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Academic literature on the topic 'SsRNA'
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Dissertations / Theses on the topic "SsRNA"
1
Powalowska, Paulina Klaudyna. "Development of ssRNA for therapeutic gene knock-down." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/40508/.
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Work presented in this thesis shows how chemical modifications of ssRNAs can increase their nuclease resistance, resulting in potent gene inhibition. The first part of thesis describes how a range of chemical modifications were introduced into the ssRNAs to test their effect on oligonucleotide stability and ability to inhibit gene expression. The ssRNAs were tested in a cancer cell line that stably expresses firefly luciferase. This work demonstrates that DNA nucleotides can be incorporated into ssRNA sequences without loss of potency in the RNAi mechanism. By using additional chemical modifications (2’-OMe and 2’-F) further improvement of ssRNA stability and a significant inhibition of firefly luciferase activity, reaching 91%, was achieved. Moreover, we show that the level of inhibition can be improved when the DNA dinucleotide linked by the (E)-vinylphosphonate is used as a protection of the 5’-end of the ASO (95% KD). The use of ssRNAs rather than dsRNAs is beneficial, since the possibility of off-target effects caused by the remaining sense strand is eliminated and the cost of production of such a medicine is reduced. The second part of this thesis focuses on development of an experimental system that will allow detection of the inhibition of a disease relevant target. Several approaches were used in order to detect KD of the target by the unmodified dsRNAs. First a western blotting technique was employed for detection. Although different ASOs and concentrations were used no KD was observed. More attempts were taken in order to generate target KD, but unfortunately they were unsuccessful so far.
2
McCauley, Sephen Jude. "The annotation and evolutionary analysis of overlapping CDS in ssRNA viral genomes." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670151.
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Ha, Cuong Viet. "Detection and identification of potyviruses and geminiviruses in Vietnam." Thesis, Queensland University of Technology, 2007. https://eprints.qut.edu.au/16540/1/Cuong_Viet_Ha_Thesis.pdf.
Full textAbstract:
Prior to the commencement of this project, few plant viruses had been identified from Vietnam despite virus-like symptoms being commonly observed on many crops and weeds. In limited surveys in the late 1990's, preliminary evidence was obtained indicating that potyviruses and geminiviruses were causing significant diseases. As a result, this study was aimed at developing generic PCR-based methods for the rapid detection of viruses belonging to viruses in the families Potyviridae and Geminiviridae in plant samples collected from Vietnam, and to characterise the viruses at the molecular level.
Novel degenerate PCR primers were developed for the identification of begomoviruses. Using these primers, 17 begomoviruses species infecting seven crop and nine weed species in Vietnam were identified and characterised. Sequence analyses showed that ten of the viruses (six monopartite and four bipartite) were new species. Of the seven previously characterized begomoviruses, five were identified in Vietnam for the first time. Additionally, eight DNA-ß and three nanovirus-like DNA-1 molecules were also found associated with the monopartite viruses. Five of the DNA-β molecules were putatively novel.
Two novel bipartite begomoviruses, named Corchorus yellow vein virus (CoYVV) and Corchorus golden mosaic virus (CoGMV), were isolated from jute plants. Analysis of these viruses showed that they were more similar to New World begomoviruses than to viruses from the Old World. This was based on the absence of an AV2 open reading frame, the presence of an N-terminal PWRLMAGT motif in the coat protein and phylogenetic analysis of the DNA A and DNA B nucleotide and deduced amino acid sequences. This is the first known occurrence of Old World viruses bearing features of New World viruses, and their presence in Vietnam suggests the presence of a "New World" virus in the Old World prior to Gondwana separation. Other interesting features relating to begomoviruses identified in Vietnam were; (i) the detection of several recombination events, particularly between the newly identified Tomato yellow leaf curl Vietnam virus (TYLCVNV), and the previously characterised, Tomato leaf curl Vietnam virus (ToLCVV), (ii) the identification of new natural hosts of Sida leaf curl virus (SiLCV), Papaya leaf curl China virus (PaLCuCNV) and Alternanthera yellow vein virus (AlYVV), (iii) the first report of variation in the geminivirus stem-loop nonanucleotide sequence (CoGMV sequence was TATTATTAC rather than TAATATTAC) and (iv) the first report of different stem sequences in the stem-loop structure of two genomic components from a bipartite begomovirus, Kudzu mosaic virus (KuMV). The sequence and phylogenetic analyses of the begomoviruses and begomovirus-associated DNAs identified in this study suggested that South East Asia, and Vietnam in particular, may be a centre of begomovirus diversity.
Two pairs of degenerate primers, designed in the CI gene (CIFor/CIRev) and HC-Pro gene (HPFo/HPRev), were developed for the detection of viruses in the genus Potyvirus. Using these primers, three novel potyviruses from Vietnam were detected, namely Telosma mosaic virus (TelMV) infecting telosma (Telosma cordata), Peace lily mosaic virus (PeLMV) infecting peace lily (Spathiphyllum patinii) and Wild tomato mosaic virus (WTMV) infecting wild tomato (Solanum torvum). The fragments amplified by the two sets of primers enabled additional PCR and complete genomic sequencing of these three viruses and a Banana bract mosaic virus (BBrMV) isolate from the Philippines. All four viruses shared genomic features typical of potyviruses. Sequence comparisons and phylogenetic analyses indicated that WTMV was most closely related to Chilli veinal mottle virus (ChiVMV) and Pepper veinal mottle virus (PVMV) while PeLMV, TelMV were related to different extents with members of the BCMV subgroup.
The incidence of potyviruses infecting plants in Vietnam was investigated using the potyvirus-specific primers. Fifty two virus isolates from 13 distinct potyvirus species infecting a broad range of crops were identified in Vietnam by PCR and sequence analysis of the 3' region of the genome. The viruses were Bean common mosaic virus (BCMV), Potato virus Y (PVY), Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Chilli veinal mottle virus (ChiVMV), Zucchini yellow mosaic virus (ZYMV), Leek yellow stripe virus (LYMV), Shallot yellow stripe virus (SYSV), Onion yellow dwarf virus (OYDV), Turnip mosaic virus (TuMV), Dasheen mosaic virus (DsMV), Sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, which was tentatively named Chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the Peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses, based on the nucleotide sequence of the entire CP-coding region of all 52 virus isolates, revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. The phylogenetic analyses also suggested the possible presence of ancestral groups of BCMV, SCMV and ZYMV in Vietnam.
4
Ha, Cuong Viet. "Detection and identification of potyviruses and geminiviruses in Vietnam." Queensland University of Technology, 2007. http://eprints.qut.edu.au/16540/.
Full textAbstract:
Prior to the commencement of this project, few plant viruses had been identified from Vietnam despite virus-like symptoms being commonly observed on many crops and weeds. In limited surveys in the late 1990's, preliminary evidence was obtained indicating that potyviruses and geminiviruses were causing significant diseases. As a result, this study was aimed at developing generic PCR-based methods for the rapid detection of viruses belonging to viruses in the families Potyviridae and Geminiviridae in plant samples collected from Vietnam, and to characterise the viruses at the molecular level. Novel degenerate PCR primers were developed for the identification of begomoviruses. Using these primers, 17 begomoviruses species infecting seven crop and nine weed species in Vietnam were identified and characterised. Sequence analyses showed that ten of the viruses (six monopartite and four bipartite) were new species. Of the seven previously characterized begomoviruses, five were identified in Vietnam for the first time. Additionally, eight DNA-ß and three nanovirus-like DNA-1 molecules were also found associated with the monopartite viruses. Five of the DNA-β molecules were putatively novel. Two novel bipartite begomoviruses, named Corchorus yellow vein virus (CoYVV) and Corchorus golden mosaic virus (CoGMV), were isolated from jute plants. Analysis of these viruses showed that they were more similar to New World begomoviruses than to viruses from the Old World. This was based on the absence of an AV2 open reading frame, the presence of an N-terminal PWRLMAGT motif in the coat protein and phylogenetic analysis of the DNA A and DNA B nucleotide and deduced amino acid sequences. This is the first known occurrence of Old World viruses bearing features of New World viruses, and their presence in Vietnam suggests the presence of a "New World" virus in the Old World prior to Gondwana separation. Other interesting features relating to begomoviruses identified in Vietnam were; (i) the detection of several recombination events, particularly between the newly identified Tomato yellow leaf curl Vietnam virus (TYLCVNV), and the previously characterised, Tomato leaf curl Vietnam virus (ToLCVV), (ii) the identification of new natural hosts of Sida leaf curl virus (SiLCV), Papaya leaf curl China virus (PaLCuCNV) and Alternanthera yellow vein virus (AlYVV), (iii) the first report of variation in the geminivirus stem-loop nonanucleotide sequence (CoGMV sequence was TATTATTAC rather than TAATATTAC) and (iv) the first report of different stem sequences in the stem-loop structure of two genomic components from a bipartite begomovirus, Kudzu mosaic virus (KuMV). The sequence and phylogenetic analyses of the begomoviruses and begomovirus-associated DNAs identified in this study suggested that South East Asia, and Vietnam in particular, may be a centre of begomovirus diversity. Two pairs of degenerate primers, designed in the CI gene (CIFor/CIRev) and HC-Pro gene (HPFo/HPRev), were developed for the detection of viruses in the genus Potyvirus. Using these primers, three novel potyviruses from Vietnam were detected, namely Telosma mosaic virus (TelMV) infecting telosma (Telosma cordata), Peace lily mosaic virus (PeLMV) infecting peace lily (Spathiphyllum patinii) and Wild tomato mosaic virus (WTMV) infecting wild tomato (Solanum torvum). The fragments amplified by the two sets of primers enabled additional PCR and complete genomic sequencing of these three viruses and a Banana bract mosaic virus (BBrMV) isolate from the Philippines. All four viruses shared genomic features typical of potyviruses. Sequence comparisons and phylogenetic analyses indicated that WTMV was most closely related to Chilli veinal mottle virus (ChiVMV) and Pepper veinal mottle virus (PVMV) while PeLMV, TelMV were related to different extents with members of the BCMV subgroup. The incidence of potyviruses infecting plants in Vietnam was investigated using the potyvirus-specific primers. Fifty two virus isolates from 13 distinct potyvirus species infecting a broad range of crops were identified in Vietnam by PCR and sequence analysis of the 3' region of the genome. The viruses were Bean common mosaic virus (BCMV), Potato virus Y (PVY), Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Chilli veinal mottle virus (ChiVMV), Zucchini yellow mosaic virus (ZYMV), Leek yellow stripe virus (LYMV), Shallot yellow stripe virus (SYSV), Onion yellow dwarf virus (OYDV), Turnip mosaic virus (TuMV), Dasheen mosaic virus (DsMV), Sweet potato feathery mottle virus (SPFMV) and a novel potyvirus infecting chilli, which was tentatively named Chilli ringspot virus (ChiRSV). With the exception of BCMV and PVY, this is first report of these viruses in Vietnam. Further, rabbit bell (Crotalaria anagyroides) and typhonia (Typhonium trilobatum) were identified as new natural hosts of the Peanut stunt virus (PStV) strain of BCMV and of DsMV, respectively. Sequence and phylogenetic analyses, based on the nucleotide sequence of the entire CP-coding region of all 52 virus isolates, revealed considerable variability in BCMV, SCMV, PVY, ZYMV and DsMV. The phylogenetic analyses also suggested the possible presence of ancestral groups of BCMV, SCMV and ZYMV in Vietnam.
5
ANTUNES, T. F. S. "Associação do papaya meleira virus e de um segundo vírus de ssRNA à meleira do mamoeiro." Universidade Federal do Espírito Santo, 2017. http://repositorio.ufes.br/handle/10/7135.
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6
Boine, Barbara. "A study of the interaction between the plant pathogenic fungus Botrytis cinerea and the filamentous ssRNA mycoviruses Botrytis virus X and Botrytis virus F." Thesis, University of Auckland, 2012. http://hdl.handle.net/2292/16777.
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The ecological significance of mycoviruses is becoming increasingly recognised, not just for
their potential as biocontrol agents but also as driving forces in the evolution and diversification
of fungi. Therefore, it is important to understand how mycoviruses and fungi interact on the
molecular and biochemical level. To this end the interaction between Botrytis cinerea and the
mycoviruses Botrytis virus F and Botrytis virus X was studied. Relative and absolute real time
PCR protocols were developed for monitoring the titres of BVX and BVF during transfection
studies to monitor changes in virus titre in relation to phenotypic and metabolic changes in the
fungal host. Phenotypic changes included severe phenotypical alterations, which were
associated with extreme up regulation of carbohydrate, amino acid and lipid metabolism, and
induction of stress responses (vacuolisation/cell lysis, increased pigmentation). To study the
location and distribution of BVX in infected Botrytis the BVX coat protein was recombinantly
expressed in E. coli, BVX specific polyclonal antibodies produced, and protocols developed for
the serological detection and visualisation of BVX. Immuno-fluorescence microscopy was used
to studying the distribution of BVX within growing Botrytis cultures indicated that the virus is
present in aggregates located attached to the cell membrane, the septum, in spores, and in
hyphal tips. A combination of light and electron microscopy showed that BVX is often closely
associated with cell walls, suggesting that the virus may be moving across the cell wall by
altering cell wall composition. If this is shown to be the case then it provides an alternative
method to transmission via hyphal anastomosis, which is currently considered to be the only
method of horizontal transfer.
7
Croci, R. "RNA DEPENDENT RNA POLYMERASE: A VALUABLE TARGET TO BLOCK VIRAL REPLICATION IN SINGLE-STRANDED (+)SENSE RNA VIRUSES." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/243352.
Full textAbstract:
The (+)strand RNA viruses include a very large group of viruses that cause epidemic diseases in humans, including dengue fever and gastroenteritis. The human (+)RNA viruses include Flaviviruses (FV) and Norovirus (NV). Both encode for proteins essential for viral replication, such as the RNA dependent RNA polymerase (RdRp). Since human cells lack RdRp, it appears as one of the most promising targets for antivirals development. I worked on the identification of new non-nucleotide inhibitors against FV and NV, using RdRp as the main target. In this context, suramin and NF023 have been identified in my lab as NV RdRp inhibitors that, however both are hampered in their application by pharmacokinetics problems. To overcome such problems, I analyzed the potential inhibitory role of Naf2, a fragment derived from these two molecules. Although Naf2 showed a low inhibitory activity, the crystal structures of NV RdRp/Naf2 complex revealed a new binding site. To further map this new site, I tested a Naf2 related molecule, PPNDS. The crystal structures of the RdRp/PPNDS complex revealed interesting features about the new binding site. I also focused on structurally related molecules synthesized following structure-driven information. NV RdRp crystal structures in complex with one of these compounds (Cpd6) were analyzed, providing new knowledge on the interactions between a small fragment and NV RdRps, establishing a platform for structure-guided drug optimization. In parallel to the NV work, I screened in silico a library of compounds against FV RdRp. One of the best compounds identified (HeE1-2Tyr) was able to inhibit the RdRp activity and several FVs in cell-based assays. Although the crystallographic analyses don't reveal clear enough electron density for the inhibitor, indirect evidence suggests that HeE1-2Tyr interferes with the RdRp priming loop that appears disordered.
8
Panzarino, Nicholas J. "The ssDNA Theory of BRCAness and Genotoxic Agents." eScholarship@UMMS, 2021. https://escholarship.umassmed.edu/gsbs_diss/1131.
Full textAbstract:
Cancers that are deficient in BRCA1 or BRCA2 are thought to be hypersensitive to genotoxic agents because they cannot prevent or repair DNA double strand breaks, but observations in patients suggest this dogma may no longer agree with experiment. Here, we propose that single stranded DNA underlies the hypersensitivity of BRCA deficient cancers, and that defects in double strand break repair and prevention do not. Specifically, in BRCA deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. In addition, we observed gaps could be suppressed by either restored fork restraint or by gap filling, both of which conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored double strand break repair and prevention did not confer therapy resistance when gaps were present. Critically, double strand breaks were not detected after therapy when apoptosis was inhibited, supporting a framework in which double strand breaks are not directly induced by genotoxic agents, but instead are created by cell death nucleases and are not fundamental to genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapy.
9
Ramsey, Jon. "Biophysical Characterization of the Sequsingle-Stranded DNA-Binding Properties of Mouse Pur : a Repressor of Smooth Muscle -Actin Gene Expression." ScholarWorks @ UVM, 2008. http://scholarworks.uvm.edu/graddis/189.
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ABSTRACT Regulation of gene transcription by structural interconversions of genomic DNA is an emerging biochemical and genetic paradigm that adds to the already diverse repertoire of eukaryotic gene regulatory mechanisms. The appearance of paranemic structures coincident with changes in gene activity, as well as participation of transcription factors that recognize and bind single-stranded DNA at numerous gene promoters in vivo illustrates the authenticity of this concept and its importance in cellular homeostasis. Despite its acceptance, this concept has been minimally described at the biochemical and biophysical levels, as the means by which sequence-specific single-stranded DNAbinding proteins exert transcriptional influence in double-stranded genomes remains largely undefined. Pur is a sequence-specific single-stranded DNA/RNA-binding protein that acts as a repressor of smooth muscle -actin (SM A) gene transcription, and mRNA translation. SM A is an important cytoskeletal protein that contributes contractile, antimigratory, and nonproliferative functions in smooth muscle. In concert with Pur protein family member Pur , and Y-box protein MSY1, Pur enacts repression of SM A gene expression by interacting with a cryptic cis-regulatory element in the 5’ region of the SM A promoter that has been shown to transiently adopt single-stranded conformations in vivo, and to confer transcriptional activation when trans-activator occupied while in a doublestranded conformation. Downregulation of SM A gene expression has been identified to be a contributing factor to cardiovascular disease progression; therefore a thorough understanding of SM A repression mechanisms is critical for clinical management of these conditions. Although highly homologous at the primary sequence level, Pur and Pur display significant conserved regions of sequence divergence that suggest these paralogs exert distinct cellular functions in various vertebrate classes. A goal of the studies presented herein was to delineate exhibited functional differences with respect to SM A repression in pertinent mouse cell lines. Loss-of-function and chromatin immunoprecipitation studies verified that Pur differs from Pur in that Pur is the dominant Pur protein repressor of SM A expression in embryonic fibroblasts and vascular smooth muscle cells, although by different, cell type-specific mechanisms. Biophysical assessment of Pur single-stranded DNA binding properties showed that despite the ability of Pur to self-dimerize in the absence of nucleic acid, Pur binds to the cryptic SM A enhancer by a sequential and cooperative mechanism, with remarkable affinity and a terminal stoichiometry of 2 to 1. Footprinting and in vitro binding site characterization confirms two Pur binding sites exist within this element and display slight degeneracy from a proposed Pur protein-binding consensus motif. These findings delineate binding mechanisms adopted by Pur and provide a means to identify putative Pur binding sites throughout the genome.
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Labonté, Jessica. "Diversity and evolution of ssDNA viruses in marine environments." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44583.
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