Toxic effects of brake wear particles on epithelial lung cells in vitro

Background: Fine particulate matter originating from traffic correlates with increased morbidity and mortality. An important source of traffic particles is brake wear of cars which contributes up to 20% of the total traffic emissions. The aim of this study was to evaluate potential toxicological effects of human epithelial lung cells exposed to freshly generated brake wear particles. Results: An exposure box was mounted around a car's braking system. Lung cells cultured at the air-liquid interface were then exposed to particles emitted from two typical braking behaviours ("full stop" and "normal deceleration"). The particle size distribution as well as the brake emission components like metals and carbons was measured on-line, and the particles deposited on grids for transmission electron microscopy were counted. The tight junction arrangement was observed by laser scanning microscopy. Cellular responses were assessed by measurement of lactate dehydrogenase (cytotoxicity), by investigating the production of reactive oxidative species and the release of the pro-inflammatory mediator interleukin-8. The tight junction protein occludin density decreased significantly (p < 0.05) with increasing concentrations of metals on the particles (iron, copper and manganese, which were all strongly correlated with each other). Occludin was also negatively correlated with the intensity of reactive oxidative species. The concentrations of interleukin-8 were significantly correlated with increasing organic carbon concentrations. No correlation was observed between occludin and interleukin-8, nor between reactive oxidative species and interleukin-8. Conclusion: These findings suggest that the metals on brake wear particles damage tight junctions with a mechanism involving oxidative stress. Brake wear particles also increase pro-inflammatory responses. However, this might be due to another mechanism than via oxidative stress. [Authors]

KAP1 regulates gene networks controlling T-cell development and responsiveness.

Chromatin remodeling at specific genomic loci controls lymphoid differentiation. Here, we investigated the role played in this process by Kruppel-associated box (KRAB)-associated protein 1 (KAP1), the universal cofactor of KRAB-zinc finger proteins (ZFPs), a tetrapod-restricted family of transcriptional repressors. T-cell-specific Kap1-deleted mice displayed a significant expansion of immature thymocytes, imbalances in CD4(+)/CD8(+) cell ratios, and altered responses to TCR and TGFβ stimulation when compared to littermate KAP1 control mice. Transcriptome and chromatin studies revealed that KAP1 binds T-cell-specific cis-acting regulatory elements marked by the H3K9me3 repressive mark and enriched in Ikaros/NuRD complexes. Also, KAP1 directly controls the expression of several genes involved in TCR and cytokine signaling. Among these, regulation of FoxO1 seems to play a major role in this system. Likely responsible for tethering KAP1 to at least part of its genomic targets, a small number of KRAB-ZFPs are selectively expressed in T-lymphoid cells. These results reveal the so far unsuspected yet important role of KAP1-mediated epigenetic regulation in T-lymphocyte differentiation and activation.

Brownian dynamics simulation of DNA condensation.

DNA condensation observed in vitro with the addition of polyvalent counterions is due to intermolecular attractive forces. We introduce a quantitative model of these forces in a Brownian dynamics simulation in addition to a standard mean-field Poisson-Boltzmann repulsion. The comparison of a theoretical value of the effective diameter calculated from the second virial coefficient in cylindrical geometry with some experimental results allows a quantitative evaluation of the one-parameter attractive potential. We show afterward that with a sufficient concentration of divalent salt (typically approximately 20 mM MgCl(2)), supercoiled DNA adopts a collapsed form where opposing segments of interwound regions present zones of lateral contact. However, under the same conditions the same plasmid without torsional stress does not collapse. The condensed molecules present coexisting open and collapsed plectonemic regions. Furthermore, simulations show that circular DNA in 50% methanol solutions with 20 mM MgCl(2) aggregates without the requirement of torsional energy. This confirms known experimental results. Finally, a simulated DNA molecule confined in a box of variable size also presents some local collapsed zones in 20 mM MgCl(2) above a critical concentration of the DNA. Conformational entropy reduction obtained either by supercoiling or by confinement seems thus to play a crucial role in all forms of condensation of DNA.

Extremal climatic states simulated by a 2-dimensional model Part I: Sensitivity of the model and present state

The criterion, based on the thermodynamics theory, that the climatic system tends to extremizesome function has suggested several studies. In particular, special attention has been devoted to the possibility that the climate reaches an extremal rate of planetary entropy production.Due to both radiative and material effects contribute to total planetary entropy production,climatic simulations obtained at the extremal rates of total, radiative or material entropy production appear to be of interest in order to elucidate which of the three extremal assumptions behaves more similar to current data. In the present paper, these results have been obtainedby applying a 2-dimensional (2-Dim) horizontal energy balance box-model, with a few independent variables (surface temperature, cloud-cover and material heat fluxes). In addition, climatic simulations for current conditions by assuming a fixed cloud-cover have been obtained. Finally,sensitivity analyses for both variable and fixed cloud models have been carried out

Convergència mediàtica digital: el consum de continguts i l'ús de nous mitjans per dones a Catalunya

Des de fa 20 anys, el sector audiovisual viu una important transformació, tant de l’oferta com del consum, en el marc de la convergència digital. La convergència anuncia la coexistència dels nous mitjans digitals amb l’apogeu d’una cultura participativa, protagonitzada per comunitats d’usuaris amb una activitat quasi frenètica (Jenkins,2008). Noves modalitats de treball cooperatiu que permeten la creació i recreació grupal de continguts, i la creació de comunitats d’usuaris que utilitzen i reutilitzen les noves modalitats de serveis. En aquest context, augmenta la segmentació, la fragmentació i l’abonament dels usuaris (Tous, 2009), perquè la tipologia de les plataformes de continguts ha variat de manera significativa, s’han incorporat els dispositius mòbils i s’han diversificat i sofisticat les ofertes a Internet.

Control d'un sistema de marcatge làser de caixes

RESUM Avui en dia, l’alta competitivitat que existeix al mercat, fa que les empreses hagin d’esprémer al màxim les seves possibilitats per no quedar-se enrere. Un dels processos en que aquest fet hi és més present és el productiu. L’empresa JCM Technologies també engloba aquest camp i és en un dels seus processos productius on aquest projecte pren part. L’objectiu d’aquest projecte final de carrera ha estat desenvolupar un sistema per poder marcar caixes mitjançant un làser de CO2 i un automatisme manipulador de caixes. D’aquesta manera aquest procés productiu té una durada molt inferior a l’antic procés, que consistia en enganxar una etiqueta al lloc on ara és marcat pel làser. Per satisfer els objectius, s’ha creat una aplicació de Windows que per mitjà d’una interfície gràfica, permet a l’usuari realitzar els passos necessaris per fer el marcatge. Primerament es recullen les dades procedents de la comanda; seguidament es seleccionen les que s’han de marcar a les caixes i s’envien al làser mitjançant comunicació sèrie; una vegada aquesta inicialització ha finalitzat correctament, s’engega la seqüència de marcatge de les caixes, que en marcarà la quantitat indicada. Aquest procés de marcatge consisteix en supervisar l’estat en que es troben certes senyals, procedents de l’automatisme i del làser, i depenent d’aquestes generar-ne unes altres. Aconseguint així realitzar el procés de marcatge de cada caixa. Com a conclusions cal dir, que els objectius s’han complert, ja que s’ha aconseguit un procés de marcatge ràpid i robust. També s’ha aconseguit que les parts de configuració de l’aplicació, i de les caixes siguin de fàcil manipulació. Per tant, amb l’acompliment dels objectius d’aquest projecte, aconseguim completar el sistema de marcatge, i permetre que les caixes siguin marcades de forma correcta, ràpida i eficient.

Bacterial subfamily of LuxR regulators that respond to plant compounds.

Pseudomonas fluorescens are rhizobacteria known for their biocontrol properties. Several antimicrobial functions are crucial for this process, and the experiments described here investigate the modulation of their expression during the plant-bacterium interaction. The role of a LuxR family regulator in interkingdom signaling has been investigated using genome-scale transcriptome analysis, gene promoter studies in vivo and in vitro, biocontrol assays, and response to plant compounds. PsoR, a LuxR solo or orphan regulator of P. fluorescens, was identified. PsoR is solubilized and activates a lux-box-containing promoter only in the presence of macerated plants, suggesting the presence of a plant molecule(s) that most likely binds to PsoR. Gene expression profiles revealed that genes involved in the inhibition of plant pathogens were affected by PsoR, including a chitinase gene, iron metabolism genes, and biosynthetic genes of antifungal compounds. 2,4-Diacetylphloroglucinol production is PsoR dependent both in vitro and in vivo. psoR mutants were significantly reduced for their ability to protect wheat plants from root rot, and damping-off caused by Pythium ultimum infection. PsoR most likely senses a molecule(s) in the plant and modulates expression of genes that have a role in biocontrol. PsoR and related proteins form a subfamily of LuxR family regulators in plant-associated bacteria.

Identification of novel functional TBP-binding sites and general factor repertoires.

Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome.

Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human.

Diversidade genética e eficiência simbiótica de rizóbios noduladores de acácia-negra de solos do Rio Grande do Sul

A acácia-negra é a terceira espécie florestal mais cultivada no Brasil. Além de sua importância econômica, é utilizada na recuperação de áreas degradadas, nas quais o solo geralmente apresenta pH baixo e altos teores de Al. O presente trabalho objetivou avaliar a diversidade genética de rizóbios naturais de solos do Rio Grande do Sul e selecionar isolados eficientes na fixação de N2 em condições de pH baixo. Um total de 50 isolados de Bradyrhizobium sp. foi obtido, os quais, juntamente com as estirpes recomendadas BR 3067 e BR 3068, foram caracterizados com o marcador BOX A 1-R. O padrão de bandas dos isolados foi utilizado na construção de um dendrograma, a partir do qual se calculou o índice de diversidade de Shannon. Dez isolados foram testados quanto à tolerância a pH baixo e à presença de Al, selecionando-se oito para o teste de eficiência simbiótica em casa de vegetação. Observou-se diversidade genética elevada entre os isolados, com a formação de 10 grupos, a partir do ponto de corte de 70 % de similaridade e com o índice de diversidade de 4,30. A presença de Al não afetou os isolados avaliados, que tiveram seu crescimento reduzido em pH 4,5. Quanto à eficiência simbiótica, os isolados T6-16 e V-7 foram os mais eficientes, assemelhando-se à estirpe recomendada BR 3068.

ON THE ROLE OF PERIOD-2 IN THE CIRCADIAN AND HOMEOSTATIC REGULATION OF SLEEP

Humans spend one third of their life sleeping, then we could raise the basic question: Why do we sleep? Despite the fact that we still don't fully understand its function, we made much progress in understanding at different levels how sleep is regulated. One model suggests that sleep is regulated by two processes: a homeostatic process that tracks the need for sleep and by a circadian rhythm that determines the preferred time-of-day sleep occurs. At the molecular level circadian rhythms are a property of interlocking transcriptional regula-tors referred to as clock genes. The heterodimeric transcription factors BMAL1::CLOCK/NPAS2 drive the transcription of many target genes including the clock genes Cryptochome1 (Cry1), Cry2, Period1 (Per1), and Per2. The encoded CRY/PER proteins are transcriptional inhibitors of BMAL1::CLOCK/NPAS2 thereby providing negative feedback to their own transcription. These genes seem, however, also involved in sleep homeostasis because the brain expression of clock genes, es-pecially that of Per2, increase as a function of time-spent-awake and because mice lacking clock genes display altered sleep homeostasis. The aim of first part of my doctoral work has been to advance our understanding the link that exists between sleep homeostasis and circadian rhythms investigating a possible mechanism by which sleep deprivation could alter clock gene expression by quantifying DNA-binding of the core-clock genes BMAL1, CLOCK and NPAS2 to their target chromatin loci including the E-box enhancers of the Per2 promoter. We made use of chromatin immunoprecipitation (ChIP) and quantitative poly-merase chain reaction (qPCR) to show that DNA-binding of CLOCK and BMAL1 to their target genes changes as a function of time-of-day in both liver and cerebral cortex. We then performed a 6h sleep deprivation (SD) and observed a significant decrease in DNA-binding of CLOCK and BMAL1 to Dbp. This is consistent with a decrease in Dbp mRNA levels after SD. The DNA-binding of NPAS2 and BMAL1 to Per2 was similarly decreased following SD. However, SD has been previously shown to in-crease Per2 expression in the cortex which seems paradoxical. Our results demonstrate that sleep-wake history can affect the molecular clock machinery directly at the level of the chromatin thereby altering the cortical expression of Dbp and Per2, and likely other targets. However, the precise dy-namic relationship between DNA-binding and mRNA expression, especially for Per2, remains elusive. The second aim of my doctoral work has been to perform an in depth characterization of cir-cadian rhythmicity, sleep architecture, analyze the response to SD in full null-Per2 knock-out (Per2-/-) mice, and Per1-/- mice, as well as their double knock-out offspring (Per1,2-/-) and littermate wildtype (Wt) mice. The techniques used include locomotor activity recording by passive infrared (PIR) sen-sors, EEG/EMG surgery, recording, and analysis, and cerebral cortex extraction and quantification of mRNA levels by qPCR. Under standard LD12:12 conditions, we found that wakefulness onset, as well as the time courses of clock gene expression in the brain and corticosterone plasma levels were ad-vanced by about 2h in Per2-/- mice compared to Wt mice. When released under constant dark condi-tions almost all Per2-/- mice (97%) became arrhythmic immediately. From these observations, we conclude that while Per2-/- mice seem to be able to anticipate dark onset, this does not result from a self-sustained circadian clock. Our results suggest instead that the earlier onset of activity results from a labile, not-self sustained 22h rhythm linked to light onset suggesting the existence of a light-driven rhythm. Analyses of sleep under LD12:12 conditions revealed that in both Per2-/- and Per1,2-/- mice the same sleep phenotypes are observed compared to Wt mice: increased NREM sleep frag-mentation and inability to adequately compensate the loss of NREM sleep. That suggests a possible role of PER2 in sleep consolidation and recovery.

Nutritional and insulin regulation of fatty acid synthetase and leptin gene expression through ADD1/SREBP1.

The ability to regulate specific genes of energy metabolism in response to fasting and feeding is an important adaptation allowing survival of intermittent food supplies. However, little is known about transcription factors involved in such responses in higher organisms. We show here that gene expression in adipose tissue for adipocyte determination differentiation dependent factor (ADD) 1/sterol regulatory element binding protein (SREBP) 1, a basic-helix-loop-helix protein that has a dual DNA-binding specificity, is reduced dramatically upon fasting and elevated upon refeeding; this parallels closely the regulation of two adipose cell genes that are crucial in energy homeostasis, fatty acid synthetase (FAS) and leptin. This elevation of ADD1/SREBP1, leptin, and FAS that is induced by feeding in vivo is mimicked by exposure of cultured adipocytes to insulin, the classic hormone of the fed state. We also show that the promoters for both leptin and FAS are transactivated by ADD1/SREBP1. A mutation in the basic domain of ADD1/SREBP1 that allows E-box binding but destroys sterol regulatory element-1 binding prevents leptin gene transactivation but has no effect on the increase in FAS promoter function. Molecular dissection of the FAS promoter shows that most if not all of this action of ADD1/SREBP1 is through an E-box motif at -64 to -59, contained with a sequence identified previously as the major insulin response element of this gene. These results indicate that ADD1/SREBP1 is a key transcription factor linking changes in nutritional status and insulin levels to the expression of certain genes that regulate systemic energy metabolism.

Avaliação da biodiversidade de rizóbios simbiontes do feijoeiro (Phaseolus vulgaris L.) em Santa Catarina

Os solos brasileiros, em geral, apresentam uma população abundante de rizóbios capazes de nodular e fixar N2 em simbiose com o feijoeiro (Phaseolus vulgaris L.); contudo, a diversidade dessas bactérias ainda é pouco conhecida. Este estudo teve por objetivo conhecer a biodiversidade de microssimbiontes do feijoeiro em Santa Catarina e, para isso, foram obtidos 117 isolados de nódulos de plantas coletadas em campo, em 23 áreas do extremo oeste, do meio oeste e do planalto sul catarinense. Com base nos atributos morfofisiológicos, os isolados foram classificados em nove grupos. Pela análise dos perfis de DNA após a amplificação (PCR) com o "primer" BOX, que codifica regiões conservadas e repetidas do genoma, 107 perfis distintos foram agrupados em um nível final de similaridade de apenas 26,9 %. Os perfis obtidos pela amplificação do gene 16S ribossômico - referência na taxonomia atual de procariotos - seguida pela digestão com três enzimas de restrição (técnica de RFLP-PCR), resultaram em seis agrupamentos principais e cinco bactérias isoladas. As populações consistiram de 17,1 % de Rhizobium tropici, 35,9 % de R. etli, 32,5 % de R. leguminosarum, 1,7 % de R. giardinii e 12,8 % com perfis distintos das espécies descritas de rizóbios de feijoeiro. R. tropici predominou em solos ácidos do meio oeste e do planalto sul, R. leguminosarum não foi detectado no extremo oeste e R. etli ocorreu nas três regiões, essas duas últimas espécies em solos menos ácidos. Os resultados enfatizam a diversidade genética elevada de rizóbios, inter e intra-específica, nos solos catarinenses, inclusive com a indicação de novas espécies.