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  • (2006) Hitchins, Megan; Suter, C; Wong, Jenny; Cheong, Kay; Hawkins, Nicholas; Leggett, B; Scott, R; Spigelman, Allan; Tomlinson, Ian; Martin, David; Ward, Robyn
    Journal Article

  • (2003) Welsh, John; Sapinoso, Lisa; Kern, Suzanne; Brown, David; Liu, Tao; Bauskin, Asne; Ward, Robyn; Hawkins, Nicholas; Quinn, David; Russell, Pamela; Sutherland, Robert; Breit, Samuel; Moskaluk, Christopher; Frierson Jr, Henry; Hampton, Garret
    Journal Article
    Genetic alterations in tumor cells often lead to the emergence of growth-stimulatory autocrine and paracrine signals, involving overexpression of secreted peptide growth factors, cytokines, and hormones. Increased levels of these soluble proteins may be exploited for cancer diagnosis and management or as points of therapeutic intervention. Here, we combined the use of controlled vocabulary terms and sequence-based algorithms to predict genes encoding secreted proteins from among ≈12,500 sequences represented on oligonucleotide microarrays. Expression of these genes was queried in 150 carcinomas from 10 anatomic sites of origin and compared with 46 normal tissues derived from the corresponding sites of tumor origin and other body tissues and organs. Of 74 different genes identified as overexpressed in cancer tissues, several encode proteins with demonstrated clinical diagnostic application, such as α-fetoprotein in liver carcinoma, and kallikreins 6 and 10 in ovarian cancer, or therapeutic utility, such as gastrin-releasing peptide/bombesin in lung carcinomas. We show that several of the other candidate genes encode proteins with high levels of tumor-associated expression by immunohistochemistry on tissue microarrays and further demonstrate significantly elevated levels of another novel candidate protein, macrophage inhibitory cytokine 1, a distant member of the tranforming growth factor-β superfamily, in the serum of patients with metastatic prostate, breast, and colorectal carcinomas. Our results suggest that the combination of annotation/protein sequence analysis, transcript profiling, immunohistochemistry, and immunoassay is a powerful approach for delineating candidate biomarkers with potential clinical significance and may be broadly applicable to other human diseases.

  • (2003) Brown, David; Ward, Robyn; Buckhaults, Philip; Liu, Tao; Romans, Katherine; Hawkins, Nicholas; Bauskin, Asne; Kinzler, Kenneth; Vogelstein, Bert; Breit, Samuel
    Journal Article
    Purpose: Macrophage inhibitory cytokine-1 (MIC-1) is a divergent member of the tumor growth factor ß (TGF-ß) superfamily. Several observations suggest that it plays a role in colorectal carcinoma (CRC). In particular, MIC-1 is markedly up-regulated in colorectal cancers as well as in premalignant adenomas. This study examines the relationship of serum MIC-1 levels and genotypes to clinical and pathologic features of colonic neoplasia. Experimental Design: We confirmed the presence of MIC-1 in CRC tissue and the cell line CaCo-2. The normal range for serum MIC-1 levels was defined in 260 healthy blood donors, and the differences between normal subjects and 193 patients having adenomatous polyps or CRC were then determined. In a separate cohort of 224 patients, we evaluated the relationship of MIC-1 serum level and genotype to standard tumor parameters and outcome measures. Results: MIC-1 was expressed in CRC tissue and the cancer cell line CaCo-2. There was a progressive increase in serum MIC-1 levels between normal individuals [mean (M) = 495 pg/ml, SD = 210), those with adenomatous polyps (M = 681 pg/ml, SD = 410), and those with CRC (M = 783 pg/ml, SD = 491)]. Serum MIC-1 level was correlated with the extent of disease so that the levels were higher in patients with higher Tumor-Node-Metastasis stage. There were significant differences in time to relapse and overall survival between subjects with different MIC-1 levels and genotypes. Conclusions: This study identifies a strong association between MIC-1 serum levels and neoplastic progression within the large bowel. We suggest that the measurement of serum MIC-1 levels and determination of MIC-1 genotype may have clinical use in the management of patients with CRC.

  • (2008) Shen, Yansong; Guo, Baoyu; Yu, Aibing; Maldonado, Daniel; Austin, Patricia; Zulli, Paul
    Journal Article
    Pulverized coal injection technology is widely used in blast furnace ironmaking due to economic, operational and environmental benefits. High burnout within the tuyere and raceway is required for high coal injection rate operation. In order to analyze the flow and combustion in the tuyere and raceway more accurately and reliably, a three-dimensional model of coal combustion is developed. This model is validated against the measurements from two pilot scale test rigs in terms of gas species composition and coal burnout. The gas-solid flow and coal combustion are simulated and analysed. The results indicate that compared to our previous model, the present model is able to provide more detailed gas species distributions and better describe the evolutions of coal particles. It is more sensitive to various parameters and hence more robust in examining various blast furnace operations.

  • (2004) Jackson, Katherine; Gaeta, Bruno; Sewell, William; Collins, Andrew
    Journal Article
    BACKGROUND: Immunoglobulin rearrangement involves random and imprecise processes that act to both create and constrain diversity. Two such processes are the loss of nucleotides through the action of unknown exonuclease(s) and the addition of P nucleotides. The study of such processes has been compromised by difficulties in reliably aligning immunoglobulin genes and in the partitioning of nucleotides between segment ends, and between N and P nucleotides. RESULTS: A dataset of 294 human IgM sequences was created and partitioned with the aid of a probabilistic model. Non-random removal of nucleotides is seen between the three IGH gene types with the IGHV gene averaging removals of 1.2 nucleotides compared to 4.7 for the other gene ends (p < 0.001). Individual IGHV, IGHD and IGHJ gene subgroups also display statistical differences in the level of nucleotide loss. For example, within the IGHJ group, IGHJ3 has average removals of 1.3 nucleotides compared to 6.4 nucleotides for IGHJ6 genes (p < 0.002). Analysis of putative P nucleotides within the IgM and pooled datasets revealed only a single putative P nucleotide motif (GTT at the 3` D-REGION end) to occur at a frequency significantly higher then would be expected from random N nucleotide addition. CONCLUSIONS: The loss of nucleotides due to the action of exonucleases is not random, but is influenced by the nucleotide composition of the genes. P nucleotides do not make a significant contribution to diversity of immunoglobulin sequences. Although palindromic sequences are present in 10% of immunologlobulin rearrangements, most of the `palindromic` nucleotides are likely to have been inserted into the junction during the process of N nucleotide addition. P nucleotides can only be stated with confidence to contribute to diversity of less than 1% of sequences. Any attempt to identify P nucleotides in immunoglobulins is therefore likely to introduce errors into the partitioning of such sequences. [Journal Article; In English;

  • (2006) Lee, Cathryn; Gaeta, Bruno; Malming, H; Bain, Michael; Sewell, William; Collins, Andrew
    Journal Article
    We have used a bioinformatics approach to evaluate the completeness and functionality of the reported human immunoglobulin heavy-chain IGHD gene repertoire. Using the hidden Markov-model-based iHMMune-align program, 1,080 relatively unmutated heavy-chain sequences were aligned against the reported repertoire. These alignments were compared with alignments to 1,639 more highly mutated sequences. Comparisons of the frequencies of gene utilization in the two databases, and analysis of features of aligned IGHD gene segments, including their length, the frequency with which they appear to mutate, and the frequency with which specific mutations were seen, were used to determine the reliability of alignments to the less commonly seen IGHD genes. Analysis demonstrates that IGHD4-23 and IGHD5-24, which have been reported to be open reading frames of uncertain functionality, are represented in the expressed gene repertoire; however, the functionality of IGHD6-25 must be questioned. Sequence similarities make the unequivocal identification of members of the IGHD1 gene family problematic, although all genes except IGHD1-14*01 appear to be functional. On the other hand, reported allelic variants of IGHD2-2 and of the IGHD3 gene family appear to be nonfunctional, very rare, or nonexistent. Analysis also suggests that the reported repertoire is relatively complete, although one new putative polymorphism (IGHD3-10*p03) was identified. This study therefore confirms a surprising lack of diversity in the available IGHD gene repertoire, and restriction of the germline sequence databases to the functional set described here will substantially improve the accuracy of IGHD gene alignments and therefore the accuracy of analysis of the V-D-J junction.

  • (2020) Masand, Natasha
    Thesis
    DNA cytosine methylation is an important epigenetic modification that plays a key role in gene expression. DNA methylation has been shown to be involved in numerous processes, including X-chromosome inactivation in mammals, retrotransposon silencing, genomic imprinting, carcinogenesis and the regulation of tissue specific gene expression during development. Gene expression is tightly regulated via DNA methylation (5mC) and the aberrant expression of meiotic genes in mitotic cells via CpG promoter hypomethylation has been proposed to cause cancer. Cancer/Testis Antigens (CTAs) are a group of genes that encode tumour specific antigens and are expressed in the testis, certain cancers but not in normal post-natal somatic tissues. CpG island methylation and histone modifications appear to play a role in the epigenetic regulation of CTA expression, however, very little is known about their functions in vivo. A widely studied but poorly understood question to date is the mechanisms behind aberrant CTA reactivation in cancer. Given that 5mC mediated gene repression has been found to exist in vertebrate genomes and CTAs have also been identified to be a subset of highly evolutionarily conserved genes, it is critical to understand the role of 5mC mediated CTA silencing in vertebrates. By gaining a deeper understanding into the mechanisms behind this highly conserved pattern of gene repression on a specific subset of genes, we would be able to identify methods to prevent aberrant gene expression. In this study, I analysed publicly available whole genome bisulfite sequencing (WGBS), RNA-seq and chromatin immuno-precipitation followed by massively parallel sequencing (ChIP-seq) data of developing embryonic and adult somatic tissue of 3 vertebrate species to elucidate the evolutionary epigenetic regulation of CTAs in vertebrate genomes. Integrative WGBS, RNA-seq and ChIP-seq analysis revealed that CTAs are evolutionarily conserved in zebrafish, mice and humans and mechanisms of their epigenetic regulation are also conserved. I observed that histone modifications could potentially serve as an indicator of the methylation status of CTA gene promoters and that the expression of CTAs was inversely related to gene promoter 5mC levels. I demonstrate that CTAs when over-expressed cause embryonic lethality in zebrafish and the same genes are aberrantly hypomethylated at their CpG islands in a subset of human cancers. Overall, my work shows that CTAs are epigenetically regulated in an evolutionarily conserved manner and possibly via a conserved transcription factor, ETS1, that is expressed both in embryonic and cancerous tissue.

  • (2021) Begik, Oguzhan
    Thesis
    RNA modifications, collectively referred to as the ‘epitranscriptome’, are not mere decorations of RNA molecules, but can be dynamically regulated upon environmental queues and changes in cellular conditions. This dynamic behaviour is achieved through the RNA modification machinery, which comprises “writer”, “reader” and “eraser” proteins that modify, recognize and remove the modification, respectively. Chapter1 presents a comprehensive analysis of the RNA modification machinery (readers, writers and erasers) across species, tissues and cancer types, revealing gene duplications during eukaryotic evolution, changes in substrate specificity and tissue- and cancer-specific expression patterns. Chapters 2 and 3 presents the exploration and development of novel methods to map and analyze RNA modifications transcriptome-wide. Nanopore direct-RNA sequencing technology was used to provide RNA modification maps in full-length native RNA molecules. Firstly, it is shown that RNA modifications can be detected in the form of base-calling ‘errors’, thus allowing us to train Support Vector Machine models that can distinguish m6A-modified from unmodified sites, both in vitro and in vivo. Secondly, it is demonstrated that distinct RNA modification types have unique base-calling ‘error’ signatures, allowing us to exploit these signatures to distinguish different RNA modification types. It is found that pseudouridine has one of the most distinct signatures, appearing in the form of C-to-U mismatches. Finally, this information was used to predict novel pseudouridine sites on ncRNAs and mRNAs transcriptome-wide, as well as to obtain quantitative measurements of the stoichiometry of modified sites. Chapter 4 presents the development of a novel nanopore-based method, which is termed ‘Nano3P-seq’, to simultaneously quantify RNA abundance and tail length dynamics in individual molecules in both the coding and non-coding transcriptome, from cDNA reads. It is demonstrated that Nano3P-seq offers a simple approach to study the coding and non-coding transcriptome at single molecule resolution regardless of the tail ends. Together, this work provides a comprehensive framework for the study of RNA modifications and polyA tail dynamics using third generation sequencing technologies, opening novel avenues for future works that aim to characterize their dynamics and biological roles both in health and in disease.

  • (2021) Ross, Samuel
    Thesis
    Upon fertilisation of vertebrate embryos, the epigenomes of the responsible gametes need to be reconfigured into a state that is compatible with totipotency and zygotic transcriptional programs. Furthermore, the epigenomes of differentiating cells then need to be remodelled again in order to form the complex structures of the body, such as the vastly intricate nervous system. This includes, but is not limited to, the remodelling of DNA methylation, the most abundant DNA modification in vertebrates with critical roles in embryogenesis and neurodevelopment. In mammals, methylation of cytosines in cytosine-guanine dinucleotides (mCG) is almost completely erased after fertilization before it is re-established during gastrulation. Similarly, methylation of cytosines outside the CG context (mCH; H = A,T,C) is diluted in the early mammalian embryo before it is re-established mainly in the nervous system. However, in non-mammalian vertebrates, it appears that no global erasure of mCG takes place, raising questions about their propensity for transgenerational epigenetic inheritance. Additionally, the conservation of mCH in non-mammalian vertebrates is largely unexplored. In this thesis, I look to expand our knowledge on the developmental dynamics, evolutionary conservation and the molecular components of DNA methylome remodelling in vertebrates by studying methylome dynamics in two distantly related teleost species (ray-finned, protruding jawed fish). I functionally explore how DNA methylation is regulated during the development of zebrafish (Danio rerio), medaka (Oryzias latipes), and zebrafish-medaka hybrids, in both the CG and CH context. I employ CRISPR/cas9 technology, whole-genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and RNA sequencing (RNA-seq), to interrogate a wide range of developmental time points and adult tissues. Overall, I have: i) developed a system to functionally test for regulators of developmental DNA methylation; ii) revealed a novel form of developmentally remodelled mCH in zebrafish and medaka which is deposited by the teleost specific DNMT3BA enzyme, iii) demonstrated evolutionary conservation of mammalian-like mCH features in the developing zebrafish nervous system, and iv) shown that DNA methylome dynamics in medaka and zebrafish embryos are highly comparable and compatible during the first 24 hours of zebrafish-medaka hybrid development. Altogether, this work greatly expands our understanding of the form and function of a critical DNA modification during development.

  • (2022) Young, Clara
    Thesis
    A breakdown in B cell self-tolerance can lead to antibody-mediated autoimmune disease. This thesis aims to explore how B cell tolerance can be broken in two distinct, but complementary projects within the context of the Goodnow somatic mutation hypothesis. In both scenarios, B cells that escape self-tolerance and generate autoantibodies are referred to as “rogue” B cells. First, this thesis aimed to elucidate the precise steps undertaken by expanded rogue B cell clones in patients with chronic Hepatitis C virus (HCV)-associated cryoglobulinemic vasculitis, an autoimmune disease characterised by the production of a rheumatoid factor cryoglobulin autoantibody. The rogue B cell clones in the HCV cryoglobulinemic vasculitis patients were confirmed to be the source of the autoantibody. The rogue B cell clone precursor antibodies failed to bind the HCV envelope glycoprotein E2, yet bound multimerised self-antigen IgG relative to membrane IgM density. These findings disfavour a molecular mimicry hypothesis, and instead indicate IgG immune complexes may be sufficient to drive recruitment of the rogue B cell clone precursors. Finally, the rogue B cells clones were found to carry somatic lymphoma-associated, non-immunoglobulin gene mutations and chromosomal aberrations, predicted to cause hyperactivation of the NF-kB signalling pathway and escape of B cell tolerance. This finding provides additional evidence in support of the Goodnow somatic mutation hypothesis. Second, this thesis examined rogue germinal centre (GC) B cells that arise in the absence of the receptor FAS. Rogue GC B cells loose specificity for the foreign antigen and incidentally generate autoantibodies. However, the accumulation of rogue GC B cells cannot be explained by our current understanding of affinity-based selection in the GC. This work revealed rogue GC B cells, unlike “conventional” GC B cells undergoing affinity maturation to the foreign antigen, can be identified by low expression of CD21 and high expression of B220 (CD21loB220hi). Moreover, rogue GC B cells were found to be rapidly entering cell cycle, enriched for a dark zone phenotype and T-cell dependent, reminiscent of positively selected GC B cells. Thus, rogue GC B cells typically removed by FAS, likely persist in the competitive GC microenvironment despite their loss of BCR specificity to foreign antigen, because they retain the capacity to undergo T-cell dependent positive GC selection.