Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus, often acquired during childhood, that currently infects more than 50% of the human population. The symptoms of infection are herpetic lesions that frequently appear throughout a host's life in response to stress in the orofacial or genital region. As a pathogen, HSV-1 replicates rapidly in epithelial cells, but it is also capable of infecting neurons where it can pursue a lytic or latent infection. Latency is a state of viral quiescence where the virus can persist indefinitely yet remain poised to reactivate. Latency is unique to herpesviruses and key to HSV's success, but the molecular mechanisms that govern this state are unclear. A virus-encoded E3-ubiquitin ligase, Infected Cell protein 0 (ICP0), is often correlated with latency establishment but is detected in opposition to the state of latency. During lytic infection, ICP0 has many biological roles but primarily catalyzes the addition of ubiquitin to target substrate, marking proteins for degradation or altering their function. This ubiquitination ability allows ICP0 to alter the intracellular environment making neurons conducive to lytic or latent HSV-1 infection. ICP0's neuron-specific targets, however, are unknown, representing a significant gap in knowledge. Through the studies presented in this dissertation, we identified some of the neuron-specific ubiquitination targets of ICP0 in neurons. We utilized primary adult sensory neurons of the dorsal root ganglia and HSV-1 viral strains KOS, wild-type virus encoding a fully functional ICP0, and HSV-1 n212, encoding a truncated ICP0 protein, to illuminate the mechanisms involved in establishing and maintaining HSV latency. By using adult primary neurons and functional HSV-1 strains with and without ICP0, we were able to show that ICP0 regulates host and viral proteins during the initial onset of neuronal infection. We also show that based on neuronal conditions set forth before HSV-1 initial infection, host proteins will influence HSV-1 viral proteins to repress viral gene expression, thereby promoting the establishment of latency. / Doctor of Philosophy / Herpes simplex virus (HSV-1) is a virus, often acquired during childhood, that more than 50% of people have. Those who are infected with HSV-1 often have cold sores that appear in response to stress on the face or on the genitals. As a virus, HSV-1 replicates around the eyes, nose, and mouth but can also infect neurons where it can continue to replicate or establish latency. Latency is when the virus is inside the neurons but is unnoticeable and can reappear in response to stress. The state of latency is unique to herpesviruses and key to the success of HSV-1, but scientists are unsure of how it works. A protein made by the virus, Infected Cell Protein 0 (ICP0), is often correlated with the state of latency but is often present when the virus is not latent. ICP0 does a lot to support HSV-1, but it primarily destroys proteins that prevent the virus from replicating. By destroying proteins that prevent HSV-1 replication, ICP0 can help the virus make more viruses. The proteins that are destroyed by ICP0 are currently unknown, which represents a significant gap in knowledge. Through the research conducted in this dissertation, we identified some of the proteins that ICP0 destroys in neurons. We utilized neurons from the dorsal root ganglia and HSV-1 viral strain KOS, which encoded a functional ICP0, and n212, which encodes a nonfunctional ICP0, to study the mechanisms used by the virus to infect neurons. By using HSV-1 viruses with and without ICP0, we were able to show what proteins ICP0 destroys during infection in neurons. We were also able to show that HSV-1's ability to establish latency is dependent on how the neurons handle the initial onset of infection. Overall, a combination of host and viral proteins coordinates the virus's ability to establish latency and persist within a host.

Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are major human pathogens. HSV establishes latency in the nervous system and reactivates to cause recurrent disease, resulting in transmission of progeny virions to naïve individuals. Though HSV-1 and HSV-2 share similar structure and genes, they have distinctive recurrence profiles. Generally, HSV-1 reactivation is associated with disease 'above the waist' and HSV-2 reactivation is associated with disease 'below the waist'. This phenomenon was described decades ago but still remains unexplained. The mechanism of HSV latent infection in the peripheral nervous system (PNS) has been extensively investigated, especially with in sensory neurons. Another component of the peripheral nervous system (PNS), autonomic neurons, were also known to be infected with HSV productively and latently, but largely ignored because of the assumption that there is no difference in the pathogenesis of HSV in the neurons and that both HSV-1 and HSV-2 behave in the same way in different types of neurons. However, autonomic neurons differ in physiological function compared to sensory neurons. Activation factors of autonomic neurons, such as emotional stress, trauma and hormonal fluctuation, are also known HSV reactivation triggering factors. Therefore, I hypothesized that autonomic neurons innervating the site of HSV infection are responsible the different reactivation frequencies of HSV-1 and HSV-2 after peripheral invasion. In this report, the role of autonomic neurons in HSV pathogenesis were examined using the female guinea pig reactivation model. Major findings of this report are that 1) parasympathetic ganglia innervating the ocular region support latent infection of HSV-1 selectively, thus contributing the more frequent HSV-1 reactivation, 2) mixed autonomic ganglia in the genital area support HSV-2 latent infection selectively, and 3) sympathetic neurons in the genital region supported productive and latent infection of HSV-1 and HSV-2 differently. All of the results in this report indicate that autonomic neurons play a distinctive role in HSV pathogenesis compared to the sensory neurons and are responsible for the different reactivation frequencies of HSV-1 and HSV-2. This report raises the importance of autonomic neurons in HSV pathogenesis and challenges the paradigm of HSV pathogenesis. / Ph. D.

Os vírus são responsáveis por infecções de distribuição mundial com elevadas prevalência e morbimortalidade sendo, muitas das vezes, sem tratamento efetivo disponível. Existem medicamentos fitoterápicos com atividade antibacteriana ou antiviral comprovadas, porém seu uso na prática clínica ainda é limitado. O objetivo deste trabalho foi investigar a presença ou ausência de atividade antiviral in vitro de extratos brutos e fracionados de Cocos nucifera L. em cultura de células Vero infectadas com herpes simplex vírus tipo 1 (HSV-1). Foram escolhidas doses não tóxicas dos extratos aquoso e hidroetanólico da fibra do C. nucifera, de 4 frações derivadas daquele com diferentes solventes (hexano, acetato de etila, metanol e água) e de duas substâncias isoladas a partir desta fibra (Substâncias CN1A e CN342B). O HSV-1 foi adicionado à cultura de células Vero uma hora após estas serem incubadas com os dois extratos e seus derivados. As placas foram analisadas em microscópio óptico a cada 24 horas, sendo o material colhido quando o efeito citopático viral no controle negativo atingisse mais de 80% das células e congelado para posterior titulação viral por TCID50. Utilizamos o aciclovir como droga de referência para o HSV-1. A substância CN342B foi capaz de inibir a replicação do HSV-1, com efeito antiviral comparável ao do aciclovir, enquanto que os extratos brutos, as quatro frações e a substância CN1A não foram efetivas. Em conclusão, a substância CN342B isolada das fibras do C. nucifera foi eficaz contra o HSV-1 in vitro / Viruses are responsible for infections worldwide with high prevalence and morbimortality, often with no effective treatment available. There are herbal medicines with proven antibacterial and antiviral activities; however, their use in clinical practice is still limited. The aim of this study was to investigate the presence or absence of in vitro antiviral activity of aqueous and hydroethanolic extracts from Cocos nucifera L. husk fiber, in culture of Vero cells infected with herpes simplex virus (HSV)-1. Non-toxic dosages of aqueous and hydroethanolic extracts, as well as the hexane, ethyl-acetate, methanol and end-aqueous fractions were used. In addition, two isolated substances (CN1A and CN342B) were tested. HSV-1 was added to the cell culture after one hour of incubation with the extracts and fractions. The plates were analyzed in optical microscopes each 24 hours, and cells were harvested when the cytopathic effect in negative controls were above 80% of the cells. The cells were then frozen for viral titulation (TCID50). Acyclovir was the reference drug for HSV-1. Substance CN342B, but none of the other drugs, was able to reduce HSV-1 replication, similarly to acyclovir. In conclusion, the substance CN342B isolated from C. nucifera husk fibers was effective against HSV-1 in vitro

Os vírus são responsáveis por infecções de distribuição mundial com elevadas prevalência e morbimortalidade sendo, muitas das vezes, sem tratamento efetivo disponível. Existem medicamentos fitoterápicos com atividade antibacteriana ou antiviral comprovadas, porém seu uso na prática clínica ainda é limitado. O objetivo deste trabalho foi investigar a presença ou ausência de atividade antiviral in vitro de extratos brutos e fracionados de Cocos nucifera L. em cultura de células Vero infectadas com herpes simplex vírus tipo 1 (HSV-1). Foram escolhidas doses não tóxicas dos extratos aquoso e hidroetanólico da fibra do C. nucifera, de 4 frações derivadas daquele com diferentes solventes (hexano, acetato de etila, metanol e água) e de duas substâncias isoladas a partir desta fibra (Substâncias CN1A e CN342B). O HSV-1 foi adicionado à cultura de células Vero uma hora após estas serem incubadas com os dois extratos e seus derivados. As placas foram analisadas em microscópio óptico a cada 24 horas, sendo o material colhido quando o efeito citopático viral no controle negativo atingisse mais de 80% das células e congelado para posterior titulação viral por TCID50. Utilizamos o aciclovir como droga de referência para o HSV-1. A substância CN342B foi capaz de inibir a replicação do HSV-1, com efeito antiviral comparável ao do aciclovir, enquanto que os extratos brutos, as quatro frações e a substância CN1A não foram efetivas. Em conclusão, a substância CN342B isolada das fibras do C. nucifera foi eficaz contra o HSV-1 in vitro / Viruses are responsible for infections worldwide with high prevalence and morbimortality, often with no effective treatment available. There are herbal medicines with proven antibacterial and antiviral activities; however, their use in clinical practice is still limited. The aim of this study was to investigate the presence or absence of in vitro antiviral activity of aqueous and hydroethanolic extracts from Cocos nucifera L. husk fiber, in culture of Vero cells infected with herpes simplex virus (HSV)-1. Non-toxic dosages of aqueous and hydroethanolic extracts, as well as the hexane, ethyl-acetate, methanol and end-aqueous fractions were used. In addition, two isolated substances (CN1A and CN342B) were tested. HSV-1 was added to the cell culture after one hour of incubation with the extracts and fractions. The plates were analyzed in optical microscopes each 24 hours, and cells were harvested when the cytopathic effect in negative controls were above 80% of the cells. The cells were then frozen for viral titulation (TCID50). Acyclovir was the reference drug for HSV-1. Substance CN342B, but none of the other drugs, was able to reduce HSV-1 replication, similarly to acyclovir. In conclusion, the substance CN342B isolated from C. nucifera husk fibers was effective against HSV-1 in vitro

Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [toliau žr. visą tekstą] / Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [to full text]

Herpes simplex virus (HSV) causes recurrent orofacial and genital infections and establishes latent infection in sensory neurons. During latency all virus genes are supressed except the latency associated transcripts which are transcribed from latency associated gene (LAT). It is established that HSV LAT promoter mutants have lower levels of spontaneous reactivation rates in small animal models compared to wild virus. However, the variation in the LAT promoter has not been studied in viruses from clinical samples in humans. The aim of the sudy was to evaluate the sequence variation in herpes simplex virus latency associated gene promoter from clinical samples by developing and applying molecular methods and correlate with herpes infection clinical features. In this study a new PCR method specific for HSV LAT promoter was developed and HSV LAT promoter DNA sequences from Lithuanian and Swedish mucocutaneous and cerebrospinal fluid clinical samples were analyzed. HSV type 2 was found to be the main cause of genital herpes in the population of the Lithuanian patients. All cases of orofacial herpes simplex infection were caused by HSV type 1. The structure of the LAT promoter region was studied in 145 HSV clinical samples. HSV LAT promoter was found to be G+C rich and contained variable homopolimer tracts. An inter- and intrastrain variability of homopolimer tracts in the promoter region was detected, potentially giving rise to a large variation at the protein level, leading to... [to full text] / Herpes simplex virusas sukelia recidyvuojančią burnos-veido ir lytinių organų infekciją. Latentinėje būklėje šis virusas glūdi sensoriniuose ganglijuose. Latencijos metu visi HSV genai yra supresuoti, išskyrus su latencija susijusį geną (LAT). Tyrimais nustatyta, kad tarp HSV LAT promotoriaus mutantų reaktyvacijos dažnis laboratorinių gyvūnėlių modeliuose yra mažesnis nei laukinių virusų. Nėra atlikta tyrimų, kurie nagrinėtų LAT promotoriaus sekų variaciją herpes simplex virusuose, išskirtuose iš žmonių klinikinių mėginių. Šio tyrimo tikslas buvo įvertinti herpes simplex viruso LAT promotoriaus sekų įvairovę molekulinės diagnostikos metodais bei palyginti su infekcijos klinikiniais požymiais. Tuo tikslu buvo sukurtas PGR metodas HSV LAT promotoriaus analizei atlikti. Buvo ištirta Lietuvos ir Švedijos klinikiniuose odos-gleivinių bei cerebrospinalinio skysčio mėginiuose rasto herpes simplex viruso promotoriaus DNR sekų įvairovė. Tyrimo metu rasta, kad 2 tipo herpes simplex virusas buvo pagrindinė lytinių organų HSV infekcijos priežastis tarp Lietuvos pacientų. Visuose veido srities bėrimuose rasta 1 tipo HSV. HSV LAT promotoriaus sekos ištirtos 145 klinikiniuose mėginiuose. Nustatyta, kad HSV LAT promotoriaus sekos yra gausios GC ir turi variabilias homopolimerinių nukleotidų sritis, kurios varijuoja tarp viruso padermių ir pačių padermių viduje. Ši variacija gali turėti įtakos baltymų sintezei, o drauge ir fenotipo pokyčiams. Nenustatytas ryšys tarp HSV LAT promotoriaus... [toliau žr. visą tekstą]

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.

Le virus Herpes Simplex de type 1 (HSV-1) possède un mode d’infection particulier dit bimodal. Il peut soit se répliquer de manière active lors d’une phase dite lytique soit migrer dans les neurones et rester en latence. Il peut réactiver pour rétablir une infection lytique. Une protéine virale majeure dans la réactivation de HSV-1 est ICP0. C’est une protéine nucléaire à activité E3 ubiquitine ligase, qui possède la particularité d’induire la dégradation par le protéasome de plusieurs protéines centromériques constitutives, ce qui provoque une déstabilisation du centromère interphasique. Mon équipe a découvert une réponse cellulaire à l’instabilité centromérique, induite par la protéine ICP0, et nommée iCDR (pour interphase Centromere Damage Response.). L’objectif général de ma thèse est de déterminer les modifications structurales que subissent les centromères endommagés par ICP0 à l’origine de l’iCDR et probablement de la réactivation. J’ai pu démontrer qu’ICP0 affectait toute la structure protéique étroitement associée aux centromères durant l’interphase. Suite à ces résultats, j’ai pu démontrer, par des analyses de digestion de chromatine à la nucléase microccocale (MNAse), que l’occupation nucléosomique de la chromatine centromérique suite à l’activité d’ICP0 était affectée de façon significative. Une étude in vivo effectuée à partir de tissus nerveux provenant de souris infectées de manière latente, a permis de démontrer une co-localisation entre les génomes HSV-1 latents et les centromères. Cette co-localisation est associée à une répression transcriptionnelle du virus. Les résultats de ma thèse montrent donc que les effets d’ICP0 sur la déstabilisation des centromères sont en relation avec un rôle de ces centromères durant la latence. Ceci suggère fortement une implication de la déstabilisation des centromères dans le processus de réactivation contrôlé par ICP0. / The Herpes Simplex type 1 (HSV-1) virus possesses a bimodal mode of infection. It can either replicates in an active way during the lytic cycle, or it can infect neurons and stay in latency. HSV-1 reactivates from latently infected neurons for re-establishing a lytic infection. A major viral protein implicated in reactivation is ICP0. It is a nuclear E3 ubiquitin-ligase, which has the particularity to induce the proteasome-mediated degradation of several constitutive centromeric proteins. This activity severely destabilizes the interphase centromere. My team has discovered a novel cellular response triggered by the estabilization of centromeres by ICP0, called iCDR (interphase Centromere Damage Response). The general aim of my thesis is to determine the centromere structural modifications induced by ICP0 that can trigger the iCDR and probably the reactivation. I was able to demonstrate that ICP0 affected the entire proteinacious structure of interphase centromeres. Following this, I showed by micrococcal nuclease (MNase) digestion approach that the nucleosomal organization of centromeric chromatin was significantly affected by ICP0. An in vivo study in nervous tissues coming from latently infected mice enabled to show a co-localization between latent HSV-1 genomes and centromeres. This co-localisation is linked to a transcriptional repression of the virus. The results of my thesis show that the destabilization of centromere by ICP0 correlates with a role of the centromeres during latency. This strongly suggests an implication of centromere destabilization in the ICP0-controlled reactivation process.

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.