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The AGATA collaboration has a long-standing leadership in the development of front-end electronics for high resolution γ -ray spectroscopy using large volume high purity germanium detectors. For two decades, the AGATA collaboration has been developing state-of-the-art digital electronics processing with high resolution sampling ADC, high-speed signal transfer and fast readout to a high throughput computing (HTC) farm for on-line pulse shape analysis. The collaboration is presently addressing the next challenge of equipping a 4 π array with more than 6000 channels in high resolution mode, generating approximately 10 MHz of total trigger requests, coupled to a large variety of complementary instruments. A next generation of front-end electronics, presently under design, is based on industrial products (System on Module FPGA’s), has higher integration and lower power consumption. In this contribution, the conceptual design of the new electronics is presented. The results of the very first tests of the pre-production electronics are presented as well as future perspectives., The European Physical Journal A, 59 (6), ISSN:1434-6001, ISSN:1434-601X

Millan, Vicente Gonzalez/0000-0001-6014-2586; Huyuk, Tayfun/0000-0003-0597-9767; GOASDUFF, Alain/0000-0003-3453-3297; carturan, sara maria/0000-0002-6702-2867; Sison, Andres Illana/0000-0003-0274-3388; SAYGI, BAHADIR/0000-0001-5406-506X; Jaworski, Grzegorz/0000-0003-2241-0329; Aliaga, Ramon J./0000-0002-2513-7711; Vidal, Rosa Maria Perez/0000-0002-4075-4152; Di Nitto, Antonio/0000-0002-9319-366X; Lazarus, Ian/0000-0003-1235-4984; Hadynska Klek, Katarzyna/0000-0003-1244-9561; Sanchis Peris, Enrique/0000-0002-9689-9131; Moszynski, Marek/0000-0002-1267-2838; Soderstrom, Par-Anders/0000-0002-9504-2814, WOS: 000462142700010, The NEutron Detector Array, NEDA, will form the next generation neutron detection system that has been designed to be operated in conjunction with gamma-ray arrays, such as the tracking-array AGATA, to aid nuclear spectroscopy studies. NEDA has been designed to be a versatile device, with high-detection efficiency, excellent neutron-gamma discrimination, and high rate capabilities. It will be employed in physics campaigns in order to maximise the scientific output, making use of the different stable and radioactive ion beams available in Europe. the first implementation of the neutron detector array NEDA with AGATA 1 pi was realised at GANIL. This manuscript reviews the various aspects of NEDA., Swedish Research Council, SwedenSwedish Research Council [VR 2014-6644]; Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [117F114, 114F473]; Polish National Science Centre, Poland [2017/25/B/ST2/01569, 2016/22/M/ST2/00269 COPIN-IN2P3]; COPIGAL projects, Poland; UK STFCScience & Technology Facilities Council (STFC) [ST/J000124/1, ST/L005727/1, STL005735/1, ST/P003885/1]; Generalitat ValencianaGeneralitat Valenciana [PROMETEO II/2014/019, FPA2017-84756-C4]; MICIU, Spain [PROMETEO II/2014/019, FPA2017-84756-C4]; Severo Ochoa, Spain [SEV-2014-0398]; E.C. FEDER, Spain funds, This study is supported by the Swedish Research Council, Sweden (contract number VR 2014-6644), the Scientific and Technological Research Council of Turkey (TUBITAK Project No: 117F114 and 114F473), the Polish National Science Centre, Poland, grants nos. 2017/25/B/ST2/01569 and 2016/22/M/ST2/00269 COPIN-IN2P3 and COPIGAL projects, Poland, the UK STFC under grant nos. (ST/J000124/1, ST/L005727/1, STL005735/1, ST/P003885/1), the Generalitat Valenciana and MICIU, Spain, grants PROMETEO II/2014/019, FPA2017-84756-C4, Severo Ochoa, Spain SEV-2014-0398 and by the E.C. FEDER, Spain funds.

International audience; The Myocardin-related transcription factor A [MRTFA, also known as Megakaryoblastic Leukemia 1 (MKL1))] is a major actor in the epithelial to mesenchymal transition (EMT). We have previously shown that activation and nuclear accumulation of MRTFA mediate endocrine resistance of estrogen receptor alpha (ERα) positive breast cancers by initiating a partial transition from luminal to basal-like phenotype and impairing ERα cistrome and transcriptome. In the present study, we deepen our understanding of the mechanism by monitoring functional changes in the receptor’s activity. We demonstrate that MRTFA nuclear accumulation down-regulates the expression of the unliganded (Apo-)ERα and causes a redistribution of the protein localization from its normal nuclear place to the entire cell volume. This phenomenon is accompanied by a shift in Apo-ERα monomer/dimer ratio towards the monomeric state, leading to significant functional consequences on ERα activities. In particular, the association of Apo-ERα with chromatin is drastically decreased, and the remaining ERα binding sites are substantially less enriched in ERE motifs than in control conditions. Monitored by proximity Ligation Assay, ERα interactions with P160 family coactivators are partly impacted when MRTFA accumulates in the nucleus, and those with SMRT and NCOR1 corepressors are abolished. Finally, ERα interactions with kinases such as c-src and PI3K are increased, thereby enhancing MAP Kinase and AKT activities. In conclusion, the activation and nuclear accumulation of MRTFA in ERα positive breast cancer cells remodels both ERα location and functions by shifting its activity from nuclear genome regulation to extra-nuclear non-genomic signaling.

Three different Artificial Neural Network architectures have been applied to perform neutron/? discrimination in NEDA based on waveform and time-of-flight information. Using the coincident ?-rays from AGATA, we have been able to measure and compare on real data the performances of the Artificial Neural Networks as classifiers. While the general performances are quite similar for the data set we used, differences, in particular related to the computing times, have been highlighted. One of the Artificial Neural Network architecture has also been found more robust to time misalignment of the waveforms. Such a feature is of great interest for online processing of waveforms. Narodowe Centrum Nauki: 2017/25/B/ST2/01569 Narodowym Centrum Nauki One of the author acknowledges support of the National Science Centre, Poland (NCN) (grant no. 2017/25/B/ST2/01569 ).

Zakopane Conference on Nuclear Physics - Extremes of the Nuclear Landscape -- AUG 26-SEP 02, 2018 -- Zakopane, POLAND, WOS: 000463866500047, A new neutron multiplicity filter NEDA, after a decade of design, R&D and construction, was employed in its first physics campaign with the AGATA spectrometer. Properties and performance of the array are discussed., Swedish Research CouncilSwedish Research Council [VR 2014-6644]; Scientific and Technological Research Council of Turkey (TUBITAK project)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [117F114, 114F473]; National Science Centre, Poland (NCN) [2017/25/B/ST2/01569, 2016/22/M/ST2/00269, 2014/14/M/ST2/00738, 2013/08/M/ST2/00257]; UK STFCScience & Technology Facilities Council (STFC) [ST/J000124/1, ST/L005727/1, STL005735/1, ST/P003885/1]; Generalitat Valenciana, SpainGeneralitat Valenciana; MICIU, Spain [PROM-ETEO II/2014/019, FPA2017-84756-C4, SEV-2014-0398]; E.C. FEDER fundsEuropean Union (EU), This study was supported by the Swedish Research Council (contract number VR 2014-6644), the Scientific and Technological Research Council of Turkey (TUBITAK project Nos. 117F114 and 114F473), the National Science Centre, Poland (NCN) (grants Nos. 2017/25/B/ST2/01569, 2016/22/M/ST2/00269, 2014/14/M/ST2/00738 and 2013/08/M/ST2/00257) the UK STFC under grant Nos. ST/J000124/1, ST/L005727/1, STL005735/1, ST/P003885/1, the Generalitat Valenciana and MICIU, Spain, grants PROM-ETEO II/2014/019, FPA2017-84756-C4, Severo Ochoa SEV-2014-0398 and by the E.C. FEDER funds.1

Livestock farms have recently experienced a significant growth in their size and this is due to the increase in animal products. This has a technical impact on farmers' daily activities. As there are more animals, it becomes more difficult for the farmers to monitor, take care of and ensure the animals are treated in a healthy manner. The purpose of this study is to examine how farmers are affected by the use of the Precision Livestock Farming (PLF) systems and how they have come to accept the technology. The study will take up an important aspect of the impact and effect of PLF. The aim of the study is to find out how farmers are affected by PLF systems in regard to Ease of Use (EOU) and Usefulness. A total of five semi-structured interviews consisting of two researchers, one product advisor, one support worker and one farmer were conducted. An observation was also conducted along with the farmer and support worker. During the interviews, the participants answered our questions regarding PLF and its EOU and Usefulness. The data was analyzed using a thematic analysis and the results showed that PLF systems are important and useful but need further EOU development. Furthermore, the results showed that the poorly designed user interfaces impact the farmers’ attitude and intention towards using the system, which also affects their attitude towards the Usefulness of the PLF systems. The research confirms the importance of including farmers under the development phase of PLF systems. Lastly, this research might be the foundation for further research on how to improve and develop a more user-friendly PLF system.

Dans certaines maladies métaboliques héréditaires, les vomissements peuvent apparaître comme un symptôme étant au premier plan, en particulier les anomalies du cycle de l'urée, qui sont habituellement diagnostiqués en période néonatale ou dans l'enfance. Nous en rapportons un cas de révélation tardive par un état de mal convulsif. Nous rapportons le cas d'une patiente âgée de 13 ans, qui a été hospitalisé pour prise en charge d'un état de mal convulsif et un retard staturo-pondéral. L'interrogatoire a révélé la notion de vomissements chroniques avec des troubles du comportement, ralentissement idéomoteur et céphalées. L'examen a trouvé une ataxie. La ponction lombaire et le scanner cérébral sont normaux. Une ammoniémie nettement augmentée est mise en évidence 75 micromoles/l (11-50). La chromatographie des acides aminés dans le sang a montré une augmentation de la glutamine et de l'alanine, La chromatographie des acides aminés dans les urines a montré une augmentation des acides aminés basiques évoquant un déficit du cycle de l'urée par déficit de l'enzyme Argininosuccinate lyase. La patiente a été traité en urgence par une alimentation exclusivement glucidolipidique, et par benzoate de sodium permettant une amélioration de l'état clinique, et une reprise de poids. Les crises convulsives ont été maîtrisées par le phénobarbital. L'enquête familiale a trouvé une sœur âgée de 20 ans suivie depuis l'âge de 3 ans pour crises convulsives traité par le phénobarbital dont le bilan métabolique réalisé dans notre service a objectivé la même anomalie du cycle de l'urée que sa sœur. A tout âge, devant une encéphalopathie avec épilepsie, vomissement, stagnation pondérale et hyperammoniémie, il faut penser à un déficit du cycle de l'urée. Le diagnostic est très souvent posé lors d'un accès neuro-digestif aigue associant vomissements, troubles de conscience et/ou crises convulsives.

The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17β-E2 stimulates, via its receptor human estrogen receptor α 66 (hERα66), type II collagen expression in differentiated and dedifferentiated (reflecting the OA phenotype) articular chondrocytes. Transactivation of type II collagen gene (COL2A1) by ligand-independent transactivation domain (AF-1) of hERα66 was mediated by "GC" binding sites of the -266/-63-bp promoter, through physical interactions between ERα, Sp1/Sp3, Sox9, and p300, as demonstrated in chromatin immunoprecipitation (ChIP) and Re-Chromatin Immuno-Precipitation (Re-ChIP) assays in primary and dedifferentiated cells. 17β-E2 and hERα66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3, and Sox-9 small interfering RNAs (siRNAs) prevented hERα66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hERα66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17β-E2 and hERα66 modulate Sp1/Sp3 heteromer binding activity and simultaneously participate in the recruitment of the essential factors Sox-9 and p300 involved respectively in the chondrocyte-differentiated status and COL2A1 transcriptional activation. These novel findings could therefore be attractive for tissue engineering of cartilage in OA, by the fact that 17β-E2 could promote chondrocyte redifferentiation.17β-E2 up-regulates type II collagen gene expression in articular chondrocytes. An ERα66/Sp1/Sp3/Sox-9/p300 protein complex mediates this stimulatory effect. This heteromeric complex interacts and binds to Col2a1 promoter and enhancer in vivo. Our findings highlight a new regulatory mechanism for 17β-E2 action in chondrocytes. 17β-E2 might be an attractive candidate for cartilage engineering applications.

19th Real Time Conference (RT) -- MAY 26-30, 2014 -- Nara, JAPAN WOS: 000356458000028 This paper presents the final design and results of the FADC Mezzanine for the EXOGAM (EXOtic GAMma array spectrometer) and NEDA (Neutron Detector Array) detectors. The measurements performed include those of studying the effective number of bits, the energy resolution using HP-Ge detectors, as well as timing histograms and discrimination performance. Finally, the conclusion shows how a common digitizing device has been integrated in the experimental environment of two very different detectors which combine both low-noise acquisition and fast sampling rates. Not only the integration fulfilled the expected specifications on both systems, but it also showed how a study of synergy between detectors could lead to the reduction of resources and time by applying a common strategy. Osaka Univ, Res Nucl Phys Generalitat Valenciana, Spain [PROMETEO/2010/101]; INFN, Italy; Spanish MINECO [AIC-D-2011-0746, FPA2011-29854, FPA2012-33650]; Swedish Research Council; Scientific and Technological Research Council of Turkey (TUBITAK); UK STFC; Science and Technology Facilities Council [ST/L005735/1, ST/J000124/1, ST/L005727/1] This work was supported by the Generalitat Valenciana, Spain, under grant PROMETEO/2010/101. Some authors were supported in part by INFN, Italy, and by the Spanish MINECO under grants AIC-D-2011-0746, FPA2011-29854 and FPA2012-33650. This work was also supported by the Swedish Research Council, the Scientific and Technological Research Council of Turkey (TUBITAK) and the UK STFC.

19th IEEE-NPSS Real Time Conference (RT) -- MAY 26-30, 2014 -- Nara, JAPAN, WOS: 000380588000136, The present paper aims to present the final design and results of the FADC Mezzanine for EXOGAM and NEDA detectors, focusing on the resolution performance. Finally, the conclusion shows how a common digitizing device has been integrated in an experimental environment involving low-noise and fast sampling rates. Not only the integration fulfilled the expected specifications on both systems, but also, it shows how a study of synergy between detectors has led to a reduction of resources and time by applying a common strategy., Osaka Univ, Res Nucl Phys, IEEE Nucl & Plasma Sci Soc

20th IEEE Real Time Conference, 5–10 Haz 2016, Padova, Italy -- F.J. Egea is with INFN Sezione di Padova, Padova, Italy. E-mail: javiegea@pd.infn.it. -- V. González and E. Sanchis are with Departament d’Enginyeria Electrònica. Universitat de València. Burjassot (Spain). -- C. Houarner, A. Boujrad, M. Tripon and G. de France are with Grand Accélératéur National d’Ions Lourds (GANIL), Caen, France. -- A. Gadea and T. Hüyük are with Institut de Física Corpuscular (IFIC), Paterna, Spain. -- J.J. Valiente-Dobón and G. Jaworski are with LNL (Laboratori Nazionali di Legnaro), Legnaro, Italy. -- J. Nyberg is with Department of Physics and Astronomy, Uppsala University, Uppsala, Sweden. -- M. Moszyński is with National Centre for Nuclear Research, A. Soltana 7, PL 05-400 Otwock-Swierk, Poland. -- M.N. Enduran is with Faculty of Engineering and Natural Sciences, Istanbul Sabahattin Zaim University Istanbul, Turkey. -- S. Ertürk is with Nigde Universitesi, Fen-Edebiyat Falkultesi, Fizik Bölümü, Nigde, Turkey. -- M. Jastrząb is with the Niewodniczański Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland. -- I. Lazarus is with STFC Daresbury Laboratory, Warrington, United Kingdom. -- P-A. Söderström is with RIKEN Nishina Center, Wako-shi, Japan. -- V. Modamio is with Universitet I Oslo, Oslo, Norway. -- X. L. Luo is with Department of Instrument Science and Technology, National University of Defense Technology, Changsha, China. -- M.Palacz are with the Heavy Ion Laboratory, University of Warsaw, Warszawa, Poland. -- R. Wadsworth is with Department of Physics, University of York, York, United Kingdom. The detection of rare events close to the proton dripline involve the utilization of high-efficient neutron detectors with excellent neutron-gamma discrimination (NGD) performance. Even though in the literature, several off-line algorithms focus on the maximization of the NGD accuracy, our aim is to look for an on-line efficient implementation capable to discard a majority of the gamma-rays, therefore allowing the system to work to counting rates up to 50 kHz, optimizing hence the overall electronics infrastructure in terms of data throughput. In this paper we analyze two of the most widespread techniques for NGD based on PSA, i.e. the charge-comparison method (CC) and the zero crossover (ZCO). Then, based on the NGD performance and FPGA resource utilization, we select one to be implemented in an FPGA. Finally we present the preliminary results obtained up to now.

International audience; The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC50 ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfER alpha, zfER beta 1 or zfER beta 2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation.

Caspase-independent programmed necrosis is a highly regulated cellular demise that displays morphological and biochemical necrotic hallmarks, such as an earlier permeability of the plasma membrane and lactate dehydrogenase (LDH) leakiness. This form of programmed cell death (PCD) is regulated by AIF, a FAD-dependent oxidoreductase, which is released from the mitochondria to the nucleus where it induces chromatin tcondensation and DNA fragmentation. Some years ago, it was established that the sequential activation of poly(ADP-ribose) polymerase- 1 (PARP-1), calpains and Bax regulates the mitochondrial AIF release associated to programmed necrosis. But, what happens when AIF is in the nucleus? How does this protein induce chromatinolysis and programmed necrosis? Recently, we have unraveled some of the mechanisms underlying the nuclear action of AIF in this type of caspase-independent cell death. Indeed, AIF plays a key role in programmed necrosis by its ability to organize a DNA-degrading complex with H2AX and Cyclophiline A (CypA). The AIF/H2AX link is indeed a critical event and explains the nuclear AIF apoptogenic action. In the present article, we outline the current knowledge on cell death by programmed necrosis and discuss the relevance of the AIF/H2AX/CypA DNA-degrading complex in the regulation of this original form of cell death.

International audience; Although involved in processes leading to the emergence and development of hormone-dependent breast cancers, the estrogen receptor alpha (ERalpha) also prevents transformed cells from progressing toward a more aggressive phenotype. The transcriptional activity of ERalpha is mediated through two transactivation functions, called activation function 1 and 2, whose respective involvement varies in a cell-specific manner. Here, we identify the Rho/megakaryoblastic leukemia 1 (MKL1) signaling pathway as a main actor in controlling the cell-specific activity of both transactivation functions of ERalpha. Notably, we show that, when the coregulator MKL1 is sequestered in an inactive form by unpolymerized actin, the transcriptional activity of ERalpha mainly relies on the activation function 1. The activation of MKL1, which results from its dissociation from unpolymerized actin, promoted by the ability of Rho to support polymeric actin accumulation, silences the activation function 1 of ERalpha and allows the receptor to mainly act through its activation function 2. Importantly, this switch in the respective contribution exerted by both transactivation functions is correlated with an impaired ability of ERalpha to efficiently transactivate estrogen-regulated reporter genes. MKL1 is further shown to be present on estrogen-responsive genes in vivo. Interestingly, the Rho/MKL1 signaling pathway is activated during the epithelial-mesenchymal transition. A reduced transactivation efficiency of ERalpha, resulting from the activation of this pathway, may therefore suppress the protective role exerted by ERalpha toward tumor progression and invasiveness.

This paper presents the design of the NEDA (Neutron Detector Array) electronics, a first attempt to involve the use of digital electronics in large neutron detector arrays. Starting from the front-end modules attached to the PMTs (PhotoMultiplier Tubes) and ending up with the data processing workstations, a comprehensive electronic system capable of dealing with the acquisition and pre-processing of the neutron array is detailed. Among the electronic modules required, we emphasize the front-end analog processing, the digitalization, digital pre-processing and communications firmware, as well as the integration of the GTS (Global Trigger and Synchronization) system, already used successfully in AGATA (Advanced Gamma Tracking Array). The NEDA array will be available for measurements in 2016.

This paper presents the final design and results of the FADC Mezzanine for the EXOGAM (EXOtic GAMma array spectrometer) and NEDA (Neutron Detector Array) detectors. The measurements performed include those of studying the effective number of bits, the energy resolution using HP-Ge detectors, as well as timing histograms and discrimination performance. Finally, the conclusion shows how a common digitizing device has been integrated in the experimental environment of two very different detectors which combine both low-noise acquisition and fast sampling rates. Not only the integration fulfilled the expected specifications on both systems, but it also showed how a study of synergy between detectors could lead to the reduction of resources and time by applying a common strategy.

Background Testicular Leydig cells use either exogenous or de novo synthesized cholesterol as the substrate for the production of testosterone with hormone stimulation. Although the long-term effect of trophic hormones on Leydig cell cholesterol uptake, storage, and deesterification has been well documented, the early effects of the human choriogonadotropin (hCG) on cell cholesterol/lipid distribution are not yet known. Methods Sections of cells treated with hCG for 15 sec to 30 min were examined by electron microscopy (EM) for the surface density of lipid moieties in the cytoplasm. In addition, the time-dependent distribution of lipids within the cytoplasmic inclusions and the ultimate destination of this substrate were evaluated by EM. The results were analyzed with standard morphometric methods. Results and Conclusion The surface density of cytoplasmic lipid pools increased significantly within the 15 sec following the exposure of cells to hCG, and it tapered off to the control level in the subsequent 30 min. Such a fluctuation in the amount of cytoplasmic lipids may be due to (1) the quantity of released substrate from the reticular compartment or (2) the rate of its transport across the outer mitochondrial membrane to the inner mitochondrial cytochrome P450 side-chain cleavage, where steroidogenesis begins with the conversion of cholesterol to pregnenolone. These two processes were not quantitatively coordinated in the stimulated cell during the intitial 30 min, resulting in a surplus of cytoplasmic lipid pools. To compensate for such uneven metabolic balance, the cell apparently disposed of the excess substrate by a mechanism of molecular regrouping from a micellar configuration to a bilayer structure followed by exocytosis. Anat. Rec. 248:374-379, 1997. © 1997 Wiley-Liss, Inc.

We examined the topography of the MA-10 Leydig tumor cell mitochondrial peripheral-type benzodiazepine receptor (PBR). In previous studies, the 18 kDa PBR was found to be functionally associated with the voltage-dependent anion channel, located in the junctions between outer and inner membranes. Transmission electron (TEM) and atomic force microscopy (AFM) of immunogold labeled PBR on Leydig cell mitochondrial preparations showed that the 18 kDa PBR protein is organized in clusters of 4-6 molecules. Addition of hCG to Leydig cells induces a rapid, within 30 sec, increase in PBR ligand binding and morphological changes, namely redistribution of PBR molecules in large clusters (7 particles). These hCG-induced changes were inhibited by a cAMP-dependent protein kinase inhibitor and by the benzodiazepine flunitrazepam. AFM further demonstrated the rapid reorganization of the mitochondrial membrane, where the formation of contacts between the outer and the inner mitochondrial membrane may facilitate cholesterol transfer.

2012 18th IEEE-NPSS Real Time Conference, RT 2012 -- 9 June 2012 through 15 June 2012 -- Berkeley, CA -- 95754 This board will be part of the upgrade for the new electronics for the EXOGAM2 (HP-Ge detector array) and NEDA (BC501A-based neutron detector array), therefore it was necessary to deal with the problem of providing a sampling card with high resolution for new gamma spectroscopy experiments while sampling at very high rates, with a broad bandwidth in order to preserve the shape for further analysis. Pulse shape analysis is of paramount importance in neutron detectors, such as NEDA, based on scintillators that are sensitive to ?-rays as well. High resolution and high speed are often two parameters which conform a trade-off and it is hard to achieve both simultaneously. The aforementioned constraints and the urge of building new sampling electronics to improve the signal analysis in nuclear physics experiments, led to the development of this FADC mezzanine This involves sampling rates up to 250 Msps preserving a high resolution of 11.3 effective bits in order to satisfy the experiment demands. In this work is described the design and the test bench proposed for a proper high speed ADC characterization system and the results obtained up to now. © 2012 IEEE.

Native MA-10 mouse Leydig tumor cell mitochondrial preparations were examined by transmission electron (TEM) and atomic force (AFM) microscopic procedures in order to investigate the topography and organization of the peripheral-type benzodiazepine receptor (PBR). Mitochondria were immunolabeled with an anti-PBR antiserum coupled to gold-labeled secondary antibodies. Results obtained indicate that the 18,000 MW PBR protein is organized in clusters of 4-6 molecules. Moreover, on many occasions, the interrelationship among the PBR molecules was found to favor the formation of a single pore. Taking into account recent observations that the 18,000 MW PBR protein is functionally associated with the pore forming 34,000 MW voltage-dependent anion channel (VDAC) these results suggest that (i) the mitochondrial PBR complex could function as a pore, thus allowing the translocation of cholesterol and other molecules to the inner mitochondrial membrane, and (ii) the native receptor is a multimeric complex of an approximate 140,000 MW composed on an average of five 18,000 PBR subunits, one 34,000 VDAC subunit, and associated lipids.

Purpose: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage whose expression is strongly decreased during osteoarthritis (OA) process. Specific proteins (Sp) and Sox proteins have been previously characterized to be major regulators of this gene. Besides, the existence of a link between estrogen deprivation and osteoarthritis in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a critical modulator of cartilaginous matrix homeostasis. The aim of present study was to investigate the molecular mechanisms regulating type II collagen gene expression under the effect of 17β-E2, and to characterize the genomic pathway via estrogen receptors (ER). Methods: Articular chondrocytes were isolated from 3-week old rabbits and incubated for 24 hours with increasing concentrations of 17β-E2 (0 to 10 nM). Nuclear proteins were extracted and submitted to gel retardation assays in order to determine Sp and Sox proteins binding activities on COL2A1 gene. In some experiments, relative expression of Sp1/Sp3 or Sox-9 was inhibited by siRNAs strategies. Moreover, chromatin immunoprecipitation was used to study protein-protein interactions on COL2A1 promoter and enhancer sequences. Results: We have previously shown that 17β-E2 could stimulate type II collagen neosynthesis, protein level and corresponding steady-state levels of COL2A1 mRNAs in primary and in vitro dedifferentiated rabbit articular chondrocytes. 17β-E2 action was mediated, in part, by an ERα/Sp transactivating effect on the -266/-63 bp region of COL2A1 proximal promoter. Our new data showed that 17β-E2 also increases the DNA-binding activities of Sp1/Sp3 and Soxs proteins to the first intron region including COL2A1 specific enhancer. In addition, Sp1, Sp3 and Sox-9 siRNAs prevented hERα66-induced transactivation of COL2A1, suggesting that these factors are required for COL2A1 up-regulation. Moreover, chromatin immunoprecipitation assays indicated that ERα physically interacts in vivo with Sp1, Sp3, Sox-9 and p300 transcription factors, on both COL2A1 promoter and enhancer sequences. Thus, considering that all these transcription factors are known to act in concert, we proposed that 17β-E2 in vivo effect could be mediated by a bridging complex, composed of ERα, Sp1, Sp3, Sox-9 and p300 transcription factors, which could establish a physical link between the two main cisregulatory regions of the gene, and consequently, may facilitate COL2A1 transcription process. Besides, this transcriptional activation of COL2A1 gene requires the ERα transactivation domains, AF-1 and AF-2, as demonstrated by using ERα mutants lacking these functional domains. Conclusions: Understanding the molecular basis for 17β-E2/ERα induction of COL2A1 transcription provides new insights into molecular mechanisms of OA and could be therefore attractive for some applications in the tissue engineering of cartilage.

International audience; Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

International audience; OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

Historically, two main forms of cell death have been distinguished: apoptosis and necrosis. Apoptosis was initially considered as the only physiological and programmed form of cell death. This type of death is recurrently associated with caspases, a family of cysteine proteases activated in apoptotic conditions. However, it is now widely recognized that programmed cell death (PCD) can also occur in the complete absence of caspase activation. The existence of non-caspase PCD pathways was corroborated by the discovery of caspase-independent executioners, such as the mitochondrial protein Apoptosis-Inducing Factor (AIF). Necrosis has often been viewed as an accidental and uncontrolled cell death process. Nevertheless, increasing evidence shows that, like apoptosis, necrosis could be a highly orchestrated type of PCD. Indeed, apoptosis and necrosis present more similarities than it has been originally thought. Here, we summarize the different classifications of PCD and the current knowledge of a necrotic PCD pathway mediated by AIF: alkylating DNA-damage mediated death. We also outline the molecular mechanisms controlling this form of PCD and discuss their potential relevance in physiological and pathological settings. These emerging data on the molecular mechanisms regulating programmed necrosis may certainly have potent therapeutic consequences in treating both apoptotic-resistant tumors and degenerating adult neurons.

11 pages; International audience; The expression of two human estrogen receptor-alpha (hERalpha) isoforms has been characterized within estrogen receptor-alpha-positive breast cancer cell lines such as MCF7: the full-length hERalpha66 and the N terminally deleted hERalpha46, which is devoid of activation function (AF)-1. Although hERalpha66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hERalpha46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hERalpha46 is mainly expressed in the nucleus at relatively low levels, whereas hERalpha66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hERalpha46 accumulating within the nucleus. Although hERalpha46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G(0)/G(1) phases. To gain further details on the influence of hERalpha46 on cell growth, we used PC12 estrogen receptor-alpha-negative cell line, in which stable transfection of hERalpha66 but not hERalpha46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hERalpha46 inhibits the hERalpha66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hERalpha46 antagonizes the proliferative action of hERalpha66 in MCF7 cells in part by inhibiting hERalpha66 AF-1 activity.

In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PPactivated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheraltype benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPAR and PPAR/, but not for PPAR. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPAR-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARdependent inhibition of Leydig cell PBR gene expression. (Endocrinology 143: 2571–2583, 2002)

In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PP-activated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheral-type benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPARα and PPARβ/δ, but not for PPARγ. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPARα-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARα-dependent inhibition of Leydig cell PBR gene expression.

In nuclear physics, the interaction between the ion beam and the target produces a a large amount of events. Some of these events have no interest for the studied physical phenomenon ; the useful events are sorted using a trigger. We have studied and realized a new trigger suitable for the GANIL (Grands Accélérateur National d'Ions Lourds) experiments. After an overview of the triggers used up to now at GANIL, we give the main features of the new trigger (GANIL Master Trigger, GMT) such as modularity, universality and versality. After a description of the trigger operating modes, we depict the trigger realization steps. The trigger informs about fired detectors in a beam to target collision but gives no information about timing, nevertheless the timing is a very important piece of information if the trigger analysis duration in several times greater than the beam period. We suggest a timing measurement structure (Time to Digital Converter, TDC) able to eliminate this imprecision. The Dead time, the low level intégration and the consumption contraisnts lead us to prefer a digital architecture based on a digital counter associated with delay lines. Simple equations are given in order to define the operating area of the TDC. This area depends on the clock duty cycle and the delay line taps. Measures of Differential Non Linearity (DNL) for different time résolutions (1 ,2, 5 and 10 ns) allow to establish the limits of this system and to underline some solutions to improve these features., En physique nucléaire, l'interaction faisceau-cible engendre une multitude d'événements qui ne sont pas tous d'intérêt pour le phénomène physique étudié. Il est donc important de disposer d'un système de sélection nommé trigger. Nous avons étudié et réalisé un nouveau trigger adapté aux expériences menées au GANIL. Nous avons présenté un historique des différents triggers utilisés aux GANIL (Grand Accélérateur National d'Ions Lourds) et avons montré la nécessité d'un nouveau trigger modulaire, universel et ouvert. Après une description des différents modes de fonctionnement du nouveau trigger (GANIL Master Trigger, GMT) nous avons décrit les phases de sa conception et de sa réalisation. Le trigger renseigne sur la configuration des détecteurs temporels, or cette information peut être fondamentale dans le cas où la fenêtre d'analyse du trigger couvre plusieurs périodes du faisceau. Nous avons donc proposé une structure de mesure de temps (Time to Digital Converter, TDC) qui permet de lever cette indétermination. Les contraintes de temps mort, d'intégration et de consommation nous ont conduit à proposer une architecture numérique basée sur un compteur associé à une ligne A Retard (LAR). Des calculs simples ont permis de définir la zone de fonctionnement du TDC. Cette zone dépend du rapport cyclique de l'horloge et des retards de la LAR. Des mesures de Non Linéarité Différentielle (NLD) pour des résolutions différentes (1,2,5 et 10 ns) ont permis d'établir les limites de ce système et de mettre en évidence des solutions d'amélioration de ces caractéristiques.

The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

To evaluate the role of the mitochondrial peripheral-type benzodiazepine receptor (PBR) in steroidogenesis, we developed a molecular approach based on the disruption of the PBR gene, by homologous recombination, in the constitutive steroid producing R2C rat Leydig tumor cell line. Inactivation of one allele of the PBR gene resulted in the suppression of PBR mRNA and ligand binding expression. Immunoblot and electron microscopic immunogold labeling analyses confirmed the absence of the 18-kDa PBR protein in the selected clone. Although mitochondria from the PBR-negative cells contained high levels of the constitutively expressed 30-kDa steroidogenic activity regulator protein, these cells produced minimal amounts of steroids compared with normal cells (5%). Moreover, mitochondria from PBR-negative cells failed to produce pregnenolone when supplied with exogenous cholesterol. Addition of the hydrosoluble cholesterol derivative, 22R-hydroxycholesterol, increased steroid production by the PBR-negative R2C cells, indicating that the cholesterol transport mechanism was impaired. Stable transfection of the PBR-negative R2C Leydig cells with a vector containing the PBR cDNA resulted in the recovery of the steroidogenic function of the cells. These data demonstrate that PBR is an indispensable element of the steroidogenic machinery, where it mediates the delivery of the substrate cholesterol to the inner mitochondrial side chain cleavage cytochrome P-450.

Glucocorticoid excess has broad pathogenic potential including neurotoxicity, neuroendangerment, and immunosuppression. Glucocorticoid synthesis is regulated by ACTH, which acts by accelerating the transport of the precursor cholesterol to the mitochondria where steroidogenesis begins. Ginkgo biloba is one of the most ancient trees, and extracts from its leaves have been used in traditional medicine. A standardized extract of Ginkgo biloba leaves, termed EGb 761 (EGb), has been shown to have neuroprotective and antistress effects. In vivo treatment of rats with EGb, and its bioactive components ginkgolide A and B, specifically reduces the ligand binding capacity, protein, and messenger RNA expression of the adrenocortical mitochondrial peripheral-type benzodiazepine receptor (PBR), a key element in the regulation of cholesterol transport, resulting in decreased corticosteroid synthesis. As expected, the ginkgolide-induced decrease in glucocorticoid levels resulted in increased ACTH release, which in turn induced the expression of the steroidogenic acute regulatory protein. Because ginkgolides reduced the adrenal PBR expression and corticosterone synthesis despite the presence of high levels of steroidogenic acute regulatory protein, these data demonstrate that PBR is indispensable for normal adrenal function. In addition, these results suggest that manipulation of PBR expression could control circulating glucocorticoid levels, and that the antistress and neuroprotective effects of EGb are caused by to its effect on glucocorticoid biosynthesis.

Gonadal steroidogenesis is regulated by pituitary gonadotropins and a locally produced, unidentified factor. A 70-kilodalton (kD) protein complex secreted from rat Sertoli cells was isolated. The complex, composed of 28- and 38-kD proteins, stimulated steroidogenesis by Leydig cells and ovarian granulosa cells in a dose-dependent and adenosine 3',5'-monophosphate-independent manner. The follicle-stimulating hormone-induced 28-kD protein appeared to be responsible for the bioactivity, but the 38-kD protein was indispensable for maximal activity. The 28- and 38-kD proteins were shown to be identical to the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the proenzyme form of cathepsin L, respectively. Thus, a TIMP-1-procathepsin L complex is a potent activator of steroidogenesis and may regulate steroid concentrations and, thus, germ cell development in both males and females.

In order to clarify the role of germ cells in the regulation of Sertoli cell secretions, three experimental models of germ cell depletion were used : hypodactyl rat mutation (testis weight [TW] : 55% less than controls), in utero busulfan treatment (TW : 88% less than controls), and neonatal experimental cryptorchidism (TW : 72% less than controls). The aim of this work was to compare the numbers of Leydig and Sertoli cells and the production of germ cells in each experimental model to the in vitro secretions of Leydig and Sertoli cells in conditioned media and to the hormonal serum profiles of the same animal in vivo. In the three models, serum levels of hypophyseal and testosterone hormones were significantly increased and decreased, respectively. In the absence of germ cells, the total length of seminiferous tubules, the total numbers of Sertoli and Leydig cells, and the daily production of germ cells were significantly diminished. The addition of both control and damaged seminiferous tubule culture media (STM : media conditioned by 10 cm of seminiferous tubules) to 10 5 control or damaged Leydig cells led to a further increase of testosterone production after ovine LH stimulation. However, expressed per Sertoli cell, testosterone production by control Leydig cells was reduced by addition of damaged STM as compared to addition of control STM, and, similarly, the addition of control STM to damaged Leydig cells enhanced testosterone production more than did the addition of damaged STM. Secretions of transferrin per Sertoli cell in STM were reduced as compared to controls by the absence of germ cells but to a lesser extent than was production of spermatocytes and of spermatids. In conclusion, secretions by Sertoli cells of the paracrine factor involved in the control of testosterone production by Leydig cells and of transferrin are modified by germ cells.