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Parkinson's disease is the second commonest neurodegenerative disease currently treated symptomatically. It is a multifactorial disease involving mechanisms ranging from protein aggregation to mitochondrial dysfunction, oxidative stress and dopamine dysregulation. The levels of α-synuclein have been causatively linked to the development and progression of Parkinson's disease. Therefore α-synuclein lowering strategies are valid approaches in Parkinson's disease. Neuropathologically, Lewy Bodies in the vulnerable substantia nigra of Parkinson's disease patients are less ubiquitinated and specifically less K-63 ubiquitinated than Lewy bodies in the cortex, suggesting differential activation or regulation of ubiquitin interactors. A targeted screen for such interactors revealed that the Deubiquitinating enzyme Usp8 is upregulated in the substantia nigra of Parkinson's disease brains and is inversely correlated with the degree of total and K-63 ubiquitination. Using genetic knockdown and overexpression techniques, Usp8 was found to colocalize and directly interact with α-synuclein. It was found to de-ubiquitinate α-synuclein and increase its half-life. Its knockdown increased the total and K-63 α-synuclein ubiquitination and decreased its levels by 35% at least partly by increasing its degradation via the lysosome. In vivo in the Drosophila melanogaster, Usp8 knockdown demonstrated protection against α-synuclein toxicity. It rescued in a specific manner the rough eye phenotype, the age-dependent locomotive defect and the loss of dopaminergic neurons caused by the expression of α-synuclein. Specific and effective pharmacological Usp8 inhibition also has the potential to lower α-synuclein levels. Collectively, the evidence produced in my thesis suggests that Usp8 could be a potential target for the future disease-modifying therapies in Parkinson's disease.

CD31 mediates homophilic interactions between leukocytes and macrophages during inflammation, apoptotic cells remain attached and are engulfed whereas viable cells actively detach. We hypothesised that differential recruitment of signalling and adapter molecules to the cytoplasmic domain were responsible for the disengagement of the viable leukocyte from macrophages. Investigation with a static attachment assay using THP-1 as a macrophage model showed the ITIM of CD31 on the leukocyte was important for viable cell detachment. Our data also implicated a role for the recruitment of SHP-2 which we attempted to knock-down by siRNA delivered by lentivirus. SHP-2, in addition to its phosphatase activity, also acts as a docking protein. To examine for potential interacting partners we fused the cytoplasmic domain of CD31 to GST which we used in pulldown assays from lysates of viable and apoptotic leukocytes. We demonstrated that the recruitment of SHP-1 and SHP-2 were dependent on an intact ITIM (immunoreceptor tyrosine-based inhibitory motif). Interestingly, apoptotic cell lysates promoted dephosphorylation of the in vitro phosphorylated GST-CD31, suggesting an increase in phosphatase activity in aged neutrophils. We were unable to demonstrate an interaction between the cytoplasmic domain of CD31 with putative binding partners β-catenin, src, RasGAPp120, RhoGAPp190, talin or calmodulin. A proteomic approach by MALDI-TOF and MS/MS identified Hsp90 as a novel binding partner of CD31 irrespective of the phosphorylation state. In contrast, 14-3-3ε bound to phosphorylated CD31, whereas eIF3 specifically bound to an ITIM double tyrosine mutant. The binding of Hsp90 to CD31 was proposed to occur via a TPR motif within the cytoplasmic domain of CD31 which comprises a surface fold of basic amino acids complexing a highly acidic carboxy tail of Hsp90. Truncation and site directed mutagenesis of the cytoplasmic domain revealed multiple binding sites for Hsp90; specifically two regions containing the sequences (KAFYLRKAKAK), previously shown to be the calmodulin binding region, and a novel area (SNNEKMSDMEANSHY) which has significant homology with other TPR-containing proteins. Systematic mutagenesis of the putative basic charged amino acids within the cytoplasmic domain of CD31 which may mediate the interaction with Hsp90 also supports the presence of a TPR motif. The importance of Hsp90, 14-3-3ε and eIF3 is currently unknown, although it is interesting to note that CD31 was recently found within this laboratory to associate with the voltage-gated potassium channel HERG which also binds 14-3-3ε and Hsp90. eIF3 is an RNA helicase that may link CD31 and leukocyte motility to spreading initiation centres where motility can be viewed as a rapid turnover of focal adhesion complexes. Together, these studies have identified novel binding partners of CD31 may form a macromolecular complex to promote CD31-dependent leukocyte motility and detachment from macrophages.

Elucidating the molecular determinants of aggressive prostate cancer has major implications for developing molecular diagnostics that, in conjunction with clinicopatholgic variables, can be used to risk-stratify patients more accurately. We hypothesized that DNA methylation can distinguish indolent from aggressive prostate cancer through aberrant gene regulation. This thesis presents the novel bioinformatics tools needed to pursue this hypothesis, a methylome analysis in aggressive prostate cancer, evidence for genomic context-specific functional interpretations of DNA methylation, data from a model system regarding a role for DNA methylation in regulating alternative cleavage and polyadenylation (APA), and integration of differential methylation data with a machine learning predictor of prostate cancer histology. We found focal DNA hypermethylation can distinguish indolent, low Gleason grade prostate cancer from aggressive, high grade disease using the genome-wide MBD-isolated genome sequencing technique. Notably, high grade DMRs occurred not only in the promoters of genes, but also in gene bodies and intergenic regions highly enriched for DNaseI hypersensitivity, annotated enhancers, and DNA-protein binding sites. DNA methylation also correlates with reduced gene expression for genes where low expression associates with poorer outcomes. By analyzing genic DMRs in a pathway network, we detected common cancer-related signatures between shared and high grade DMR genes. We further extended our analysis to consider connections with alternative transcript usage. Using RNA-seq data from The Cancer Genome Atlas (TCGA), we correlated DNA methylation changes with alternative exon usage in aggressive prostate cancer. Following an observation that prostate cancer DMRs show enrichment in gene 3' ends, we used a model system with genomic depletion of DNA methylation to consider if DNA methylation can regulate alternative cleavage and polyadenylation (APA). We related candidate genes from this analysis back to prostate cancer via correlations based on TCGA RNA-seq data, and further found evidence for these associations by using data across developmental lineages in the Epignome Roadmap Project. We also employed machine learning methods to identify if any DMRs occur proximal to genes that can predict Gleason score. This catalog of high grade DMRs can be used to guide mechanistic studies into how DNA methylation alters gene regulation that leads to aggressive cancer.

Hyaluronan (HA) is a glycosaminoglycan polymer found in the extracellular matrix of all mammalian tissues and a component of human milk. Breast-feeding is associated with enhanced protection from gastrointestinal infection in infants. However, the function of naturally produced milk hyaluronan relevant to antimicrobial function was unknown. Recent work has suggested a role for small, fragmented HA polymers in initiating innate defense responses through interaction with innate molecular pattern recognition receptors such as TLR4. It was hypothesized that hyaluronan from human breast milk enhances innate intestinal epithelial antimicrobial defense. Synthetically produced HA fragments promote expression of the innate antimicrobial peptide human ß-defensin 2 (HßD2) in intestinal epithelial cells. Treatment of HT-29 colonic epithelial cells with HA fragment preparations resulted in time- and dose-dependent upregulated expression of HßD2 protein in a fragment size-specific manner, with 35 kDa HA fragment preparations emerging as the most potent inducers of intracellular HßD2. Furthermore, oral administration of specific-sized HA fragments promotes the expression of an HßD2 ortholog in the colonic epithelium of both wild-type and CD44-deficient mice, but not in TLR4-deficient mice. Thus, a highly size-specific, TLR4-dependent, innate defense response to fragmented HA contributes to intestinal epithelium antimicrobial defense through the induction of intracellular HßD2 protein. Having examined the induction of antimicrobial defense by synthetically produced HA, functional antimicrobial defense was evaluated following treatment of epithelium with HA isolated from human milk. The expression of milk HA was defined during the first 6 months postpartum among a cohort of 44 healthy donors. Treatment of HT-29 epithelium with preparations of human milk HA at physiologic concentrations resulted in a time- and dose-dependent induction of the antimicrobial peptide HßD2 that was abrogated by digestion of milk HA with a specific hyaluronidase. In addition, oral administration of human milk-derived HA to adult, wild-type mice resulted in induction of murine HßD2 ortholog in intestinal mucosa. Finally, treatment of cultured colonic epithelium with human milk HA preparations significantly enhanced resistance to infection by the enteric pathogen Salmonella enterica. Together, these observations suggest that maternally provided HA mediates one of the multiple protective antimicrobial defense mechanisms delivered through milk to the newborn.