Use of Random Peptide Reactivities to Analyze Host Immune Responses of African Swine Fever Virus Infection and ImmunizationJanuary 2011 (has links)
abstract: African Swine Fever (ASF), endemic in many African countries, is now spreading to other continents. Though ASF is capable of incurring serious economic losses in affected countries, no vaccine exists to provide immunity to animals. Disease control relies largely on rapid diagnosis and the implementation of movement restrictions and strict eradication programs. Developing a scalable, accurate and low cost diagnostic for ASF will be of great help for the current situation. CIM's 10K random peptide microarray is a new high-throughput platform that allows systematic investigations of immune responses associated with disease and shows promise as a diagnostic tool. In this study, this new technology was applied to characterize the immune responses of ASF virus (ASFV) infections and immunizations. Six sets of sera from ASFV antigen immunized pigs, 6 sera from infected pigs and 20 sera samples from unexposed pigs were tested and analyzed statistically. Results show that both ASFV antigen immunized pigs and ASFV viral infected pigs can be distinguished from unexposed pigs. Since it appears that immune responses to other viral infections are also distinguishable on this platform, it holds the potential of being useful in developing a new ASF diagnostic. The ability of this platform to identify specific ASFV antibody epitopes was also explored. A subtle motif was found to be shared among a set of peptides displaying the highest reactivity for an antigen specific antibody. However, this motif does not seem to match with any antibody epitopes predicted by a linear antibody epitope prediction. / Dissertation/Thesis / M.S. Biological Design 2011
Ballantyne, Maeve Kellett
Livestock animals are an ancient, vital renewable natural resource. Many livestock species have the ability to convert inedible crops and waste food into food fit for human consumption, in the form of meat, eggs and dairy products. As the global demand for high value animal protein is ever increasing, the livestock market continues to play a major role in worldwide economics. Animal disease has the potential to be a huge burden on the livestock industry, impacting both welfare and production. Major outbreaks of transboundary diseases, such as foot and mouth disease, rinderpest and classical swine disease, have resulted in devastating global economic losses. As a result, scientific research is engaged in lowering this impact by generating effective preventative measures and treatments. One way to reduce livestock disease is to select animals that are genetically resistant, traditionally carried out through selective animal breeding programs; however, this is a time-consuming process and requires that appropriate genetic variation exists within the population. Advances in genome engineering technologies offer us an alternative approach, with the capability to make genetic improvements in livestock within a single generation. It is hypothesised that resilience to a disease, known as African swine fever (ASF), could be genetically engineered into the domestic pig. ASF is a highly contagious disease of domestic pigs and is a re-emerging global threat to the swine industry. It is a lethal haemorrhagic disease caused by a virus, known as the African swine fever virus (ASFV). At present, there is no vaccine or treatment for ASF, and disease control relies on rapid diagnosis, quarantine and the mass slaughter of animals. Unlike the domestic pig, swine indigenous to Sub-Saharan Africa, such as the warthog, show no clinical signs of disease following infection with ASFV. A comparative study was carried out to identify host genetic variation that could underlie the difference in response to ASFV, with candidate genes selected based on their potential involvement with the viral protein A238L, involved in immune evasion. Functional polymorphisms where identified in the porcine RELA gene, encoding RelA, a subunit of the NF-κB transcription factor family. This evolutionary conserved protein family plays a vital role in mediating inflammatory and immune responses. The specific RELA polymorphisms identified alter potential phosphorylation sites within the C-terminal transactivation domain of RelA which have been found to modulate NF-κB transcriptional activity in vitro. We set out to investigate whether genome editing tools could be employed to engineer the RELA sequence of domestic pigs. Initial attempts targeted the final exon of RELA, producing animals with a truncated RelA protein; modified animals lack the final 60 amino acids of the C-terminal transactivation domain. The aim of this thesis was to genotype and characterise the effects of this RELA modification at a molecular, cellular, morphological and whole organism level. The ultimate goal of this project was to investigate whether this RELA modification altered the domestic pig’s response to ASFV in vitro and in vivo. Unlike rela-/- mice which have an embryonic lethal phenotype, these RELA-edited pigs were born healthy and were fully viable when housed in a typical farm environment. Phenotypic analysis of lymphoid tissues from the RELA-edited pigs demonstrated no significant anatomical or histological changes compared to unmodified counterparts. Pigs homozygous for the RELA mutation had a significantly lower body weight compared to wild-type pigs. Molecular studies of samples from these pigs have shown that the modified RelA has an altered activity; however, the RELA modified pigs do develop the characteristic disease phenotype when challenged with ASFV. Finally, genome editors have been developed to introduce a specific warthog allele into the domestic pig RELA locus, these editors are currently being taken forward to produce a novel pig line.
Tapia-Santos, Aixa S.
31 March 2011
No description available.
Evaluation of the new Power & Biomass to Liquid (PBtL) concept for production of biofuels from woody biomass / Utvärdering av det nya Power & Biomass to Liquid (PBtL) konceptet för produktion av biobränslen från träbaserad biomassaDahl, Robert January 2021 (has links)
I den här rapporten utvärderas det nya konceptet Power & Biomass to Liquid (PBtL). PBtL är ett alternativ till den tidigare och mer etablerade Biomass to Liquid (BtL) processen. Med PBtL förbättras utbytet av kol jämfört med BtL genom att elektricitet läggs till i processen. Elektriciteten används för att producera H2, som används för att höja H2/CO förhållandet istället för att använda WGS som i vanlig BtL process. Rapporten är en del i ett större PBtL projekt som bedrivits vid Institutt for kjemisk prosessteknologi vid NTNU och på SINTEF. Utvärderingen utfördes genom flera simuleringar av lågtemperaturs Fischer-Tropsch reaktorer i simuleringsprogrammet Aspen Plus. Omvandling och katalytiska reaktorer utvecklades och togs fram i programmet. Produktfördelningen i omvandlingsreaktorn modellerades med ASF distribution theory tillsammans med en metod för sammanslagning av högre kolväten. Fördelningen av paraffiner, olefiner och oxygenater baserades på experimentella resultat från Shafer et al. som studerade en slurryreaktor under liknande förhållanden. Den kinetiska reaktorn modellerades med en variant av ASF fördelningsteori kallad ”consorted vinylene mechanism” från Rytter och Holmen. Reaktorerna adderades till förgasningsprocess, som utvecklats tidigare av PBtL gruppen, I förgasningsprocessen förgasas biomassa till syntesgas, dvs H2 och CO. För att möjliggöra en utvärdering av det efterföljande steget med separering av vax, mellandestillat och lättare kolväten så antogs en väl fungerande separation av Fischer-Tropsch produkterna. En enklare separation av med flash förångning gjordes också, dels för fortsättningen av PBtL processen och för att kunna studera tailgasrecirkulering. Ett mindre bidrag var studier av en torkningsprocess för biomassa innan inloppet till förgasningsprocessen. PBtL konceptet diskuterades även ur ett praktiskt perspektiv. Resultaten visar att vid driftbetingelser på 210 °C, 25 bar och H2/CO = 1,95 så gav omvandlingsreaktorn en kolselektivitet för CH4 respektive C5+ på 14,77 respektive 75,40 mol% C. Högre temperatur, tryck och H2/CO förhållande i reaktorn resulterar i en högre kolselektivitet mot lägre kolväten. Vid samma driftbetingelser gav den katalysreaktorn en kolselektivitet för CH4 respektive C5+ på 7,612 respektive 86,00 mol% C. Resultaten visar att C8-C16 produktionen var högre än C17+ med avseende på molflöde men lägre beträffande massflöde för katalysreaktorn. Generellt så ökar kolselektiviteten med ökande kolnummer till ett maximum runt 13 för att sedan minska. / In this report, the new Power & Biomass to Liquid (PBtL) concept was evaluated. The PBtL concept is a new alternative to the more well-established Biomass to Liquid (BtL) concept where electricity is added to the process. The main purpose for developing the PBtL is that the BtL process exhibits poor carbon efficiency compared to the PBtL process. The electricity here is used to produce H2 in electrolysis. The report is part of a larger PBtL project pursued for several years at the Department of Chemical Engineering at NTNU and SINTEF. The evaluation was done by simulating different types of low temperature Fischer-Tropsch reactors in simulation software Aspen Plus. A conversion reactor and a kinetic reactor was developed. A conversion reactor based on the result from the kinetic reactor was also developed. The conversion-based reactor was modeled with the ASF distribution theory which describes the distribution of products formed in Fischer-Tropsch synthesis along with a method of lumping higher hydrocarbons. The distribution between paraffins, olefins and oxygenates was based on experimental data from Shafer et al. with similar operating condition with a Slurry reactor. The kinetic-based reactor was modeled with ASF distribution theory with a consorted vinylene mechanism previously described in Rytter and Holmen. The reactors were added to a process for which the biomass gasification section had previously been developed by the PBtL group. The Fischer-Tropsch products were as well separated in order to evaluate the subsequent step of separation of waxes, middle distillate and lighter hydrocarbons. This enabled the option of recycling of tail gas to the Fischer-Tropsch reactor to be evaluated. A smaller contribution included addition of a biomass dryer prior the biomass gasification section. The PBtL concept is also shortly discussed from a practical point-of-view. It was found that for the operating condition of 210 °C, 25 bar and H2/CO = 1.95 for the conversion-based reactor yielded a carbon selectivity towards CH4 and C5+ of 14.77 and 75.40 mol C% respectively. For the same operating condition, the kinetic-based reactor yield a carbon selectivity towards CH4 and C5+ of 7.612 and 86.00 mol C% respectively. It could be seen from the conversion-based reactor that elevating temperature, pressure and H2/CO (to a certain extent) results in higher carbon selectivity towards lower hydrocarbons. From the product separation with the kinetic reactor, it was observed that C8-C16 production was higher than the C17+ production in terms of mole flow but lower in terms of mass flow. For both models, carbon selectivity increases with carbon number and peaks around carbon number 13 and then starts to decrease.
Hepatitis Delta Virus: Identification of Host Factors Involved in the Viral Life Cycle, and the Investigation of the Evolutionary Relationship Between HDV and Plant ViroidsSikora, Dorota 19 June 2012 (has links)
Hepatitis delta virus (HDV) is the smallest known human RNA pathogen. It requires the human hepatitis B virus (HBV) for virion production and transmission, and is hence closely associated with HBV in natural infections. HDV RNA encodes only two viral proteins - the small and the large delta antigens. Due to its limited coding capacity, HDV needs to exploit host factors to ensure its propagation. However, few human proteins are known to interact with the HDV RNA genome. The current study has identified several host proteins interacting with an HDV-derived RNA promoter by multiple approaches: mass spectrometry of a UV-crosslinked ribonucleoprotein complex, RNA affinity chromatography, and screening of a library of purified RNA-binding proteins. Co-immunoprecipitation, both in vitro and ex vivo, confirmed the interactions of eEF1A1, p54nrb, PSF, hnRNP-L, GAPDH and ASF/SF2 with both polarities of the HDV RNA genome. In vitro transcription assays suggested a possible involvement of eEF1A1, GAPDH and PSF in HDV replication. At least three of these proteins, eEF1A1, GAPDH and ASF/SF2, have also been shown to associate with potato spindle tuber viroid (PSTVd) RNA. Because HDV’s structure and mechanism of replication share many similarities with viroids, subviral helper-independent plant pathogens, I transfected human hepatocytes with RNA derived from PSTVd. Here, I show that PSTVd RNA can replicate in human hepatocytes. I further demonstrate that a mutant of HDV, lacking the delta antigen coding region (miniHDV), can also replicate in human cells. However, both PSTVd and miniHDV require the function of the small delta antigen for successful replication. Our discovery that HDV and PSTVd RNAs associate with similar RNA-processing pathways and translation machineries during their replication provides new insight into HDV biology and its evolution.
25 September 2009
In the presented study, we demonstrate that the alternative splicing of caspase 9 was dysregulated in a large percentage of NSCLC tumors and cell lines. These findings led to the hypothesis that survival pathways activated by oncogenic mutation regulated this mechanism. Indeed, the oncogenic PI3-Kinase/Akt pathway was demonstrated to regulate the alternative splicing of caspase 9. Further mechanistic studies demonstrate that multiple Akt isoforms can regulate the alternative splicing of caspase 9 in NSCLC. Akt was additionally shown to mediate the exclusion of the exon 3,4,5,6 cassette of caspase 9 via the phospho-state of the RNA trans-factor, SRp30a. Mutagenesis studies identified serine 199, serine 201, serine 227, and serine 234 as critical residues regulating the alternative splicing of caspase 9, as well as playing a role in the anchorage-independent growth of A549 cells. Since dysregulation of this splicing mechanism correlated with NSCLC tumors/cell lines and constitutively active Akt, oncogenic factors for NSCLC known to activate the PI3-Kinase/Akt pathway were examined in HBEC-3KT cells. In contrast to k-ras V12 expression, the overexpression/mutation of EGFR affected the alternative splicing of caspase 9 in a pro-oncogenic manner, dramatically lowering the caspase 9a/9b mRNA ratio. Stable downregulation of caspase 9b by shRNA blocked the ability of E746-A750 del EGFR expressing HBEC-3KTs to induce anchorage-independent growth, suggesting a role for caspase 9b as a cooperative oncogenic factor. These findings were further corroborated by the ability of caspase 9b expression to completely block the inhibition of clonogenic colony formation by erlotinib. Therefore, this study demonstrates that oncogenic factors activating the PI3-Kinase/Akt pathway regulate the alternative splicing of caspase 9, to produce caspase 9b, via a coordinated mechanism involving the phosphorylation of SRp30a. In additional studies, we demonstrate that the PI3-Kinase/PKCι pathway, a pathway important for cancer cell survival and transformation of lung epithelial cells, regulates the alternative splicing of Bcl-x pre-mRNA via modulation of SAP155 expression to produce an anti-apoptotic phenotype in NSCLC. Therefore, these studies link oncogenic mechanisms in NSCLC to the therapeutically relevant and distal target mechanisms of caspase 9 and Bcl-x pre-mRNA splicing.
Analyse structurale et fonctionnelle de la région des A-repeats de l'ARN Xist impliqué dans l'inactivation du chromosome X dans les mammifères femelles / Structural and functional analysis of the A region of the Xist RNA involved in the X-chromosome inactivation in mammals female cellsSavoye, Anne 14 December 2012 (has links)
L'inactivation du chromosome X correspond au silence transcriptionnel de l'un des deux chromosomes X dans les cellules des mammifères femelles. Il s'agit d'un mécanisme de compensation du dosage du chromosome X qui assure un taux d'expression des gènes liés aux chromosomes X équivalent entre organismes mâles (XY) et femelles (XX). Elle débute par une accumulation de l'ARN Xist (X inactive specific transcript) sur le chromosome X qui sera inactivé (Xi). Elle est suivie très rapidement par des modifications des histones qui assurent l'établissement, le maintien et la transmission de l'état transcriptionnel inactif de la chromatine. L'ARN Xist comprend plusieurs régions d'éléments répétés et notamment la région des A-repeats, essentielle pour la mise en place de l'inactivation. Mes recherches se sont portées sur l'étude de cette région singulière : sa structure et ses interactions protéiques. La technique de FRET (Fluorescence Resonance Energy Transfer) appliquée à l'ARN nous a permis de confirmer la structure de cette région parmi 3 modèles possibles. Elle se structure en deux tiges-boucles formée par l'appariement 2 à 2 de 4 répétitions successives. Dans une seconde partie, j'ai caractérisé l'interaction de cette région avec certains de ses partenaires protéiques in vitro. La région des A-repeats interagit notamment de manière directe avec les protéines PTB, KSRP et ASF/SF2. Les 2 premières protéines pourraient avoir un rôle dans la stabilité de l'ARN tandis qu'ASF/SF2 serait impliquée dans la maturation de l'ARN X / X-chromosome inactivation is the transcriptional silencing of one of the two X chromosomes in female mammal cells. This mechanism of dosage compensation ensures an equal level of the X-linked genes expression between males (XY) and females (XX). It initiates with the accumulation of the Xist RNA (X inactive specific transcript) on the futur inactive X chromosome (Xi). It is followed by the apposition of epigenetic marks such as histone modifications, that ensure establishment, maintenance and transmission of the inactive state of the chromatin. Xist RNA comprises a number of repeated regions and, in particular to its 5' end the A region, absolutely necessary for the establishment of the X-inactivation. My research was focused on the study of this singular region: its structure and its protein interactions. The FRET method (Fluorescence Resonance Energy Transfer) applied to RNA allowed us to ascertain that the RNA is structured in two long stem-loop structures each including four repeats. In a second part, I characterized the in vitro interaction of this region with some of its protein partners. The A region interacts directly with PTB, KSRP and ASF/SF2 proteins. The first two proteins may have a role in RNA stability whereas ASF/SF2 could be involved in the splicing process
<p>Viruses depend on their hosts for the production and spread of new virus particles. For efficient virus replication, the viral genes have adapted the strategy of being recognized and processed by the cellular biosynthetic machineries. Viruses therefore provide an important tool to study the cellular machinery regulating gene expression. In this thesis, we have used two model DNA viruses; herpes simplex virus (HSV) and adenovirus, to study RNA processing at the level of pre-mRNA splicing in mammalian cells. </p><p>During a lytic infection, HSV cause an almost complete shut-off of host cell gene expression. Importantly, HSV infection cause inhibition of pre-mRNA splicing which is possibly advantageous to the virus, as only four HSV genes contain introns. </p><p>The HSV immediate early protein, ICP27, has been shown to modulate several post-transcriptional processes such as polyadenylation and pre-mRNA splicing. We have studied the role of ICP27 as an inhibitor of pre-mRNA splicing.</p><p>We show that ICP27 inhibits pre-mRNA splicing <i>in vitro</i> in the absence of other HSV proteins. We further show that ICP27 inhibits splicing at the level of spliceosome assembly. Importantly, ICP27 induced inhibition of splicing can be reversed, either by the addition of purified SR proteins or by the addition of an SR protein specific kinase, SRPK1. We propose that SR proteins are prime candidates as mediators of the inhibitory effect of ICP27 on pre-mRNA splicing. </p><p>In order to learn more about how splicing is organized in the cell nucleus <i>in vivo</i>, we investigated how cellular splicing factors are recruited to sites of transcription and splicing in adenovirus infected cells using confocal microscopy. Our results showed that the SR proteins, ASF/SF2 and SC35, are efficiently recruited to sites in the nucleus where adenovirus genes are transcribed and the resulting pre-mRNAs are processed. Our results demonstrate that only one of the two RNA recognition motifs (RRMs) present in the ASF/SF2 protein is required for its recruitment to active sites of splicing. The arginine/serine rich (RS) domain in ASF/SF2 is redundant and insufficient for the translocation of the protein to active viral polymerase II genes in adenovirus infected cells.</p>
Adenovirus vector systems permitting regulated protein expression and their use for in vivo splicing studiesMolin, Magnus January 2001 (has links)
<p>We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and the progesterone antagonist RU 486-regulated gene expression systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing a broad species-variety and the regulation of protein expression was shown to be tightly controlled in cells not permissive for virus replication. Furthermore, the adenovirus-Tet-ON system was shown to perform in mice after intramuscular administration.</p><p>The novel adenovirus-vector systems were then used to study the effects of overexpression of selected proteins on adenovirus replication during a lytic infection, with focus on regulation of adenovirus alternative splicing. Expression of adenovirus transcription units is to a large extent temporally regulated at the level of alternative pre-mRNA splicing, where viral splice site usage shifts from proximal to distal splice site selection as infection proceeds. This makes adenovirus an appropriate model for mechanistic studies of regulated splicing. We show that overexpression of the essential host cell splicing factor ASF/SF2 inhibits this shift by promoting usage of proximal splice sites. As a consequence, the virus displayed a markedly inhibited growth. Interestingly, mRNA expression from the adenovirus major late promoter was almost completely lost as a consequence of ASF/SF2 overexpression. Collectively, the cellular splicing factor ASF/SF2 prevents adenovirus from entering the late phase of infection. This strongly argues for a need for the virus to block the splicing enhancer activity of ASF/SF2 for establishment of a lytic infection. Further, from analysis of the strict inhibition of late region 1 late pre-mRNA splicing we propose that the temporal regulation of alternative splicing is merely a consequence of fitness rather than profoundly deleterious effects of an unregulated expression. During our studies we noted that in 293 cells, which are used for growth of E1-deficient Ad vectors, an unwanted background reporter gene expression was evident in our vector systems. We therefore introduced an additional regulatory element, functioning as a transcriptional road-block, and showed that this methodological innovation represents a way to overcome the potentially deleterious effects of background reporter gene expression. This modified viral vector system should make it possible to reconstruct recombinant viruses expressing highly toxic proteins.</p><p>In conclusion, this work presents a new <i>in vivo </i>model system to study proteins involved in RNA splicing and other gene regulatory mechanisms.</p>
Adenovirus vector systems permitting regulated protein expression and their use for in vivo splicing studiesMolin, Magnus January 2001 (has links)
We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and the progesterone antagonist RU 486-regulated gene expression systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing a broad species-variety and the regulation of protein expression was shown to be tightly controlled in cells not permissive for virus replication. Furthermore, the adenovirus-Tet-ON system was shown to perform in mice after intramuscular administration. The novel adenovirus-vector systems were then used to study the effects of overexpression of selected proteins on adenovirus replication during a lytic infection, with focus on regulation of adenovirus alternative splicing. Expression of adenovirus transcription units is to a large extent temporally regulated at the level of alternative pre-mRNA splicing, where viral splice site usage shifts from proximal to distal splice site selection as infection proceeds. This makes adenovirus an appropriate model for mechanistic studies of regulated splicing. We show that overexpression of the essential host cell splicing factor ASF/SF2 inhibits this shift by promoting usage of proximal splice sites. As a consequence, the virus displayed a markedly inhibited growth. Interestingly, mRNA expression from the adenovirus major late promoter was almost completely lost as a consequence of ASF/SF2 overexpression. Collectively, the cellular splicing factor ASF/SF2 prevents adenovirus from entering the late phase of infection. This strongly argues for a need for the virus to block the splicing enhancer activity of ASF/SF2 for establishment of a lytic infection. Further, from analysis of the strict inhibition of late region 1 late pre-mRNA splicing we propose that the temporal regulation of alternative splicing is merely a consequence of fitness rather than profoundly deleterious effects of an unregulated expression. During our studies we noted that in 293 cells, which are used for growth of E1-deficient Ad vectors, an unwanted background reporter gene expression was evident in our vector systems. We therefore introduced an additional regulatory element, functioning as a transcriptional road-block, and showed that this methodological innovation represents a way to overcome the potentially deleterious effects of background reporter gene expression. This modified viral vector system should make it possible to reconstruct recombinant viruses expressing highly toxic proteins. In conclusion, this work presents a new in vivo model system to study proteins involved in RNA splicing and other gene regulatory mechanisms.
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