CLOCK-controlled polyphonic regulation of circadian rhythms through canonical and noncanonical E-boxes.

In mammalian circadian clockwork, the CLOCK-BMAL1 complex binds to DNA enhancers of target genes and drives circadian oscillation of transcription. Here we identified 7,978 CLOCK-binding sites in mouse liver by chromatin immunoprecipitation-sequencing (ChIP-Seq), and a newly developed bioinformatics method, motif centrality analysis of ChIP-Seq (MOCCS), revealed a genome-wide distribution of previously unappreciated noncanonical E-boxes targeted by CLOCK. In vitro promoter assays showed that CACGNG, CACGTT, and CATG(T/C)G are functional CLOCK-binding motifs. Furthermore, we extensively revealed rhythmically expressed genes by poly(A)-tailed RNA-Seq and identified 1,629 CLOCK target genes within 11,926 genes expressed in the liver. Our analysis also revealed rhythmically expressed genes that have no apparent CLOCK-binding site, indicating the importance of indirect transcriptional and posttranscriptional regulations. Indirect transcriptional regulation is represented by rhythmic expression of CLOCK-regulated transcription factors, such as Krüppel-like factors (KLFs). Indirect posttranscriptional regulation involves rhythmic microRNAs that were identified by small-RNA-Seq. Collectively, CLOCK-dependent direct transactivation through multiple E-boxes and indirect regulations polyphonically orchestrate dynamic circadian outputs.

Nuclear factor I-C links platelet-derived growth factor and transforming growth factor beta1 signaling to skin wound healing progression.

Transforming growth factor beta (TGF-beta) and platelet-derived growth factor A (PDGFAlpha) play a central role in tissue morphogenesis and repair, but their interplay remain poorly understood. The nuclear factor I C (NFI-C) transcription factor has been implicated in TGF-beta signaling, extracellular matrix deposition, and skin appendage pathologies, but a potential role in skin morphogenesis or healing had not been assessed. To evaluate this possibility, we performed a global gene expression analysis in NFI-C(-/-) and wild-type embryonic primary murine fibroblasts. This indicated that NFI-C acts mostly to repress gene expression in response to TGF-beta1. Misregulated genes were prominently overrepresented by regulators of connective tissue inflammation and repair. In vivo skin healing revealed a faster inflammatory stage and wound closure in NFI-C(-/-) mice. Expression of PDGFA and PDGF-receptor alpha were increased in wounds of NFI-C(-/-) mice, explaining the early recruitment of macrophages and fibroblasts. Differentiation of fibroblasts to contractile myofibroblasts was also elevated, providing a rationale for faster wound closure. Taken together with the role of TGF-beta in myofibroblast differentiation, our results imply a central role of NFI-C in the interplay of the two signaling pathways and in regulation of the progression of tissue regeneration.

Impact of preoperative risk factors on morbidity after esophagectomy: is there room for improvement?

BACKGROUND: Despite progress in multidisciplinary treatment of esophageal cancer, oncologic esophagectomy is still the cornerstone of therapeutic strategies. Several scoring systems are used to predict postoperative morbidity, but in most cases they identify nonmodifiable parameters. The aim of this study was to identify potentially modifiable risk factors associated with complications after oncologic esophagectomy. METHODS: All consecutive patients with complete data sets undergoing oncologic esophagectomy in our department during 2001-2011 were included in this study. As potentially modifiable risk factors we assessed nutritional status depicted by body mass index (BMI) and preoperative serum albumin levels, excessive alcohol consumption, and active smoking. Postoperative complications were graded according to a validated 5-grade system. Univariate and multivariate analyses were used to identify preoperative risk factors associated with the occurrence and severity of complications. RESULTS: Our series included 93 patients. Overall morbidity rate was 81 % (n = 75), with 56 % (n = 52) minor complications and 18 % (n = 17) major complications. Active smoking and excessive alcohol consumption were associated with the occurrence of severe complications, whereas BMI and low preoperative albumin levels were not. The simultaneous presence of two or more of these risk factors significantly increased the risk of postoperative complications. CONCLUSIONS: A combination of malnutrition, active smoking and alcohol consumption were found to have a negative impact on postoperative morbidity rates. Therefore, preoperative smoking and alcohol cessation counseling and monitoring and improving the nutritional status are strongly recommended.

The role of the hypothalamic kisspeptin/GPR54 system in the control of GnRH neurons

Summary : The hypothalamus represents less than 1 % of the total volume of the brain tissue, yet it plays a crucial role in endocrine regulations. Puberty is defined as a process leading to physical, sexual and psychosocial maturation. The hypothalamus is central to this process, via the activation of GnRH neurons. Pulsatile GnRH secretion, minimal during childhood, increases with the onset of puberty. The primary function of GnRH is to regulate the growth, development and function of testes in boys and ovaries in girls, by stimulating the pituitary gland secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Several factors contribute to the timing of puberty, including sex and ethnicity, genetics, dietary intake and energy expenditure. Kisspeptins constitute a family of small peptides arising from the proteolytic cleavage of metastin, a peptide with 54 amino acids initially purified from human placenta. These kisspeptins were the subject of much attention following their discovery because of their antimetastatic properties, but it was more recently that their determining role in the reproductive function was demonstrated. It was shown that kisspeptins are ligands of a receptor, GPR54, whose natural inactivating mutation in humans, or knockout in the mouse, lead to infertility. GnRH neurons play a pivotal role in the central regulation of fertility. Kisspeptin greatly increases GnRH release and GnRH neuron firing activity, but the neurobiological mechanisms for these actions are unknown. Gprotein-coupled receptor 54, the receptor for kisspeptin, is expressed by GnRH neurons as well as other hypothalamic neurons, suggesting that both direct and indirect effects are possible. In the first part of my thesis, we investigated a possible connection between the acceleration of sexual development induced by leptin and hypothalamic metastin neurons. However, the data generated by our preliminary experiments confirmed that the commercially available antibodies are non-specific. This finding constituted a major drawback for our studies, which relied heavily upon the neuroanatomical study of the hypothalamic metastinergic pathways to elucidate their sensitivity to exogenous leptin. Therefore, we decided to postpone any further in vivo experiment until a better antibody becomes available, and focused on in vitro studies to better understand the mechanisms of action of kisspeptins in the modulation of the activity of GnRH neurons. We used two GnRH-expressing neuronal cell lines to investigate the cellular and molecular mechanisms of action of metastin in GnRH neurons. We demonstrated that kisspeptin induces an early activation of the MAP kinase intracellular signaling pathway in both cell lines, whereas the SAP/JNK or the Akt pathways were unaffected. Moreover, we found an increase in GnRH mRNA levels after 6h of metastin stimulation. Thus, we can conclude that kisspeptin regulates GnRH neurons both at the secretion and the gene expression levels. The MAPK pathway is the major pathway activated by metastin in GnRH expressing neurons. Taken together, these data provide the first mechanism of action of kisspeptin on GnRH neurons. Résumé : L'hypothalamus est une zone située au centre du cerveau, dont il représente moins de 1 du volume total. La puberté est la période de transition entre l'enfance et l'age adulte, qui s'accompagne de transformations somatiques, psychologiques, métaboliques et hormonales conduisant à la possibilité de procréer. La fonction principale de la GnRH est la régulation de la croissance, du développement et de la fonction des testicules chez les hommes, et des ovaires chez les femmes en stimulant la sécrétion de l'hormone lutéinisante (LH) et de l'hormone folliculostimulante (FSH) par la glande hypophysaire. Plusieurs facteurs contribuent au déclanchement de la puberté, y compris le sexe et l'appartenance ethnique, la génétique, l'apport alimentaire et la dépense énergétique. Les Kisspeptines constituent une famille de peptides résultant de la dissociation proteolytique de la métastine, un peptide de 54 acides aminés initialement purifié à partir de placenta humain. Ces kisspeptines ont fait l'objet de beaucoup d'attention à la suite de leur découverte en raison de leurs propriétés anti-metastatiques, et c'est plus récemment que leur rôle déterminant dans la fonction reproductive a été démontré. Les kisspeptines sont des ligands du récepteur GPR54, dont la mutation inactivatrice chez l'homme, ou le knockout chez la souris, conduisent à l'infertilité par hypogonadisme hypogonadotrope. Les neurones à GnRH jouent un rôle central dans le règlement des fonctions reproductrices et la kisspeptine stimule l'activité des neurones à GnRH et la libération de GnRH par ces neurones. Toutefois, les mécanismes neurobiologiques de ces actions ne sont pas connus. Dans la première partie de ma thèse, nous avons étudié le lien potentiel entre l'accélération du développement sexuel induite par la leptine et les neurones hypothalamiques à metastine. Les données générées dans cette première série d'expériences ont malheureusement confirmé que les anticorps anti-metastine disponibles dans le commerce sont aspécifiques. Ceci a constitué un inconvénient majeur pour nos études, qui devaient fortement s'appuyer sur l' étude neuroanatomique des neurones hypothalamiques à metastine pour évaluer leur sensibilité à la leptine exogène. Nous avons donc décidé de focaliser nos travaux sur une étude in vitro des mécanismes d'action de la kisspeptine pour moduler l'activité des neurones à GnRH. Nous avons utilisé deux lignées de cellules neuronales exprimant la GnRH pour étudier les mécanismes d'action cellulaires et moléculaires de la metastine dans des neurones. Nous avons ainsi pu démontrer que la kisspeptine induit une activation précoce de la voie f de signalisation de la MAP kinase dans les deux lignées cellulaires, alors que nous n'avons observé aucune activation de la voie de signalisation de la P13 Kinase et de la SAP/JNK. Nous avons en outre démontré une augmentation de l'expression de la GnRH par la stimulation avec la Kisspeptine. L'ensemble de ces données contribue à élucider le mécanisme d'action avec lequel la kisspeptine agit dans les neurones à GnRH, en démontrant un effet sur l'expression génique de la GnRH. Nous pouvons également conclure que la voie de la MAPK est la voie principale activée par la metastine dans les neurones exprimant la GnRH.

IB1/JIP-1 controls JNK activation and increased during prostatic LNCaP cells neuroendocrine differentiation.

The scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1) is a modulator of the c-Jun N-terminal kinase (JNK) activity, which has been implicated in pleiotrophic cellular functions including cell differentiation, division, and death. In this study, we described the presence of IB1/JIP-1 in epithelium of the rat prostate as well as in the human prostatic LNCaP cells. We investigated the functional role of IB1/JIP-1 in LNCaP cells exposed to the proapoptotic agent N-(4-hydroxyphenyl)retinamide (4-HPR) which induced a reduction of IB1/JIP-1 content and a concomittant increase in JNK activity. Conversely, IB1/JIP-1 overexpression using a viral gene transfer prevented the JNK activation and the 4-HPR-induced apoptosis was blunted. In prostatic adenocarcinoma cells, the neuroendocrine (NE) phenotype acquisition is associated with tumor progression and androgen independence. During NE transdifferentiation of LNCaP cells, IB1/JIP-1 levels were increased. This regulated expression of IB1/JIP-1 is secondary to a loss of the neuronal transcriptional repressor neuron restrictive silencing factor (NRSF/REST) function which is known to repress IB1/JIP-1. Together, these results indicated that IB1/JIP-1 participates to the neuronal phenotype of the human LNCaP cells and is a regulator of JNK signaling pathway.

La influència del dolor i els factors relacionats amb el dolor sobre el nivell de funcionament dels joves entre 8 i 16 anys d'edat

Projecte de recerca elaborat a partir d’una estada al Pain Management Unit de la University of Bath-Royal National Hospital for Rheumatic Disease, a Gran Bretanya, entre juliol i octubre del 2006. El dolor i la discapacitat que hi va associada són experiències comunes. Les relacions entre variables de tipus biològic, social i psicològic han estat rarament investigades en mostres de nens que informen de dolor però extrets de contextos no clínics. L’objectiu d'aquest estudi va ser identificar els predictors de funcionament ens nens escolaritzats, que informaven que el dolor era una experiència força freqüent. Mig miler de nens d'entre 8 i 16 anys i els seus pares van participar en aquest estudi de disseny transversal. Informació de tipus biològic, social i psicològic era recollida a través d'entrevistes individualitzades amb els nens i la complementació de qüestionaris auto-informats per pares. El nivell funcionament del nen va ser avaluat a partir de 4 indicadors: auto-informes de qualitat de vida, assistència a l'escola, implicació en activitats físiques i/o esportives fòra de l'escola, i implicació en activitats de tipus sedentari. Es van recollir diversos tipus de variables psicològiques referents a l’afrontament del nen davant del dolor i tipus d'atenció dels pares a les conductes de dolor dels seu fill. Els resultats mostren un patró variable en funció de la mesura de funcionament específica utilitzada. Més específicament, s'ha proposat que la resposta de por a les experiències de dolor diàries pot ser un important predictor de funcionament. Són necessaris més estudis sobre la relació entre dolor i nivell d'implicació en conductes actives o sedentàries en nens.

Desenvolupament d'hidrogels superficials que contenen motius d'adhesió cel·lular

Projecte de recerca elaborat a partir d’una estada al Departament d’Enginyeria Química del Massachusetts Institute of Technology entre abril i octubre del 2006. S’ha dissenyat i sintetitzat uns nous films polimèrics, amb aplicacions en l’àmbit de l’enginyeria de teixits, utilitzant la tècnica anomenada iCVD (initiated Chemical Vapor Deposition), prèviament desenvolupada pel grup receptor. Es tracta d’uns hidrogels superficials de gruix controlable, que incorporen un monòmer fluorat, el qual s’havia estudiat extensament en el grup d’origen. Aquest monòmer es caracteritza per reaccionar molt fàcilment amb pèptids, de manera que aquests queden units covalentment a la superfície. Diferents estratègies pel desenvolupament d’aquests copolímers han estat avaluades, tant des del punt de vista purament sintètic com de la pròpia aplicació. Les condicions de polimerització han estat optimitzades i els hidrogels s’han caracteritzat químicament per tècniques espectroscòpiques (FTIR, XPS), i físicament per angle de contacte i el·lipsometria. D’aquesta manera, s’ha estudiat la capacitat dels hidrogels d’absorbir aigua i alhora augmentar el seu gruix, depenent de la quantitat d’agent reticulant introduït i de la incorporació del nou monòmer. A continuació, s’han optimitzat les condicions de reacció d’aquestes superfícies amb pèptids que incorporen una molècula fluorescent, la qual permet detectar fàcilment per microscòpia de fluorescència si la reacció ha tingut lloc. Una vegada la plataforma ha estat posada a punt, s’han iniciat assajos cel·lulars tant amb fibroblasts embriònics de ratolí com amb cèl·lules humanes umbilicals. Els resultats preliminars suggereixen una morfologia diferent de les cèl·lules segons si es cultiven sobre films modificats amb pèptids que promouen l’adhesió cel·lular o sobre les seves seqüències permutades no actives. Però, el més interessant és que també s’han observat certes diferències depenent si els films contenen el component hidrogel o no, fet que suggeriria un paper actiu d’aquests noves superfícies en el comportament cel·lular.

Host factors and genetic susceptibility to infections due to intracellular bacteria and fastidious organisms.

While genetic polymorphisms play a paramount role in tuberculosis (TB), less is known about their contribution to the severity of diseases caused by other intracellular bacteria and fastidious microorganisms. We searched electronic databases for observational studies reporting on host factors and genetic predisposition to infections caused by intracellular fastidious bacteria published up to 30 May 2014. The contribution of genetic polymorphisms was documented for TB. This includes genetic defects in the mononuclear phagocyte/T helper cell type 1 (Th1) pathway contributing to disseminated TB disease in children and genome-wide linkage analysis (GWAS) in reactivated pulmonary TB in adults. Similarly, experimental studies supported the role of host genetic factors in the clinical presentation of illnesses resulting from other fastidious intracellular bacteria. These include IL-6 -174G/C or low mannose-binding (MBL) polymorphisms, which are incriminated in chronic pulmonary conditions triggered by C. pneumoniae, type 2-like cytokine secretion polymorphisms, which are correlated with various clinical patterns of M. pneumoniae infections, and genetic variation in the NOD2 gene, which is an indicator of tubal pathology resulting from Chamydia trachomatis infections. Monocyte/macrophage migration and T lymphocyte recruitment defects are corroborated to ineffective granuloma formation observed among patients with chronic Q fever. Similar genetic polymorphisms have also been suggested for infections caused by T. whipplei although not confirmed yet. In conclusion, this review supports the paramount role of genetic factors in clinical presentations and severity of infections caused by intracellular fastidious bacteria. Genetic predisposition should be further explored through such as exome sequencing.

The study of insect blood-feeding behaviour: 1. Feeding equipment, physical and endogenous factors, dose effect analysis, and diet destination

Experimental techniques that we have found useful during our studies of insect blood-feeding behaviour are reviewed. Some of the principal findings resulting from these techniques are discussed. Where directly applicable, the work of others is included, but no complete review of the subject has been attempted.

The cell biology of mosquito vitellogenesis

Insect vitellogenesis involves coordinated activities of the fat body and oocytes. We have studied these activities at the cellular level in the mosquito. During each vitellogenic cycle, the fat body undergoes three successive stages: 1) proliferation of biosynthetic organelles, 2) vitellogenin synthesis, 3) termination of vitellogenin synthesis and degradation of biosynthetic organelles by lysosomes. Analysis with monoclonal antibodies and radiolabelling demonstrated that the mosquito yolk protein consists of two subunits (200-kDa and 65-kDa). Both subunits are glycosylated, their carbohydrate moieties are composed of high-mannose oligosaccharides. The yolk protein subunits are derived from a single 220 kDa precursor detected by an in vitro translation. Oocytes become competent to internalize proteins as a result of juvenile hormone-mediated biogenesis of endocytotic organelles. The yolk protein is then accumulated by receptor-mediated endocytosis. A pathway of the yold protein and factors determining its routing in the oocyte have been studied.

Cell-matrix interactions in Schistosomal portal fibrosis: a dynamic event

In recent years, one of the most significant progress in the understanding of liver diseases was the demonstration that liver fibrosis is a dynamic process resulting from a balance between synthesis and degradation of several matrix components, collagen in particular. Thus, fibrosis has been found to be a very early event during liver diseases, be it of toxic, viral or parasitic origin, and to be spontaneously reversible, either partially or totally. In liver fibrosis cell matrix interactions are dependent on the existence of the many factors (sometimes acting in combination) which produce the same events at the cellular and molecular levels. These events are: (i) the recruitment of fiber-producing cells, (ii) their proliferation, (iii) the secretion of matrix constituents of the extracellular matrix, and (iv) the remodeling and degradation of the newly formed matrix. All these events represent, at least in principle, a target for a therapeutic intervention aimed at influencing the experimentally induced hepatic fibrosis. In this context, hepatosplenic schistosomiasis is of particular interest, being an immune cell-mediated granulomatous disease and a model of liver fibrosis allowing extensive studies in human and animals as well as providing original in vitro models.

Developmental expression of glial fibrillary acidic protein and glutamine synthetase in serum-free aggregating cell cultures of fetal rat telencephalon.

Serum-free aggregating cell cultures of fetal rat telencephalon were examined by a combined biochemical and double-labeling immunocytochemical study for the developmental expression of glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS). It was found that these two astroglial markers are co-expressed at different developmental stages in vitro. During the phase of cellular maturation (i.e. between days 14 and 34), GFAP levels and GS activity increase rapidly and in parallel. At the same time, the number of immunoreactive cells increase while the long and thick processes staining in early cultures gradually disappear. The present results demonstrate that in this particular cell culture system only one type of astrocytes develops which expresses both GFAP and GS and which attains a relatively high degree of maturation.