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Analysis of retroviral production in murine leukaemia virusYap, Wee Ching Melvyn January 2000 (has links)
No description available.
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Etude de la régulation de l’export nucléaire de l’ARN non-épissé du virus de la leucémie murine / Study of the unspliced RNA nuclear export regulation of the murine leukemia virus (MLV)Pessel-Vivares, Lucie 09 October 2014 (has links)
Les cellules eucaryotes ont évolué de façon à s'assurer qu'aucun ARNm contenant des introns ne soit exporté du noyau. Néanmoins, les rétrovirus ont besoin d'exporter leur ARN non-épissé vers le cytoplasme afin de servir de matrice pour la traduction des protéines virales, ou d'être encapsidé comme ARN génomique dans les néo-virions. Différentes stratégies ont alors été mises en place par ces virus dans le but de détourner les voies d'export nucléaire cellulaires pour mener à bien l'export de leur ARN non-épissé. Le virus de la leucémie murine (MLV), l'un des premier rétrovirus de mammifère découvert, possède un génome qui, à l'inverse d'autres rétrovirus ne permet pas de coder pour des protéines accessoires pouvant aider à l'export de son ARN non-épissé. Bien que différentes séquences cis-régulatrices présentes sur l'ARN non-épissé ont été montrées comme favorisant l'export nucléaire de cet ARN, la/(les) voie(s) d'export détournée(s) par le MLV reste(nt) jusqu'alors inconnue(s).Dans les travaux présentés ici nous démontrons que le virus du MLV est capable de détourner la voie d'export cellulaire Tap pour exporter ses ARN épissé et non-épissé du noyau. Cet export permet notamment au virus d'exprimer ses protéines structurales et enzymatiques. De plus, nos résultats suggèrent également que l'export de l'ARN non-épissé du MLV est aussi possible par la voie d'export cellulaire CRM1, afin de favoriser leur encapsidation. En résumé, nos données révèlent un mode de régulation complexe, mis en place par le MLV, afin d'exporter l'ARN non-épissé et ainsi de distinguer sa destinée. / Eukaryotic cells have evolved to ensure that intron-containing mRNA do not leave the nucleus. However, retroviruses must export their intron-containing RNA in the cytoplasm to be either translated in viral proteins or packaged as genomic RNA in progeny viruses. Then, retroviruses are using different mechanisms in order to hijack cellular nuclear export pathway to export their unspliced RNA. Murine leukemia virus (MLV) is a simple retrovirus, one of the first discovered, which do not have the possibility to encode accessory proteins to help its unspliced RNA export. Although several cis-elements of the MLV unspliced RNA have been identified to regulate the cytoplasmic accumulation of this RNA, the pathway(s) hijacked by the MLV is(are) unrevealed until today. The researches present in this manuscript show that the MLV is able to hijack the cellular export pathway Tap dependant to export its spiced and unspliced RNA from the nucleus. This export leads to the expression of structural and enzymatic viral proteins. Moreover, our results suggest that the MLV can also hijack the cellular export factor CRM1 to export its unspliced RNA in order to package them. Finally, our data reveal the existence of a complex regulation mechanism use by the MLV to export and distinguish unspliced RNA regarding their destiny.
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Construction of a modified live HP-PRRS virus vaccine and an attenuated listeria vaccine vector using reverse geneticsRen, Jie January 1900 (has links)
Master of Science / Department of Anatomy and Physiology / Jishu Shi / The development of reverse genetics systems for the manipulation of viral and bacterial genomes has provided platforms for identifying virulence genes, studying pathogenesis and developing vaccines. Replication-competent vaccines (e.g., modified live virus (MLV) vaccines and replicating viral/bacterial vectors) are considered the most efficacious approach for vaccine development. We constructed replication-competent candidate vaccines for two viral diseases in pigs via reverse genetics. The first vaccine we designed is to protect against highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). HP-PRRSV can cause high mortality in pigs of all ages. Vaccines to protect pigs from HP-PRRSV are not commercially available in the US. According to previous studies, the non-structural protein (NSP) coding region of HP-PRRSV is closely related to the high mortality rate and the structural protein (SP) coding region contributes to the induction of broadly protective neutralizing antibodies. We created a chimeric PRRSV, of which the SP coding region was derived from HP-PRRSV and NSP coding region was derived from a low-pathogenic strain. This chimeric PRRSV caused similar CPE in cells as parental viruses, but had slower growth kinetics. We hypothesize that this chimeric virus will have a low pathogenicity and could serve as a candidate vaccine that can provide protection against HP-PRRV. The second vaccine vector is a modified Listeria innocua (L.inn), a non-pathogenic strain of Listeria. Genetically related Listeria monocytogenes (L.m) is a well-known intracellular pathogen that encodes specialized virulent determinants facilitating its intracellular growth and spread. Our goal is to make L.inn a vaccine vector that can deliver classical swine fever (CSF) viral antigen into intracellular environments by complementation of L.inn with selected L.m virulence genes necessary for intracellular survival and induction of a robust immune response. In this study, we constructed a shuttle vector pHT-E2 that can express CSFV antigen E2 in L.inn. We cloned the plcA-prfA operon of L.m virulence gene cluster (vgc) into pHT-E2, which enhanced the expression of E2 in L.inn. In future studies, we plan to clone additional L.m virulence genes into the shuttle vector to increase immunogenicity of this recombinant L.inn and test its ability to protect pigs from CSFV.
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Möjligheter att samnyttja parallellväg vid mötesfri landsväg för drift, underhåll och cykeltrafik / Opportunities for the combined use of parallel roads on median divided carriageway for operation, maintenance and bicycle trafficKilefors, John January 2016 (has links)
Purpose: The introduction of median divided carriageways, MDC, has brought difficulties to the performing of maintenance tasks and operational improvements without disrupting traffic or compromising work safety. Meanwhile, cyclists have suffered deteriorating conditions along these roads. The aim of this study was to investigate whether the operational and maintenance tasks on this road type can be facilitated by the use of a parallel road, which can also be used for bicycle traffic. Method: The work is mostly based on qualitative semi-structured interviews with both operating entrepreneurs and representatives from the client side. Alongside this, some literaturestudy has been made. Findings: Work safety is a concern when working at MDC. For major planned actions, redirecting traffic is always the first option, but it can be difficult to find suitable diversion routes. It is more expensive to operate MDC than conventional carriageways, but there are no key formulas that tell you how much more expensive. Along some difficult stretches, especially 1 + 1 sections, some measures may be withheld or delayed. The benefits of a parallel road are many, especially for rerouting traffic. Some operations could also be conducted from a parallel road if it is close enough to the main road. If the parallel road is to be used for operation and maintenance as well as bicycle traffic, the road cannot be converted into cycling way as other vehicles can be expected to appear. Implications: The benefit of a parallel road is primarily the ability to reroute traffic. Diversion will probably become even more relevant in the future, with more narrow median divided carriageways sections combined with increased focus on work safety. The tasks that can be conducted from the parallel road makes such demands on the placement of the parallel road, that the benefits in relation to this are probably limited. The combined use for traffic redirection and cycling can be a good alternative, but this must be looked at from a project-specific perspective as the conditions are so different. Limitations: Conditions differ hugely in all projects. This report is intended for general application and can be viewed as a tool where one or more solutions can be applicable to the project. The financial aspect is not considered in this project. Maximum improvement for cyclists has not been studied in this project, operational and maintenance aspects have been prioritised. / Syfte: Införandet av mötesfria landsvägar, MLV, har inneburit svårigheter att utföra drift- och underhållsarbeten utan att störa trafiken eller äventyra arbetsmiljön. Samtidigt har cyklisterna ofta fått försämrade förutsättningar längs dessa vägar. Målet med detta examensarbete var att utreda hur drift och underhåll av MLV kan underlättas av en parallellväg som även kan användas för cykeltrafiken. Metod: Arbetet bygger till stor del på kvalitativa undersökningar i form av semistrukturerade intervjuer med både driftsentreprenörer och representanter från beställarsidan. Vid sidan om detta har litteraturstudier gjorts. Resultat: Arbetsmiljön är ett bekymmer vid arbete på MLV. Vid större planerade åtgärder är alltid omledning av trafiken första alternativet, men det kan vara svårt att finna lämpliga omledningsvägar. Det är dyrare att sköta MLV än vanlig motsvarande väg, men man har inga nyckeltal som talar om hur mycket dyrare. Längs vissa komplicerade sträckor, i synnerhet med 1+1-sektion, kan vissa drift- och underhållsåtgärder utebli eller försenas. Nyttan av en parallellväg är stor, framför allt för omledning av trafiken. Vissa arbetsmoment skulle också kunna utföras från parallellvägen om den ligger tillräckligt nära huvudvägen. För att samnyttja parallellvägen för drift, underhåll och cykeltrafik kan vägen inte antas som cykelväg i vägplan då även andra trafikslag kan förväntas förekomma. Konsekvenser: Nyttan av en parallellväg är framförallt möjligheten att leda om trafiken. Omledning kommer antagligen bli än mer aktuellt framöver, med fler smala MLV-sektioner i kombination med ökat fokus på arbetsmiljön. De arbeten som kan göras från parallellvägen ställer sådana krav på placeringen av vägen att nyttan i förhållande till detta antagligen är begränsad. Att samnyttja omledningstrafiken med cykeltrafik kan vara ett gott alternativ, men detta måste ses ur ett projektspecifikt perspektiv då förutsättningarna är så olika. Begränsningar: Förutsättningarna är så olika i alla projekt. Rapporten är skriven för att gälla generellt och kan ses som en verktygslåda där en eller flera lösningar är applicerbara i projektet. Den ekonomiska aspekten är inte beaktad i detta projekt. Vad som innebär hög måluppfyllelse för cyklister har inte studerats i detta projekt, drift- och underhållsaspekten har prioriterats.
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Assessment and Analysis of the Restriction of Retroviral Infection by the Murine APOBEC3 ProteinAydin, Halil Ibrahim 26 August 2011 (has links)
Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.
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Assessment and Analysis of the Restriction of Retroviral Infection by the Murine APOBEC3 ProteinAydin, Halil Ibrahim 26 August 2011 (has links)
Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.
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Interaction Of The Non Steroidal Anti-inflammatory Drug Celecoxib With Pure And Cholesterol-containing Model MembranesSade, Asli 01 July 2009 (has links) (PDF)
The interactions of the non steroidal anti-inflammatory drug celecoxib with pure and cholesterol containing distearoyl phosphatidylcholine multilamellar vesicles were studied using Fourier transform infrared spectroscopy, differential scanning calorimetry and turbidity technique at 440 nm.
The results reveal that celecoxib exerts opposing effects on membrane order in a concentration dependent manner while cholesterol disorders and orders the membrane in the gel and liquid crystalline phase, respectively. Ternary mixtures of DSPC/Cholesterol/celecoxib behave similar to cholesterol with a small effect of celecoxib. While celecoxib decreases fluidity of the DSPC membranes, cholesterol shows an opposite effect, and in ternary mixtures, a dominant effect of cholesterol is observed. Celecoxib induces opposite effects on the hydration status of the carbonyl groups in the binary system whereas / cholesterol induces hydrogen bonding around this group. An evidence of phase separation has also been observed for all three systems (DSPC/celecoxib, DSPC/Chol, and DSPC/Chol/celecoxib). In addition, a possible location of celecoxib in the interfacial region of the membrane has been proposed. Finally, penetration of celecoxib into the hydrophobic core of the ternary system at high cholesterol concentrations and formation of a new phase has also been suggested.
Thus, depending on the concentration used, celecoxib induces significant changes in the biophysical properties of membranes that may aid in understanding its mechanism of action. Furthermore, highly complex interactions take place in ternary membrane systems and further investigations are needed to explore them in detail.
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Assessment and Analysis of the Restriction of Retroviral Infection by the Murine APOBEC3 ProteinAydin, Halil Ibrahim 26 August 2011 (has links)
Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.
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Assessment and Analysis of the Restriction of Retroviral Infection by the Murine APOBEC3 ProteinAydin, Halil Ibrahim January 2011 (has links)
Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.
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Analysis of the Antigenic Composition and Differential Incorporation of Host Membrane Proteins into Murine Leukemia Virus by Flow VirometryMaltseva, Mariam 29 September 2020 (has links)
Traditionally, viral particles have been primarily analyzed as a whole population according to their biochemical, genetic, and biophysical properties. Here, we describe single particle phenotypic analysis using surface markers found on Murine Leukemia Virus (MLV) by flow virometry. We used this technology to show differential incorporation of host surface markers between wild type MLV and glycosylated Gag (glycogag) deficient MLV. Moreover, we analyzed differential uptake efficiency of host proteins between two cell lines and primary lymphocytes. We hypothesize that the phenotypic profiling and quantification of antigens on the surface of individual viral particles will provide crucial information on the identity of the infected parental cells. Furthermore, we demonstrate that the MLV accessory protein glycogag is associated with the upregulation of surface antigen incorporation during assembly and release. Aside from possible evolutionary implications of glycogag, we demonstrate presence and varying antigenic composition on the surface of MLV viral particles reflective of the cell phenotype that they were released from.
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