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Giuditta De Lorenzo, 2016, doktorska disertacija

Opis: Studies on rotavirus biology have always suffered for the lack of a reverse genetics system applicable to all genome segments and independent from the use of helper virus. One of the main reasons proposed to explain the difficulties encountered is the poor expression of the transfected cDNAs. In this work we investigated the role of 5’UTRs in rotavirus cDNAs expression in un-infected cells. We showed that in the 5’UTR of several (but not all) genome segments of rotavirus is present a 5’-terminal inhibitory motif (IM = 5’-GGY(U/A)UY-3’) that, independently from the ORF or the 3’UTR, downregulates both transcription and translation of rotavirus cDNAs when expressed exploiting T7 polymerase-encoding recombinant vaccinia virus. We mapped two mutations (insertion of a G upstream 5’UTR and the U to A mutation of the fifth nucleotide of IM) that are capable of making the inhibitory motif non-functional restoring a satisfying yield of protein synthesis. After the analysis of all genome segment 5’UTR of SA11 strain, we created two distinct sets of mutated rotavirus genome segments containing non-functional IM. We planned to employ these sets in the development of a plasmid-based reverse genetics system that exploit vaccinia virus-encoded T7 polymerase. In an attempt to produce in vivo biotinylated Tripled-Layered Particles (TLPs), we created a recombinant VP4 fused to the Biotin Acceptor Peptide (BAP) that, when co-expressed with the biotin ligase BirA, is efficiently in vivo biotinylated. We exploited recombinant vaccinia virus system to achieve high levels of VP4-BAP and the enzyme BirA in cells. Upon infection with rotavirus, VP4-BAP would be incorporated in the newly forming viral particles. In addition, we constructed a full-length genome segment 4 cDNA encoding the recombinant VP4-BAP to be used with a VP4 temperature sensitive rotavirus in order to generate a recombinant rotavirus encoding VP4-BAP. Thus, the results obtained allowed us to design two distinct possible ways of generating recombinant rotavirus. The first was a genetic strategy for the incorporation of an exogenous genomic segment, with the characteristic of being helper-virus free and applicable to all genomic segments. This was the consequence of the description of an inhibitory motif present in many viral mRNAs and the identification of two mutations that abrogate its inhibitory activity allowing expression of viral proteins. The second focused on the expression of a recombinant viral structural protein expressed in virus-infected cells for the consequent incorporation into newly made virions.
Najdeno v: ključnih besedah
Ključne besede: rotavirus, UTR, reverse genetics, T7 polymerase, in vivo biotinylation
Objavljeno: 08.06.2016; Ogledov: 1112; Prenosov: 101
.pdf Polno besedilo (2,87 MB)

José Manuel Carita Gonçalves, 2018, doktorska disertacija

Opis: The available classical diagnostic methods, due to many disadvantages, do not allow effective detection of pathogenic enteric viruses in environmental samples. Due to low concentrations of pathogenic viruses in the sea, it is important to develop an effective concentration procedure for their successful detection. In the first part of the doctoral thesis, we focused primarily on the development of a protocol for an effective concentration of pathogenic enteric viruses in coastal water samples. Monolithic chromatographic columns (BIAseparations) were used for the concentration of rotaviruses and noroviruses, prior to the detection with reverse transcription quantitative PCR in real time (RT-qPCR). We tested the efficiency of concentration using columns of various chemical properties and selected pathogenic enteric viruses (rotavirus and norovirus). Among them, hydrophobic interaction monolithic column (CIM® C4) was the most effective. CIM C4 was used to optimize the concentration step and tested in waters with different salinities. The presence of concentrated viruses was confirmed by RT-qPCR and transmission electron microscope. We have developed a protocol that enables rapid concentration of viruses in coastal waters of various salinities and can be used on-site. The presence of RoV and NoV was surveyed, using the developed concentration protocol, prior to one-step RT-qPCR molecular detection, in the inner part of the Bay of Koper, in mussel farming areas and a swimming area. Rotaviruses, noroviruses and fecal indicator bacteria were frequently detected in the inner part of the Bay of Koper. Rotaviruses and noroviruses were detected in the studied area, with higher rates close to the outfall of the wastewater treatment plant in the estuary of river Rižana and were also detected in the middle of the Bay of Koper and in areas used for recreation and mussel farming. The results show that water bodies, which are otherwise defined as suitable for bathing or mussel farming, based on the results of fecal indicator bacteria, still contain low concentrations of pathogenic enteric viruses. In addition to human pathogenic enteric viruses and faecal coliforms, changes in abundance of bacteria and virus particles were studied in relation to temperature, salinity, inorganic and organic nutrient concentrations in the organically polluted Rižana estuary. Preliminary results showed spatially and seasonally changes in bacterial and viral particles abundance, and bacterial composition spatially and seasonally. However, seasonality plays a greater role in bacterial dynamics.
Najdeno v: ključnih besedah
Povzetek najdenega: ...(BIAseparations) were used for the concentration of rotaviruses and noroviruses, prior to the detection with...
Ključne besede: Concentration of viruses, Enteric viruses, Rotavirus, Norovirus, Feacal coliforms, Feacal contamination, qPCR, RT-qPCR, Monolithic columns, Sewage, Seasonal dynamics, Concentration, Coastal environment, Gulf of Trieste
Objavljeno: 02.07.2018; Ogledov: 128; Prenosov: 28
.pdf Polno besedilo (1,95 MB)

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