http://hdl.handle.net/10468/4 2013-05-24T08:16:03Z http://hdl.handle.net/10468/872 Cyclodextrins for non-viral gene and siRNA delivery O'Mahony, Aoife M.; O'Driscoll, Caitríona M.; O'Neill, Martin J.; Godinho, Bruno M. D. C.; Darcy, Raphael; Cryan, John F. Considerable research is focused on the development of non-viral vectors for gene and RNA interference therapies, with significant advancements in this field over the past number of years. Cationic lipids and polymers have been extensively investigated for these purposes, but there still remains a need for alternative vectors. Cyclodextrins (CDs) are cyclic oligosaccharides derived from starch and are well characterised pharmaceutical excipients. They offer many advantages as potential non-viral vectors for gene and siRNA delivery, in particular the ease with which they can be chemically modified and their limited toxicity. In recent years, there has been a surge in the number of publications concerning CDs in this field. In this paper, we will review the two main approaches to the use of CDs for gene and siRNA delivery. In the first instance, CDs are used as a scaffold, to which various chemical groups can be grafted, yielding monodisperse functionalised CDs which can self-assemble in the presence of oligonucleotides. CDs are particularly amenable to chemical modification and this approach enables specific and precise design of CD vectors for targeting to various cell and tissue types. In the second approach, CDs can be included as a component of a delivery system, for example, as part of a polymer backbone, appended to a dendrimeric vector, or in polyrotaxane systems. Here, the inclusion of CDs facilitates post-modification of the vector through the formation of inclusion complexes with adamantane and, in some instances, reduces toxicity of the vector. Lastly, we will consider the development of in vivo CD vectors for therapeutic use and other novel applications including siRNA delivery in neurons and the CNS. 2013-01-01T00:00:00Z http://hdl.handle.net/10468/927 Mediating effects of coping style on associations between psychological factors and self-harm among adolescents McMahon, Elaine M.; Corcoran, Paul; McAuliffe, Carmel; Keeley, Helen S.; Perry, Ivan J.; Arensman, Ella There is evidence for an association between suicidal behaviour and coping style among adolescents. The aims of this study were to examine associations between coping style, mental health factors and self-harm thoughts and acts among Irish adolescents, and to investigate whether coping style mediates associations between mental health factors (depression, anxiety and self-esteem) and self-harm. A cross-sectional school-based survey was carried out. Information was obtained on history of self-harm, life events, demographic, psychological and lifestyle factors. Emotion-oriented coping was strongly associated with poorer mental health and self-harm thoughts and acts, while problem-oriented coping was associated with better mental health. A mediating effect of emotion-oriented coping on associations between mental health factors and DSH was found for both genders and between problem-oriented coping and mental health factors for girls. Similar mediating effects of coping style were found when risk of self-harm thoughts was examined. 2013-01-01T00:00:00Z http://hdl.handle.net/10468/1092 Ryanodine receptor expression in trophoblasts Zheng, Limian Trophoblasts of the placenta are the frontline cells involved in communication and exchange of materials between the mother and fetus. Within trophoblasts, calcium signalling proteins are richly expressed. Intracellular free calcium ions are a key second messenger, regulating various cellular activities. Transcellular Ca2+ transport through trophoblasts is essential in fetal skeleton formation. Ryanodine receptors (RyRs) are high conductance cation channels that mediate Ca2+ release from intracellular stores to the cytoplasm. To date, the roles of RyRs in trophoblasts have not been reported. By use of reverse transcription PCR and western blotting, the current study revealed that RyRs are expressed in model trophoblast cell lines (BeWo and JEG-3) and in human first trimester and term placental villi. Immunohistochemistry of human placental sections indicated that both syncytiotrophoblast and cytotrophoblast cell layers were positively stained by antibodies recognising RyRs; likewise, expression of RyR isoforms was also revealed in BeWo and JEG-3 cells by immunofluorescence microscopy. In addition, changes in [Ca2+]i were observed in both BeWo and JEG-3 cells upon application of various RyR agonists and antagonists, using fura-2 fluorescent videomicroscopy. Furthermore, endogenous placental peptide hormones, namely angiotensin II, arginine vasopressin and endothelin 1, were demonstrated to increase [Ca2+]i in BeWo cells, and such increases were suppressed by RyR antagonists and by blockers of the corresponding peptide hormone receptors. These findings indicate that 1) multiple RyR subtypes are expressed in human trophoblasts; 2) functional RyRs in BeWo and JEG-3 cells response to both RyR agonists and antagonists; 3) RyRs in BeWo cells mediate Ca2+ release from intracellular store in response to the indirect stimulation by endogenous peptides. These observations suggest that RyR contributes to trophoblastic cellular Ca2+ homeostasis; trophoblastic RyRs are also involved in the functional regulation of human placenta by coupling to endogenous placental peptide-induced signalling pathways. 2013-01-01T00:00:00Z http://hdl.handle.net/10468/873 Characterisation of cationic amphiphilic cyclodextrins for neuronal delivery of siRNA: effect of reversing primary and secondary face modifications O'Mahony, Aoife M.; Doyle, D.; Darcy, Raphael; Cryan, John F.; O'Driscoll, Caitríona M. Significant research is focused on the development of non-viral vectors for delivery of siRNA to neurons and the central nervous system. Cyclodextrins (CDs) have shown great promise as efficient and low toxicity gene delivery vectors in various cell types. Here, we investigate two CDs for siRNA delivery in a neuronal cell model. These CDs were substituted on opposite faces (primary and secondary) with amphiphilic and cationic groups. Physical properties of CD.siRNA complexes, including size, charge and stability were measured. In vitro investigations were carried out in immortalised hypothalamic neurons. Neuronal cell uptake was measured by flow cytometry and cytotoxicity was assessed by MTT assay. Knockdown of a luciferase reporter gene was used as a measure of gene silencing efficiency. Both CDs interacted with siRNA, yielding nanosized cationic complexes which exhibited good stability on storage. A favourable toxicity profile was demonstrated for the CD.siRNA complexes. However, only one of the two CDs mediated high levels of neuronal uptake and efficient gene silencing, equivalent to those achieved with a commercial lipid-based vector. Despite the suitability of both CDs as siRNA delivery vectors in terms of their ability to complex siRNA and the properties of the complexes yielded, only one CD achieved good transfection efficiency. This was likely due to the differences in their chemical structures. The effective CD offers great potential as a novel non-toxic vector for neuronal siRNA delivery. 2012-12-18T00:00:00Z