Biochemistry and Pharmacology - Theses

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Investigating features and interactions of the childhood respiratory microbiome

Watts, Stephen ( 2022)

The human microbiome is closely linked with the health of an individual and is implicated in numerous complex diseases including diabetes, inflammatory bowel disease, cancer, cystic fibrosis (CF), and asthma. There is growing evidence to suggest the respiratory microbiome influences risk and trajectory of respiratory disease from an early age. Hence, unravelling the biology of the childhood respiratory microbiome is critical to gain a comprehensive understanding of respiratory disease, and requires characterisation of both the aggregate community and individual community members. This thesis strengthens our understanding of the childhood respiratory microbiome through i) investigation of specific community members, Haemophilus influenzae and Haemophilus parainfluenzae, in the context of CF, and ii) exploration of upper respiratory tract (URT) microbiome development during the first year of life with a particular focus on microbe-microbe interactions. While morbidity and mortality of CF principally result from repeated respiratory infections by Pseudomonas aeruginosa, there is emerging evidence that respiratory tract colonisation by Haemophilus species during childhood induces early disease progression. I describe the detection, antimicrobial resistance (AMR), and genome sequencing of H. influenzae and H. parainfluenzae isolated from airway samples of children enrolled in the AREST CF program. This work revealed H. influenzae and H. parainfluenzae carriage rates and strain persistence among participants. Haemophilus isolates were genetically diverse and commonly resistant to antimicrobials with several putative novel resistance determinants identified. Finally, genomic data identified transmission of Haemophilus strains between participants. The association between the respiratory microbiome and respiratory disease has been established in several cohort studies. However, no work has been undertaken to compare preservation of respiratory microbiome dynamics or to reconcile differences between cohort studies. This thesis explores 16S rRNA gene survey data from four longitudinal childhood cohorts, with a focus on microbe-microbe interactions. The URT microbiome composition dynamics during the first year of life are shown to be well preserved across cohorts, and the aggregate data set is leveraged to reveal associations between specific community members and symptoms of acute respiratory illness. A foundation for microbe-microbe interactions during the first year of life is established, which facilitated discovery of two communities that dominate the URT microbiome. For both areas of focus presented in this thesis I additionally developed two novel software tools to support and enhance analysis: hicap, a tool for robust inference of H. influenzae serotype and cap locus structure from WGS data, and FastSpar, a tool for rapid and scalable correlation estimation from compositional data. Collectively, this thesis contributes to our understanding of the childhood URT microbiome in the context of CF and normal development. The results in this thesis provide the first insights into the population dynamics and genomic AMR determinants of H. influenzae and H. parainfluenzae strains in a paediatric CF cohort. The presented findings further recapitulate the most complete overview of URT microbiome development during the first year of life and provide the first foundation for microbe-microbe interaction dynamics. The tools developed and the analyses performed in this thesis provide an important framework for future studies to investigate features and interactions of the respiratory microbiome.
Measuring intolerance to missense variation within the human genome and proteome

Silk, Michael Aanand ( 2021)

This thesis summarises an experimental investigation of the measurement and application of intolerance to missense variation in the human genome, and its use in predicting the functional consequences of variants, as well as its ability to identify novel functionally relevant protein features. Using gnomAD, currently the largest publicly available dataset of sequenced human exomes, as well as UK Biobank and DiscovEHR population variation databases, I have systematically measured the proportion of missense variation across over 18,000 human genes and 80,000 gene transcripts over a sliding window of 31 codons, named the Missense Tolerance Ratio (MTR), and observed that known pathogenic variants in epilepsy patients preferentially exist in regions estimated as intolerant. We further validated the MTR using the set of known pathogenic variants in ClinVar and observed a significant difference in the MTR distribution between these and novel control missense variant datasets. Intolerant regions within a gene’s sequence have also been observed to cluster within the protein tertiary structure. We anticipate that the MTR therefore has extraordinary potential in identifying important functional domains within protein structures. Current methods of estimating the functional importance of regions within structures largely rely on conservation, however this is heavily dependent on the depth and appropriateness of the alignment where functionality is often not fully preserved between species. Missense intolerance within tertiary structures was measured using the MTR3D and shown to provide complementary information to the MTR. By combining the MTR and MTR3D with additional structural properties such as residue depth, we also developed the MTRX, a combined measure of intolerance that incorporates the predictions from the different scores. This was shown to have high predictive power towards known pathogenic variants. To assist in the prediction of variant consequences and inform on research in drug design, protein biochemistry and gene analyses, we are providing these intolerance estimates in their sequence-based and structure-based formulations to be freely available as user-friendly web-servers.
Investigation of alternative splicing in apicomplexan parasites

Lee, V Vern ( 2021)

Alternative splicing is the phenomenon by which coding and non-coding regions of pre-mRNA molecules can be differentially spliced to yield multiple mRNA isoforms from a single gene. In metazoans, alternative splicing occurs to a substantial degree, contributing to protein diversity and the post-transcriptional regulation of gene expression. However, to what extent this occurs in apicomplexan parasites is much less understood. This thesis examines the landscape, regulators and function of alternative splicing in two apicomplexan parasites, T. gondii and P. falciparum. Technological advances in the short read sequencing of nucleic acids at unprecedented depths have enabled deep profiling of the transcriptome. However, the short reads present a limitation in the analysis of complex splicing events that span beyond the length of the reads. We evaluated the capability of a third generation long read sequencing technology, Oxford Nanopore Technologies (ONT) sequencing, in sequencing full-length native mRNA from T. gondii and P. falciparum, and established a method to analyse the alternative splicing landscape from the long reads. We successfully identified full-length transcripts spanning annotated and non-annotated junctions, implying a suitability in exploring complex splicing events. The analysis reveals an unusually high level of intron retained transcripts with premature terminating codons (PTCs). This suggests that most alternative splicing events in T. gondii and P. falciparum are unlikely to be productive. Alternative splicing in metazoans is modulated by alterative splicing factors, most notably the SR (serine-arginine–rich) protein family. We characterised the suite of SR proteins and two putative kinases/regulators of SR proteins in T. gondii. The proteins were found localised to sub-nuclear compartments characteristic of splicing factors. We demonstrated through genetic ablation and whole-transcriptome sequencing that the SR proteins modulate distinct but overlapping subsets of mostly non-productive alternative splicing events, as well as impacting transcript abundance. Alternatively spliced junctions were also enriched in characteristic SR binding motifs. The putative kinases of SR proteins were found to be essential to parasite survival and modulate extensive splicing events, but the events poorly mirrored that modulated by the SR proteins. This suggests a complex system of splicing regulation that do not conform to other eukaryotic models. The targeting of non-productive alternatively spliced transcripts for degradation through the nonsense mediated decay (NMD) pathway is one mechanism by which metazoans post-transcriptionally regulate gene expression. To explore if this was the case for T. gondii, we characterised the three core NDM proteins- UPF1, UPF2 and UPF3. The three proteins were found to co-immunoprecipitate with one another, implying a conservation of the core NMD complex. However, when we conditionally ablated the UPF proteins, parasite growth and survival was not impacted. We sequenced the parasite mRNA and found that only UPF2 impacted global intron retention rates. Moreover, a link between intron retention and gene expression regulation could not be established. Our results show that the fitness cost of mis- splicing determines intron retention rates, rather than targeted regulation. Hence, this thesis has shown that although non-productive alternative splicing is widespread and regulated in T. gondii, it is not a mechanism for post-transcriptional regulation of gene expression through the NMD pathway.
Detecting horizontal co-transfer of antimicrobial resistance genes in bacteria: a network approach

Wan, Yu ( 2019)

Antimicrobials have been widely using as a major resource to treat bacterial infections for almost a century. However, it is not unusual to see antimicrobial resistance emerges in a bacterial species due to natural selection under the usage of antimicrobials. Moreover, numerous studies show that bacteria can accumulate genes encoding resistance to different classes of antimicrobials and share them with other bacteria regardless of ancestry via a biological process called horizontal gene transfer, causing emergence and fast transmission of multidrug resistance. As such, antimicrobial resistance becomes an urgent and global threat to public health, pushing us backwards to the pre-antimicrobial era. In this thesis, I focus on horizontal co-transfer of resistance genes between bacteria of the same species, which is usually caused by co-localisation of resistance genes in mobile genetic elements, also known as physical linkage between these genes. This kind of linkage plays a pivotal role in the evolution of multidrug resistance, because the mobile elements can translocate, recombine and aggregate, rapidly rendering their host bacteria resistant to a wide spectrum of antimicrobials. By far there is nonetheless not an approach identifying horizontally co-transferred genes in a single bacterial species. Yet most authors of literature reported a few co-mobilised resistance genes each time following biological experiments, and some researchers only applied simple association analysis to representative bacterial isolates of distinct species so as to minimise the possibility that a specific combination of genes is inherited from their most-recent common ancestor. In contrast, intra-species association analysis is severely confounded by strong sample relatedness because of bacterial clonal reproduction. This obstacle leaves a gap between the known high frequency of intra-species horizontal gene transfer and our understandings of this process. This thesis presents a scalable computational approach that uses whole-genome sequencing data to identify co-transferred antimicrobial resistance genes in bacteria collected in a few decades from the same species. Moreover, it demonstrates applications of the approach to three clinically important pathogens and reports key players, patterns and dynamics underlying the horizontal co-transfer of resistance genes within each species. In the first chapter, I provide a background to antimicrobial resistance, horizontal gene transfer, whole-genome sequencing and contemporary bioinformatic techniques. I also summarise outcomes of horizontal gene co-transfer for characteristics that we can utilise for inference of physical linkage. Finally, I compare several statistical approaches determining pairwise association between presence-absence status of genes or alleles in bacteria to justify the necessity of controlling for sample relatedness in association analysis. For the second chapter, I derived a methodology inferring co-transferred genes by integrating gene detection, de novo genome assembly, core-genome and phylogenetic analysis, linear mixed models, hypothesis tests for effects of sample relatedness and evaluation of consistency in pairwise physical distances between resistance alleles in bacterial genomes. This methodology is designed to overcome limitations of existing methods summarised in the first chapter. Moreover, I show interpretations of expected outcomes and discuss constraints of this approach. The next three chapters present an implementation of my methodology and its applications on antimicrobial resistance genes in three clinically important species of Enterobacteriaceae. First, I conducted an empirical study following a simulation-and-validation strategy on finished-grade full genomes of six strains of Klebsiella pneumoniae to find out an optimal method that measures the pairwise physical distances between alleles in de novo genome assemblies. I found that for each assembly graph, the most accurate measurements are obtained via setting up constraints for both the number of nodes in the graph and the maximum of distance measurements. Second, I developed GeneMates, a computational and integrative software package that implements my methods proposed in the second chapter for the identification of physically linked resistance alleles or for analysing associations between a large number of resistance alleles when controlling for individual relatedness. In particular, GeneMates leverages network topology to identify potential physical linkage between the alleles. For validation, I applied this tool to whole-genome sequencing data of Escherichia coli and Salmonella Typhimurium, whose acquired resistance genes and relevant mobile genetic elements have been well characterised in publications. In result sections, I illustrate clusters of physically linked resistance alleles and discoveries of their vectors. For the last result chapter, I applied GeneMates to genomes of a large global collection of K.pneumoniae strains, which are adept to uptake DNA from various environments. Furthermore, I compared structure and contents of co-localised allele clusters across time and geography and discovered patterns underlying the evolution of multidrug resistance in this species. To conclude, I have developed and implemented a network approach that performs association tests on presence-absence of resistance alleles in a large collection of bacterial isolates of the same species and infers potential horizontally co-transferred alleles. I have validated this approach using known co-mobilisable resistance genes and the approach showed higher statistical power than existing methods. The GeneMates package will become a powerful tool contributing to routine surveillance of antimicrobial resistance and identifications of known and novel mobile genetic elements. In addition, applications of this package to other kinds of bacterial genes is also feasible and convenient.
Transcriptional regulation and functional differences driven by STAT3 isoforms, STAT3α and STAT3β

Tano, Vincent ( 2018)

The Signal transducer and activator of transcription 3 (STAT3) protein, a member of the STAT family of transcription factors, plays important roles in the regulation of critical biological processes. STAT3 is best known for its role in the JAK-STAT pathway, mediating the transcriptional regulation of target gene expression following its activation by extracellular signal, and continues to be the subject of intense research efforts due to its essential roles in crucial physiological processes, such as embryonic development and inflammation, as well as its involvement in numerous types of cancer. While the genome-wide DNA-binding and transcriptional regulation activities of the full-length STAT3α have been widely studied, those of a naturally occurring shorter STAT3β spliceform are less understood. Despite having identical DNA-binding domains, the STAT3 spliceforms display significant differences in their transcriptional regulation activities. Furthermore, STAT3α and STAT3β can drive opposing oncogenic and tumour suppression outcomes, respectively. Thus, the major focus of this study is to explore STAT3α- and STAT3β-specific genome-wide DNA-binding, transcriptional regulation of non-coding micro-RNAs (miRNAs), and functional outcomes in cancer cell biology. By employing ChIP-seq and miRNA expression profiling assays, this study presents results revealing that STAT3α and STAT3β display clear differences in genome-wide DNA-binding and regulation of miRNA expression. Bioinformatic analyses of transcription factor binding sites also uncovered the possible roles of co-transcription factors in the distinct STAT3 spliceform-specific transcriptional regulation activities. In addition, a Morpholino-directed splicing modulation approach to drive STAT3 knockdown and STAT3α-to-β splicing switch in cancer cells showed that the STAT3 spliceforms can differentially alter the tumourigenic properties of cancer cells, with STAT3β expression being associated with tumour suppression outcomes by driving the suppression of cell proliferation, survival and migration. Taken together, this study highlights the distinct properties of the STAT3 spliceforms in genome-wide DNA-binding which possibly underlie their different gene transcriptional regulation activities in both protein-coding and non-coding genes, and further provides evidence that STAT3β can drive distinct tumour suppression outcomes.
Population structure and carriage-infection dynamics of Klebsiella pneumoniae

Gorrie, Claire Louise ( 2018)

Klebsiella pneumoniae is an opportunistic pathogen and global cause of hospital-associated (HA) infections. K. pneumoniae is also part of the healthy human microbiome, providing a potential reservoir for infection. However, the frequency of colonisation and its contribution to infections are not well characterised. A prospective, hospital-wide surveillance of all infections attributed to K. pneumoniae – across the Alfred Health network, Melbourne, Australia – was conducted over a one-year period in 2013. Concurrently, patients in the intensive care unit (Alfred Hospital; AH) and geriatric care wards (Caulfield Hospital; CH) were screened for asymptomatic colonisation. Isolates were characterised using whole genome sequencing and antimicrobial susceptibility profiling. This study aimed to address several objectives: i) To investigate the frequency of colonisation in intensive care unit (ICU) patients and whether colonising strains are a source of infection; ii) To investigate the frequency and source of antimicrobial-resistant colonisation or infection in geriatric patients; iii) To characterise the population structure and genetic diversity of K. pneumoniae causing infections in hospital patients. This study estimated community-associated (CA) asymptomatic gastrointestinal carriage among ICU patients was at 6%, rising significantly to 19% among HA individuals, the latter including all identified multi-drug resistant carriage isolates. Many patients had their own unique colonising and infecting strains, often matching within a patient, though there were instances suggestive of transmission. The combination of genomic and epidemiological data supported five clusters of recent patient-to-patient transmission, frequently involving carriage. Among the CH geriatric patients screened, GI carriage rates were 10.8% and 1.7% of patients had extended-spectrum beta-lactamase (ESBL) producing carriage strains. There were three variably MDR lineages observed among multiple patients, and though there was no direct transmission within the CH, there was evidence supporting transmission at the AH prior to CH admission. The major MDR plasmids were identified; all had regions of clustered AMR genes on transposons and/or integrons. Two of the lineages shared variants of the same plasmid indicating transmission of AMR mobile elements between strains. Across all Klebsiella infections extensive diversity was observed including multiple species, lineages, capsule types, and O-antigen types. Most patients had their own unique carriage and infection strains but there were a small number of transmission chains detected. In most transmission cases, the lineages responsible were ESBL, MDR, or carbapenemase producers. Therefore, patients with antimicrobial-resistant strains pose a greater threat to those around them, as these strains were most strongly associated with transmission. This study showed that the majority of hospital Klebsiella infections arise from the host microbiome, though there is also a smaller burden of infection due to transmission of typically antimicrobial-resistant strains. This has important implications for infection prevention and control: people who are not colonised with Klebsiella have low risk of subsequent infection, except for when infections arise from acquisition of other patients’ antimicrobial-resistant strains. This work revealed that screening for colonisation can elucidate; i) which patients are at risk from infection through self-contamination, and ii) the AMR status of patients’ strains and therefore which individuals pose a risk to other patients through potential transmission.
Probing the immunopeptidome: enhanced epitope discovery through sHLA technology and bioinformatics

Scull, Katherine Elise ( 2018)

Human leukocyte antigen (HLA) molecules are cell-surface glycoproteins that present peptides, derived from diverse protein antigens, for surveillance by T lymphocytes. This immunosurveillance seeks signs of disease or abnormality, facilitating the eradication of infected or malignant cells. Collectively, the vast array of HLA-bound peptides (including immunogenic epitopes) is termed the immunopeptidome. Many research groups seek to identify the peptides which comprise the immunopeptidome of particular HLA allomorphs or cell types using tandem mass spectrometry. However, the nature of the immunopeptidome presents particular challenges for such discovery studies; the peptides are not only hugely diverse both qualitatively and quantitatively, but their complex biological origins render standard proteomic methodologies problematic. For example, conventional analysis software typically identify peptides by matching tandem mass spectra with sequences from genome-based protein databases, but HLA-bound peptides can have sequences not found in such databases, causing the software to ignore or misidentify such peptides. My thesis aims to facilitate epitope discovery in two ways. Firstly, I investigated an existing experimental technique, secreted HLA (sHLA) technology, in which a secreted form of a chosen HLA allele is transfected into cells, allowing straightforward and specific purification of the HLA allotype of choice. I validated the use of sHLA technology by showing that sHLA resembles natural membrane-bound HLA in terms of the molecules’ maturation kinetics and peptide repertoires. Secondly, I used computational methods and novel bioinformatics to aid immunopeptidomics studies. This involved the development and implementation of several computer programs. The major program is ‘Mmers’, which aids identification of peptides with non-standard sequences, in addition to those with sequences found in standard genome-based protein databases. Mmers generates comprehensive ‘artificial databases’ which include all of the possible permutations of amino acids for peptides of a given length, then searches spectra using Mascot-based scoring. To test Mmers, I obtained tandem mass spectrometric data from complex SW480 and SW620 colon cancer immunopeptidome samples, and searched for peptides of 8-11 amino acids in length using Mmers, alongside conventional software. I showed that Mmers can identify many sequences in agreement with Mascot, ProteinPilot and PEAKS DB. Furthermore, despite statistical challenges necessitating more manual inspection than desired, Mmers allowed me to identify four novel peptides with non-standard sequences, which I validated in comparison to synthetic peptides by MRM. I wrote two minor programs, Shifty and Spliceprot, to start investigating the possible biological origins of the four peptides. I found that two of the peptides could derive from unusual transcription or translation from the WAPL oncogene and the PTEN tumour suppressor gene, respectively. In conclusion, my thesis confronts the challenging complexity of the immunopeptidome, and seeks to provide novel tools to help researchers understand it more fully, with potential benefits for developing cancer immunotherapies and vaccines, and for determining the causes of autoimmune disease.
Alternative splicing and stage differentiation in apicomplexan parasites

Yeoh, Lee Ming ( 2017)

Alternative splicing is the phenomenon by which single genes code for multiple mRNA isoforms. This is common in metazoans, with alternative splicing observed in over 90% of human genes (Wang et al., 2008). However, the full extent of alternative splicing in apicomplexans has been previously under-reported. Here, I address this deficiency by transcriptomic analysis of two apicomplexan parasites: Toxoplasma gondii, which causes toxoplasmosis; and Plasmodium berghei, which is a murine model for human malaria. I identified apicomplexan homologues to SR (serine-arginine–rich) proteins, which are alternative-splicing factors in humans. I then localised a homologue, which I named TgSR3, to a subnuclear compartment in T. gondii. Conditional overexpression of TgSR3 was deleterious to growth. I detected perturbation of alternative splicing by qRT-PCR. Parasites were sequenced with RNA-seq, and 2000 genes were identified as constitutively alternatively spliced. Overexpression of TgSR3 perturbed alternative splicing in over 1000 genes. Previously, computational tools were poorly suited to compacted parasite genomes, making these analyses difficult. I alleviated this by writing a program, GeneGuillotine, which deconvolutes RNA-seq reads mapped to these genomes. I wrote another program, JunctionJuror, which estimates the amount of constitutive alternative splicing in single samples. Most alternative splicing in humans is tissue specific (Wang et al., 2008; Pan et al., 2008). However, unicellular parasites including Apicomplexa lack tissue. Nevertheless, I have shown that alternative splicing can still be common. I hypothesised that the tissue-specific alternative splicing of metazoans is analogous to stage-specific alternative splicing in unicellular organisms. I purified female and male gametocytes of P. berghei and sequenced these stages, with the aim of investigating alternative splicing and its relationship to stage differentiation. As a reference point, I first established the wild-type differences between female and male gametocytes. I detected a trend towards downregulation of transcripts in gametocytes compared to asexual erythrocytic stages, with this phenomenon more marked in female gametocytes. I was also able to identify many female- and male-specific genes, some previously-characterised, and some novel. My findings were further placed in an evolutionary context. Sex-specific genes were well conserved within the Plasmodium genus, but relatively poorly conserved outside this clade, suggesting that many Plasmodium sex-related genes evolved within this genus. This trend is least pronounced in male-specific genes, which suggests that sexual development of male gametocytes may have preferentially evolved from genes already present in organisms outside this genus. I then analysed these transcriptomes, now focusing on changes in alternative splicing. While non-gendered gametocyte differentiation is modulated by known transcription factors such as AP2-G (Sinha et al., 2014), I provide evidence that alternative splicing adds another level of regulation, which is required for differentiation into specific genders. I ablated a Plasmodium SR-protein homologue, which I named PbSR-MG. By transcriptomic analysis, I show that it regulates alternative splicing, predominantly in male gametocytes. Ablation was also associated with a drastic reduction in the viability of male gametocytes. Hence, I have shown that alternative splicing is common in apicomplexan parasites, is regulated by specific genes, and acts on specific targets. Alternative splicing is important for parasite viability and fundamental to stage differentiation in Plasmodium.
Defining the roles of long and small transcriptomes in neurodegenerative diseases

Quek, Camelia ( 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.
Protein localisation in a divergent eukaryotic parasite

Woodcroft, Benjamin James ( 2013)

Malaria infects 200 million people every year, with more than half a million of those cases resulting in death. Understanding the cell biology of Plasmodium falciparum remains largely an unsolved and unexplored problem. This doctoral assertion furthers understanding by investigating the sub-cellular localisation of Plasmodium falciparum proteins. A comprehensive literature review was undertaken, and the sub-cellular localisations of hundreds of proteins has been recorded in a transparent, traceable, publicly accessible and tactile fashion, and this serves as the basis for the further studies undertaken. It has been named ApiLoc. Using this curated set of protein localisations, the first Plasmodium falciparum-specific algorithm to predict sub-cellular localisation globally was created. Amino-acid based protein features were useful for prediction, but the predictor leveraged other predictive data types, such as microarray information and evolutionary conservation of the protein. Predictions were rigorously validated using both computational and epitope tagging approaches. ApiLoc also served as the basis for studies into the evolutionary conservation of protein localisation itself. This showed a remarkable lack of conservation, with only ~50% of protein localisations conserved across Apicomplexa, and ~20% throughout Eukaryota. The nucleus was studied in particular detail, given my involvement in a project to analyse isolated nuclei using a proteomic approach. Biochemical and bioinformatic studies were undertaken, providing further evidence that a classical nuclear import system involving basic-rich nuclear localisation signals is functional in Plasmodium falciparum and the evolutionarily related parasite Toxoplasma gondii.

Biochemistry and Pharmacology - Theses

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