Understanding the genetic basis of biological processes is a fundamental component of modern ecology and evolutionary biology studies. With the recent advent of next generation sequencing (NGS) technologies, it is now possible to perform large genome and transcriptome projects for ecologically important non-model species. In this thesis, I focused on the development and application of genomic resources of two non-model bird species, the black grouse (Tetrao tetrix) and the great snipe (Gallinago media). Using the chicken genome as a reference, I developed a reference guided NGS pipeline to assemble the complete draft genome of black grouse. The draft genome has a good coverage of the main 29 chromosomes of the chicken genome. The genome was used to develop a vast number of genetic markers. Comparing this genome with that of other species, I identified the genomic regions which were important for the lineage specific evolution of black grouse. I also sequenced and characterised the spleen transcriptome of the black grouse. I identified and validated a large number of gene-based microsatellite markers from the transcriptome and identified and confirmed the expression of immune related genes. Using a similar RNA-Seq approach, I also sequenced the blood transcriptomes of 14 great snipe males with different mating success. I identified genes and single nucleotide polymorphisms (SNPs) which might be related to male mating success in this species, both in terms of gene expression levels and genetic variation structure. For the immunologically important major histocompatibility complex (MHC) gene region of black grouse, I constructed a fosmid library and used it to sequence the complete core MHC region of this species. This resource allowed me to perform a comprehensive comparative genomics analysis of the galliform MHC, by which I found that some genes in this region were affected by selective forces. I was also able to develop a single locus genotyping protocol for the duplicated MHC BLB (class IIB) genes and found that the two black grouse BLB loci followed different evolutionary trajectories. This thesis set an example of developing genomic resources in non-model species and applying them in addressing questions relevant to ecology and evolutionary biology.

Flesh colour and fillet fat percentage are the two most important attributes to salmon fillet quality. A medium genetic component to body lipid percentage within commercial lines has previously been shown (h2 = 0.17-0.24). A low level of heritability (h2 = 0.16) has also been reported for flesh colour in Atlantic salmon. To investigate whether this genetic component includes loci of major effect, a genome-wide QTL scan was performed with commercially bred Atlantic salmon (Landcatch Natural Selection). Five large full-sib families (10 parents with 153 offspring) were genotyped using microsatellite markers. To utilize the large difference between sire and dam recombination rate, a two-stage genotyping was employed. Initially, the parents and offspring were genotyped for two microsatellite markers per linkage group, and sire based QTL analysis was used to detect linkage groups with significant effects on those flesh quality traits. A linear-regression based interval as analytical method was applied for QTL detection. The results revealed evidence of QTLs affecting percentage fat percentage and flesh colour on linkage groups LNS16 and LNS1 respectively. To confirm the QTL and to provide an improved estimate of position, a dam-based analysis was then employed. One major QTL was located on the genome-wide significance level for percentage fat percentage. Microsatellite marker Ssa0016NVH (at position of 1.3 cM) was found to be tightly linked to QTL affecting percentage fat percentage. In addition, a QTL affecting flesh colour was found to be flanked by microsatellite markers Ssa9.44NUIG at position of 68.7 cM and Ssa0021NVH at position of 50.6 on linkage group LNS16. The evidence for suggestive QTL affecting flesh colour on linkage group LNS1 was also revealed. In order to increase marker density within these and other linkage groups, AFLP markers were employed, 24 primer combinations resulted in a total of 489 polymorphic fragments. Among 11 fragments that were found to be linked to the microsatellite markers on linkage group LNS16, four fragments (AAG-CAC328, AGG-CAG447, AGG-CTA237 and AGG-CTC237) were tightly linked to microsatellite marker Ssa9.44NUIG, but none were found to be linked to microsatellite Ssa0021NVH. Moreover, none of the AFLP markers were found to be linked to microsatellites residing on linkage group LNS1. Using a constructed map of microsatellite and AFLP markers for linkage group LNS16, the dam based analysis revealed a significant QTL for flesh colour at the location of 189 cM, while the sire based analysis detected a significant QTL for fat percentage at the location of 80 cM. Considering the dominant nature and clustering character of AFLP markers, it was concluded that a certain primer combination in AFLP markers could be of limited use for fine mapping and QTL detection in Atlantic salmon.

No description available.

No description available.

A next generation nanosatellite bus is under development at the University of Toronto’s Space Flight Laboratory (SFL), and is being used for the first time in an ambitious Earth observation mission to identify and monitor atmospheric aerosol species. The spacecraft system brings together novel advanced designs that expand the capability envelope of nanosatellites, with heritage SFL technology that is presently defining the state-of-the-art in microspace applications. The work presented in this thesis pertains primarily to the development of the structural subsystem of the Nanosatellite for Earth Monitoring and Observation – Aerosol Monitor (NEMO-AM). Described extensively are the design and analysis efforts made by the author to validate and finalize the structural design in order to bring it to a manufacturing-ready stage. Subsequent work to meet the mechanical requirements of ground operations during the assembly and testing of the spacecraft is also presented.

A next generation nanosatellite bus is under development at the University of Toronto’s Space Flight Laboratory (SFL), and is being used for the first time in an ambitious Earth observation mission to identify and monitor atmospheric aerosol species. The spacecraft system brings together novel advanced designs that expand the capability envelope of nanosatellites, with heritage SFL technology that is presently defining the state-of-the-art in microspace applications. The work presented in this thesis pertains primarily to the development of the structural subsystem of the Nanosatellite for Earth Monitoring and Observation – Aerosol Monitor (NEMO-AM). Described extensively are the design and analysis efforts made by the author to validate and finalize the structural design in order to bring it to a manufacturing-ready stage. Subsequent work to meet the mechanical requirements of ground operations during the assembly and testing of the spacecraft is also presented.

Les lésions tumorales cortico-surrénaliennes sont majoritairement des adénomes bénins et très rarement des carcinomes. Les altérations génétiques impliquées dans le développement des tumeurs cortico-surrénaliennes sporadiques, plus particulièrement au stade malin, demeurent à ce jour très peu connues. Lors de travaux récents menant à l’identification d’altérations génétiques de β-CATÉNINE nous avons constaté que plusieurs tumeurs présentaient une accumulation nucléo/cytoplasmique de la protéine β-CATÉNINE sans toutefois contenir de mutations pour ce gène. Nous avons donc émis l’hypothèse que, comme pour d’autres types de cancers, d’autres composants de la voie de signalisation Wnt/β-CATÉNINE, tel qu’AXIN2, pourrait être impliqués dans le développement des tumeurs du cortex surrénalien. De plus, plusieurs aberrations dans l’expression d’AXIN2 et de β-CATÉNINE sont associées à des tumeurs présentant de l’instabilité microsatellite dans d’autres types de cancer, notamment le cancer gastrique et colorectal. Nous avons donc étudié une cohorte de 30 adénomes, 6 carcinomes, 5 AIMAH, 3 hyperplasies ACTH-dépendante et 5 PPNAD ainsi que les lignées cellulaires de carcinomes cortico-surrénaliens humains H295R et SW13. Une étude préliminaire du statut MSI a également été réalisée sur 10 tumeurs contenant une mutation pour AXIN2 et/ou β-CATÉNINE. Nous avons trouvé des mutations d’AXIN2 dans 7% des adénomes (2/30) et 17% des carcinomes (1/6) cortico-surrénaliens. L’analyse fonctionnelle des mutations par immunohistochimie, analyse western blot et analyse de RT-PCR en temps réel a révélé une diminution de l’expression d’AXIN2 associée à cette mutation. L’analyse préliminaire MSI a démontré 1 échantillon AIMAH MSI-H, c’est-à-dire instable pour le locus BAT-25 et BAT-26 et 3 autres adénomes sécrétant de l’aldostérone instables seulement pour le locus BAT-26. Ainsi, ces travaux permirent d’identifier une nouvelle altération génétique associée au développement des tumeurs du cortex surrénalien en plus de rapporter pour la première fois la présence de MSI-H dans ce type de tumeurs. / Adrenocortical lesions are mostly benign tumors and rarely carcinomas. From now on, genetic alterations implicated in sporadic adrecocortical tumour development remains largely unknown. In our previous work leading to identification of genetic alterations in β-catenin, we observed that many tumors presented a nucleo/cytoplasmic accumulation of β-catenin protein without β-catenin mutations. Thus, we hypothesised that, as for many others cancers, others components of the Wnt/ β-catenin signalling pathway, as AXIN2, are implicated in development of adrenocortical tumors. Also, many aberrations in AXIN2 and β-catenin expression have been reported in association with microsatellite instability in other types of cancers like gastroinstestinal and colorectal cancer. We have studied 30 adenomas, 6 carcinomas, 5 AIMAH, 3 ACTH-dependant hyperplasias and 5 PPNAD as well as the human carcinoma cancer cells lines H295R and SW13. Preliminary study for MSI was also realised on 10 tumors harbouring AXIN2 and/or Β-CATENIN mutations. We have found AXIN2 mutations in 7% of adrenocortical adenomas (2/30) and 17% of adrenocortiocal carcinomas. Functional analysis of this mutation by immunohistochemical, western blot and real-time RT-PCR analysis revealed a down-regulation of AXIN2 expression associated with this mutation. Preliminary analysis of MSI results in 1 AIMAH sample MSI-H, which means instable for BAT-25 and BAT-26 locus, and 2 aldosterone adenomas were unstable for BAT-26 locus. This work identified a new genetic alteration involved in adrenocortical tumour development and report for the first time MSI-H in this type of tumor.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Background Colorectal cancer (CRC) is the second most common malignancy in developed countries. The mortality is high, with nearly half of patients dying from the disease. The primary treatment of CRC is surgery, and decisions about additional treatment with chemotherapy are based mainly on tumor stage. Novel prognostic markers that identify patients at high risk of recurrence and cancer-related death are needed. The development of CRC has been described in terms of two different pathways; the microsatellite instability (MSI) and chromosomal instability (microsatellite stable, MSS) pathway. More recently, the CpG island methylator phenotype (CIMP), characterized by frequent DNA hypermethylation, has been described as an alternative pathway of tumorigenesis. The event of DNA methylation is dependent on one-carbon metabolism, in which folate and vitamin B12 have essential functions. The purpose of this thesis was to study CIMP in CRC. The specific aims were to investigate the potential role of components of one-carbon metabolism as risk factors for this subgroup of tumors, and the prognostic importance of CIMP status, taking into consideration important confounding factors, such as MSI and tumor-infiltrating T cells. Methods CRC cases and referents included in the Northern Sweden Health and Disease Study (NSHDS, 226 cases and 437 referents) and CRC cases in the Colorectal Cancer in Umeå Study (CRUMS, n=490) were studied. Prediagnostic plasma concentrations of folate and vitamin B12 were analyzed in NSHDS. In both study groups, CIMP status was determined in archival tumor tissue by real-time quantitative PCR using an eight-gene panel (CDKN2A, MLH1, CACNA1G, NEUROG1, RUNX3, SOCS1, IGF2 and CRABP1). MSI screening status and the density of tumor-infiltrating T cells were determined by immunohistochemistry.  Results An inverse association was found between plasma concentrations of vitamin B12 and rectal, but not colon, cancer risk. We also found a reduced risk of CIMP-high and CIMP-low CRC in study subjects with the lowest levels of plasma folate. We found that patients with CIMP-low tumors in both NSHDS and CRUMS had a poorer prognosis compared with CIMP-negative, regardless of MSI screening status. We also found that MSS CIMP-high patients had a poorer prognosis compared with MSS CIMP-negative. The density of tumor-infiltrating T cells and CIMP status were both found to be independent predictors of CRC patient prognosis. A particularly poor prognosis was found in patients with CIMP-low tumors poorly infiltrated by T cells. In addition, the density of T cells appeared to be more important than MSI screening status for predicting CRC patient prognosis. Conclusion Rather than being one disease, CRC is a heterogeneous set of diseases with respect to clinico-pathological and molecular characteristics. We found that the association between risk and plasma concentration of vitamin B12 and folate depends on tumor site and CIMP status, respectively. Patient prognosis was found to be different depending on CIMP and MSI screening status, and the density of tumor-infiltrating T cells.

DNA is the code for all life. Via intermediary RNA the information encoded by the genome is relayed to proteins executing the various functions in a cell. Together, this repertoire of inherently linked biological macromolecules determines all characteristics and features of a cell. Technological advancements during the last decades have enabled the pursuit of novel types of studies and the investigation of the cell and its constituents at a progressively higher level of detail. This has shed light on numerous cellular processes and on the underpinnings of several diseases. For the majority of studies focusing on nucleic acids, an amplification step has to be implemented before an analysis, scoring or interrogation method translates the amplified material into relevant biological information. This information can, for instance, be the genotype of particular SNPs or STRs, or the abundance level of a set of interesting transcripts. As such, amplification plays a significant role in nucleic acid assays. Over the years, a number of techniques – most notably PCR – has been devised to meet this amplification need, specifically or randomly multiplying desired regions. However, many of the approaches do not scale up easily rendering comprehensive studies cumbersome, time-consuming and necessitating large quantities of material.Trinucleotide threading (TnT) – forming the red thread throughout this thesis – is a multiplex amplification method, enabling simultaneous targeted amplification of several nucleic acid regions in a specific manner. TnT begins with a controlled linear DNA thread formation, each type of thread corresponding to a segment of interest, by a gap-fill reaction using a restricted trinucleotide set. The whole collection of created threads is subsequently subjected to an exponential PCR amplification employing a single primer pair. The generated material can thereafter be analyzed with a multitude of readout and detection platforms depending on the issue or characteristic under consideration.TnT offers a high level of specificity by harnessing the inherent specificities of a polymerase and a ligase acting on a nucleotide set encompassing three out of the four nucleotide types. Accordingly, several erroneous events have to occur in order to produce artifacts. This necessitates override of a number of control points.The studies constituting this thesis demonstrate integration of the TnT amplification strategy in assays for analysis of various aspects of DNA and RNA. TnT was adapted for expression profiling of intermediately-sized gene sets using both conventional DNA microarrays and massively parallel second generation 454 sequencing for readout. TnT, in conjunction with 454 sequencing, was also employed for allelotyping, defined as determination of allele frequencies in a cohort. In this study, 147 SNPs were simultaneously assayed in a pool comprising genomic DNA of 462 individuals. Finally, TnT was recruited for parallel amplification of STR loci with detection relying on capillary gel electrophoresis. In all investigations, the material generated with TnT was of sufficient quality and quantity to produce reliable and accurate biological information.Taken together, TnT represents a viable multiplex amplification technique permitting parallel amplification of genomic segments, for instance harboring polymorphisms, or of expressed genes. In addition to these, this versatile amplification module can be implemented in assays targeting a range of other features of genomes and transcriptomes. / QC 20100819