Biochemistry and Pharmacology - Theses
Permanent URI for this collection
Search Results
1 - 1 of 1
-
ItemDefining the roles of long and small transcriptomes in neurodegenerative diseases( 2015)Neurodegenerative diseases belong to a group of disorders that are characterised by progressive degeneration of neurons, and often exhibit aberrant transcriptional regulatory programs prior to the manifestation of clinical symptoms. Owing to the evidence that RNAs are functionally rich class of molecules that dictates phenotypic changes, the identification of aberrant RNA signatures potentially provides mechanistic insights into disease pathogenesis. In this thesis, I present a comprehensive study of long and small transcriptomes in neurodegenerative diseases, with the objective of restructuring the approach of disease diagnosis and treatment. I determine the underlying disease mechanisms associated with misfolded protein aggregation and neuronal death using murine models. I then investigate whether these transcripts can be used as biomarkers and therapeutics to monitor disease progression. The first aspect of this thesis highlighted the challenges of large-scale transcriptomic data processing for biological analysis. I developed iSRAP, an integrated small RNA analysis pipeline, for rapid profiling of small RNA sequences generated from next-generation sequencing. Data sets from several small RNA studies were used to demonstrate iSRAP workflow, which covers from data quality assessment to differential expression analysis. The results revealed iSRAP features, including high-throughput capability, graphical result representations, convenience and reliability. iSRAP can serve as a platform for rapid analysis of transcriptomic data so that informed decision can be made on the downstream analyses of small RNA studies. The second aspect of my work emphasised the significant interest in utilising exosomes to identify small RNA biomarkers for diagnostics purposes. Exosomes are nano-sized extracellular vesicles of endocytic origin that involves in shuttling of RNA between cells within a biological system, facilitating disease spreading and pathogenesis. There are currently different methods available to isolate exosomes for studying small RNA profiles. In order to investigate the feasible exosome isolation method for biomarker discovery that required simple workflow, I implemented a combined approach of RNA sequencing and bioinformatics to profile a wide range of small RNA species in exosomes isolated by two different methods: differential ultracentrifugation and OptiPrep velocity ultracentrifugation. The analysis showed that these methods yielded exosomes with similar small RNA profiles to each other, suggesting that the higher purity of exosomes by OptiPrep method did not influence the small RNA profiles. Therefore, these findings revealed that the conventional ultracentrifugation-based method posed as a simple and sufficient protocol for biomarker discovery in exosomes. To better understand the disease mechanisms and therapeutic intervention in neurodegenerative diseases, a mouse model was used to recapitulate human prion diseases and Parkinson’s disease respectively. In the context of prion diseases, the study focused on the clinical relevance of microRNA (miRNA), which is a class of small RNAs that negatively regulates gene targets controlling fundamental pathways such as cellular signalling and neuronal development. The temporal distribution of miRNA expression profiles over the course of prion infection was determined. I established an analytical workflow to detect pre-clinical and clinical miRNA signatures in mice that were infected with prions over a time course. There were several pre-clinical and clinical miRNA signatures, such as let-7b, miR-223-3p and miR-362-5p, which were implicated in the early impairment of neurogenesis or terminal-stage disease progression. The detected miRNA signatures at different stages of prion disease can potentially serve as promising diagnostic and therapeutic measures for inhibiting and screening of prion infectivity. The final aspect of this thesis detailed the therapeutic relevance of the CuII(atsm) compound in Parkinson’s disease using whole transcriptomes analysis. I employed several computational and statistical methods to show a panel of protein-coding RNAs associated with neuronal development, dopamine synthesis and synaptic neurotransmission in the substantia nigra of the brain. Upon CuII(atsm) treatment, the expression of 40 genes involved in promoting dopamine synthesis, calcium signalling and synaptic plasticity were restored. To my knowledge, this is the first transcriptomic study that comprehensively reported the key therapeutic pathways targeted by CuII(atsm) compound that may provide therapeutic benefits for Parkinson’s disease and other neurodegenerative diseases. In summary, the collective findings from this thesis provide neurologists with mechanistic insights into the neuronal pathways, and thereby enabling the development of diagnostic and therapeutic measures that aim at inhibiting the progression of degenerating neurons in the brain. The present studies will therefore provide more informed treatment alternatives for neurodegenerative diseases.