Characterisation of the non-muscle α-actinins

Actinins are cytoskeleton proteins that cross-link actin filaments. Evolution of the actinin family resulted in the formation of Ca++-insensitive muscle isoforms (actinin-2 and- 3) and Ca++-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Despite high sequence similarity, unique properties have been ascribed to actinin-4 compared with actinin-1. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney and and is able to translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions I have comprehensively compared these proteins in terms of localisation, migration, alternative splicing, actin-binding properties, heterodimer formation and molecular interactions for the first time. This work characterises a number of commercially available actinin antibodies and in doing so, identifies actinin-1, -2 and -4 isoform-specific antibodies that enabled studies of actinin expression and localisation. This work identifies the actinin rod domain as the predominant domain that influences actinin localisation however localisation is likely to be effected by the entire actinin protein. si-RNA- mediated knockdown of actinin-1 and -4 did not affect migration in a number of cell lines highlighting that migration may only require a fraction of total non-muscle actinin levels. This work finds that the Ca++-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca++-insensitive variant of actinin-1. This work also identifies a previously unreported exon 19a+19b expressing variant of actinin-4 in human skeletal muscle. This work finds that alternative splice variants of actinin-1 and -4 are co-expressed in a number of tissues, in particular the brain. In contrast to healthy brain, glioblastoma cells express Ca++-sensitive variants of both actinin-1 and -4. Actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, this work reveals that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cancer cell lines. Taken together this data suggests that actinin-1 and -4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. Finally, this work employs yeast two-hybrid and proteomic approaches to identify actinin-interacting proteins. In doing so, this work identifies a number of putative actinin-4 specific interacting partners that may help to explain some of the unique functions attributed the actinin-4. The observation of alternative splice variants of actinin-1 and -4 combined with the observed potential of these proteins to form homodimers and heterodimers suggests that homodimers and heterodimers with novel actin-binding properties and interaction networks may exist. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion. This significantly alters our view of the non-muscle actinins.

The right to humanitarian assistance in natural and human-made disasters: progress and challenges for an emerging international legal framework

Natural and human-made disasters cause on average 120,000 deaths and over US$140 billion in damage to property and infrastructure every year, with national, regional and international actors consistently responding to the humanitarian imperative to alleviate suffering wherever it may be found. Despite various attempts to codify international disaster laws since the 1920s, a right to humanitarian assistance remains contested, reflecting concerns regarding the relative importance of state sovereignty vis-à-vis individual rights under international law. However, the evolving acquis humanitaire of binding and non-binding normative standards for responses to humanitarian crises highlights the increasing focus on rights and responsibilities applicable in disasters; although the International Law Commission has also noted the difficulty of identifying lex lata and lex ferenda regarding the protection of persons in the event of disasters due to the “amorphous state of the law relating to international disaster response.” Therefore, using the conceptual framework of transnational legal process, this thesis analyses the evolving normative frameworks and standards for rights-holders and duty-bearers in disasters. Determining the process whereby rights are created and evolve, and their potential internalisation into domestic law and policy, provides a powerful analytical framework for examining the progress and challenges of developing accountable responses to major disasters.

Modified cyclodextrins as novel non-viral vectors for neuronal siRNA delivery: focus on Huntington’s disease

Huntington’s Disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a mutant Huntingtin (muHTT) protein. Therefore, preventing the expression of muHTT by harnessing the specificity of the RNA interference (RNAi) pathway is a key research avenue for developing novel therapies for HD. However, the biggest caveat in the RNAi approach is the delivery of short interfering RNA (siRNAs) to neurons, which are notoriously difficult to transfect. Indeed, despite the great advances in the field of nanotechnology, there remains a great need to develop more effective and less toxic carriers for siRNA delivery to the Central Nervous System (CNS). Thus, the aim of this thesis was to investigate the utility of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as non-viral vectors for siRNA delivery for HD. Modified CDs were able to bind and complex siRNAs forming nanoparticles capable of delivering siRNAs to ST14A-HTT120Q cells and to human HD fibroblasts, and reducing the expression of the HTT gene in these in vitro models of HD. Moreover, direct administration of CD.siRNA nanoparticles into the R6/2 mouse brain resulted in significant HTT gene expression knockdown and selective alleviation of rotarod motor deficits in this mouse model of HD. In contrast to widely used transfection reagents, CD.siRNA nanoparticles only induced limited cytotoxic and neuroinflammatory responses in multiple brain-derived cell-lines, and also in vivo after single direct injections into the mouse brain. Alternatively, we have also described a PEGylation-based formulation approach to further stabilise CD.siRNA nanoparticles and progress towards a systemic delivery nanosystem. Resulting PEGylated CD.siRNA nanoparticles showed increased stability in physiological saltconditions and, to some extent, reduced protein-induced aggregation. Taken together, the work outlined in this thesis identifies modified CDs as effective, safe and versatile siRNA delivery systems that hold great potential for the treatment of CNS disorders, such as HD.

Identification of RNA editing in the human exome and development of DARNED DatabaseSF

RNA editing is a biological phenomena that alters nascent RNA transcripts by insertion, deletion and/or substitution of one or a few nucleotides. It is ubiquitous in all kingdoms of life and in viruses. The predominant editing event in organisms with a developed central nervous system is Adenosine to Inosine deamination. Inosine is recognized as Guanosine by the translational machinery and reverse-transcriptase. In primates, RNA editing occurs frequently in transcripts from repetitive regions of the genome. In humans, more than 500,000 editing instances have been identified, by applying computational pipelines on available ESTs and high-throughput sequencing data, and by using chemical methods. However, the functions of only a small number of cases have been studied thoroughly. RNA editing instances have been found to have roles in peptide variants synthesis by non-synonymous codon substitutions, transcript variants by alterations in splicing sites and gene silencing by miRNAs sequence modifications. We established the Database of RNA EDiting (DARNED) to accommo-date the reference genomic coordinates of substitution editing in human, mouse and fly transcripts from published literatures, with additional information on edited genomic coordinates collected from various databases e.g. UCSC, NCBI. DARNED contains mostly Adenosine to Inosine editing and allows searches based on genomic region, gene ID, and user provided sequence. The Database is accessible at http://darned.ucc.ie RNA editing instances in coding region are likely to result in recoding in protein synthesis. This encouraged me to focus my research on the occurrences of RNA editing specific CDS and non-Alu exonic regions. By applying various filters on discrepancies between available ESTs and their corresponding reference genomic sequences, putative RNA editing candidates were identified. High-throughput sequencing was used to validate these candidates. All predicted coordinates appeared to be either SNPs or unedited.