Introduction: Le dalcétrapib est un inhibiteur de la protéine de transfert des esters de cholestérol (CETP) qui augmente le niveau du cholestérol-HDL. Des études d’association pangénomiques ont révélé une association entre les polymorphismes du gène adénylate cyclase de type 9 (ADCY9) et les réponses au dalcétrapib. Le but de cette étude était d’identifier le polymorphisme nucléotidique (SNP) causal, ce qui pourrait mener à comprendre le mécanisme moléculaire modifiant les effets du dalcétrapib sur les bénéfices cardiovasculaires. Méthodes: Des essais d’EMSA (electrophoretic mobility shift assay) ont été réalisés afin d’analyser les effets modificateurs de douze SNPs candidats sur la liaison de protéines nucléaires, provenant de cellules monocytaires THP-1. Ensuite, des essais de transfections avec un gène rapporteur ont été utilisées pour évaluer l’effet transcriptionnel de ces SNPs. La liaison des protéines au SNP rs12920508 a par la suite été étudiée par des chromatographies d’affinité d’ADN suivies par des spectrométries de masse et par MC-EMSA (multiplexed competitor EMSA). Résultats: Sept sur douze SNPs ont démontré une liaison spécifique à un allèle qui n’a pas été influencée par l’exposition des cellules au dalcétrapib. Le résultat des transfections de vecteurs rapporteurs dans les cellules THP-1 a montré que les constructions plasmidiques portant les variants rs1967309 et rs12920508 augmentaient l’activité transcriptionnelle. Onze protéines ont été identifiées comme des candidats potentiels pouvant se lier à la région du SNP rs12920508. De plus, la région contenant les deux variants rs1967309 et rs12920509 a présenté une activité transcriptionnelle accrue et significativement plus élevée pour l’haplotype délétère. Conclusion: Le polymorphisme rs1967309 semble causer la majorité des effets fonctionnels dans la lignée cellulaire THP-1. Cependant, une interaction avec le SNP rs12920508 ou la présence de la région de ce SNP pourrait être nécessaire pour l’activité optimale de rs1967309. Des travaux supplémentaires sont nécessaires pour élucider le lien entre le SNP potentiellement causal et les réponses cardiovasculaires induites par le dalcétrapib. / Introduction: Dalcetrapib is a cholesteryl ester transfer protein (CETP) inhibitor that increases the circulating level of HDL-cholesterol. Genome-wide association studies have revealed an association between polymorphisms found in the adenylate cyclase type 9 (ADCY9) gene and responses to dalcetrapib, including its cardiovascular benefits. The purpose of this study was to identify the causal single nucleotide polymorphisms (SNP) which could lead to understand the molecular mechanisms altering dalcetrapib effects on cardiovascular outcomes. Methods: Electrophoretic mobility shift assays (EMSA) were performed to analyze the allele-specific effects of the best causal SNP candidates on binding with nuclear proteins obtained from a THP-1 monocytic cell line. Afterwards, a dual luciferase reporter assay was used to assess the effect of selected genetic variants on gene transcription. Protein binding to SNP rs12920508 was investigated by DNA-affinity chromatography followed by mass spectrometry and multiplexed competitor EMSA. Results: Seven out of 12 SNPs demonstrated allele-specific protein binding, which was not influenced by dalcetrapib exposure of the cells. Results from dual luciferase reporter assay showed that plasmid constructs bearing variants rs12920508 and rs1967309 increased transcriptional activity when transfected into THP-1 undifferentiated monocytic cells. Eleven proteins were identified as potential candidates binding to region of SNP rs12920508. Additionally, region containing both SNPs rs1967309 and rs12920508 displayed increased transcriptional activity with significantly higher activity for deleterious haplotype. Conclusion: Polymorphism rs1967309 seems to be causing most functional effects in the THP-1 monocytic cell line. However, an interaction with rs12920508 or presence of the DNA region of this SNP may be necessary for optimal activity of rs1967309. Further work is required to elucidate the link between potentially causal SNPs and cardiovascular responses induced by dalcetrapib.

No description available.

This diploma thesis aims to contribute to the proposed project concerning the update of the multi-media application EMSA (Elektronická mluvnice současné angličtiny, The electronic grammar of contemporary English). This project is based on the text of Mluvnice současné angličtiny na pozadí češtiny [Grammar of contemporary English against the background of Czech] by Professor PhDr. Libuše Dušková, DrSc. et al., which was digitalized and can be accessed at the website www.mluvniceanglictiny.cz. In the future, this application aims to contain supplementary texts, relevant bibliographic information, exercises, authentic examples acquired from electronic corpora, etc. The particular task of this diploma thesis is the critical revision of chapter 8.4 Modal verbs. The first step was the survey of relevant literature and identification of parts which are not explicitly covered by Dušková's original text. These findings are presented in the theoretical part of the thesis (chapter 1.); they are concerned mainly with theoretical definitions (e.g. the delimitation of central and marginal modals or kinds of modality) and information concerning the distribution and recent developments of modal verbs. The second main step was to compile these findings into texts which would be suitable for the use in the project...

Nuclear receptors (NRs) are important transcription factors involved in the regulation of a variety of physiological processes such as embryonic development, cell differentiation and homeostasis (for review, see Mangelsdorf et al., 1995 TenBaum and Baniahrned, 1997). In contrast to membrane bound receptors, they bind small lipophilic ligands and function in the nucleus as ligand-modulated transcription factors. The ligands for nuclear receptors include steroids (glucocorticoids, progestins, mineralocorticoids, androgens and estrogens), vitamin D3, retinoids, thyroid hormone, prostaglandins, farnesoids etc. Several other nuclear receptors are classified as orphan receptors for which no ligand has yet been identified. More than 300 nuclear receptors have now been identified and together these proteins comprise the single largest family of metazoan transcription factors, the nuclear receptor superfamily. Recently, a unified nomenclature has been evolved (nuclear receptor nomenclature committee, 1999), a summary of which is presented in Table 1.

No description available.

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

L'IRES du VHC est organisé en une structure secondaire, complexe et hautement conservée, qui comprend quatre domaines distincts (I-IV). Cette organisation permet la liaison directe du facteur d'initiation eucaryote eIF3 et de la sous-unité 40S du ribosome. Le facteur d'initiation eucaryote eIF3 est un complexe de 13 protéines (~ 800 kDa). Certaines de ses sous-unités montrent une affinité pour l'ARN, et parmi elles deux possèdent des motifs de reconnaissance à l'ARN (MRR) : eIF3b (aa : 185-268) et eIF3g (aa : 239-317). Afin de déterminer l'implication de ces motifs dans l'interaction ARN-protéine, nous avons cloné les domaines contenant ces motifs MRR, ainsi que les protéines entières eIF3b et eIF3g. Nous avons ensuite étudié l'interaction de l'ARN et/ou domaines d'ARN avec le complexe eIF3 et/ou protéines ou domaines protéiques en utilisant la méthode de rétention sur filtre. Nous avons ainsi calculé les différentes constantes apparentes de dissociation entre : eIF3/IRES, eIF3b/IRES et MRR-eIF3b/IRES. Ces résultats montrent que la sous-unité eIF3b se lie directement au domaine III de l'IRES via son motif MRR situé dans le domaine N-terminal. Dans un deuxième temps, nous avons utilisé la spectroscopie RMN pour identifier précisément les acides aminés impliqués dans la reconnaissance de l'ARN viral. En utilisant différentes techniques telles que FBA, RMN et SAXS, il s'avère que le domaine IIId (30 nucléotides) représente le motif minimum impliqué dans l'interaction MRR-eIF3b/IRES. Enfin, la technique de SAXS a été mise à profit pour étudier la structure tridimensionnelle de l'IRES libre en solution.

Genetic variation is a strong risk factor for many human diseases, including diabetes, cancer, cardiovascular disease, depression, autoimmunity and asthma. Most of the disease genes identified so far alter the amino acid sequences of encoded proteins. However, a significant number of genetic variants affecting complex diseases may alter the regulation of gene transcription. The map of the regulatory elements in the human genome is still to a large extent unknown, and it remains a challenge to separate the functional regulatory genetic variations from linked neutral variations. The objective of this thesis was to develop methods for the identification of genetic variation with a potential to affect the transcriptional regulation of human genes, and to analyze potential regulatory polymorphisms in the CD36 glycoprotein, a candidate gene for cardiovascular disease. An in silico tool for the prediction of regulatory polymorphisms in human genes was implemented and is available at www.cisreg.ca/RAVEN. The tool was evaluated using experimentally verified regulatory single nucleotide polymorphisms (SNPs) collected from the scientific literature, and tested in combination with experimental detection of allele specific expression of target genes (allelic imbalance). Regulatory SNPs were shown to be located in evolutionary conserved regions more often than background SNPs, but predicted transcription factor binding sites were unable to enrich for regulatory SNPs unless additional information linking transcription factors with the target genes were available. The in silico tool was applied to the CD36 glycoprotein, a candidate gene for cardiovascular disease. Potential regulatory SNPs in the alternative promoters of this gene were identified and evaluated in vitro and in vivo using a clinical study for coronary artery disease. We observed association to the plasma concentrations of inflammation markers (serum amyloid A protein and C-reactive protein) in myocardial infarction patients, which highlights the need for further analyses of potential regulatory polymorphisms in this gene. Taken together, this thesis describes an in silico approach to identify putative regulatory polymorphisms which can be useful for directing limited laboratory resources to the polymorphisms most likely to have a phenotypic effect.

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

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.