Orientador: Laymert Garcia dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Filosofia e Ciencias Humanas / Made available in DSpace on 2018-08-05T01:28:26Z (GMT). No. of bitstreams: 1 Leite_Marcelo_D.pdf: 18137235 bytes, checksum: d2ccf296709649c706ae95e568a4a4e8 (MD5) Previous issue date: 2005 / Resumo: A tese central deste trabalho é que a aceitação pública despertada pelo Projeto Genoma Humano só se explica pelo uso político e retórico de um determinismo genético crescentemente irreconciliável com os resultados empíricos da pesquisa genômica atual. A complexidade verificada no genoma humano e em suas interações com o meio desautoriza a manutenção de uma noção simples e unidirecional de causalidade, contrariamente ao pressuposto na idéia de gene como único portador de informação, esteio da doutrina do determinismo genético. Um complexo de metáforas informacionais e/ou lingüísticas continuo vivo nos textos publicados por biólogos moleculares na literatura científica, notadamente nos artigos veiculados nos periódicos de alto impacto Nature e Science de 15 e 16 fevereiro de 2001, respectivamente. Tais metáforas inspiram um tipo de discurso ambíguo que modula nuances variadas de retórica determinista, conforme se dirija aos próprios pares ou ao público leigo" O campo da genômica ainda está longe de rejeitar a conjunção problemática das noções de gene pré-formacionista e de gene como recurso desenvo/vimenta/ na base da metáfora do gene como informação. Essa fusão inspirada pela terminologia cibernética propicia uma versão asséptica de gene, distanciada da natureza, puramente sintática, móvel e virtual o bastante para circular desimpedida nos circuitos de produção de valor como recurso genético passível de garimpagem e de patenteamento. Críticos dã tecnociência devem desafiar o campo da genômica a reformular drasticamente as metáforas que dão suporte a seu programa hegemônico de pesquisa / Abstract: The central thesis of this work is that the public support generated for the Human Genome Project and the hype surrounding it can be explained only by the political and rhetorical uses of genetic determinism, a notion which increasingly cannot be reconciled with the empirical results of on-going genomic research. The complexity that has been uncovered in the human genome and in its interactions with the environment implies that a simple and unidirectional notion of causality cannot be maintained, contrary to a presupposition of the idea of the gene as the sole carrier of iliformation, an idea that contributes to sustain the doctrine of genetic determinism. A complex of informational and/or linguistic metaphors lives on in the texts published by molecular biologists in the scientific press, most notably in the issues published February 15thand 16thof 2001 ofthe high impact journals Nature and Science, respectively. These metaphors generate an ambiguous type of discourse that modulates various nuances of deterministic rhetoric, depending on whether it addresses peers or the lay publico The field of genomics is still a long way ITom rejecting the questionable conflation of the notions of gene as preformation and gene as developmental resource which underpins the metaphor of gene as information. This conflation inspired by cybernetics terminology enables an aseptic version of the gene, separated ITom nature, portable and virtual enough to flow unimpeded through the channels ofvalue production as genetic resource suitable for mining and patenting. Critics of technoscience should challenge the field of genomics to drastically reshape the metaphors which have supported its hegemonic research agenda / Doutorado / Doutor em Ciências Sociais

Two mechanisms, intrinsic and factor-dependent, have evolved for accomplishing the termination of transcription in eubacteria. In this thesis, the first chapter is an introduction to the topic that presents what is known about the mechanisms of termination. The properties of the primary and secondary ‘players’- intrinsic terminators, Rho protein, rho-dependent terminators, RNA polymerse and Nus factors - are presented and the known mechanisms by which termination functions are discussed. In Chapter 2, a detailed analysis of intrinsic terminators – their differential distribution, similarity and divergence - has been penned. The database, compiled using the program GeSTer (Genome Scanner for Terminators), comprises ~2000 sequences and is one of the largest of its kind. Furthermore, analyzing the data from over 700 bacteria reveals how different species have fine-tuned intrinsic terminators to suit their cellular needs. Non-canonical intrinsic terminators emerge to be a significant fraction of the observed structures. The conserved structural features of identified intrinsic terminators are discussed and the relationship between the two modes of termination is assessed. Chapter 3 deals with the importance of transcription termination in regulating horizontally acquired DNA. The results show that genomic islands are scarce in intrinsic terminators and thus constitute most likely sites for Rho-dependent termination. Plausible reasons for why such a scenario has evolved are discussed and a generally applicable model is presented. Chapters 4 and 5 focus on Rho protein from Mycobacterium tuberculosis. In silico identification of M. tuberculosisgenes that rely on MtbRho-dependent termination is followed by experimental validation. The data show that Rho-dependent termination is the predominant mechanism in this species.MtbRho is a majorly expressed protein that governs termination of protein-coding and non-protein coding genes. Further, MtbRho can productively interact with RNA that has considerable secondary structure. Such interactions cause conformational changes in the enzyme. Given that MtbRho has to function with a GC-rich transcriptome, the altered properties could have evolved for optimal function. Taken together, the thesis extends our current understanding of both modes of termination. The importance of non-canonical intrinsic terminators in mycobacteria and other organisms is discussed. The unusual function of Rho and its predominant role in mycobacteria is elucidated. Finally, the inter-relationship between the two modes of termination is also discussed.

HCV genome is a positive sense single-stranded RNA containing a single open reading frame (ORF) flanked by untranslated regions (UTRs), 5’UTR and 3’UTR.Initiation of HCV RNA translation is mediated by internal ribosome entry site (IRES) present in 5’ UTR and this process is independent of cap-structure and requires only a small subset of canonical initiation factors. Hence, HCV IRES-mediated translation initiation mechanism is quite different from canonical cellular mRNA translation initiation. The IRES is organized into highly structured domains, namely domain II, III and IV. High affinity interactions between structured RNA elements present in the IRES and 40S ribosomal proteins mediate 40S recruitment to HCV IRES. However, details of the RNA elements and region of ribosomal proteins involved in these interactions are poorly understood. In recent days, RNA-based molecules like siRNAs, antisense RNAs and RNA decoys have become promising candidates for antiviral molecules. So designing short RNA molecules that target unique HCV translation initiation mechanism might help in developing novel anti-HCV molecules. HCV 3’UTR and antisense-5’ UTRs serve as sites for replication initiation to synthesize negative and positive strand and this process is catalyzed by NS5B protein (RNA-dependent RNA polymerase). Hence, host proteins binding to both 3’UTR and antisense-5’UTR might play important role in HCV replication. This puts the study of HCV RNA–host protein interactions and its role in viral translation and replication in perspective. Studying the HCV IRES-ribosomal protein S5 interactions and its role in HCV IRES function Previous studies from our laboratory have demonstrated that binding of La protein to GCAC close to initiator AUG enhances ribosomal protein S5 (RPS5) binding with HCV IRES and stimulates HCV translation. However in-detail study on HCV IRES–RPS5 interactions and its implication on HCV translation initiation were lacking. In present study computational modelling suggested that domain II and IV interact majorly with the beta hairpin structure and C-terminal helix of RPS5. Filter-binding and UV cross-linking studies with peptides derived from predicated RNA-binding region of RPS5 and mutational studies with RPS5 demonstrated that beta hairpin structure present in RPS5 is critical for IRES–RPS5 interaction. In parallel, we have studied RNA elements involved in the IRES–RPS5 interactions using deletions and substitution mutations, which we had generated on the basis of the computational model. Direct and competition UV cross-linking experiments performed with these IRES mutants and 40S subunits as a source of RPS5 suggested that structure and sequence of both domain II and IV play crucial role in IRES–RPS5 interactions. We further investigated the effect of these mutations on IRES activity by in vitro translation assay and found that all the mutants that were compromised in binding to RPS5 showed reduced IRES activity. Moreover, ribosome assembly experiments on HCV IRES demonstrated that mutations affecting IRES–RPS5 interactions result in reduction of 80S peak and slight increase of 48S peak. Since the 40S subunit had been previously reported to bind with HCV 3’UTR, we explored the possible interaction of RPS5 with HCV 3’UTR. From direct and competition UV cross-linking assays, we found that RPS5 does not bind to 3’UTR and the interaction is unique to IRES (5’UTR). Interestingly, partial silencing of RPS5 preferentially inhibited HCV translation with marginal effect on cap-dependent translation. Recently, reduction in 40S subunit abundance was reported to preferentially inhibit HCV translation. So, we investigated the abundance of free 40S subunit upon silencing RPS5 and results showed reduction in free 40S subunit level. So, we hypothesize that silencing of RPS5 reduces free 40S abundance to inhibit HCV translation. Taken together, results identified specific RNA elements present in HCV IRES that are critical for IRES–RPS5 interactions and demonstrated the role of these interactions in HCV translation initiation. Targeting ribosome assembly on HCV IRES using short RNAs Stem-loops (SL) IIIe and IIIf of HCV IRES are known to play an important role in stable IRES–40S complex formation. However interaction of these stem-loops with 40S subunit in isolation, independent of other regions of HCV IRES, was not studied. In this study, using electrophoretic mobility shift assay (EMSA) and sucrose gradient centrifugation experiments, we demonstrate that short RNA containing both SLIIIe and SLIIIf together (SLRef RNA) binds to 40S subunit, while short RNAs containing either of the stem-loops (SLRe RNA and SLRf RNA) lose their ability to interact with 40S subunit. Further, SLRef RNA inhibited ribosome assembly on the IRES, whereas SLRe and SLRf RNA failed to inhibit the same. Since SLRef RNA is derived from IRES, we investigated the interaction SLRef RNA with IRES–trans-acting factors (ITAFs). UV cross-linking of radio-labelled HCV IRES with cytoplasmic extract (S10) in presence of unlabelled short RNAs suggested possible interactions of La and RPS5 proteins with SLRef RNA. Studies with recombinant La protein and RPS5 further confirmed their interaction with SLRef RNA. Ex vivo experiments with HCV bicistronic RNA suggested that SLRef RNA specifically inhibits HCV translation. In addition to that SLRef RNA inhibited the HCV RNA synthesis in JFH1 HCV cell culture system. Moreover, specific delivery of pSUPER construct expressing SLRef RNA (pSUPERSLRef) to mice liver along with HCV bicistronic construct using Sendai virosomes demonstrated specific inhibition of HCV IRES activity by SLRef RNA in mice hepotocytes. In summary, short RNA derived from HCV IRES was shown to bind with La protein and RPS5 to inhibit ribosome assembly on HCV IRES. Further, targeted delivery of SLRef RNA into mice liver using Sendai virosome resulted in inhibition of HCV RNA translation in mice hepatocytes. Characterizing the interaction of host proteins with antisense-5’UTR and 3’UTR and its significance in HCV replication Antisense-5’UTR and 3’UTR of HCV RNA are the sites of replication initiation. Hence, host proteins binding to both of these RNA sequences are potential candidates for regulation of HCV replication. In this study, we have investigated host proteins binding with antisense-5’UTR and 3’UTRof HCV RNA by performing UV cross-linking experiments with cytoplasmic extract of Huh7 cells, and found that a protein of ~42kDa protein interacts with both antisense-5’UTR and 3’UTR. Based on earlier report, we predicted that the ~42kDa protein could be hnRNPC1/C2. Results of UV cross-linking followed by immuno pull-down (UV-IP assay) and UV cross-linking experiments with recombinant hnRNPC1 protein confirmed that hnRNPC1 indeed binds to antisense-5’UTR and 3’UTR. Further, filter-binding experiments demonstrated that hnRNPC1 protein binds to 3’UTR with higher affinity compared to antisense-5’UTR. Subsequently, we investigated the regions within 3’UTR and antisense-5’UTR that interact with hnRNPC1protein. Results demonstrated that poly-(U/UC) region of 3’UTR and region containing stem-loops SL-IIIa’, SL-IIIb’, SL-IIIcdef’ and SL-IV’ in antisense-5’UTR were mostly involved in the interaction. Interestingly, studies with confocal microscopy suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection, which might in turn influence HCV replication. To investigate the role of hnRNPC1/C2 in HCV replication, partial silencing of hnRNPC1/C2 was performed in HCV cell culture system (JFH1) and results demonstrated that hnRNPC1/C2 is critical for HCV RNA synthesis. However experiments with HCV bicistronic RNA suggested that hnRNPC1/C2 does not play significant role in HCV translation. Taken together, results suggested that hnRNPC1/C2 re-localizes from nucleus to cytoplasm upon JFH1 infection and binds to HCV 3’UTR and antisense- 5’UTR to regulate HCV replication. In summary, this thesis provides novel insights into the interaction of host proteins with HCV RNA and its significance in HCV translation and replication. Inhibition of the ribosome assembly and consequent reduction in HCV translation with mutations interfering with IRES–RPS5 interaction, reported in the present study, unfolds the novel role of this interaction in HCV translation. Further, results obtained in the present study with a small RNA SLRef, derived from HCV IRES, provide proof of concept for using short RNAs to specifically inhibit HCV translation. In addition, studies of interaction of hnRNPC1/C2 with HCV RNA and its re-localization upon HCV infection sheds light on the significance of host–virus interaction in viral RNA replication.

Maintenance of genomic integrity and stability is of prime importance for the survival of an organism. Upon exposure to different damaging agents, DNA acquires various lesions such as base modifications, single-strand breaks (SSBs), and double-strand breaks (DSBs). Organisms have evolved specific repair pathways in order to efficiently correct such DNA damages. Among various types of DNA damages, DSBs are the most serious when present inside cells. Unrepaired or misrepaired DSBs account for some of the genetic instabilities that lead to secondary chromosomal rearrangements, such as deletions, inversions, and translocations and consequently to cancer predisposition. Nonhomologous DNA end joining (NHEJ) is one of the major DSB repair pathways in higher organisms. Mitochondrial DNA (mtDNA) deletions identified in humans are flanked by short directly-repeated sequences, however, the mechanism by which these deletions arise are unknown. mtDNA deletions are associated with various types of mitochondrial disorders related to cancer, aging, diabetes, deafness, neurodegenerative disorders, sporadic and inherited diseases. Compared to nuclear DNA (nDNA), mtDNA is highly exposed to oxidative stress due to its proximity to the respiratory chain and the lack of protective histones. DSBs generated by reactive oxygen species, replication stalling or radiation represents a highly dangerous form of damage to both nDNA and mtDNA. However, the repair of DSBs in mitochondria and the proteins involved in this repair are still elusive. Animals deficient for any one of the known Classical-NHEJ factors are immunodeficient. However, DSB repair (DSBR) is not eliminated entirely in these animals suggesting evidence of alternative mechanism, ‘alternative NHEJ’ (A-NHEJ/A-EJ). Several lines of evidence also suggest that alternative and less well-defined backup NHEJ (B-NHEJ) pathways play an important role in physiological and pathological DSBR. We studied NHEJ in different tissue mitochondrial protein extracts using oligomeric DNA substrates which mimics various endogenous DSBs. Results showed A-EJ, as the predominant pathway in mitochondria. Interestingly, immunoprecipitation (IP) studies and specific inhibitor assays suggested, mitochondrial end joining (EJ) was dependent on A-EJ proteins and independent of C-NHEJ proteins. Further, colocalization studies showed A-EJ proteins localize into mitochondria in HeLa cells. More importantly knockdown experiments showed the involvement of DNA LIGASE III in mitochondrial A-EJ. These observations highlight the central role of A-EJ in maintenance of the mammalian mitochondrial genome. By using oligomeric DNA substrates mimicking various endogenous DSBs, NHEJ in different cancer cell lines were studied. We found that the efficiency of NHEJ varies among cancer cells; however, there was no remarkable difference in the mechanism and expression of NHEJ proteins. Interestingly, cancer cells with lower levels of BCL2 possessed efficient NHEJ and vice versa. Removal of BCL2 by immunoprecipitation and protein fractionation using size exclusion column chromatography showed elevated levels of EJ. Most importantly, the overexpression of BCL2 in vivo or the addition of purified BCL2 in vitro led to the downregulation of NHEJ in cancer cells. Further, we found that BCL2 interacts with KU proteins both in vitro and in vivo using immunoprecipitation and immunofluorescence, respectively. Hence, NHEJ in cancer cells is negatively regulated by the anti-apoptotic protein, BCL2, and this may contribute towards increased chromosomal abnormalities in cancer. In summary, our study showed that the efficiency of EJ in cancers could be regulated by the antiapoptotic protein BCL2. However, it may not affect the mechanistic aspect of EJ. BCL2 instead may interfere with EJ by sequestering KU and preventing it from binding to DNA ends. This may help in better understanding towards increased chromosomal abnormalities in cancer. Study of mitochondrial DSBR in mammalian system highlights the central role of microhomology-mediated A-EJ in the maintenance of the mammalian mitochondrial genome and this knowledge will helpful for the development of future therapeutic strategies against variety of mitochondria associated diseases.

No description available.

Determination of cell signalling behaviour is crucial for understanding the physiological response to a specific stimulus or drug treatment. Current approaches for large-scale data analysis do not effectively incorporate critical topological information provided by the signalling network. We herein describe a novel model- and data-driven hybrid approach, or signal transduction score flow algorithm, which allows quantitative visualization of cyclic cell signalling pathways that lead to ultimate cell responses such as survival, migration or death. This score flow algorithm translates signalling pathways as a directed graph and maps experimental data, including negative and positive feedbacks, onto gene nodes as scores, which then computationally traverse the signalling pathway until a pre-defined biological target response is attained. Initially, experimental data-driven enrichment scores of the genes were computed in a pathway, then a heuristic approach was applied using the gene score partition as a solution for protein node stoichiometry during dynamic scoring of the pathway of interest. Incorporation of a score partition during the signal flow and cyclic feedback loops in the signalling pathway significantly improves the usefulness of this model, as compared to other approaches. Evaluation of the score flow algorithm using both transcriptome and ChIP-seq data-generated signalling pathways showed good correlation with expected cellular behaviour on both KEGG and manually generated pathways. Implementation of the algorithm as a Cytoscape plug-in allows interactive visualization and analysis of KEGG pathways as well as user-generated and curated Cytoscape pathways. Moreover, the algorithm accurately predicts gene-level and global impacts of single or multiple in silico gene knockouts. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

The elucidation of the composition and organization of genomes of higher plants is a fundamental problem of modern molecular biology. The genus Beta containing 14 species assigned to the sections Beta, Corollinae, Nanae and Procumbentes provides a suitable system for the comparative study of the nuclear genomes. Sugar beet Beta vulgaris has a genome size of 758 Mbp DNA with estimated 63 % repetitive sequences and the number of chromosomes n=9. The wild beet Beta procumbens is an important natural pool of resistance against pests and tolerance to unfavorable growth conditions. The subject of this research was the isolation and description of new repetitive DNA families from genomes of this Beta species. This work presents the molecular investigation and cytogenetic characterization by high-resolution multicolor fluorescent in situ hybridization (FISH) of the satellite and dispersed repetitive sequences in wild and cultivated beet species and in their hybrids. New repetitive sequences were isolated from the B. procumbens genome. The AluI restriction satellite repeats pAp11 are 229-246 bp long and form subfamilies. The satellite is amplified in the section Procumbentes, but also found in distantly related section Beta. Thus, pAp11 is probably an ancient component of Beta genomes. It could be the ancestor of the satellite subfamily pEV4 in B. vulgaris based on sequence analysis, Southern hybridization and comparative FISH. pAp11 was found at centromeric and a few intercalary sites in B. procumbens and formed intercalary blocks on B. vulgaris chromosomes where it co-localized with pEV4. These remarkable differences in the chromosomal position of pAp11 between Procumbentes and Beta species indicate that both satellites were likely involved in the expansion or rearrangement of the intercalary heterochromatin of B. vulgaris. Other two sequence families characterized on molecular, genomic and chromosomal levels are the non-homologous repeats pAp4 and pAp22, 1354 and 582 bp long. They have a dispersed organization in the genome and are widely scattered along B. procumbens chromosomes. pAp4 and pAp22 are specific for the section Procumbentes and can be used as DNA probes to discriminate parental genomes in interspecific hybrids. High-resolution FISH on meiotic chromosomes showed that the both sequences mostly co-localize. The PCR analysis of their flanking regions revealed that pAp22 is a part of a Long Terminal Repeat (LTR) of an Athila-like env-class retrotransposon. This is the first indication that the retrovirus-like DNA elements exist in Beta. An ancient family of subtelomeric satellite DNA pAv34 was isolated from all four sections of the genus Beta and from spinach, a related Chenopodiaceae. Five clones were analyzed from each of the five species. The genomic organization and species distribution of the satellites were studied by sequencing and Southern hybridization. The repeating units in all families are 344-362 bp long and share 46.2-98.8 % similarity. Each monomer consists of two subunits SU1 and SU2 of 165-184 bp. The maximum likelihood and neighbor joining analyses of the 25 subtelomeric satellite monomers and their subunits indicated, that the duplication leading to the emergence of the 360 bp satellite should have occurred early in the phylogeny. The two directions of diversification are the clustering of satellites in two groups of subunits SU1 and SU2 and the arrangement of satellite repeats in section-specific groups. The comparative chromosomal localization of the telomeric repeat, pAv34 and rDNA was investigated by multicolor FISH. B. vulgaris chromosome termini showed unique physical organization of telomeric repeat and the subtelomeric satellite, as studied by high-resolution FISH on extended DNA fibers. The estimated length of the telomeric array was 0.55 - 62.65 kb, the length of pAv34 was 5.0-125.25 kb, the spacer between these sequences spanned 1.0-16.60 kb. Eight various classes of repeats were used to characterize the minichromosomes of the sugar beet fragment addition lines PRO1 and PAT2 by comparative multi-color FISH. The study allowed to propose a schematic pattern of repetitive DNA organization on the PRO1 and PAT2 minichromosomes. PRO1 has an acrocentric minichromosome, while PAT2 possesses a metacentric or submetacentric chromosome fragment. The functional integrity of the fragment addition line centromeres was confirmed by an immunostaining localization of the proteins specific to the active kinetochore. The serine 10-phosphorylated histone H3 was detected in pericentromeric regions of the PRO1 chromosomes. The microtubuli attachment sites were visualized as parts of kinetochore complexes.

Only 40 years ago, just three individuals made the journey from Finland/Russia to found the current wolf population in the southwest of Sweden. This population, that to this date descends from less than 10 founders, has a substantial increased extinction risk due to inbreeding. Several previous studies have used SNPs to monitor the level of inbreeding and homozygosity in the population, as well as measure immigration and the inflow of new genetic material. This study uses both short- and long-read data to discover structural variants (SVs) and small indels in the population, so that they may be used to extend the analyses and provide more insight into the current state of the Scandinavian wolf population. After the calling of the SVs, strict filtering and manual curation were applied to the data, thereby removing many false positive calls and increasing confidence in the remaining SVs. Short-read and long-read SV-callers found 31,800 and 57,821 SVs respectively, with relatively little overlap between the two sets. By far, the most common SV-types were deletions and insertions, at about 30,000 each with varying length ranging from a 50 base pairs to several tens of Mbp. Analyses on the data, such as PCAs and parent-offspring trio analyses, reveal high-confidence calls and consistent results between SV-types and SV-callers, as well as a low estimated genotyping error rate. PCAs performed on the SVs resembled those performed on SNPs, which strengthens the credibility of the identified variants. Finally, this study suggests several alternative steps for possible improvement to the dataset, along with some proposals for subsequent research topics that may use the variants discovered in this study.

Ankylosing spondylitis (AS) is a common inflammatory arthritis of the spine and other affected joints, which is highly heritable, being strongly influenced by the HLA-B27 status, as well as hundreds of mostly unknown genetic variants of smaller effect. The aim of my research was to confirm some of the previously observed genetic associations and to identify new associations, many of which are in biological pathways relevant to AS pathogenesis, most notably the IL-23/T_H17 axis (IL23R) and antigen presentation (ERAP1 and ERAP2). Studies presented in this thesis include replication and refinement of several potential associations initially identified by earlier GWAS (WTCCC-TASC, 2007 and TASC, 2010). I conducted an extended study of IL23R association with AS and undertook a meta-analysis, confirming the association between AS and IL23R (non-synonymous SNP rs11209026, p=1.5 x 10-9, OR=0.61). An extensive re-sequencing and fine mapping project, including a meta-analysis, to replicate and refine the association of TNFRSF1A with AS was also undertaken; a novel variant in intron 6 was identified and a weak association with a low frequency variant, rs4149584 (p=0.01, OR=1.58), was detected. Somewhat stronger associations were seen with rs4149577 (p=0.002, OR=0.91) and rs4149578 (p=0.015, OR=1.14) in the meta-analysis. Associations at several additional loci had been identified by a more recent GWAS (WTCCC2-TASC, 2011). I used in silico techniques, including imputation using a denser panel of variants from the 1000 Genomes Project, conditional analysis and rare/low frequency variant analysis, to refine these associations. Imputation analysis (1782 cases/5167 controls) revealed novel associations with ERAP2 (rs4869313, p=7.3 x 10-8, OR=0.79) and several additional candidate loci including IL6R, UBE2L3 and 2p16.3. Ten SNPs were then directly typed in an independent sample (1804 cases/1848 controls) to replicate selected associations and to determine the imputation accuracy. I established that imputation using the 1000 Genomes Project pilot data was largely reliable, specifically for common variants (genotype concordence~97%). However, more accurate imputation of low frequency variants may require larger reference populations, like the most recent 1000 Genomes reference panels. The results of my research provide a better understanding of the complex genetics of AS, and help identify future targets for genetic and functional studies.