Entre 10% et 30% des maladies humaines sont liées à l'apparition d'une mutation non-sens (PTC). La synthèse protéique est alors arrêté prématurément. Cet arrêt peut être inhibé par des molécules inductrices de translecture qui permettent l’incorporation d’un ARNt suppresseur naturel au niveau du PTC (translecture). Le ribosome peut alors franchir le PTC et restaurer l’expression de la protéine.Au cours de ma thèse, je me suis intéressé à la suppression des codons stop en caractérisant de nouvelles molécules inductrices de translecture et en analysant les mécanismes de la fidélité de la traduction.J’ai tout d’abord mis au point un système de criblage innovant avec lequel j’ai testé plus de 17 000 molécules et identifié la molécule TLN468. J’ai pu mettre en évidence que cette molécule est capable d’induire la réexpression d’une protéine p53 active.J'ai aussi caractérisé de nouveaux composés dérivés d’aminoglycosides. J’ai pu montré que le NB124 est capable d’induire l’apoptose de cellules tumorales via la réexpression de la protéine p53 tout ayant une toxicité bien plus faible que la gentamicine.En parallèle, j’ai développé une approche en molécule unique permettant d’étudier les erreurs programmées du ribosome (recodage). J’ai ainsi pu analyser la cinétique d’élongation des ribosomes eucaryotes et montré que l’initiation de la traduction sur un site d’entrée interne (IRES) ralentit le ribosome lors des premiers cycles d’élongation. / Nonsense mutations, also known as premature termination codons (PTCs) are responsible for 10% to 30% of all human genetic diseases. Nonsense translation suppression can be induced by readthrough inducers. The presence of such PTC leads to premature translation termination. These stop therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at the PTC (readthrough). The, translation continue in the same reading frame until the next stop codon. I first developed an innovative screening system I used to test more than 17,000 molecules and have identified one hit, TLN468 molecule. I have shown that this molecule is able to induce re-expression of an active p53 protein.I also characterized new compounds derived from aminoglycosides. I have shown that the NB124 induces apoptosis of tumor cells by re-expressing p53 protein while having a much lower toxicity than gentamicin.I developed a single molecule approach for studying the ribosome programmed errors (recoding). I was able to analyze the kinetics of elongation eukaryotic ribosomes and showed that the initiation of translation at an internal entry site (IRES) slows the ribosome during the first elongation cycle.

Brinjal shoot and fruit borer (Leucinodes orbonalis Guenee) is a major limiting factor in commercial cultivation of eggplant in southeast Asia. Extensive use of pesticides as well as the conventional breeding methods have been ineffective in controlling the borer so there is a need for Integrated Pest Management (IPM) strategies for its control. Bacillus thuringiensis (Bt) is known to produce a variety of insecticidal crystal proteins toxic to lepidopteran, dipteran and coleopteran pests. The Cry2Aa protein has been found to be more toxic to brinjal shoot and fruit borer than Cry1Ab. My objective was to develop eggplant cultivars that express a codon-optimized cry2Aa gene, the sequence of which is based on that of an Indian isolate of Bt, with the eventual goal of producing fully resistant cultivars. The cry2Aa gene was modified for optimal expression in eggplant using the codon usage frequencies based on solanaceous sequences (eggplant, tomato and pepper). The GC content was increased from 34.3% in the native gene to 41.3% in the optimized gene, thus removing the AT-rich regions that are typical for Bt cry genes. Also, other mRNA destabilizing and hairpin forming structure sequences were removed. The gene was synthesized in four different parts with complementary restriction sites. A total of 152 oligonucleotides (oligos) was used to assemble the 1.9 kb gene using dual asymmetric (DA) and overlap extension (OE) PCR techniques. The individual parts were subsequently ligated using the complementary restriction sites and inserted into vector pCAMBIA 1302. Also, the transformation efficiency of 12 different eggplant cultivars was tested using plasmid pHB2892 to predict utility for transformation with the synthetic cry2Aa. / Master of Science

In this thesis work, I have used a comparative genomics approach to study a fungal model organism, Neurospora tetrasperma. My specific focus has been on genomic introgression, intron evolution, chromosomal structural rearrangements and codon usage. All of the studies are based on large-scale dataset generated by next-generation sequencing technology (NGS), combined with other techniques, such as Optical Mapping. In the introgression study, we detected large-scale introgression tracts in three N. tetrasperma lineages, and the introgression showed allele-specific and chromosomal-specific pattern. In the study of introns, we found indications of mRNA mediated intron loss and non-homologous end joining (NHEJ) mediated intron gains in N. tetrasperma. We found that selection is involved in shaping intron gains and losses, and associated with intron position, intron phase and GC content. In the study of chromosomal structural rearrangements, we found a lineage specific chromosomal inversion pattern in N. tetrasperma, which indicates that inversions are unlikely to associate with the origin of the suppressed recombination and the mating system transition in N. tetrasperma. The result suggests inversions are the consequences, rather than the causes, of suppressed recombination on the mating-type chromosome of N. tetrasperma. In the final study, analyses of codon usage indicated that the region of suppressed recombination in N. tetrasperma is subjected to genomic degeneration, and selection efficiency has been much reduced in this region.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Developing microbial systems capable of converting low cost lipids into value added products depends on the ability to acquire substrates from the growth media. Saccharomyces cerevisiae can acquire free fatty acids from the growth media and a portion of these lipids can be converted into new lipid products. However, they cannot acquire complex lipids from the growth media unless a nonspecific lipase is included. To circumvent lipase addition, we are genetically engineering S. cerevisiae to secrete a lipase into the growth media. We selected the LIP2 gene from Yarrowia lipolytica, which encodes a nonspecific lipase. Several modifications were made to the LIP2 gene to improve processing. Results identified strains secreting the most lipase. From these results, high producing strains were inserted into an oil inducible vector. Halo assays confirmed lipase secretion, while measuring the fatty acid composition confirmed triacylglycerol breakdown, and yeast uptake of the free fatty acids released.

Le NMD (nonsense-mediated mRNA decay) est un mécanisme qui reconnaît et dégrade les ARNm portant un codon stop prématuré afin d’empêcher la synthèse de protéines tronquées qui pourraient avoir des effets néfastes pour la cellule ou tout simplement être non fonctionnelles. Cependant, dans un certain nombre de cas, selon la position du codon stop prématuré, la protéine tronquée qui serait synthétisée si le NMD n’existait pas, pourrait remplir complètement ou partiellement la fonction de la protéine sauvage. Il faut noter qu’un codon stop prématuré est retrouvé dans le gène responsable d’une pathologie dans un tiers des maladies génétiques et de nombreuses formes de cancer. Dans la plus grande majorité des cas, la maladie se développe non pas parce qu’une protéine tronquée non fonctionnelle ou instable est synthétisée, mais plutôt parce que le gène muté n’est pas exprimé du fait de l’intervention du NMD sur l’ARNm qui en dérive. Une nouvelle approche thérapeutique de ces maladies serait d’inhiber le NMD afin de permettre la synthèse de protéines tronquées fonctionnelles et sauver le phénotype clinique. Nous avons donc décidé de rechercher des inhibiteurs du mécanisme du NMD parmi des petites molécules chimiques. Pour cela, nous avons mis au point un système de criblage en culture cellulaire reliant l’efficacité du NMD dans une cellule avec une activité luciférase mesurable directement sur les cultures cellulaires, au moyen d’un luminomètre. A partir d’un premier criblage d’environ 1500 composés chimiques, nous avons identifié une nouvelle molécule capable d’inhiber efficacement le NMD. De façon intéressante, cette nouvelle molécule est capable également d’induire la synthèse de protéines entières à partir d’un ARNm portant un codon stop prématuré. Nous avons utilisé cet inhibiteur dans des expériences pour déterminer son potentiel thérapeutique sur des modèles cellulaires de maladies génétiques tels que la dystrophie musculaire de Duchenne, la mucoviscidose et le cancer. Nos résultats démontrent que l’inhibition du NMD peut être en effet envisagée comme une nouvelle approche thérapeutique pour des maladies causées par l’apparition d’une mutation non sens. Nous avons aussi identifié une autre molécule chimique capable d’inhiber le NMD et permettant de faire un lien entre efficacité du NMD et intégrité du cytosquelette. / MRNAs harboring a premature termination codon are rapidly degraded by a mechanism called nonsense-mediated mRNA decay (NMD). NMD is a surveillance pathway that prevents the synthesis of truncated proteins that could be harmful for the cell or simply be non-functional. However in some cases, depending on the position of the premature stop codon, the truncated protein that would be synthesized if there were no NMD would be partially or fully as functional as the wild-type protein. It is noteworthy that premature termination codons are found in approximately one-third of inherited genetic disorders and several forms of cancer. In most of cases the disease arises not because a non-functional or unstable truncated protein is synthesized, but instead because the degradation of the transcript by NMD leads to complete loss of protein production. Therefore, NMD inhibition could be an interesting therapeutic approach in some cases of nonsense-related genetic diseases in which functional truncated proteins can restore the clinical phenotype. We decided to search for NMD inhibitors among thousands of small molecules. We developed a cell-based screening method which couples NMD efficiency into the cell to a luciferase activity that can be measured directly into cells by a luminometer. From a screening of approximately 1500 compounds, we have identified one molecule capable of efficiently inhibit NMD. Interestingly, this compound is also able to induce the synthesis of full-length proteins from an mRNA bearing a premature termination codon. We evaluated the therapeutic potential of this compound in different cellular models of genetic disorders such as Duchenne’s muscular dystrophy, cystic fibrosis and cancer. Our results demonstrate that NMD inhibition in general can be considered as an useful therapeutic approach to rescue PTC consequences in genetic diseases provoked by the apparition of a nonsense mutation. We have also identified another compound that inhibits NMD and uncovers a relationship between the NMD efficiency and the integrity of the cytoskeleton.

Made available in DSpace on 2016-08-10T10:39:20Z (GMT). No. of bitstreams: 1 Rodrigo Egidio da Silva.pdf: 422179 bytes, checksum: 83dd8949e51efd8441440005afe1be0c (MD5) Previous issue date: 2009-02-17 / Glaucoma primário de ângulo aberto (GPAA) é o tipo de glaucoma mais comum. O polimorfismo Arg-Pro (CGC para CCC) no exon 4 do codon 72 do gene p53 afeta várias propriedades biológicas; recentemente foi publicado que este afeta a indução da apoptose in vitro. Vários genótipos têm sido associados significantemente ao GPAA. Nós analisamos a distribuição deste polimorfismo em 104 pacientes com GPAA e em 58 pessoas saudáveis sem história de glaucoma na Pronto Clínica de Olhos em Goiânia, Brasil. Os controles foram recrutados entre os indivíduos em acompanhamento oftalmológico. O polimorfismo do codon 72 do gene p53 foi analisado no DNA genômico por PCR. O alelo Arg72 foi mais comum do que o alelo Pro72 em ambos os grupos. Não houve diferença significante na distribuição do polimorfismo do codon 72 entre os grupos (P= 0.3311). A distribuição de genótipo no grupo com GPAA foi de 23,07% homozigotos Arg, 75% heterozigotos e 1,93% homozigotos Pro, enquanto que no grupo controle foi de 31,04% homozigotos Arg, 68,96% heterozigotos e 0% homozigotos Pro. Concluímos que o polimorfismo do codon 72 Arg/Pro não está associado com o glaucoma em pacientes brasileiros.

Made available in DSpace on 2016-08-10T10:39:21Z (GMT). No. of bitstreams: 1 CIRCONCISTO LAURENTINO RIBEIRO JUNIOR.pdf: 2083186 bytes, checksum: bfcfede933b252abac1e5de63aa561f2 (MD5) Previous issue date: 2009-03-13 / Endometriosis it is considered as the presence of ectopic endometrial tissue, with similar histology and function to the endometrium usually located. However, the explanation for the implantation of the endometrial tissue in certain women is still unknown. The determination of the degree of compromising of the endometriosis is based on a system of points proposed by American Fertility Society finds of the laparoscopy. Endometriosis is a disease that occurs mainly in women in reproductive age. The disease can be related with infertility in 30% to 40% of the cases. The possible mechanisms as the endometriosis as cause of the infertility are: (a) interference with the sexual function (dyspareunia, reduction of the frequency of sexual intercourse). (b) Interference with the ovulation: (anovulation; deficient luteal phase and luteinization phase. The aim of this study was to determine the frequency of p53 polymorphism codon 72, in two groups of patients with endometriosis and other symptoms of the disease. The study included 38 peripheral blood samples from women submitted to videolaparoscopy that confirmed endometriosis. The patients were divided into two groups of 19 women each, one with infertility and the other not. The polymorphism was evaluated by PCR. The data were submitted to statistical analysis using the chi-square and odds ratio tests. Of the patients who only had pain and/or bleeding without infertility, 09 (47.3%) were arginine homozygous and 10 (52.6%) were proline homozygote or heterozygote. Of those who presented with infertility associated with pain and/or bleeding, 12 (100%) were proline homozygous or heterozygous, and in those with only complaint of infertility, 05 (71.4%) were arginine homozygous and 02 (28.6%) proline homozygous or heterozygous). In correlating pain intensity, 04 (23.5%) women with the proline allele (homozygous or heterozygous) reported slight or moderate pain and 20 (95.5%) intense pain. All these differences were statistically significant (p= 0.003). In relating pain intensity to laparoscopic classification, the following was found: 08 (50%) patients who showed slight or moderate pain had a classification I or II and 08 (50%) III or IV. Of those with intense pelvic pain, 08 (36.4%) were included in classification I or II and 14 (63.6%) in III or IV. The proline allele (homozygous or heterozygous) is linked more to patients with infertility and with a more severe clinical picture. / A endometriose é conceituada como a presença de tecido endometrial ectópico, com histologia e função semelhante ao endométrio normalmente situado. No entanto, a explicação para a implantação do tecido endometrial em determinadas mulheres ainda é desconhecida. O grau do comprometimento da endometriose baseia-se num sistema de pontos proposta pela American Fertility Society (1978), hoje denominada American Society for Reproductive Medicine com base nos achados de laparoscópica. A intenção deste estudo foi determinar a freqüência do polimorfismo do p53, códon 72, em dois grupos de pacientes com endometriose e outros sintomas da doença. O estudo incluiu 38 amostras de sangue periférico de mulheres submetidas à videolaparoscopia com diagnóstico confirmado de endometriose. As pacientes foram divididas em dois grupos de 19 mulheres cada, um com infertilidade e o outro não. O polimorfismo foi avaliado por PCR. Os dados foram submetidos a análise estatística sendo usado o teste de qui-quadrado e Odds Ratio. Dos pacientes que só tiveram dor e/ou sangramento sem infertilidade, 09(47,3 %) eram arginina homozigoto e 10(52,6%) eram prolina homozigoto ou heterozigoto. Das pacientes que apresentaram infertilidade associado à dor e/ou sangramento 12(100%) era prolina homozigoto ou heterozigoto. Das pacientes que queixavam apenas de infertilidade, 05 (71,4%) eram arginina homozigoto e 02 (28,6%) prolina homozigoto ou heterozigoto. Correlacionando intensidade da dor, 04(23,5%) com alelo prolina (homozigoto ou heterozigoto) informaram dor leve e/ou moderada e 20(95,5%) dor intensas. Todas essas diferenças foram estatisticamente significativas (p=0,003). Em intensidade da dor relacionado com a classificação laparoscópica foi encontrado o seguinte: 08(50%) pacientes que tiveram dor leve e/ou moderada tiveram classificação I ou II e 08(36,4%) foram incluídos na classificação III ou IV. Das pacientes com dor intensa, 08(36,4%) foram incluídas na classificação I ou II e 14 (63,6%) em III ou IV. Conclui-se que a presença do alelo prolina (homozigoto ou heterozigoto) está mais relacionado com pacientes com endometriose e com quadro clínico mais severo da doença.

This thesis aims to address issues relating to genome architecture and base composition. The first part of this thesis addresses questions relating to codon usage. Initially I will investigate thousands of bacterial species using a detailed analysis of strengths of selection acting upon codons usage while also investigating patterns of optimal codon changes with respect to genomic base composition and tRNA abundance. I report that selection on codon usage increases throughout the length of highly expressed genes, in particular, the first quarter of genes have significantly lower selection. Further, it is clear that factors affecting genomic base composition can eventually lead to changes in optimal codons if the change in base composition is strong enough, however these patterns differ substantially between amino acids. The debate over translational efficiency vs. accuracy was addressed by comparing sites of differing conservation. Differing conservation were defined using a phylogenetic method, allowing sites to change in their extent of conservation throughout the tree. The results show that translational accuracy acts strongly on the top 10% of conserved sites, however is relatively weak when compared to the efficiency for other sites. Also detected is a reduction in apparent selection on codon usage on the bottom 10% of conserved sites which is likely to be caused by conflicting positive selection on amino acids. Finally, although differences in patterns are observed between amino acids, the general relationship to conservation is similar. As much of the variation in codon usage is determined by variation in base composition, this aspect of base composition is investigated in the second part of the thesis. The observed variation in intragenomic base composition in bacteria was found to be far higher than expected for GC-rich bacteria. The non-core part of the genome contributes to this variation to a greater extent than the core part, suggesting that processes such as AT-rich horizontal gene transfer may be involved. Secondly, base composition is modelled under Brownian motion and as an extension, the Ornstein- Uhlenbeck process, which allows for multiple optima throughout the tree. The model including optima fits the data better than standard Brownian motion or Brownian motion with multiple diffusion coefficients. Finally, I investigate a case where a previous codon usage analysis has been seriously confounded by an unusual genome architecture of abnormal regional base composition in two species of eukaryotic parasites in the genus Theileria. In both species, the background G+C content is 37% at most, out of the four syntenic chromosomes. In many orthologous regions however, T.annulata has a decreased G+C content of 28% while T.parva has an increased G+C content of 41%. Various factors coincide with this remarkable divergence: increased divergence at all types of site, recombination hot spots in T.parva, an increased frequency of tandem repeats and DNA sequence motifs in both species. The evolutionary origins of these unusual patterns will be discussed.

The genetic code is redundant, with most amino acids coded by multiple codons. In many organisms, codon usage is biased towards particular codons. A variety of adaptive and non-adaptive explanations have been proposed to explain these patterns of codon usage bias. Using mechanistic models of protein translation and population genetics, I explore the relative importance of various evolutionary forces in shaping these patterns. This work challenges one of the fundamental assumptions made in over 30 years of research: codons with higher tRNA abundances leads to lower error rates. I show that observed patterns of codon usage are inconsistent with selection for translation accuracy. I also show that almost all the variation in patterns of codon usage in S. cerevisiae can be explained by a model taking into account the effects of mutational biases and selection for efficient ribosome usage. In addition, by sampling suboptimal mRNA secondary structures at various temperatures, I show that melting of ribosomal binding sites in a special class of mRNAs known as RNA thermometers is a more general phenomenon.