1,721,065 research outputs found
Gene expression in Rhipicephalus (Boophilus) microplus: Identification of tick antigens and investigation of the tick RNA interference pathway
Get PDFThe cattle tick Rhipicephalus microplus is a hematophagous ectoparasite of major veterinary importance both globally and in Australia. Australian beef cattle producers in the tropical and subtropical regions of the continent suffer estimated annual losses of AU$175m. The development of a novel tick vaccine has therefore been identified as a high priority research goal by all stakeholders of the Australian beef industry (Playford 2005). A novel bioinformatics methodology for the identification of potential vaccine targets in the R. microplus expressed sequence tags sequences was developed and applied resulting in the identification of 68 potential tick vaccine candidates. Furthermore, the applicability of RNA interference (RNAi) for tick gene functional studies was assessed. This study comprehensively characterized the major components of the RNAi pathway in both R. microplus and Ixodes scapularis, which resulted in the identification of an I. scapularis homologue of Dicer protein, as well as the identification of Argonaute-1 and -2 homologues in both I. scapularis and R. microplus. Most notably, homologues of a C. elegans RNA-dependent RNA polymerase were found in both R. microplus and I. scapularis, a protein involved in the amplification of the RNAi signal and previously not identified in any other arthropod species. A comparative RNAi study targeting 10 highly conserved R. microplus homologues of Drosophila genes with known RNAi phenotypes was conducted. The R. microplus homologue of D. melanogaster Ubiquitin-63E showed the strongest phenotypic changes in both cell culture and in adult female ticks injected with dsRNA. This Ubiquitin-63Esilencing effect in fully engorged females was subsequently investigated by microarray analysis. The contribution of potential off-target effects to phenotypic changes was assessed by developing an in silico analysis and combining the prediction results with the microarray data. A total of 224 unique transcripts were detected as differentially expressed. The off-target analysis revealed that 11 off-targets predicted by the in silico analysis were present in the list of differentially expressed genes. The research presented in this thesis has contributed to the development of novel approaches for reverse vaccinology in ectoparasites and it has furthermore improved the understanding of the mechanisms of RNAi and the delivery of double-stranded RNA in ticks
Data integration for decision making in wheat breeding
Get PDFPlant breeding is a production process requiring the creation of germplasm through taking existing successful cultivars and crossing them with new parental lines with agronomic and quality attributes of interest. After crossing, F2 generations generally display all possible combinations between the parental lines. The process from this step is to identify elite crossbred lines and backcross these several times to the parental lines in order to generate new elite lines that are predominately equivalent to the cultivar but with specific novel and desirable attributes present.
Plant breeding continually requires judgements to identify elite plant germplasm containing traits that maximise plant performance. These judgements are often made using incomplete information resulting from the greater complexity in modern plant breeding decision making. Judgements can be improved through the utilisation of new technologies and a stronger scientific basis. This thesis uses decision and information management processes to contribute to:
• Pioneering the application of unbalanced datasets to wheat breeding. The methodologies were derived from tree and animal breeding experience and successfully applied to data sets from a wheat breeding program.
• Providing the first integration of molecular data into a decision-matrix framework.
• Building on the molecular integration in output-2 by establishing a more sophisticated integration of complex NIR spectral data with molecular data.
• Providing inputs into decision matrices for breeding using the outputs discussed above.
This thesis establishes the methodology to make use of new technologies to use unbalanced datasets with decision matrix methodology to make better decisions. This thesis has utilised multivariate methodologies more broadly to include complex data such as NIR fingerprint to differentiate flour samples between controls and breeding germplasm. These differences appear to be related to genetic factors as demonstrated after variability relating to the environment had been removed. This thesis first reviews the literature and then addresses this breeding processes through the use of decision and information management processes, and makes significant contributions in using these methodologies
Developing a bioinformatics framework for proteogenomics
Get PDFIn the last 15 years, since the human genome was first sequenced, genome sequencing and annotation have continued to improve. However, genome annotation has not kept up with the accelerating rate of genome sequencing and as a result there is now a large backlog of genomic data waiting to be interpreted both quickly and accurately. Through advances in proteomics a new field has emerged to help improve genome annotation, termed proteogenomics, which uses peptide mass spectrometry data, enabling the discovery of novel protein coding genes, as well as the refinement and validation of known and putative protein-coding genes.
The annotation of genomes relies heavily on ab initio gene prediction programs and/or mapping of a range of RNA transcripts. Although this method provides insights into the gene content of genomes it is unable to distinguish protein-coding genes from putative non-coding RNA genes. This problem is further confounded by the fact that only 5% of the public protein sequence repository at UniProt/SwissProt has been curated and derived from actual protein evidence.
This thesis contends that it is critically important to incorporate proteomics data into genome annotation pipelines to provide experimental protein-coding evidence. Although there have been major improvements in proteogenomics over the last decade there are still numerous challenges to overcome. These key challenges include the loss of sensitivity when using inflated search spaces of putative sequences, how best to interpret novel identifications and how best to control for false discoveries.
This thesis addresses the existing gap between the use of genomic and proteomic sources for accurate genome annotation by applying a proteogenomics approach with a customised methodology. This new approach was applied within four case studies: a prokaryote bacterium; a monocotyledonous wheat plant; a dicotyledonous grape plant; and human. The key contributions of this thesis are: a new methodology for proteogenomics analysis; 145 suggested gene refinements in Bradyrhizobium diazoefficiens (nitrogen-fixing bacteria); 55 new gene predictions (57 protein isoforms) in Vitis vinifera (grape); 49 new gene predictions (52 protein isoforms) in Homo sapiens (human); and 67 new gene predictions (70 protein isoforms) in Triticum aestivum (bread wheat). Lastly, a number of possible improvements for the studies conducted in this thesis and proteogenomics as a whole have been identified and discussed
Comparative genomics to investigate genome function and adaptations in the newly sequenced Brachyspira hyodysenteriae and Brachyspira pilosicoli
Get PDFBrachyspira hyodysenteriae and Brachyspira pilosicoli are anaerobic intestinal spirochaetes that are the aetiological agents of swine dysentery and intestinal spirochaetosis, respectively. As part of this PhD study the genome sequence of B. hyodysenteriae strain WA1 and a near complete sequence of B. pilosicoli strain 95/1000 were obtained, and subjected to comparative genomic analysis. The B. hyodysenteriae genome consisted of a circular 3.0 Mb chromosome, and a 35,940 bp circular plasmid that has not previously been described. The incomplete genome of B. pilosicoli contained 4 scaffolds. There were 2,652 and 2,297 predicted ORFs in the B. hyodysenteriae and B. pilosicoli strains, respectively. Of the predicted ORFs, more had similarities to proteins of the enteric Clostridium species than they did to proteins of other spirochaetes. Many of these genes were associated with transport and metabolism, and they may have been gradually acquired through horizontal gene transfer in the environment of the large intestine.
A reconstruction of central metabolic pathways of the Brachyspira species identified a complete set of coding sequences for glycolysis, gluconeogenesis, a non-oxidative pentose phosphate pathway, nucleotide metabolism and a respiratory electron transport chain. A notable finding was the presence of rfb genes on the B. hyodysenteriae plasmid, and their apparent absence from B. pilosicoli. As these genes are involved in rhamnose biosynthesis it is likely that the composition of the B. hyodysenteriae lipooligosaccharide O-sugars is different from that of B. pilosicoli. O-antigen differences in these related species could be associated with differences in their specific niches, and/or with their disease specificity. Overall, comparison of B. hyodysenteriae and B. pilosicoli protein content and analysis of their central metabolic pathways showed that they have diverged markedly from other spirochaetes in the process of adapting to their habitat in the large intestine.
The presence of overlapping genes in the two spirochaetes and in other spirochaete species also was investigated. The number of overlapping genes in the 12 spirochaete genomes examined ranged from 11-45%. Of these, 80% were unidirectional. Overlapping genes were found irregularly distributed within the Brachyspira genomes such that 70-80% of them occurred on the same strand (unidirectional, ->->/). The remaining 4-6% of overlapping genes were convergent (->50% of the total observations overlapping by >4 bp. A small number of overlapping gene-pairs were duplicated within each genome and there were some triplet overlapping genes. Unique orthologous overlapping genes were identified within the various spirochaete genera. Over 75% of the overlapping genes in the Brachyspira species were in the same or related metabolic pathway. This finding suggests that overlapping genes are not only likely to be the result of functional constraints but also are constrained from a metabolomic context. Of the remaining 25% overlapping genes, 50% contained one hypothetical gene with unknown function. In addition, in one of the orthologous overlapping genes in the Brachyspira species, a promoter was shared, indicating the presence of a novel class of overlapping gene operon in these intestinal spirochaetes
Assembly of a Complex Genome: Defining Elements of Structure and Function
Get PDFThe post-human genome sequencing project era has seen an influx of genome sequencing projects established to investigate the structure, composition and characteristics of plant genomes. While the genome sequences of smaller plant genomes (ie. Rice) are currently available, there has been a lack of progress on the study of large, complex genomes such as barley (Hordeum vulgare) and wheat (Triticum aestivum), due to the difficulties in their sequencing and assembly. The aim of this study is to assemble and annotate targeted regions of chromosome 3B from Triticum aestivum cv. Chinese Spring (CS) and Hope. This study also aimed to complete a comprehensive, inter- and intra-species comparative analysis using Bioinformatics tools and strategies, in order to define structural and functional elements within the genome.
Genome sequences totalling 2.7Mb from two different loci of chromosome 3B in two different cultivars (ctg11 from the short arm of CS, ctg1034 from the long arm of CS and three assembled sequences over the equivalent ctg11 region of Hope) were assembled using a novel ‘two-phase’ process that integrated information from a genome sequence assembler and a Triticeae-specific transposable element database. Through comparative genomics analysis a gene island was identified within a highly repetitive, heterochromatic region on 3BL that was highly conserved over four other cereal genomes (Brachypodium distachyon, Oryza sativa, Sorghum bicolor and Zea mays). Chromodomain-containing long terminal repeats from the gypsy family of retrotransposons were identified adjacent to the gene island and may suggest an involvement in the targeted insertion of transposable elements at the loci, protecting the gene-island from dynamic evolutionary change. Characterisation of the ctg11 (Sr2 region) genome sequence on 3BS, identified a large ~60kb mitochondrial genome insert and three members of the multi-gene beta-expansin family, with sequence analysis indicating local duplication within the sequence and rearrangements when compared to the equivalent region in a different wheat cultivar. In silico and real-time transcription analysis of the individual gene was also confirmed. Within the equivalent ctg11 in Hope, a germin-like protein (GLP) cluster was identified and characterised that distinguishes between the two wheat cultivars. The genes in this GLP cluster were identified to belong to a sub-gene family that conferred broad level basal resistance in transient over-expressed systems in rice and barley.
The main outcome of this study was the development of a novel strategy of genome sequence assembly by utilising the complex component of the wheat genome that made assembly difficult: transposable elements. The complex genome sequence assembly methodology outlined in this thesis is suitable to be used as a model for future sequence assembly studies. The assembly of large pseudomolecule sequences (among the largest and most complete ever assembled in the wheat genome) enabled the Bioinformatics analysis of a representative sample of wheat chromosome 3B, providing valuable in silico outputs for future functional analyses and allowing an in-depth intra- and inter-species comparative analysis with related genomes
Bioinformatics approaches for functional predictions in diverse informatics environments
Get PDFBioinformatics is the scientific discipline that collates, integrates and analyses data and information sets for the life sciences. Critically important in agricultural and biomedical fields, there is a pressing need to integrate large and diverse data sets into biologically significant information. This places major challenges on research strategies and resources (data repositories, computer infrastructure and software) required to integrate relevant data and analysis workflows. These challenges include:
The construction of processes to integrate data from disparate and diverse resources and legacy systems that have variable data formats, qualities, availability and accessibility constraints.
Substantially contributing to hypothesis driven research for biologically significant information.
The hypothesis proposed in this thesis is that in organisms from divergent origins, with differing data availability and analysis resources, in silico approaches can identify genomic targets in a range of disease systems. The particular aims were to:
1. Overcome data constraints that impact analysis of different organisms.
2. Make functional genomic predictions in diverse biological systems.
3. Identify specific genomic targets for diagnostics and therapeutics in diverse disease mechanisms.
In order to test the hypothesis three case studies in human cancer, pathogenic bacteria, and parasitic arthropod were selected, the results are as follows.
In case study 1 sequence information was integrated to make novel predictions, and generate novel findings for the role of the Alu repeat element in cancer. An under representation of Alu was found in cancerous transcript and most noncancerous Alu transcript found were of an unknown function. These findings led to an Alu-mediated siRNA model for the down regulation of Alu containing mRNA in cancer.
Case study 2, comparative genomic analyses identified venereal diagnostic targets that discriminated Campylobacter fetus subspecies venerealis from other Campylobacter species and subspecies. Plasmid borne virulence Type IV secretory pathway genes specificity however varied for biovars, compromising their use for diagnostics. These findings resulted in the targeted sequencing of Campylobacter fetus subspecies venerealis biovar genomes.
Case study 3, in cattle tick ectoparasite (Rhipicephalus microplus), a large highly complex and under researched genome, transcript sequence was analysed and tick vaccination targets identified. These vaccine candidates successfully imparted immunity in the bovine host. The developed high throughput vaccine target identification system is now being applied to other disease systems.
Through the shared bioinformatics approaches, novel functional targets and models in disease were determined.
This thesis has developed and demonstrated in silico approaches for:
1. The collation, annotation and integration of data from divergent organisms with variable data constraints.
2. Novel functional predictions in diverse biological systems.
3. Novel vaccine and diagnostic candidate identification, in diverse disease mechanisms, substantially contributing to hypothesis driven research
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Studies on polymorphic alu insertions and genomic diversity within the major histocompatibility complex
Get PDFAfter the initiation of the human genome sequencing project and the introduction of the field of 'bioinformatics', interest in human genetic diversity studies has been increased. Sequence diversity has helped define differences between genes and genomic regions that were previously unknown or difficult to determine. In this thesis I have undertaken to study sequence diversity in the human genome in three areas; 1) investigated diversity in the MHC as represented by the MICA alleles with respect to the known HLA alleles, 2) investigated the structure and diversity in the intergenic region from an MHC related (paralogous) genomic region and related the structural and diversity findings to the knowledge available on the MHC and the wider genome, and 3) described the identification of three and characterization of five new MHC class I polymorphic markers (Alu) and their polymorphic characteristics in worldwide populations and their associations with skin cancer.
1. Phylogenetic analysis of MICA alpha-domain (extracellular) sequences demonstrated relationships with HLA-B cross-reactive serogroups. The HLA-B and MICA loci are in linkage disequilibrium. The data indicated that MICA and HLA-B have evolved in concert from their common ancestors and that the transmembrane polymorphisms have arisen independently and more recently.
2. Sequence analysis of the CD1 genomic region confirmed the presence of five CD1 genes and revealed that there are four unrelated intergenic regions (IGRs). The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans.
3. Five polymorphic Alu insertions (POALINs) were identified (two from previous studies) and located within the 1.8 megabase of the MHC class I genomic region. All five POALINs are polymorphic, and are positively associated with the HLA-A and HLA-B alleles. The AluyHJ insertion was found most frequently associated with HLA-A1 or A24, AluyHG with HLA-A2, AluyHF with HLA-A2, A-10 or -A26 and AluyTF showed a marginal association with HLA-A29. The AluyMICB insertion was strongly associated with HLA-B17 (HLA-B57, HLA-B58) and HLA-B13. The presence of three Alu insertions (AluyHJ, AluyHG and AluyHF) was found in only one HLA class I haplotype (HLA-A1, -B57, -Cw6) in the 10th IHW cell lines. A novel positive association between the presence of AluyMICB and the 'MICAdel/MICBnull/HLA-B48' haplotype was determined. The AluyMICB insertion was also associated with at least three different MICB alleles (*0102, *0107N and *0105) and three different HLA-B alleles (B13, B48 and B57). Based on the analysis of associations between different polymorphic markers within the beta block, the MICB*0102 allele was inferred to be the ancestral form of the MICB*0105 and MICB*0107N alleles. The AluyMICB polymorphism can be used to further investigate haplotype relationship and consequently their lineage origins. Some of the MHC POALINs are haplospecific and associate strongly with certain groups of HLA class I alleles and MHC ancestral haplotypes. The AluyTF frequency was significantly associated with skin cancer (p<0.005).
MICA gene diversity is derived from two different evolving paths, therefore one or the other alone cannot reliably mark an ancestral haplotype. The CD1 duplicons originated well before the HLA class I duplicons. The MHC POALINs provide new lineage and linkage markers for the fine mapping study of different haplotypes and variations in linkage groups across 1.8 Mb of the MHC class I region. The POALINs may also prove useful in investigating the origins and history of human populations and in determining the role of human genetic diversity in disease risk
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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