Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Made available in DSpace on 2018-06-27T18:56:08Z (GMT). No. of bitstreams: 2 Adrianne Del Fabro Ceccim.pdf: 4991781 bytes, checksum: d1128fe71a2a03c2beaa42a7847dfedb (MD5) Adrianne Del Fabro Ceccim.pdf.jpg: 3245 bytes, checksum: 583d4670e00da5b265b326517cddf626 (MD5) Previous issue date: 2011-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Thousands of people die annually due to infections caused by some kind of virus, despite the large investment on development of technologies that improve the diagnostics and prognostics of virus diseases. However, there is a lack of methods for diagnosis that are more accurate, low cost and that handle problems such as the immunologic window, the bounds of different infection phases, as well as the low amount of virions. The Hsps 70 are molecular chaperones that are expressed in various situations of stress or heat, forming a highly preserved immunologic system. They are highly expressed in different kind of tumors and in some in almost all viral infections. In the same way, its co-chaperona, the heat chock protein 70 linked to protein 1 (HspBP1) is involved in tumors processes, such as the lung cancer and the neuroblastoma. Recently, it was identified as a marker for the prognostic of breast cancer. Despite having studies related to the expression of HspBP1 on cancers and on its prognostics, the studies of its expression in viral infections are rare. Due to the fact that this protein can be used as an indicator of cellular metabolic alteration and that the viral infection will induce the cellular stress, the modification of its expression during the viral cycle can predict a higher or lower replicative index. This property can be used as a tool in the viral diagnostic. The mainly objective of this study was to determine the pattern of expression of protein HspBP1 in cells that hosts HSV-1 (vero cells) in vitro using the Western-blotting and determine increasing or reduction on its expression. In addition, the levels of HspBP1 and antibodies agains HspBP1 in the serum of HIV infected people were analized by ELISA. The results in vitro showed that after the viral infection there was no different in the pattern of expression of the total protein, when analyzed by SDS-PAGE. However, using the Western-Blotting the expression of HspBP1 increased in the 48 hours after the infection, comparing with non-infected Vero cells. Seventy-two hours after infection the expression decreased. The results of HspBP1 protein by ELISA showed a significant increasing of this protein in the HIV infected group that also presented a high viral load and low number of T CD4+ lymphocytes, when compared with the groups with an undetected load of HIV infected and uninfected. Regarding the research of anti-HspBP1 the results showed an increase in serum protein from HIV infected people. In conclusion these results suggest that the expression of HspBP1 is increased in HIV infected people and it is followed by an increasing in antibody production against HspBP1 and decreasing in T CD4+ lymphocytes for high viral load. / Milhares de pessoas morrem anualmente por infecções causadas por algum tipo de vírus, mesmo que, nos últimos anos, se invista muito no desenvolvimento de tecnologias para aprimorar o diagnóstico e prognóstico de viroses. Entretanto, ainda existe uma carência de métodos diagnósticos mais sensíveis, de baixo custo, que contornem problemas como a janela imunológica, os limites de detecção em determinadas fases da infecção, assim como a baixa quantidade de vírions. As Hsps 70 são chaperonas moleculares que se expressam em várias situações de estresse e ou calor, constituindo um sistema genético altamente conservado, sendo altamente expressas em diversos tipos de tumores e em certos tipos de viroses. Do mesmo modo, a sua co-chaperona, a proteína de choque térmico 70 ligada à proteína 1 (HspBP1), está envolvida em processos tumorais, como no câncer de pulmão e no neuroblastoma, e recentemente, foi identificada como um marcador de prognóstico no câncer de mama. Embora existam estudos relacionando à expressão da HspBP1 no câncer e o prognóstico deste, os estudos de sua expressão em infecções virais são raros. Em razão dessa proteína poder ser utilizada como um indicador de alteração metabólica celular, e a infecção viral induzir estresse celular, a alteração de sua expressão durante o ciclo viral pode predizer um maior ou menor índice replicativo. Assim, essa propriedade pode ser utilizada como ferramenta no diagnóstico viral. Dessa forma, este trabalho teve como objetivo determinar o padrão de expressão da proteína HspBP1 em células hospedeiras do vírus HSV-1 (células Vero) in vitro através da técnica de Western-blotting, avaliando um provável aumento ou diminuição da sua expressão. Além disso, quantificar a proteína HspBP1 e os anticorpos IgG anti-HspBP1 no soro de indivíduos HIV positivos através da técnica de ELISA. Os resultados demonstraram que após a infecção viral não houve diferença no padrão de expressão de proteína total, quando analisados por SDS-PAGE. No entanto, pela técnica de Western-Blotting, a expressão de HspBP1 apresentou aumento nas 48 horas após a infecção em comparação com células Vero não infectadas. Setenta e duas horas após a infecção, a expressão diminuiu. Os resultados da pesquisa da proteína HspBP1 pelo teste ELISA mostraram que houve um aumento significativo dessa proteína no grupo dos indivíduos infectados pelo HIV apresentando carga viral alta e contagem de linfócitos T CD4+ baixos quando comparado com os grupos dos infectados pelo HIV com carga viral indetectável e com os não infectados. Em relação a pesquisa de anticorpos IgG anti-HspBP1 os resultados mostraram que houve um aumento da proteína em indivíduos infectados pelo HIV. Assim, esses resultados sugerem que a expressão HspBP1 está aumentada em indivíduos infectados pelo HIV, seguidos por um aumento na produção de anticorpos contra a HspBP1 e redução dos linfóctios T CD4+ para uma carga viral elevada.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-16T20:02:22Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_LilianeMedianeiraMayerPossani.pdf: 1773175 bytes, checksum: 0fea418092d5f10f54b9e5790226b20b (MD5) / Made available in DSpace on 2018-08-16T20:02:22Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_LilianeMedianeiraMayerPossani.pdf: 1773175 bytes, checksum: 0fea418092d5f10f54b9e5790226b20b (MD5) Previous issue date: 2015-03-26 / Herpes is a condition caused by Herpes Simplex Virus types 1 (HSV-1) and 2 (HSV-2) which largely affects the global population. Both species can be transmitted through direct contact with infected lesions or biological fluids such as saliva and genital fluids. However, asymptomatic carriers can also transmit and excrete virus. Control of herpes is accomplished through the use of antiviral drugs, however, its extensive use has led to the emergence of resistant virus strains, particularly in immunocompromised patients. Because of that the development of an effective vaccine will not only control the disease, but also its etiologic agent. HSV-1 is an enveloped DNA viruses, and in this envelope there are viral glycoproteins that are responsible for the process of entering into the host cell. The mainly glycoproteins are B (gB) glycoprotein D (gD), glycoprotein H (gH) and glycoprotein L (gL). Glycoprotein B is the most studied protein and the reason for that is because its sequence is conserved among all herpesviruses and also is essential for the cell infection process. In this glycoprotein there is a fragment of 8 amino acid residues referred SSIEFARL. It is an immunodominant peptide responsible for inducing a strong T cell response upon infection process. This peptide is poorly immunogenic when adminstered without protection and or adjuvant. Thus, the nanobiotechnology can help for the development of a potent adjuvant to protect SSIEFARL and increase its immunogenicity. For this propouse, the production of previous developed polymeric nanocapsules (NCPs) with aqueous core was otimized and its physical and chemical parameters determined. After otimization, the encapsulation efficiency of SSIEFARL was evaluated and the interaction/compatibility of unloaded NCPs with cell in vitro were also determined. The NCPs presented unimodal distribution, low PDI (0.23±0.03), mean diameter of 267.5±51.6 and zeta potential -29.6±2.2, pH 7.2±0.3. The analyzes showed that 98% (97.7±1.5) of the particles in the sample were in the nanoscale and the a concentration of NCPs 6,07x1010/cm3. In vitro tests showed no cytotoxicity, and unload NCPs and in vivo tested suggest a cell migration to the site of injection and draning to the reagional lymph nodes, suggesting some adjuvant propertie. Taken together, the data suggest that the NCPs can be used in the development of an adjuvant system for the protection of SSIEFARL and development of a vaccine against HSV-1. / A herpes é uma patologia causada pelo Herpes Simplex Vírus dos tipos 1 (HSV-1) e 2 (HSV-2) que afeta grande parte da população mundial. Ambas as espécies podem ser transmitidas através de contato direto com lesões ou fluidos biológicos infectados, como saliva e fluidos genitais. No entanto, portadores assintomáticos também podem excretar e transmitir os vírus. O controle da herpes é realizado através do uso de medicamentos antivirais, porém seu uso extensivo, levou ao aparecimento de linhagens de vírus resistentes, principalmente em pacientes imunocomprometidos. Esse fato torna imprescindível o desenvolvimento de uma vacina que seja eficaz não só no controle da doença, mas também de seu agente etiológico. O HSV-1 é um vírus de DNA envelopado e, nesse envelope estão ancoradas glicoproteínas virais que são responsáveis pelo processo de entrada na célula hospedeira, sendo as principais glicoproteína B (gB), glicoproteína D (gD), glicoproteína H (gH) e glicoproteína L (gL). A gB é um dos principais alvos nas pesquisas por ser uma sequência conservada em todos os herpesvírus e essencial no processo de infecção da célula alvo. Nessa glicoproteína, há um fragmento constituído por 8 resíduos de aminoácidos denominado SSIEFARL. Trata-se de um peptídeo imunodominante responsável por induzir forte resposta de células T no processo de infecção. Porém, esse peptídeo, quando administrado livre, é fracamente imunogênico. Sendo assim, busca-se na nanobiotecnologia, o desenvolvimento de um adjuvante potente para proteger o peptídeo e aumentar sua imunogenicidade. Para isso, nanocápsulas poliméricas (NCPs) de núcleo aquoso previamente desenvolvidas, tiveram sua produção otimizada e seus parâmetros físico-químicos avaliados. Após o estabelecimento das condições ótimas de produção, foram analisados o encapsulamento do SSIEFARL e testes para verificar a interação das NCPs brancas com cultura celular. As NCPs apresentaram distribuição unimodal, baixo PDI (0,23 ± 0,03), diâmetro médio de 267,5±51,6 e potencial zeta -29,6±2,2, com pH 7,2±0,3. As análises mostraram que aproximadamente 98% (97,7±1,5) das partículas presentes na amostra estão na escala nanométrica em uma concentração de 6,07x1010 NCPs/cm3. Os testes in vitro não evidenciaram citotoxicidade, e as NCPs brancas apresentaram capacidade de induzir migração celular ao local da injeção com tendência à drenagem dessas células aos linfonodos, sugerindo bom efeito adjuvante. A partir desses resultados pode-se sugerir que as NCPs podem ser utilizadas no desenvolvimento de um sistema adjuvante para a proteção do SSIEFARL e desenvolvimento de uma vacina contra o HSV-1.

Herpes Zoster é uma doença causada pela reativação do vírus Varicela Zoster nos gânglios sensoriais, caracterizada pelo desenvolvimento de lesões na pele e dor. Não há modelos animais disponíveis para estudo da patofisiologia da doença. No entanto, um modelo murino que utiliza o HSV-1 tem sido usado para tal fim, visto que os animais desenvolvem lesões zosteriformes e desenvolvem hipersensibilidade na pata infectada. Não há dados na literatura acerca da resposta imune que se desenvolve nos gânglios da raiz dorsal destes animais. Logo, o objetivo deste trabalho foi o de avaliar células e mediadores inflamatórios presentes nos gânglios da raiz dorsal e sua relação com a hiperalgesia durante a infecção cutânea por HSV-1. Durante a fase aguda da infecção, os camundongos desenvolveram hiperalgesia nas patas ipsilaterais a partir do 3 dia pós-infecção, que perdurou até o 7 dia pós-infecção. A maior carga viral foi detectada nos gânglios L4, L5 e L6, os quais compõem o nervo ciático, que inerva a área infectada. O tratamento dos animais infectados com dexametasona ou fucoidina resultou na redução do comportamento de hiperalgesia, a partir do 5 dia pós-infecção, que corresponde ao período em que a migração de leucócitos passa a aumentar nos gânglios da raiz dorsal. Macrófagos, neutrófilos e linfócitos T CD4 foram detectados nos gânglios durante a infecção aguda. No entanto, linfócitos T CD8 estavam ausentes. A expressão do mRNA de TNF- e COX-2 estava aumentada nos gânglios, e o tratamento de animais infectados com drogas inibidoras de ambos resultou na redução da hiperalgesia. Os receptores do tipo Toll-like e da IL-1 não participam da geração da hipersensibilidade herpética. Após 50 dias da infecção, constatou-se que alguns animais apresentavam comportamento de hiperalgesia irreversível, semelhante à neuralgia pós-herpética humana (NPH). Não houve diferença significativa na incidência da NPH em animais de linhagens ou sexos diferentes. Ainda, o tratamento com drogas anticonvulsivantes e antidepressivas, mas não com morfina e anti-inflamatórios, resultou na redução transiente da hiperalgesia. Neste período, não há participação da inflamação na manutenção da hiperalgesia. A expressão de TNF- e COX-2 retorna aos níveis basais, e não são mais detectados neutrófilos e macrófagos. No entanto, a migração de linfócitos T CD4+ e CD8+ aos gânglios aumenta de maneira tempo-dependente. Durante a NPH, detectou-se uma intensa ativação das células satélites gliais, que contribuem para a manutenção da hiperalgesia pós-herpética. Nossos resultados demonstram que a manutenção hiperalgesia herpética é resultado da intensa resposta inflamatória que ocorre nos gânglios da raiz dorsal infectados, com aumento da produção de TNF- e COX-2, importantes mediadores para a hipersensibilidade. No entanto, durante a neuralgia pós-herpética, não há participação de células ou mediadores inflamatórios, mas de células da glia, as quais são importantes na manutenção da hiperalgesia. / Herpes Zoster is a disease caused by reactivation of varicella zoster virus in sensory ganglia, characterized by dermal rash and pain. There are no animal models available to study the pathophysiology of the disease. A murine model of HSV-1 infection on the hind paw skin has been used to study HZ, since mice develop HZ-like skin lesions and pain-related responses. There are no data available about the immune response in dorsal root ganglion (DRG) of these mice. Thus, the aim of this study was to evaluate cells and inflammatory mediators present in DRGs and its relationship with hiperalgesia during HSV-1 cutaneous infection. During the acute phase of infection, mice developed hyperalgesia in ipsilateral paws from 3 days post-infection, which persisted until 7 days post-infection. The highest viral load was detected in ganglia L4, L5 and L6. Treatment of infected mice with fucoidin or dexamethasone resulted in the reduction of hyperalgesic behavior, from the 5th post-infection day, which corresponds to the period in which leukocyte migration increase in the dorsal root ganglia. Macrophages, neutrophils and CD4 + T lymphocytes were detected in the ganglia during acute infection. However, CD8 + T lymphocytes were absent. The mRNA expression of TNF- and COX-2 was increased in dorsal root ganglia, and the treatment of infected mice with drugs that inhibits both mediators resulted in reduced hyperalgesia. The Toll-like receptors and IL-1 does not participate in the generation of herpetic hypersensitivity. After 50 days of infection, it was found that some animals presented irreversible hyperalgesic behavior, like human post-herpetic neuralgia (PHN). There was no significant difference in the incidence of PHN in animals of different genders or strains. Furthermore, treatment with anticonvulsant and antidepressant drugs, but not morphine and anti-inflammatory, resulted in transient reduction of hyperalgesia. In this period, there is no participation of inflammation in the hyperalgesia maintenance of. The expression of TNF- and COX-2 returns to baseline levels, and neutrophils and macrophages are no longer detected. However, the migration of CD4 + and CD8 + to ganglia increases in a time-dependent manner. During NPH, an intense activation of glial cells satellites was detected, that contributes to the maintenance of post-herpetic hyperalgesia. Our results demonstrate that herpetic hyperalgesia maintenance is a result of an intense inflammatory response that occurs in the infected dorsal root ganglia, with increased production of TNF- and COX-2. However, during post-herpetic neuralgia, there is involvement of glial cells, which are important in hyperalgesia maintenance.

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Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

DNA damage response (DDR) pathways are essential in maintaining genomic integrity in cells, but many DDR proteins have other important functions such as in the innate immune sensing of cytoplasmic DNA. Some DDR proteins are known to be beneficial or restrictive to viral infection, but most remain uncharacterised in this respect. Non-homologous end joining (NHEJ) is a mechanism of double stranded DNA (dsDNA) repair that functions to rapidly mend broken DNA ends. The NHEJ machinery is well characterised in the context of DDR but recent studies have linked the same proteins to innate immune DNA sensing and, hence, anti-viral responses. The aim of this thesis is to further investigate the interplay between herpes simplex virus 1 (HSV-1), a dsDNA virus, and two NHEJ proteins, DNA protein kinase catalytic subunit (DNA-PKcs) and paralogue of XRCC4 and XLF (PAXX). PAXX was first described in the literature as a NHEJ protein in 2015, but whether it has any role in the regulation of virus infection has not been established. Here we show that PAXX acts as a restriction factor for HSV-1 because PAXX-/- (KO) cells produce a consistently higher titre of HSV-1 than the respective wild type (WT) cells. We hypothesised that this could be due to a role of PAXX binding viral DNA and directly inhibiting HSV-1 replication or activating an anti-viral innate immune response. We have been able to, at least partially, rule out both of these initial hypotheses by showing that there was a reduced number of viral genomes present in KO cells during active lytic infection, and that an identical level of type I interferons are produced from WT and KO cells during HSV-1 infection. Although further characterisation of HSV-1 infection in WT and KO cells has not defined the molecular mechanism of restriction of HSV-1 by PAXX, we have uncovered a potential role for PAXX in mitogen-activated protein kinase (MAPK) signalling. In addition, and consistent with its function in restriction of HSV-1 infection, we show that infection with this virus in WT cells induces a loss of nuclear PAXX protein. Preliminary data suggest that these changes in localisation may occur as a result of stimulation of the cells with DNA, but not the RNA analogue poly(I:C). The role of PAXX in the regulation of HSV-1 infection in vivo was investigated by studying KO mice. Despite previous observations that mice lacking NHEJ proteins have brain defects related to autoinflammatory pathology, there were no obvious defects in the development of Paxx-/- mice, and they had brains of normal weight. No significant difference in viral spread or viral protein expression was observed between WT and KO HSV-1 infected mice, and KO mice did not exhibit abnormal pathology. There were, however, small but significant differences in the cellular immune response to infection which might be explained by reduced MAPK signalling in KO cells. DNA-PKcs is another component of the NHEJ machinery that acts to assist in dsDNA break repair in the nucleus and as an innate sensor of cytoplasmic viral DNA, but the effect of DNA-PKcs on HSV-1 infection has not been fully explored. Murine skin fibroblasts (MSFs) derived from wild type and PRKDC-/- (DNA-PKcs deficient) mice were cultured ex vivo and used for innate immune studies. Although HSV-1 was able to infect and stimulate these cells, no differences in the stimulation of innate immune gene expression between the two genotypes was observed, suggesting that DNA-PKcs does not contribute to HSV-1 sensing in MSFs. It has previously been reported that the HSV-1 protein ICP0 targets DNA-PKcs for degradation, although the reason for this is unknown. We confirmed these data, although found it to be cell-type specific and explored this interaction further using PRKDC-/- RPE-1 cells created using CRISPR/Cas9. HSV-1 infection in these cells followed unusual dynamics, and the development of cytopathic effect was accelerated as compared to WT cells. Together these observations confirm that DNA-PKcs regulates HSV-1 infection, but more work is required to fully understand the mechanisms involved.

The egress pathway of herpes simplex virus-1 (HSV-1) is a complicated process mediated by co-ordinated activity of several virus glycoproteins. The virions are first assembled and enveloped at trans-Golgi-network (TGN) or endosome membranes and then travel through a guided pathway that is directed towards the cell adherent points for secretion. Once secreted the vast majority of virions remain associated with the extracellular membrane of cells and very few free virions are released into the culture medium (< 1%). The mechanisms that mediate both the targeted secretion of newly assembled virions at cell contact points and post-secretion attachment of virions with the extracellular surface of cells are poorly understood, and were the topics of this research. In this thesis, an HSV-1 passage mutant of increased virion secretion phenotype had been studied. Genome sequencing of the mutant virus identified mutations in three viral envelope proteins. Study of recombinant viruses that were constructed based on those three mutations revealed that a single amino acid change in glycoprotein I (gI) of glycine to arginine at residue 39 is responsible for the increased release of virus. The result suggests the principal effect of this mutation is to modify the secretory pathway used by virions during their release from infected cells. Data also suggests a role of gC in the attachment of virions to the extracellular surface of cells after egress. In the context of HSV-1 envelopment and egress glycoprotein E (gE), which forms a heterodimeric complex with gI (gE/gI), is known to be important. The gE/gI complex has been shown to interact with many tegument proteins and have a redundant role in secondary envelopment. The gE/gI complex has been also proposed to colocalise with various cellular components and sort the nascent virions to cell contact points. However, there is little understanding of the cellular proteins that gE/gI interact with, or the mechanisms that mediate targeted secretion of virions. This research has identified a novel interactome of gE/gI by mass-spectrometric analysis utilising stable isotope labelling with amino acids in cell culture (SILAC) medium. Among the cellular interactome obtained, Nipsnap1 was validated by co-precipitation assays from both infected and transfected cells, and furthermore using cell free systems, suggesting gE and Nipsnap1 directly interact. Nipsnap1 and its homologue Nipsnap2 have been proposed to contribute in vesicle transport and membrane fusion in cells. Using CRISPR-Cas9 technology these proteins were knocked out in a keratinocyte cell line (HaCaT) to investigate their role in HSV-1 egress. However, little or no effect on HSV-1 egress could be observed upon loss of either or both of these proteins suggesting the biological significance of gE-Nipsnap1 interaction may not be directly linked to any egress function of gE/gI. Two further interesting 'hits' from the gE/gI interactome were interferon-induced transmembrane protein type-2 (IFITM2), a virus restriction factor, and Myoferlin that has a putative role in endocytic vesicle recycling. This study could validate gE-Myoferlin interaction and co-localisation in infected or transfected cells however, functional significance of this interaction remains to be determined. Overall, the research of this thesis has provided a better understanding of the role of the gE/gI complex in HSV-1 egress and investigated the role of some interesting cellular proteins in the context of virion egress.

The herpesvirus life cycle involves a step where newly formed capsids leave the nucleus by translocating across the intact nuclear envelope (NE). Little is known about the role of cellular factors during nuclear egress. We sought to identify novel cellular proteins that interact with the conserved herpes simplex virus-1 (HSV-1) pUL34 by performing a yeast two-hybrid screen. pUL34 was chosen due to its crucial and multifunctional role during nuclear egress. From 42 cellular factors that interacted with pUL34 in yeast, twelve were further evaluated in mammalian cells by co-localization studies using immunofluorescence. No specific co-location between the tested cellular factors and pUL34 was observed in infected cells, thus the screen failed to convincingly identify novel pUL34 interactors. In the second part of the thesis we addressed the functional significance of the cellular protein torsinA (TA) in the HSV-1 life cycle. We became interested in TA due to its role in maintaining normal NE morphology. We showed that perturbing the normal function of TA through overexpression impaired HSV-1 replication and caused a defect in capsid nuclear egress. In mouse embryonic fibroblasts that failed to express TA (TA-/-MEFs), HSV-1 replication was also inhibited, but a defect in capsid nuclear egress was not apparent. Strikingly, infection in TA-null MEFs induced a NE breakdown, the extent of which was dependent on viral products involved in nuclear egress. The viral growth defect and NE envelope breakdown, however, seem to be TA-null cell line specific rather than a functional consequence of TA loss as indicated by TA-/-MEFs reconstituted with TA and 293T with reduced TA levels. In conclusion, overexpression and loss of TA have different effects on the HSV-1 life cycle.