Recruitment of hepatocyte nuclear factor 4 into specific intranuclear compartments depends on tyrosine phosphorylation that affects its DNA-binding and transactivation potential.

Hepatocyte nuclear factor 4 (HNF-4) is a prominent member of the family of liver-enriched transcription factors, playing a role in the expression of a large number of liver-specific genes. We report here that HNF-4 is a phosphoprotein and that phosphorylation at tyrosine residue(s) is important for its DNA-binding activity and, consequently, for its transactivation potential both in cell-free systems and in cultured cells. Tyrosine phosphorylation did not affect the transport of HNF-4 from the cytoplasm to the nucleus but had a dramatic effect on its subnuclear localization. HNF-4 was concentrated in distinct nuclear compartments, as evidenced by in situ immunofluorescence and electron microscopy. This compartmentalization disappeared when tyrosine phosphorylation was inhibited by genistein. The correlation between the intranuclear distribution of HNF-4 and its ability to activate endogenous target genes demonstrates a phosphorylation signal-dependent pathway in the regulation of transcription factor activity.

A dominant-negative mutant of human poly(ADP-ribose) polymerase affects cell recovery, apoptosis, and sister chromatid exchange following DNA damage.

Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.

O \"si\" da técnica psicanalítica: uma análise institucional do discurso de O mal-estar na civilização

Partindo das considerações de que os termos sujeito e subjetividade permeiam o discurso psicanalítico contemporâneo e de que são, direta e indiretamente, atribuídos a Freud a despeito de ele próprio nunca tê-los conceituado, esta pesquisa tem como objetivo caracterizar um perfil de sujeito a partir do discurso freudiano. O trabalho orientou-se pela metodologia da Análise Institucional do Discurso, uma analítica do domínio subjetivo que toma o discurso em seu caráter de ato e acontecimento. Primeiramente se realizou um estudo de As técnicas de si, de Michel Foucault, de modo a permitir o circunstanciamento da psicanálise como uma técnica que produz um si, um sujeito este circunstanciamento permitiu, então, tomar sujeito e subjetividade na qualidade de produções histórica, geográfica e analiticamente contextualizadas, não como formas de imanência ou transcendência. A partir desse pressuposto, elaborou-se uma análise institucional do discurso de O mal-estar na civilização que privilegiou não apenas seu conteúdo, mas principalmente seu modo de produção, colocando em relevo o contexto presente no texto, as interlocuções que se criam, os lugares atribuídos e ocupados, as expectativas assim mobilizadas, as estratégias discursivas utilizadas, os jogos de poder e verdade exercidos, bem como os efeitos de reconhecimento e desconhecimento então facultados. Esta análise mostrou que Freud exerce uma perspectiva de interioridade, pois o mal-estar que acomete a civilização é compreendido em analogia à concepção psicanalítica de desenvolvimento individual, explicando, em suma, a cultura pelo prisma do indivíduo; evidenciou que as teorizações sobre a vida instintiva são a principal sustentação do discurso sobre o mal-estar da civilização; apontou como as estratégias discursivas utilizadas por Freud promovem a subjetivação, por parte do leitor, daquilo que seu discurso produz como verdade; e que o conceito de indivíduo é ocasião de exercício daquela perspectiva de interioridade e de atualização dos pressupostos teóricos. Com base nisso, pôde-se caracterizar um sujeito universal; psicologizado; determinado sobretudo pelos movimentos da sexualidade e da agressividade; cuja tônica recai sobre o dito mundo interno; dotado de origens e propósitos concernentes à vida instintiva; e cujo perfil é delimitado pela tarefa de administração dos instintos, isto é, cujo perfil se dá entre os imperativos superegóicos de renúncia e a margem de liberdade de que dispõe para satisfazer as exigências do princípio de prazer. Observou-se também, na esteira do pontuado por Guirado (2010), que em geral Freud naturaliza os termos do discurso teórico, fazendo de sua universalização a condição e o limite para se pensar o domínio subjetivo e a singularidade; diferentemente de Foucault, que compreende esse domínio em referência às relações de poder e saber, de forma contextualizada. Do ponto de vista da análise institucional do discurso freudiano, concluiu-se, finalmente, que o si ao qual a técnica psicanalítica dá lastro é efeito da perspectiva exercida por Freud, que promove o reconhecimento da interioridade instintiva como crivo da civilização, de um modo de vida e de si mesmo

Contribuição das células-tronco mesenquimais para as propriedades tumorigênicas de células de glioblastoma humano

Células-tronco mesenquimais (CTM) apresentam tropismo a tumores, sendo importantes componentes do estroma tumoral. No cérebro, o nicho perivascular é uma importante fonte de CTM, as quais podem contribuir direta e/ou indiretamente para o desenvolvimento de tumores, embora os mecanismos envolvidos sejam pouco conhecidos. No presente trabalho, investigou-se a influência de CTM sobre a proliferação, capacidade invasiva e tumorigenicidade de células de Glioblastoma (GBM) humano. Sabe-se que CTM produzem TGFB1, uma citocina multifuncional envolvida em imunomodulação, proliferação, migração e transição epitelial-mesenquimal de células tumorais. Experimentos in vitro, realizados com meios condicionados de CTM de cordão umbilical humano com silenciamento permanente do gene TGFB1, demonstraram que o TGFB1 secretado por CTM é capaz de aumentar significativamente a proliferação e viabilidade de células de GBM humano da linhagem U87FP635. Esses resultados revelam uma importante ação parácrina dessa citocina regulatória, quando produzida por outros tipos celulares contidos no microambiente tumoral. Entretanto, sob condições experimentais que melhor mimetizam o microambiente tumoral, detectou-se que CTM também afetam o comportamento de células tumorais por um mecanismo alternativo, dependente de contato celular, mas independente dos níveis de TGFB1 secretados pelas CTM. Sob condições de cocultivo celular, envolvendo contato físico entre CTM e células de GBM U87FP635, detectou-se um aumento significativo na quantidade de células tumorais viáveis. Quando cultivadas na forma de esferoides tumorais, o contato com CTM aumentou a capacidade invasiva das células U87FP635. Finalmente, em modelo in vivo ectópico de GBM, células U87FP635 geraram tumores mais desenvolvidos quando coinjetadas com CTM. Esses efeitos pró-tumorigênicos foram observados tanto em contato com CTM controles, quanto com CTM contendo o gene TGFB1 permanentemente silenciado. Assim, esses achados indicam que CTM podem exercer efeitos pró-tumorigênicos por dois mecanismos alternativos e independentes: ação parácrina de TGFB1 secretado por CTM e ação mediada por contato célula-célula. Nas condições experimentais testadas, o mecanismo dependente de contato célula-célula demonstrou ser predominante. O estudo proteômico do secretoma dessas células identificou 126 proteínas diferencialmente expressas além de 10 proteínas exclusivamente detectadas em meios condicionados de cocultivos de CTM com células de GBM U87FP635. Cerca de 80% dessas proteínas exclusivamente secretadas pelo contato célula-célula são componentes de exossomos e estão envolvidas em proliferação celular e desenvolvimento tecidual. Esses resultados apontam uma interação dinâmica de comunicação entre CTM e células tumorais, e revelam algumas proteínas interessantes potencialmente envolvidas em uma ação pró-tumorigênica de CTM mediada por contato celular

Manipulation of the Quorum Sensing Signal AI-2 Affects the Antibiotic-Treated Gut Microbiota

The mammalian gut microbiota harbors a diverse ecosystem where hundreds of bacterial species interact with each other and their host. Given that bacteria use signals to communicate and regulate group behaviors (quorum sensing), we asked whether such communication between different commensal species can influence the interactions occurring in this environment. We engineered the enteric bacterium, Escherichia coli, to manipulate the levels of the interspecies quorum sensing signal, autoinducer-2 (AI-2), in the mouse intestine and investigated the effect upon antibiotic-induced gut microbiota dysbiosis. E. coli that increased intestinal AI-2 levels altered the composition of the antibiotic-treated gut microbiota, favoring the expansion of the Firmicutes phylum. This significantly increased the Firmicutes/Bacteroidetes ratio, to oppose the strong effect of the antibiotic, which had almost cleared the Firmicutes. This demonstrates that AI-2 levels influence the abundance of the major phyla of the gut microbiota, the balance of which is known to influence human health.

Maternal separation followed by isolation-housing differentially affects prepulse inhibition of the acoustic startle response in C57BL/6 mice

Exposure to chronic stress is associated with an increased incidence of neuropsychiatric dysfunction. The current study evaluated two competing hypotheses, the cumulative stress and the match/mismatch hypothesis of neuropsychiatric dysfunction, using two paradigms relating to exposure to “stress”: pre-weaning maternal separation and post-weaning isolation-housing. C57BL/6 offspring were reared under four conditions: typical animal facility rearing (AFR, control), early handling (EH, daily 15 min separation from dam), maternal separation (MS, daily 4 hr separation from dam), and maternal and peer separation (MPS, daily 4 hr separation from dam and from littermates). After weaning, mice were either housed socially (2–3/cage) or in isolation (1/cage) and then tested for prepulse inhibition in adulthood. Isolation-housed MPS subjects displayed greater deficits in prepulse inhibition relative to socially-housed MPS subjects while socially-housed AFR subjects displayed greater deficits in prepulse inhibition relative to isolation-housed AFR subjects. The results indicate that these treatment conditions represent a potentially valuable model for evaluating the match/mismatch hypothesis in regards to neuropsychiatric dysfunction.

Genetic selection to increase bone strength affects prevalence of keel bone damage and egg parameters in commercially housed laying hens.

The prevalence of keel bone damage as well as external egg parameters of 2 pure lines divergently selected for high (H) and low (L) bone strength were investigated in 2 aviary systems under commercial conditions. A standard LSL hybrid was used as a reference group. Birds were kept mixed per genetic line (77 hens of the H and L line and 201 or 206 hens of the LSL line, respectively, per pen) in 8 pens of 2 aviary systems differing in design. Keel bone status and body mass of 20 focal hens per line and pen were assessed at 17, 18, 23, 30, 36, 43, 52, and 63 wk of age. External egg parameters (i.e., egg mass, eggshell breaking strength, thickness, and mass) were measured using 10 eggs per line at both 38 and 57 wk of age. Body parameters (i.e. tarsus and third primary wing feather length to calculate index of wing loading) were recorded at 38 wk of age and mortality per genetic line throughout the laying cycle. Bone mineral density (BMD) of 15 keel bones per genetic line was measured after slaughter to confirm assignment of the experimental lines. We found a greater BMD in the H compared with the L and LSL lines. Fewer keel bone fractures and deviations, a poorer external egg quality, as well as a lower index of wing loading were found in the H compared with the L line. Mortality was lower and production parameters (e.g., laying performance) were higher in the LSL line compared with the 2 experimental lines. Aviary design affected prevalence of keel bone damage, body mass, and mortality. We conclude that selection of specific bone traits associated with bone strength as well as the related differences in body morphology (i.e., lower index of wing loading) have potential to reduce keel bone damage in commercial settings. Also, the housing environment (i.e., aviary design) may have additive effects.

Wie rasch ändert sich die Waldvegetation als Folge von raschen Klimaänderungen?

How rapidly does forest vegetation change due to rapid climatic change? Current predictions of future climates show a global increase of mean temperatures of 1.4 to 5.8 °C. How rapidly can forest vegetation adapt to such predicted large changes, and in which way? We looked for answers in three different disciplines: ecological modelling, palaeoecology and succession theory. We found that changes of forest vegetation after rapid climatic changes can be continuous or abrupt. Rapid or abrupt changes may result within years to decades, among others, from marked drought as a direct effect of climate warming, limiting tree growth in the driest parts of Switzerland within a few years or decades. Indirectly, climate warming affects forest vegetation by forest fires, windstorms and, consequently, insect outbreaks. Questions relevant to forestry arise from these considerations: What is the most suitable combination of tree species for which management should aim in the future, and how do we adequately manage protection forests so that they can resist or adapt to the climatic change?

How unequal and unjust taxation affects the interests of the citizens of Pennsylvania.

Mode of access: Internet.

John Bull and his unemployed. A plain statement on the law of England as it affects the unemployed.

Mode of access: Internet.

List of theses written at Cornell relating directly or indirectly to the history of New York State.

Mode of access: Internet.

Informational hand book : prepared in response to numerous inquiries for statistical and other data relating directly or indirectly to the the activities of federal agencies.

Mode of access: Internet.

Mixed-Member Electoral Systems in Constitutional Context : Taiwan, Japan, and Beyond /

After the electoral reform in 1994, Japan saw a gradual evolution from a multi-party system toward a two-party system over the course of five House of Representatives election cycles. In contrast, after Taiwan’s constitutional amendment in 2005, a two-party system emerged in the first post-reform legislative election in 2008. Critically, however, Taiwan’s president is directly elected while Japan’s prime minister is indirectly elected. The contributors conclude that the higher the payoffs of holding the executive office and the greater degree of cross-district coordination required to win it, the stronger the incentives for elites to form and stay in the major parties. In such a context, a country will move rapidly toward a two-party system. In Part II, the contributors apply this theoretical logic to other countries with mixed-member systems to demonstrate its generality. They find the effect of executive competition on legislative electoral rules in countries as disparate as Thailand, the Philippines, New Zealand, Bolivia, and Russia. The findings presented in this book have important implications for political reform. Often, reformers are motivated by high hopes of solving some political problems and enhancing the quality of democracy. But, as this group of scholars demonstrates, electoral reform alone is not a panacea. Whether and to what extent it achieves the advocated goals depends not only on the specification of new electoral rules per se but also on the political context—and especially the constitutional framework—within which such rules are embedded.

Fundamental research on raw materials used for electron emissivity on indirectly heated cathodes. Interim technical report.

1st has subtitle: First quarterly progress report; 2d-6th, Second-Sixth quarterly periodic status report.