Porphyromonas gingivalis é um dos principais organismos associados à periodontite crônica e apresenta intensa diversidade, que poderia refletir em sua virulência. A maioria dos estudos sobre a virulência de P. gingivalis foi realizada com cepas de referência, e pouco se conhece sobre este aspecto em isolados clínicos. A capacidade de indução de abscessos difusos em modelos animais experimentais parece estar associada a cepas capsuladas, enquanto a expressão de fímbrias e a capacidade de internalização em células epiteliais não fagocíticas, foram relacionadas à cepa não capsulada. Em P. gingivalis, o lócus de biossíntese do polissacarídeo capsular (BPC) apresenta características de ter sido adquirido por transferência horizontal de genes. O objetivo do presente estudo foi testar a hipótese de que a estrutura populacional de P. gingivalis relaciona-se com a variabilidade do lócus BPC e características fenotípicas como produção de cápsula e hidrofobicidade. Foram analisadas 28 cepas de P. gingivalis pertencentes aos 5 genótipos fimA quanto a, presença da cápsula por microscopia óptica, hidrofobicidade e detecção de genes do lócus BPC por PCR. A análise filogenética foi realizada por tipagem através de seqüenciamento de genes housekeeping (multilocus sequence typing, MLST). Dezesseis entre 28 amostras estudadas apresentaram cápsula, e não foram detectadas diferenças na hidrofobicidade dos isolados clínicos capsulados e não capsulados. O gene pg0106 foi detectado por PCR em 78% das amostras capsuladas e em 80% das amostras não capsuladas, enquanto pg0111 foi detectado em apenas 25% de amostras capsuladas. Apenas um isolado clínico e a amostra padrão W83 foram classificados como K1, por apresentarem o gene pg0118. Através do MLST foi possível identificar grande variabilidade entre as amostras de P.gingivalis. Foi observada relação entre STs e o tipo fimA ou hidrofobicidade, mas nenhum cluster foi associado à presença da cápsula ou aos genes do lócus de biossíntese do polissacarídeo capsular. Os dados indicam associação entre o lócus BPC e produção de cápsula, porém a diversidade deste lócus parece ser maior do que a relatada na literatura. O lócus BPC e a produção de cápsula não se relacionam com a origem filogenética da cepa, indicando a intensa recombinação que ocorre em P. gingivalis. / Porphyromonas gingivalis is one of main organisms associated with chronic periodontitis and is largely diverse, which could reflect in its virulence. Most studies on the virulence of P.gingivalis were performed with reference strains, and little is known about this aspect in clinical isolates. The ability to induce diffuse abscesses in experimental animal models seems to be associated with encapsulated strains, while expression of fimbriae and the ability to internalize into non phagocytic epithelial cells were related to noncapsulated strains. In P.gingivalis, the locus of biosynthesis of the capsular polysaccharide (GP) has characteristics of having been acquired by horizontal gene transfer. This study aimed to test the hypothesis that the structure population of P.gingivalis is related to the variability of the GPC locus and phenotypic characteristics such as capsule production and hydrophobicity. 28 P.gingivalis isolates representing fimA genotypes were screened for presence of capsule by light microscopy, hydrophobic properties and detection of genes in the GPC locus by PCR. The phylogenetic analysis was performed by sequencing of housekeeping genes typing (multilocus sequence typing, MLST). Sixteen of 28 studied samples had capsule, and there were no differences in the hydrophobic properties of capsulated and non capsulated clinical isolates. The pg0106 gene was detected by PCR in 78% of capsulated isolates and in 80% of not capsulated while pg011 was detected in only 25% of encapsulated isolates. One clinical isolate and reference strain W83 were classified as K1, due to the presence of pg0118 gene. MLST detected large variations within the P.gingivalis population. MLST clustering analysis revealed a relation between sequence type (STs) and fimA genotype or hydrophobic property, but there was no association of STs with the presence of capsule or the genes encoding for the biosynthesis of capsular polysaccharide. The data indicated an association between GPC locus and capsule production but the diversity of this locus appeared to be greater than that reported in the literature. The GPC locus and capsule production were not related to the phylogenetic origin of the strain, indicating intense recombination in P. gingivalis.

A presente pesquisa teve a finalidade de estudar a hematologia de bezerros clonados da raça Nelore, originados por meio da técnica de transferência nuclear de células somáticas (TNCS) por meio da avaliação do quadro eritrocitário, da dinâmica dos tipos de hemoglobina e do metabolismo do ferro destes animais durante o primeiro mês de vida. O delineamento experimental envolveu a colheita de 260 amostras de sangue e soro sanguíneo de 20 bezerros distribuídas nos seguintes momentos: imediatamente após o nascimento, 12 horas após o nascimento, 1 dia de vida, 2 , 3 , 4, 5, 7, 10, 15, 20 e 30 dias de vida. Os animais foram divididos em quatro grupos experimentais: 12 bezerros obtidos por meio da técnica de TNCS pelos laboratórios A e 8, 4 por meio de fertilização in vitro (FIV) e 4 por monta natural (MN). A ocorrência de anemia de grau moderado a grave, do tipo normocítico e normocrômico, foi observada em 100 % (5/5) dos 5 bezerros clonados pelo Laboratório A, enquanto a incidência nos bezerros clonados pelo Laboratório B foi igual a 14,2 % (1/7), nos bezerros obtidos por fertilização in vitro foi igual 50,0 % (2/4) e em bezerros obtidos por monta natural foi igual a 50,0 % (2/4). A avaliação do eritrograma dos bezerros cio nados pelo Laboratório A evidenciou que a anemia instalou-se gradualmente a partir das 12 horas de vida atingindo ao final da primeira semana, a, sua intensidade máxima, sendo observada a recuperação gradativa dos valores eritrograma a partir do 15°dia de vida. Os valores médios obtidos para o eritrograma dos bezerros clonados do Laboratório A no 7º dia de vida foram os seguintes: Hemácias - 4,33X106/mm3-; Volume Globular - 23 %, Taxa de Hemoglobina - 7,25 g/dL; VCM - 52,89 µ3-; HCM - 16,65 pg; CHCM - 31,47%. A anemia observada nos bezerros clonados pelo Laboratório A era de origem ferropriva, pois se evidenciou nesses animais uma significativa diminuição dos teores séricos de ferro associada à diminuição do índice de saturação da transferrina (1ST), enquanto os valores da capacidade total de ligação do ferro (CTLF) não sofreram influência durante o período. Os valores médios obtidos para o metabolismo de ferro dos bezerros clonados pelo Laboratório A no 7º dia de vida foram os seguintes: teores séricos de ferro - 47,35 mg/dL; capacidade total de ligação de ferro - 455,90 mg/dL, índice de saturação da transferrina - 9,64%. Durante o estudo dos tipos de hemoglobinas, utilizando-se técnica de eletroforese foram identificados três fenótipos de hemoblogina adulta (Hb-A; Hb-B e Hb-AB) e a presença de hemoglobina fetal (Hb-F), não sendo observadas anomalias que pudessem sugerir a ocorrência de hemoglobinopatias hereditárias e/ ou congênitas. Verificou-se que as taxas de Hb-A, nos clones com fenótipo Hb-AB e Hb-A, permaneceram estáveis durante todo o período experimental, enquanto nos bezerros obtidos por fertilização in vitro ou monta natural com os mesmos fenótipos (Hb-A e Hb-AB) observou-se a partir de 120 horas de vida um gradativo aumento das taxas de Hb-A. Durante a avaliação da dinâmica da hemoglobina do tipo fetal (Hb-F) no primeiro mês de vida observou-se, que todos os grupos animais apresentaram comportamento similar, caracterizado por sua diminuição com o desenvolvimento etário. / The present work aimed to study the hematology of cloned Nelore calves produced using the technique of somatic cell nuclear transfer (SCNT), by evaluating erythrocyte parameters, hemoglobin dynamics, and iron metabolism in the animals during the first month after birth. The experimental design included the collection of 260 blood and blood serum samples from 20 calves in the following times: immediately after birth, 12 hours after birth, 1st of life, 2nd, 3rd, 4th, 5th, 7th, 20th and 30th day of life. The animals were classified in four experimental groups: 12 calves produced, using SCNT for two commercial laboratories (laboratory A=5 calves and laboratory B= 7 calves), four calves produced by in vitro fertilization (IVF) and four calves produced by natural mating (NM). Mild to severe normocytic and normochromic anemia was observed in 100% (5/5) cloned calves from laboratory A, and 14.2% (117) cloned calves from laboratory B. In both IVF and NM calves, anemia was observed in 50% (214) of the calves. Erytrogram evaluation of cloned calves from laboratory A showed that anemia developed gradually from 12 hours after birth, was most intense at the end of the first week, and then erytrogram normal values were recovered after the 15th day of life. Mean values for the laboratory A cloned calves erytrogram in the 7th day of life were the following: Red cells 4033X106/mm3, hematocrit 23%, hemoglobin 7.25g/dL, MCV 52.89µ3-; MCH 16.65, MCHC 31.47%. Anemia observed in cloned calves from laboratory A was caused by iron deficiency, since a significant decrease in iron se rum levels together with a decrease in transferrin saturation index (TSI) was confirmed. At the same time, Total iron-binding capacity (TIBC) was not changed in this period of time. Mean iron metabolism values for cloned calves from laboratory A were the following: serum iron amount: 47,35mg/dL, TIBC 455,90 mg/dL and TSI 9,64%. Hemoglobin identification by eletrophoresis identified three adult hemolglobin phenotypes (A-Hb; B-Hb e AB-Hb) and the fetal hemoglobin (F-HB), and there was no sign of hereditary hemoglobin disorders were observed. The rate of A-Hb in cloned calves with A-Hb and AB-Hb phenotypes was maintained during the experimental period. Nonetheless, for the IVF calves with the same phenotypes (A-Hb and AB-Hb) a constant increase in the A-Hb was noticed. For the evaluation of F-Hb dynamics in the first month of life, results showed similar pattern, characterized by its decrease with age.

A síndrome dos ovários policísticos (SOP) é o distúrbio endócrino-metabólico mais frequente na menacme, com prevalência de 7 a 10 %, contribuindo com o aumento do risco cardiovascular e/ou diabetes mellitus tipo II nessas mulheres. OBJETIVOS: Identificar as características epidemiológicas e os diferentes fenótipos da SOP, a prevalência da síndrome metabólica encontrada em cada fenótipo e os fatores associados ao risco metabólico dessas pacientes. CASUÍSTICA E MÉTODO: Estudo observacional com 566 mulheres entre 14 e 39 anos portadoras de SOP, segundo o consenso de Rotterdam. O risco metabólico foi avaliado pela análise descritiva com intervalo de confiança de 95%. As variáveis quantitativas foram testadas pelo método de Shapiro-Wilk e teste não paramétrico de Mann-Whitney. Para a análise multivariada usou-se a razão de prevalências entre as diversas variáveis independentes e o desfecho risco metabólico. Identificamos os fatores associados ao risco metabólico empregando a regressão de Cox com variância robusta. RESULTADOS: Das 566 pacientes, 27,9% tinham entre 20 e 24 anos; 84,5% eram afrodescendentes; 90,6% apresentavam irregularidade menstrual; 91,8% hirsutismo; 77,7% ovários aumentados e/ou policísticos; 15,7% com pelo menos um filho; IMC elevado em 66,5%; CA superior a 88 em 51%; pressão arterial sistólica e diastólica elevadas em 38,9% e 20% das pacientes respectivamente; 7,7% intolerância a carboidratos, 40,8% de HDL-colesterol reduzido, 8,8% de triglicerídeos elevados. Encontramos risco metabólico em 21%, com predomínio dos fenótipos E (28,4%), B (25%) e A (22%). Antecedentes familiares de diabete, hipertensão arterial, câncer ginecológico e câncer não ginecológico não contribuíram, com significância estatística, para o aumento de eventos metabólicos. O acréscimo de um ano na idade elevou o risco em 5%. A cada subida de uma unidade no IMC foram adicionados 8%. A presença de hirsutismo triplicou o risco. Pacientes com pelo menos um filho apresentaram duas vezes mais síndrome metabólica do que as sem filhos. CONCLUSÕES: Foi observada maior frequência de síndrome metabólica entre os fenótipos que apresentam em comum oligoanovulação e hirsutismo (E, B e A). Em pacientes com SOP a idade, a paridade, a presença de hirsutismo e obesidade foram os fatores independentemente relacionados ao aumento do risco metabólico / Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder that is more frequent in premenopausal, affecting 7 to 10% of women, contributing to the increase of cardiovascular and/or type II diabetes mellitus risk. OBJECTIVES: To identify the epidemiological characteristics and different phenotypes of PCOS, the prevalence of metabolic syndrome found in each phenotype and metabolic risk factors associated with these patients. PATIENTS AND METHODS: Observational study of 566 women between 14 and 39 years with PCOS according to the Rotterdam criteria. The metabolic risk was assessed by descriptive analysis with a confidence interval of 95%. Quantitative variables were tested by using Shapiro-Wilk method and nonparametric Mann-Whitney test. For the multivariate analysis the prevalence ratio between several independent variables and the outcome metabolic risk were used. Factors associated with the metabolic risk were identified by using Cox regression with a robust variance. RESULTS: Of 566 patients, 27.9% were between 20 and 24 years, 84.5% were of African descents; 90.6% had oligoanovulation; 91.8% hirsutism; 77.7% enlarged ovaries and/or polycystic, 15.7% with at least one child in high BMI 66.5%, CA 88 exceeding 51%; systolic and diastolic blood pressure elevated by 38.9% and 20% of patients, respectively, 7.7% carbohydrate intolerance, 40.8% HDL-cholesterol changed, 8.8% triglyceride levels. Metabolic risk found in 21%, with a predominance of E phenotypes (28.4%), B (25%) and A (22.1%). Family history of diabetes, hypertension, gynecological cancer and gynecological cancer does not contribute with statistical significance for increased metabolic events. The one-year increase in age raised the risk by 5%. Every increase of one unit in BMI 8% were added. Presence of hirsutism tripled the risk. Patients with at least one child were twice as metabolic syndrome than those without children. CONCLUSIONS: A higher frequency of metabolic syndrome phenotypes that have in common oligoanovulation and hirsutism (E, B and A) were observed. Independently associated factors with the metabolic risk in PCOS patients were age, parity, hirsutism and obesity

Les phénotypes de l’asthme sont affectés par l’exposition à de multiples facteurs durant la grossesse. Pour évaluer cette hypothèse, deux études ont été menées : l’une en France, l’autre au Liban. Dans l'étude française, l'objectif était de décrire les phénotypes respiratoires de sifflement chez l’enfant entre l’âge de deux mois et d’un an, et d'évaluer les facteurs de risque associés à ces phénotypes de sifflement dans une grande cohorte de naissance. Dans l'étude libanaise, les objectifs étaient d'évaluer les associations entre l'utilisation de médicaments, d'alcool, de cigarettes et/ou de narguileh, et l'exposition aux pesticides/détergents pendant la grossesse avec l'asthme infantile au Liban et de valider la version arabe du test de contrôle de l'asthme (ACT) chez ces enfants et d’identifier les facteurs de risque qui pourraient affecter le contrôle de l'asthme.Méthodes: Nous avons étudié 18 041 nourrissons de la cohorte de naissance ELFE (Étude Longitudinale Française depuis l'Enfance). Les parents ont signalé une respiration sifflante et des symptômes respiratoires à deux et 12 mois, et ont répondu à un questionnaire complet (exposition pendant la grossesse, allergie parentale). Le plan d'étude du projet libanais consistait en une étude cas-témoins, menée entre Décembre 2015 et Avril 2016, recrutant 1503 enfants, âgés de 3 à 17 ans. Le questionnaire administré évaluait les caractéristiques sociodémographiques (âge, sexe, niveau d'éducation des deux parents), les antécédents familiaux d'asthme et d'autres facteurs de risque connus de l'asthme (système de chauffage à domicile, antécédents d'otites récidivantes, humidité dans la maison, enfant allant à une garderie, fumer et boire de l'alcool pendant la grossesse, exposition aux pesticides et aux détergents).Résultats: Les enfants sans symptômes (témoins) représentaient 77,2%, 2,1% avaient une respiration sifflante à deux mois mais pas de respiration sifflante à un an (sifflement intermittent), 2,4% avaient une respiration sifflante persistante et 18,3% avaient une respiration sifflante à un an. En comparant les sifflements persistants aux contrôles, on a observé qu’avoir un frère ou une sœur (ORa = 2,19) ou deux frères et sœurs (ORa = 2,23) contre aucun, une toux nocturne (OR = 5,2), une détresse respiratoire (OR = 4,1) et un excès de sécrétions bronchiques (OR = 3,47 ) à deux mois, un reflux chez l'enfant à 2 mois (OR = 1,55), des antécédents d'asthme maternel (OR = 1,46) et le tabagisme maternel pendant la grossesse (OR = 1,57) étaient significativement associés à une respiration sifflante persistante. Ces mêmes facteurs, avec en sus une éruption cutanée chez l'enfant à 2 mois (OR = 1,13) et des antécédents paternels d'asthme (OR = 1,32) étaient significativement associés à une augmentation de la probabilité d'une respiration sifflante. Avoir un frère (ORa = 1,9) en comparaison à ne pas en avoir, une toux nocturne à 2 mois (OR = 1,76) et un excès de sécrétions bronchiques à 2 mois (OR = 1,65) étaient significativement associés à une respiration sifflante persistante par rapport à une respiration sifflante intermittente.... / Asthma in childhood seems affected by exposure to various factors in early life. To assess this hypothesis, we conducted 2 studies: one in France, and the other in Lebanon. In the French study, we aimed at describing wheezing phenotypes between the ages of two months and one year, and assess risk factors associated with these wheezing phenotypes in a large birth cohort. In the Lebanese study, the aims were to evaluate the associations between caregiver-reported use of medications, alcohol, cigarette and/or waterpipe (WP), and exposure to pesticides/detergents during pregnancy with childhood-onset asthma in Lebanon and to validate the Arabic version of the Asthma Control Test (ACT) among these children and identify risk factors that might affect asthma control.Methods: We studied 18,041 infants from the ELFE (French Longitudinal Study of Children) birth cohort. Parents reported wheezing and respiratory symptoms at two and 12 months, and answered a complete questionnaire (exposure during pregnancy, parental allergy).The study design of the Lebanese project consisted of a case-control study, conducted between December 2015 and April 2016, recruited 1503 children, aged between 3-16 years old. A questionnaire assessed the sociodemographic characteristics (age, gender, education level of both parents), the family history of asthma, and other known risk factors of asthma (heating system at home, child history of recurrent otitis, humidity in the house, child went to a daycare, smoking and drinking alcohol during pregnancy, exposure to pesticides and detergents).Results: Children with no symptoms (controls) accounted for 77.2%, 2.1% had had wheezing at two months but no wheezing at one year (intermittent wheezing), 2.4% had persistent wheezing, while 18.3% had incident wheezing at one year. Comparing persistent wheezing to controls showed that having one sibling (ORa=2.19) or 2 siblings (ORa=2.23) compared to none, nocturnal cough (OR=5.2), respiratory distress (OR=4.1) and excess bronchial secretions (OR=3.47) at two months, reflux in the child at 2 months (OR=1.55), maternal history of asthma (OR=1.46) and maternal smoking during pregnancy (OR=1.57) were significantly associated with persistent wheezing. These same factors, along with cutaneous rash in the child at 2 months (OR=1.13) and paternal history of asthma (OR=1.32) were significantly associated with increased odds of incident wheezing. Having one sibling (ORa=1.9) compared to none, nocturnal cough at 2 months (OR=1.76) and excess bronchial secretions at 2 months (OR=1.65) were significantly associated with persistent compared to intermittent wheezing.In the Lebanese study, the multivariate analysis showed that children living in North and South Lebanon and the children living in areas where pesticides are frequently used had an increased risk of asthma (ORa=1.625; ORa=13.65; ORa=3.307) respectively. Smoking WP during pregnancy and cigarette during lactation would increase the risk of asthma in children (ORa=6.11; ORa=3.44 respectively). A high Cronbach’s alpha was found for the full scale (0.959). As for the asthma control scale (ACT), the correlation factors between each item of the ACT scale and the whole scale ranged between 0.710 and 0.775 (p<0.001 for all items). Mother’s low educational level as well as the history of asthma in the mother and the father would significantly increase the risk of uncontrolled asthma (Beta= 1.862; Beta= 3.534; and Beta= 1.885 respectively). Cigarette smoking during breastfeeding and waterpipe smoking by the mother during pregnancy were both significantly associated with uncontrolled asthma (Beta= 2.105; Beta=2.325 respectively). Mother’s high educational level was significantly associated with more asthma control (Beta= -0.715).

Over the past ten years, more than 4,000 genome-wide association studies (GWAS) have helped to shed light on the genetic architecture of complex traits and diseases. In recent years, phenotyping of the samples has often gone beyond single traits and it has become common to record multi- to high-dimensional phenotypes for individu- als. Whilst these rich datasets offer the potential to analyse complex trait structures and pleiotropic effects at a genome-wide level, novel analytic challenges arise. This thesis summarises my research into genetic associations for high-dimensional phen- otype data. First, I developed a novel and computationally efficient approach for multivari- ate analysis of high-dimensional phenotypes based on linear mixed models, com- bined with bootstrapping (LiMMBo). Both in simulation studies and on real data, I demonstrate the statistical validity of LiMMBo and that it can scale to hundreds of phenotypes. I show the gain in power of multivariate analyses for high-dimensional phenotypes compared to univariate approaches, and illustrate that LiMMBo allows for detecting pleiotropy in a large number of phenotypic traits. Aside from their computational challenges in GWAS, the true dimensionality of very high-dimensional phenotypes is often unknown and lies hidden in high-dimen- sional space. Retaining maximum power for association studies of such phenotype data relies on using an appropriate phenotype representation. I systematically ana- lysed twelve unsupervised dimensionality reduction methods based on their per- formance in finding a robust phenotype representation in simulated data of different structure and size. I propose a stability criteria for choosing low-dimensional phen- otype representations and demonstrate that stable phenotypes can recover genetic associations. Finally, I analysed genetic variants for associations to high-dimensional cardiac phenotypes based on MRI data from 1,500 healthy individuals. I used an unsuper- vised approach to extract a low-dimensional representation of cardiac wall thickness and conducted a GWAS on this representation. In addition, I investigated genetic associations to a trabeculation phenotype generated from a supervised feature ex- traction approach on the cardiac MRI data. In summary, this thesis highlights and overcomes some of the challenges in per- forming genetic association studies on high-dimensional phenotypes. It describes new approaches for phenotype processing, and genotype to phenotype mapping for high-dimensional datasets, as well as providing new insights in the genetic structure of cardiac morphology in humans.

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

Equine arteritis virus (EAV) is the causal agent of equine viral arteritis (EVA), a disease of equids. Following EAV infection, up to 70% of stallions may become carriers and continuously shed the virus in their semen for varying time periods. The long-term carrier stallion has an important role in the transmission and maintenance of EAV in horse populations. Recently, it has been demonstrated a correlation between in vitro susceptibility of CD3+ T lymphocytes to EAV infection and establishment of long-term persistent infection among stallions following natural infections. In this study, we investigated whether stallions with in vitro EAV susceptible CD3+ T lymphocytes are at higher risk of becoming long-term carriers compared to those with the resistant phenotype following experimental infection with the KY84 strain of EAV. Furthermore, we investigated whether there is a significant effect of EAV infection on semen quality during acute phase of the infection. The data suggested that the establishment of the long-term carrier state seems to be associated with the in vitro CD3+ T lymphocyte susceptible phenotypes and that reduced semen quality resulted from the combined effect of fever and scrotal edema observed following EAV infection rather than the direct effect of the virus.

Η ΧΑΠ είναι νόσος με πολλούς φαινοτύπους, με παθοφυσιολογία που διαφέρει σε κάθε έναν από αυτούς, με κοινό χαρακτηριστικό την πτώση του λόγου FEV1/FVC. Παρόλα ταύτα πολύ πρόσφατες μελέτες δείχνουν πως δεν υπάρχει σαφής συσχέτιση μεταξύ της προσεκτικά μετρούμενης φλεγμονής στους αεραγωγούς και της πτώσης της FEV1 σε ομάδες ασθενών με ΧΑΠ, κάτι που δυνητικά μπορεί να ανατρέψει βιβλιογραφία 40 ετών [Roy K, et al 2009]. Στόχος όλων των γενετικών μελετών στη ΧΑΠ είναι ο διαχωρισμός των φαινοτύπων και η δημιουργία του γενετικού προφίλ της νόσου. Γνωρίζοντας πως η ΧΑΠ είναι πολυγονιδιακή νόσος και σε συνδυασμό με τη μεγάλη ποικιλότητα των φαινοτύπων της, οι γενετικές αλλοιώσεις φαίνεται πως οδηγούν σε διαφορετικό φαινότυπο, που τώρα ολιστικά ορίζεται ως νόσος «ΧΑΠ», αλλά σίγουρα στο εγγύς μέλλον θα διαχωριστεί σε επιμέρους ομάδες στα πλαίσια μιας πιο αποτελεσματικής και εξατομικευμένης θεραπευτικής προσέγγισης. Η παρούσα μελέτη μεταξύ άλλων, συνέβαλε στα εξής: α. Σχεδιασμός και αξιολόγηση εκκινητών και ιχνηθέτων για τον +134InsA/DelA πολυμορφισμό, με υψηλή διακριτική ικανότητα έναντι των αλληλίων 3Α και 4Α. β. Ανέδειξε την εμπλοκή των πολυμορφισμών+134InsA/DelA και G198T στην εμφάνιση αλλά και στη βαρύτητα της ΧΑΠ (όπως αυτή αξιολογείται με την FEV1), ενώ σκιαγραφήθηκε και το γενετικό προφίλ του ευαίσθητου στον καπνό του τσιγάρου καπνιστή, με λεπτομερή συσχέτιση των απλοτύπων των πολυμορφισμών της ΕΤ-1 που εμπλέκονται στη ΧΑΠ. γ. Ανέδειξε πιθανή εμπλοκή του +134InsA/DelA πολυμορφισμού στην στατική υπερδιάταση και στις αυξημένες αντιστάσεις στη ροή του αέρα στους πνεύμονες δ. Σκιαγράφησε τη σχέση των +134InsA/DelA και G198T με την ανοχή στην άσκηση και συνέκρινε τα αποτελέσματα αυτά με όσα ήδη υπάρχουν στη βιβλιογραφία. Η παρούσα μελέτη είναι η πρώτη που συσχετίζει πολυμορφισμούς με πολλαπλές αξιολογήσεις της αναπνευστικής λειτουργίας, πέραν της FEV1, κάτι που σκιαγραφεί με λεπτομέρεια το φαινότυπο της ΧΑΠ. / Chronic Obstructive Pulmonary Disease (COPD) is an entity with many phenotypes, different pathophysiological characteristics, that exhibits in all cases a diminished FEV to FVC ratio. Nevertheless, recent studies show that there is not a strict relationship between airway inflammation and FEV1 decline in patients with COPD, contrasting a 40 year literature [Roy K, et al 2009]. The aim of all recent studies dealing with genetics in COPD is the distinction of different phenotypes and the elucidation of the genetic profile of the disease. COPD is a multi-gene disorder, and knowing that it is composited by many phenotypes, one can say that, in the near future, “holistic COPD phenotype” will be unraveled in many distinguished phenotypes, leading to a personalized and patient-targeted diagnostic and therapeutic approach. The current study contributed in: a. Designing primers and probes for the +134InsA/DelA polymorphism, that could clearly distinguish both 3A and 4A alleles. b. Exhibiting that both +134InsA/DelA & G198T polymorphisms are implicated in COPD progression and severity (as defined by FEV1 values). At the same time, we managed to highlight the genetic profile of the susceptible to smoke smoker, associating haplotypes and polymorphisms of Endothelin-1 (ET-1) gene (+134InsA/DelA & G198T ) with COPD. c. Showing the implication of the +134InsA/DelA polymorphism with static lung hyperinflation and increased airway resistance. d. Revealing the association of +134InsA/DelA & G198T polymorphisms with exercise tolerance. According to our knowledge, the current study is the first in the literature showing association of ET-1 gene with lung function deterioration.

Genome-wide associations studies (GWASs) have identified hundreds of loci that are strongly associated with coronary artery disease and its risk factors. However, the causal variants and genes remain unknown for the vast majority of the identified loci. Zebrafish model systems coupled with clustered regularly interspaced short palindromic repeats-C–associated 9 (CRISPR Cas-9) mutagenesis have enabled the possibility to systematically characterize candidate genes in GWAS-identified loci. In this thesis, canonical correlation analysis (CCA) was used to identify putative causal genes in multiplexed genetic screens for atherogenic traits in zebrafish larvae in an efficient manner. The two datasets used in this thesis contained genes and phenotypes obtained through sequencing and high-throughput imaging of fish larvae. Dataset 1 contained (7 genes, 11 phenotypes, n = 384) and dataset 2 (4 genes, 11 phenotypes, n = 384). CCA’s multiple genes vs. multiple phenotype analysis in dataset 1 identified the genes met, pepd, timd4 and vegfa to have an association with the total cholesterol, triglycerides, glucose, corrected lipid disposition, as well as co- localization of (macrophage and lipid deposition,) (neutrophils and lipid deposition) and (macrophage and neutrophils). In dataset 2, CCA found previously reported correlation of genes apobb1 and apoea with total cholesterol, low-density lipoprotein and triglycerides as well as co localization of neutrophils and lipids. In comparison with hierarchical linear model, CCA represents a powerful and promising tool to identify causal genes for cardiovascular diseases in data from zebrafish model systems.

A major challenge in biology is to understand and predict the effect of mutations on protein structure, stability and function. Chapter 1 provides a general introduction on protein sequence-structure relationships and use of the CcdAB toxin-antitoxin system as a model to study molecular determinants of mutant phenotypes. In Chapter 2, we describe the use of saturation mutagenesis combined with deep sequencing to determine phenotypes for 1664 single-site mutants of the E. coli cytotoxin, CcdB. We examined multiple expression levels, effects of multiple chaperones and proteases and employed extensive in vitro characterization to understand how mutations affect these phenotypes. While general substitution preferences are known, eg polar residues preferred at exposed positions and non-polar ones at buried positions, we show that depth from the surface is important and that there are distinctly different energetic penalties for each specific polar, charged and aromatic amino acid introduced at buried positions. We also show that over-expression of ATP independent chaperones can rescue mutant phenotypes. Other studies have primarily looked at effects of ATP dependent chaperone expression on phenotype, where it is not possible to say whether mutational effects on folding kinetics or thermodynamic stability are the primary determinant of altered phenotypes, since there is energy input with these chaperones. The data suggest that mutational effects on folding rather than stability determine the in vivo phenotype of CcdB mutants. This has important implications for efforts to predict phenotypic effects of mutations and in protein design. While looking at the mutational landscape of a given gene from an evolutionary perspective, it is important to establish the genotype-phenotype relationships under physiologically relevant conditions. At the molecular level, the relationship between gene sequence and fitness has implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Chapter 3 describes a methodology to test the fitness of individual CcdB mutants in E.coli over several generations by monitoring the rate of plasmid loss. We also propose a methodology for high throughput analysis of a pool of CcdB mutants using deep sequencing to quantitate the relative population of each mutant in a population of E.coli cells, grown for several generations and build the fitness landscape. While the F-plasmid based CcdAB system is known to be involved in plasmid maintenance through post-segregational killing, recent identification of ccdAB homologs on the chromosome, including in pathogenic strains of E.coli and other bacteria, has led to speculations on their functional role on the chromosome. In Chapter 4, we show that both the native ccd operon of the E.coli O157 strain as well as the ccd operon from the F- plasmid when inserted on the E.coli chromosome lead to protection from cell death under multiple antibiotic stress conditions through formation of persisters. Both the ccdF and ccdO157 operons may share common mechanisms for activation under stress conditions and also display weak cross activation. The chromosomal toxin shows weaker activity as compared to the plasmidic counterpart and is therefore less efficient in causing cell death. This has important implications in generation of potential therapeutics that target these TA systems. Chapter 5 describes the use of site-saturation mutagenesis coupled with deep sequencing to infer mutational sensitivity for the intrinsically disordered antitoxin, CcdA. The data allows us to make comparisons between overall as well as residue specific mutational sensitivity patterns with that of globular proteins, like CcdB (described in Chapter 2) and study toxin- antitoxin interaction and regulation through saturation suppressor mutagenesis. Interestingly, we found several examples of synonymous point mutations in CcdA that lead to loss of its activity. In Chapter 6 we attempt to explore the molecular bases for some of these synonymous mutations. In most cases the mutated codon has a similar overall codon preference to the WT one. Initial findings suggest a change in mRNA structure leading to change in CcdB: CcdA ratio, thereby causing cell death. These observations have important implications, because TA systems are ubiquitous, highly regulated and are known to be involved in multiple functions including drug tolerance. However a role for RNA structure in their regulation has not been shown previously. Appendix–I lists the mutational sensitivity scores for the CcdB mutants. Phenotypes for CcdA mutants obtained through deep sequencing have been tabulated in Appendix-II. Overall, we provide extensive datasets for mutational sensitivities of a globular (CcdB) and an intrinsically disordered protein (CcdA). Exploration of the molecular determinants of these mutant phenotypes not only provides interesting insights into CcdAB operon function but is also useful in understanding various aspects of protein stability, folding and activity as well as regulation of gene expression in bacteria.