Developing genomic models for cancer prevention and treatment stratification

Malignant tumors remain one of the leading causes of mortality with over 8.2 million deaths worldwide in 2012. Over the last two decades, high-throughput profiling of the human transcriptome has become an essential tool to investigate molecular processes involved in carcinogenesis. In this thesis I explore how gene expression profiling (GEP) can be used in multiple aspects of cancer research, including prevention, patient stratification and subtype discovery. The first part details how GEP could be used to supplement or even replace the current gold standard assay for testing the carcinogenic potential of chemicals. This toxicogenomic approach coupled with a Random Forest algorithm allowed me to build models capable of predicting carcinogenicity with an area under the curve of up to 86.8% and provided valuable insights into the underlying mechanisms that may contribute to cancer development. The second part describes how GEP could be used to stratify heterogeneous populations of lymphoma patients into therapeutically relevant disease sub-classes, with a particular focus on diffuse large B-cell lymphoma (DLBCL). Here, I successfully translated established biomarkers from the Affymetrix platform to the clinically relevant Nanostring nCounter© assay. This translation allowed us to profile custom sets of transcripts from formalin-fixed samples, transforming these biomarkers into clinically relevant diagnostic tools. Finally, I describe my effort to discover tumor samples dependent on altered metabolism driven by oxidative phosphorylation (OxPhos) across multiple tissue types. This work was motivated by previous studies that identified a therapeutically relevant OxPhos sub-type in DLBCL, and by the hypothesis that this stratification might be applicable to other solid tumor types. To that end, I carried out a transcriptomics-based pan-cancer analysis, derived a generalized PanOxPhos gene signature, and identified mTOR as a potential regulator in primary tumor samples. High throughput GEP coupled with statistical machine learning methods represent an important toolbox in modern cancer research. It provides a cost effective and promising new approach for predicting cancer risk associated to chemical exposure, it can reduce the cost of the ever increasing drug development process by identifying therapeutically actionable disease subtypes, and it can increase patients’ survival by matching them with the most effective drugs.2016-12-01T00:00:00

Investigations into the contributions of mitochondrial dynamics and function to platelet ageing and reactivity

PhD ThesisPlatelets are essential for the physiological process of haemostasis, but also drive pathological thrombosis. Platelet lifespan is a tightly controlled process through which platelets exist for approximately 10 days within the circulation of healthy individuals. However, in a number of disease states this process is dysregulated leading to an accelerated platelet turnover. Indeed, there are a number of reports suggesting that newly formed platelets are hyper-reactive and their presence has been associated with a higher risk of thrombosis. Whilst there are these indications of hyper-reactivity in young platelets, there are few systematic studies. Here I have used proteomics coupled with functional studies and immunofluorescence to show that there is a progressive decline in mitochondrial and cytoskeletal proteins as platelets age and an increase in apoptotic pathways. Given the apparent importance of mitochondria in supporting the predetermined platelet lifespan, it raised the question as to whether mitochondria are important for other platelet functional processes. Therefore, I sought to elucidate the impact of platelet activation on mitochondrial function and dynamics. Physiological stimulation causes an increase in mitochondrial respiration, consistent with an increase in energy demand. Interestingly, P2Y12 receptor inhibition causes a reduction in basal oxygen consumption, suggesting a dysregulation in mitochondrial function. Furthermore, this work highlights a role for mitochondria beyond energy production, with indications that stimulation causes platelets to package and release their mitochondria into microvesicles. Interestingly, these mitochondria-containing microvesicles have high P-selectin expression suggesting they may be more likely to interact with neutrophils than the rest of the microvesicle population. Indeed, incubation of neutrophils with mitochondria-positive microvesicles but not mitochondria-negative microvesicles causes alterations in the expression of surface markers; CD11b, CD66b and CXCR2, indicative of neutrophil activation potentially as a result of phagocytosis. This work highlights an important role of mitochondria in both platelet ageing and activation

Functional Classification of Skeletal Muscle Networks. I. Normal Physiology

Extensive measurements of the parts list of human skeletal muscle through transcriptomics and other phenotypic assays offer the opportunity to reconstruct detailed functional models. Through integration of vast amounts of data present in databases and extant knowledge of muscle function combined with robust analyses that include a clustering approach, we present both a protein parts list and network models for skeletal muscle function. The model comprises the four key functional family networks that coexist within a functional space; namely, excitation-activation family (forward pathways that transmit a motoneuronal command signal into the spatial volume of the cell and then use Ca2+ fluxes to bind Ca2+ to troponin C sites on F-actin filaments, plus transmembrane pumps that maintain transmission capacity); mechanical transmission family (a sophisticated three-dimensional mechanical apparatus that bidirectionally couples the millions of actin-myosin nanomotors with external axial tensile forces at insertion sites); metabolic and bioenergetics family (pathways that supply energy for the skeletal muscle function under widely varying demands and provide for other cellular processes); and signaling-production family (which represents various sensing, signal transduction, and nuclear infrastructure that controls the turn over and structural integrity and regulates the maintenance, regeneration, and remodeling of the muscle). Within each family, we identify subfamilies that function as a unit through analysis of large-scale transcription profiles of muscle and other tissues. This comprehensive network model provides a framework for exploring functional mechanisms of the skeletal muscle in normal and pathophysiology, as well as for quantitative modeling

MALDI-ToF mass spectrometry biomarker profiling via multivariate data analysis application in the biopharmaceutical bioprocessing industry

PhD ThesisMatrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) is a technique by which protein profiles can be rapidly produced from biological samples. Proteomic profiling and biomarker identification using MALDI-ToF MS have been utilised widely in microbiology for bacteria identification and in clinical proteomics for disease-related biomarker discovery. To date, the benefits of MALDI-ToF MS have not been realised in the area of mammalian cell culture during bioprocessing. This thesis explores the approach of ‘intact-cell’ MALDI-ToF MS (ICM-MS) combined with projection to latent structures – discriminant analysis (PLS-DA), to discriminate between mammalian cell lines during bioprocessing. Specifically, the industrial collaborator, Lonza Biologics is interested in adopting this approach to discriminate between IgG monoclonal antibody producing Chinese hamster ovaries (CHO) cell lines based on their productivities and identify protein biomarkers which are associated with the cell line productivities. After classifying cell lines into two categories (high/low producers; Hs/Ls), it is hypothesised that Hs and Ls CHO cells exhibit different metabolic profiles and hence differences in phenotypic expression patterns will be observed. The protein expression patterns correlate to the productivities of the cell lines, and introduce between-class variability. The chemometric method of PLS-DA can use this variability to classify the cell lines as Hs or Ls. A number of differentially expressed proteins were matched and identified as biomarkers after a SwissProt/TrEMBL protein database search. The identified proteins revealed that proteins involved in biological processes such as protein biosynthesis, protein folding, glycolysis and cytoskeleton architecture were upregulated in Hs. This study demonstrates that ICM-MS combined with PLS-DA and a protein database search can be a rapid and valuable tool for biomarker discovery in the bioprocessing industry. It may help in providing clues to potential cell genetic engineering targets as well as a tool in process development in the bioprocessing industry. With the completion of the sequencing of the CHO genome, this study provides a foundation for rapid biomarker profiling of CHO cell lines in culture during recombinant protein manufacturing.Lonza Biologics

Cellular and molecular investigations of undiagnosed neurometabolic disorders

Inborn errors of metabolism (IEM) affect 1 in 500 newborns causing significant disease-burden and mortality throughout childhood. However, despite extensive genetic and biochemical investigations the cause of disease remains unknown in up to 50% of patients with neurological symptoms; so-called neurometabolic disorders (NMD). The overarching aim of this thesis was to determine the cellular and molecular aetiologies for the clinical phenotypes seen in patients with undiagnosed NMD. In order to improve the diagnosis of these disorders in clinical practice, a comprehensive targeted gene panel of 614 genes known to cause IEM was designed and a cohort of 44 patients was analysed. A definitive or probable genetic diagnosis was achieved in 53% of patients without a prior genetic diagnosis. Method optimisation and validation, comparison to other diagnostic strategies and the advantages and disadvantages of targeted sequencing are reviewed. Case reports, novel mutations/phenotypes and their contribution to the expansion of the literature are described. Whole exome sequencing and functional characterisation was also undertaken for patients who had been extensively clinically investigated previously. Five patients identified with mutations in the mitochondrial glutamate transporter, SLC25A22, presenting with novel biochemical phenotypes are described and novel transporter functions are postulated. One patient diagnosed with a potassium channelopathy with biochemical abnormalities and anticonvulsant responses suggestive of an inborn error of vitmain B6 metabolism is documented and the mechanisms underlying the generalised anticonvulsant effects of vitamin B6 are postulated. Characterisation of a possible novel inborn error of lysine metabolism in a patient presenting with hyperlysinaemia and motor neuron disease is also discussed. These studies also demonstrate the complexity of unravelling the relationship between genotype and phenotype and highlight the need for novel functional assays to assess the pathogenicity of sequence variants. Mass spectrometry-based assays were developed to enable characterisation of disorders affecting vitamin B6 homeostasis, including pyridox(am)ine 5'-phosphate oxidase (PNPO), antiquitin and PROSC deficiency, the latter being a novel disorder. The differences between pyridoxine- and pyridoxal phosphate-responsive PNPO deficiency and fibroblast vitamer profiles in all patients were all investigated. Finally, multiple methodologies were employed with the aim of understanding the biological function of PROSC

A-to-I RNA Editing in Human Cells

RNA editing is a means of diversifying the transcriptome and regulating innate immunity. Among the different classes of enzymes that modify RNA, adenosine deaminase acting on RNA (ADAR) is a type that catalyzes adenosine-to-inosine editing on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Of the three ADAR homologs in humans, dysregulation of ADAR1 editing due to inherited mutations leads to disorders such as Aicardi-Goutieres syndrome, an inflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. ADAR1 is the primary A-to-I editor of RNA in humans, and the majority of edit sites are found in a class of repetitive elements called Alu, many of which are located in introns and 3’ untranslated regions of RNA. The functional consequences of A-to-I editing are varied, although a complete lack of functional ADAR1 is usually not tolerated, as revealed by the MDA5-mediated embryonic lethality in mice lacking functional ADAR1. In human neural progenitor cells, loss of ADAR1 causes spontaneous upregulation of interferon and cell death, although the RNA triggers remain unknown. Given the importance of ADAR1-editing in maintaining homeostasis in various contexts, there is a need to understand in more detail how ADAR1 isoforms are regulated and how they individually contribute to the A-to-I RNA editome. Two ADAR1 protein isoforms, p110 (110 kDa) and p150 (150 kDa), are expressed constitutively and in response to interferon, respectively, but the contribution of each isoform to the editing landscape remains incompletely characterized, largely because of the challenges in expressing p150 without p110. We revealed that the p110 isoform can be expressed from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. Synonymous mutations introduced in the region between the p150 and p110 start codons reduce leaky scanning and usage of the p110 start codon, and cells expressing p150 constructs with these mutations produce significantly reduced levels of p110. With the ability to express p150 with significantly reduced levels of p110, the A-to-I editome can be classified in terms of p150-selective and p110-selective sites, allowing evaluation of the relative contributions of either isoform to global editing levels. Our editing analysis revealed that the majority of ADAR1-edit sites are p150-selective, although a significant proportion of ADAR1-edit sites are also shared between p150 and p110, being not dependent on presence of either isoform for editing to occur. Of the sites that are putatively p110- selective, the majority are located in introns. Finally, the ability of p150 mRNA to give rise to p110 means that p110 is also an interferon-inducible protein alongside the canonical interferon-stimulated ADAR1 isoform: p150. During the interferon response, the transcriptome changes, and many new mRNA structures, perhaps some immunogenic ones, will enter the nucleus and cytoplasm. The distribution of ADAR1 isoforms is such that p110 is mostly present in the nucleus, and p150 mostly in the cytoplasm. We propose that optimal editing in the nucleus and cytoplasm during the interferon response is achieved by the inducibility of p110 and p150, both of which share a large number of target sites