results: In examining Aim 1, the majority of participants (80.4%) disclosed to their last sexual partner. Age, relationship length, type of relationship, and expectations of a partner's response were significantly associated with the decision to disclose at the bivariate level. Expectations of a partner's reaction (AOR = .20, 95% CI .074-.539) and relationship type (AOR = 8.31, 95% CI 1.96-35.32) remained significant in multivariable modeling, explaining 45.2% of the variance in disclosure. Respondents who reported being in socially committed relationships and those who expected more positive partner reactions to a disclosure were more likely to disclose. Disclosure was also significantly associated with many romantic relationship building activities (e.g., establishing an exclusive relationship) but largely not associated with the sexual progression of a relationship. The decision to disclose was commonly multi-faceted, with the majority of participants reporting more than one reason that they did or did not disclose. Primary reasons for disclosure included "I wanted to be honest", "To protect my partner from getting herpes", and "It's my partner's right to know", while the most common reasons for non-disclosure were "I was concerned my partner would react badly", "I was ashamed", and "I was concerned that my partner would have rejected me". Regarding Aim 2, participants reported low levels of negative reactions and perceived rejection in response to their last disclosure experience. Intentions to disclose in the future were high among those who anticipated future sex partners. Discussion: The decision to disclose is often multi-faceted, and relationship characteristics play a key role in the decision to disclose. Among those who did disclose in this study, the majority did not report negative repercussions, including bad partner reactions and rejection. Future studies should examine if individuals are able to accurately assess potential partner reactions in order to better understand the differences between those who choose not to disclose and those who choose to disclose but experience a negative partner reaction or rejection.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that establishes life-long latent infection in sensory neurones. This makes it potentially useful as a gene therapy vector to target neuronal cells. HSV-1 enters cells by membrane fusion, the viral envelope and most tegument proteins dissociate, and the capsid is transported to the cell nucleus to establish infection. There is increasing evidence that the retrograde transport of HSV-1 along sensory axons is mediated by cytoplasmic dynein, but the viral and cellular proteins involved are not known. Cytoplasmic dynein is the major molecular motor involved in minus-end-directed cellular transport along microtubules. It is a large complex molecule, with heavy chains providing motility, while intermediate and light chains are involved in specific cargo binding. A library of HSV-1 capsid and tegument structural genes was constructed and tested for interaction with dynein subunits in a yeast two-hybrid system. A strong interaction was demonstrated between the HSV-1 outer capsid protein VP26 (UL35), as well as the tegument protein VP11/12 (UL46), with the homologous 14 kDa dynein light chains rp3 and Tctex1. In vitro pull-down assays confirmed binding of VP26 to rp3, Tctex1 and cytoplasmic dynein complexes. Recombinant HSV-1 capsids +/- VP26 were used in similar pull-down assays. Only VP26+ capsids bound to rp3. Recombinant HSV-1 capsids were microinjected into living cells and incubated at 37�C. After 1 h capsids were observed to co-localise with rp3, Tctex1 and microtubules. After 2 or 4 h VP26+ capsids had moved closer to the cell nucleus, while VP26- capsids remained in a random distribution. Our results suggest that the HSV-1 outer capsid protein VP26 mediates binding of incoming capsids to the retrograde motor cytoplasmic dynein during cellular infection, through interactions with dynein light chains. It is hoped that these findings will help in the development of a synthetic viral vector, which may allow targeted gene therapy in patients with neurological diseases.

Bibliography: leaves 92-118. / xii, 118, [20] leaves, [12] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports a method for the study of HSV-1 transcripts during latency. High resolution non-isotopic in situ hybridization (ISH) is used to study the intracellular location of HSV-1 latency associated transcripts (LATs) in primary sensory neurons of latently infected mice and humans. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995?

<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>