Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

Selective neuronal toxicity of cocaine in embryonic mouse brain cocultures.

Cocaine exposure in utero causes severe alterations in the development of the central nervous system. To study the basis of these teratogenic effects in vitro, we have used cocultures of neurons and glial cells from mouse embryonic brain. Cocaine selectively affected embryonic neuronal cells, causing first a dramatic reduction of both number and length of neurites and then extensive neuronal death. Scanning electron microscopy demonstrated a shift from a multipolar neuronal pattern towards bi- and unipolarity prior to the rounding up and eventual disappearance of the neurons. Selective toxicity of cocaine on neurons was paralleled by a concomitant decrease of the culture content in microtubule-associated protein 2 (MAP2), a neuronal marker measured by solid-phase immunoassay. These effects on neurons were reversible when cocaine was removed from the culture medium. In contrast, cocaine did not affect astroglial cells and their glial fibrillary acidic protein (GFAP) content. Thus, in embryonic neuronal-glial cell cocultures, cocaine induces major neurite perturbations followed by neuronal death without affecting the survival of glial cells. Provided similar neuronal alterations are produced in the developing human brain, they could account for the qualitative or quantitative defects in neuronal pathways that cause a major handicap in brain function following in utero exposure to cocaine.

Subunit interactions in coordination of Ni2+ in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels present a unique model for studying the molecular mechanisms of channel gating. We have studied the mechanism of potentiation of expressed rod CNG channels by Ni2+ as a first step toward understanding the channel gating process. Here we report that coordination of Ni2+ between histidine residues (H420) on adjacent channel subunits occurs when the channels are open. Mutation of H420 to lysine completely eliminated the potentiation by Ni2+ but did not markedly alter the apparent cGMP affinity of the channel, indicating that the introduction of positive charge at the Ni(2+)-binding site was not sufficient to produce potentiation. Deletion or mutation of most of the other histidines present in the channel did not diminish potentiation by Ni2+. We studied the role of subunit interactions in Ni2+ potentiation by generating heteromultimeric channels using tandem dimers of the rod CNG channel sequence. Injection of single heterodimers in which one subunit contained H420 and the other did not (wt/H420Q or H420Q/wt) resulted in channels that were not potentiated by Ni2+. However, coinjection of both heterodimers into Xenopus oocytes resulted in channels that exhibited potentiation. The H420 residues probably occurred predominantly in nonadjacent subunits when each heterodimer was injected individually, but, when the two heterodimers were coinjected, the H420 residues could occur in adjacent subunits as well. These results suggest that the mechanism of Ni2+ potentiation involves intersubunit coordination of Ni2+ by H420. Based on the preferential binding of Ni2+ to open channels, we suggest that alignment of H420 residues of neighboring subunits into the Ni(2+)-coordinating position may be associated with channel opening.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Avaliação de impacto do Proagro Mais: um estudo de caso

Esta pesquisa avalia o impacto do \"Programa de Garantia da Atividade Agropecuária\" para agricultores familiares, conhecido como Proagro Mais. A relevância do trabalho fundamenta-se no considerável tamanho do Programa dentro do contexto das políticas de gestão de risco agrícola no Brasil. Além disso, é a primeira pesquisa desse tipo na literatura científica do país. A amostra é formada por produtores de milho do Estado do Paraná, tendo como linha base o ano de 2003, uma vez que é o ano anterior ao lançamento do Proagro Mais, e o ano de 2005 como ano de impacto. A base de dados utilizada neste estudo foi fornecida pelo Tribunal de Contas da União (TCU), cujas variáveis relevantes incluem características da cultura e dos agricultores familiares, como área financiada, atividades agrícolas complementares, educação e rendimento esperado. Adicionalmente, a partir de outras fontes públicas, foram adicionadas variáveis meteorológicas e regionais para controlar a localização da fazenda. O objetivo da pesquisa é avaliar o impacto do Proagro Mais sobre o montante de crédito por hectare concedido aos beneficiários do Programa. As metodologias usadas incluem o Propensity Score Matching (PSM), a Diferença das Diferenças (DID) e dois estimadores condicionais do DID com PSM usando dados em painel e repeated cross-section. As estimativas econométricas mostram que o Efeito Médio do Tratamento nos Tratados (EMTT) teve sinal negativo na maioria dos modelos revelando que, após o período de perda de rendimento, o grupo de controle teve um valor médio mais elevado de crédito por hectare do que os beneficiários do Proagro Mais. Os resultados sugerem a existência de mecanismos que poderiam complementar ou substituir o Proagro Mais como instrumento de gestão de risco agrícola, mas também podem sugerir que o Programa avaliado não cubra todos os riscos do setor.

Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon

Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons.

Essays on Firm Heterogeneity with Empirical Applications in Economic History and Agricultural Economics

This thesis uses models of firm-heterogeneity to complete empirical analyses in economic history and agricultural economics. In Chapter 2, a theoretical model of firm heterogeneity is used to derive a statistic that summarizes the welfare gains from the introduction of a new technology. The empirical application considers the use of mechanical steam power in the Canadian manufacturing sector during the late nineteenth century. I exploit exogenous variation in geography to estimate several parameters of the model. My results indicate that the use of steam power resulted in a 15.1 percent increase in firm-level productivity and a 3.0-5.2 percent increase in aggregate welfare. Chapter 3 considers various policy alternatives to price ceiling legislation in the market for production quotas in the dairy farming sector in Quebec. I develop a dynamic model of the demand for quotas with farmers that are heterogeneous in their marginal cost of milk production. The econometric analysis uses farm-level data and estimates a parameter of the theoretical model that is required for the counterfactual experiments. The results indicate that the price of quotas could be reduced to the ceiling price through a 4.16 percent expansion of the aggregate supply of quotas, or through moderate trade liberalization of Canadian dairy products. In Chapter 4, I study the relationship between farm-level productivity and participation in the Commercial Export Milk (CEM) program. I use a difference-in-difference research design with inverse propensity weights to test for causality between participation in the CEM program and total factor productivity (TFP). I find a positive correlation between participation in the CEM program and TFP, however I find no statistically significant evidence that the CEM program affected TFP.

Triangular Geopolitics in Europe’s Eastern Neighbourhood. CEPS Commentary, 2 December 2014

Over the last few months, Russia has employed a number of economic and security measures to derail the Deep and Comprehensive Free Trade Agreement (DCFTA) between the EU and Ukraine. Russia’s opposition to the Agreement was based on the argument that it would damage its economy and weaken its trade ties with Ukraine. Russia’s actions ultimately led to war in Ukraine, but did not succeed in reversing Ukraine’s EU integration policies; instead there are now trilateral negotiations between the EU, Ukraine and Russia on mere technical trade aspects of the DCFTA. The Kremlin is using similar rhetoric and, to some extent, similar coercive measures against the DCFTAs with Moldova and Georgia. But the small scale of Moldovan and Georgian trade with Russia is not a legitimate reason for the EU to replicate the Ukraine ‘trialogue’ on the DCFTAs in these countries. Instead, Moldova’s heavy dependence on Russia’s energy and the former’s transit role for the EU offers a greater possibility to set up trilateral negotiations, similar to the recently finalised gas trialogue between the EU, Ukraine and Russia.

Activities of fenbendazole in comparison with albendazole against Echinococcus multilocularis metacestodes in vitro and in a murine infection model.

The current chemotherapeutic treatment of alveolar echinococcosis (AE) in humans is based on albendazole and/or mebendazole. However, the costs of treatment, life-long consumption of drugs, parasitostatic rather than parasiticidal activity of chemotherapy, and high recurrence rates after treatment interruption warrant more efficient treatment options. Experimental treatment of mice infected with Echinococcus multilocularis metacestodes with fenbendazole revealed similar efficacy to albendazole. Inspection of parasite tissue from infected and benzimidazole-treated mice by transmission electron microscopy (TEM) demonstrated drug-induced alterations within the germinal layer of the parasites, and most notably an almost complete absence of microtriches. On the other hand, upon in vitro exposure of metacestodes to benzimidazoles, no phosphoglucose isomerase activity could be detected in medium supernatants during treatment with any of these drugs, indicating that in vitro treatment did not severely affect the viability of metacestode tissue. Corresponding TEM analysis also revealed a dramatic shortening/retraction of microtriches as a hallmark of benzimidazole action, and as a consequence separation of the acellular laminated layer from the cellular germinal layer. Since TEM did not reveal any microtubule-based structures within Echinococcus microtriches, this effect cannot be explained by the previously described mechanism of action of benzimidazoles targeting β-tubulin, thus benzimidazoles must interact with additional targets that have not been yet identified. In addition, these results indicate the potential usefulness of fenbendazole for the chemotherapy of AE.

Risk of Cerebral Venous Thrombosis in Obese Women.

IMPORTANCE Obesity is a risk factor for deep vein thrombosis of the leg and pulmonary embolism. To date, however, whether obesity is associated with adult cerebral venous thrombosis (CVT) has not been assessed. OBJECTIVE To assess whether obesity is a risk factor for CVT. DESIGN, SETTING, AND PARTICIPANTS A case-control study was performed in consecutive adult patients with CVT admitted from July 1, 2006 (Amsterdam), and October 1, 2009 (Berne), through December 31, 2014, to the Academic Medical Center in Amsterdam, the Netherlands, or Inselspital University Hospital in Berne, Switzerland. The control group was composed of individuals from the control population of the Multiple Environmental and Genetic Assessment of Risk Factors for Venous Thrombosis study, which was a large Dutch case-control study performed from March 1, 1999, to September 31, 2004, and in which risk factors for deep vein thrombosis and pulmonary embolism were assessed. Data analysis was performed from January 2 to July 12, 2015. MAIN OUTCOMES AND MEASURES Obesity was determined by body mass index (BMI). A BMI of 30 or greater was considered to indicate obesity, and a BMI of 25 to 29.99 was considered to indicate overweight. A multiple imputation procedure was used for missing data. We adjusted for sex, age, history of cancer, ethnicity, smoking status, and oral contraceptive use. Individuals with normal weight (BMI <25) were the reference category. RESULTS The study included 186 cases and 6134 controls. Cases were younger (median age, 40 vs 48 years), more often female (133 [71.5%] vs 3220 [52.5%]), more often used oral contraceptives (97 [72.9%] vs 758 [23.5%] of women), and more frequently had a history of cancer (17 [9.1%] vs 235 [3.8%]) compared with controls. Obesity (BMI ≥30) was associated with an increased risk of CVT (adjusted odds ratio [OR], 2.63; 95% CI, 1.53-4.54). Stratification by sex revealed a strong association between CVT and obesity in women (adjusted OR, 3.50; 95% CI, 2.00-6.14) but not in men (adjusted OR, 1.16; 95% CI, 0.25-5.30). Further stratification revealed that, in women who used oral contraceptives, overweight and obesity were associated with an increased risk of CVT in a dose-dependent manner (BMI 25.0-29.9: adjusted OR, 11.87; 95% CI, 5.94-23.74; BMI ≥30: adjusted OR, 29.26; 95% CI, 13.47-63.60). No association was found in women who did not use oral contraceptives. CONCLUSIONS AND RELEVANCE Obesity is a strong risk factor for CVT in women who use oral contraceptives.

Three Essays on the Economics of International Migration [electronic resource].

requirements for Filipino performing artists, effectively closing this migration channel. I employ a difference-in-differences estimation strategy using the share of performing artists at baseline in each Philippine province as a continuous policy variable. International migration falls in response to the policy change by 1.2%. The effect on international migration is larger than the policy change itself would suggest, indicating the importance of spillovers across migrant occupations. Domestically, more children are employed, and adults are more likely to be unemployed or engaged in short-term work.

Methylomic and Transcriptomic Insights Linking Air Pollution and Atherosclerosis

Thesis (Ph.D.)--University of Washington, 2016-06

The relationship between diurnal type and time duration estimation at morning and evening times of day

This study investigates whether different diurnal types (morning versus evening) differ in their estimation of time duration at different times of the day. Given that the performance of morning and evening types is typically best at their preferred times of day, and assuming different diurnal trends in subjective alertness (arousal?) for morning and evening types, and adopting the attentional gate model of time duration estimation, it was predicted that morning types would tend to underestimate and be more accurate in the morning compared to evening types where the opposite pattern was expected. Nineteen morning types, 18 evening types and 18 intermediate types were drawn from a large sample (N=1175) of undergraduates administered the Early/Late Preference Scale. Groups performed a time duration estimation task using the production method for estimating 20-s unfilled intervals at two times of day: 0800/1830. The median absolute error, median directional error and frequency of under- and overestimation were analysed using repeated-measures ANOVA. While all differences were statistically non-significant, the following trends were observed: morning types performed better than evening types; participants overestimated in the morning and underestimated in the evening; and participants were more accurate later in the day. It was concluded that the trends are inconsistent with a relationship between subjective alertness and time duration estimation but consistent with a possible relationship between time duration estimation and diurnal body temperature fluctuations. (C) 2002 Elsevier Ltd. All rights reserved.

Neighborhood Change after Investment in Light-rail Transit (LRT)

Thesis (Master's)--University of Washington, 2016-06

"mind the gap": The size-distance dissociation in visual neglect is a cueing effect

There is a growing body of evidence that the processes mediating the allocation of spatial attention within objects may be separable from those governing attentional distribution between objects. In the neglect literature, a related proposal has been made regarding the perception of (within-object) sizes and (between-object) distances. This proposal follows observations that, in size-matching and bisection tasks, neglect is more strongly expressed when patients are required to attend to the sizes of discrete objects than to the (unfilled) distances between objects. These findings are consistent with a partial dissociation between size and distance processing, but a simpler alternative must also be considered. Whilst a neglect patient may fail to explore the full extent of a solid stimulus, the estimation of an unfilled distance requires that both endpoints be inspected before the task can be attempted at all. The attentional cueing implicit in distance estimation tasks might thus account for their superior performance by neglect patients. We report two bisection studies that address this issue. The first confirmed, amongst patients with left visual neglect, a reliable reduction of rightward error for unfilled gap stimuli as compared with solid lines. The second study assessed the cause of this reduction, deconfounding the effects of stimulus type (lines vs. gaps) and attentional cueing, by applying an explicit cueing manipulation to line and gap bisection tasks. Under these matched cueing conditions, all patients performed similarly on line and gap bisection tasks, suggesting that the reduction of neglect typically observed for gap stimuli may be attributable entirely to cueing effects. We found no evidence that a spatial extent, once fully attended, is judged any differently according to whether it is filled or unfilled.