Les variations en nombre de copies (CNV) sont des gains ou pertes d’une séquence d’ADN et peuvent avoir un rôle dans la susceptibilité à certaines maladies. Les CNVs peuvent être détectés par les puces de SNPs de haute résolution en analysant les intensités des allèles avec des algorithmes de détection des CNVs tels que CNV partition, PennCNV et QuantiSNP. Dans cette thèse, nous avons évalué les performances de ces outils pour la détection des CNVs au niveau pangénomique et pour les tests d'association. Nous avons également étudié des stratégies d'association combinant les informations de l'allèle et du nombre de copies pour des SNP situés dans des CNV. Nous avons appliqué ces outils pour mener une étude d’association pan-génomique avec les CNV en utilisant les données de l'étude espagnole du cancer de lavessie (SBC)/EPICURO générées par la puce Illumina 1M.Nos résultats montrent une faible fiabilité et une faible sensibilité des algorithmes de détection des CNV. Dans la région du gène GSTM1 où un CNV très fréquent existe qui est associé au risque de cancer de la vessie, nous avons constaté que les algorithmes de détection des CNV ont de faibles performances. Néanmoins, l’utilisation de la mesure d'intensité des allèles dans les tests d'association peut alors être une alternative intéressante car cela nous a permis de détecter cette association connue. Pour les SNPs situés dans des CNVs, nous avons étudié plusieurs stratégies de tests d'association et nous avons montré que la plus puissante était d’utiliser un modèle avec deux termes correspondant respectivement à la somme et à la différence du nombre de copies des deux allèles. Finalement, en appliquant ces stratégies à l'étude (SBC)/EPICURO, nous avons identifié des CNVs potentiellement associés au risque de cancer de la vessie, ainsi que des SNP dont l'allèle et le nombre de copies pourraient être impliqués dans le risque de cancer de la vessie. / Copy number variations (CNVs) are losses or gains of DNA sequences that may play a role in specific disease susceptibility. CNVs can be detected by high-resolution SNP-arrays through the analysis of allele intensities with CNV calling algorithms such as CNVpartition, PennCNV and QuantiSNP. In this thesis, we identified and assessed the performances of available tools for CNV calling and for association testing, at the genome-wide level. We also investigatedassociation strategies that combine information on both the allele and the number of copies for SNPs located in CNV regions. We applied these tools to conduct a genome-wide association study with CNV using data from the Spanish Bladder Cancer (SBC)/EPICURO Study generated by the Illumina 1M SNP-array. Our results showed a low reliability and a low sensitivity of the investigated CNV calling algorithms applied to SNP-array data. The GSTM1 locus shows a very frequent CNV that is associated with bladder cancer (BC) risk. We reported that the calling algorithms performed very poorly in identifying this CNV. We proposed using allele intensity measures (LRR) as a screening step to assess association as it allowed the detection of the GSTM1 CNV association with BC. To combine the allele and the number of copies for SNPs located in CNV regions, we investigated several strategies of association testing and we showed that the more powerfulone used a two-term model with the sum and the difference of the number of copies of both alleles. Finally, by applying these strategies to the (SBC)/EPICURO Study, we identified CNV regions potentially associated with BC risk, as well as SNPs for which both the allele and the number of copies could be involved in BC risk.

No description available.

The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

This PhD aimed to elucidate the mechanisms by which polymorphisms may alter androgen-induced transactivation of androgen receptor (AR) target genes which may be important in prostate cancer aetiology. The second aspect of this PhD focused on identifying and characterising functional polymorphisms that may have utility as predictive risk indicators for prostate cancer and which may aid in earlier therapeutic intervention and better disease management. Analyses were carried out on the kallikrein-related peptidase 3 (KLK3), also known as the prostate specific antigen (PSA), gene and the kallikrein-related peptidase 4 (KLK4) gene. The PSA and KLK4 genes are part of the serine protease family that have trypsin or chymotrypsin like activity and are thought to play a role in the development of hormone-dependent cancers in tissues such as those in the prostate, breast, endometrium and ovaries. In the prostate, PSA is regulated by androgens and three androgen response elements (AREs) have been described in the promoter and upstream enhancer region. The PSA ARE I harbours a polymorphism at -158 bp from the transcription initiation site (TIS) that results in a G to A transition (G-158A). This PhD investigated the functional significance of the PSA G-158A polymorphism which has been reported to be associated with prostate cancer risk. Electromobility shift assays (EMSAs) investigating the interaction of ARE I variants with the AR DNA binding domain (AR-DBD) demonstrated that the A allele had a two-fold increased binding affinity for the AR-DBD when compared with the G allele. This was confirmed with endogenous AR in limited proteolysis-EMSA experiments. The limited proteolysis-EMSA experiments also demonstrated differential sensitivities of PSA ARE I alleles to trypsin digestion, which suggests that the G-158A polymorphism has an allosteric effect on the AR that alters AR/ARE I complex stability. Furthermore, Chromatin Immunoprecipitation (ChIP) assays suggest that the A allele more readily recruited the AR in vivo when compared with the G allele and is consistent with the in vitro binding data. Luciferase reporter assays carried out in both LNCaP and 22Rv1 prostate cancer cells, and using the natural (dihydrotestosterone; DHT) ligand demonstrated that the A allele was more responsive to androgens in LNCaP cells. Hence, this study has elucidated the potential mechanisms by which the G-158A polymorphism may differentially regulate PSA expression (of which up-regulation of PSA is thought to be important in prostate cancer development and progression). KLK4 has similar tissue-restricted expression as PSA and is up-regulated by steroid hormones in many endocrine cells including those in the prostate. A putative ARE (KLK4-pARE) located at -1,005 to -1019 relative to the more predominantly used transcription initiation site, TIS3, was initially found in supershift assays using AR antibodies to interact with endogenous AR. However, subsequent EMSA analysis using purified AR-DBD suggest that KLK4-pARE may be interacting with the AR indirectly. To investigate this hypothesis, a tandem construct of KLK4-pARE was cloned into the pGL3-Promoter vector for hormone-induced reporter assays. However, reporter assays did not demonstrate any responsiveness of KLK4-pARE to androgens, estradiol or progestins. Consequently, Real-Time PCR was carried out to reassess the hormonal regulation of KLK4 at the mRNA level. Consistent with the literature, data from this study suggests that KLK4 may be up-regulated by androgens, progestins and estradiol in a cyclical manner. Hormone-induced luciferase reporter assays were then carried out on seven promoter constructs that span 2.8 kb of the KLK4 promoter from TIS3. However, none of the seven promoter constructs demonstrated any significant responsiveness to androgens, estradiol or progestins. This study suggests that hormone response elements (HREs) that may drive the hormonal regulation of KLK4 in prostate cancer may be located further upstream from the promoter region investigated in this PhD, or alternatively, may lie 3' of TIS3. The characterisation of KLK4 promoter polymorphisms and their flanking sequences were also carried out in parallel to the functional work with the intent to assess the functional significance of any polymorphisms that may be located within HREs. In total 19 polymorphisms were identified from the public databases and from direct sequencing within 2.8 kb of the KLK4 promoter from TIS3. However, the functional and clinical significance of these 19 polymorphisms were not further pursued given the negative findings from the functional work. The PSA AR enhancer region was also assessed for potential polymorphisms that may be associated with prostate cancer risk. A total of 12 polymorphisms were identified in the PSA enhancer of which two (A-4643G and T-5412C) have been reported to alter functionality of the enhancer region and thus, prioritised for further analysis. Association analysis for prostate cancer risk was then carried out on these PSA enhancer polymorphisms as none of the KLK4 promoter polymorphisms were found in functional HREs. No significant association for either the A-4643G or T-5412C polymorphism with prostate cancer risk was found at the P = 0.05 level. However, under an age-adjusted dominant model a 1.22- (95% CI = 1.16-1.26) and 1.23-fold (95% CI = 1.17-1.29) increased risk for prostate cancer was found for the A-4643G or T-5412C polymorphisms, respectively. Both polymorphisms were also assessed for association with tumour grade and stage and PSA levels. Genotypes were significantly different for the A-4643G and T-5412C polymorphisms with tumour stage and PSA levels, respectively. However, these results are likely to be biased by the case population which consist primarily of men who presented with incidental (pT1) and organ-confined (pT2) tumours. To summarise, the A-4643G and T-5412C polymorphisms are unlikely to be associated with prostate cancer risk, PSA levels or stage/grade of disease. However, further analyses in a larger cohort is warranted given that these polymorphisms alter androgen responsiveness of the PSA enhancer and that elevated PSA levels are indicative of men with prostate cancer. To summarise, this PhD has elucidated the functional significance of the PSA G-158A polymorphism in prostate cancer and which may be important in prostate cancer patho-physiology. This PhD has also furthered the understanding of the hormonal regulation of KLK4 in prostate cancer cells. Finally, this PhD has carried out a pilot study on two functional PSA enhancer polymorphisms (A-4643G and T-5412C) with prostate cancer risk.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen that is endemic in hospitals all over the world. It has more recently emerged as a serious threat to the general public in the form of community-acquired MRSA. MRSA has been implicated in a wide variety of diseases, ranging from skin infections and food poisoning to more severe and potentially fatal conditions, including; endocarditis, septicaemia and necrotising pneumonia. Treatment of MRSA disease is complicated and can be unsuccessful due to the bacterium's remarkable ability to develop antibiotic resistance. The considerable economic and public health burden imposed by MRSA has fuelled attempts by researchers to understand the evolution of virulent and antibiotic resistant strains and thereby improve epidemiological management strategies. Central to MRSA transmission management strategies is the implementation of active surveillance programs, via which unique genetic fingerprints, or genotypes, of each strain can be identified. Despite numerous advances in MRSA genotyping methodology, there remains a need for a rapid, reproducible, cost-effective method that is capable of producing a high level of genotype discrimination, whilst being suitable for high throughput use. Consequently, the fundamental aim of this thesis was to develop a novel MRSA genotyping strategy incorporating these benefits. This thesis explored the possibility that the development of more efficient genotyping strategies could be achieved through careful identification, and then simple interrogation, of multiple, unlinked DNA loci that exhibit progressively increasing mutation rates. The baseline component of the MRSA genotyping strategy described in this thesis is the allele-specific real-time PCR interrogation of slowly evolving core single nucleotide polymorphisms (SNPs). The genotyping SNP set was identified previously from the Multi-locus sequence typing (MLST) sequence database using an in-house software package named Minimum SNPs. As discussed in Chapter Three, the genotyping utility of the SNP set was validated on 107 diverse Australian MRSA isolates, which were largely clustered into groups of related strains as defined by MLST. To increase the resolution of the SNP genotyping method, a selection of binary virulence genes and antimicrobial resistance plasmids were tested that were successful at sub typing the SNP groups. A comprehensive MRSA genotyping strategy requires characterisation of the clonal background as well as interrogation of the hypervariable Staphylococcal Cassette Chromosome mec (SCCmec) that carries the β-lactam resistance gene, mecA. SCCmec genotyping defines the MRSA lineages; however, current SCCmec genotyping methods have struggled to handle the increasing number of SCCmec elements resulting from a recent explosion of comparative genomic analyses. Chapter Four of this thesis collates the known SCCmec binary marker diversity and demonstrates the ability of Minimum SNPs to identify systematically a minimal set of binary markers capable of generating maximum genotyping resolution. A number of binary targets were identified that indeed permit high resolution genotyping of the SCCmec element. Furthermore, the SCCmec genotyping targets are amenable for combinatorial use with the MLST genotyping SNPs and therefore are suitable as the second component of the MRSA genotyping strategy. To increase genotyping resolution of the slowly evolving MLST SNPs and the SCCmec binary markers, the analysis of a hypervariable repeat region was required. Sequence analysis of the Staphylococcal protein A (spa) repeat region has been conducted frequently with great success. Chapter Five describes the characterisation of the tandem repeats in the spa gene using real-time PCR and high resolution melting (HRM) analysis. Since the melting rate and precise point of dissociation of double stranded DNA is dependent on the size and sequence of the PCR amplicon, the HRM method was used successfully to identify 20 of 22 spa sequence types, without the need for DNA sequencing. The accumulation of comparative genomic information has allowed the systematic identification of key MRSA genomic polymorphisms to genotype MRSA efficiently. If implemented in its entirety, the strategy described in this thesis would produce efficient and deep-rooted genotypes. For example, an unknown MRSA isolate would be positioned within the MLST defined population structure, categorised based on its SCCmec lineage, then subtyped based on the polymorphic spa repeat region. Overall, by combining the genotyping methods described here, an integrated and novel MRSA genotyping strategy results that is efficacious for both long and short term investigations. Furthermore, an additional benefit is that each component can be performed easily and cost-effectively on a standard real-time PCR platform.

No description available.

Neste estudo, investigamos o envolvimento de galectina-3 na fisiologia e no câncer de tiróide usando vários modelos biológicos e metodologias. Observamos que o gene LGALS3 apresenta um SNP no códon 98, mas não observamos correlação entre os genótipos deste SNP e fenótipo de câncer de tiróide. Na linhagem de tiróide de rato PCCl3, mostramos que a indução da expressão do oncogene RET/PTC promove o aumento da expressão de galectina-3, no entanto, a expressão de galectina-3, por si só, não confere vantagem de proliferação à célula. Por outro lado, na linhagem de carcinoma papilífero de tiróide TPC-1, a galectina-3 contribui para a sobrevivência da célula tumoral e progressão do ciclo celular, aumentando a expressão de c-Myc, diminuindo a expressão de p21 e caspase-3, e favorecendo a ativação de importantes vias envolvidas no controle do ciclo celular. Além disto, em modelos in vivo e in vitro, a galectina-3 interferiu na função e diferenciação da célula folicular tiroidiana, exercendo um papel indireto na regulação da expressão da tireoglobulina e atividade de TTF-1. / In this study, we investigate the involvement of galectin-3 in thyroid physiology and cancer using several biological models and methodologies. We observed that LGALS3 gene presents a SNP in codon 98, but no correlation between the genotype and the phenotype of benign or malignant thyroid tumor was observed. In the rat thyroid cell line PCCl3, we showed that the conditional induction of RET/PTC oncogene expression promotes the increase of galectin-3 expression, however, galectin-3 expression itself did not confer a proliferative advantage to cell. On the other hand, in papillary thyroid carcinoma cell line TPC-1 the galectin-3 contributes to tumor cell survival and cell cycle progression, increasing c-Myc expression, decreasing p21 and caspase-3 expression and cooperating to activation of important signaling pathways which are involved in the cell cycle control. In addition, in vitro and in vivo models the galectin-3 interferes in the differentiation and function of thyroid follicular cell, playing an indirect role in the regulation of thyroglobulin expression and TTF-1 activity.

The ability to identify germplasm and select traits accurately is fundamental to successful plant breeding. Pedigrees and molecular markers facilitate these processes; however misleading experimental results can occur when incorrect relationships and/or cultivar names are recorded. Molecular markers can identify these inconsistencies, and with advances in genotyping technology these diagnostics can be done faster and more objectively. This study aimed to develop molecular marker assays and graphical genotyping methodologies for cultivar identification, seed purity assessment and trait selection in oat (Avena sativa L.). KBioscience’s Allele-Specific PCR (KASP™) and genotyping-by-sequencing (GBS) technologies were applied to a set of current Canadian oat cultivars to evaluate their utility for identifying cultivars and detecting intra-cultivar variation. Both KASP™ and GBS detected different extents of heterogeneity among a set of 160 seeds that originated from four seed sources of four cultivars. In both cases, the detected variation did not appear to be limited to a specific cultivar or seed source, reinforcing that all cultivars are heterogeneous. Graphical genotyping localized heterogeneity to specific chromosome regions, thereby distinguishing physical contamination from true genetic heterogeneity and heterozygosity. Pre-existing genotype data for 700 oat cultivars and breeding lines were also used to construct graphical genotypes for pedigree validation and discovery of potential sources for favourable quantitative trait loci (QTL) alleles. This methodology used historical QTLs and anchoring markers to identify 25 putative “high oil” allele carriers. The results from this study will provide diagnostic tools for cultivar identification and pedigree validation, in addition to meaningful information about existing heterogeneity and possible QTL locations in current cultivars.

Bermudagrass (Cynodon spp.) breeding and cultivar development is hampered by limited information regarding its genetic and phenotypic diversity. A germplasm collection of 206 bermudagrass accessions from 29 countries was genotyped with high-throughput genotyping-by-sequencing technique. Genomic diversity in this diverse germplasm panel was assessed with multifaceted approaches including population structure, phylogenetic analysis, principal component analysis, and genetic diversity parameters. This study revealed substantial genetic variation in the Cynodon accessions, demonstrating the potential of this germplasm panel for further genetic studies and cultivar development in breeding programs. Another critical issue in turfgrass breeding is the lack of information regarding the genetic architecture of traits. Four agronomic traits leaf length, leaf width, internode distance and stem diameter were evaluated in a germplasm panel of common bermudagrass accessions. Then genome-wide association study was performed to dissect the genetic basis of the traits.