Cardiac remodelling is associated with differential expression of microRNAs (miRNAs ) and down-regulation of inward rectifier K+ current (lKl) , of which Kir2.1 is the predominant molecular component . miR-212, miR-132 and miR-320 have been reported to be pro-hypertrophic miRNAs. Furthermore they are predicted to bind Kir2 .1 3' untranslated region (3'UTR) by miRNA target prediction algorithms, indicating a potential role in IKI down-regulation. The aims of this study were to examine which miRNAs overexpressed in hearts undergoing remodelling, en route to failure, are involved in the downregulation of Kir2.1 and IKI. MiRNAs up- regulated in failing and hypertrophic hearts were cross-referenced to miRNAs predicted to target Kir2 .1 by prediction algorithms . The interaction of the shortlisted miRNAs, miR-132, miR-212 and miR-320, with Kir2.1 3'UTR was investigated by a duallight luciferase assay expressing a luciferase gene with fragments of Kir2.1 3'UTR. Co - transfection of HEK293T cells with a luciferase construct, a Bgalactosidase construct and a plasmid encoding miR-212 (pmiR-212) reduced luciferase activity compared to control. MiR-132 and miR-320 did not show any significant reduction of luciferase activity. The HeLa cell line was chosen as an experimental model to study Kir2.1 expression under the regulation of miR- 212 . HeLa cells were confirmed to express Kir2.1 with a negligible contribution by Kir2 .2 at mRNA and protein levels. Transfection of HeLa with miR-212 or control indicated a strong down-regulation of IKl, confirmed by protein decrease. HL-l cells were characterized for the first time to pass IKI and to express Kir2.1, Kir2.2 and Kir2 .3. However, Kir2.1 expression was apparent l y dependent on the serum used for cell culture . miR-212 was transfected into HL-l expression was very low Kir2.1 compared to Kir2.2 and Kir2 . 3. IKI was not reduced, suggesting that miR- 212 reduces IKI by down - regulating Kir2.1 expression, suggesting miR-212 to be a regulator of IKI in cardiac hypertrophy and heart failure.

miRNAs are an important class of small non-protein coding RNAs whose specific functions in animals are rapidly being elucidated. miRNAs regulate the expression of many animal genes by post-transcriptional gene silencing and play vital roles in stem cell maintenance, differentiation and apoptosis. In this study, planarians were used as a model system in order to study whether miRNAs have a regulatory role in their stem cell dynamics. Planarians are well known for their remarkable regenerative ability and their amazing capacity for constant re-patterning owing to a population of somatic pluripotent stem cells known as neoblasts. In particular, the aim of the study was to investigate the regulatory role miRNAs may have in planarian stem cell self renewal, proliferation and differentiation. Recently, the differential spatial patterns of expression of miRNAs in whole and regenerating planarians have been characterized by in situ hybridization to nascent miRNA transcripts. These miRNA expression patterns were the first to be determined for a Lophotrocozoan animal. The expression patterns of 42 miRNAs in adult planarians have been characterized, constituting a complete range of tissue specific expression patterns. The majority of planarian miRNAs were expressed either in areas where stem cells (neoblasts) are located and/or in the nervous system. Some miRNAs were definitively expressed in stem cells and dividing cells and moreover miRNAs were found to be expressed in germ stem cells of the sexual strain. Taken the facts that cellular proliferation and differentiation must be controlled during planarian regeneration and tissue homeostasis as well as that miRNAs have been implicated in the control stem cell functions in other organisms, aim of this study was to disrupt the canonical miRNA biosynthesis pathway. In that way, information about the global impact of miRNA regulation in planarian regeneration and tissue homeostasis would be gained.

Mitochondria are present in all nucleated eukaryotic cells and generate virtually all the energy for cellular processes. Mitochondria possess their own genome, and mutations in mtDNA or in nuclear genes encoding mitochondrial proteins have been associated with a number of human disorders, and the decline of the activity and efficiency of the oxidative phosphorylation has been linked to the majority of age- related degenerative diseases. Investigation of the mechanisms that govern mtDNA expression is therefore critical to understand their role in physiological and disease conditions. Human mt-mRNA turnover remains one of the unsolved puzzles. Neither the possible pathways nor the enzymes involved have been described. Mitochondrial mRNAs are polyadenylated, but the role ofpoly(A) tails and the existence of a protective poly(A)-binding protein is uncertain. In the studies presented in this thesis the extended biochemical fractionation of mitochondria from rat liver or human cell line combined with in vitro enzymatic analyses was used to isolate putative ribonucleases involved in the metabolism of mitochondrial mRNA. This resulted in identification ofHRSP12 as a potential candidate. The intracellular localisation ofHRSP12 could not be determined unequivocally, and further study will assess whether any of the several splicing variants ofHRSP12 is indeed a mitochondrial enzyme involved in mRNA degradation. To improve future research into factors involved in mitochondrial mRNA degradation a modified purification strategy was initiated and optimised during the course of this work, with the aim to preserve the native interactions of putative multiprotein machineries controlling and facilitating mitochondrial gene expression. In an alternative approach to studying the mechanism of mt-mRNA degradation the role of polyadenylation in mitochondrial transcript stability was investigated. The exogenous poly(A)-exoribonuclease PARN was targeted to the mitochondria of human cell line. Ensuing deadenylation of mitochondrial mRNAs revealed that while the effect of deadenylation on the steady-state levels of transcripts varies between the mRNA species, polyadenylation is not an absolute requirement for stability of mt-mRNA. Moreover, following transcript deadenylation by exogenous PARN, synthesis of the mitochondrially-encoded polypeptides appeared to be compromised, therefore the results of this study strongly suggest that polyadenylation of mitochondrial mRNA is essential in regulating protein synthesis in human mitochondria.

At the beginning of the 2oth century Paul Ehrlich proposed the concept of 'magic bullets' to fight disease in the human body. At the start of the 21st century there are now a range of highly selective biologics. Amongst these are the emerging RNAi therapeutics that offer gene specific treatment. There is a need for delivery systems that will localise these RNAi therapeutics at the disease site and facilitate their cellular internalisation. To fully capitalise on the potential of RNAi therapeutics the delivery systems will need to be platform technologies that can be applied to a broad range of diseases. This project explored the potential of the bacteriophage MS2 as a platform delivery technology for RNAi therapeutics. The 19 nucleotide translational repressor sequence of the bacteriophage MS2 genome was synthesised as part of a si RNA. The double stranded 21 nucleotide siRNA was specific for a 21 nucleotide sequence in the mRNA of the onco-protein Bcl2. The chimeric siRNA triggered the assembly of recombinant MS2 coat protein into virus-like particles in vitro. The siRNA was stable and protected from nuclease-mediated degradation inside the capsids:The human transferrin protein was then covalently linked to the virus-like particles to form synthetic virions. The effect of these synthetic virions on human epithelial cancer cells was investigated in vitro using flow cytometry. The synthetic virions were targeted for receptor- mediated endocytosis and the siRNA accumulated in the cancer cells where it reduced Bcl2 expression. All the cells that internalised a detectable quantity of the siRNA in the initial 24 hours were apoptotic within 48 hours. Increasing the synthetic virion dose improved the number of cancer cells with the lethal intracellular quantity of siRNA, but also caused off-target effects. In order to test synthetic virions incorporating other siRNA sequences, a high- throughput screening system was developed. It used automated confocal microscopy technology and image recognition software to observe and map individual cells from adherent monolayers. Different targeter and siRNA combinations can now be tested as part of the MS2 platform technology in a high-throughput manner.

This work focuses on the replicase translational operator (TR) stem loop of the RNA MS2 bacteriophage. Single stranded RNA (ss RNA) viruses are important organisms as they represent pathogenic species in all walks of life including a number of clinically important species such as HIV, Marburg Virus and Ebola. MS2 provides a great model system in which to study regulatory pathways affected by RNA folds due to the extensive biochemical and structural data available. In MS2 the TR stem loop acts as an allosteric switch, via a sequence specific recognition event involving the phage coat protein. Binding of the coat protein dimer to TR signals a switch in the life cycle of the phage from replication to assembly and release. Binding of the coat protein dimer leads to translational repression the viral replicase cistron and also leads to an allosteric conformational change in the protein. TR also contains the AUG start codon for replicase gene and thus is involved in the regulatory pathway of the cistron. Thus any insight to how the folds in the RNA effect binding or translational repression are important for understanding the MS2 virus and other ssRNA viruses like it. The folding ensembles present in solution for a series of RNA oligonucleotides that encompass TR have been examined. Single molecule fluorescence assays suggest that these RNAs exist in solution as differentially base paired/stacked states at equilibrium. What emerges from the experimental is data consistent with an unfolding mechanism in which interruptions to the A- form duplex such as the single stranded loop, the free ends and bulged base of TR, act as nucleation points of unfolding. Additionally substitution of a uracil to a cytosine in the loop causes heterogeneity in the folded and unfolded ensembles relative to the wild-type molecule. This data is used to study folding of the RNAs by 'steered' molecular dynamics (MD) simulations. These show remarkable agreement with experimental data and provide atomistic detail on the molecular interactions which take place during the unfolding pathways studied. The adopted conformers for each simulation provide details about hydrogen bonding between the base pairs of the stem and confirm predicted conformers from apparent dye separation in FRET experiments.

Replication of poliovirus involves complexes between the viral RNA-dependent-RNA polymerase 3Dpol, its precursor 3CD and the primer Vpg, although the details of replication initiation and elongation are not fully understood. Much less is known about replication complexes of other picornaviruses, especially FMDV. To gain insight into these mechanisms, we carried out in vitro selection to produce RNA aptarners to FMDV 3Dpol. Aptamers are small nucleic acid molecules that can bind targets with affinities and specificities to rival those of antibodies, but offer significant advantages as molecular tools. They are generated by an in vitro selection process termed SELEX and can be modified chemically for use both in vitro and in live cells and to facilitate their detection.

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve the understanding of subunit roles and enzyme architecture, protein-protein and protein-RNA interactions have been examined in vitro, complementing existing yeast two-hybrid data.

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Long non-coding RNAs (IncRNAs) are a biological entity defined by what they are not, rather than by what they are. This indicates that our knowledge about them is sensibly limited. The aim of my PhD is to gain insights into the evolution and the functions of IncRNAs through computational approaches and the usage of large scale functional genomics dataset. I developed an annotation pipeline, which can effectively identify IncRNAs in entire transcriptomes. The pipeline is able to accurately annotate the coding genes while predicting a conservative estimate of the IncRNA population. It allowed me to show, for the first time, the presence of lncRNA transcription in a diverse range of organisms. Further, I analysed sequence and positional conservation of lncRNAs, demonstrating the presence of short segments of conserved sequence in IncRNAs and the existence of several syntenically conserved non-coding transcripts over large evolutionary distances. However, I also demonstrate that positional conservation of lncRNAs with a flanking coding gene is generally independent from the conservation of the lncRNA expression with respect to the coding gene. Finally, I have characterised the diversity of lncRNA transcription in specific cells and developmental stages of two teleost fishes. In summary, the work presented in the thesis provides novel findings and contributions in the field of lncRNAomics.