Dissertations / Theses on the topic 'Virus overexpression'

Fusarium graminearum is a major fungal pathogen of wheat and other small grain cereal crops globally, causing Fusarium ear blight (FEB) disease. Like many other plant pathogens, F. graminearum is predicted to produce in planta secreted effector proteins that modulate plant metabolism to suppress or re-programme plant defences. Understanding the molecular functions of Fg effectors will help to elucidate the processes underlying wheat spike colonisation and fungal pathogenicity. With the aim of identifying Fg effector proteins that can suppress host plant defences, I selected using next generation sequencing and bioinformatic analysis, a set of small secreted proteins (SSP) to express in planta using the Barley stripe mosaic virus over-expression system (BSMV-VOX). I then tested whether expression of any of these SSPs enhanced Fg fungal infection of susceptible wheat spikes. Amongst the set of Fg SSP tested, FgSSP8, which encodes a ribonuclease protein, induced strong symptoms of necrosis in N. benthamiana leaves when infiltrated via the BSMV:FgSSP8. Three other genes tested (FgSSP7, FgSSP6 and FgSSP5) enhance FEB disease formation in the majority of the experiments when overexpressed in wheat ears prior to infecting with F. graminearum. FgSSP6 and FgSSP7 belong to the cerato-platanin protein (CPP) family. In several other plant pathogenic fungi, CPPs have been implicated in a number of virulence and plant protection mechanisms, including induction of host plant cell death, binding specific polymers and/or expansin-like activity. FgSSP5 encodes a protein that possesses the pfam domain RALF (Rapid alkalinization factor; PF05498.6). RALF domain-containing proteins are predominately found in plants and play a role in plant development regulating tissue expansion and/or negatively regulating pollen tube elongation. BLAST analyses identified RALF domain containing proteins in a restricted range of different pathogen species. Based on the VOX results and biochemical tests, our hypothesis is that pre-elevated cerato-platanins (FgSSP6 and FgSSP7) levels in the apoplast/surrounding the hyphae could initially shield the hyphae from detection by the plant, but late induce an intense defence response culminating in cell death to benefit the necrotrophic phase of Fg by increasing nutrient availability. FgSSP5 may be a specific virulence factor that manipulates a key plant process, by alkalinising the plant environment during infection, and using the same plant receptor repertoire used to recognise plant proteins. Once the mechanisms are further understood, these genes/proteins could potentially be novel intervention targets either for conventional chemistries and/or for methods such as host-induced gene silencing to achieve FEB disease and/or mycotoxin control. The characterisation of single and double gene deletion F. graminearum mutants is in progress.

Thèse doctorat : Neurosciences : Strasbourg 1 : 2007. Thèse doctorat : Neurosciences : Freiburg - Allemagne : 2007.
Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. p. 204-228.

Quelques régions stables ont été découvertes sur les gp d’env du VIH-1 contre lesquelles des patients produisent des anticorps neutralisants. Les épitopes les plus prometteurs se trouvent dans la MPER et sont probablement exposés durant la fusion. Alors que les peptides isolés à partir de cette région ne sont pas parvenus à induire une réaction immunogène neutralisante, des études antérieures suggèrent que la membrane lipidique joue un rôle dans la structuration des antigènes et dans la réponse immunogénique.C’est pourquoi nous étudions la structure de ces épitopes. Cela nécessite leur surexpression, leur purification et leur reconstitution dans des liposomes. Une étude de CD montre qu’ils pourraient changer de conformation, cela sera confirmé par RMN. En outre, leur immunogénicité sera vérifiée par vaccination des souris. En plus, nous trouvons que le cholestérol peut modifier l’orientation des peptides englobant le motif CRAC de la gp41
A few stable regions have been discovered on the HIV-1 env gp against which some patients produce neutralizing antibodies. The most promising ones are located in the MPER and are probably exposed transiently during the fusion. Whereas the peptides isolated from this region failed to induce immunogenic response, previous studies suggest the lipid membrane plays a role in antigens structure and in the immunogenic response.That is why we investigate the structure of these épitopes in membrane models. This requires the production of these épitopes by bacterial overexpression, their purification and their reconstitution in liposomes. A CD study shows that they could undergo a conformational change; this will be confirmed by NMR. Also their immunogenicity will be checked by mice immunization. In addition, we find that cholesterol could change the orientation of peptides encompassing a gp41 CRAC motif

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

碩士
中興大學
生物化學研究所
94
The triple gene block (TGB) of Bamboo mosaic virus (BaMV) encodes three movement proteins, TGBp1, TGBp2 and TGBp3, which are involved in cell-to-cell movement of viral RNA. However, detailed mechanism of the three TGB proteins to assist viral movement remains to be uncovered. The purpose of this study is to analyze the biochemical properties of TGBp3 as well as the possibility of interaction between TGBp3 and other viral movement components. Amino acid sequence analysis of TGBp3 revealed that it is a 6 kDa protein with an N-terminal hydrophobic transmembrane segment and a hydrophilic C-terminal region. Moreover, about 25% of the amino acid codons in TGBp3 are rarely used by E. coli. Therefore, E. coli Rosetta (DE3) / pLysSRARE carrying plasmid-borne tRNA complementary to the rare codons was used as host for TGBp3 expression. In addition, mutant TGBp3 with the rare codons in the whole coding sequence replaced with the frequently used ones was created. The overexpressed TGBp3 was purified by TALON immobilized metal affinity chromatography and a subsequent gel purification protocol. The identity of the purified protein was confirmed by N-terminal sequencing to be TGBp3. To confirm that TGBp3 is really an integral membrane protein, a micelle-vesicle transition theory was adopted to reconstitute TGBp3-containing proteoliposomes. Ficoll gradient centrifugation of the reconstituted sample revealed that TGBp3 was able to co-migrate with phospholipid, suggesting that it is membrane associated. Chemical treatments of TGBp3-containing liposomes revealed that most of TGBp3 is integrated in lipid-bilayer. In the future, the membrane topology of TGBp3 and the interaction between TGBp3 and other viral movement components will be studied.

碩士
國立中興大學
農業生物科技學研究所
85
The product of open-reading frame 1(ORF1) of bamboo mosaicvirus(BaMV) has been postulated to be a replicase that is involved in the replication of viral genome and in the capping reaction of the positive strand RNA according to the analysis of nucleotide sequence. In this study, the corresponding cDNA was inserted into pET-32a or pET29a and transformed intoEscherichia coli strain BL21(DE3). The recombinant viral protein, fused at N-terminus with thioredoxin , was over-expressed under the induction of isopropyl b-D-thiogalactopyranoside, and partial purified by differential S*Tag affinity chromatography or glycerol gradient centrifugation. The purified enzyme was able to produce P32-labeled RNA fragments upon the addition of particular RNA substrates. Among the RNA molecules tested in this study, such reaction was observed when the substrate was 3*-terrminal fragment of positive strand or negative strand RNA of BaMV. The sensitivity of the P32-labeled RNA products to nuclease S1 digestion suggests that they were arise from the incorporation of a-P32-GTP or a-P32-UTP moiety at 3*-end of RNA substrates. Neither guanylyltransferase or methyltransferase was observed in this study.

Vaccinia virus plays an important role in human medicine and molecular biology ever since the 18th century after E. Jenner discovered its value as a vaccination virus against smallpox. After the successful eradication of smallpox, vaccinia virus, apart from its use as a vaccine carrier, is today mainly used as a viral vector in molecular biology and increasingly in cancer therapy. The capability to specifically target and destroy cancer cells makes it a perfect agent for oncolytic virotherapy. Furthermore, the virus can easily be modified by inserting genes encoding therapeutic or diagnostic proteins to be expressed within the tumor. The emphasis in this study was the diagnosis of tumors using different vaccinia virus strains. Viruses with metal-accumulating capabilities for tumor detection via MRI technology were generated and tested for their usefulness in cell culture and in vivo. The virus strains GLV-1h131, GLV-1h132, and GLV-1h133 carry the gene encoding the two subunits of the iron storage protein ferritin under the control of three different promoters. GLV-1h110, GLV-1h111, and GLV-1h112 encode the bacterial iron storage protein bacterioferritin, whereas GLV-1h113 encodes the codon-optimized version of bacterioferritin for more efficient expression in human cells. GLV-1h22 contains the transferrin receptor gene, which plays an important role in iron uptake, and GLV-1h114 and GLV-1h115 contain the murine transferrin receptor gene. For possibly better iron uptake the virus strains GLV-1h154, GLV-1h155, GLV-1h156, and GLV-1h157 were generated, each with a version of a ferritin gene and a transferrin receptor gene. GLV-1h154 carries the genes that encode bacterioferritin and human transferrin receptor, GLV-1h155 the human ferritin H-chain gene and the human transferrin receptor gene. GLV-1h156 and GLV-1h157 infected cells both express the mouse transferrin receptor and bacterioferritin or human ferritin H-chain, respectively. The virus strains GLV-1h186 and GLV-1h187 were generated to contain a mutated form of the ferritin light chain, which was shown to result in iron overload and the wildtype light chain gene, respectively. The gene encoding the Divalent Metal Transporter 1, which is a major protein in the uptake of iron, was inserted in the virus strain GLV-1h102. The virus strain GLV-1h184 contains the magA gene of the magnetotactic bacterium Magnetospirillum magnetotacticum, which produces magnetic nanoparticles for orientation in the earth’s magnetic field. Initially the infection and replication capability of all the virus strains were analyzed and compared to that of the parental virus strain GLV-1h68, revealing that all the viruses were able to infect cells of the human cancer cell lines A549 and GI-101A. All constructs exhibited a course of infection comparable to that of GLV-1h68. Next, to investigate the expression of the foreign proteins in GI-101A and A549 cells with protein analytical methods, SDS-gelelectrophoresis, Western blots and ELISAs were performed. The proteins, which were expressed under the control of the strong promoters, could be detected using these methods. To be able to successfully detect the protein expression of MagA and DMT1, which were expressed under the control of the weak promoter, the more sensitive method RT-PCR was used to at least confirm the transcription of the inserted genes. The determination of the iron content in infected GI-101A and A549 cells showed that infection with all used virus strains led to iron accumulation in comparison to uninfected cells, even infection with the parental virus strain GLV-1h68. The synthetic phytochelatin EC20 was also shown to enhance the accumulation of different heavy metals in bacterial cultures. In vivo experiments with A549 tumor-bearing athymic nude mice revealed that 24 days post infection virus particles were found mainly in the tumor. The virus-mediated expression of recombinant proteins in the tumors was detected successfully by Western blot. Iron accumulation in tumor lysates was investigated by using the ferrozine assay and led to the result that GLV-1h68-infected tumors had the highest iron content. Histological stainings confirmed the finding that iron accumulation was not a direct result of the insertion of genes encoding iron-accumulating proteins in the virus genome. Furthermore virus-injected tumorous mice were analyzed using MRI technology. Two different measurements were performed, the first scan being done with a seven Tesla small animal scanner seven days post infection whereas the second scan was performed using a three Tesla human scanner 21 days after virus injection. Tumors of mice injected with the virus strains GLV-1h113 and GLV-1h184 were shown to exhibit shortened T2 and T2* relaxation times, which indicates enhanced iron accumulation. In conclusion, the experiments in this study suggest that the bacterioferritin-encoding virus strain GLV-1h113 and the magA-encoding virus strain GLV-1h184 are promising candidates to be used for cancer imaging after further analyzation and optimization
Das Vaccinia Virus spielt in der Humanmedizin und Molekularbiologie eine wichtige Rolle seit E. Jenner im 18. Jahrhundert seinen Nutzen als Impfvirus entdeckt hat. Nach der erfolgreichen Ausrottung der Pocken, wird das Vaccinia Virus heutzutage neben der Anwendung als Impfstoffträger hauptsächlich als viraler Vektor in der Molekularbiologie und in zunehmendem Maße in der Krebstherapie verwendet. Die Fähigkeit Krebszellen gezielt zu zerstören, macht es zu einem perfekten Wirkstoff für die onkolytische Virotherapie. Des Weiteren kann das Virus durch das Inserieren von Genen, die für therapeutische oder diagnostische Proteine kodieren, und im Tumor exprimiert werden, modifiziert werden. Der Schwerpunkt dieser Arbeit war die Tumordiagnose mit Hilfe verschiedener Vaccinia Virusstämme. Viren mit der Fähigkeit, Metalle anzureichern wurden zur Tumordetektion mittels Kernspintomographie hergestellt und auf ihre Nutzbarkeit in Zellkultur und in vivo getestet. Die Virusstämme GLV-1h132, GLV-1h132 und GLV-1h133 tragen das Gen, welches für die zwei Untereinheiten des Eisenspeicherproteins Ferritin kodieren unter der Kontrolle von drei verschiedenen Promotoren. GLV-1h110, GLV-1h111, und GLV-1h112 tragen das Gen, welches für das bakterielle Eisenspeicherprotein Bacterioferritin kodiert, wohingegen das inserierte Gen in GLV-1h113 für die codon-optimierte Version dieses Proteins kodiert, die eine effizientere Expression in humanen Zellen ermöglichen soll. GLV-1h22 beinhaltet das Transferrin-Rezeptor-Gen, welches eine wichtige Rolle in der Eisenaufnahme spielt, und GLV-1h114 und GLV-1h115 beinhalten das murine Transferrin-Rezeptor-Gen. Für eine möglicherweise bessere Eisenaufnahme wurden die Virusstämme GLV-1h154, GLV-1h155, GLV-1h156 und GLV-1h157 mit je einer Version eines Ferritin-Gens und eines Transferrin-Rezeptor-Gens generiert. GLV-1h154 trägt die Gene, die für Bacterioferritin und den humanen Transferrin Rezeptor kodieren, GLV-1h155 trägt die Gene für die humane Ferritin H-Untereinheit und den humanen Transferrin Rezeptor. Zellen, die mit GLV-1h156 und GLV-1h157 infiziert wurden, exprimierten den Maus-Transferrin-Rezeptor und Bacterioferritin beziehungsweise die humane Ferritin-H-Untereinheit. Die Virusstämme GLV-1h186 und GLV-1h187 wurden mit einer mutierten Form der leichten Untereinheit von Ferritin, für die eine Überladung mit Eisen gezeigt wurde, beziehungsweise mit der leichten Untereinheit des wildtypischen Gens ausgestattet. Das Gen, das für den Divalenten Metal Transporter 1 kodiert, welches ein bedeutendes Protein für die Aufnahme von Eisen darstellt, wurde in den Virusstamm GLV-1h102 inseriert. Der Virusstamm GLV-1h184 trägt das magA Gen des magnetotaktischen Bakteriums Magnetospirillum magnetotacticum, welches magnetische Nanopartikel zur Orientierung im Erdmagnetfeld produziert. Zunächst wurde die Infektions- und Replikationsfähigkeit aller Viren analysiert und mit der des Ausgangsstammes GLV-1h68 verglichen, was zeigte, dass alle Viren in der Lage waren humane Krebszellen der Zelllinien GI-101A und A549 zu infizieren. Alle Konstrukte zeigten einen vergleichbaren Infektionsverlauf zu GLV-1h68. Als nächstes, um die Expression der fremden Proteine in GI-101A und A549 Zellen zu untersuchen, wurden SDS-Gelelektrophorese, Western Blots und ELISAs durchgeführt. Die Proteine, welche unter der Kontrolle von starken Promotoren exprimiert wurden, konnten mit diesen Methoden detektiert werden. Um die Expression von MagA und DMT1 zu detektieren, welche unter der Kontrolle des schwachen Promotors exprimiert wurden, wurde die sensitivere Methode RT-PCR angewendet, mit der zumindest die Transkription der Gene nachgewiesen werden konnte. Die Bestimmung des Eisengehaltes in infizierten GI-101A und A549 Zellen zeigte, dass die Infektion mit allen Viren im Vergleich zu uninfizierten Zellen zu einer Eisenanreicherung führte, sogar die Infektion mit dem Ausgangsstamm GLV-1h68. Für das synthetische Phytochelatin EC20 wurde auch eine Anhäufung von verschiedenen Schwermetallen in Bakterienkulturen gezeigt. In vivo Experimente mit A549 tumor-tragenden athymischen Nacktmäusen ergaben, dass Viruspartikel 24 Tage nach der Infektion hauptsächlich im Tumor gefunden wurden. Die von den Viren vermittelte Expression der rekombinanten Proteine in den Tumoren wurde erfolgreich mit Hilfe von Western Blots detektiert. Die Eisenansammlung in Tumorlysaten wurde mit dem Ferrozine Assay untersucht und führte zu dem Ergebnis, dass Tumore, die mit GLV-1h68 infiziert wurden, den höchsten Eisengehalt vorwiesen. Histologische Färbungen bestätigten die Erkenntnis, dass die Eisenansammlung nicht ein direktes Resultat der Insertion von eisenansammelnden Genen in das Virusgenom war. Darüberhinaus wurden tumortragende Mäuse, denen Virus injiziert wurde, mittels Kernspintomographie analysiert. Zwei verschiedene Messungen wurden durchgeführt, wobei die erste Messung sieben Tage nach Virusinjektion mit einem sieben Tesla Kleintier-Scanner durchgeführt wurde und die zweite Messung mit einem humanen drei Tesla Scanner 21 Tage nach Virusinjektion. Tumore von Mäusen, die mit den Virusstämmen GLV-1h113 und GLV-1h184 injiziert wurden, zeigten verkürzte T2- und T2*-Relaxationszeiten, was auf eine verbesserte Eisenakkumulation hinweist. Zusammenfassend deuten die Experimente dieser Studie darauf hin, dass der Virusstamm GLV-1h113, welcher für Bacterioferritin kodiert, und der Virusstamm GLV-1h184, welcher für MagA kodiert, nach weiterer Untersuchung und Optimierung vielversprechende Kandidaten für die Krebs Bildgebung sind

博士
國立成功大學
基礎醫學研究所
96
Dengue virus infection may cause self-limited dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). The viral load is the key point of the pathogenesis of dengue virus infection. Moreover, vascular leakage, thrombocytopenia and hemoconcentration are the hallmarks of dengue hemorrhage fever (DHF) and dengue shock syndrome (DSS). Permeability aberration may contribute to the plasma leakage, which is most likely affected by certain soluble mediators. Cytokines may play an important role in the vascular leakage. Autophagy, a cellular response against stresses, functions to recycle protein and organelles. However, the roles of autophagy in disease pathogenesis and microbial infection remain unclear. Several viral infections could induce autophagy and replicate using the autophagosome. We found that dengue virus-2 (DV2) infection can induce punctate GFP-microtubule-associated protein light chain (GFP-LC3) and increased expression of the proteolytic derivate LC-II in DV2 infected cells. DV2 infection induced autophagosome formation revealed by immuno-gold labeling of LC3 protein under the transsimission electron microscopy. The mTOR/p70S6K signaling pathway may be involved in DV2-induced autophagy. Further study reveals that DV2 infection-induced autophagy can enhance the replication of the virus in the infected cells. Furthermore, high levels of monocyte chemoattractant protein-1 (MCP-1) were detected in the plasma of DHF patients. DV2 infection can induce MCP-1 production in monocytes, but not in hepatoma and endothelium cells. The production of MCP-1 is dengue virus-dose dependent. Exposure of human umbilical vein endothelial cells (HUVEC) to recombinant human MCP-1 (rhMCP-1) or the cultured supernatant of DV2-infected human monocytes increased vascular permeability of HUVECs. Neutralizing monoclonal antibody of MCP-1 partially reduced vascular permeability. The distribution of the tight junction protein ZO-1 on the cellular membrane of HUVEC was disrupted either by pretreatment of recombinant MCP-1 or the conditioned medium from DV2-infected monocytes. All together, we unravel that dengue virus infection can induce autophagosome formation and increase viral replication. Moreover, increased viral load sustains the overexpression of MCP-1, which further increases the degree of the vascular permeability. Our findings open a new direction toward understanding the pathogenesis of DHF.

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.

Le criblage par siRNA de 49 protéines de l'hôte qui sont incorporées dans les particules matures du virus herpès simplex de type 1 (VHS-1) a révélé l'importance d'au moins 15 d’entre elle pour infectivité du virus (Stegen, C et al. 2013). Parmi celle-ci figure la protéine humaine DDX3X, qui est une ARN hélicase ATP-dépendante. Cette protéine multifonctionnelle participe à différents stages de l'expression génique, tels que la transcription, la maturation et le transport d'ARNm ainsi que la traduction. DDX3X est impliquée dans la réplication de plusieurs virus tels que le Virus de l’immunodéficience humaine de type 1 (VIH-1), l'hépatite B (VHB), le virus de la vaccine (VACV) et le virus de l'hépatite C (VHC). Le rôle exact de DDX3X dans le cycle de réplication du VHS-1 est toutefois inconnu. Ce mémoire consiste en l’étude détaillée de l'interaction de DDX3X avec le virus. De manière surprenante, tant l’inhibition que la surexpression de DDX3X réduit de manière significative l'infectivité du VHS-1. Fait intéressant, lorsque nous avons restauré la déplétion de DDX3X par une construction résistante aux ARNi utilisés, le virus pouvait de nouveau infecter les cellules efficacement, indiquant que le virus est sensible aux quantités de cette protéine de son hôte. Nos résultats indiquent de plus que le virus modifie la localisation de DDX3X et cause son agrégation tôt dès les premiers temps de l'infection. Cependant, le virus ne modifie pas les niveaux cellulaires de DDX3X dans deux des trois lignées cellulaires examinées. Nous avons également pu établir que cette protéine n'a pas d'effet sur l'entrée du VHS-1, suggérant qu’elle agit à un stade ultérieure de l’infection. En examinant cette relation plus en détail, nos résultats ont démontré que l’inhibition ou la surexpression de DDX3X inhibent toutes deux la production de nouvelles particules virales en réduisant l'expression des diverses classes cinétiques des protéines virales et ce au niveau de leur transcription. Malgré le rôle connu DDX3X dans la stimulation de la réponse immunitaire innée et la production d’interférons de type I, l’impact de DDX3X sur la réplication du VHS-1 est ici indépendante de cette fonction. Ces travaux démontrent donc une nouvelle voie d’action de DDX3X sur les virus en agissant directement sur la transcription de gènes viraux et la réplication du génome d’un virus à ADN. En comprenant mieux cette interactions hôtepathogène, il est maintenant envisageable de concevoir des nouvelles approches thérapeutiques contre ce virus.
siRNA screening of 49 host proteins that are known to be incorporated in the mature virions of herpes simplex virus type 1 (HSV-1) revealed the importance of at least 15 cellular proteins for viral infectivity (Stegen, C et al. 2013). Among these, was the human protein DDX3X, a DEAD-box ATP-dependent RNA helicase. This multifunctional protein participates in different stages of gene expression such as mRNA transcription, maturation, mRNA export and translation. DDX3X has been shown to be involved in the replication of several viruses such as human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) vaccinia virus (VACV) and hepatitis C virus (HCV). The exact role of DDX3X in HSV-1 replication cycle is not known. Here we sought to find the detailed interaction between DDX3X with HSV-1. Surprisingly, the down-regulation as well as overexpression of DDX3X, significantly reduced the infectivity of HSV-1, indicating that the virus is sensitive to the precise levels of DDX3X. Accordingly, when we rescued DDX3X back to its normal cellular levels by sequential transfection of DDX3X siRNA and siRNA resistant DDX3X plasmid, the virus was able to infect cells efficiently compare to wild-type conditions. Furthermore, the virus changes the localization of DDX3X and causes its aggregation at early times in the infection. However, the virus does not change the cellular levels of DDX3X in at least two of three different cell lines tested. Using a luciferase assay we were able to establish that this protein has no effect on the entry of HSV-1. In fact, depleting or overexpressing DDX3X impaired the production on newly assembled viral particles by blocking the expression of all classes of viral proteins at the transcription level. Despite the known role of DDX3X in the stimulation of innate immune response and interferon type I production, DDX3X appears to act on HSV-1 replication independently of this pathway. This highlights a novel route of action of DDX3X by acting at the transcription level and the consequent genome replication of a DNA virus. By better understanding such pathogen interactions, it might now be possible to design novel therapeutic approaches.

博士
國立臺灣大學
微生物學研究所
88
Overexpression of HER-2/neu proto-oncogene frequently occurs in many different types of human cancers, including ovarian carcinoma, and is known to enhance tumor metastasis and chemoresistance. Previous studies showed that inhibition of HER-2/neu expression by various agents, such as adenovirus E1A protein and simian virus 40 large T antigen, can lead to suppression of tumorigenicity of HER-2/neu-overexpressing cancer cells. In an effort to search for other agents that can repress HER-2/neu expression, we found that T/t-common, which contains the N-terminal common domain of simian virus 40 large T and small t antigens, could specifically repress the HER-2/neu promoter. When the coding sequence of T/t-common was stably transfected into the HER-2/neu-overexpressing human ovarian carcinoma SK-OV-3 cells, the expression of HER-2/neu was dramatically reduced by the expression of T/t-common. Accordingly the tumorigenic potential of these T/t-common-expressing clones, including the ability to grow anchorage-independently and the ability to induce tumor in nu/nu mice, was also drastically suppressed. Furthermore, when T/t-common was transiently cotransfected with the activated genomic neu into NIH/3T3 cells, the transforming activity of the latter was suppressed by T/t-common in soft-agarose microcolony formation assays. Taken together, these data suggest that T/t-common may act as a transformation suppressor of the HER-2/neu oncogene. To develop an effective HER-2/neu-targeting cancer gene therapy with T/t-common, we have constructed a recombinant adenovirus carrying the T/t-common gene, rAdNT/t, and a control adenovirus, rAdV, which is similar to rAdNT/t except lacking the T/t-common sequence. The ability of the T/t-common-carrying adenovirus to suppress the tumorigenicity of HER-2/neu-overexpressing human breast cancer cells, AU565, was tested by soft-agarose colony formation assays. We found that rAdNT/t-infected AU565 cells gave much reduced number of colonies than the mock infected and control virus infected AU565 cells. These results suggests that this T/t-common-carrying adenovirus, rAdNT/t, has the ability to suppress the tumorigenic potential of the HER-2/neu-overexpressing cancer cells, and may be used in gene therapy for treating cancers with HER-2/neu overexpression .

碩士
國立臺灣大學
微生物學研究所
106
There are 15-20 million people worldwide chronically coinfected with Hepatitis delta virus (HDV) and Hepatitis B virus (HBV). Simultaneous infection of HBV and HDV may progress easier into the most severe form of hepatitis, fulminant hepatitis, than infection of HBV only. Fulminant hepatitis causes rapid death of hepatocytes and liver failure. HDV is a satellite virus of HBV and requires HBV surface proteins (HBs Ag) to serve as viral envelopes. Since HDV is the smallest virus in animal and encodes only one protein, delta antigen, which does not possess the activity to replicate HDV genome, it must utilize host enzymes or other host factors to fulfill its life cycle. To study the role of host factors in HDV life cycle, researchers use primary human hepatocytes (PHH) and HepaRG cells in cell infection model. However, limited supply and difficult management of these cells render experiments hard to be manipulated and reproduced. The discovery of sodium-taurocholate cotransporting polypeptide (NTCP) as a HBV or HDV receptor opens a gate for researchers. Some studies indicate that overexpression of hNTCP makes cells susceptible to HBV or HDV infection. However, HDV infection rate is less than 1% even in hNTCP-overexpressing hepatoma cell line. In order to optimize the infection model in our laboratory, I used suspension infection, treatment of DMSO and PEG8000 and different clones of HDV. In our work, HDV infection rate increases from 0.1% to about 30% in the infection model. I also examine the expression of HDV genotype I, II and IV in the infection model. Since this infection model is established, the relationship between host and HDV can be further examined and eventually, find an effective antiviral therapy.

碩士
國立臺灣大學
微生物學研究所
93
HER-2/neu proto-oncogene belongs to epidermal growth factor receptor families. Overexpression or gene amplification of HER-2/neu has been detected frequently in many types of human cancers, including cancers of breast, ovary, lung, kidney, colon, bladder, stomach and salivary gland. Overexpression of HER-2/neu enhances metastatic potential, chemoresistance and tumor angiogenesis. Previous studies showed that inhibition of HER-2/neu gene expression leads to suppression of tumorigenicity of HER-2/neu-overexpressing cancer cells. Therefore, HER-2/neu gene represents a suitable target for cancer therapy. Our lab previously reported that T/t-common, which contains the N-terminal common domain of simian virus 40 large T and small t antigens, can specifically repress the HER-2/neu promoter and its expression in HER-2/neu-overexpressing ovarian carcinoma SK-OV-3 cells. This phenomenon may presumably contribute to T/t-common suppression of the tumorigenic potential of these cancer cells. Furthermore, we discovered that the ability of T/t-common to suppress the tumorigenic potential of HER-2/neu-overexpressing cells is through induction of apoptosis. T/t-common specifically induces apoptosis in HER-2/neu-overexpressing cells but not in HER-2/neu low-expressing or non-transformed cells. Radiotherapy and chemotherapy are most common ways to treat cancer, but the overdose of drugs usually causes severe side effects such as vomits, alopecia, leucopenia, and susceptible to bacterial infections. In addition, chemoresistances often occurred during the prolonged period of treatment. Therefore, based on previous studies in our lab, we further investigated the possibilities of combination therapy using T/t-common and chemotherapeutic agents or irradiation in treatment of HER-2/neu-overexpressing cancers. Our data indicate that T/t-common can specifically enhance cell death induced by either etoposide or irradiation in HER-2/neu-overexpressing breast cancer cells SK-OV-3 and AU565 but not in HER-2/neu low-expressing breast cancer cells MDA-MB-231 and laryngeal cancer cells HEp-2. Further, this sensitization of HER-2/neu-overexpressing cancer cells to etoposide or irradiation is through the induction of apoptosis.

碩士
淡江大學
化學學系碩士班
101
PartⅠ Tobacco mosaic virus is the most common virus in the plant. The genome of this virus can be translated to protein which contains methyltransferase activity. In the past, a lot of researches focused on the viral nucleic acid methylation of the host. The host protein methylation has not yet been studied. In this study, we used recombinant E. coli to express heterologous protein. At first, E. coli. expressed the his-tag protein with inclusion body. We tried a number of conditions, such as cooling or adding surfactants, but it still expressed the protein with inclusion body. Some studies have found that GST tag could help protein fold with correct form. Therefore, we used pGEX4T-1 as a carrier vector to construct pGEX4T-1-MT and finally expressed the protein present in the supernatant. We purified the GST fusion protein with GSH column and reacted with Hot-SAM and tobacco crude protein. Experimental results showed that it had more substrate signal in the infected plants than uninfected plants. Therefore, E. coli can express heterologous proteins with activity and may allow host protein methylation. PartⅡ Previous studies have found that ADH3 protein in Saccharomyces cerevisiae affects morphology of mitochondria. The putative reason is that ADH3 protein can influence NADH/NAD+ ratio. It has been shown that GPD1 protein could synthesize glycerol and oxidize NADH to NAD+. Therefore, we constructed pYes2-GPD1-GFP and transformed it to S. cerevisiae. Experimental results show that after we induced the construct gene, pYes2-GPD1-GFP, the mitochondrial morphology of S. cerevisiae appeared fragmented for 83 percent. With non-induced genes, pYes2-GPD1-GFP and pYes2, the mitochondrial morphology of S. cerevisiae appeared tubular. When we replaced the medium glucose to glycerol, we found that both constructs, pYes2-GPD1-GFP and pYes2, made mitochondrial morphology to patterns appear fragmented. The result showed that the morphology of mitochondria may be influenced by NADH/NAD+ ratio.

Indiana University-Purdue University Indianapolis (IUPUI)
After spinal cord injury (SCI), poor axonal regeneration of the central nervous system, which mainly attributed to glial scar and low intrinsic regenerating capacity of severely injured neurons, causes limited functional recovery. Combinatory strategy has been applied to target multiple mechanisms. Schwann cells (SCs) have been explored as promising donors for transplantation to promote axonal regeneration. Among the central neurons, descending propriospinal neurons (DPSN) displayed the impressive regeneration response to SCs graft. Glial cell line-derived neurotrophic factor (GDNF), which receptor is widely expressed in nervous system, possesses the ability to promote neuronal survival, axonal regeneration/sprouting, remyelination, synaptic formation and modulate the glial response. We constructed a novel axonal permissive pathway in rat model of thoracic complete transection injury by grafting SCs over-expressing GDNF (SCs-GDNF) both inside and caudal to the lesion gap. Behavior evaluation and histological analyses have been applied to this study. Our results indicated that tremendous DPSN axons as well as brain stem axons regenerated across the lesion gap back to the caudal spinal cord. In addition to direct promotion on axonal regeneration, GDNF also significantly improved the astroglial environment around the lesion. These regenerations caused motor functional recovery. The dendritic plasticity of axotomized DPSN also contributed to the functional recovery. We applied a G-mutated rabies virus (G-Rabies) co-expressing green fluorescence protein (GFP) to reveal Golgi-like dendritic morphology of DPSNs and its response to axotomy injury and GDNF treatment. We also investigated the neurotransmitters phenotype of FluoroGold (FG) labeled DPSNs. Our results indicated that over 90 percent of FG-labeled DPSNs were glutamatergic neurons. DPSNs in sham animals had a predominantly dorsal-ventral distribution of dendrites. Transection injury resulted in alterations in the dendritic distribution, with dorsal-ventral retraction and lateral-medial extension of dendrites. Treatment with GDNF significantly increased the terminal dendritic length of DPSNs. The density of spine-like structures was increased after injury and treatment with GDNF enhanced this effect.