Deciphering causal genetic determinants of red blood cell traits

Les études d’association pan-génomiques ont révélé plusieurs variants génétiques associés à des traits complexes. Les mesures érythrocytaires ont souvent fait l’objet de ce genre d’études, étant mesurées de façon routinière et précise. Comprendre comment les variations génétiques influencent ces phénotypes est primordial étant donné leur importance comme marqueurs cliniques et leur influence sur la sévérité de plusieurs maladies. En particulier, des niveaux élevés d’hémoglobine fœtal chez les patients atteints d’anémie falciforme est associé à une réduction des complications et une augmentation de l’espérance de vie. Néanmoins, la majorité des variants génétiques identifiés par ces études tombent à l’intérieur de régions génétiques non-codantes, augmentant la difficulté d’identifier des gènes causaux. L’objectif premier de ce projet est l’identification et la caractérisation de gènes influençant les traits complexes, et tout particulièrement les traits sanguins. Pour y arriver, j’ai tout d’abord développé une méthode permettant d’identifier et de tester l’effet de gènes knockouts sur les traits anthropométriques. Malgré un échantillon de grande taille, cette approche n’a révélé aucune association. Ensuite, j’ai caractérisé le méthylome et le transcriptome d’érythroblastes différentiés à partir de cellules souches hématopoïétiques et identifié plusieurs gènes potentiellement impliqués dans les programmes érythroïdes fœtaux et adultes. Par ailleurs, j’ai identifié plusieurs micro-ARNs montrant des motifs d’expression spécifiques entre les stages fœtaux et adultes et qui sont enrichis pour des cibles exprimées de façon opposée. Finalement, j’ai identifié plusieurs variants génétiques associés à l’expression de gènes dans les érythroblastes (eQTL). Cette étude a permis d’identifier des variants associés à l’expression du gène ATP2B4, qui encode le principal transporteur de calcium des érythrocytes. Ces variants, qui sont également associés à des traits sanguins et à la susceptibilité à la malaria, tombent dans un élément d’ADN spécifique aux cellules érythroïdes. La délétion de cet élément par le système CRISPR/Cas9 induit une forte diminution de l’expression du gène et une augmentation des niveaux de calcium intracellulaires. En conclusion, des échantillons de génotypages exhaustifs seront nécessaires pour étudier l’effet de gènes knockouts sur les traits complexes. Les érythroblastes montrent de grandes différences au niveau de leur méthylome et transcriptome entre les différents stages développementaux. Ces différences influencent potentiellement la régulation de l’hémoglobine fœtale et impliquent de nombreux micro-ARNs et régions régulatrices non-codantes. Finalement, l’exemple d’ATP2B4 montre qu’intégrer des études épigénomiques, transcriptomiques et des expériences d’édition de génome est une approche puissante pour caractériser des variants génétiques non-codants. Par ailleurs, ces résultats impliquent ATP2B4 dans l’hydratation des érythroblastes, qui est associé à la susceptibilité à la malaria et la sévérité de l’anémie falciforme. Cibler ATP2B4 de façon thérapeutique pourrait avoir un impact majeur sur ces maladies qui affectent des millions d’individus à travers le monde.Genome-wide association studies (GWAS) have revealed several genetic variants associated with complex phenotypes. This is the case for red blood cell (RBC) traits, which are particularly amenable to GWAS as they are routinely and accurately measured. Understanding RBC trait variation is important given their significance as clinical markers and modifiers of disease severity. Notably, increased fetal hemoglobin (HbF) production in sickle cell disease (SCD) patients is associated with a higher life expectancy and decreased morbidity. Nonetheless, most variants identified through GWAS fall in non-coding regions of the human genome, increasing the difficulty of identifying causal links. The main goal of this project was to identify and characterize genes influencing complex traits, and in particular RBC phenotypes. First, I developed an approach to identify and test potential gene knockouts affecting anthropometric traits in a large sample from the general population, which did not yield significant associations. Then, I characterized the DNA methylome and transcriptome of erythroblasts differentiated ex vivo from hematopoietic progenitor stem cells (HPSC), and identified several genes potentially implicated in fetal and adult-stage erythroid programs. I also identified microRNAs (miRNA) that show specific developmental expression patterns and that are enriched in inversely expressed targets. Finally, I mapped expression quantitative trait loci (eQTL) in erythroblasts, and identify erythroid-specific eQTLs for ATP2B4, the main calcium ATPase of RBCs. These genetic variants are associated with RBC traits and malaria susceptibly, and overlap an erythroid-specific enhancer of ATP2B4. Deletion of this regulatory element using CRISPR/Cas9 experiments in human erythroid cells minimized ATP2B4 expression and increased intracellular calcium levels. In conclusion, large and comprehensive genotyping datasets will be necessary to test the role of rare gene knockouts on complex phenotypes. The transcriptomes and DNA methylomes of erythroblasts show substantial differences correlating with their developmental stages and that may be implicated in HbF production. These results also suggest a strong implication of erythroid enhancers and miRNAs in developmental stage specificity. Finally, characterizing the erythroid-specific enhancer of ATP2B4 suggest that integrating epigenomic, transcriptomic and gene editing experiments can be a powerful approach to characterize non-coding genetic variants. These results implicate ATP2B4 in erythroid cell hydration, which is associated with malaria susceptibility and SCD severity, suggesting that therapies targeting this gene could impact diseases affecting millions of individuals worldwide

La dépendance européenne au gaz naturel russe : analyse comparée de la sécurité de l'approvisionnement en Allemagne, en Ukraine et en Turquie

En 1968, les premières exportations de gaz naturel soviétique pénètrent le marché européen. Quarante ans plus tard, le marché gazier russo-européen représente le plus grand bassin d'échanges gaziers au monde. Toutefois, malgré les immenses réserves russes et les capacités financières des États européens, une crainte demeure: celle de voir la Russie accentuer sa présence, déjà complète en amont, en aval du marché gazier. Ainsi, Gazprom serait utilisée par le Kremlin pour acquérir le contrôle d'entreprises européennes de distribution de gaz naturel, afin de développer une menace plus efficace encore que la menace nucléaire parce qu'applicable à des durées et degrés divers: la menace énergétique. Ce mémoire se limite aux questions gazières parce que l'étendue aux autres énergies eût représenté un travail colossal inadéquat pour un mémoire de maîtrise. Mais le marché gazier russo-européen implique suffisamment de questionnements sociaux, politiques et économiques pour construire une recherche complète et précise. Le but de ce mémoire sera de déterminer s'il existe des schèmes de relations gazières avec la Russie, et, le cas échéant, comment ces schèmes se développent et influencent les relations qu'entretient Gazprom avec chacun des États partenaires. Parallèlement, et en utilisant ces schèmes, cette recherche tentera aussi de déconstruire le mythe du « danger russe » hérité de la Guerre Froide et qui politise les relations gazières. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Russie, Europe, Politique énergétique, Gaz Naturel, Sécurité de l'approvisionnement

Trends in the Frequency, Patient Characteristics, Management, and in-Hospital Outcomes of Diabetic Patients Presenting with Acute Myocardial Infarction

Background: Diabetic patients have more complications and higher hospital mortality rates after an acute myocardial infarction (AMI) than patients without diabetes (DM). Increased morbidity and mortality among diabetic patients suffering an AMI is especially concerning given the increasing prevalence of obesity and diabetes in the U.S. and worldwide. The objectives of this study were to describe recent trends in the frequency, patient characteristics, treatment practices, and in-hospital outcomes associated with STEMI and NSTEMI in diabetic compared with non-diabetic patients hospitalized with AMI. Methods: We reviewed the medical records of 6,903 persons, known to be either diabetic (n =2,329) or non-diabetic (n=4,574 ) who were hospitalized for STEMI or NSTEMI between 1997 and 2009 at all 11 greater Worcester medical centers. Results: Diabetic patients presenting with both STEMI and NSTEMI were more likely to be older, female, and obese, and to have a higher prevalence of comorbidities compared with non-diabetics. Diabetic patients were more likely to develop important in-hospital complications including heart failure (39% vs.27%),and atrial fibrillation (18% vs.16%), and had a longer hospital stay (6.3 days vs.5.4 days) compared to non-diabetics. Diabetic patients were significantly more likely to be treated with an angiotensin converting enzyme inhibitor or angiotensin receptor blocker and a diuretic. The proportion of patients undergoing cardiac catheterization during their index hospitalization for AMI approximately doubled during the period under study, while the proportion treated with PCI increased by 3 to 4-fold. The proportion of diabetic and non-diabetic patients undergoing cardiac catheterization was similar, though diabetics were less likely to be treated with PCI and more likely to receive CABG than non-diabetics. In-hospital mortality was significantly higher among diabetics than non-diabetics for both STEMI (13% vs. 10%) and NSTEMI (11% vs. 9%) Conclusions: During the period 1997 to 2009, the use of effective therapies for all patients presenting with AMI has improved, with a concomitant decrease in in-hospital complications and mortality . Nonetheless, diabetic patients experienced , more complications, and worse in-hospital outcomes compared to non-diabetics

Trends in the medical management of patients with heart failure

BACKGROUND: Despite the availability of effective therapies, heart failure (HF) remains a highly prevalent disease and the leading cause of hospitalizations in the U.S. Few data are available, however, describing changing trends in the use of various cardiac medications to treat patients with HF and factors associated with treatment. The objectives of this population-based study were to examine decade-long trends (1995 - 2004) in the use of several cardiac medications in patients hospitalized with acute decompensated heart failure (ADHF) and factors associated with evidence-based treatment. METHODS: We reviewed the medical records of 9,748 residents of the Worcester, MA, metropolitan area who were hospitalized with ADHF at all 11 central Massachusetts medical centers in 1995, 2000, 2002, and 2004. RESULTS: Between 1995 and 2004, respectively, the prescription upon hospital discharge of beta-blockers (23%; 67%), angiotensin pathway inhibitors (47%; 55%), statins (5%; 43%), and aspirin (35%; 51%) increased markedly, while the use of digoxin (51%; 29%), nitrates (46%; 24%), and calcium channel blockers (33%; 22%) declined significantly; nearly all patients received diuretics. Patients in the earliest study year, those with a history of obstructive pulmonary disease or anemia, incident HF, non-specific symptoms, and women were less likely to receive beta blockers and angiotensin pathway inhibitors than respective comparison groups. In 2004, 82% of patients were discharged on at least one of these recommended agents; however, only 41% were discharged on medications from both recommended classes. CONCLUSIONS: Our data suggest that opportunities exist to further improve the use of HF therapeutics

Improved Survival after Heart Failure: A Community-based Perspective

Background: Heart failure is a highly prevalent, morbid, and costly disease with a poor long-term prognosis. Evidence-based therapies utilized over the past 2 decades hold the promise of improved outcomes, yet few contemporary studies have examined survival trends in patients with acute heart failure. Objectives: The primary objective of this population-based study was to describe trends in short and long-term survival in patients hospitalized with acute decompensated heart failure (ADHF). A secondary objective was to examine patient characteristics associated with decreased long-term survival. Methods and Results: We reviewed the medical records of 9,748 patients hospitalized with ADHF at all 11 medical centers in central Massachusetts during 1995, 2000, 2002, and 2004. Patients hospitalized with ADHF were more likely to be elderly and to have been diagnosed with multiple comorbidities in 2004 compared with 1995. Over this period, survival was significantly improved in-hospital, and at 1, 2, and 5 years post-discharge. Five-year survival rates increased from 20% in 1995 to 28% in 2004. Although survival improved substantially over time, older patients and patients with chronic kidney disease, chronic obstructive pulmonary disease, anemia, low body mass index, and low blood pressures had consistently lower post-discharge survival rates than patients without these comorbidities. Conclusion: Between 1995 and 2004, patients hospitalized with ADHF have become older and increasingly comorbid. Although there has been a significant improvement in survival among these patients, their long-term prognosis remains poor, as fewer than 1 in 3 patients hospitalized with ADHF in 2004 survived more than 5 years

Spoken word recognition of novel words, either produced or only heard during learning

This document is the Accepted Manuscript Version of the following article: Tania S. Zamuner, Elizabeth Morin-Lessard, Stephanie Strahm, and Michael P. A. Page, 'Soke word recognition of novel words, either produced or only heard during learning', Journal of Memory and Language, Vol. 89, August 2016, pp. 55-67, doi: 10.1016/j.jml.2015.10.003. Under embargo. Embargo end date: 1 December 2017. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Psycholinguistic models of spoken word production differ in how they conceptualize the relationship between lexical, phonological and output representations, making different predictions for the role of production in language acquisition and language processing. This work examines the impact of production on spoken word recognition of newly learned non-words. In Experiment 1, adults were trained on non-words with visual referents; during training, they produced half of the non-words, with the other half being heard-only. Using a visual world paradigm at test, eye tracking results indicated faster recognition of non-words that were produced compared with heard-only during training. In Experiment 2, non-words were correctly pronounced or mispronounced at test. Participants showed a different pattern of recognition for mispronunciation on non-words that were produced compared with heard-only during training. Together these results indicate that production affects the representations of newly learned words.Peer reviewedFinal Accepted Versio

Domestication of different varieties in the cheese-making fungus Geotrichum candidum

Domestication is an excellent model for studying adaptation processes, involving recent adaptation and diversification, convergence following adaptation to similar conditions, as well as degeneration of unused functions. Geotrichum candidum is a fungus used for cheese making and is also found in other environments such as soil and plants. By analyzing whole-genome data from 98 strains, we found that all strains isolated from cheese formed a monophyletic clade. Within the cheese clade, we identified three genetically differentiated populations and we detected footprints of recombination and admixture. The genetic diversity in the cheese clade was similar as that in the wild clade, suggesting the lack of strong bottlenecks. Commercial starter strains were scattered across the cheese clade, thus not constituting a single clonal lineage. The cheese populations were phenotypically differentiated from other populations, with a slower growth on all media, even cheese, a prominent production of typical cheese volatiles and a lower proteolytic activity. One of the cheese clusters encompassed all soft goat cheese strains, suggesting an effect of cheese-making practices on differentiation. Another of the cheese populations seemed to represent a more advanced stage of domestication, with stronger phenotypic differentiation from the wild clade, harboring much lower genetic diversity, and phenotypes more typical of cheese fungi, with denser and fluffier colonies and a greater ability of excluding cheese spoiler fungi. Cheese populations lacked two beta lactamase-like genes present in the wild clade, involved in xenobiotic clearance, and displayed higher contents of transposable elements, likely due to relaxed selection. Our findings suggest the existence of genuine domestication in G. candidum, which led to diversification into different varieties with contrasted phenotypes. Some of the traits acquired by cheese strains indicate convergence with other, distantly related fungi used for cheese maturation

P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells

Adipose tissue contains self-renewing multipotent cells termed mesenchymal stromal cells. In situ, these cells serve to expand adipose tissue by adipogenesis, but their multipotency has gained interest for use in tissue regeneration. Little is known regarding the repertoire of receptors expressed by adipose-derived mesenchymal stromal cells (AD-MSCs). The purpose of this study was to undertake a comprehensive analysis of purinergic receptor expression. Mesenchymal stromal cells were isolated from human subcutaneous adipose tissue and confirmed by flow cytometry. The expression profile of purinergic receptors was determined by quantitative real-time PCR and immunocytochemistry. The molecular basis for adenine and uracil nucleotide-evoked intracellular calcium responses was determined using Fura-2 measurements. All the known subtypes of P2X and P2Y receptors, excluding P2X2, P2X3 and P2Y12 receptors, were detected at the mRNA and protein level. ATP, ADP and UTP elicited concentration-dependent calcium responses in mesenchymal cells (N = 7–9 donors), with a potency ranking ADP (EC50 1.3 ± 1.0 μM) > ATP (EC50 2.2 ± 1.1 μM) = UTP (3.2 ± 2.8 μM). Cells were unresponsive to UDP (< 30 μM) and UDP-glucose (< 30 μM). ATP responses were attenuated by selective P2Y2 receptor antagonism (AR-C118925XX; IC50 1.1 ± 0.8 μM, 73.0 ± 8.5% max inhibition; N = 7 donors), and UTP responses were abolished. ADP responses were attenuated by the selective P2Y6 receptor antagonist, MRS2587 (IC50 437 ± 133nM, 81.0 ± 8.4% max inhibition; N = 6 donors). These data demonstrate that adenine and uracil nucleotides elicit intracellular calcium responses in human AD-MSCs with a predominant role for P2Y2 and P2Y6 receptor activation. This study furthers understanding about how human adipose-derived mesenchymal stromal cells can respond to external signalling cues