Revised annotation and characterization of novel Aedes albopictus miRNAs and their potential functions in dengue virus infection

The Asian tiger mosquito, Ae. albopictus, is a highly invasive species that transmits several arboviruses including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV). Although several studies have identified microRNAs (miRNAs) in Ae. albopictus, it is crucial to extend and improve current annotations with both the newly improved genome assembly and the increased number of small RNA-sequencing data. We combined our high-depth sequence data and 26 public datasets to re-annotate Ae. albopictus miRNAs and found a total of 72 novel mature miRNAs. We discovered that the expression of novel miRNAs was lower than known miRNAs. Furthermore, compared to known miRNAs, novel miRNAs are prone to expression in a stage-specific manner. Upon DENV infection, a total of 44 novel miRNAs were differentially expressed, and target prediction analysis revealed that miRNA-target genes were involved in lipid metabolism and protein processing in endoplasmic reticulum. Taken together, the miRNA annotation profile provided here is the most comprehensive to date. We believed that this would facilitate future research in understanding virus–host interactions, particularly in the role of miRNAs

Assessment of Aedes albopictus reference genes for quantitative PCR at different stages of development.

Members of the Aedes genus of mosquitoes are widely recognized as vectors of viral diseases. Ae.albopictus is its most invasive species, and are known to carry viruses such as Dengue, Chikugunya and Zika. Its emerging importance puts Ae.albopictus on the forefront of genetic interaction and evolution studies. However, a panel of suitable reference genes specific for this insect is as of now undescribed. Nine reference genes, namely ACT, eEF1-γ, eIF2α, PP2A, RPL32, RPS17, PGK1, ILK and STK were evaluated. Expression patterns of the candidate reference genes were observed in a total of seventeen sample types, separated by stage of development and age. Gene stability was inferred from obtained quantification data through three widely cited evaluation algorithms i.e. BestKeeper, geNorm, and NormFinder. No single gene showed a satisfactory degree of stability throughout all developmental stages. Therefore, we propose combinations of PGK and ILK for early embryos; RPL32 and RPS17 for late embryos, all four larval instars, and pupae samples; eEF1-γ with STK for adult males; eEF1-γ with RPS17 for non-blood fed females; and eEF1-γ with eIF2α for both blood-fed females and cell culture. The results from this study should be able to provide a more informed selection of normalizing genes during qPCR in Ae.albopictus

MicroRNA biogenesis and cytoophidia in Drosophila melanogaster

This thesis contains two separate projects. The first project involves the study of Argonaute 1 (Ago1) which is a member of the Argonaute/PIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Ago1 plays a specific role in microRNA (miRNA) function. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspects of oogenesis is still elusive. Here, the function of Ago1 was analysed in the female germline and follicle cells. The Ago1 protein is enriched in the oocyte and also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles shows that mutant egg chambers often contain only eight nurse cells and lack an oocyte which is phenocopied in dicer-1, pasha and drosha mutants. This implies a general role of miRNAs in this process. Further analysis of the clones shows that Ago1, Dicer-1, Drosha and miR-124a play a role in maintaining follicle cell integrity. Additionally, it was found that Ago1 protein levels were lowered when miRNAs were not present and that overexpression of miR-124a increased the steady state level of Ago1 proteins. These were believed to be the effect of Ago1 protein stability. In the meantime, a second project was carried studying a novel filamentous structure called cytoophidia which contains CTP synthase (CTPS) was analysed. Previous studies have shown that the cytoophidia is prominent in the egg chambers but how and why CTPS forms cytoophidia is still unclear. Here, the different CTPS isoforms were analysed and only CTPS isoform C was found to form cytoophidia. This isoform could also induce cytoophidia formation in other tissues that does not have obvious cytoophidia formation. Then, it was identified that the first 56 amino acid of CTPS isoform C is the important region for cytoophidia formation. Furthermore, ctps mutants were generated and analysed. CTPS mutations caused lethality during the larval stage and when maternal contribution was removed, lethality was seen during embryogenesis.</p

Heritextuality : a study in an emergent discipline of touristic discource

This thesis defines a new transdisciplinary field o fheritextuality as cultural productions designed for touristic consumption containing elements of historical information. Its focus is on touristic discourse in and about Tasmania in the twentieth century. As such, the thesis identifies for analysis and study, heritexts like travelogues, tourist guides, promotional films, photographic essays, advertising media, and postcards employing a methodological approach that combines archival research, historical survey and textual analysis. In its discussion of heritexts, the thesis examines the convergence between history and tourism as evidenced in the variety of textual forms, including the visual, produced for touristic consumption. It argues that such touristically constructed heritexts constantly generate and inform distinctive and discrete versions of the past that influence and inform popular conceptions of history, and that define a touristic sense-of-place independent of scholarly historical discourse. Heritextuality facilitates specific negotiations of historical elements that are not accessible to, and that operate outside of, the usual constraints of conventional historical discourse. This study highlights the arbitrary relationship that allows heritexts to reconfigure historical events, particularly those otherwise considered problematic, for its own touristic purposes. The thesis examines how this process occurs. The study focuses on the inner workings of the Tasmanian tourist bureaucracy, highlighting early collaborative projects between the Tasmanian Government Tourist Bureau and producers of heritexts like Frank Hurley, Bill Beatty, E.T. Emmett, Charles Barrett and Michael Sharland. This research identifies the ways in which the production and consolidation of heritextuality continues to shape and inform contemporary uses of history in touristic contexts

MicroRNA biogenesis and cytoophidia in Drosophila melanogaster

This thesis contains two separate projects. The first project involves the study of Argonaute 1 (Ago l) which is a member of the ArgonautelPIWI protein family involved in small RNA-mediated gene regulation. In Drosophila, Agol plays a specific role in microRNA (miRNA) function. Previous studies have demonstrated that Ago 1 regulates the fate of germline stem cells. However, the function of Ago 1 in other aspects of oogenesis is still elusive. Here, the function of Ago 1 was analysed in the female germline and follicle cells. The Ago 1 protein is enriched in the oocyte and also highly expressed in the cytoplasm of follicle cells. Clonal analysis of multiple ago] mutant alleles shows that mutant egg chambers often contain only eight nurse cells and lack an oocyte which is phenocopied in dicer-I , pasha and drosha mutants. This implies a general role of miRNAs in this process. Further analysis of the clones shows that Agol, Dicer-l, Drosha and miR-l24a play a role in maintaining follicle cell integrity. Additionally, it was found that Ago 1 protein levels were lowered when miRNAs were not present and that overexpression of miR-124a increased the steady state level of Ago 1 proteins. These were believed to be the effect of Ago 1 protein stability. In the meantime, a second project was carried studying a novel filamentous structure called cytoophidia which contains CTP synthase (CTPS) was analysed. Previous studies have shown that the cytoophidia is prominent in the egg chambers but how and why CTPS forms cytoophidia is still unclear. Here, the different CTPS isoforms were analysed and only CTPS isoform C was found to form cytoophidia. This isoform could also induce cytoophidia formation in other tissues that does not have obvious cytoophidia formation. Then, it was identified that the first 56 amino acid of CTPS isoform C is the important region for cytoophidia formation. Furthermore, ctps mutants were generated and analysed. CTPS mutations caused lethality during the larval stage and when maternal contribution was removed, lethality was seen during embryogenesis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Average stability values (M) of genes in individual developmental stages and cell culture.

<p>Lower values indicate better stability.</p

Rankings of candidate genes by geNorm.

<p>Rankings of candidate genes by geNorm.</p

Rankings from all three algorithms and resulting consensus.

<p>Rankings from all three algorithms and resulting consensus.</p