Homéostasie des histones en réponse au dommage à l’ADN et étude d’inhibiteurs de désacétylases d’importance clinique

La chromatine possède une plasticité complexe et essentielle pour répondre à différents mécanismes cellulaires fondamentaux tels la réplication, la transcription et la réparation de l’ADN. Les histones sont les constituants essentiels de la formation des nucléosomes qui assurent le bon fonctionnement cellulaire d’où l’intérêt de cette thèse d’y porter une attention particulière. Un dysfonctionnement de la chromatine est souvent associé à l’émergence du cancer. Le chapitre II de cette thèse focalise sur la répression transcriptionnelle des gènes d’histones par le complexe HIR (HIstone gene Repressor) en réponse au dommage à l'ADN chez Saccharomyces cerevisiae. Lors de dommage à l’ADN en début de phase S, les kinases du point de contrôle Mec1, Tel1 et Rad53 s’assurent de bloquer les origines tardives de réplication pour limiter le nombre de collisions potentiellement mutagéniques ou cytotoxiques entre les ADN polymérases et les lésions persistantes dans l'ADN. Lorsque la synthèse totale d’ADN est soudainement ralentie par le point de contrôle, l’accumulation d'un excès d'histones nouvellement synthétisées est néfaste pour les cellules car les histones libres se lient de manière non-spécifique aux acides nucléiques. L'un des mécanismes mis en place afin de minimiser la quantité d’histones libres consiste à réprimer la transcription des gènes d'histones lors d'une chute rapide de la synthèse d'ADN, mais les bases moléculaires de ce mécanisme étaient très mal connues. Notre étude sur la répression des gènes d’histones en réponse aux agents génotoxiques nous a permis d’identifier que les kinases du point de contrôle jouent un rôle dans la répression des gènes d’histones. Avant le début de mon projet, il était déjà connu que le complexe HIR est requis pour la répression des gènes d’histones en phase G1, G2/M et lors de dommage à l’ADN en phase S. Par contre, la régulation du complexe HIR en réponse au dommage à l'ADN n'était pas connue. Nous avons démontré par des essais de spectrométrie de masse (SM) que Rad53 régule le complexe HIR en phosphorylant directement une de ses sous-unités, Hpc2, à de multiples résidus in vivo et in vitro. La phosphorylation d’Hpc2 est essentielle pour le recrutement aux promoteurs de gènes d’histones du complexe RSC (Remodels the Structure of Chromatin) dont la présence sur les promoteurs des gènes d'histones corrèle avec leur répression. De plus, nous avons mis à jour un nouveau mécanisme de régulation du complexe HIR durant la progression normale à travers le cycle cellulaire ainsi qu'en réponse aux agents génotoxiques. En effet, durant le cycle cellulaire normal, la protéine Hpc2 est très instable durant la transition G1/S afin de permettre la transcription des gènes d’histones et la production d'un pool d'histones néo-synthétisées juste avant l'initiation de la réplication de l’ADN. Toutefois, Hpc2 n'est instable que pour une brève période de temps durant la phase S. Ces résultats suggèrent qu'Hpc2 est une protéine clef pour la régulation de l'activité du complexe HIR et la répression des gènes d’histones lors du cycle cellulaire normal ainsi qu'en réponse au dommage à l’ADN. Dans le but de poursuivre notre étude sur la régulation des histones, le chapitre III de ma thèse concerne l’analyse globale de l’acétylation des histones induite par les inhibiteurs d’histone désacétylases (HDACi) dans les cellules normales et cancéreuses. Les histones désacétylases (HDACs) sont les enzymes qui enlèvent l’acétylation sur les lysines des histones. Dans plusieurs types de cancers, les HDACs contribuent à l’oncogenèse par leur fusion aberrante avec des complexes protéiques oncogéniques. Les perturbations causées mènent souvent à un état silencieux anormal des suppresseurs de tumeurs. Les HDACs sont donc une cible de choix dans le traitement des cancers engendrés par ces protéines de fusion. Notre étude de l’effet sur l’acétylation des histones de deux inhibiteurs d'HDACs de relevance clinique, le vorinostat (SAHA) et l’entinostat (MS-275), a permis de démontrer une augmentation élevée de l’acétylation globale des histones H3 et H4, contrairement à H2A et H2B, et ce, autant chez les cellules normales que cancéreuses. Notre quantification en SM de l'acétylation des histones a révélé de façon inattendue que la stœchiométrie d'acétylation sur la lysine 56 de l’histone H3 (H3K56Ac) est de seulement 0,03% et, de manière surprenante, cette stœchiométrie n'augmente pas dans des cellules traitées avec différents HDACi. Plusieurs études de H3K56Ac chez l’humain présentes dans la littérature ont rapporté des résultats irréconciliables. Qui plus est, H3K56Ac était considéré comme un biomarqueur potentiel dans le diagnostic et pronostic de plusieurs types de cancers. C’est pourquoi nous avons porté notre attention sur la spécificité des anticorps utilisés et avons déterminé qu’une grande majorité d’anticorps utilisés dans la littérature reconnaissent d’autres sites d'acétylation de l’histone H3, notamment H3K9Ac dont la stœchiométrie d'acétylation in vivo est beaucoup plus élevée que celle d'H3K56Ac. De plus, le chapitre IV fait suite à notre étude sur l’acétylation des histones et consiste en un rapport spécial de recherche décrivant la fonction de H3K56Ac chez la levure et l’homme et comporte également une évaluation d’un anticorps supposément spécifique d'H3K56Ac en tant qu'outil diagnostic du cancer chez l’humain.The chromatin is a complex structure and its plasticity is essential to complete different fundamental cellular processes such as DNA replication, transcription and repair. Furthermore, chromatin malfunction is often associated with cancer emergence. The focus of this thesis will be on the function and regulation of histones, as they are essential components of nucleosomes and they ensure proper chromatin formation. Chapter II of this thesis focuses on the transcriptional repression of histone genes by the HIR (HIstone gene Repressor) complex in response to DNA damage in Saccharomyces cerevisiae. When DNA damage occurs in early S phase, the DNA damage checkpoint kinases Mec1, Tel1 and Rad53 block late origins of replication to limit potentially mutagenic or cytotoxic collisions between DNA polymerases and remaining DNA lesions. When the total DNA synthesis rate drops suddenly in S- phase, following the checkpoint control activation, accumulation of newly synthesized histones becomes detrimental for the cells because free histones bind non-specifically to nucleic acids. One mechanism that contributes to a reduction in free histones at this time is the repression of histone gene transcription; however, the molecular basis of this repression was not known. Our study on histone gene repression in response to genotoxic agents allowed us to identify the checkpoint kinases as major players in the repression of histone genes. Before initiating this project, it was known that the HIR complex is required to repress histone genes in G1 and G2/M phases and during DNA damage. Nonetheless, HIR complex regulation was not well characterized. We demonstrated by mass spectrometry (MS) analyses that Rad53 regulates the HIR complex by directly phosphorylating one of its subunits, Hpc2, at many residues in vivo and in vitro. Hpc2 phosphorylation is essential to recruit the RSC complex (Remodels the Structure of Chromatin) to histone gene promoters where its presence correlates with histone gene repression. Moreover, we uncovered a novel mechanism for the HIR complex regulation during a normal cell cycle progression and in response to genotoxic agents. Indeed, during a normal cell cycle, the Hpc2 protein is very unstable at the G1/S transition to allow histone gene transcription and production of a pool of newly synthesized histones just before DNA replication initiation. These results suggest that Hpc2 is a key player in the regulation of HIR complex activity and can repress histone gene expression both during a normal cell cycle and in response to DNA damage. In order to pursue our study on histone regulation, chapter III of this thesis covers histone acetylation induced by histone deacetylase inhibitors (HDACi) in normal and cancer cells. Histone deacetylases (HDACs) are enzymes that remove acetyl groups from lysine residues on histones, condensing the chromatin and effectively repressing local transcription. Several types of cancers are characterized by epigenetic abnormalities and HDACs contribute to oncogenesis by aberrant fusion with oncogenic protein complexes. The disruptions often lead to an abnormal silent state of tumour suppressors. HDACs are then targets of interest in cancer treatment caused by those fusion proteins. Our study of the effects of two clinically relevant HDAC inhibitors, vorinostat (SAHA) and entinostat (MS-275) on acetylation of histones demonstrated an obvious increase of histones H3 and H4 acetylation, unlike histones H2A and H2B in both normal and cancer cells. Unexpectedly, our MS quantification of histone acetylation revealed that the stoichiometry of histone H3 lysine 56 acetylation (H3K56Ac) was only 0.03% and, surprisingly, this stoichiometry did not increase upon HDACi treatments. Several reported studies in the literature of H3K56Ac in humans are irreconcilable. Furthermore, H3K56Ac was considered as a potential biomarker in diagnosis and prognosis in many cancer types. Therefore we focussed on antibody specificity and determined that the majority of antibodies used in the literature recognize other acetylation sites in histone H3, especially H3K9Ac whose stoichiometry of acetylation in vivo is much higher than H3K56Ac. Additionally, chapter IV is a follow-up of our study on histone acetylation and consists of a special report describing the function of H3K56Ac in yeast and human and also contains an evaluation of a supposedly specific H3K56Ac antibody as a diagnostic tool in human cancers

Facteurs de localisation des immigrants entrepreneurs francophones et potentiel attractif du Saguenay-Lac-St-Jean

Le bilan économique du Saguenay-Lac-St-Jean est alarmant. À titre d'exemple, l'agglomération urbaine du haut Saguenay possède le plus haut taux de chômage au Canada. La situation exige de trouver des moyens pour dynamiser la région. La régionalisation des immigrants entrepreneurs en est un. Pour assurer le succès de la régionalisation, il doit y avoir concordance entre les besoins des immigrants et les possibilités régionales. À cet effet, il était nécessaire de savoir si le Saguenay-Lac-St-Jean disposait du potentiel nécessaire pour attirer les immigrants entrepreneurs. Pour y arriver, nous devions d'abord connaître les facteurs d'attraction qui incitent les immigrants entrepreneurs à se localiser en un lieu donné. Notre recherche inclut cinq chapitres de présentation: le premier définit la problématique, le deuxième introduit le corpus théorique et la recension des écrits, le troisième expose la méthodologie suivie pour vérifier les hypothèses soulevées. Les quatrième et cinquième chapitres présentent, quant à eux, les résultats de notre recherche en fonction des objectifs fixés. Deux enquêtes furent réalisées pour recueillir les données de notre recherche. Une s'est d'abord effectuée auprès de 29 immigrants entrepreneurs francophones pour connaître les facteurs d'attraction qui les incitent à se localiser en un lieu donné. Un questionnaire devait être préalablement construit avant d'amorcer l'enquête. Aucune des études recensées n?offrait référence sur ce plan. Une autre fut produite subséquemment pour vérifier, à l'aide de documentations régionales et à partir des résultats de localisation obtenus, les potentialités du Saguenay-Lac-St-Jean. L'analyse descriptive a été privilégiée pour traiter l'ensemble de nos données. Les résultats obtenus sur les facteurs de choix de localisation ont confirmé notre première hypothèse de recherche. Les facteurs incitatifs de localisation sont de nature diverse. Ils se définissent en terme de qualité de vie, de prix de revient, de famille et de réseaux de contacts. Les résultats montrent aussi toute l'importance accordée par nos répondants aux facteurs extraéconomiques de localisation. Les facteurs économiques sont, pour leur part, des agents incitatifs de second ordre. Les résultats obtenus sur le potentiel régional ont confirmé que partiellement notre seconde hypothèse. Ils ont démontré que le Saguenay-Lac-St-Jean ne dispose pas de tout le potentiel nécessaire pour attirer les immigrants entrepreneurs. L'enquête a tout de même dénombré plusieurs atouts qui rendent cette région attractive. Les atouts découlent surtout de la qualité de vie mais aussi des ressources économiques. Une concordance s'est ainsi faite entre le vouloir des immigrants entrepreneurs et les possibilités du Saguenay-Lac-St-Jean rendant ainsi la régionalisation possible

Dynamique et modélisation de l’oxygène dissous en rivière

La concentration en oxygène dissous en milieu fluvial varie selon un cycle diurne (24 h) qu’il est essentiel de considérer dans l’évaluation de l’état d’oxygénation d’un cours d’eau. En principe, seules des mesures en continu recueillies au cours de cycles de 24 h permettent d’évaluer correctement l’état d’oxygénation d’une rivière, ce que, en pratique, les contraintes logistiques et budgétaires ne permettent pas de réaliser. Le présent article vise à faire la synthèse des connaissances sur les facteurs de contrôle et la modélisation des variations diurnes de la concentration en oxygène dissous en rivière. Parmi les facteurs biologiques et physico-chimiques, les activités autotrophe et hétérotrophe sont les facteurs dominants responsables des variations diurnes de l’oxygène dissous. Certains modèles de qualité de l’eau permettent de modéliser la teneur en oxygène et, dans certains cas, la variation diurne. Toutefois, ces modèles sont souvent complexes, d’utilisation ardue et impliquent la mesure directe sur des cycles de 24 h des variables qui régissent la concentration en oxygène dans le milieu. Une démarche est proposée pour l’élaboration d’un modèle et a été appliquée dans une étude de cas réalisée dans la rivière Saint-Charles (Québec, Canada). Un modèle simple (une fonction sinusoïdale) dont les paramètres ont été corrélés à la température et à la concentration moyenne en nitrate a permis de générer des valeurs simulées d’oxygène dissous très proches des valeurs observées in situ. Un modèle alternatif utilisant des valeurs ponctuelles de température et de concentration de nitrate a donné des résultats équivalents. L’approche proposée constitue donc une alternative simple et pratique à la mesure en continu de l’oxygène et permet une évaluation plus réaliste de l’état réel d’oxygénation d’une rivière que la prise de mesures ponctuelles.In rivers, dissolved oxygen concentrations typically show diel variations with maximum values during daytime and minimum values at night. The diel cycle must be taken into account when assessing the state of oxygenation of a watercourse. However, in water quality monitoring programs, dissolved oxygen concentrations are usually obtained from single measurements taken during daytime. The resulting data do not represent the real overall oxygen levels of a watercourse and thus can lead to erroneous conclusions regarding the oxygen status of a river. Continuous data collected over 24‑hour cycles are required for an accurate oxygen status assessment, but in practice, logistic and budget constraints do not allow such samplings. Modelling can be a convenient alternative to direct measurements. However, the water quality models that take into account the diel cycle of oxygen are generally complex to run. The purpose of this study was to review the information relating to the dynamics and the modelling of the diel variations in dissolved oxygen in rivers and to apply a simple model in a case study involving dissolved oxygen, nutrients, temperature and chlorophyll a data collected over 24‑hour cycles in the St. Charles River near Quebec City (Canada).Photosynthesis by algae, both benthic and planktonic, as well as by macrophytes, is an important and sometimes dominant factor in the oxygen budget of a river. Sediments and heterotrophic activity by bacteria, particularly in rivers receiving important loads of wastewaters, can be important sinks for oxygen. Temperature determines the solubility of oxygen, thereby directly influencing oxygen concentrations. Diel variations in oxygen thus reflect diel variations in temperature. Temperature also has an effect on biological processes such as respiration and photosynthesis. Reaeration varies not only with temperature, but also with the type of flow (laminar vs. turbulent) and current velocity. In rivers with important slopes and current velocities, reaeration can be sufficient to make up for oxygen losses due to high heterotrophic activity. Nevertheless, light is the first causative factor for the diel variations in oxygen, determining both autotrophic activity and water temperature. However, suspended matter in the water column reduces light penetration. Higher levels of suspended matter result in lower levels of photosynthesis and oxygen production. The sudden or large influx of runoff waters after heavy rain or snowmelt can also have an important impact on the oxygen budget of a river. Finally, chemical factors can have an influence on the diel variations in oxygen: nutrient inputs, in particular, can stimulate the rate of photosynthesis and oxygen production. Overall, oxygen dynamics are determined by the relative importance of biological, physical and chemical factors, which vary in time and space. However, biological processes often dominate over the other causative factors affecting diel variations in oxygen. In temperate climates, biological processes have a controlling function only during warmer months, with temperature and flow being the dominant controlling factors during colder months.Water quality models that take into account diel variations in oxygen are often designed to assess primary production and respiration. These models are based on either ODUM’s (1956) concept of oxygen curves or on direct and continuous measurements of dissolved oxygen. Periodic functions or Fourier series are also used to simulate diel variations in oxygen. The widely used USEPA QUAL2e model predicts diel variations in oxygen from different measurements including light intensity and from computation of the rates of photosynthesis and respiration. Several other modelling approaches use various combinations of indirect methods to predict the variations in oxygen, based on light intensity, algal biomass, primary production, and reaeration. Specific models are sometimes necessary due to particular regional characteristics or environmental issues.In general, water quality models designed for water management purposes are complex and require the measurement of a large number of parameters, which necessitates elaborate and costly logistics. Estimating the parameters controlling the oxygen budget in a river thus ends up being more time and labour consuming than the direct measurement of diel variations in oxygen. A simpler model leading to the estimation of the concentrations and the amplitude of the variations of dissolved oxygen was developed and applied to the St. Charles River.The St. Charles River flows from the Laurentians north of Quebec City to the St. Lawrence River. The vast upper watershed is mostly forested, but the lower part is heavily urbanized. Two stations were located in the upper watershed and one in the section of the river within Quebec City. Water quality was excellent at the upstream stations and poor at the downstream station, due to wastewater inputs and low flow rates. Sampling was carried out in the months of July and August of 1996 and 1997. Physico-chemical and light measurements were made every two hours for periods of 24 hours. Nutrient and chlorophyll a samples were collected every four hours. Small diel variations in oxygen (amplitude: 1.48 mg/L) were observed at the upstream station, while much larger ones (amplitude: 4.23 mg/L) were measured at the downstream station. Modelling of the diel variations in oxygen was carried out using a sine function. Oxygen concentrations over 24 hours were successfully predicted from average concentration of dissolved oxygen, amplitude, and phase of the cycle. Average oxygen concentrations and amplitude can be derived from physico-chemical and/or biological variables easily measured in standard water quality monitoring programs. Average oxygen concentrations showed a very strong correlation with temperature (r2 = 0.91) and amplitude of oxygen level variations was strongly correlated with average nitrate concentration (r2 = 0.58). These relations were used in the sine function and resulted in significantly correlated modelled and measured oxygen concentrations for six of the seven cycles. Overall correlation between modelled and observed values was high (r2 = 0.77). Modelled values obtained with single measurements of temperature (taken at 2:30 P.M.) and nitrate concentration (which shows no diel variation) were also highly correlated with observed data (r2 = 0.75). Absolute and relative bias as well as root-mean-square error also showed the validity and the equivalence of the two approaches.This study shows that simple models based on available water quality data may generate realistic oxygen values over 24‑hour cycles. These models would be a valuable diagnostic and decision making tool for the management of water quality in rivers

Relationship between smoothing temperature, storage time, syneresis and rheological properties of stirred yogurt

Six different smoothing temperatures were compared for nonfat yogurt and the changes in syneresis and rheological properties observed for up to 22 days. Multiple linear regressions were used to describe the syneresis, firmness, flow time, viscosity, and flow resistance and the relationship between these properties, the smoothing temperature and the storage time. During storage, viscosity, firmness, and flow time increased; syneresis and flow resistance remained stable. Syneresis increased significantly (P ≤ 0.05) with smoothing temperature (10–35 °C). Other properties increased slightly (P > 0.05), and properties started to decrease above 30 °C. Syneresis, viscosity, and flow resistance were more sensitive to smoothing temperature; firmness and flow time were more sensitive to storage time. Lower smoothing temperature (10 °C) should be used to minimize syneresis while smoothing temperature ranging from 25 to 30 °C is better to improve rheological properties. Storage time must be considered to optimize these properties

How do smoothing conditions and storage time change syneresis, rheological and microstructural properties of nonfat stirred acid milk gel?

Nonfat acid milk gel, acidified by GDL, was used to simulate microbial fermentation of milk to produce stirred yoghurt. Acid milk gel preparation at laboratory scale included stirring, pumping, smoothing and cooling operations. Two filters (pre-smoothed, 1 mm; smoothed, 500 μm), three smoothing temperatures (13, 22 and 35 °C) and two storage times (1 and 22 days) were studied. Syneresis, microgels size and smoothness of microgels were analysed for pre-smoothed and smoothed gels; viscosity, storage modulus, firmness and total pore area were only analysed for smoothed gel. After 1 and 22 days of storage, pre-smoothed gels developed lower syneresis and smaller microgels than smoothed gels at 22 °C. For smoothed gels, regardless of the smoothing temperature, syneresis, firmness, microgels size and smoothness increased during storage, while total pore area decreased and viscosity remained stable. Viscosity was lower when smoothing was performed at 35 °C and was correlated to rougher microgels

Soil and land use research in Europe: lessons learned from INSPIRATION bottom-up strategic research agenda setting

We introduce the INSPIRATION bottom-up approach for the development of a strategic research agenda for spatial planning, land use and soil-sediment-water-system management in Europe. Research and innovation needs were identified by more than 500 European funders, endusers, scientists, policy makers, public administrators and consultants. We report both on the concept and on the implementation of the bottom-up approach, provide a critique of the process and draw key lessons for the development of research agendas in the future. Based on identified strengths and weaknesses we identified as key opportunities and threats 1) a high ranking and attentiveness for the research topics on the political agenda, in press and media or in public awareness, 2) availability of funding for research, 3) the resources available for creating the agenda itself, 4) the role of the sponsor of the agenda development, and 5) the continuity of stakeholder engagement as bases for identification of windows of opportunity, creating ownership for the agenda and facilitating its implementation. Our derived key recommendations are 1) a clear definition of the area for which the agenda is to be developed and for the targeted user, 2) a conceptual model to structure the agenda, 3) making clear the expected roles, tasks, input formats regarding the involvement and communication with the stakeholders and project partners, 4) a sufficient number of iterations and checks of the agenda with stakeholders to insure completeness, relevance and creation of co-ownership for the agenda, and 5) from the beginning prepare the infrastructure for the network to implement the agenda

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Sacré serpent, magie et mythologie

International audienc

Sacré serpent, magie et mythologie

International audienc