Estudi de la interpretació entre els factors de transcripció de Drosophila GAGA i Tramtrack

Consultable des del TDXTítol obtingut de la portada digitalitzadaEls factors de transcripció de Drosophila GAGA i Tramtrack (TTK) tenen en comú la presència d'un domini N-terminal de tipus POZ/BTB, un motiu estructural altament conservat present en factors de transcripció, proteïnes «kelch» i proteïnes de poxvirus que media interaccions proteïna-proteïna específiques. En algunes proteïnes, el domini POZ/BTB s'ha observat que participa en la formació d'homooligòmers i, basant-se en experiments de co-expressió in vitro, s'ha proposat que els dominis POZ/BTB també poden formar heterooligòmers. A més a més, en alguns factors de transcripció, el domini POZ/BTB s'ha demostrat que està involucrat en interaccions amb varis co-repressors i desacetilases d'histones. A la primera part d'aquest treball s'ha pogut demostrar que les isoformes GAGA519 i TTK69 interaccionen tant in vitro com in vivo, en llevats i en cèl·lules de Drosophila Schneider SL2, i que els seus dominis POZ/BTB són necessaris i suficients perquè aquesta interacció tingui lloc. Com molts promotors de Drosophila, la regió reguladora even-skipped (eve) stripe 2 conté alguns llocs d'unió per GAGA i TTK els quals estan relativament propers, suggerint que la interacció GAGA-TTK descrita podria participar en la seva regulació funcional. GAGA i TTK semblaria que tenen efectes oposats en la transcripció: l'expressió de molts gens de Drosophila està regulada positivament per GAGA i a més s'ha demostrat que GAGA actua com un activador transcripcional en condicions in vitro. Per altra banda, durant el desenvolupament embrionari primerenc, TTK actua com un repressor transcripcional d'alguns gens pair-rule, incloent-hi eve. Mitjançant experiments de transfecció transitòria en cèl·lules SL2 hem pogut demostrar que GAGA activa la transcripció del promotor eve stripe 2 i que TTK inhibeix aquesta activació mediada per GAGA. La repressió per TTK del promotor eve requereix de l'activació per GAGA i depèn de la presència d'ambdós dominis POZ de TTK i GAGA, suggerint que aquesta repressió és una conseqüència de la interacció GAGA-TTK. Interessantment, el domini POZ de TTK és capaç de reprimir per sí sol l'activació mediada per GAGA, mentre que la regió C-terminal de TTK, que conté la seqüència consens P-DLS d'unió al co-repressor dCtBP, no és necessària per una repressió eficient. Consistent amb això, la substitució del domini POZ de GAGA pel domini POZ de TTK aboleix per complet la capacitat transactivadora de GAGA i converteix la proteïna de fusió POZTTKDPOZGAGA en un repressor de l'activació de GAGA, un resultat que dóna suport a la consideració del domini POZ de TTK com un motiu repressor. Com els dominis POZ dels factors de transcripció PLZF i Bcl-6 reprimeixen la transcripció per un reclutament de desacetilases, es va testar aquesta possibilitat pel domini POZ de TTK. Per aquesta raó, es varen realitzar assajos transcripcionals a cèl·lules SL2 en presència de TSA, un inhibidor de les desacetilases de classe I i II. Els resultats obtinguts indiquen que la repressió de TTK sobre l'activació mediada per GAGA no involucra un reclutament de desacetilases al promotor, ja que la presència de TSA no té cap efecte sobre l'activitat de TTK. No obstant, no es pot descartar la possibilitat que la repressió per TTK pogués estar mediada per desacetilases insensibles a TSA. Utilitzant delecions del promotor eve stripe 2, s'ha observat que la repressió per TTK pot tenir lloc en absència de llocs d'unió per TTK al promotor, indicant que aquesta repressió no requereix unió directa de TTK al DNA. Amb aquesta observació ens vàrem plantejar si TTK pot interferir en la unió de GAGA al DNA, per la qual cosa vàrem realitzar assajos in vitro EMSA i de footprinting amb DNasa I utilitzant un fragment eve sense llocs d'unió per TTK emprant proteïnes recombinants GAGA i TTK. Els resultats obtinguts mostren que TTK no interfereix en la unió de GAGA al DNA.; contràriament, els complexes GAGA-TTK semblen tenir una major afinitat d'unió al DNA. En resum, aquestes observacions suggereixen un model en el qual la repressió per TTK de l'activació mediada per GAGA implica una interacció directa GAGA-TTK la qual facilita el reclutament de TTK al promotor. No obstant, els nostres resultats no permeten diferenciar si la repressió per TTK és conseqüència d'una interferència en l'activació mediada per GAGA o a un reclutament actiu de co-repressors.The Drosophila transcription factors GAGA and TTK share in common the presence of a N-terminal POZ/BTB-domain, a highly conserved structural motif present in transcription factors, kelch proteins and poxviruses proteins that mediates specific protein-protein interactions. In several proteins, the POZ/BTB-domain has been found to participate in the formation of homo-oligomers and, based on in vitro co-expression experiments, it has been proposed that POZ/BTB-domains can also form hetero-oligomers. In addition, in several transcription factors, the POZ/BTB-domain has been shown to be involved in interactions with various co-repressor proteins and HDACs. In the first part of this work, we have demonstrated that GAGA519 and TTK69 isoforms interact in vitro as well as in vivo, both in yeast and Schneider S2 cells, and that their POZ/BTB-domains are necessary and sufficient for this interaction to occur. As many Drosophila promoters, the even-skipped (eve) stripe 2 regulatory region contains several binding sites for GAGA and TTK which are in relatively close proximity, suggesting that the GAGA-TTK interaction described might participate in its functional regulation. GAGA and TTK appear to have opposite effects on transcription: the expression of many genes in Drosophila is positively regulated by GAGA and it has been shown that GAGA acts as a transcription activator in vitro. On the other hand, during early embryo development, TTK was shown to function as a transcription repressor of several pair-rule genes, including eve. Based on transient expression experiments in SL2 cells we have shown that GAGA activates transcription from the eve stripe 2 promoter and that TTK inhibits this GAGA-dependent activation. Repression by TTK of the eve promoter requires its activation by GAGA and depends on the presence of the POZ/BTB-domains of TTK and GAGA, suggesting that this repression is a consequence of GAGA-TTK interaction. Interestingly, the POZ domain of TTK appears to be able to repress GAGA-mediated activation, while the C-terminal region of TTK, that contains the P-DLS consensus sequence for dCtBP corepressor binding, is not necessary for an efficient repression. Consistent with this, the substitution of the POZ domain of GAGA by the POZ domain of TTK fully abolishes the transactivation activity of GAGA and converts the fusion protein POZTTKDPOZGAGA in a repressor of GAGA-activation. This result argues in favour of the repression activity of the POZ domain of TTK. As the POZ domains of PLZF and Bcl-6 transcription factors repress transcription by recruiting HDACs to the promoter, we tested this possibility for the POZ domain of TTK. For this reason, we have performed transfection assays in the presence of TSA, an inhibitor of HDACs class I and II. The results obtained indicated that TTK repression of GAGA-mediated activation doesn't involve recruitment of HDACs to the promoter, since the presence of TSA hasn't any effect in TTK activity. However, we can't discard the possibility that TTK repression should be mediated by HDACs insensitive to TSA. Using deletions of the eve stripe 2 promoter, we have observed that TTK repression could take place in the absence of TTK-binding sites in the promoter, indicating that TTK repression doesn't require direct binding of TTK to DNA. Then, we asked if TTK could interfere in GAGA-binding to DNA, so we performed in vitro EMSA and DNase I footprinting assays using an eve fragment without TTK binding sites and recombinant GAGA and TTK. The results obtained have shown that TTK doesn't interfere in the binding of GAGA to DNA.; on the contrary, GAGA-TTK complexes appear to have a higher affinity to bind DNA. In summary, these observations suggest a model in which TTK repression of GAGA-mediated activation involves a direct interaction of GAGA and TTK that facilitates TTK recruitment to the promoter. However, our results don't allow to differentiate if TTK repression is a consequence of an interference of GAGA-dependent activation or an active recruitment of co-repressors

Efficacy of lung volume optimization maneuver monitored by optoelectronic pletismography in the management of congenital diaphragmatic hernia

Newborns affected by congenital diaphragmatic hernia (CDH) need cardio-respiratory stabilization before undergoing surgical repair. Open lung strategy is a well-established approach to optimize lung volume in preterm infants with Respiratory Distress Syndrome (RDS), using both High Frequency Oscillatory Ventilation (HFOV) and Conventional Mechanical Ventilation (CMV). We report a case of left CDH with severe lung hypoplasia, managed applying open lung strategy in HFOV (pre-surgery period) and in Assist-Control with Volume Guarantee (post-surgery period), guided by SpO2changes, TcPO2and TcPCO2monitoring. Opto-electronic plethysmography was used to measure end-expiratory chest wall volume changes (ΔEEcw) related to lung volume variations occurring during pressure changes. OEP confirmed the efficacy of using SpO2and transcutaneous gas monitoring during this recruitment maneuver

…que nos tenemus a dicto domino rege pro camera assignata. Desarrollo, administración y significado de los bienes reginales de Leonor de Sicilia (1349-1375)

Queens dowries were of central importance for their capacity of action. So far, however, they have not been studied extensively or comparatively. The case of queen Elionor of Sicily, wife of Peter IV the “Ceremonious”, allows a detailed reconstruction of the development and functions of her estate based on a wide documentary corpus which has not yet been analysed systematically. Also the concrete administration of her domain can be reconstructed very precisely in this particular case, allowing us to determine its position and functions within the framework of the monarchy as well as the agency of the queen.Los bienes dotales de una reina eran de una importancia fundamental para determinar su margen de acción. Pese a ello, no disponemos de estudios sistemáticos y comparativos al respecto. El caso de la reina Leonor de Sicilia, esposa de Pedro IV el Ceremonioso, permite la reconstrucción detallada del desarrollo y de las funciones de su patrimonio sobre la base de un amplio corpus documental que no ha sido analizado hasta ahora. A partir de él, se puede también analizar claramente cómo se desarrolló la administración concreta de su patrimonio en este caso particular. De esta manera, se pueden determinar las funciones concretas dentro del sistema de la monarquía y, por lo tanto, también el margen de acción reginal

Prospective Multicenter Study of Community-Associated Skin and Skin Structure Infections due to Methicillin-Resistant Staphylococcus aureus in Buenos Aires, Argentina

Background. Community-associated methicillin-resistant Staphylococcus aureus(CAMRSA) is now the most common cause of skin and skin structure infections (SSSI) in several world regions. In Argentina prospective, multicenter clinical studies have only been conducted in pediatric populations. Objective. Primary: describe the prevalence, clinical and demographic characteristics of adult patients with community acquired SSSI due to MRSA; secondary: molecular evaluation of CA-MRSA strains. Patients with MRSA were compared to those without MRSA. Material and Methods. Prospective, observational, multicenter, epidemiologic study, with molecular analysis, conducted at 19 sites in Argentina (18 in Buenos Aires)between March 2010 and October 2011. Patients were included if they were ≥ 14 years, were diagnosed with SSSI, a culture was obtained, and there had no significant healthcare contact identified. A logistic regression model was used to identify factors associated with CA-MRSA. Pulse field types, SCCmec, and PVL status were also determined. Results. A total of 311 patients were included. CA-MRSA was isolated in 70% (218/311) of patients. Clinical variables independently associated with CA-MRSA were: presence of purulent lesion (OR 3.29; 95%CI 1.67, 6.49) and age <50 years (OR 2.39; 95%CI 1.22, 4.70). The vast majority of CA-MRSA strains causing SSSI carried PVL genes (95%) and were SCCmec type IV. The sequence type CA-MRSA ST30 spa t019 was the predominant clone. Conclusions. CA-MRSA is now the most common cause of SSSI in our adult patients without healthcare contact. ST30, SCCmec IV, PVL+, spa t019 is the predominant clone in Buenos Aires, Argentina.Fil: Lopez Furst, Maria Jose. Sanatorio Municipal Dr. Julio Méndez, Ciudad Autónoma de Buenos Aires; Argentina;Fil: de Vedia, Lautaro. Gobierno de la Ciudad de Buenos Aires. Htal.de Infecciosas F.j. Muñiz; Argentina;Fil: Fernandez, Silvina. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Quimica Biologica. Cat.de Microbiologia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Gardella, Noella Mariel. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Quimica Biologica. Cat.de Microbiologia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Ganaha, Cristina. Pcia. de Buenos Aires. Hospital Vicente López y Planes, Gral. Rodríguez; Argentina;Fil: Prieto, Sergio. Provincia de Buenos Aires. Hospital Nuestra Señora de Luján; Argentina;Fil: Carbone, Edith. Hospital Aeronautico Central; Argentina;Fil: Lista, Nicolás. Gobierno de la Ciudad de Buenos Aires. Htal.de Infecciosas F.j. Muñiz; Argentina;Fil: Rotryng, Flavio. Universidad Abierta Interamericana; Argentina;Fil: Morera, Graciana I.. Hospital Dr. Jose Cullen; Argentina;Fil: Mollerach, Marta Eugenia. Universidad de Buenos Aires. Facultad de Cs.exactas y Naturales. Departamento de Quimica Biologica. Cat.de Microbiologia; Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina;Fil: Stryjewski, Martin E.. Centro de Educaciones Medicas E Investig.Clinica "Norberto Quirno"; Argentina

High rate, fast timing Glass RPC for the high {\eta} CMS muon detectors

The HL-LHC phase is designed to increase by an order of magnitude the amount of data to be collected by the LHC experiments. To achieve this goal in a reasonable time scale the instantaneous luminosity would also increase by an order of magnitude up to $6.10^{34} cm^{-2} s^{-1}$ . The region of the forward muon spectrometer ($|{\eta}| > 1.6$) is not equipped with RPC stations. The increase of the expected particles rate up to $2 kHz/cm^{2}$ (including a safety factor 3) motivates the installation of RPC chambers to guarantee redundancy with the CSC chambers already present. The actual RPC technology of CMS cannot sustain the expected background level. The new technology that will be chosen should have a high rate capability and provides a good spatial and timing resolution. A new generation of Glass-RPC (GRPC) using low-resistivity (LR) glass is proposed to equip at least the two most far away of the four high ${\eta}$ muon stations of CMS. First the design of small size prototypes and studies of their performance in high-rate particles flux is presented. Then the proposed designs for large size chambers and their fast-timing electronic readout are examined and preliminary results are provided.Comment: 14 pages, 11 figures, Conference proceeding for the 2016 Resistive Plate Chambers and Related Detector

Surfactant lung delivery with LISA and InSurE in adult rabbits with respiratory distress

Background: In preterm infants, InSurE (Intubation\u2013Surfactant\u2013Extubation) and LISA (less invasive surfactant administration) techniques allow for exogenous surfactant administration while reducing lung injury associated with mechanical ventilation. We compared the acute pulmonary response and lung deposition of surfactant by LISA and InSurE in surfactant-depleted adult rabbits. Methods: Twenty-six spontaneously breathing surfactant-depleted adult rabbits (6\u20137 weeks old) with moderate RDS and managed with nasal continuous positive airway pressure were randomized to 3 groups: (1) 200 mg/kg of surfactant by InSurE; (2) 200 mg/kg of surfactant by LISA; (3) no surfactant treatment (Control). Gas exchange and lung mechanics were monitored for 180 min. After that, surfactant lung deposition and distribution were evaluated monitoring disaturated-phosphatidylcholine (DSPC) and surfactant protein C (SP-C), respectively. Results: No signs of recovery were found in the untreated animals. After InSurE, oxygenation improved more rapidly compared to LISA. However, at 180\u2019 LISA and InSurE showed comparable outcomes in terms of gas exchange, ventilation parameters, and lung mechanics. Neither DSPC in the alveolar pool nor SP-C signal distributions in a frontal lung section were significantly different between InSurE and LISA groups. Conclusions: In an acute setting, LISA demonstrated efficacy and surfactant lung delivery similar to that of InSurE in surfactant-depleted adult rabbits. Impact: Although LISA technique is gaining popularity, there are still several questions to address. This is the first study comparing LISA and InSurE in terms of gas exchange, ventilation parameters, and lung mechanics as well as surfactant deposition and distribution.In our animal study, three hours post-treatment, LISA method seems to be as effective as InSurE and showed similar surfactant lung delivery.Our findings provide some clarifications on a fair comparison between LISA and InSurE techniques, particularly in terms of surfactant delivery. They should reassure some of the concerns raised by the clinical community on LISA adoption in neonatal units

Surfactant lung delivery with LISA and InSurE in adult rabbits with respiratory distress

Background In preterm infants, InSurE (Intubation–Surfactant–Extubation) and LISA (less invasive surfactant administration) techniques allow for exogenous surfactant administration while reducing lung injury associated with mechanical ventilation. We compared the acute pulmonary response and lung deposition of surfactant by LISA and InSurE in surfactant-depleted adult rabbits. Methods Twenty-six spontaneously breathing surfactant-depleted adult rabbits (6–7 weeks old) with moderate RDS and managed with nasal continuous positive airway pressure were randomized to 3 groups: (1) 200 mg/kg of surfactant by InSurE; (2) 200 mg/kg of surfactant by LISA; (3) no surfactant treatment (Control). Gas exchange and lung mechanics were monitored for 180 min. After that, surfactant lung deposition and distribution were evaluated monitoring disaturated-phosphatidylcholine (DSPC) and surfactant protein C (SP-C), respectively. Results No signs of recovery were found in the untreated animals. After InSurE, oxygenation improved more rapidly compared to LISA. However, at 180’ LISA and InSurE showed comparable outcomes in terms of gas exchange, ventilation parameters, and lung mechanics. Neither DSPC in the alveolar pool nor SP-C signal distributions in a frontal lung section were significantly different between InSurE and LISA groups. Conclusions In an acute setting, LISA demonstrated efficacy and surfactant lung delivery similar to that of InSurE in surfactant-depleted adult rabbits. Impact Although LISA technique is gaining popularity, there are still several questions to address. This is the first study comparing LISA and InSurE in terms of gas exchange, ventilation parameters, and lung mechanics as well as surfactant deposition and distribution. In our animal study, three hours post-treatment, LISA method seems to be as effective as InSurE and showed similar surfactant lung delivery. Our findings provide some clarifications on a fair comparison between LISA and InSurE techniques, particularly in terms of surfactant delivery. They should reassure some of the concerns raised by the clinical community on LISA adoption in neonatal units

Optimization of Italian CMS Computing Centers via MIUR funded Research Projects

In 2012, 14 Italian Institutions participating LHC Experiments (10 in CMS) have won a grant from the Italian Ministry of Research (MIUR), to optimize Analysis activities and in general the Tier2/Tier3 infrastructure. A large range of activities is actively carried on: they cover data distribution over WAN, dynamic provisioning for both scheduled and interactive processing, design and development of tools for distributed data analysis, and tests on the porting of CMS software stack to new highly performing / low power architectures