Genomes are constantly challenged by agents that promote DNA damage, with interstrand crosslinks (ICLs) representing a particularly dangerous lesion. Ongoing work in the Wilkinson laboratory aimed at identifying novel agents that target Trypanosoma brucei, the causative agent of African trypanosomiasis, identified several prodrugs that once activated form ICLs in this protozoan parasite. To understand the complexity of ICL repair systems that T. brucei employs to resolve such damage, a variety of null mutant lines were generated that lack activities postulated to fix such lesions. Phenotypic screens using various DNA damaging agents revealed that TbMRE11, TbEXO1, TbCSB, TbCHL1, TbFAN1, TbBRCA2 and TbRAD51 all help to resolve ICLs, implicating components of the homologous recombination, nucleotide excision repair and mismatch repair pathways in resolving this form of damage: This approach demonstrated that components of the translesion synthesis pathway (TbREV2 and TbREV3) do not play a significant role in ICL repair. In many organisms, nucleases belonging to the SNM1/PSO2 family play a key and specific role in the repair of ICLs with this property extending to the T. brucei homologue, TbSNM1. To assess whether there is a functional linkage between the DNA repair factors noted above and TbSNM1, a series of double null mutants were constructed and the susceptibility of these lines to ICL inducing agents determined. Identification of their epistatic/non-epistatic interactions revealed that T. brucei expresses at least two ICL repair systems with one pathway involving the concerted activities of TbSNM1/TbCSB/TbEXO1, that we postulate functions to repair ICLs encountered by the transcriptional machinery, while the other is centred upon TbMRE11/TbFAN1/TbEXO1 that may help resolve lesions which cause stalling of DNA replication forks. By unravelling how T. brucei repairs ICLs, specific inhibitors against key components of these pathways could be developed and used in combination with DNA damaging agents to target trypanosomal infections.

Wang, Xingyan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 126-131). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.V / 摘要 --- p.VIII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- CLINICAL DISEASE CAUSED BY M.TB --- p.1 / Chapter 1.1.1 --- Tuberculosis (TB) --- p.1 / Chapter 1.1.2 --- Pathogen: Mycobacteria tuberculosis (M. TB) --- p.2 / Chapter 1.2 --- HOST DEFENSE AGAINST M.TB --- p.4 / Chapter 1.2.1 --- Overview --- p.4 / Chapter 1.2.2 --- Specific pathways --- p.6 / Chapter 1.3 --- GENETIC PREDISPOSITION OF HOST TO INFECTION --- p.12 / Chapter CHAPTER 2 --- OVERVIEW AND AIM OF THIS PROJECT --- p.14 / Chapter 2.1 --- GWAS REPLICATION --- p.14 / Chapter 2.2 --- CANDIDATE GENES REVEALED IN GWAS OF OTHER GRANULOMATOUS INFLAMMATORY DISEASES (GLD) --- p.14 / Chapter 2.3 --- CHROMOSOME 17 CHEMOKINE CLUSTER REGION --- p.15 / Chapter CHAPTER 3 --- REPLICATION STUDY OF TB GWAS --- p.16 / Chapter 3.1 --- INTRODUCTION --- p.16 / Chapter 3.1.1 --- TB GWAS study --- p.16 / Chapter 3.1.2 --- Aims of this part --- p.16 / Chapter 3.2 --- MATERIAL AND METHODS --- p.17 / Chapter 3.2.1 --- Case and control samples --- p.17 / Chapter 3.2.2 --- DNA extraction --- p.18 / Chapter 3.2.3 --- Genotyping of the SNPs --- p.19 / Chapter 3.2.4 --- Statistical analysis --- p.21 / Chapter 3.3 --- RESULTS --- p.23 / Chapter 3.3.1 --- Description of studied samples --- p.23 / Chapter 3.3.2 --- Results of case-control study for replication studies of TB GWAS --- p.23 / Chapter 3.4 --- DISCUSSION --- p.28 / Chapter CHAPTER 4 --- GENETIC VARIANTS IN GRANULOMATOUS INFLAMMATORY DISEASES --- p.32 / Chapter 4.1 --- INTRODUCTION --- p.32 / Chapter 4.1.1 --- Granulomatous inflammation --- p.32 / Chapter 4.1.2 --- Diseases characterized by granulomatous inflammatory --- p.34 / Chapter 4.1.3 --- Shared immune mechanisms in GiDs --- p.38 / Chapter 4.1.4 --- Genome-wide Association Studies (GWAS) in GiD --- p.38 / Chapter 4.1.5 --- Hypothesis of this part --- p.41 / Chapter 4.2 --- MATERIAL AND METHODS --- p.43 / Chapter 4.2.1 --- Case and control samples --- p.43 / Chapter 4.2.2 --- DNA extraction --- p.44 / Chapter 4.2.3 --- Tag SNP selection --- p.44 / Chapter 4.2.4 --- Genotyping of tagging SNPs --- p.45 / Chapter 4.2.5 --- Statisitical analysis --- p.45 / Chapter 4.3 --- RESULTS --- p.55 / Chapter 4.3.1 --- Description of TB case samples --- p.55 / Chapter 4.3.2 --- Primary endpoint case-control results --- p.56 / Chapter 4.3.3 --- Secondary endpoint case-only studies results --- p.67 / Chapter 4.3.4 --- Haplotype analysis --- p.78 / Chapter 4.4 --- DISCUSSION --- p.83 / Chapter 4.4.1 --- ATG16L1 gene with TB susceptibility --- p.83 / Chapter 4.4.2 --- Associations in case-only studies (Interaction effects) --- p.83 / Chapter 4.4.2.1 --- Age and pathogenesis of TB --- p.83 / Chapter CHAPTER 5 --- STUDIES IN THE CHEMOKINE-GENE CLUSTER AND A MIRNA SNP STUDY --- p.89 / Chapter 5.1 --- INTRODUCTION --- p.89 / Chapter 5.1.1 --- Genetic susceptibility to TB in familial cases --- p.89 / Chapter 5.1.2 --- Familial studies suggested linkage at 17qll.2 --- p.89 / Chapter 5.1.3 --- Chemokines --- p.90 / Chapter 5.1.4 --- Studies of SNP rs2910164 of microRNA-146a (miRNA-146a) --- p.91 / Chapter 5.2 --- MATERIAL AND METHODS --- p.92 / Chapter 5.2.1 --- Case and control samples --- p.92 / Chapter 5.2.2 --- DNA extraction --- p.92 / Chapter 5.2.3 --- TagSNP selection --- p.92 / Chapter 5.2.4 --- Genotyping of tagging SNPs --- p.93 / Chapter 5.2.5 --- PCR-RFLP --- p.93 / Chapter 5.2.6 --- Statistical analysis --- p.94 / Chapter 5.3 --- RESULTS --- p.100 / Chapter 5.3.1 --- PCR-RFLP results of the three SNPs --- p.100 / Chapter 5.3.2 --- Description of TB case samples --- p.102 / Chapter 5.3.3 --- Primary endpoint case-control results --- p.103 / Chapter 5.3.4 --- Secondary endpoint case-only studies results of CCL genes --- p.109 / Chapter 5.4 --- DISCUSSION --- p.120 / Chapter 5.4.1 --- Genetic association of SNPs with severity of TB --- p.120 / Chapter 5.4.2 --- Smoking and immunity --- p.121 / Chapter CHAPTER 6 --- FINAL CONCLUSION AND PROSPECT FOR FUTURE WORK --- p.122 / Chapter 6.1 --- CONCLUSION --- p.122 / Chapter 6.2 --- LIMITATION OF THE STUDIES --- p.124 / Chapter 6.3 --- FUTURE WORKS AND PROSPECT --- p.125 / REFERENCES --- p.126

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 76-83.

Clostridium scatologenes is an anaerobic bacterium that demonstrates some unusual metabolic traits such as the production of 3-methyl indole. The availability of genome level sequencing has lent itself to the exploration and elucidation of unique metabolic pathways in other organisms such as Clostridium botulinum. The Clostridium scatologenes genome, with an estimated length 4.2 million bp, was sequenced by the Applied Biosystems Solid method and the Roche 454 pyrosequencing method. The resulting DNA sequences were combined and assembled into 8267 contigs with an average length of 1250 bp with the Newbler Assembler program. Comparision of published subunits of csd gene and assembled contigs identified that one contig contained all three subunits. In addition a gene with similarity to clostridium carboxidivorans butyrate kinase was found lined next to csd gene. An alignment of the contig and csdgene sequences identified three deletions in the contig within the 4066 bases of the alignment. This implies that there is about 0.07% error rate in the sequencing itself requiring more finishing. Even without finishing the genome assembly into single contig, contigs were annotated in RAST pipeline predicting 2521 protein encoding genes (PEGs). The PEGs were classified by their metabolic function and compared to classified PEGs found in the closely related clostridium species, Clostridium carboxidivorans and Clostridium. ljungdahlii, which have similarly sized genomes. According to the RAST analysis, Clostridium scatologenes had 35% subsystem coverage of all known metabolic processes with its 2521 PEGs. This compares to 41% for Clostridium carboxidivorans with 4174 PEGs (29) and 42% for Clostridium ljungdahlii with 4184 PEGs (30), indicating that Clostridium scatologenesmay still have more genes to be identified. Comparison of the percent genes found in the metabolic subsystems was similar except in motility and chemotaxis. The contigs, on which the csd gene and tryptophan metabolizing genes lay, were examined to see if additional genes might support these metabolic pathways. Butyrate kinase was associated with the csd genes but no other associations were found for the two tryptophan metabolizing genes. The tryptophan biosynthesis operon genes were all found on one contig (contig 6771) and were syntenic with other bacterial species.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 105-129). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Microbiology / Master / Master of Philosophy

RNA-seq sequencing has revolutionized the landscape of whole transcriptome sequencing and analysis. With its capacity of sequencing in a high-throughput and low-cost way, it produced ever increasingly amount of RNA-seq reads that are mines of treasure in biological and therapeutic studies. However, due to the complex nature and relatively un-developed knowledge base of transcription process, many challenges exist in the modeling and investigation of RNA-seq read data. It is of high importance to develop efficient computational tools to satisfy these needs. The first part of this thesis concentrates on algorithms for both upstream and downstream analysis of RNA-seq data. For the upstream, we aim to tackle down the problems of RNA-seq reads alignment where the segmental alignment causes the major difficulty. By employing a strategy of rigid extensive tries on read segmentations indices, we implemented an accurate algorithm for returning two-segmental alignments based on bi-directional BWT. For the downstream analysis, we study two types of gene fusion events which play a critical role in the formation of cancers. Unlike previous down-scoping-search methods, we applied a search-validate approach to design the framework. By introducing key techniques such as masking, two-segmental alignment and retention of multiple maps, we developed an efficient and robust tool for detecting gene fusions with high accuracy that proved by extensive simulation and real data tests. Optical mapping is a cutting edge technique for the study of genomic structural variations which address the defect and limitation of paired-end sequencing. It was designed with great improvement in accuracy, resolution and throughput than current techniques. Also, it produces much longer molecules which enables us to explore genomic regions rich in repetitive sequences. Optical mapping has the potential to enable us to draw a complete picture of the genome structure polymorphism and it is important for us to design tools for analysis of the data. The second part of the thesis is dedicated to the algorithms for both upstream and downstream analysis of optical map data. For the upstream, we formulated a robust scoring function, which combines the effectiveness of heuristic functions and the accuracy of statistical functions. Based on it, we implemented the high performance OMDP algorithm. For the downstream, we developed BP-OMDP which makes use of both split-mapping and disparity of coverage depth to call inversions in NA12878 human genome sample. / published_or_final_version / Computer science / Doctoral / Doctor of Philosophy

Whether certain Epstein-Barr virus (EBV) strains are associated with pathogenesis of nasopharyngeal carcinoma (NPC) is still an unresolved question. In the present study, we aimed to sequence the complete EBV genomes harbored in NPC tumor biopsies and compare against the non-NPC EBV strains to identify NPC-specific EBV variations. In the first part of the study, EBV genome contained in one primary NPC tumor biopsy was PCR-amplified and sequenced using next-generation and dideoxy-DNA sequencing. The EBV genome, designated HKNPC1 (Accession number JQ009376), was generated by reference mapping and it appears to be a uniform strain in general despite minor heterogeneity. Phylogenetic analysis with the four published EBV strains, B95-8, AG876, GD1, and GD2, indicated HKNPC1 was more closely related to the Chinese NPC strains. HKNPC1 contains 1,589 single nucleotide variations (SNVs) and 132 insertions or deletions (indels). We found 76 non-synonymous SNVs shared amongst the Chinese GD1, GD2 and HKNPC1 isolates, while another 88 nonsynonymous SNVs were shared only by the two NPC tumor-derived strains HKNPC1 and GD2. In the second part of the study, SureSelect target enrichment technology was used instead of PCR to capture EBV DNA from total DNA. The study was scaled-up to sequence EBV strains in cell lines, saliva and NPC tumor, using the MiSeq Personal Sequencer and the Genome Analyzer IIx platforms. The reads were de novo assembled to generate 17 complete EBV genomes, out of which 9 were NPC-EBV strains. Phylogenetic analysis of all available EBV strains has demonstrated that all NPC strains were type 1 EBV. Phylogeny predicted by LMP-1 gene showed clear geographical pattern of where the EBV strains were isolated. A total of 5,011 variations were identified by comparing every EBV strain against the reference. MicroRNAs and EBERs are generally well conserved across all genomes. Comparative analysis of variations between NPC and non-NPC EBV strains discovered 904 NPC-specific variations, out of which 112 appeared in more than one NPC strains. Among these recurrent variations, 39 non-synonymous substitutions and seven deletions in coding region were found. About half of these recurrent variations were located in EBNA-3A, -3B and -3C, while the rest was found in latent, tegument, capsid and packaging-related proteins and transcription factors. There were two NPC EBV strains isolated from the primary tumors which later diagnosed to have distant metastasis. Unique variations were shared in these two EBV strains in regions between IR2 and IR3, where genes such as BPLF1, BOLF1 and EBNA-3A, -3B and -3C were located, and leftward of IR3, where BBLF2/3 and BBRF1 were found. In conclusion, we have demonstrated the feasibility of target capture and next-generation sequencing in whole genome sequencing of EBV. Comparison of reference mapping and de novo assembly of EBV sequences illustrated that both are feasible approaches, though de novo assembly is preferred since the method is less dependent on the reference genome. Large-scale sequencing of NPC and non-NPC EBV strains may facilitate the discovery of previously unknown variations of biological significance and reveal the diverse role of EBV in NPC pathogenesis. words) / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

Not available / text

Not available / text