Daily cycling of nitric oxide synthase (NOS) in the hippocampus of pigeons (C. livia)

Abstract: Background: Nitric oxide synthase (NOS) is essential for the synthesis of nitric oxide (NO), a non-conventional neurotransmitter with an important role in synaptic plasticity underlying processes of hippocampus-dependent memory and in the regulation of biological clocks and circadian rhythms. Many studies have shown that both the NOS cytosolic protein content and its enzymatic activity present a circadian variation in different regions of the rodent brain, including the hippocampus. The present study investigated the daily variation of NOS enzymatic activity and the cytosolic content of nNOS in the hippocampus of pigeons. Results: Adult pigeons kept under a skeleton photoperiod were assigned to six different groups. Homogenates of the hippocampus obtained at six different times-of-day were used for NOS analyses. Both iNOS activity and nNOS cytosolic protein concentrations were highest during the subjective light phase and lowest in the subjective dark phase of the circadian period. ANOVA showed significant time differences for iNOS enzymatic activity (p < 0.05) and for nNOS protein content (p < 0.05) in the hippocampus. A significant daily rhythm for both iNOS and nNOS was confirmed by analysis with the Cosinor method (p < 0.05). The present findings indicate that the enzymatic activity of iNOS and content of nNOS protein in the hippocampus of pigeons exhibit a daily rhythm, with acrophase values occurring during the behavioral activity phase. Conclusions: The data corroborate the reports on circadian variation of NOS in the mammalian hippocampus and can be considered indicative of a dynamic interaction between hippocampus-dependent processes and circadian clock mechanisms.

Daily cycling of nitric oxide synthase (NOS) in the hippocampus of pigeons (C. livia)

Background: Nitric oxide synthase (NOS) is essential for the synthesis of nitric oxide (NO), a non-conventional neurotransmitter with an important role in synaptic plasticity underlying processes of hippocampus-dependent memory and in the regulation of biological clocks and circadian rhythms. Many studies have shown that both the NOS cytosolic protein content and its enzymatic activity present a circadian variation in different regions of the rodent brain, including the hippocampus. The present study investigated the daily variation of NOS enzymatic activity and the cytosolic content of nNOS in the hippocampus of pigeons. Results: Adult pigeons kept under a skeleton photoperiod were assigned to six different groups. Homogenates of the hippocampus obtained at six different times-of-day were used for NOS analyses. Both iNOS activity and nNOS cytosolic protein concentrations were highest during the subjective light phase and lowest in the subjective dark phase of the circadian period. ANOVA showed significant time differences for iNOS enzymatic activity (p < 0.05) and for nNOS protein content (p < 0.05) in the hippocampus. A significant daily rhythm for both iNOS and nNOS was confirmed by analysis with the Cosinor method (p < 0.05). The present findings indicate that the enzymatic activity of iNOS and content of nNOS protein in the hippocampus of pigeons exhibit a daily rhythm, with acrophase values occurring during the behavioral activity phase. Conclusions: The data corroborate the reports on circadian variation of NOS in the mammalian hippocampus and can be considered indicative of a dynamic interaction between hippocampus-dependent processes and circadian clock mechanisms.

The Effect of Load Follow Operation on Fuel Behaviour in Loviisa NPP

Työn tarkoituksena oli analysoida polttoainesauvojen käyttäytymistä Loviisan ydinvoimalaitoksen tehonsäätöajossa. Sähkömarkkinoiden vapautuminen Pohjoismaissa sekä tämän seurauksena vaihteleva sähkön markkinahinta ovat ajaneet sähkötuottajat tilanteeseen, jossa tuotanto aiempaa enemmän mukautuu markkinatilanteeseen. Näin ollen myös Loviisan ydinvoimalaitoksen osallistuminen sähkön tuotannon säätelyyn saattaa tulevaisuudessa olla ajankohtaista. Ennen kuin reaktorin tehonsäätöajoa voidaan alkaa toteuttaa, tulee varmistua siitä, että polttoainesauvassa tehonsäätöjen seurauksena tapahtuvat muutokset eivät aiheuta epäsuotuisia käyttäytymisilmiöitä. Työssä tarkastellaan kahden Loviisan ydinvoimalaitoksen polttoainetoimittajan, British Nuclear Fuels plc:n ja venäläisen TVEL:n ensinippujen polttoainesauvan käyttäytymistä tehonsäätötapauksissa. Työssä tarkastellut tehonsäätötapaukset on pyritty valitsemaan niin, että ne kuvaisivat tulevaisuudessa mahdollisesti toteutettavia tehonsäätöjä. Laskentatapauksien sauvatehohistoriat on generoitu HEXBU-3D sydänsimulaattoriohjelmalla lasketun nelivuotisen perustehohistorian pohjalta lisäämällä säätösauvan aiheuttama reaktoritehon muutos, säätösauvan viereisen polttoainenipun aksiaalitehon muutos sekä säätösauvan rakenteen aiheuttama paikallinen tehopiikki säätösauvan vieressä. Työssä tarkastellaan tehonsäätöjen toteuttamista eri tehotasoille ja vaihtelevilla määrillä tehonsäätösyklejä. Työssä käsitellyt laskentatapaukset on jaoteltu reaktorin ajotavan mukaan seuraavasti: peruskuorma-ajo, viikonloppusäätö ja päiväsäätö. Laskenta suoritettiin ydinpolttoaineen käyttäytymistä kuvaavaa ENIGMA-B 7.3.0 ohjelmaa apuna käyttäen. Laskelmien tulokset osoittavat, että molempien polttoainetoimittajien ensinippujen sauvat kestävät reaktorin tehonsäätöajoa rajoituksetta tarkastelluissa laskentatapauksissa. ENIGMA-ohjelman sisältämät mallit, jotka ennustavat polttoainesauvan suojakuoren vaurioitumistodennäköisyyden jännityskorroosion tai väsymismurtuman kautta, eivät näytä mitään merkkejä vaurioitumisesta. BNFL:n polttoainesauva saavuttaa kuitenkin suurempia väsymismurtumatodennäköisyyden arvoja. Tämä johtuu siitä, että polttoainepelletin ja suojakuoren välinen mekaaninen vuorovaikutus syntyy BNFL:n sauvassa aikaisemmin, joka taas johtaa suurempaan määrään sauvaa rasittavia muodonmuutoksia tehonnostotilanteissa. TVEL:n Zr1%Nb -materiaalista valmistetun suojakuoren käyttäytymistä ei voida kuitenkaan suoraan näiden laskujen perusteella arvioida, sillä ENIGMA-ohjelman mallit perustuvat Zircaloy-suojakuorimateriaaleilla suoritettuihin kokeisiin.

Characterization of the molecular clockwork in the cockroach Rhyparobia maderae

Der Wechsel von Tag und Nacht erzeugt einen regelmäßigen Rhythmus von verschiedenen Umweltreizen, allen voran Licht und Temperatur. Fast jedes bis zum heutigen Tage untersuchte Lebewesen besitzt einen endogenen Mechanismus zur Zeitwahrnehmung, und diese "innere Uhr" befähigt Lebewesen dazu, sich vorausschauend an rhythmische Umwelt-Änderungen anzupassen. Circadiane Rhythmen bestehen auch ohne jegliche äußere Reize und basieren auf einem molekularen Rückkopplungs-Mechanismus, der Rhythmen in Genexpression und Proteinkonzentration von etwa 24 Stunden erzeugt. Obwohl sich die grundsätzlichen Mechanismen und Komponenten dieses molekularen Uhrwerks in allen Insekten ähneln, zeigte sich jedoch immer mehr, dass es im Detail doch wesentliche Unterschiede zwischen verschiedenen Insektengruppen gibt. Während das molekulare Uhrwerk der Fruchtfliege Drosophila melanogaster inzwischen sehr gut untersucht ist, fehlen bei den meisten Insektengruppen immernoch eingehende Untersuchungen. Fast nichts ist über die molekulare Basis von circadianen Rhythmen bei der Schabe Rhyparobia maderae bekannt, obwohl diese Art bereits seit Langem als Modellorganismus in der Chronobiologie dient. Um mit der Forschung am molekularen, circadianen System von R. maderae zu beginnen, wurde die Struktur und das Expressionsprofil der core feedback loop Gene per, tim1 und cry2 analysiert. Mittels degenerierten Primern und RACE konnte das vollständige offene Leseraster (OLR) von rmPer und rmCry2, und ein Teil des rmTim1 OLR kloniert werden. Eine phylogenetische Analyse gruppierte rmPER und rmCRY2 gemeinsam mit den Orthologa hemimetaboler Insekten. Viele bei D. melanogaster funktionell charakterisierte Domänen sind bei diesen Proteinen konserviert, was auf eine ähnliche Funktion in der inneren Uhr von R. maderae hinweist. Mittels quantitativer PCR konnte gezeigt werden, dass die mRNA von rmPer, rmTim1 und rmCry2 in verschiedenen Lichtregimen in der gleichen Phasenlage Tageszeit-abhängig schwankt. Die Phasenlage stellte sich bei unterschiedlichen Photoperioden jeweils relativ zum Beginn der Skotophase ein, mit Maxima in der ersten Hälfte der Nacht. Auch im Dauerdunkel zeigen sich Rhythmen in der rmTim1 und rmCry2 Expression. Die Amplitude der rmPer Expressionsrhythmen war jedoch so gering, dass keine signifikanten Unterschiede zwischen den einzelnen Zeitgeberzeiten (ZT) festgestellt werden konnten. Mittels Laufrad-Assays wurde untersucht wie Kurz- und Langtag Lichtregime die Verhaltensrhythmen beeinflussen. Es konnten nur Unterschiede in der Periodenlänge unter freilaufenden Bedingungen festgestellt werden, wenn höhere Lichtintensitäten (1000lx) zur Synchronisation (entrainment) genutzt wurden. Die Periode des freilaufenden Rhythmus war bei Tieren aus dem Kurztag länger. Die photoperiodische Plastizität zeigte sich also auch auf Verhaltensebene, obwohl höhere Lichtintensitäten notwendig waren um einen Effekt zu beobachten. Basierend auf den Sequenzen der zuvor klonierten OLR wurden gegen rmPER, rmTIM1 und rmCRY2 gerichtete Antikörper hergestellt. Die Antikörper gegen rmPER und rmTIM1 erkannten in western blots sehr wahrscheinlich spezifisch das jeweilige Protein. Zeitreihen von Gehirngewebe-Homogenisaten zeigten keinen offensichtlichen circadianen Rhythmus in der Proteinkonzentration, wahrscheinlich auf Grund einer Oszillation mit niedriger Amplitude. In Immunhistochemischen Färbungen konnte nur mit dem gegen rmPER gerichteten Antikörper aus Kaninchen ein Signal beobachtet werden. Beinahe jede Zelle des Zentralnervensystems war rmPER-immunreaktiv im Zellkern. Es konnten keine Unterschiede zwischen den untersuchten ZTs festgestellt werden, ähnlich wie bei den western blot Zeitreihen. In dieser Studie konnten erstmals molekulare Daten der circadianen Uhr von R. maderae erfasst und dargestellt werden. Die Uhrgene per, tim1 und cry2 werden in dieser Schabenart exprimiert und ihre Domänenstruktur sowie das circadiane Expressionsmuster ähneln dem hypothetischen ursprünglichen Insektenuhrwerk, welches der circadianen Uhr von Vertebraten nahesteht. Das molekulare Uhrwerk von R. maderae kann sich an unterschiedliche Photoperioden anpassen, und diese Anpassungen manifestieren sich im Expressionsprofil der untersuchten Uhrgene ebenso wie im Verhalten.

Cycling Dof Factors: Molecular and functional characterization of Arabidopsis thaliana AtCDF3 and tomato (Solanum lycopersicum L.) SlCDF3 in response to abiotic stress

El tomate (Solanum lycopersicum L.) es considerado uno de los cultivos hortícolas de mayor importancia económica en el territorio Español. Sin embargo, su producción está seriamente afectada por condiciones ambientales adversas como, salinidad, sequía y temperaturas extremas. Para resolver los problemas que se presentan en condiciones de estrés, se han empleado una serie de técnicas culturales que disminuyen sus efectos negativos, siendo de gran interés el desarrollo de variedades tolerantes. En este sentido la obtención y análisis de plantas transgénicas, ha supuesto un avance tecnológico, que ha facilitado el estudio y la evaluación de genes seleccionados en relación con la tolerancia al estrés. Estudios recientes han mostrado que el uso de genes reguladores como factores de transcripción (FTs) es una gran herramienta para obtener nuevas variedades de tomate con mayor tolerancia a estreses abióticos. Las proteínas DOF (DNA binding with One Finger) son una familia de FTs específica de plantas (Yangisawa, 2002), que están involucrados en procesos fisiológicos exclusivos de plantas como: asimilación del nitrógeno y fijación del carbono fotosintético, germinación de semilla, metabolismo secundario y respuesta al fotoperiodo pero su preciso rol en la tolerancia a estrés abiótico se desconoce en gran parte. El trabajo descrito en esta tesis tiene como objetivo estudiar genes reguladores tipo DOF para incrementar la tolerancia a estrés abiotico tanto en especies modelo como en tomate. En el primer capítulo de esta tesis se muestra la caracterización funcional del gen CDF3 de Arabidopsis, así como su papel en la respuesta a estrés abiótico y otros procesos del desarrollo. La expresión del gen AtCDF3 es altamente inducido por sequía, temperaturas extremas, salinidad y tratamientos con ácido abscísico (ABA). La línea de inserción T-DNA cdf3-1 es más sensible al estrés por sequía y bajas temperaturas, mientras que líneas transgénicas de Arabidopsis 35S::AtCDF3 aumentan la tolerancia al estrés por sequía, osmótico y bajas temperaturas en comparación con plantas wild-type (WT). Además, estas plantas presentan un incremento en la tasa fotosintética y apertura estomática. El gen AtCDF3 se localiza en el núcleo y que muestran una unión específica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activación transcripcional en ensayos de protoplastos de Arabidopsis. El dominio C-terminal de AtCDF3 es esencial para esta localización y su capacidad activación, la delección de este dominio reduce la tolerancia a sequía en plantas transgénicas 35S::AtCDF3. Análisis por microarray revelan que el AtCDF3 regula un set de genes involucrados en el metabolismo del carbono y nitrógeno. Nuestros resultados demuestran que el gen AtCDF3 juega un doble papel en la regulación de la respuesta a estrés por sequía y bajas temperaturas y en el control del tiempo de floración. En el segundo capítulo de este trabajo se lleva a cabo la identificación de 34 genes Dof en tomate que se pueden clasificar en base a homología de secuencia en cuatro grupos A-D, similares a los descritos en Arabidopsis. Dentro del grupo D se han identificado cinco genes DOF que presentan características similares a los Cycling Dof Factors (CDFs) de Arabidopsis. Estos genes son considerados ortólogos de Arabidopsis CDF1-5, y han sido nombrados como Solanum lycopersicum CDFs o SlCDFs. Los SlCDF1-5 son proteínas nucleares que muestran una unión específica al ADN con diferente afinidad a secuencias diana y presentan diversas capacidades de activación transcripcional in vivo. Análisis de expresión de los genes SlCDF1-5 muestran diferentes patrones de expresión durante el día y son inducidos de forma diferente en respuesta a estrés osmótico, salino, y de altas y bajas temperaturas. Plantas de Arabidopsis que sobre-expresan SlCDF1 y SlCDF3 muestran un incremento de la tolerancia a la sequía y salinidad. Además, de la expresión de varios genes de respuesta estrés como AtCOR15, AtRD29A y AtERD10, son expresados de forma diferente en estas líneas. La sobre-expresión de SlCDF3 en Arabidopsis promueve un retardo en el tiempo de floración a través de la modulación de la expresión de genes que controlan la floración como CONSTANS (CO) y FLOWERING LOCUS T (FT). En general, nuestros datos demuestran que los SlCDFs están asociados a funciones aun no descritas, relacionadas con la tolerancia a estrés abiótico y el control del tiempo de floración a través de la regulación de genes específicos y a un aumento de metabolitos particulares. ABSTRACT Tomato (Solanum lycopersicum L.) is one of the horticultural crops of major economic importance in the Spanish territory. However, its production is being affected by adverse environmental conditions such as salinity, drought and extreme temperatures. To resolve the problems triggered by stress conditions, a number of agricultural techniques that reduce the negative effects of stress are being frequently applied. However, the development of stress tolerant varieties is of a great interest. In this direction, the technological progress in obtaining and analysis of transgenic plants facilitated the study and evaluation of selected genes in relation to stress tolerance. Recent studies have shown that a use of regulatory genes such as transcription factors (TFs) is a great tool to obtain new tomato varieties with greater tolerance to abiotic stresses. The DOF (DNA binding with One Finger) proteins form a family of plant-specific TFs (Yangisawa, 2002) that are involved in the regulation of particular plant processes such as nitrogen assimilation, photosynthetic carbon fixation, seed germination, secondary metabolism and flowering time bur their precise roles in abiotic stress tolerance are largely unknown. The work described in this thesis aims at the study of the DOF type regulatory genes to increase tolerance to abiotic stress in both model species and the tomato. In the first chapter of this thesis, we present molecular characterization of the Arabidopsis CDF3 gene as well as its role in the response to abiotic stress and in other developmental processes. AtCDF3 is highly induced by drought, extreme temperatures, salt and abscisic acid (ABA) treatments. The cdf3-1 T-DNA insertion mutant was more sensitive to drought and low temperature stresses, whereas the AtCDF3 overexpression enhanced the tolerance of transgenic plants to drought, cold and osmotic stress comparing to the wild-type (WT) plants. In addition, these plants exhibit increased photosynthesis rates and stomatal aperture. AtCDF3 is localized in the nuclear region, displays specific binding to the canonical DNA target sequences and has a transcriptional activation activity in Arabidopsis protoplast assays. In addition, the C-terminal domain of AtCDF3 is essential for its localization and activation capabilities and the deletion of this domain significantly reduces the tolerance to drought in transgenic 35S::AtCDF3 overexpressing plants. Microarray analysis revealed that AtCDF3 regulated a set of genes involved in nitrogen and carbon metabolism. Our results demonstrate that AtCDF3 plays dual roles in regulating plant responses to drought and low temperature stress and in control of flowering time in vegetative tissues. In the second chapter this work, we carried out to identification of 34 tomato DOF genes that were classified by sequence similarity into four groups A-D, similar to the situation in Arabidopsis. In the D group we have identified five DOF genes that show similar characteristics to the Cycling Dof Factors (CDFs) of Arabidopsis. These genes were considered orthologous to the Arabidopsis CDF1 - 5 and were named Solanum lycopersicum CDFs or SlCDFs. SlCDF1-5 are nuclear proteins that display specific binding to canonical DNA target sequences and have transcriptional activation capacities in vivo. Expression analysis of SlCDF1-5 genes showed distinct diurnal expression patterns and were differentially induced in response to osmotic, salt and low and high temperature stresses. Arabidopsis plants overexpressing SlCDF1 and SlCDF3 showed increased drought and salt tolerance. In addition, various stress-responsive genes, such as AtCOR15, AtRD29A and AtERD10, were expressed differently in these lines. The overexpression of SlCDF3 in Arabidopsis also results in the late flowering phenotype through the modulation of the expression of flowering control genes such CONSTANS (CO) and FLOWERING LOCUS T (FT). Overall, our data connet SlCDFs to undescribed functions related to abiotic stress tolerance and flowering time through the regulation of specific target genes and an increase in particular metabolites.

Thermal Cycling Stability of Silica Membranes for Gas Separation

Hydrogen is being seen as an alternative energy carrier to conventional hydrocarbons to reduce greenhouse gas emissions. High efficiency separation technologies to remove hydrogen from the greenhouse gas, carbon dioxide, are therefore in growing demand. Traditional thermodynamic separation systems utilise distillation, absorption and adsorption, but are limited in efficiency at compact scales. Molecular sieve silica (MSS) membranes can perform this separation as they have high permselectivity of hydrogen to carbon dioxide, but their stability under thermal cycling is not well reported. In this work we exposed a standard MSS membrane and a carbonised template MSS (CTMSS) membrane to thermal cycling from 100 to 450°C. The standard MSS and carbonised template CTMSS membranes both showed permselectivity of helium to nitrogen dropping from around 10 to 6 in the first set of cycles, remaining stable until the last test. The permselectivity drop was due to small micropore collapse, which occurred via structure movement during cycling. Simulating single stage membrane separation with a 50:50 molar feed of H2:CO2, H2 exiting the permeate stream would start at 79% and stabilise at 67%. Higher selectivity membranes showed less of a purity drop, indicating the margin at which to design a stable membrane separation unit for CO2 capture.

Folic acid supplementation during early hepatocarcinogenesis: cellular and molecular effects

Folic acid (FA) supplementation during carcinogenesis is controversial. Considering the impact of liver cancer as a public health problem and mandatory FA fortification in several countries, the role of FA supplementation in hepatocarcinogenesis should be elucidated. We evaluated FA supplementation during early hepatocarcinogenesis. Rats received daily 0.08 mg (FA8 group) or 0.16 mg (FA16 group) of FA/100 g body weight or water (CO group, controls). After a 2-week treatment, animals were subjected to the ""resistant hepatocyte"" model of hepatocarcinogenesis (initiation with diethylnitrosamine, selection/promotion with 2-acetylaminofluorene and partial hepatectomy) and euthanized after 8 weeks of treatment. Compared to the CO group, the FA16 group presented: reduced (p < 0.05) number of persistent and increased (p < 0.05) number of remodeling glutathione S-transferase (GST-P) positive preneoplastic lesions (PNL); reduced (p < 0.05) cell proliferation in persistent GST-P positive PNL; decreased (p < 0.05) hepatic DNA damage; and a tendency (p < 0.10) for decreased c-myc expression in microdissected PNL. Regarding all these parameters, no differences (p > 0.05) were observed between CO and FA8 groups. FA-treated groups presented increased hepatic levels of S-adenosylmethionine but only FA16 group presented increased S-adenosylmethionine/S-adenosylhomocysteine ratio. No differences (p > 0.05) were observed between experimental groups regarding apoptosis in persistent and remodeling GST-P positive PNL, and global DNA methylation pattern in microdissected PNL. Altogether, the FA16 group, but not the FA8 group, presented chemopreventive activity. Reversion of PNL phenotype and inhibition of DNA damage and of c-myc expression represent relevant FA cellular and molecular effects.

Reversible current power supply for fast-field cycling nuclear magnetic resonance equipment

The Fast Field-Cycling Nuclear Magnetic Resonance (FFC-NMR) is a technique used to study the molecular dynamics of different types of materials. The main elements of this equipment are a magnet and its power supply. The magnet used as reference in this work is basically a ferromagnetic core with two sets of coils and an air-gap where the materials' sample is placed. The power supply should supply the magnet being the magnet current controlled in order to perform cycles. One of the technical issues of this type of solution is the compensation of the non-linearities associated to the magnetic characteristic of the magnet and to parasitic magnetic fields. To overcome this problem, this paper describes and discusses a solution for the FFC-NMR power supply based on a four quadrant DC/DC converter.

Classic and molecular study of Giardia duodenalis in children from a daycare center in the region of Presidente Prudente, São Paulo, Brazil

Epidemiological studies on giardiasis by using molecular techniques such as RAPD (Randomly Amplified Polymorphic DNA) may give information on factors related to the transmission of Giardia duodenalis. The aim of this work was to assess the epidemiology of G. duodenalis in 101 children attended at a daycare center in Presidente Bernardes, SP, Brazil. After parasitological examinations in feces samples, 15 children presented cysts of G. duodenalis. Their respective parents, brothers and pets, besides the daycare center workers, also had their feces submitted to parasitological analysis. Seven mothers and nine brothers also presented G. duodenalis cysts, while fathers, daycare workers and pets (dogs) did not presented the parasite. Besides the 15 cases with G. duodenalis, other 23 children presented other enteroparasites (Entamoeba coli, Endolimax nana, Enterobius vermicularis, Ascaris lumbricoides and Trichuris trichiura). Samples of G. duodenalis cysts from children and their relatives were submitted to molecular typing by RAPD after genomic DNA extraction and amplification of a fragment of the 18S rDNA region by PCR. After examining 31 isolates of G. duodenalis (children and their respective mothers and brothers), it was concluded that the parasite transmission occurred in children, probably during daily cohabitation at the daycare center, but not at home among their relatives or pets.

Evaluation of inflammatory response induced by different types of daily contact lenses

Dissertação de mestrado em Genética Molecular

Significant molecular and systemic adaptations after repeated sprint training in hypoxia

While intermittent hypoxic training (IHT) has been reported to evoke cellular responses via hypoxia inducible factors (HIFs) but without substantial performance benefits in endurance athletes, we hypothesized that repeated sprint training in hypoxia could enhance repeated sprint ability (RSA) performed in normoxia via improved glycolysis and O(2) utilization. 40 trained subjects completed 8 cycling repeated sprint sessions in hypoxia (RSH, 3000 m) or normoxia (RSN, 485 m). Before (Pre-) and after (Post-) training, muscular levels of selected mRNAs were analyzed from resting muscle biopsies and RSA tested until exhaustion (10-s sprint, work-to-rest ratio 1ratio2) with muscle perfusion assessed by near-infrared spectroscopy. From Pre- to Post-, the average power output of all sprints in RSA was increased (p<0.01) to the same extent (6% vs 7%, NS) in RSH and in RSN but the number of sprints to exhaustion was increased in RSH (9.4+/-4.8 vs. 13.0+/-6.2 sprints, p<0.01) but not in RSN (9.3+/-4.2 vs. 8.9+/-3.5). mRNA concentrations of HIF-1alpha (+55%), carbonic anhydrase III (+35%) and monocarboxylate transporter-4 (+20%) were augmented (p<0.05) whereas mitochondrial transcription factor A (-40%), peroxisome proliferator-activated receptor gamma coactivator 1alpha (-23%) and monocarboxylate transporter-1 (-36%) were decreased (p<0.01) in RSH only. Besides, the changes in total hemoglobin variations (Delta[tHb]) during sprints throughout RSA test increased to a greater extent (p<0.01) in RSH. Our findings show larger improvement in repeated sprint performance in RSH than in RSN with significant molecular adaptations and larger blood perfusion variations in active muscles.

Cost-effectiveness of low-molecular-weight heparin for treatment of pulmonary embolism.

BACKGROUND: Low-molecular-weight heparin (LMWH) appears to be safe and effective for treating pulmonary embolism (PE), but its cost-effectiveness has not been assessed. METHODS: We built a Markov state-transition model to evaluate the medical and economic outcomes of a 6-day course with fixed-dose LMWH or adjusted-dose unfractionated heparin (UFH) in a hypothetical cohort of 60-year-old patients with acute submassive PE. Probabilities for clinical outcomes were obtained from a meta-analysis of clinical trials. Cost estimates were derived from Medicare reimbursement data and other sources. The base-case analysis used an inpatient setting, whereas secondary analyses examined early discharge and outpatient treatment with LMWH. Using a societal perspective, strategies were compared based on lifetime costs, quality-adjusted life-years (QALYs), and the incremental cost-effectiveness ratio. RESULTS: Inpatient treatment costs were higher for LMWH treatment than for UFH (dollar 13,001 vs dollar 12,780), but LMWH yielded a greater number of QALYs than did UFH (7.677 QALYs vs 7.493 QALYs). The incremental costs of dollar 221 and the corresponding incremental effectiveness of 0.184 QALYs resulted in an incremental cost-effectiveness ratio of dollar 1,209/QALY. Our results were highly robust in sensitivity analyses. LMWH became cost-saving if the daily pharmacy costs for LMWH were < dollar 51, if > or = 8% of patients were eligible for early discharge, or if > or = 5% of patients could be treated entirely as outpatients. CONCLUSION: For inpatient treatment of PE, the use of LMWH is cost-effective compared to UFH. Early discharge or outpatient treatment in suitable patients with PE would lead to substantial cost savings.

Cycling of nutrients and silicon in pigeonpea and pearl millet monoculture and intercropping

In a no-tillage system, cover crops must be used that combine shoot dry matter production and nutrient recycling. The aim of this study was to evaluate shoot dry matter production, decomposition rate and macronutrient and silicon release from pigeonpea and pearl millet in monoculture and intercropping systems. A randomized block design was used with a 3 x 6 factorial arrangement, with four replications. The first factor consisted of three cover crops (pigeonpea, pearl millet and intercropping of these cover crops) and the second consisted of six sampling times [0, 18, 32, 46, 74 and 91 days after desiccation (DAD)]. Pearl millet produced greater amounts of shoot dry matter and content of N, P, K, Ca, Mg, S, C and Si and had a higher decomposition rate and macronutrient and Si release than the other cover crops. The rates of decomposition and daily nutrient release from shoot dry matter were highest in the first period of evaluation (0-18 DAD). Over time, the C/N, C/P and C/S ratios increased, while C/Si and the decomposition rate decreased. Potassium was the nutrient most quickly released to the soil, especially from pearl millet residue. Silicon had the lowest release rate, with 62, 82 and 74 % of the total content in the shoot dry matter remaining in the last evaluation of pearl millet, pigeonpea and in the intercrop system, respectively. The shoot dry matter from the intercrop system had a different decomposition rate than that from the pearl millet monoculture and pigeonpea. Plants with greater shoot dry matter production and lower C/Si ratio are more effective in a no-tillage system for providing a more complete and persistent soil cover.

Low molecular weight carboxylic acids in oxidizing porphyry copper tailings

The distribution of low molecular weight carboxylic acids (LMWCA) was investigated in pore water profiles from two porphyry copper tailings impoundments in Chile (Piuquenes at La Andina and Cauquenes at El Teniente mine). The objectives of this study were (1) to determine the distribution of LMWCA, which are interpreted to be the metabolic byproducts of the autotroph microbial community in this low organic carbon system, and (2) to infer the potential role of these acids in cycling of Fe and other elements in the tailings impoundments. The speciation and mobility of iron, and potential for the release of H+ via hydrolysis of the ferric iron, are key factors in the formation of acid mine drainage in sulfidic mine wastes. In the low-pH oxidation zone of the Piuquenes tailings, Fe(III) is the dominant iron species and shows high mobility. LMWCA, which occur mainly between the oxidation front down to 300 cm below the tailings surface at both locations (e.g., max concentrations of 0.12 mmol/L formate, 0.17 mmol/L acetate, and 0.01 mmol/L pyruvate at Piuquenes and 0.14 mmol/L formate, 0.14 mmol/L acetate, and 0.006 mmol/L pyruvate at Cauquenes), are observed at the same location as high Fe concentrations (up to 71.2 mmol/L Fe(II) and 16.1 mmol/L Fe(III), respectively). In this zone, secondary Fe(111) hydroxides are depleted. Our data suggest that LMWCA may influence the mobility of iron in two ways. First, complexation of Fe(III), through formation of bidentate Fe(III)-LMWCA complexes (e.g., pyruvate, oxalate), may enhance the dissolution of Fe(III) (oxy)hydroxides or may prevent precipitation of Fe(III) (oxy)hydroxides. Soluble Fe(III) chelate complexes which may be mobilized downward and convert to Fe(II) by Fe(III) reducing bacteria. Second, monodentate LMWCA (e.g., acetate and formate) can be used by iron-reducing bacteria as electron donors (e.g., Acidophilum spp.), with ferric iron as the electron acceptor. These processes may, in part, explain the low abundances of secondary Fe(III) hydroxide precipitates below the oxidation front and the high concentrations of Fe(II) observed in the pore waters of some low-sulfide systems. The reduction of Fe(III) and the subsequent increase of iron mobility and potential acidity transfer (Fe(II) oxidation can result in the release of H+ in an oxic environment) should be taken in account in mine waste management strategies.

Post-transcriptional regulation of circadian gene expression by miRNAs

La grande majorité des organismes vivants ont développé un système d'horloges biologiques internes, appelées aussi horloges circadiennes, contrôlant l'expression de gênes impliqués dans de nombreux processus moléculaires et comportementaux. Au cours de la dernière décennie, des analyses « microarray » et séquençages à haut débit sur divers tissus de mammifères, indiquent que jusqu'à 20% du transcriptome serait sous contrôle circadien. Il était jusqu'à présent admis que la majorité des ARNm ayant une accumulation rythmique était générée par une transcription qui était elle-même rythmique. Toutefois, de récentes études ont suggéré qu'une proportion considérable des ARNm cycliques serait en fait générée par des mécanismes post-transcriptionnelles, incluant une régulation par micro-ARN (miARN). Lorsque j'ai débuté mon travail de thèse, l'influence des miARN sur l'expression des gènes circadiens, au niveau pangénomique, était encore méconnue. Par l'utilisation d'un modèle murin, dont la biogenèse des miARN a été spécifiquement désactivée au niveau des cellules hépatiques (knockout conditionnel pour Dicer), je me suis donc intéressée au rôle que jouaient ces molécules régulatrices sur la rythmicité de l'expression génique dans le foie. Des séquençages sur l'ensemble du transcriptome révèlent que l'horloge interne du foie est étonnement résistante à la perte totale des miARN. Nous avons cependant trouvé que les miARN agissent de façon importante sur la régulation de l'expression des gènes contrôlés par l'horloge moléculaire. La corégulation par les miARN, affectant jusqu'à 30% des gènes transcrits de façon rythmiques, conduit ainsi à une modulation de phase et d'amplitude du rythme de l'abondance des ARNm. En revanche, seuls peu de transcrits dépendent uniquement des miARN pour la rythmicité de leur accumulation. Enfin, mon travail met en évidence plusieurs miARN spécifiques, qui semblent préférentiellement moduler l'expression des gènes cycliques et permet l'identification de voies hépatiques particulièrement sujettes à une double régulation par les miARN et l'horloge biologique interne. La première masse d'analyses a essentiellement porté sur le rôle que jouent les miARN au niveau de l'expression des gènes contrôlés par l'horloge interne. Dans deux études de suivi, je me suis penchée sur deux aspects supplémentaires et complémentaires de la manière dont les miARN et l'oscillation de l'expression des gènes interagissent. Dans les hépatocytes murins, spécifiquement privés de Dicer, je me suis demandée si un phénotype horloge avait pu être masqué, dû à un entraînement stable de l'horloge du foie par l'horloge maîtresse du cerveau. J'ai donc commencé une série d'expériences ambitieuses (impliquant la mesure de la rythmicité du foie in vivo, chez l'animal vivant) afin de déséquilibrer l'entrainement de l'horloge hépatique via l'utilisation d'un protocole nutritionnel spécifique. Les premiers résultats suggèrent que dans des conditions où l'animal subit une restriction alimentaire pendant la journée, les miARN sont importants dans la cinétique d'adaptation des organes périphériques à un nouvel horaire de sustentation. Dans une deuxième ligne de recherche, j'ai plus profondément étudié quels seraient les miARN responsables des rythmes post-transcriptionnels des ARNm, en utilisant le séquençage de « small » ARN sur 24h. L'analyse est en cours et se poursuivra après l'obtention de mon diplôme. De façon générale, mon travail révèle d'importants et nouveaux rôles des miARN dans la modulation de l'expression circadienne des gènes hépatiques. De plus, le set de données générées dans l'étude déjà publiée, peut dorénavant servir de ressource valable pour de prochaines investigations sur le rôle physiologique que les miARN jouent au niveau du foie. -- Most living organisms have developed internal timing systems, called circadian clocks, to drive the rhythmic expression of genes involved in many molecular and behavioral processes. Over the last decade, microarray analyses and high- throughput sequencing from various mammalian tissues have indicated that up to 20% of the transcriptome are under circadian control. It was generally assumed that the majority of rhythmic mRNA accumulation is generated by rhythmic transcription. However, recent studies have suggested that a considerable proportion of mRNA cycling may actually be generated by post-transcriptional mechanisms, including by microRNAs. When I started my thesis work, it was still unknown how miRNAs influence circadian gene expression in a genome-wide fashion. Using a mouse model in which miRNA biogenesis can be inactivated in hepatocytes (conditional Dicer knockout mouse), I have thus addressed the role that these regulatory molecules play in rhythmic gene expression in the liver. Whole transcriptome sequencing revealed that the hepatic core clock was surprisingly resilient to total miRNA loss. However, we found that miRNAs acted as important regulators of clock-controlled gene expression. Co- regulation by miRNAs, which affected up to 30% of rhythmically transcribed genes, thus led to the modulation of phases and amplitudes of mRNA abundance rhythms. By contrast, only very few transcripts were strictly dependent on miRNAs for their rhythmic accumulation. Finally, my work highlights several specific miRNAs that appear to preferentially modulate cyclic gene expression, and identifies pathways in the liver that are particularly prone to dual regulation through miRNAs and the clock. The first bulk of analyses mainly dealt with the role that miRNAs play at the level of rhythmic clock output gene expression. In two follow-up studies I further delved into two additional, complementary aspects of how miRNAs and gene expression oscillations interact. First, I addressed whether a core clock phenotype in the hepatocyte-specific Dicer knockout could have been masked due to the stable entrainment of the liver clock by the animals' master clock in the brain. I thus started a series of ambitious experiments (involving the in vivo recording of liver rhythms in live animals) to bring the stable entrainment of the liver clock out of equilibrium using specific feeding protocols. My first results suggest that under conditions when animals are challenged by food restriction to daytime, miRNAs are important for the kinetics of adapting to unusual mealtime in peripheral tissue. In a second line of research, I have more carefully investigated which miRNAs are responsible for post- transcriptional mRNA rhythms using small RNA sequencing around-the-clock. The analyses are ongoing and will be continued after my graduation. Overall, my work uncovered important and novel roles of miRNA activity in shaping hepatic circadian gene expression; moreover, the datasets collect in the published studies can serve as a valuable resource for further investigations into the physiological roles that miRNAs play in liver. -- L'alternance du jour et de la nuit dirige depuis longtemps la vie quotidienne des êtres humains et de la plupart des organismes sur terre. Ce cycle de 24 heures façonne beaucoup de changements comportementaux et physiologiques tels que la vigilance, la température corporelle et le sommeil. Les rythmes journaliers, appelés rythmes circadiens, sont dirigés par des horloges biologiques tournant dans presque chaque cellule du corps. Une structure dans le cerveau agit en tant qu'horloge maitresse pour synchroniser les horloges internes entre elles et en fonction des signaux de jour/nuit extérieurs. Dans les cellules "les gènes de l'horloge" sont activés et désactivés une fois par jour ce qui déclenche des cycles dans lesquels des protéines sont produites de manière circadienne. Ces rythmes protéiques sont spécialisés pour chaque tissu ou organe et peuvent les aider à réaliser leurs tâches quotidiennes. Les rythmes circadiens peuvent être générés d'autres manières n'impliquant pas directement les composants des gènes de l'horloge. Les ARN messagers (ARNm) sont des molécules intermédiaires dans la production de protéines à partir d'ADN. Dans le foie des souris jusqu'à 20% des molécules d'ARNm sont produites suivant des rythmes circadiens. Le foie réalise des tâches essentielles dans le contrôle du métabolisme incluant celui des hydrates de carbone, des graisses et du cholestérol. Un timing précis est important afin de traiter les substances nutritives correctement lors des repas il en résulte une variation des quantités de certains ARNm et protéines coïncidant avec les repas. Les microARNs constituent une autre classe de molécules ARN de très petite taille qui régulent l'efficacité de traduction des ARNm en protéines et la stabilité des ARNm. Lors de mon travail de thèse, j'ai exploré de manière approfondie l'influence de ces petits régulateurs sur les rythmes circadiens du foie de souris. Ces expériences qui impliquaient le "Knock-out" d'un gène essentiel à la production de microARNs montrent qu'au lieu de générer les rythmes des ARNm, les microARNs les ajustent pour répondre aux besoins spécifiques du foie comme assurer leur pic au bon moment de la journée. Le ciblage de microARNs spécifiques peut révéler de nouvelles stratégies pour rectifier ces rythmes lorsque par exemple les fonctions métaboliques ne fonctionnent plus normalement. -- The rising and setting of the sun have long driven the daily schedules of humans and most organisms on the earth. This 24-hr cycle shapes many behavioural and physiological changes, such as alertness, body temperature, and sleep. These daily rhythms, which are called circadian rhythms, are dictated by biological clocks that are ticking in almost every single cell of the body. A region in the brain acts as a master clock to synchronize the internal clocks with each other and with the outside light/dark cycles. In cells, "core clock genes" are turned on and off once per day, which triggers cycles that cause some proteins to be produced in a circadian manner. The protein rhythms are specialized to a particular tissue or organ, and may help them to carry out their designated daily tasks. However, circadian rhythms might also be produced by other ways that do not involve these core clock components. Messenger RNAs (mRNAs) are intermediate molecules in the production of proteins from DNA. In the mouse liver, up to 20% of mRNA molecules are produced in circadian cycles. The liver performs essential tasks that control metabolism-including that of carbohydrates, fats, and cholesterol. Precisely timing when certain mRNAs and proteins reach peaks and troughs in their activities to coincide with mealtimes is important for nutrients to be properly processed. Other RNA molecules called microRNAs, i.e. RNAs of very small size, regulate at which rate mRNA molecules are translated into proteins. In my thesis work, I have explored at the influence of these small regulators on circadian rhythms in the mouse liver in greater detail. These experiments, which involved "knocking out" a gene that is essential for the production of microRNAs, show that rather than generating the mRNA rhythms, the microRNAs appear to adjust them to meet the specific needs of the liver, such as ensuring that they peak at the right time-of-day. Targeting specific microRNA molecules may reveal new strategies to tweak these rhythms, which could help to improve conditions when metabolic functions go wrong.