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Introduction Asthma is a common and heterogeneous disease in primary care. Asthma phenotypes are recognisable clusters of for example clinical characteristics. Current asthma symptoms and previous exacerbations are used to assess the level of asthma control. Asthma control is used clinically to plan follow-up strategies. Aim The aim of this study is to examine if an data-driven algorithm based on sex and age of onset can categorize an asthma population at a primary care center into three phenotypes with different risk of disease. To investigate if the results can be generalized by comparing to an epidemiological survey in Sweden. Secondary aims are to investigate if these phenotypes predict the level of follow-up and which factors influence asthma control. Methods In this cross-sectional study, 335 participants from one primary care site and 1442 participants from an epidemiological study were compared on sex, age, medical treatment, respiratory allergy, smoking, asthma symptoms and exacerbations. Logistic regression analyses focusing on factors affecting asthma control were performed in a consolidated dataset. Results An adult asthma population can easily be categorized according to the data-driven algorithm. However, these phenotypes do not predict follow-up strategies. Clinical follow-up based on level of asthma control, did not differ between the phenotypes. There were statistically significant differences between the phenotypes regarding respiratory allergy and smoking. In the logistic regression, smoking has the highest odds for poor asthma control. Conclusion The clinical use of the data-driven phenotypes were limited. Follow-up strategies are probably best based on traditional clinical outcomes like asthma control.

DNA microarray has become a powerful tool in genetics, molecular biology, and biomedical research. DNA microarray can be used for measuring the genotypes, structural changes, and gene expressions of human genomes. Detection and characterization of multilevel, high-throughput microarray genomic data pose new challenges to statistical pattern recognition and machine learning research. In this dissertation, we propose novel computational methods for analyzing DNA copy number changes and learning the trees of phenotypes using DNA microarray data. DNA copy number change is an important form of structural variations in human genomes. The copy number signals measured by high-density DNA microarrays usually have low signal-to-noise ratios and complex patterns due to inhomogeneous composition of tissue samples. We propose a robust detection method for extracting copy number changes in a single signal profile and consensus copy number changes in the signal profiles of a population. We adapt a solution-path algorithm to efficiently solve the optimization problems associated with the proposed method. We tested the proposed method on both simulation and real CGH and SNP microarray datasets, and observed competitively improved performance as compared to several widely-adopted copy number change detection methods. We also propose a chromosome instability measure to summarize the extracted copy number changes for assessing chromosomal instabilities of tumor genomes. The proposed measure demonstrates distinct patterns between different subtypes of ovarian serous carcinomas and normal samples. Among active research on complex human diseases using genomic data, little effort and progress have been made in discovering the relational structural information embedded in the molecular data. We propose two stability analysis based methods to learn stable and highly resolved trees of phenotypes using microarray gene expression data of heterogeneous diseases. In the first method, we use a hierarchical, divisive visualization approach to explore the tree of phenotypes and a leave-one-out cross validation to select stable tree structures. In the second method, we propose a node bandwidth constraint to construct stable trees that can balance the descriptive power and reproducibility of tree structures. Using a top-down merging procedure, we modify the binary tree structures learned by hierarchical group clustering methods to achieve a given node bandwidth. We use a bootstrap based stability analysis to select stable tree structures under different node bandwidth constraints. The experimental results on two microarray gene expression datasets of human diseases show that the proposed methods can discover stable trees of phenotypes that reveal the relationships between multiple diseases with biological plausibility. / Ph. D.

Les dystrophies héréditaires vitelliformes de transmission autosomique dominante représentent la 2ème cause de maculopathie après la maladie de Stargardt, maladie récessive monogénique (ABCA4). BEST1 et PRPH2 sont les deux gènes connus associés aux dépôts vitellins. L'étude d'une large cohorte de 88 patients ayant une dystrophie vitelliforme juvénile ou de l'adulte avec un criblage systématique des deux gènes BEST1 et PRPH2 nous a permis d'établir des recommandations en fonction des trois critères : l'âge, l'histoire familiale et le rapport d'Arden. Nous avons ensuite recherché de larges réarrangements (délétions, insertions) dans les familles négatives par MLPA. Deux cas de délétion exonique ont été retrouvés (délétion de l'exon 4 du gène BEST1, délétion de l'exon 2 du gène PRPH2). L'étude de l'exome d'une grande famille (3 générations, 10 sujets atteints) n'ayant pas de mutations exoniques ou de réarrangements, a permis de démontrer l'implication du gène IMPG1 qui code pour une glycoprotéine de la matrice interphotoréceptrice. La même mutation faux-sens hétérozygote a été retrouvée dans deux autres familles. Nous avons ensuite testé son paralogue IMPG2 qui code également une protéine de la matrice interphotoréceptrice. Une seule famille avec une forme modérée de dystrophie vitelliforme a une mutation faux-sens hétérozygote dans ce second gène. IMPG1 et IMPG2, deux gènes de la matrice interphotoréceptrice sont désormais à ajouter à la liste des gènes des dystrophies vitelliformes après BEST1 le gène majeur et PRPH2. / Vitelliform dystrophies represent the second cause of inherited macular dystrophies after Stargardt disease (monogenic disease linked to ABCA4). To date, BEST1 and PRPH2 are the only known genes involved in vitellin deposits. Considering a large cohort of 88 unrelated patients with juvenile or adult form of vitelliform dystrophy and after a systematic screening of both genes, we propose a rational for BEST1 and PRPH2 analysis according to age of onset, positive family history and Arden ratio. The second step was to consider large deletions or insertions in these genes in patients negative for BEST1 and PRPH2. Exonic deletions are rare: one exon 4 deletion of BEST1 and one exon 2 deletion of PRPH2. Whole exome sequencing in a large family (3 generations, 10 affected patients) revealed a hetezogygous missense variation in IMPG1 an interphotoreceptor matrix gene. IMPG1 was the causal gene in two additionnal families. In the same way, its paralog IMPG2 have been tested : only one family with an heterozygous missense mutation was found. IMPG1 and IMPG2 are two new genes involved in vitelliform dystrophies after BEST1 the main gene and PRPH2.

Ensaios clínicos indicam haver diferenças regionais na eficácia do tratamento da doença de Chagas com (BZ) e Nifurtimox (NFX). Transportadores ABC desempenham um papel importante na resistência a drogas. Evidências indicam que o transportador TcABCG1 é superexpresso em cepas resistentes a BZ. O objetivo geral foi obter evidências para o envolvimento de TcABCG1 no fenótipo de resistência a BZ. Determinamos a classificação de seis cepas em suscetíveis e resistentes a BZ, e o efeito de três inibidores de transportadores ABC de eucariotos. Transfectamos em CL Brener os genes TcABCG1 de cepas resistentes. Nas culturas transfectadas com os genes de Silvio e YuYu verificamos aumento da resistência a BZ e a NFX, e aumento da abundância relativa de transcritos de TcABCG1. Genes ABC ortólogos aos de T. cruzi foram identificados em tripanossomas africanos e Leishmania spp. Dados referentes à análise filogenética e classificação dos transportadores ABC de T. cruzi nas oito subfamílias das proteínas ABC de eucariotos são apresentados. / Clinical trials indicate regional differences in the efficacy of Chagas disease treatment with (BZ) and nifurtimox (NFX). ABC transporters play an important role in drug resistance. Evidences indicate that TcABCG1 is overexpressed in BZ-resistant strains. The overall aim was to obtain evidence for the involvement of TcABCG1 in the BZ resistance phenotype. We determined the classification of six BZ susceptible and resistant strains and the effect of three eukaryotic ABC transporters inhibitors. We transfected TcABCG1 genes from resistant strains in CL Brener. In cultures transfected with Silvio and YuYu genes we observed an increased resistance to BZ and NFX, and increased relative abundance for TcABCG1 transcripts. ABC orthologue genes to those of T. cruzi were identified in African trypanosomes and Leishmania spp. Data relating to classification and phylogenetic analysis of T. cruzi ABC transporters in eight eukaryotic ABC proteins subfamilies are presented.

Introdução: Os mecanismos fisiopatológicos da asma grave resistente ao tratamento (STRA) em crianças não está totalmente elucidado e parece diferir do observado em adultos, justificando investigações específicas neste grupo de pacientes. O escarro induzido é método útil para identificar fenótipos e endotipos de asma grave através de marcadores inflamatórios. O objetivo deste estudo foi investigar os padrões inflamatórios de crianças com STRA através escarro induzido e comparar com um grupo de crianças com asma grave que atingiram o controle. Métodos: Crianças (6-18 anos) com diagnóstico de asma grave (critério GINA) em tratamento a pelo menos 6 meses em um centro de referência foram avaliadas em um coorte prospectivo por 3 meses (3 visitas consecutivas). Foi averiguada técnica inalatória, adesão ao tratamento e investigado as principais comorbidades. Realizado coleta de escarro induzido para análise citológica e avaliação quantitativa de citocinas do sobrenadante, espirometria, pletismografia e medidas da FeNO. Após período de seguimento, os pacientes foram classificados em dois grupos: asma grave controlada e asma grave resistente ao tratamento conforme critérios da ATS/ERS. Resultados: Foram incluídos 40 pacientes (idade média 12,8 anos; 62,5% sexo masculino), sendo 13 (32,5%) classificados como STRA após o período de seguimento. A mediana do número de exacerbações foi maior e do escore de ACT menor nos pacientes STRA e esta diferença foi significativa. Não foram encontradas diferenças significativas: nos dados demográficos, nos parâmetros funcionais espirométricos e de pletismografia (CVF, VEF1, VEF/CV, FEF 25-75%, LTC, RV, RV/LTC, resistência e condutância das vias aéreas) e nos valores de FeNO quando comparado o grupo de pacientes controlados com o de STRA. O padrão inflamatório eosinofílico foi predominante nos dois grupos de pacientes, entretanto, o grupo STRA apresentou porcentagem proporcionalmente maior de neutrófilos no escarro comparados com o grupo de asma grave controlada, na visita 3 e também na visita 1 quando analisados retrospectivamente (p < 0,05). As medianas nos níveis das citocinas IL10, GM-CSF, INFy e TNFalfa no escarro foram significativamente maiores no grupo STRA quando comparado ao grupo controlado (p < 0,05) e o GM-CSF e TNF-alfa apresentaram correlação inversa com escore de ACT. Conclusão: Nesta coorte prospectiva, os parâmetros funcionais e a FeNO não discriminaram crianças com STRA dos que atingiram o controle. A presença de neutrófilos no escarro e das citocinas IL10, INFy e, particularmente, GM-CSF e TNFalfa podem ter para um papel na resistência ao tratamento da asma grave em crianças e adolescentes. Antagonistas específicos dessas citocinas podem no futuro representar uma estratégia na terapêutica / Background: The pathophysiological mechanisms of severe therapyresistant asthma (STRA) in children are not fully elucidated and seem to differ from findings in adults, thus justifying specific research on children. Induced sputum is useful for detecting phenotypes and endotypes of severe asthma via inflammatory markers. The aim of the present study was to investigate the inflammatory patterns of children with STRA by the induced sputum method and to compare them with a group of children who achieved control of severe asthma. Methods: A prospective cohort of children (6-18 years old) diagnosed with severe asthma (Global Initiative for Asthma - GINA criteria) and in treatment for at least 6 months at a reference center was assessed for 3 months (3 consecutive visits). Inhalation technique, adherence to treatment and main comorbidities were assessed. Induced sputum samples were collected for cytology analysis and quantitative assessment of cytokines in the supernatant; the participants were also subjected to spirometry, plethysmography and fractional exhaled nitric oxide (FeNO) measurements. At the end of follow-up, the patients were classified into two groups: controlled severe asthma and STRA according to the European Respiratory Society and American Thoracic Society (ERS/ATS) criteria. Results: Forty patients were included (average age 12.8 years old; 62.5% male); 13 (32.5%) were classified as STRA at the end of follow up. The median number of exacerbations was higher and the Asthma Control Test (ACT) score was lower in the STRA group; these differences were significant. Significant differences were not found relative to demographic data, spirometry and plethysmography function parameters [forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), ratio of FEV1 to slow vital capacity (FEV1/SCV), forced expiratory flow at 25-75% of FVC (FEF 25-75%), total lung capacity (TLC), residual volume (RV), RV/TLC, airway resistance and conductance] and FeNO after comparison of the STRA and controlled asthma groups. The eosinophilic inflammatory pattern predominated in both groups; however, the STRA group showed a proportionally higher percentage of sputum neutrophils compared with the controlled asthma group at visit 3 and visit 1 upon retrospective analysis (p<0.05). The median sputum levels of the cytokines IL-10, GM-CSF, IFN-y and TNF-alpha were significantly higher in the STRA group compared with the controlled asthma group (p < 0.05); GM-CSF and TNF-? showed inverse correlations with ACT scores. Conclusion: In the analyzed prospective cohort, functional parameters and FeNO did not discriminate between children with STRA and children with controlled asthma. The presence of neutrophils and the cytokines IL-10, IFN-y and, more particularly, TNF-alpha and GM-CSF in the sputum might have a role in resistance to treatment for severe asthma among children and adolescents. Antagonists specific for these cytokines might represent a therapeutic strategy in the future

Les maladies cardiovasculaires (MCV) sont d’une étiologie complexe et elles sont soumises à de nombreux facteurs environnementaux ainsi que génétiques. Malgré les succès obtenus, pendant la dernière décennie, et pour réduire la mortalité CV il est nécessaire l’identification de nouveaux biomarqueurs en utilisant des approches différentes. Cette thèse propose une approche intégrative pour découvrir de nouvelles associations génétiques associés avec les MCV. Nous avons d’abord réuni les résultats existants grâce à des GWAS précédents, puis nous avons recherché la pléiotropie de ces gènes et nous avons dirigé nos efforts vers une possible traduction des résultats obtenus dans l’application clinique. Nous avons détecté les effets pléiotropiques de différent gènes (IL-6R et ABO) avec différents phénotypes lipidiques et inflammatoires. Par ailleurs, nous avons trouvé quelques associations gène-genre intéressantes pour certains gènes étudiés (ABO et GNB3). Concernant l’implémentation clinique des connaissances obtenues par cette thèse, une SNP dans le gène TREM-1, pourrait être utilisé comme un marqueur de risque pour différentes maladies, et nous avons déposé un brevet Européen et nous envisageons de mener des essais cliniques de chez les patients. D’autre part, nous avons détecté une haplotype du gène IL6R qui pourrait être utilisés dans la médecine personnalisée. Nos résultats aident à mieux comprendre comment les gènes étudiés exercent leurs effets au niveau moléculaire, en influant finalement sur l’état des patients souffrant de MCV. Nous espérons que nos résultats vont être pris en compte pour faire progresser la médecine personnalisée / Cardiovascular diseases (CVD) are complex diseases where many environmental and genetic factors are involved. Although the genetic aetiology of the CVD has been extensively investigated the last two decades, alternative approaches are needed in order to keep advancing in the pathophysiology of CVD. In this thesis, we propose an integrative approach to discover new genetic associations potentially involved in CVD. We chose previous GWAS hits and we centred our efforts in studying the pleiotropic and gene-gender interaction effects. Finally, we focused on the implementation of personalized genome-based therapy of the results obtained. New pleiotropic effects were discovered in the IL-6R and ABO genes relating them with different inflammatory and lipid phenotypes. In addition, we studied the gene-gender interaction effects, finding some sex-specific associations in two of the genes studied (ABO and GNB3). Further, we centered our efforts in implementing the results obtained during the thesis at the clinical level. One SNP within the TREM-1 gene was associated with increased levels of its protein and could be used as a predictor or risk biomarker for different diseases. Due to the high potential of this SNP, we applied a European patent and we are planning to start clinical trials in patients. Also, one haplotype in the IL-6R gene could be used in the treatment of personalized medicine. During this thesis, we discovered new gene-phenotype associations involved in CVD and other diseases. Our results help to better understand how the studied genes are exerting their effects at the molecular level. Our results will hopefully be taken into account in future personalized treatments

A clonagem bovina por transferência nuclear de células somáticas (SCNT) é uma biotécnica ainda pouco eficiente devido aos elevados custos em sua aplicação e às altas perdas gestacionais oriundas de alterações do desenvolvimento embrionário e placentário. Desse modo, o uso comercial da clonagem bovina por SCNT é pouco viável atualmente, sendo aplicada majoritariamente em pesquisa. Nesse contexto, o presente trabalho objetivou estudar a eficiência de um programa de clonagem por SCNT, identificando pontos críticos no desenvolvimento do concepto, assim como detectar diferentes apresentações gestacionais induzidas por esta biotécnica. Para isso, 215 embriões clonados foram transferidos para receptoras de embriões e 30 prenhezes foram confirmadas pela detecção de batimentos cardíacos no concepto por ultrassonografia transretal, totalizando 14.0% de índice de gestação. Em 6.1% das receptoras foi identificado por ultrassonografia sinais das vesículas embrionárias sem presença de batimento cardíaco (gestações anembrionadas) e em 7.4% das receptoras não foram detectados sinais do concepto embora o CL tenha sido mantido por mais de 25 dias pós-ovulação (CLs persistentes). Este estudo mostrou que os maiores índices de perdas gestacionais ocorreram antes do dia 25 (dia 0 = ovulação). Ao analisar as perdas gestacionais após o diagnóstico de gestação (dia 25 a 35), um maior número de perdas ocorreu entre os dias 30 e 40 (26.7%) e entre os dias 40 e 60 (20.0%), não sendo identificada a formação de placentônios nessas prenhezes. Além disso, as receptoras gestantes que desenvolveram placentônios prolongaram a gestação após esses períodos críticos, sendo detectado um novo aumento da frequência de perdas no terço final da gestação. Sendo assim, o presente estudo confirma a baixa eficiência da técnica de clonagem bovina por SCNT e identifica os períodos críticos do desenvolvimento do concepto, sendo eles o período imediato pós-reconhecimento materno da gestação, anteriormente aos 25 dias, o período de completo desenvolvimento placentário e formação de placentônios, entre os dias 30 e 60, e o período de aumento da exigência funcional da placenta devido ao acelerado desenvolvimento do feto no terço final da gestação. Além disso, pela primeira vez na literatura é relatada a ocorrência das apresentações gestacionais aberrantes, as prenhezes anembrionadas e aquelas com CL persistente após a transferência de embriões clonados por SCNT. Novos estudos são necessários para um melhor entendimento da origem e das causas dessas apresentações, as quais podem ser decorrentes de perdas gestacionais precoces ou mau funcionamento de tecidos do concepto como o trofoectoderma e a massa celular interna. / Cloning by somatic cell nuclear transfer (SCNT) in cattle is still an inefficient biotechnique due to the high costs for its application and also to the high pregnancy losses resulted from changes in embryonic and placental development. Thus, the commercial use of the bovine cloning by SCNT is not commercially feasible, being mainly applied for research interests. In this context, the present study investigated the efficiency of a cloning program by SCNT with the objective of to identify the critical periods for conceptuses development and to detect different gestational presentations induced by this biotechnique. Two hundred fifteen embryos were transferred into embryo recipient cows and 30 pregnancies were confirmed by heart beats detection in the embryo by transrectal ultrasonography, totaling 14.0% of pregnancy rate. 6.1% of the recipients were diagnosed presenting embryonic vesicles without the presence of heart beats (anembryonic gestations) and 7.4% of the recipients did not present any signal of the conceptus and the CL kept active for more than 25 days post-ovulation (persistent CL). This study showed that the higher rates of embryo loss occurred before day 25 (day 0 = ovulation). Analyzing the data of pregnancy losses after diagnosis of pregnancy (days 25 to 35), higher amount of losses occurred between days 30 and 40 (26.7%) and between 40 and 60 (20.0%). Placentomes were not identified in any of these pregnancies. In addition, the recipient in which presented developed placentonios had their pregnancies prolonged. However, a new increase in the frequency of losses was observed in the final three months of gestation. Thus, this study confirms the low efficiency of bovine cloning by SCNT and identified the critical periods for the development of the conceptus, as the period immediately after maternal recognition of pregnancy, before 25 days of pregnancy, the period of full placental development and placentomes development, between days 30 and 60, and the period of placental functional requirements increase due to the rapid development of the fetus, during the last three months of gestation. Also, this work for the first time in the literature reported the occurrence of aberrant gestational presentations, the anembryonic and persistent CL gestations, after embryo transfer of bovine cloned embryos by SCNT. Further studies are necessary to understand the origin and causes of these aberrant gestational presentations, which may be due to early pregnancy loss or malfunction of conceptus tissues as trophoectoderm and inner cell mass.

Ensaios clínicos indicam haver diferenças regionais na eficácia do tratamento da doença de Chagas com (BZ) e Nifurtimox (NFX). Transportadores ABC desempenham um papel importante na resistência a drogas. Evidências indicam que o transportador TcABCG1 é superexpresso em cepas resistentes a BZ. O objetivo geral foi obter evidências para o envolvimento de TcABCG1 no fenótipo de resistência a BZ. Determinamos a classificação de seis cepas em suscetíveis e resistentes a BZ, e o efeito de três inibidores de transportadores ABC de eucariotos. Transfectamos em CL Brener os genes TcABCG1 de cepas resistentes. Nas culturas transfectadas com os genes de Silvio e YuYu verificamos aumento da resistência a BZ e a NFX, e aumento da abundância relativa de transcritos de TcABCG1. Genes ABC ortólogos aos de T. cruzi foram identificados em tripanossomas africanos e Leishmania spp. Dados referentes à análise filogenética e classificação dos transportadores ABC de T. cruzi nas oito subfamílias das proteínas ABC de eucariotos são apresentados. / Clinical trials indicate regional differences in the efficacy of Chagas disease treatment with (BZ) and nifurtimox (NFX). ABC transporters play an important role in drug resistance. Evidences indicate that TcABCG1 is overexpressed in BZ-resistant strains. The overall aim was to obtain evidence for the involvement of TcABCG1 in the BZ resistance phenotype. We determined the classification of six BZ susceptible and resistant strains and the effect of three eukaryotic ABC transporters inhibitors. We transfected TcABCG1 genes from resistant strains in CL Brener. In cultures transfected with Silvio and YuYu genes we observed an increased resistance to BZ and NFX, and increased relative abundance for TcABCG1 transcripts. ABC orthologue genes to those of T. cruzi were identified in African trypanosomes and Leishmania spp. Data relating to classification and phylogenetic analysis of T. cruzi ABC transporters in eight eukaryotic ABC proteins subfamilies are presented.