No description available.

No description available.

No description available.

Spinal muscular atrophy is the leading genetic cause of infant death, and much research has gone into the development of potential therapies for the disease. It is caused by a loss of the SMN1 gene. However, patients have the SMN2 gene, which contains a few silent mutations causing the skipping of exon 7 during splicing. One of the most promising therapeutic strategies involves the use of antisense oligonucleotides to rescue the splicing of SMN2 exon 7, allowing for production of full length SMN protein. One successful antisense oligonucleotide strategy involves the use of a bifunctional targeted oligonucleotide enhancer of splicing (TOES) (Skordis et al., 2003). This oligonucleotide, named GGA, consists of an annealing region that targets it to SMN2 exon 7 and a non-annealed enhancer tail domain, designed to recruit activator proteins and stimulate inclusion of exon 7. However, the precise mechanism by which GGA induces exon 7 inclusion was not fully understood at the time of design. This study has focused on investigation of the mechanism of action of GGA, in order to improve the therapeutic potential for GGA and future TOES. GGA was found to bind directly to SRSF1, an activator protein, via its enhancer tail domain. The tail domain forms a G-quadruplex structure in vitro (Smith et al., manuscript submitted). The presence of this structure in nuclear extracts was confirmed, and the enhancer domain was found to bind the G-quadruplex associated proteins CNBP and nucleolin. Stabilization of this structure using ligands reduced the efficacy of GGA, indicating that GGA does not form a G-quadruplex when it is actively stimulating SMN2 exon 7 inclusion. Single molecule methods revealed that the annealing domain of GGA, which anneals over an exonic splicing silencer shown to bind hnRNP A1 and/or Sam68 (Kashima et al., 2007; Pedrotti et al., 2010), reduces the number of Sam68 proteins bound per SMN2 RNA. These findings are consistent with the fact that the annealing region of GGA promotes U2AF65 binding and the enhancer tail domain promotes U2 snRNP binding to SMN2 transcripts (Smith et al., manuscript submitted; Smith, 2012).

Mechanisms of tolerance by which the human body distinguishes self from non-self serve to protect us from pathogens, and are central to the understanding of immunology. Breach of self- tolerance can result in inappropriate immune responses targeting one's own proteins, cells 0 tissues and lead to autoimmune disease. Ankylosing Spondylitis (AS) is a systemic, rheumatic autoimmune disease, which affects up to 0. 1% of the population. Despite a strong association with the MHC class I allele, HLA-B27, the aetiology of AS is unknown. The work in this thesis describes the use of existing and the development of new proteomic methods to characterize and quantify differences in protein expression in both cellular an humoral components of the immune system in patients with AS. Monocytes from AS patients have been characterized by 2D-gel electrophoresis and label-free quantitative nano-UPLC-MSE mass spectrometry analysis, which has revealed differential expression in proteins from the Ubiquitin Proteasome Pathway (UPP) as well as an up-regulation of 13 proteins that are induced by Tumour Necrosis Factor. An in vitro proteasomal digest assay demonstrated that one protein upregulated in the UPP, the proteasomal activator PA2S, increase the generation ofHLA•B27•restricted epitopes. In parallel, novel methods for the MALDI-TOF analysis of endogenous and tryptic plasma peptides were developed with the aim of identifying diagnostic biomarkers in AS plasma. The depletion of abundant plasma proteins followed by plasma fractionalion based on isoelectric focusing or nano-UPLC•MSE mass spectrometry analysis was also employed to this end Preliminary plasma cytokine profiling showed a specific upregulation of interleukin-27 in AS patients. Levels of AS plasma matrix-metallo proteins (MMPs) analysed demonstrated a~ upregulation of two collagenases, however this find ing was not supported by plasma MM enzymatic activity assays. Finally, in collaboration with the Harvard Institute of Proteomics, the novel finding 0 autoantibodies in AS patients was characterized and quantified by the use of Nucleic Acid Programmable Protein Arrays. 44% of AS patients studied demonstrated a broad, non-specific autoantibody response and multiple AS patients showed responses to common putative autoantigens. The AS cohort autoantibody response was more specific compared to that from the RA cohort and autoantibodies from AS patients showed a bias towards antigens involved i6 skeletal and connective tissue disorders. Taken together, evidence for differential expression of proteins in the Ubiquitin Proteasome Pathway and a broad, non-specific autoantibody response in AS patients support the hypothesis ~f a role in antigen presentation for HLA-B27 in Ankylosing Spondylitis. The work in this thesis demonstrates that clinical proteomics is a viable method for the investigation of au toimmuI1f diseases, provides entry points to target cellular and molecular mechanisms for further investigation of disease pathogenesis, disease diagnosis and therapeutic benefit.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease in which low levels of survival of motor neuron (SMN) protein lead to the degeneration of alpha motor neurons (MNs) in the spinal cord. The pathological mechanism of SMA is highly controversial and until recently it was not possible to obtain human MNs to study the disease. The development of induced pluripotent stem cell (iPSC) technology has made it possible to bypass this obstacle and iPSC-based models of SMA type I have already been validated by two separate groups. Encouraged by these pioneering findings I have produced and characterized iPSCs from several members of a discordant consanguineous family in which four haploidentical siblings share the same homozygous SMNl mutation, but nonetheless show different phenotypes of the disease. I have differentiated iPSC clones from three of the siblings and the carrier unaffected mother, as well as a control unaffected clone and a clone derived from a patient with SMA type I, into ISL1+/ChAT+ MNs. No obvious phenotypic difference was observed between the MN cultures of the siblings during the period of study, but cells from the SMA type I patient did show an impaired ability to form rosettes. The study of SMN and PLS3 levels during the differentiation from iPSCs to ChAT+ MNs showed a gradual decay of these proteins during MN development in all clones. Furthermore, SMN protein levels did not correlate with the pattern of mRNA expression, c suggesting the existence of post-transcriptional and/or post-translational regulation of full-length SMN (FL-SMN) transcripts and protein. The FL/.d7-SMN mRNA ratio and total SMN (tSMN) mRNA levels were found to be possible biomarkers to distinguish unaffected individuals from SMA patients and the severity of SMA pathology, respectively. PLS3 protein level was higher in the SMA type IV/asymptomatic sibling than in two of the type III SMA siblings, but it could not be confirmed as a modifier factor in the family. These results suggest that SMN levels are regulated during MN development, and that low levels may impair the generation of rosettes but not necessarily of MNs. SMN levels in MNs only show minor differences between patients, suggesting that there may be a threshold after which reduced levels of SMN within a narrow range become suddenly and increasingly detrimental unless modifier factors can compensate for the cellular function(s) lost. 3

Low back pain is associated with degenerative changes in the intervertebral discs (IVDs) of the spine. Much interest has been placed in recent years on the development of biological therapies for the repair of damaged IVDs. In this thesis, disc cells were assessed for their suitability for this role and in particular for the presence of a progenitor cell sub-population. Emphasis was placed on the analysis of cell clusters, formed from proliferation in the degenerate disc, as a possible source of progenitor cells. Surgical samples of IVD tissue from 100 patients undergoing routine spinal surgery were used to compare cells from within clusters to cells existing in isolation in vivo. IVD cell viability in situ was shown to be greater than often previously suggested, indicating that the IVD may represent an adequate source of autologous cells for biological therapy. Immunohistochemical analysis showed that both clustered and single cells stained positively for putative progenitor and notochordal markers, but there was no significant difference between the incidences in the 2 populations. Sub-culture and flow cytometry analysis of clustered and single IVD cells showed that both populations possessed markers indicative of mesenchymal stromal cells. Further, the clustered and single IVD cells were found to behave in a similar way when exposed to nutrient deprivation (modelling the avascular IVD environment). Single cells could be isolated in a consistent manner from IVDs and proliferated faster in vitro than those originating from clusters. Indeed, . contrary to the original hypothesis, clustered cells appeared no better than single cells as a cell source for biological therapy and the results derived from this thesis may suggest that those single cells in vivo should be used solely in a biological therapy for the regeneration of degenerate IVDs.

Vertebral burst fractures are a significant health and economic concern worldwide. The optimal management of these types of fractures remains unknown. Current techniques for treating burst fractures carry a high risk of complications including neurological damage. Percutaneous vertebroplasty has become a pro~sing alternative procedure for the treatment of Osteoporotic spinal fractures. However, little is known about its effectiveness for the augmentation of burst fractures. The aim of this project was to investigate the biomechanical effectiveness of vertebroplasty as a primary intervention for the treatment of burst fractures with varying degrees of severity. Porcine thoracolumbar spines of varying fracture severity were augmented with PMMA cement. The mechanical stability of these specimens was determined dynamically with 2500 N of axial compression to a maximum of 100000 cycles. A control group of intact and of fractured-unaugmented specimens were also tested under the same conditions. The results show that there was an overall statistically significant difference in the dynamic stiffness means of augmented specimens in the different fracture groups (F (4, 44) =20.7, P < 0.05). The dynamic stiffness of the augmented specimens with the least fracture severity and that of the intact group were not significantly different over 100000 cycles of loading (P>0.05). This corresponded with a similar subsidence rate of 1.73 mm ± 0.69 SD and 1.80 mm ± 0.68 SD respectively. Conversely, the difference in the dynamic stiffness and subsidence rate between the intact and the augmented specimens of greater fracture severity was significant (P < 0.05). The results of this preliminary study indicate that the mechanical properties of the mild burst fracture can be significantly restored with vertebroplasty treatment to their intact levels. However, the technique does not restore the mechanical integrity of fractures with greater severity. The results of this study support recent clinical findings.

The purpose of this thesis was to test the hypothesis that bone marrow oedema (BMO) lesions as shown on MRI in the axial and appendicular skeleton in the spondyloarthropathies (SpA) have diagnostic and prognostic implications in this group of diseases.

No description available.