The chicken ovalbumin upstream promoter-transcription factors modulate genes and pathways involved in skeletal muscle cell metabolism

The chicken ovalbumin upstream promoter-transcription factors ( COUP-TFs) are orphan members of the nuclear hormone receptor ( NR) superfamily. COUP-TFs are involved in organogenesis and neurogenesis. However, their role in skeletal muscle ( and other major mass tissues) and metabolism remains obscure. Skeletal muscle accounts for similar to 40% of total body mass and energy expenditure. Moreover, this peripheral tissue is a primary site of glucose and fatty acid utilization. We utilize small interfering RNA ( siRNA)-mediated attenuation of Coup-TfI and II ( mRNA and protein) in a skeletal muscle cell culture model to understand the regulatory role of Coup-Tfs in this energy demanding tissue. This targeted NR repression resulted in the significant attenuation of genes that regulate lipid mobilization and utilization ( including Ppar alpha, Fabp3, and Cpt-1). This was coupled to reduced fatty acid beta-oxidation. Additionally we observed significant attenuation of Ucp1, a gene involved in energy expenditure. Concordantly, we observed a 5-fold increase in ATP levels in cells with siRNA-mediated repression of Coup-TfI and II. Furthermore, the expression of classical liver X receptor ( LXR) target genes involved in reverse cholesterol transport ( Abca1 and Abcg1) were both significantly repressed. Moreover, we observed that repression of the Coup-Tfs ablated the activation of Abca1, and Abcg1 mRNA expression by the selective LXR agonist, T0901317. In concordance, Coup-Tf-siRNA-transfected cells were refractory to Lxr-mediated reduction of total intracellular cholesterol levels in contrast to the negative control cells. In agreement Lxr-mediated activation of the Abca1 promoter in Coup-Tf-siRNA cells was attenuated. Collectively, these data suggest a pivotal role for Coup-Tfs in the regulation of lipid utilization/cholesterol homeostasis in skeletal muscle cells and the modulation of Lxr-dependent gene regulation.

Influencing water consumption at South Staffordshire Water PLC : a disaggregated behavioural analysis of contributory factors

This research identifies factors which influence the consumption of potable water supplied to customers' property. A complete spectrum of the customer base is examined including household, commercial and industrial properties. The research considers information from around the world, particularly demand management and tariff related projects from North America. A device termed the Flow Moderator was developed and proven, with extensive trials, to conserve water at a rate equivalent to 40 litres/property/day whilst maintaining standards-of-service considerably in excess of Regulatory requirements. A detailed appraisal of the Moderator underlines the costs and benefits available to the industry through deliberate application of even mild demand management. More radically the concept of a charging policy utilising the Moderator is developed and appraised. Advantages include the lower costs of conventional fixed-price charging systems coupled with the conservation and equitability aspects associated with metering. Explanatory models were developed linking consumption to a range of variables demonstrated that households served by a communal water service-pipe (known in the UK as a shared supply) are subject to associated restrictions equivalent to -180 litres/property/day. The research confirmed that occupancy levels were a significant predictive element for household, commercial and industrial customers. The occurrence of on-property leakage was also demonstrated to be a significant factor recorded as an event which offers considerable scope for demand management in its own right.

The influence of political, economic and passenger market factors on international civil aviation

Civil aviation plays an essential role in maintaining international communications. Many extraneous factors influence the daily operations of the air transport industry. This thesis begins by investigating the major categories of so­ called "external interests" in civil aviation. These are shown to have played a significant part in ensuring the need for international agreement over the adoption of regulating principles. The combination and interaction of the various influences has produced a particular type of regulatory environment in which all commercial air services have to operate. The need for such regulation and the extreme difficulty experienced in trying to define universally acceptable methods of supervision is discussed. It is shown how opportunity for the development of on-scheduled air services was created by default on the part of the European Governments.The concept of so-called "scheduled" and "non-scheduled" sectors" is considered and it is suggested that growth of the inclusive tour industry resulted from inappropriate categorisation of the air services involved. The means by which development opportunities were created for inclusive tour operations is considered and the work then investigates the importance of British air transport policy in their exploitation. The politics of British civil aviation in the post-war years is the subject of detailed examination and the process by which Independent airlines were encouraged to develop inclusive tours, is identified. This theme is expanded to demonstrate the vital contribution of British air transport policy in the restructuring of the international industry. The subsequent involvement of the United States is shown to have been directed specifically towards the satisfaction of domestic issues. British objectives, however, are considered to have been more generally concerned with improving the tariff structure. The unique opportunities for British experimentation with international fares are seen to have major influence in forcing the pace of tariff rationalisation.

Risk factors for childhood myopia:findings from the NICER study

PURPOSE. We explored risk factors for myopia in 12- to 13-year-old children in Northern Ireland (NI). METHODS. Stratified random sampling was performed to obtain representation of schools and children. Cycloplegia was achieved using cyclopentolate hydrochloride 1%. Distance autorefraction was measured using the Shin-Nippon SRW-5000 device. Height and weight were measured. Parents and children completed a questionnaire, including questions on parental history of myopia, sociodemographic factors, childhood levels of near vision, and physical activity to identify potential risk factors for myopia. Myopia was defined as spherical equivalent ≤0.50 diopters (D) in either eye. RESULTS. Data from 661 white children aged 12-to 13-years showed that regular physical activity was associated with a lower estimated prevalence of myopia compared to sedentary lifestyles (odds ratio [OR] = 0.46 adjusted for age, sex, deprivation score, family size, school type, urbanicity; 95% confidence interval [CI], 0.23–0.90; P for trend = 0.027). The odds of myopia were more than 2.5 times higher among children attending academically-selective schools (adjusted OR = 2.66; 95% CI, 1.48–4.78) compared to nonacademically-selective schools. There was no evidence of an effect of urban versus nonurban environment on the odds of myopia. Compared to children with no myopic parents, children with one or both parents being myopic were 2.91 times (95% CI, 1.54–5.52) and 7.79 times (95% CI, 2.93– 20.67) more likely to have myopia, respectively. CONCLUSIONS. In NI children, parental history of myopia and type of schooling are important determinants of myopia. The association between myopia and an environmental factor, such as physical activity levels, may provide insight into preventive strategies.

The new institutionalism in the context of Kosovo's transition:regulatory institutions in contested states

This article explores the new institutionalist literature in political economy in the context of Kosovo's contested statehood, focusing on institutional arbitrage and legitimacy. This article considers both the consequences of institutions for actors' behaviour and the norms that shape this, as well as the factors determining the legitimacy of institutions. In doing so, it combines the new institutionalist theory with documentary and interview material collected during research on energy regulation in one contested state, Kosovo. Rather than singling out one particular variety of "new institutionalism", the article attempts to blend insights from historical (or "political"), rational choice, and sociological institutionalism. © 2014 © 2014 Taylor & Francis.

The Future of EU Cohesion Policy. Endogenous Development – Added Value of Intervention – Regulatory Frameworks

The successful operation of the regional development – or cohesion – policy of the European Union has a strategic importance from the point of view of the whole integration process. Strengthening economic, social and territorial cohesion and decreasing disparities between member states and regions are not only one of the main priorities of the integration, but at the same time these are justified expectations of the people living in the member states of the union. The cohesion transfers should be spent on those factors which have the biggest contribution to the improvement of development prospects and competitiveness in the given regions. Theories on regional development have controversial conclusions about the long-term formation of development disparities. However, it has become evident that successful development policies are based on endogenous factors, innovation and well-functioning institutions. After examining theoretical considerations and regional disparities the study analyses the impacts of EU regional policy and evaluates the main elements of the proposed regulatory frameworks for the period 2014-2020.

A cash flow-kimutatás szabályozásainak nemzetközi összehasonlítása és csoportosítása (International comparison and classification of Cash Flow Statement regulations)

Az egyes nemzetek számviteli szabályozásának vizsgálatánál az adott ország sajátosságaiból eredően részben eltérő szabályozások alakultak ki. Az induktív megközelítésű vizsgálatok jellemzően a szabályozási kérdések széles körét fogják át, de csak néhány tényező mentén közelítve. A cash flow-kimutatások témakörénél a legtöbbször csak azt nézték, hogy van-e előírás a kimutatás elkészítésére, de a részletekkel már kevésbé foglalkoztak. Ebből adódóan e területen viszonylag kis különbséget mutattak ki ezek a felmérések. A szerző kutatása szerint a nemzeti cash flow-kimutatások szabályozásának részleteiben eltérések tapasztalhatók, és ezek alapján a nemzetek klaszterelemzéssel hierarchikusan csoportokba rendezhetők. _____ Research has found that as a result of their particularities, different countries have established partly different accounting frameworks. Studies with inductive approaches typically encompass a wide range of regulatory issues, but based on a limited number of factors only. In the case of Statements of Cash Flows, most studies have so far only examined the existence of rules governing the presentation of the statement, without an in-depth analysis of the details. Therefore, these studies only found relatively minor differences in this field. The author’s research shows that many differences exist in the details of national Cash Flow Statement regulations, which makes it possible to classify the countries in groups using the method of hierarchical clustering.

The role of splicing factors and small nuclear RNAs in spliceosomal formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatiotemporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

Cardiovascular risk factors in Turkish immigrants with type 2 diabetes living in the Netherlands

The cross sectional study investigated the association of tobacco smoke, vitamin D status, anthropometric parameters, and kidney function in Turkish immigrants with type 2 diabetes (T2D) living in the Netherlands. Study sample included a total of 110 participants aged 30 years and older (males= 46; females= 64). Serum cotinine, a biomarker for smoke exposure, was measured with a solid-phase competitive chemiluminescent immunoassay. Serum 25-hydroxyvitamin D [25(OH)D] was determined by electrochemiluminescence immunoassay (ECLIA). Measures of obesity including: body weight, body mass index (BMI), waist circumference (WC), and hip circumference (HC) were measured. Waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) were calculated. Urine albumin was measured by immunoturbidimetric assay. Urine creatinine was determined using the Jaffe method. All statistical analyses were performed using SPSS, version 19.0 (SPSS Inc., Chicago, IL, USA). Independent samples t-test, chi-squared tests, multiple linear regression and logistic regression analysis were used. Cotinine levels were positively associated with cholesterol to HDL ratio and atherosclerosis-index. Serum 25(OH)D levels were negatively associated with diastolic blood pressure. Gender-specific associations between anthropometric measures and high sensitivity C-reactive protein (hs-CRP) levels were observed. Hs-CRP was positively associated with WC and WHR in males and WHtR in females. Microalbuminuria (MAU), as determined by albumin-to-creatinine ratio, was present in 21% of the Turkish immigrants with T2D. Participants with hypertension were 6.58 times more likely (adjusted odds ratio) to have positive MAU as compared to normotensive participants. Our findings indicate that serum cotinine, 25(OH)D, hs-CRP, and MAU may be assessed as a standard of care for T2D management in the Turkish immigrant population. Further research should be conducted following cohorts to determine the effects of these biomarkers on CVD morbidity and mortality.

Pseudomonas aeruginosa AMPR transcriptional regulatory network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Psychological and Cognitive Factors that Influence Post-Exercise Energy Intake in Normal Weight and Overweight Sedentary Males

The primary purpose of this study was to evaluate the effects of a single bout of moderate-intensity exercise on acute (ad libitum lunch) post-exercise energy intake (PE-EI) and 12-hour energy intake in normal-weight and overweight sedentary males. Accuracy in estimating energy intake (EI) and energy expenditure (EE), solid vs. liquid carbohydrate intake, mood, and perceived hunger were also assessed. The study consisted of two conditions, exercise and rest, with each subject participating in each condition, in a counterbalanced-crossover design on two days. The participants were randomly assigned to either the exercise or resting (seated) control condition on the first day of the experiment, and then the condition was reversed on the second day. Exercise consisted of walking on a treadmill at moderate-intensity for 60 minutes. Eighty males, mean age 30+8 years were categorized into five groups according to weight status (overweight/normal-weight), dietary restraint status (high/low), and dieting status (yes/no). The main effects of condition and group, and the interaction were not significant for acute (lunch) or 12-hour PE-EI. Overall, participants estimated EE for exercise at 46% higher than actual exercise EE, and they estimated EE for rest by 45% lower than actual resting EE. Participants significantly underestimated EI at lunch on both the exercise and rest days by 43% and 44%, respectively. Participants with high restraint were significantly better at estimating EE on the exercise day, and better at estimating EI on the rest day. Mood, perceived hunger, and solid vs. liquid carbohydrate intake were not influenced by dietary restraint, weight, or dieting status. In conclusion, a single bout of moderate-intensity exercise did not influence PE-EI in sedentary males in reference to dietary restraint, weight, and dieting status. Results also suggested that among sedentary males, there is a general inability to accurately estimate calories for moderate-intensity physical activity and EI. Inaccurate estimates of EE and EI have the potential to influence how males manage their weight.

The Role of Splicing Factors and Small Nuclear RNAS in Spliceosomal Formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

IL-10-producing Regulatory B Cell Development in Human Autoimmune Disease

B cell abnormalities contribute to the development and progress of autoimmune disease. Traditionally, the role of B cells in autoimmune disease was thought to be predominantly limited to the production of autoantibodies. Nevertheless, in addition to autoantibody production, B cells have other functions potentially relevant to autoimmunity. Such functions include antigen presentation to and activation of T cells, expression of costimulatory molecules and cytokine production. Recently, the ability of B cells to negatively regulate cellular immune responses and inflammation has been described and the concept of “regulatory B cells” has emerged. A variety of cytokines produced by regulatory B cell subsets have been reported with interleukin-10 (IL-10) being the most studied. IL-10-producing regulatory B cells predominantly localize within a rare CD1dhiCD5+ B cell subset in mice and the CD24hiCD27+ B cell subset in adult humans. This specific IL-10-producing subset of regulatory B cells have been named “B10 cells” to highlight that the regulatory function of these rare B cells is primarily mediated by IL-10, and to distinguish them from other regulatory B cell subsets that regulate immune responses through different mechanisms. B10 cells have been studies in a variety of animal models with autoimmune disease and clinical settings of human autoimmunity. There are many unsolved questions related to B10 cells including their surface phenotype, their origin and development in vivo, and their role in autoimmunity.

In Chapter 3 of this dissertation, the role of the B cell receptor (BCR) in B10 cell development is highlighted. First, the BCR repertoire of mouse peritoneal cavity B10 cells is examined by single cell sequencing; peritoneal cavity B10 cells have clonally diverse germline BCRs that are predominantly unmutated. Second, mouse B10 cells are shown to have higher frequencies of λ+ BCRs compared to non-B10 cells which may indicate the involvement of BCR light chain editing early in the process of B10 cell development in vivo. Third, human peripheral blood B10 cells are examined and are also found to express higher frequencies of λ chains compared to non-b10 cells. Therefore, B10 cell BCRs are clonally diverse and enriched for unmutated germline sequences and λ light chains.

In Chapter 4 of this dissertation, B10 cells are examined in the healthy developing human across the entire age range of infancy, childhood and adolescence, and in a large cohort of children with autoimmunity. The study of B10 cells in the developing human documents a massive transient expansion during middle childhood when up to 30% of blood B cells were competent to produce IL-10. The surface phenotype of pediatric B10 cells was variable and reflective of overall B cell development. B10 cells down-regulated CD4+ T cell interferon-gamma (IFN-γ) production through IL-10-dependent pathways and IFN-γ inhibited whereas interleukin-21 (IL-21) promoted B cell IL-10 competency in vitro. Children with autoimmunity had a contracted B10 cell compartment, along with increased IFN-γ and decreased IL-21 serum levels compared to age-matched healthy controls. The decreased B10 cell frequencies and numbers in children with autoimmunity may be partially explained by the differential regulation of B10 cell development by IFN-γ and IL-21 and alterations in serum cytokine levels. The age-related changes of the B10 cell compartment during normal human development provide new insights into immune tolerance mechanisms involved in inflammation and autoimmunity.

These studies collectively demonstrate that BCR signals are the most important early determinant of B10 cell development in vivo, that human B10 cells are not a surface phenotype defined developmental B cell subset but a functionally defined regulatory B cell subset that regulates CD4+ T IFN-γ production through IL-10-dependent pathways and that human B10 cell development can be regulated by soluble factors in vivo such as the cytokine milieu. The findings of these studies provide new insights into immune tolerance mechanisms involved in human autoimmunity and the potent effects of IL-21 on human B cell IL-10 competence in vitro open new horizons in the development of autologous B10 cell-based therapies as an approach to treat human autoimmune disease in the future.

Effet préventif de la milrinone inhalée chez les patients avec hypertension pulmonaire subissant une chirurgie cardiaque sous circulation extracorporelle : une approche pharmacométrique

La circulation extracorporelle (CEC) est une technique utilisée en chirurgie cardiaque effectuée des milliers de fois chaque jour à travers le monde. L’instabilité hémodynamique associée au sevrage de la CEC difficile constitue la principale cause de mortalité en chirurgie cardiaque et l’hypertension pulmonaire (HP) a été identifiée comme un des facteurs de risque les plus importants. Récemment, une hypothèse a été émise suggérant que l'administration prophylactique (avant la CEC) de la milrinone par inhalation puisse avoir un effet préventif et faciliter le sevrage de la CEC chez les patients atteints d’HP. Toutefois, cette indication et voie d'administration pour la milrinone n'ont pas encore été approuvées par les organismes réglementaires. Jusqu'à présent, la recherche clinique sur la milrinone inhalée s’est principalement concentrée sur l’efficacité hémodynamique et l'innocuité chez les patients cardiaques, bien qu’aucun biomarqueur n’ait encore été établi. La dose la plus appropriée pour l’administration par nébulisation n'a pas été déterminée, de même que la caractérisation des profils pharmacocinétiques (PK) et pharmacodynamiques (PD) suite à l'inhalation. L'objectif de notre recherche consistait à caractériser la relation exposition-réponse de la milrinone inhalée administrée chez les patients subissant une chirurgie cardiaque sous CEC. Une méthode analytique par chromatographie liquide à haute performance couplée à un détecteur ultraviolet (HPLC-UV) a été optimisée et validée pour le dosage de la milrinone plasmatique suite à l’inhalation et s’est avérée sensible et précise. La limite de quantification (LLOQ) était de 1.25 ng/ml avec des valeurs de précision intra- et inter-dosage moyennes (CV%) <8%. Des patients souffrant d’HP pour lesquels une chirurgie cardiaque sous CEC était prévue ont d’abord été recrutés pour une étude pilote (n=12) et, par la suite, pour une étude à plus grande échelle (n=28) où la milrinone (5 mg) était administrée par inhalation pré-CEC. Dans l'étude pilote, nous avons comparé l'exposition systémique de la milrinone peu après son administration avec un nébuliseur pneumatique ou un nébuliseur à tamis vibrant. L’efficacité des nébuliseurs en termes de dose émise et dose inhalée a également été déterminée in vitro. Dans l'étude à plus grande échelle conduite en utilisant exclusivement le nébuliseur à tamis vibrant, la dose inhalée in vivo a été estimée et le profil pharmacocinétique de la milrinone inhalée a été pleinement caractérisé aux niveaux plasmatique et urinaire. Le ratio de la pression artérielle moyenne sur la pression artérielle pulmonaire moyenne (PAm/PAPm) a été choisi comme biomarqueur PD. La relation exposition-réponse de la milrinone a été caractérisée pendant la période d'inhalation en étudiant la relation entre l'aire sous la courbe de l’effet (ASCE) et l’aire sous la courbe des concentrations plasmatiques (ASC) de chacun des patients. Enfin, le ratio PAm/PAPm a été exploré comme un prédicteur potentiel de sortie de CEC difficile dans un modèle de régression logistique. Les expériences in vitro ont démontré que les doses émises étaient similaires pour les nébuliseurs pneumatique (64%) et à tamis vibrant (68%). Cependant, la dose inhalée était 2-3 fois supérieure (46% vs 17%) avec le nébuliseur à tamis vibrant, et ce, en accord avec les concentrations plasmatiques. Chez les patients, en raison des variations au niveau des facteurs liés au circuit et au ventilateur causant une plus grande dose expirée, la dose inhalée a été estimée inférieure (30%) et cela a été confirmé après récupération de la dose de milrinone dans l'urine 24 h (26%). Les concentrations plasmatiques maximales (Cmax: 41-189 ng/ml) et l'ampleur de la réponse maximale ΔRmax-R0 (0-65%) ont été observées à la fin de l'inhalation (10-30 min). Les données obtenues suite aux analyses PK sont en accord avec les données publiées pour la milrinone intraveineuse. Après la période d'inhalation, les ASCE individuelles étaient directement reliées aux ASC (P=0.045). Enfin, notre biomarqueur PD ainsi que la durée de CEC ont été identifiés comme des prédicteurs significatifs de la sortie de CEC difficile. La comparaison des ASC et ASCE correspondantes a fourni des données préliminaires supportant une preuve de concept pour l'utilisation du ratio PAm/PAPm comme biomarqueur PD prometteur et justifie de futures études PK/PD. Nous avons pu démontrer que la variation du ratio PAm/PAPm en réponse à la milrinone inhalée contribue à la prévention de la sortie de CEC difficile.

Experiment: Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H(+)-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH(e) and pH(i)) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO(2) conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO(2). Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH(e) whenever seawater pH changes. However, measurements of pH(i) demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na(+) and HCO(3)(-), suggesting a bicarbonate buffer mechanism involving secondary active Na(+)-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH(i) enables calcification to proceed despite decreased pH(e). However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.