Thesis (M. Sc.)--York University, 2007. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 92-98). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004 & res_dat=xri:pqdiss & rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation & rft_dat=xri:pqdiss:MR32030.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation.

Thesis (Ph. D.)--University of Lund, 1995. / Published dissertation.

Les analogues de nucléosides ont été largement utilisés dans le traitement d’affections d’origine virale grâce à l’activité biologique de ces dérivés. Nous nous sommes particulièrement intéressés dans ce travail à l’étude de carbonucléosides phosphonates. Le premier chapitre de cette thèse rapporte une étude bibliographique sur les virus et les analogues nucléosidiques approuvés par la FDA. Les mécanismes d’action de ces analogues ont été également abordés dans ce chapitre. Dans le deuxième chapitre, nous avons décrit la synthèse des dérivés 2’- ou 4’-hydroxy-4’-carbonucléoside méthyle phosphonates en série d’adénine et guanine. Le troisième chapitre est consacré à la mise au point d’une réaction d’hydrophosphonylation sous irradiation UV d’alcènes/alcynes conduisant aux alkyle/vinyle phosphonates correspondants. Enfin, le dernier chapitre présente la synthèse des dérivés 4’-hydroxy-4’-carbonucléosides éthyle phosphonates utilisant la méthodologie développée dans le troisième chapitre. Tous les carbonucléosides phosphonates synthétisés sont en cours d’évaluation biologique. / Nucleoside analogues have been widely used in the treatment of viral diseases owing to biological activity of these derivatives. We are particularly interested in carbonucleosides phosphonates. The first chapter of this thesis reports on a bibliographic study on viruses and on nucleosidic analogues approved by FDA for medical use. Antiviral mechanisms of action were also discussed in this chapter. In the second chapter, we described the synthesis of derivatives 2’- or 4’-hydroxy-4’-methyl carbonucleoside phosphonate in adenine and guanine series. The third chapter is devoted to the development of a hydrophosphonylation reaction under UV irradiation of alkenes/alkyne leading to the alkyl/vinyl corresponding phosphonates. Finally, the last chapter presents the synthesis of derivatives 4’-hydroxy-4’-ethyl carbonucleosides phosphonates using the methodology developed in the third chapter. All carbonucleoside phosphonates synthesized are ongoing biological evaluation.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23939号 / 農博第2488号 / 新制||農||1089(附属図書館) / 学位論文||R4||N5374(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 渡邊 隆司, 教授 梅澤 俊明, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

published_or_final_version / abstract / Microbiology / Doctoral / Doctor of Philosophy

The human respiratory syncytial virus (hRSV) infection is a global public health burden in children aged under 2 years and immunocompromised or elderly adults. The choice for prophylaxis and therapy of hRSV infection was constrained to Palivizumab and Ribavirin. Therefore, this study aimed to develop effective anti-hRSV infection agents, such as short interfering RNA (siRNA) and β-defensin-4 (β-D-4). Since there still is no compatible animal model to evaluate antiviral effect of anti-hRSV agents, we first attempted to establish suitable animal model. A clinical isolate of hRSV (KI-RSV-W) was adapted in BALB/c mice by serial passages. Old male mice (age > 8 months) were used for establishment of hRSV infection model, because the more efficient viral infection in lungs of the old male mice than that old female mice and young mice (age < 3 weeks). After the virus was propagated in old male mice for 20 passages, a virus variant KI-RSV-P70-4 exhibited more efficient infection/replication in the mice. Its viral load was about 100-fold higher than that of wild type strain KI-RSV-W. The infection of KI-RSV-P70-4 also caused more severe histopathological changes in lung tissues. Although KI-RSV-P70-4 could not result in death of the infected mice, both viral load and pathological change in lungs may be good indicators for evaluating antiviral effect. The mouse model and adapted hRSV strain solidly laid the foundation for evaluation of anti-hRSV agents. We then designed and evaluated anti-hRSV effect of siRNAs. A total of 25 siRNAs targeting 4 viral genes (M2-1, NS2, N and F) were designed and their anti-hRSV effect was assessed in vitro. The results showed that 6 siRNAs respectively targeting M2-1, N and F genes exhibited higher anti-hRSV effect than that of the positive control, whereas those targeting NS2 gene did not show significant antiviral effect. The 50% inhibitory concentrations (IC50) of three most potent siRNAs (M2-1-361, N889 and F-1143) were 0.51, 2.14 and 0.64 nM, respectively. Antiviral activity of β-D-4 against hRSV infection was evaluated in vitro and in vivo. In vitro experiments showed supreme antiviral effect with IC50 around 3.4 μg/ml when the virus was pretreated with β-D-4, but no significant inhibitory effect was observed when the cells were pretreated with β-D-4 or β-D-4 was maintained in the culture medium after viral infection. These results indicated that the inhibitory effect of β-D-4 was associated with direct interaction with the virus itself and blocked virus entry of the cells. Furthermore, a single dose (13.6 μg) of β-D-4 intratracheal (i.t.) administration in old male mice after the viral infection resulted in about 0.7 log reduce of viral load in lung tissues, while inoculation of premixed β-D-4 and the virus caused about 3 logs decrease of viral load in lungs. These results have demonstrated that β-D-4 may be an effective anti-hRSV agent. Taken together, old male BALB/c mice might be used to establish hRSV infection model. Three siRNAs and the β-D-4 were validated as the potent anti-hRSV agents, respectively. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

No description available.

Avian influenza viruses can cause devastating outbreaks in domesticated poultry, with rapid transmission of virus between birds and high mortality. Current measures for control of influenza involve surveillance, closure of live poultry markets and mass culling. However, despite implementing these measures, outbreaks continue. Considering the uncertainty over whether certain strains of avian influenza viruses will adapt to human transmission and limitations over how their spread may be controlled, this study considers the use of anti-viral peptides as protective agents against low pathogenicity avian influenza (LPAI) virus. This study investigated the activity and mode of action of two cell-penetrating anti-viral peptide families against influenza A virus infection: ‘FluPep’ (a family of short, hydrophobic peptides related to suppressor of cytokine signaling- 1 proteins) and ‘Entry Blocker’ (derived from the signal sequence of fibroblast growth factor-4). Plaque reduction assays demonstrated dose-dependent anti-viral activity of both peptide families against a panel of influenza viruses with diverse haemagglutinin subtypes. Determination of IC50 values showed strain-specific differences in sensitivity to FluPep but not Entry Blocker. The IC50 of FluPep 4 for A/PR/8/34 was reduced by reassorting in the HA and NA from a relatively sensitive avian strain using a reverse genetics approach, suggesting inhibitory effects on the viral glycoproteins. Accordingly, viral entry assays focusing on binding, internalisation, fusion and import were designed and optimised to dissect the mechanism(s) of action of the peptides. Results indicated that the peptides acted upstream of nuclear import of viral ribonucleoprotein complexes but did not reduce overall virus binding to cells. However, the peptides caused aggregation of the virus particles on the surface of the host cells and reduced their internalisation. Further work evaluated how the peptides may be delivered to the site(s) of viral replication in poultry. A screen of the current literature was completed to allow for the design of an expression cassette for poultry-derived Lactobacillus to express FluPep and Entry Blocker. Though the cassette has been reported to be suitable for expression of heterologous proteins in Lactobacilli, rescue of recombinants for expression of anti-viral peptides or a reporter protein proved challenging, possibly owing to toxicity. A stable construct for the expression of FluPep 4 in Lactobacillus was obtained but culture supernatant did not inhibit virus replication.

No description available.