Dissertations / Theses on the topic 'Propagation virale'

Les Rémorines (REM) du groupe 1 sont des protéines spécifiques du monde végétale. Malgré leur caractère hydrophile elles sont localisées à la membrane plasmique. La phosphorylation des REM serait potentiellement impliquée dans la signalisation précoce et la défense des végétaux contre les pathogènes. Benschop et al. (2007) détecte AtREM1.3 (Arabidopsis thaliana, groupe 1b) phosphorylée en réponse au traitement par l'éliciteur générale flg22, tandis que Widjaja et al. (2008) a suggéré que la phosphorylation de AtREM1.2 est potentiellement impliquée dans la signalisation précoce à l'infection par Pseudomonas syringae. La fonction précise de la phosphorylation des protéines REM du groupe 1 reste inconnue. Des travaux antérieurs dans le laboratoire ont montré que le mouvement du virus X de la pomme de terre (PVX) est inversement corrélée à l'accumulation de StREM1.3 (Solanum tuberosum) et que StREM1.3 peut interagir physiquement avec la protéine de mouvement TRIPLE GENE BLOC Protein 1 (TGBp1) du PVX (Raffaele et al., 2009). Dans ce travail, nous avons étudié les mécanismes qui sous-tendent les interactions REM-TGBp1 et nous avons essayé de caractériser biochimiquement la kinase qui phosphoryle REM. Les conséquences physiologiques de l'interaction TGBp1 / StREM1.3 et de la phosphorylation de REM en terme de propagation des virus, d’inactivation génique post-transcriptionnelle, de régulation de l’ouverture des plasmodesmes, et d’activation de kinase ont également été étudiés
The group 1 Remorin (REM) proteins are plant-specific oligomeric proteins that have been reported to localize to the plasma membrane despite their overall hydrophilic nature. There is evidence that the REM protein phosphorylation is potentially implicated in the early signaling and defense. Benschop et al. (2007) detected the AtREM1.3 (Arabidopsis thaliana group 1b of REM protein family) to be phosphorylated in response to treatment with the general elicitor flg22, while the Widjaja et al. (2008) suggested that the phosphorylation of AtREM1.2 is potentially implicated in early signaling upon infection with Pseudomonas syringae. The precise exact function of the group 1 REM protein phosphorylation remains unknown. Previous work in the laboratory showed that Potato virus X (PVX) movement is inversely correlated to potato StREM1.3 accumulation and that StREM1.3 can physically interacts with the movement protein TRIPLE GENE BLOCK PROTEIN 1 (TGBp1) from PVX (Raffaele et al., 2009). In this work, we studied the mechanism underlying the REM-TGBp1 interactions and we tried to characterise biochemically the kinase that phosphorylate REM. The physiological consequences of TGBp1/ StREM1.3 interaction and REM phosphorylation in terms of virus spreading, post-transcriptional gene silencing, plasmodesmata gating, kinase activation were also investigated

Nous avons réalisé une étude centrée sur la propagation virale du bactériophage lambda. Afin de caractériser en détail la dynamique de formations des plaques, nous avons génétiquement modifié le phage lambda de façon à rendre sa capside fluorescente en créant une fusion entre la protéine GpD de la capside du phage et la protéine EYFP. Le comportement de ce phage modifié a été caractérisé et son coefficient de diffusion au sein d’une plaque a été mesuré avec la technique de FRAPP. Ensuite nous avons étudié les profiles de population de phages au sein dúne plaque avec la technique de vidéomicroscopie en time-lapse. Des expériences supplémentaires en time-lapse ont été réalisées afin de caractériser la croissance des bactéries hôte en boîte de Petri sur un substrat d’agar. Les résultats de propagation obtenus sont comparés avec les modèles et expériences antérieurs développés afin de décrire la propagation du phage T7. Pour finir, nous avons réalisé des simulations numériques complémentaires qui sont qualitativement en accord avec les résultats expérimentaux
We have developed a study focused on the viral propagation of the lambda bacteriophage. With the purpose of a detailed characterization of the dynamics of the spread of infection during the formation of plaques, we have constructed a bacteriophage lambda carrying a fluorescent fusion between the minor protein of the capsid GpD and the Enhanced Yellow Fluorescent Protein (EYFP). The next step has been to perform the preliminary characterisation of the properties of this new fluorescent virus including measurements of its diffusion coefficient on a plaque using the technique called FRAPP. Subsequently we have measured the dynamic of the phage population profiles in time within the plaque using time-lapse videomicroscopy. Supplementary experiments have been done in order to characterize the bacterial growth in Petri dishes on an agar substrate. The results have been compared to previous experiments and theoretical works performed in order to describe the propagation of the T7 bacteriophage. Finally, we have performed complementary numerical simulations that are in qualitative agreement with the experimental results

La propagation du virus de la Diarrhée Virale Bovine est modélisée et étudiée au sein d'un troupeau laitier en tenant compte de la structure en groupes, de la conduite du troupeau et du contrôle de l'effectif. Un modèle stochastique a été développé pour représenter la variabilité des dynamiques de propagation de l'infection. Le modèle est individu-centré et représente la dynamique du troupeau, les changements d'état de santé et la transmission du virus. Le comportement du modèle est simulé. Une validation qualitative du modèle a été réalisée en confrontant les données simulées à des données d'observations agrégées au niveau troupeau. La pertinence de la prise en compte du degré de séparation entre groupes sur les dynamiques de propagation du virus a été montrée. La méthodologie utilisée va pourvoir être utilisée par la suite pour étudier l'efficacité des stratégies de maîtrise. Les études de l'influence de différents facteurs et les extensions possibles du modèle sont discutées.

Un ballon lance pendant la campagne boa viking etait equipe d'un magnetometre tri axe ainsi que d'une serie d'autres moyens d'observation. Les premieres mesures des perturbations magnetiques en zone aurorale, au niveau du ballon sont presentees et discutees. Les magnetogrammes mettent en evidence des oscillations de grandes amplitudes qui se superposent a la signature de l'electrojet. Ces oscillations sont tres attenuees au niveau du sol. Ces oscillations sont interpretees en terme de trains d'onde d'alfven qui se reflechissent sur l'ionosphere. Ces trains d'onde referment, en polarisant le plasma magnetospherique, les variations positives ou negatives des courants alignes. Le champ electrique transmis au niveau de l'ionosphere genere des courants de hall, orthogonaux a l'electrojet principal, qui tendent a charger ou a decharger les capacites de hall ionospheriques pendant la phase d'expansion ou de recouvrement et ainsi ramener le champ electrique de l'ionosphere dans la direction du regime permanent

Dans cette thèse, nous étudions deux applications des processus stochastiques au marketing internet. Le premier chapitre s’intéresse au scoring d’internautes pour les enchères en temps réel. Ce problème consiste à trouver la probabilité qu’un internaute donné réalise une action d’intérêt, appelée conversion, dans les quelques jours suivant l’affichage d’une bannière publicitaire. Nous montrons que les processus de Hawkes constituent une modélisation naturelle de ce phénomène mais que les algorithmes de l’état de l’art ne sont pas applicables à la taille des données typiquement à l’œuvre dans des applications industrielles. Nous développons donc deux nouveaux algorithmes d’inférence non-paramétrique qui sont plusieurs ordres de grandeurs plus rapides que les méthodes précédentes. Nous montrons empiriquement que le premier a de meilleures performances que les compétiteurs de l’état de l’art, et que le second permet une application à des jeux de données encore plus importants sans payer un prix trop important en terme de pouvoir de prédiction. Les algorithmes qui en découlent ont été implémentés avec de très bonnes performances depuis plusieurs années à 1000 mercis, l’agence marketing d’avant-garde étant le partenaire industriel de cette thèse CIFRE, où ils sont devenus un actif important pour la production. Le deuxième chapitre s’intéresse aux processus diffusifs sur les graphes qui constituent un outil important pour modéliser la propagation d’une opération de marketing viral sur les réseaux sociaux. Nous établissons les premières bornes théoriques sur le nombre total de nœuds atteint par une contagion dans le cadre de graphes et dynamiques de diffusion quelconques, et montrons l’existence de deux régimes bien distincts : le régime sous-critique où au maximum $O(sqrt{n})$ nœuds seront infectés, où $n$ est la taille du réseau, et le régime sur-critique ou $O(n)$ nœuds peuvent être infectés. Nous étudions également le comportement par rapport au temps d’observation $T$ et mettons en lumière l’existence de temps critiques en-dessous desquels une diffusion, même sur-critique sur le long terme, se comporte de manière sous-critique. Enfin, nous étendons nos travaux à la percolation et l’épidémiologie, où nous améliorons les résultats existants
In this thesis, we study two applications of stochastic processes in internet marketing. The first chapter focuses on internet user scoring for real-time bidding. This problem consists in finding the probability for a given user to perform an action of interest, called conversion, in the next few days. We show that Hawkes processes are well suited for modelizing this phenomena but that state-of-the-art algorithms are not applicable to the size of datasets involved. We therefore develop two new algorithms able to perform nonparametric multivariate Hawkes process inference orders of magnitude faster than previous methods. We show empirically that the first one outperforms state-of-the-art competitors, and the second one scales to very large datasets while keeping very high prediction power. The resulting algorithms have been implemented with very good performances for several years in 1000mercis, a pioneering marketing agency being the industrial partner of this CIFRE PhD, where they became an important business asset. The second chapter focuses on diffusion processes graphs, an important tool for modelizing the spread of a viral marketing operation over social networks. We derive the first theoretical bounds for the total number of nodes reached by a contagion for general graphs and diffusion dynamics, and show the existence of two well distinct regimes: the sub-critical one where at most $O(sqrt{n})$ nodes are infected, where $n$ is the size of the network, and the super-critical one where $O(n)$ nodes can be infected. We also study the behavior wrt to the observation time $T$ and reveals the existence of critical times under which a long-term super-critical diffusion process behaves sub-critically. Finally, we extend our works to different application fields, and improve state-of-the-art results in percolation and epidemiology

Hepatitis C virus (HCV) is remarkably capable of efficiently hijacking host cell pathways including lipid metabolism in the liver in order to create pro-viral environments for pathogenesis. It is becoming increasingly clear that identifying small molecule inhibitors that target host factors exploited by the virus will expand available HCV treatment options. As such, a thorough understanding of host-virus interactions is critical to the development of alternative therapeutic strategies. Hepatic lipid droplets (LDs) are recruited by HCV to play essential roles in the viral lifecycle. The intracellular location of LDs is modified upon interacting with viral structural core protein. This enables formation of platforms that support viral particle assembly. Because these interactions are non-static, capturing its dynamic processes in order to better understand viral assembly can be achieved with label-free molecular imaging enhanced with live-cell capabilities. Chemical biology approaches that includes CARS microscopy employed in a multi-modal imaging system was used to probe interactions between HCV and host LDs. By successfully tracking LD trajectories, we identified core protein’s ability to alter LD speed and control for LD directionality. Using protein expression model systems that allowed for simultaneous tracking of core protein and LDs, our data revealed that mutations in the core protein region that vary in hydrophobicity and LD binding strengths, are factors that control for differential modulation of LD kinetics. Furthermore, we measured bidirectional LD travels runs and velocities, and observed critical properties by which core protein induces LD migration towards regions of viral particle assembly. Given that many steps in the HCV lifecycle are directly linked to host lipid metabolism, it is not surprising that disrupting lipid biosynthetic pathways would negatively affect viral replication. From this outlook, we explored small molecule inhibitors that targeted several lipid metabolic pathways to study its antiviral properties. Using fluorescent probes covalently labeled to viral RNA, we captured the visualization of disrupted replication complexes upon antagonizing nuclear hormone receptors that are linked to regulating lipid homeostasis. Correspondingly, biochemistry and molecular imaging techniques were also employed to identify novel antiviral mechanisms of small molecule inhibitors that target additional HCV-dependent lipid metabolic pathways.

Introduits dans l’environnement par l’intermédiaire de sources ponctuelles et diffuses, les virus et les bactériophages entériques peuvent se propager dans les cours d’eau par l’intermédiaire de différentes voies de dissémination. Détectées à la fois dans les eaux de surface et les sédiments des rivières, ces particules virales demeurent inertes dans le milieu hydrique. Leur propagation dépend donc uniquement des nombreuses interactions qu’elles partagent avec leur environnement. Qui plus est, la contamination virale des ressources en eau semble étroitement liée aux variations hydro-climatologiques. Mais malgré les connaissances déjà acquises à ce sujet, de multiples zones d’ombre subsistent concernant les variables et facteurs contrôlant le comportement in situ des particules virales dans le milieu hydrique. L’objectif de ce travail a donc été de définir le transport et le devenir des bactériophages ARN F-spécifiques dans une rivière en fonction de leurs caractéristiques propres et des conditions hydro-climatologiques. L’application de stratégies et méthodologies originales, tirées du domaine de l’hydrologie comme l’utilisation du temps de résidence de la masse d’eau ou l’échantillonnage automatique à haute fréquence, a permis d’étudier les comportements des bactériophages ARN F-spécifiques in situ. L’influence des facteurs environnementaux et plus particulièrement de la température de l’eau et du débit de la rivière sur la propagation et la survie in situ de ces particules infectieuses dans la colonne d’eau a été démontrée. Dans les sédiments, une distribution spatiale des bactériophages ARN F-spécifiques infectieux a été mise à jour. Cette particularité a pu être comprise et déchiffrée grâce à la combinaison de la caractérisation du sédiment et de l’étude du comportement d’attachement des quatre génogroupes. Les transferts de particules virales entre la colonne d’eau et les sédiments ont également pu être mis en exergue et s’avèrent être fortement dépendants des conditions hydro-climatologiques. Ainsi, la dynamique des bactériophages ARN F-spécifiques a pu être mieux appréhendée, et de même, les origines et la nature de la pollution virale ont été mieux discernées lors d’événements de crues. L’ensemble de ces résultats permet de compléter le puzzle de la dynamique des bactériophages ARN F-spécifiques dans la rivière. Les nouvelles approches expérimentales et méthodes d’analyse mises en place devraient permettre d’aboutir à une meilleure évaluation des risques viraux pour la santé humaine liés à l’utilisation des ressources en eau
Introduced into the environment through point and diffuse sources, enteric viruses and bacteriophages can be spread in watercourses via various dissemination routes. Detected in both surface water and river sediment, these viral particles remain inert in environmental water. Their spread is governed by many interactions that they have with their direct environment. Moreover, viral contamination of water resources is closely related to hydro-climatological variations. Despite the important knowledge already reported on this subject, many grey areas remain about the variables and factors controlling the in situ behavior of viral particles in environmental water. The aim of this study was therefore to define the transport and fate of F-specific RNA bacteriophages in a river according to their intrinsic characteristics and hydro-climatological conditions. The application of innovative strategies and methodologies from the hydrological science domain, such as the use of the residence time of the river water mass or high frequency automatic sampling, allowed studying the in situ behavior of F-specific RNA bacteriophages. The influence of environmental factors, especially water temperature and flow rate, has been demonstrated to have an impact on the in situ propagation and survival of infectious viral particles in the water column. Furthermore, the spatial distribution of infectious F-specific RNA bacteriophages was underlined in sediments. The accurate characterization of sediment and the study of the attachment capacity of the four genogroups explained this specific distribution. Finally, transfers of viral particles between the water column and sediment was highlighted and appeared to be highly dependent on hydro-climatological conditions. Besides the gained knowledge of the dynamics of F-specific RNA bacteriophages, the sources and origins of viral pollution of streams during rain and flood events were elucidated. This work helps completing the jigsaw puzzle on presence and transmission of F-specific RNA bacteriophages in river systems. The novel experimental approach further enhances human health-dependent viral risk evaluation linked to water resource utilization and management

Universidade Federal do CearÃ
A participaÃÃo do setor saÃde em saneamento deve estar orientada para a universalizaÃÃo do atendimento, superando entraves tecnolÃgicos, polÃticos e gerenciais que tem dificultado a extensÃo dos benefÃcios Ãs populaÃÃes residentes em Ãreas rurais, municÃpios e localidades de pequeno porte. Constata-se assim, a nÃo consolidaÃÃo de direitos sociais bÃsicos de grupos vulnerÃveis e desassistidos, conformando um quadro e desigualdades sociais, pobreza e indigÃncia. Ao se abordar a relaÃÃo entre saÃde e saneamento, Ã vital inseri-la no contexto exposto da relaÃÃo saÃde e ambiente. A primeira constataÃÃo, nesse aspecto, Ã de que preocupaÃÃes sobre a relaÃÃo saÃde-saneamento estiveram, na verdade, na raiz da atual visÃo saÃde-ambiente. Foram quase exclusivamente as questÃes de saneamento, sobretudo antes da RevoluÃÃo Industrial, aquelas que historicamente caracterizaram os determinantes ambientais da saÃde. Esta correlaÃÃo entre saneamento bÃsico e saÃde, aqui representada pela Taxa de IncidÃncia de doenÃas de veiculaÃÃo hÃdrica, Hepatite Viral, foi estudada para os municÃpios do Estado do CearÃ, como uma forma e mensurar a influÃncia da operaÃÃo da Cagece na melhoria das condiÃÃes de saÃde da populaÃÃo em relaÃÃo aos municÃpios nÃo operados pela companhia.
The participation of the health sector in sanitation must be guided to the universality of the service, surnaunting technical, political and managerial enumbrances which have brought difficulties to extended the benefits to the population who live in rural areas, small counties and localities. Thus, we verify that the basic social rights of the vulnerable and abandonned groups donât consolidate, becoming for this reason in a picture of social inequalities, poverty and indigence. When we speak about the relation between health and sanitation is essential to include it in the spoken subject of the relation health and environment. The first confirmation, in this aspect, show preoccupations about the relation health-sanitation. They were certainly in the root of the actual sight health-environment. The questions about sanitation were almost exclusive, especially before the Industrial Revolution. They were that characterized historically the environment of the health. This correlation between basic sanitation and health represented here by the Tax of Incidence of tre sicknesses brought by the veiculation of the water, the Viral Hepatite, was studied by the counties of the State of CearÃ, as a way to measure the influence of the operation of the CAGECE to improve the peopleâs health conditions in relation to the counties that werenât operate by this company.

L'agrégation de l'α-synucléine (α-syn) est au coeur du processus pathologique observé dans la maladie de Parkinson ou l'atrophie multi systèmatisée. La β-synucléine (β-syn) ressemble à l'α-syn mais pourrait avoir un rôle neuroprotecteur en inhibant l'agrégation de l'α- syn. Le but de cette thèse était de mettre en place et de tester chez la souris une stratégie thérapeutique visant à inhiber l'agrégation de l'α-syn en faisant exprimer la β-syn humaine via des vecteurs viraux adéno-associés (AAV). Nous avons d'abord caractérisé un modèle de synucléinopathie, la souris transgénique M83. Ces souris expriment l'α-syn humaine mutée en A53T, et développent une pathologie sévère associée à l'agrégation de l'α-syn. Nos résultats confirment le fait que l'agrégation de l'α-syn peut se propager à partir de l'injection périphérique de forme agrégée de la protéine, vers le système nerveux central. De plus, ils montrent que l'accumulation de l'α-syn pathologique chez la souris M83 malade dépend de l'inoculum utilisé pour accélérer leur pathologie et du niveau d'expression de l'α-syn mutée en A53T chez ces souris. Les essais de thérapie génique réalisés dans ce modèle M83 suggèrent que, quelle que soit la stratégie d'injection du vecteur AAV utilisée, la β-syn n'a pas eu d'effet protecteur détecté contre l'agrégation de l'α-syn. Une expression durable de la protéine β-syn humaine chez les souris M83 malades inoculées avec le vecteur AAV véhiculant le gène de la β-syn a pourtant été confirmée. Cependant, la β-syn pourrait s'être agrégée dans nos conditions expérimentales, comme cela a été précédemment décrit, ce qui pourrait peut-être expliquer l'absence d'effet protecteur détecté
The aggregation of α-synuclein (α-syn) is central in the pathological process observed in Parkinson’s disease or multiple system atrophy. Β synuclein (β-syn) shares some similarities with α-syn but may have a neuroprotective effect by inhibiting the aggregation of α-syn. The goal of this thesis was to develop and to test a therapeutical strategy potentially able to inhibit the aggregation of α-syn by expressing human β-syn using adeno-associated vectors (AAV). First, we characterized a model of synucleinopathy, the M83 transgenic mouse. These mice express the human A53T mutated α-syn and develop a severe disease associated with the aggregation of α-syn. Our results confirm the ability of the aggregation of α-syn to spread through the central nervous system following a peripheral injection of aggregates of α-syn. Furthermore, they show that the accumulation of pathological α-syn in sick M83 mice depends on the inoculum used to accelerate their disease and the expression level of human A53T mutated α-syn in these mice. Gene therapy trials realized in the M83 model suggest that, regardless the strategy used to inject AAV vector, β syn did not have a protective effect against the aggregation of α-syn. A lasting expression of the human β-syn protein was however detected in sick M83 mice injected with AAV vector carrying human β-syn gene. Nevertheless, β-syn could have aggregated in our specific experimental conditions, as this has already been described, which might explain the absence of protective effect detected

Au cours d'une infection lytique, le virus de l'herpès simplex de type 1 (VHS-1) doit entreprendre plusieurs étapes de fusion afin de se répliquer et se propager correctement. Ainsi, le virus a évolué afin de tirer avantage de la machinerie cellulaire en utilisant des protéines et facteurs de l’hôte à cet effet. Dans la littérature, les processus sous-jacents à l’entrée du VHS-1 ont été largement élucidés. Cependant, on ne sait toujours pas comment les particules virales nouvellement synthétisées sortent de la cellule hôte et quels facteurs cellulaires sont impliqués dans ce processus. Des résultats publiés par notre laboratoire indiquent que la protéine cellulaire, Extended Synaptotagmin 1 (E-Syt1), a un impact négatif sur la propagation globale du virus lorsqu’inhibée par de l’ARN d’interférence. Conséquemment, la présente étude a pour objectif de confirmer et d'approfondir le rôle d’E-Syt1 sur la propagation virale, en particulier sur la sortie du virus. Étant donné que l’activation d’E-Syt1 est liée à l’augmentation de la concentration de calcium cytoplasmique, nous avons également étudié l'implication du calcium au cours des stades ultérieurs de la réplication virale. Ici, nous avons démontré que la surexpression d’E-Syt1 n’a pas d’effet détectable sur la sortie du VHS-1, mais que le calcium a effet sur la propagation virale. Alors que la séquestration précoce du calcium (4 et 6 heures post-infection) à l'aide de chélateurs réprime la sortie virale, aucun effet significatif a été détecté lorsque les chélateurs ont été ajoutés à un stade avancé de l’infection (12 et 16 heures post-infection). Nos résultats fournissent des données intéressantes sur la nécessité de l’homéostasie du calcium intracellulaire afin que VHS-1 puisse assurer une médiation adéquate de la sortie virale. Ces résultats pourraient conduire à la découverte de nouveaux mécanismes ou protéines cellulaires régulées par le calcium et utilisés par le VHS-1 lors de réplications lytiques virales.
During a lytic infection, Herpes Simplex Virus type 1 (HSV-1) must go through multiple steps of fusion to replicate and propagate properly. For this purpose, the virus has evolved consequently by taking advantage of the cellular machinery using host factors and proteins. In the literature, processes underlying HSV-1’s entry have been extensively elucidated. However, it remains unclear how newly synthesized viral particles egress from the host cell, and what cellular factors are implicated in this process. Results published by our laboratory suggest that the cellular protein, Extended Synaptotagmin 1 (E-Syt1), has a negative and global impact on the viral propagation when down regulated by RNA interference. Consequently, this study aims to confirm and deepen our understanding of E-Syt1’s role on HSV-1, particularly during viral egress. Since activation of E-Syt1 is linked to the increase in cytoplasmic calcium concentration, we also investigated calcium involvement during later stages of viral propagation. Interestingly, overexpression of E-Syt1 had no measurable effect on HSV-1 propagation whereas calcium has a dual effect on viral propagation. While early calcium sequestering (4 and 6 hours post-infection) using chelators represses viral egress, no significant effect was detected when chelators were added at later time points (12 and 16 hours post-infection). Our results give interesting insights on how HSV-1 relies on intracellular calcium homeostasis to properly mediate viral egress. These results may lead to the discovery of new mechanisms or cellular proteins that are regulated by calcium and hijacked by HSV-1 during lytic replication.

Le VHS-1 est un modèle très utilisé pour l’étude du cycle viral et des interactions hôte-pathogène des Herpesvirdidae, en partie dû à sa capacité de se répliquer de façon rapide dans plusieurs types cellulaires. La transcription, réplication de l’ADN et la formation des capsides de ce virus se produisent dans le noyau cellulaire. Une fois les capsides formées, elles doivent sortir du noyau pour continuer le cycle. Cependant, les capsides sont trop grandes (125 nm) pour passer à travers les pores nucléaires, dont la taille d’exclusion est de 40 nm, et elles entament alors un processus d’enveloppement dé-enveloppement à travers les deux membranes nucléaires. Le complexe LINC (de l’anglais Linker of the Nucleoskeleton and Cytoskeleton) est un complexe qui se retrouve dans les membranes nucléaires et l’espace périnucléaire. Il sert de liaison entre le noyau et le cytoplasme et, parmi ses fonctions on retrouve le maintien d’un espace périnucléaire constant, le positionnement du noyau et la transmission de forces entre le noyau et le cytoplasme. Le complexe LINC est composé par les protéines de la famille SUN et par les protéines de la famille KASH, qui interagissent physiquement l'une avec l'autre. L'implication du complexe LINC dans la propagation du virus de la pseudo-rage (PrV) a déjà été démontrée. Étant donné que le PrV fait partie de la même famille de virus que le VHS-1, notre hypothèse est que le complexe LINC joue aussi un rôle dans la propagation du VHS-1. Mes travaux démontrent que la surexpression d'une forme négative dominante de SUN2 ou sa déplétion montre un effet proviral sur la propagation du VHS-1. Ce résultat diffère de ce qui a été précédemment constaté pour le PrV en ce sens que SUN2 est antiviral pour le PrV. Ceci confirme notre hypothèse, mais démontre un scénario plus complexe qu'anticipé.
HSV-1 is widely used to study viral cycles and host-pathogen interactions of Herpesvirdidae, because it replicates quickly and efficiently in many cells types. The transcription, replication and capsid assembly of HSV-1 take place in the nucleus of the infected cell. The assembled HSV-1 capsid must exit the nucleus to continue the viral cycle. The nuclear membranes constitute a barrier for the nuclear egress of nucleocapsids (125 nm) given the exclusion size of the nuclear pores is approximately 40 nm. The nucleocapsids therefore pass through the nuclear membranes by an envelopement-deenvelopement process. The capsids acquire an envelope from the inner nuclear membrane when they are released into the perinuclear space. This primary viral envelope then fuses with the outer nuclear membrane, enabling the capsid to reach the cytoplasm. Situated between the two nuclear membranes is the linker of the nucleoskeleton and cytoskeleton (LINC) complex. It is involved in the maintenance of the perinuclear space, nuclear positioning and force transmission between the nucleus and the cytoplasm. The LINC complex is composed of two families of proteins, SUN and KASH proteins. SUN proteins are found in the inner nuclear membrane, their N-terminal interacts with the nucleoskeleton and the C-terminal includes a conserved domain, the SUN domain, which interacts in the perinuclear space with the conserved domain of KASH proteins. The implication of the LINC complex in the propagation of the pseudorabies virus (PrV), a member of the alphaherpesviruses, has already been demonstrated. Since PrV is part of the same family as HSV-1, we hypothesized it may also play a role in HSV-1 propagation. My work shows that overexpression of a dominant negative form of SUN2 or its depletion shows a proviral effect on HSV-1 propagation. This result differs to what has been previously found for PrV, where SUN2 displays an antiviral phenotype. This work confirms our hypothesis but reveals a more complex scenario than anticipated.

The propagation of wild-type virus and a transgenic viral vector was examined in cultured plant cells to identify factors affecting viral infection of and accumulation in cultured cells and to determine if viral vectors could be used to facilitate the expression of heterologous proteins in vitro. Tobacco mosaic virus (TMV) accumulation was examined in Nicotiana tabacum and N. benthamiana suspension and hairy root cultures. TMV accumulation was superior in N. benthamiana hairy roots. Hairy roots were infected by adding TMV to the liquid culture medium at the same time as root inoculation. Hairy root growth was unaffected by virus infection. The distribution of virus within root mats from shake-flask grown cultures was non-uniform and the concentration of virus accumulated in replicate cultures was variable. When N. benthamiana hairy roots were infected using 1.5 μg mL-1 TMV, the average maximum concentration of virus accumulated in the biomass was 1.6 ?? 0.25 mg g-1 dry weight, or 15-fold lower than in TMV infected N. tabacum leaves. Virus coat protein accumulated to a level of (26 ?? 10)% total soluble protein in the hairy roots. Inoculum virus concentration and the medium in which infection was performed affected the virus yield and the percentage of inoculated cultures that accumulated virus. When cultures were inoculated using 9.0 μg mL-1 TMV, virus accumulated in the biomass to 4.2 ?? 0.60 mg g-1 dry weight. Proportional scale-up of hairy root infection in shake flasks did not result in constant virus concentrations in the scaled cultures. TMV accumulation in bioreactor-infected and -grown hairy roots was poor. N. benthamiana hairy roots were infected with a TMV-based vector (30B-GFPC3) that encoded Cycle 3 green fluorescent protein (GFP). TMV-GFPC3 was (260 ?? 140)-fold less infectious than TMV as measured by local lesion assays. Propagation of TMV-GFPC3 could not be confirmed using mass balance. GFP was not detected in the infected hairy roots or the culture medium. Hairy roots represent a potentially viable culture-based system for the in vitro production of virus and virus products when field-grown agricultural systems do not adequately address issues of containment or product safety.

博士
國立清華大學
生命科學系
94
Mammalian cell culture is an important technology for the production of viral vaccines. Microcarrier culture introduces the possibility the practical high yield culture of anchorage-dependent mammalian cells in suspension. A new trend of microcarrier technology is to utilize serum-free culture to overcome the drawbacks of serum-containing media. These drawbacks include high serum-protein content for complicating downstream purification process and the risk for potential contamination of prions due to the bovine resource. Enterovirus 71 (EV71) and dengue virus (DEN) are two important infectious diseases in Taiwan. Effective vaccines for enterovirus 71 and dengue virus are still been developed. This research has proposed the large-scale preparation by a scalable cell culture system and established a serum-free microcarrier-based cell culture for development of inactivated enterovirus 71 vaccine and live-attenuated dengue virus vaccine. In EV71 virus research, three strains (EV71-1207, EV71-075 and EV71-117) that contain two genotypes were propagated in a serum-free microcarrier culture. Vero cells were found to produce higher titers of EV71 than WI-38 and MRC-5 cells. Microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the extracellular release of EV71s from infected Vero cells at 32oC. The large-scale preparation of EV71s can be achieved using serum-free microcarrier Vero cell culture in a 2-liter bioreactor. No significant differences were observed for the formalin-inactivation kinetics of the three EV71 strains in serum-free and serum-containing cultures. The immunogenicity of the inactivated EV71 virions produced in serum-free cultures elicited slight higher levels of neutralizing antibody response in immunized mice and exhibited a cross-neutralization ability to resist three EV71 virus strains. The inactivated virions of EV71-075 and EV71-117 strains elicited approximately 1-log increase in ID50 values than EV71-1207 strain. EV71-075 and EV71-117 has identical amino acid sequences in the VP1 protein, while EV71-1207 revealed 12 amino acid sequences differences. Nucleotide sequence analysis indicated that the VP1 protein of EV71-075 and EV71-117 belonging to the B4 subgenotype. In dengue virus research, four serotypes dengue virus strains (DEN-1 strain HAWAII, DEN-2 strain NGC, DEN-3 strain H-87 and DEN-4 strain H-241) and a DEN-4 infectious clone (strain 2A) were propagated in serum-free microcarrier cultures. Vero and MRC-5 cells were grown on microcarriers using 2g/L Cytodex 1 and propagated these dengue viruses in serum-free cultures. The virus titers of dengue virus produced in microcarrier Vero cell cultures were higher than that produced in microcarrier MRC-5 cell cultures for each of these dengue strains. The genetic quasispecises of DEN-4 infectious clone virus were observed that MRC-5 cells produced the lower sequence heterogeneity than Vero cells in microcarrier cultures. These results constitute valuable information on the development of a serum-free microcarrier cell culture process for producing inactivated EV71 vaccine and tetravalent live-attenuated dengue vaccines.

The 2 micron plasmid of the budding yeast Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number around 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-coded partitioning system and an amplification system to exhibit nearly chromosome-like stability in its host. Plasmid behavior under conditions that missegregate chromosomes suggest that the partitioning system couples plasmid segregation to chromosome segregation. However, the mechanism of this coupling has not been elucidated. A plausible model, consistent with current evidence, is the hitchhiking model, in which plasmid-chromosome tethering provides the basis for faithful plasmid partitioning. Testing this hypothesis unequivocally has been difficult, primarily because of the technical limitations posed by the small size of the budding yeast nucleus and poor resolution of chromosomes. As a result, cell biological assays based on fluorescence microscopy have had only modest success in addressing this problem. In the present study, I devised an experimental verification of the hitchhiking model using a single copy derivative of the 2 micron plasmid as a reporter. The rationale was to establish various conditions that force sister chromatids to co-segregate during mitosis in a bias-free manner or with a bias towards the daughter. The segregation patterns of plasmid sisters were followed under these conditions. The sum of the results from this analysis is accommodated by the hitchhiking model, with sister plasmids associating with sister chromatids in a one-to-one fashion. Episomes of mammalian viruses belonging to the gamma-herpes and papilloma families utilize a hitchhiking mechanism to persist in cells during the latent phase of their infection. Two of the viral partitioning systems have been reconstituted in S. cerevisiae. We wished to exploit these systems to characterize the efficiency of non-native chromosome tethering systems in promoting equal segregation of viral plasmids in S. cerevisiae. We find that the 2 micron plasmid partitioning system is considerably superior to the viral systems. This could be due to the higher efficiency of plasmid-chromosome association and/or due to the ability of plasmid sisters to tether to sister chromatids.
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The chimeric Alb-uPA SCID mouse that has been transplanted with human hepatocytes is a model to facilitate in vivo study of HCV. We explored further development of the model by using repopulation with immortalized human hepatocytes (IHH) in place of primary human hepatocyte (PHH) transplantation to support HCV infection. In vitro HCV studies typically utilize a human hepatoma cell line (Huh7) and rely on transfection with transcribed genomic RNA derived from a unique HCV strain (JFH1). Unfortunately, this system has not been successful in support of infection with serum-derived HCV (HCVser). IHH may offer an alternative since their differentiation status remains close to that of PHH. IHH transfected with HCV RNA (H77 or JFH1) or infected with HCVser showed stable intracellular and supernatant HCV RNA by real-time RT-PCR. IHH showed intracellular HCV NS3 proteins. HCV transfected or infected IHH secrete infectious HCVcc for in vivo and vitro.
Experimental Surgery