Submitted by Milena Dias (milena.dias@ufpe.br) on 2015-03-11T18:37:19Z No. of bitstreams: 2 Dissertaçao Catarina Cavalcanti.pdf: 1896576 bytes, checksum: 7233174a7425eebbde63a3852f48d1b8 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-11T18:37:19Z (GMT). No. of bitstreams: 2 Dissertaçao Catarina Cavalcanti.pdf: 1896576 bytes, checksum: 7233174a7425eebbde63a3852f48d1b8 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune que afeta diversos órgãos e sistemas. Embora os fatores que contribuem para a patogenia da doença não estejam completamente esclarecidos, sabe-se que o LES é um distúrbio multifatorial com influência de fatores genéticos e ambientais e com o envolvimento de células B e T autorreativas contra uma variedade de componentes celulares. Sabendo-se que o gene FYB (Proteína Ligante de Fyn) codifica uma proteína adaptadora que participa da cascata de ativação dos linfócitos T e que uma desordem nesse processo pode influenciar o desenvolvimento da autoimunidade, esse gene foi considerado candidato à susceptibilidade ao LES. Em um estudo anterior realizado pelo nosso grupo, foi observada a associação dos TagSNPs rs6863066 e rs358501, localizados nas regiões 5’UTR e 3’UTR do gene respectivamente, com a susceptibilidade ao LES em uma população de Ribeirão Preto/SP. Uma vez que TagSNPs representam outros SNPs que se encontram em desequilíbrio de ligação, o presente estudo analisou possíveis associações entre o desenvolvimento do LES e SNPs representados pelos TagSNPs rs6863066 e rs358501 na mesma população. Para confirmarmos a associação do desenvolvimento do LES com os TagSNPs rs6863066 e rs358501 e outros TagSNPs, também foi realizado um estudo de replica na população de Pernambuco. Para isso, sequenciamos a região promotora e parte das regiões 5’UTR e 3’UTR do gene FYB no grupo de Ribeirão Preto/SP e genotipamos 7 TagSNPs no grupo amostral de Pernambuco. O grupo de estudo foi composto por 143 pacientes e 184 controles de Ribeirão Preto/SP; e 116 pacientes e 162 controles de Pernambuco. Na população de Ribeirão Preto/SP, além da confirmação da associação com os SNPs rs6863066 e rs358501, também foi observada a associação do SNP rs13188259 G>A no promotor do gene FYB com o desenvolvimento do LES: tanto a presença do alelo A (OR = 1,77, CI = 1,27 – 2,47, p = 0,001), como dos genótipos A/A (OR = 2,6, CI = 1,19 – 5,64, p = 0,003) e G/A (OR = 2,2, CI = 1,37 – 3,52, p = 0,001) mostraram conferir risco ao desenvolvimento do LES. Na população de Pernambuco, nenhuma associação com a susceptibilidade foi observada, entretanto foi encontrada a associação entre o alelo T (OR = 3,5, CI = 1,13 – 10,83, p = 0,02) e o genótipo T/T (OR = 11,8, CI = 1,1 – 123,3, p = 0,01) do SNP rs6863066 C>T e do alelo G (OR = 3,9, CI = 1,26 – 12,03, p = 0,01) e genótipo G/G (OR = 8,15, CI = 1,3 – 49,5, p = 0,002) do SNP rs2161612 A>G com a ocorrência da Síndrome Antifosfolipídeo. Também foram observadas a associação entre o alelo G (OR = 2,4, CI = 1,28 – 4,6, p = 0,005) e o genótipo G/G (OR = 5,22, CI = 1,6 – 17,6, p = 0,004) do SNP rs2161612 A>G e o desenvolvimento da serosite e a associação entre o alelo A (OR = 4,4, CI = 1,22 – 15,96, p = 0,01) e o genótipo G/A (OR = 4,89, CI = 1,3 – 18,6, p = 0,01) do SNP rs1642515 G>A, o alelo T (OR = 2,14, CI = 1,1 – 4,2, p = 0,03) e o genótipo T/T (OR = 5,3, CI = 1,42 – 19,8, p = 0,006) do SNP rs404122 A>T e o alelo C (OR = 2,4, CI = 1,2 – 4,9, p = 0,01) e o genótipo C/C (OR = 8,6, CI = 1,8 – 40,9, p = 0,002) do SNP rs358501 T>C com a ocorrência de úlceras nos pacientes com LES. Esses resultados sugerem um possível envolvimento do gene FYB no desenvolvimento do LES e suas manifestações clínicas e laboratoriais, contribuindo para uma maior compreensão da patogênese do LES.

Les syndromes myélodysplasiques (SMD) sont des hémopathies myéloïdes clonales évoluant vers une leucémie aiguë (LA). Les SMD et LA secondaires, survenant après traitement par chimiothérapie et/ou radiothérapie, ont un pronostic très péjoratif. Cependant seule une partie des sujets exposés aux traitements cytotoxiques développent un SMD secondaire, ce qui suggère une composante génétique dans la susceptibilité individuelle au risque de développer un SMD secondaire. Les objectifs de ce travail ont été d’identifier des polymorphismes génétiques de type SNP (Single Nucleotide Polymorphism) significativement associés à des caractères cliniques et biologiques des SMD tel leur caractère secondaire. Une « puce » à façon a sélectionné 384 SNP de fréquence allélique supérieure à 10 % impliqués dans la réparation de l’ADN, le métabolisme, le transport et la détoxication des xénobiotiques. L’ADN constitutionnel de 65 patients atteints de SMD primaire et secondaire a été recueilli et génotypé pour ces 384 SNP. La seule association significative par test exact de Fisher (p = 0,009 après correction de Benjamini-Hochberg) a été observée pour le caractère secondaire des SMD et la présence de l’allèle variant de MGMT (MéthylGuanine MéthylTransférase) sur deux SNP en déséquilibre de liaison, rs2308321 (Ile143Val) et rs2308327 (Lys178Arg). Nous avons recherché le caractère prédictif de la présence de l’allèle variant de MGMT pour le risque de SMD/LA secondaire chez des patientes ayant reçu un traitement cytotoxique pour un cancer du sein, et ayant développé une LA secondaire. Enfin, nous avons construit des lignées cellulaires stables, isogéniques, exprimant soit la forme sauvage soit la forme variante de MGMT. Les études fonctionnelles par tests de cytotoxicité, co-cultures à long terme et étude des demi-vies des protéines, sous traitement alkylant, montrent respectivement des différences de sensibilité, de prolifération ou de dégradation entre les formes variante et sauvage de MGMT. / Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders evolving toward acute myeloid leukaemia (AML). Therapy-related MDS and AML occur after chemo- and/or radiotherapy for previous cancer and have a very poor outcome. However, only a minimal proportion of patients exposed to anticancer drugs develop secondary MDS, suggesting a genetic component in individual susceptibility. The aim of our study was to identify gene polymorphisms significantly associated with MDS in patients. We have selected 384 SNPs (single nucleotide polymorphisms) with allele frequency >10% in genes involved in DNA repair and drug metabolism and transport. DNA extracts were obtained from blood and cheek samples from a population of 65 MDS patients, and the 384 SNPs were genotyped. We analysed the associations existing between each genotype and several pathological features, especially the treatment-related character of MDS. The Fisher exact test with Benjamini-Hochberg correction for multiple testing was applied for statistical analysis. The only significant association (p = 0.009 after correction) was observed for the treatment-related character of MDS and the presence of a variant allele in MGMT (methylguanine methyltransferase, a gene involved in DNA repair), characterised by two SNPs in complete linkage disequilibrium: rs2308321 (Ile143Val) and rs2308327 (Lys178Arg). An epidemiological study was performed to assess the predictive value of the variant allele in MGMT for the development of secondary acute leukaemia among patients treated for breast cancer. We have constructed isogenic stable cell lines expressing either the wild-type or the variant allele of MGMT. Functional studies (analysis of response to alkylating agents, allele quantification of mixed cultures of wild-type and variant cells and half-lives study of the proteins) show differences in sensitivity to DNA-damaging agents, proliferative capacity and MGMT rate of degradation between the wild-type and the variant MGMT.

Single Nucleotide Polymorphisms (SNPs) are the most frequent genomic variations and the main basis for genetic differences among individuals and many diseases. As genotyping millions of SNPs at once is now possible with the microarrays and advanced sequencing technologies, SNPs are becoming more popular as genomic biomarkers. Like other high-throughput research techniques, genome wide association studies (GWAS) of SNPs usually hit a bottleneck after statistical analysis of significantly associated SNPs, as there is no standardized approach to prioritize SNPs or to select representative SNPs that show association with the conditions under study. In this study, a java based integrated system that makes use of major public databases to prioritize SNPs according to their biological relevance and statistical significance has been constructed. The Analytic Hierarchy Process, has been utilized for objective prioritization of SNPs and a new emerging methodology for second-wave analysis of genes and pathways related to disease associated SNPs based on a combined p-value approach is applied into the prioritization scheme. Using the subset of SNPs that is most representative of all SNPs associated with the diseases reduces the required computational power for analysis and decreases cost of following association and biomarker discovery studies. In addition to the proposed prioritization system, we have developed a novel feature selection method based on Simulated Annealing (SA) for representative SNP selection. The validity and accuracy of developed model has been tested on real life case control data set and produced biologically meaningful results. The integrated desktop application developed in our study will facilitate reliable identification of SNPs that are involved in the etiology of complex diseases, ultimately supporting timely identification of genomic disease biomarkers, and development of personalized medicine approaches and targeted drug discoveries.

The aim was to develop new methods for measuring texture of Atlantic salmon (Salmo salar L.) fillets and investigate the genetic basis of flesh quality traits. Firstly, a new tensile strength method was developed to quantify the force required to tear a standardized block of salmon muscle with the aim of identifying those samples more prone to factory downgrading as a result of gaping. The repeatability, sensitivity and predictability of the new technique was evaluated against other common instrumental texture measurement methods. Data from the new method were shown to have the strongest correlations with gaping severity r=-0.514, P<0.001) and the highest level of repeatability of data when analysing cold-smoked samples. The Warner Bratzler shear method gave the most repeatable data from fresh samples and had the highest correlations between fresh and smoked product from the same fish (r=0.811, P<0.001). It is therefore recommended that the new method be adopted for measuring gaping potential and the Warner Bratzler method become the standard for firmness assessment. Genes associated with post mortem softening in mammals were characterised in Atlantic salmon. A previously unknown ancient paralogue of calpastatin (here named CAST2) was identified. Evidence was provided for the existence of highly homologous recent paralogues of CAST2 and CTSD1. Evidence for the ancestral history of these paralogues was provided by phylogenetic analysis. Recent gene duplicates of 6 further genes were identified. In all cases, homology between recent paralogues was greater than 94%. Analysis of synonymous vs non-synonymous nucleotide substitution between the observed paralogue pairs shows a significant purifying selection in most cases. The CTSD1 gene shows significant purifying selection in a pairwise analysis between 12 teleost species (all cases P<0.0001) but a similar analysis of CTSD2 revealed no significant occurrence of purifying selection. The present study provides further support for the idea of asymmetrical selective pressure on paralogues. Genetic markers were developed that can distinguish individuals with above average fillet yield and texture. A database of firmness, tensile strength and fillet yield was made from 254 individuals from 5 batches of farmed salmon and these fish were genotyped at 7 novel SNP loci. Individuals with the combined favourable genotype at CAPN1a and MYOD1b were associated with an average increase in fillet yield of 2.7% above batch average. A combined genotype of CAPN1a, MYOD1b and MYF5 was significantly associated with an average increase in tensile strength of 9.8% above batch average (P=0.015). In both cases individuals with the combined favourable genotype occurred with a frequency of c. 6% across all batches. The favourable genotypes had no unfavourable effects on other traits. Highly polymorphic microsatellite loci were used to perform tests of assignment, which revealed an overall correct assignment rate of 92.7% to batch of origin and a minimum reference sample number of 25 was empirically determined. A phylogenetic analysis supported the results of the assignment tests. Given that 7 microsatellites is a relatively small number for a study of this nature, these results suggest that reliable assignment of unknown fish to the true batch of origin is potentially rapid and cost effective. Overall, the thesis presents molecular markers for broodstock selection, new genes of relevance to flesh quality, a new method of texture analysis and a proposal for an escapee traceability project.

Low density lipoprotein receptor (LDLR) is an apolipoprotein E (apoE) receptor and may play a role in Alzheimer’s disease (AD) development. A single nucleotide polymorphism (SNP), rs688, that has been identified to modulate the splicing efficiency of LDLR exon 12 and is associated with higher cholesterol and AD in some case-control populations. The exon 12 deleted mRNA is predicted to produce a soluble form of LDLR that fails to mediate apoE uptake. To gain additional insights, in this study, I seek to understand the regulation of LDLR splicing efficiency. To identify functional cis-elements within LDLR exon 12, I mutated several conserved putative exonic splicing enhancers (ESEs) to neutralize their affinity to serine/arginine-rich (SR) proteins. Transfection of wild type (WT) or mutant LDLR minigenes in HepG2 cells was performed, and splicing efficiency evaluated by quantitative RT-PCR. The results showed that two functional ESEs within exon 12, near rs688, are critical to LDLR splicing. To identify splicing factors that modulate exon 12 splicing, I co-transfected an LDLR minigene and vectors encoding different SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). After quantifying the splicing efficiency, I found that SRp20 and SRp38 increased exon 11- skipping. Moreover, ectopic expression of SRp38-2 and hnRNP G increased exon 11&12-skipping. Interestingly, the actions of hnRNP G did not require its RNA recognition motif (RRM). To further investigate the role of theses splicing factors on LDLR splicing, I quantified the expression level of these splicing factors as well as LDLR splicing efficiency in human brain and liver. I found that SRp38 mRNA expression is associated with LDLR splicing efficiency. In conclusion, this study discovered that rs688 is located close to the two functional ESEs within LDLR exon 12, and revealed a role of SRp38 in LDLR splicing efficiency.

The demographic history of human populations, both archaic and modern, have been the focus of extensive research. Earlier studies were based on a small number of genetic markers but technological advances have made possible the examination of data at the genome scale to answer important questions regarding the history of our species. A widely used application of single nucleotide polymorphisms (SNPs) are genotyping arrays that allow the study of several hundred thousand of these sites at the same time. However, most of the SNPs present in commercial genotyping arrays have often been discovered by sampling a small number of chromosomes from a group of selected populations. This form of non-random discovery skews patterns of nucleotide diversity and can affect population genetic inferences. Although different methods have been proposed to take into account this ascertainment bias, the challenge remains because the exact discovery protocols are not known for most of the commercial arrays. In this dissertation, I propose a demographic inference pipeline that explicitly models the underlying SNP discovery and I implement this methodology in specific examples of admixture in human populations when only SNP array data are available. In the first chapter, I describe the developed pipeline and applied it to a known example of recent population admixture in Mexico. The inferred time of admixture between Iberian and Native American populations that gave rise to admixed Mexicans was in line with historical records, as opposed to previous published underestimates. Next, I examined different demographic models on the first human settlement in Easter Island and determined that the island of Mangareva is the most likely point of origin for this migration. Finally, I investigated the dynamics of the admixture process between the ancestral Jomon and Yayoi populations in different locations across Japan. The estimates of the time of this encounter were closer to dates inferred from anthropological data, in contrast with past works. The results show that the proposed framework corrects ascertainment bias to improve inference in cases when only SNP chip data are available, and for genotype data originated from different platforms.

Lowering plasma LDL-cholesterol (LDL-C) and increasing HDL-cholesterol (HDL-C) concentrations remain the primary targets in cardiovascular disease (CVD) risk reduction. Dietary cholesterol and plant sterols differentially modulate cholesterol kinetics and lipoprotein distribution. Inter-individual variations in the rates of cholesterol absorption and synthesis, and the reciprocal interaction between them affect the responses to dietary sterols. Genetic heterogeneity profoundly influences such responsiveness. However, limited research exists on the genetic determinants of dietary cholesterol versus plant sterols responsiveness in healthy individuals, especially in a founder population, such as the Hutterites in Manitoba of European descent who practice a communal living system. Our study examined the differential effects of dietary cholesterol versus plant sterol consumption on plasma lipoprotein levels, subclasses, and cholesterol kinetics and assessed how genetic variants influenced these responses. A double-blind, randomized, crossover study with three interventional periods of 4 wk duration each was conducted. Healthy Hutterite individuals (n=49) from Manitoba consumed daily either 2 g of plant sterols or 600 mg of cholesterol incorporated into milkshakes, or a placebo during each period. Plasma lipid profile and lipoprotein subclass distribution were determined. Cholesterol absorption and synthesis were assessed by stable isotopic tracer techniques. Participants were genotyped for 38 candidate single nucleotide polymorphisms across 25 genes involved in cholesterol and lipoprotein metabolism. Dietary cholesterol consumption increased plasma TC, HDL-C concentrations and large HDL subclasses with no changes in cholesterol absorption or synthesis. In contrast, plant sterol intake failed to reduce LDL-C concentrations, with a modest reduction in cholesterol absorption, and did not affect lipoprotein subclasses. However, a large non-compensatory increase in cholesterol synthesis was observed due to plant sterol consumption. Gender and common genetic variants affected plasma HDL-C and HDL subclass distribution to dietary cholesterol and plant sterol consumption. ACAT2 and NPC1L1 gene variants affected plasma campesterol and β-sitosterol concentrations respectively, to plant sterol intake by modifying cholesterol absorption. In summary, our results demonstrate that dietary cholesterol and plant sterol intake differentially modulate cholesterol trafficking in a manner dependent on common genetic variants and gender in healthy individuals. Such knowledge facilitates the development of effective cholesterol lowering strategies for the alleviation of CVD burden. / October 2016

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Regulation of pre-mRNA splicing is a key process for most if not all eukaryotes. The process can, in the abstract, be considered as a series of trans-acting factors that interact with cis-motifs in the RNA to enable the removal of introns and joining of exons. As the cis factors need not only be the splice sites themselves, but also motifs in the exons, the splicing process has the potential to impose selective constraint on exonic sequence in addition to the normal selection on the amino acid content of the protein. To understand this more clearly, in this thesis, I mainly focus on a type of important and widely investigated cis-motifs, exonic splicing enhancers (ESEs), which bind with SR proteins to re-enforce the splice sites and so ensure splicing correctly. First, I explore splice-related cis-motif usage of the Ectocarpus genome, which is a species phylogenetically very distant from vertebrates but, like vertebrates in having abundant large introns. A deep phylogenetic conservation of exonic splice-related constraints is observed (Chapter II). Then I extend the analysis across taxa in a phylogenetically explicit framework. In this section stronger selection on exon end synonymous sites can be detected within humans when the exons are flanked by larger introns. Additionally I report evidence that reduced Ne might lead to larger introns and weakened splice sites. Thus I suggest an unusual circumstance in which selection (for cis-motifs to control error-prone splicing) might be stronger when population sizes are smaller; this is unexpected and would be a necessary complement to nearly-neutral theory (Chapter III). Third, I ask whether what we know about biases in the usage of ESEs and splicing control elements allows us to understand where in human genes pathogenic mutations tend to occur (Chapter IV). By examining the relationship between determinants of the usage of splice-associated cis-motifs and the distribution of human pathogenic SNPs, I found certain exons are vulnerable to splice disruption owing to low ESE density and a “fragile” exon model we proposed could describe and explain this phenomenon (Chapter IV). Finally I perform preliminary analysis, with a view to biotechnological optimization of transgenes, to address whether there might be such a thing as a tissue specific ESE. To this end I examine ESE usage in tissue specific genes. I find some preliminary evidence for tissue specific biased usage of certain ESEs.

Heritable retinal degenerations dramatically affect individuals across the lifespan. Heritable degenerations with onset in childhood or young adulthood, such as the ABCA4- associated maculopathies, generally obey Mendelian segregation and are attributable to mutations within a single gene. Retinal degenerations with onset in late adulthood, such as age-related macular degeneration, are usually influenced by a complex constellation of genetic and environmental factors. This thesis applies several complementary, high-throughput genotyping platforms to identify relationships between specific heritable retinal phenotypes and genetic variation. This findings of this thesis will aid in the development of guidelines for inclusion in retinal gene therapy trials and help physicians refine their prognoses based on genetic information.