Later Life Consequences of Subteratogenic Exposure to a Complex PAH Mixture in the Atlantic Killifish (Fundulus heteroclitus)

Subteratogenic and other low-level chronic exposures to toxicant mixtures are an understudied threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs) a large group of more than 100 individual compounds, which are important environmental (including aquatic) contaminants. Aquatic sediments constitute a major sink for hydrophobic pollutants, and studies show PAHs can persist in sediments over time. Furthermore, estuarine systems (namely breeding grounds) are of particular concern, as they are highly impacted by a wide variety of pollutants, and estuarine fishes are often exposed to some of the highest levels of contaminants of any vertebrate taxon. Acute embryonic exposure to PAHs results in cardiac teratogenesis in fish, and early life exposure to certain individual PAHs and PAH mixtures cause heart alterations with decreased swimming capacity in adult fish. Consequently, the heart and cardiorespiratory system are thought to be targets of PAH mixture exposure. While many studies have investigated acute, teratogenic PAH exposures, few studies have longitudinally examined the impacts of subtle, subteratogenic PAH mixture exposures, which are arguably more broadly applicable to environmental contamination scenarios. The goal of this dissertation was to highlight the later-life consequences of early-life exposure to subteratogenic concentrations of a complex, environmentally relevant PAH mixture.

A unique population of Fundulus heteroclitus (the Atlantic killifish or mummichog, hereafter referred to as killifish), has adapted to creosote-based polycyclic aromatic hydrocarbons (PAHs) found at the Atlantic Wood Industries (AW) Superfund site in the southern branch of the Elizabeth River, VA, USA. This killifish population survives in a site heavily contaminated with a mixture of PAHs from former creosote operations. They have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to compare and contrast later-life outcomes of early-life, subteratogenic PAH mixture exposure in both the Atlantic Wood killifish (AW) and a naïve reference population of killifish from King’s Creek (KC; a relatively uncontaminated tributary of the Severn River, VA). Killifish from both populations were exposed to subteratogenic concentrations of a complex PAH-sediment extract, Elizabeth River Sediment Extract (ERSE), made by collecting sediment from the AW site. Fish were reared over a 5-month period in the laboratory, during which they were examined for a variety of molecular, physiological and behavioral responses.

The central aims of my dissertation were to determine alterations to embryonic gene expression, larval swimming activity, adult behavior, heart structure, enzyme activity, and swimming/cardiorespiratory performance following subteratogenic exposure to ERSE. I hypothesized that subteratogenic exposure to ERSE would impair cardiac ontogenic processes in a way that would be detectable via gene expression in embryos, and that the misregulation of cardiac genes would help to explain activity changes, behavioral deficits, and later-life swimming deficiencies. I also hypothesized that fish heart structure would be altered. In addition, I hypothesized that the AW killifish population would be resistant to developmental exposures and perform normally in later life challenges. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from Elizabeth River and in reference killifish. As an ancillary project to the primary aims of the dissertation, I examined the toxicity of weaker aryl hydrocarbon receptor (AHR) agonists in combination with fluoranthene (FL), an inhibitor of cytochrome P4501A1 (CYP1A1). This side project was conducted in both Danio rerio (zebrafish) and the KC and AW killifish.

Embryonic gene expression was measured in both killifish populations over an ERSE dose response with multiple time points (12, 24, 48, and 144 hours post exposure). Genes known to play critical roles in cardiac structure/development, cardiac function, and angiogenesis were elevated, indicating cardiac damage and activation of cardiovascular repair mechanisms. These data helped to inform later-life swimming performance and cardiac histology studies. Behavior was assessed during light and dark cycles in larvae of both populations following developmental exposure to ERSE. While KC killifish showed activity differences following exposure, AW killifish showed no significant changes even at concentrations that would cause overt cardiac toxicity in KC killifish. Juvenile behavior experiments demonstrated hyperactivity following ERSE exposure in KC killifish, but no significant behavioral changes in AW killifish. Adult swimming performance via prolonged critical swimming capacity (Ucrit) demonstrated performance costs in the AW killifish. Furthermore, swimming performance decline was observed in KC killifish following exposure to increasing dilutions of ERSE. Lastly, cardiac histology suggested that early-life exposure to ERSE could result in cardiac structural alteration and extravasation of blood into the pericardial cavity.

Responses to AHR agonists resulted in a ranking of relative potency for agonists, and determined which agonists, when combined with FL, caused cardiac teratogenesis. These experiments showed interesting species differences for zebrafish and killifish. To probe mechanisms responsible for cardiotoxicity, a CYP1A-morpholino and a AHR2-morpholino were used to mimic FL effects or attempt to rescue cardiac deformities respectively. Findings suggested that the cardiac toxicity elicited by weak agonist + FL exposure was likely driven by AHR-independent mechanisms. These studies stand in contrast to previous research from our lab showing that moderate AHR agonist + FL caused cardiac toxicity that can be partially rescued by AHR-morpholino knockdown.

My findings will form better characterization of mechanisms of PAH toxicity, and advance our understanding of how subteratogenic mixtures of PAHs exert their toxic action in naïve killifish. Furthermore, these studies will provide a framework for investigating how subteratogenic exposures to PAH mixtures can impact aquatic organismal health and performance. Most importantly, these experiments have the potential to help inform risk assessment in fish, mammals, and potentially humans. Ultimately, this research will help protect populations exposed to subtle PAH-contamination.

Manutenção preventiva ou manutenção corretiva em linhas aéreas de média tensão? Utilização do data envelopment analysis como auxiliar de gestão dos ativos técnicos de manutenção

O uso de Data Envelopment Analysis (DEA) no setor de distribuição de energia elétrica tem dado origem à publicação de vários artigos científicos. No geral, estes artigos focam-se na comparação da eficiência das empresas de distribuição de eletricidade. Na generalidade dos artigos, o tratamento da informação tem sido predominantemente descritivo e classificatório, sem focar no processo de transformação. Em contraste, o trabalho que se apresenta aqui pretende mostrar as potencialidades do DEA na análise de variáveis do processo de transformação e procura explorar o seu potencial para a identificação dos programas e intervenções que contribuem para a melhoria efetiva no processo de distribuição de eletricidade. É nossa convicção que as avaliações de natureza formativa, com fins de aprendizagem, são mais eficazes do que os estudos sumativos porque contribuem para uma melhor compreensão das estruturas e processos, sendo portanto mais adequadas para contribuir para a melhoria do desempenho. Neste trabalho, apresenta-se uma questão importante no contexto da análise DEA: a de investigar, se as diferenças de eficiência são devidas a um programa específico de gestão ou às características de conceção. Para o efeito, o estudo recorre a dois métodos diferentes para realizar este tipo de análise. Em primeiro lugar, aplicamos a estatística de rank de Mann-Whitney aos scores do DEA, a fim de avaliar a significância estatística das diferenças observadas entre um programa de tratamento e o programa de controlo. Em segundo lugar, procedemos a uma análise dinâmica com o Índice de Produtividade de Malmquist, a fim de estudar o impacto da introdução de uma nova tecnologia num grupo de unidades. O estudo de caso desenvolvido centra-se na avaliação do desempenho de linhas de média tensão afetas a uma das regiões de serviço de uma empresa de distribuição de energia elétrica, regulada pelo Sistema Público de Distribuição de Energia em Portugal (ERSE). Os resultados do estudo de caso mostram que a aplicação do DEA tem um grande potencial para contribuir para a melhoria dos processos e deve ser explorado noutros contextos.

A evolução do défice tarifário em Portugal

Segundo as previsões da Entidade Reguladora dos Serviços Energéticos (ERSE), o défice tarifário deverá atingir em finais do 2013 um valor acumulado de 3,7 mil milhões de Euros. Na comunicação social, o défice tarifário (DT) é considerado como mais uma “renda” a alguns grupos de interesse. Na realidade o DT é justificado por uma acumulação sucessiva de diversos tipos de sobrecustos do sistema elétrico que não foi considerada nas tarifas e preços da eletricidade nos anos anteriores aos respetivos consumidores elétricos. O trabalho aqui apresentado corresponde a uma primeira tentativa de analisar as razões políticas e económicofinanceiras para a existência do défice tarifário. Nesse sentido serão abordadas: as origens e justificações dos diferentes sobrecustos (CAE’s, CMEC’s, Garantia de Potência, Produção em Regime geral (PRE), convergência tarifária com as regiões autónomas dos Açores (RAA) e da Madeira (RAM), entre outros); a análise dos diferentes tipos de mecanismos; os parâmetros no cálculo dos sobrecustos bem como as possíveis soluções e medidas já implementadas para reduzir os efeitos do défice tarifário nas tarifas nos consumidores.

Melhortarifa.pt – simulador de tarifários

Assistimos à queda de um serviço, que em muitos países, figurava como um serviço de um modelo social europeu, passando de um monopólio para um sistema de mercado liberalizado e concorrencial: o mercado energético. (EURO COOP, 2014) Em maio de 2014, o mercado livre chegou a ultrapassar os 2,8 milhões de consumidores, tendo um crescimento face a maio de 2013 de 64%, com uma taxa média mensal de 4,2%. (Entidade Reguladora dos Energéticos - ERSE, 2009). Perante a crescente adesão de consumidores para o mercado liberalizado, e suas possíveis futuras mudanças, e tendo em conta com os potenciais clientes a aderirem, foi proposto então construir a plataforma MelhorTarifa.pt . Apesar da já existência de simuladores, estes demonstram-se a ser pouco intuitivos e a não demonstrar toda a informação que o utilizador pretende bem como não existir uma imagem de marca associada a um apoio à mudança de tarifário, tendo sido estas umas das razões que levaram ao desenvolvimento do MelhorTarifa.pt. O âmbito principal deste trabalho, é fornecer uma plataforma informática grátis, imparcial, com o acréscimo até à data, em relação a outros simuladores, da disponibilização de faturas detalhadas sobre o produto que o cliente pretende adquirir, serviços de consultoria, tudo isto de forma rápida e de simples acesso. Com este trabalho pretende-se também definir o atual estado de arte de simuladores idênticos em Portugal e no estrangeiro, pretendendo-se também dar conhecimento do trabalho desenvolvido para esta dissertação, denominado como MelhorTarifa.pt.

Ética, responsabilidad social y empresa

Este texto recoge algunos de los resultados de la investigación en ética, responsabilidad social y empresa, llevada a cabo durante siete años por el grupo ERSE (Ética, Responsabilidad Social y Empresa) de la Escuela de Ciencias Humanas de la Universidad del Rosario. Desde un punto de vista, las empresas se constituyen el lugar desde donde más evidentemente se promueven o se obstaculizan las condiciones para el ejercicio real de la ciudadanía: en la medida en que ellas se convierten fácilmente en el punto de encuentro entre los proyectos de vida individuales y las posibilidades de socializar esos proyectos, también son el lugar donde más claramente convergen la vida pública y la vida privada. Cada uno de los artículos que contiene el libro es el resultado de la convicción acerca de que la investigación filosófica tiene algo para decir sobre la manera como debe ser concebido el conjunto de reglas a través de las cuales las empresas dan marcha a su gestión.

Unfolded protein response suppresses CEBPA by induction of calreticulin in acute myeloid leukaemia

The unfolded protein response (UPR) is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). The role of the UPR during leukemogenesis is unknown so far. Here, we studied the induction of mediators of the UPR in leukaemic cells of AML patients. Increased expression of the spliced variant of the X-box binding protein 1 (XBP1s) was detected in 17.4% (16 of 92) of AML patients. Consistent with activated UPR, this group also had increased expression of ER-resident chaperones such as the 78 kD glucose-regulated protein (GRP78) and of calreticulin. Conditional expression of calreticulin in leukaemic U937 cells was found to increase calreticulin binding to the CEBPA mRNA thereby efficiently blocking translation of the myeloid key transcription factor CEBPA and ultimately affecting myeloid differentiation. Consequently, leukaemic cells from AML patients with activated UPR and thus increased calreticulin levels showed in fact suppressed CEBPA protein expression. We identified two functional ER stress response elements (ERSE) in the calreticulin promoter. The presence of NFY and ATF6, as well as an intact binding site for YY1 within these ERSE motifs were essential for mediating sensitivity to ER stress and activation of calreticulin. Thus, we propose a model of the UPR being activated in a considerable subset of AML patients through induction of calreticulin along the ATF6 pathway, thereby ultimately suppressing CEBPA translation and contributing to the block in myeloid differentiation.

Hepatic Responses of Juvenile Fundulus heteroclitus from Pollution-adapted and Nonadapted Populations Exposed to Elizabeth River Sediment Extract.

Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood Industries region of the Elizabeth River, Virginia, have passed polycyclic aromatic hydrocarbon (PAH) resistance to their offspring as evidenced by early life stage testing of developmental toxicity after exposure to specific PAHs. Our study focused on environmentally relevant PAH mixtures in the form of Elizabeth River sediment extract (ERSE). Juvenile (5 month) F1 progeny of pollution-adapted Atlantic Wood (AW) parents and of reference site (King's Creek [KC]) parents were exposed as embryos to ERSE. Liver alterations, including nonneoplastic lesions and microvesicular vacuolation, were observed in both populations. ERSE-exposed KC fish developed significantly more alterations than unexposed KC fish. Interestingly, unexposed AW killifish developed significantly more alterations than unexposed KC individuals, suggesting that AW juveniles are not fully protected from liver disease; rapid growth of juvenile fish may also be an accelerating factor for tumorigenesis. Because recent reports show hepatic tumor formation in adult AW fish, the differing responses from the 2 populations provided a way to determine whether embryo toxicity protection extends to juveniles. Future investigations will analyze older life stages of killifish to determine differences in responses related to chronic disease.

Developmental exposure to a complex PAH mixture causes persistent behavioral effects in naive Fundulus heteroclitus (killifish) but not in a population of PAH-adapted killifish.

Acute exposures to some individual polycyclic aromatic hydrocarbons (PAHs) and complex PAH mixtures are known to cause cardiac malformations and edema in the developing fish embryo. However, the heart is not the only organ impacted by developmental PAH exposure. The developing brain is also affected, resulting in lasting behavioral dysfunction. While acute exposures to some PAHs are teratogenically lethal in fish, little is known about the later life consequences of early life, lower dose subteratogenic PAH exposures. We sought to determine and characterize the long-term behavioral consequences of subteratogenic developmental PAH mixture exposure in both naive killifish and PAH-adapted killifish using sediment pore water derived from the Atlantic Wood Industries Superfund Site. Killifish offspring were embryonically treated with two low-level PAH mixture dilutions of Elizabeth River sediment extract (ERSE) (TPAH 5.04 μg/L and 50.4 μg/L) at 24h post fertilization. Following exposure, killifish were raised to larval, juvenile, and adult life stages and subjected to a series of behavioral tests including: a locomotor activity test (4 days post-hatch), a sensorimotor response tap/habituation test (3 months post hatch), and a novel tank diving and exploration test (3months post hatch). Killifish were also monitored for survival at 1, 2, and 5 months over 5-month rearing period. Developmental PAH exposure caused short-term as well as persistent behavioral impairments in naive killifish. In contrast, the PAH-adapted killifish did not show behavioral alterations following PAH exposure. PAH mixture exposure caused increased mortality in reference killifish over time; yet, the PAH-adapted killifish, while demonstrating long-term rearing mortality, had no significant changes in mortality associated with ERSE exposure. This study demonstrated that early embryonic exposure to PAH-contaminated sediment pore water caused long-term locomotor and behavioral alterations in killifish, and that locomotor alterations could be observed in early larval stages. Additionally, our study highlights the resistance to behavioral alterations caused by low-level PAH mixture exposure in the adapted killifish population. Furthermore, this is the first longitudinal behavioral study to use killifish, an environmentally important estuarine teleost fish, and this testing framework can be used for future contaminant assessment.

Adolescent depression in schools: + Contigo Project

epidemiological data. It involves a high degree of mortality, namely by suicide, which is the third leading cause of death in the 15-24 age group. Objectives: To assess the presence and severity of depressive symptoms in a non-clinical population of adolescents. Methodology: This is a quantitative, descriptive and cross-sectional study, using the Portuguese version of the Beck Depression Inventory (BDI-II). The sample was composed of 741 adolescents. Results: The results show that 31.2% of the adolescents have depression and, of these, 17.7% have moderate to severe depressive symptoms. Girls have higher levels of depression (p=.00). The total mean score in the BDI-II was 12. Conclusion: Given the adolescents' high vulnerability to depression and suicide, it is essential to implement prevention programs in schools to promote the early detection of depression and suicidal behaviors, and the referral to mental health services.

Saúde Mental em adolescentes portugueses: contributos do +Contigo

INTRODUÇÂO A prevalência de doença mental é uma das mais altas do mundo (Caldas de Almeida & Xavier, 2013) em adultos, desconhecendo-se os dados epidemiológicos para os adolescentes. O + Contigo é um projecto que visa promover a saúde mental e prevenir comportamentos suicidários na comunidade educativa (Santos et al 2013). OBJETIVOS Os objectivos deste estudos são caracterizar o bem-estar, autoconceito, coping e depressão numa amostra de 3500 adolescentes portugueses, estudantes do 7º ao 10 ano. Comparar os diversos indicadores tendo em conta o género. Comparar os diversos indicadores tendo em conta o ano de escolaridade. METODOLOGIA A recolha de dados foi feita através de questionário, preenchido em sala de aula, autorizado pela DSPE (inquérito n.º 0224900002). Do questionário faziam parte os seguintes instrumentos: Índice de Bem-estar da Organização Mundial de Saúde (1998), Escala Toulosiana de Coping elaborada por (Esparbés et 1993 e validada para Portugal, por Tap, Costa & Alves (2005), Inventário de Depressão de Beck criado por Beck & Steer (1987) e validado por Martins, (2000), Escala de auto-conceito de Piers (Piers-Harris Children's Self-Concept Scale 2, Piers & Hertzberg, 2002, validada por Veiga (2006 ) RESULTADOS Foram considerados válidos 3150 questionários, com 50,1% de raparigas, com média etária de 13,56 anos, distribuídos pelo 7º ano (43%); 8ºano (24,6%); 9º ano (15,1%); 10º ano (17,3%). Os resultados indicam níveis elevados de depressão 26,7%, com 14,4% com depressão moderada ou severa. O índice de bem-estar apresenta uma média de 18,75, o autoconceito uma média de 42,03 e o coping de 153,46. Nas variáveis protetoras de comportamentos suicidários as raparigas apresentam scores inferiores relativamente aos rapazes, apresentando níveis superiores na sintomatologia depressiva, considerado fator de risco para os comportamentos suicidários.Considerando o ano de escolaridade verificou-se, de forma consistente, um agravamento das variáveis estudadas ao longo dos anos, mas com uma diferença estatisticamente significativa entre os alunos do 10º ano e os restantes. CONCLUSÕES Cerca de um quarto dos adolescentes apresentam níveis de sintomatologia depressiva, em que 14,4% apresentam níveis moderados ou severos, mais evidentes nas raparigas e agravando-se ao longo da sua progressão académica, entre o 7º e o 10º ano. As adolescentes apresentam maiores vulnerabilidades em saúde mental. Estes achados reforçam a necessidade de privilegiar a promoção da saúde mental junto da comunidade educativa e, particularmente, junto dos adolescentes. Tendo em conta os dados recolhidos, a supervisão, monitorização e acompanhamento de comportamentos de risco assume particular importância na promoção de saúde mental, evidenciando assim a relevância do projecto + Contigo. BIBLIOGRAFIA- Caldas de Almeida, J., & Xavier, M. (2013). Estudo Epidemiológico Nacional de Saúde Mental (Vol. 1). Lisboa. Faculdade de Ciências Médicas, da Universidade Nova de Lisboa. Santos, José; Erse, Maria; Simões, Rosa; Façanha, Jorge; Marques, Lucia; (2013) "+ Contigo na promoção da saúde mental e prevenção de comportamentos suicidários em meio escolar" - Revista de Enfermagem Referência, Número: 10, Série: III série, 1ª Edição, UICISA-E, Coimbra, p203 - 207.

The +Contigo Project in the Voice of Eight-Grade Students

The +Contigo Project is a longitudinal research project based on a multilevel network intervention aimed at promoting mental health and well-being and preventing suicidal behaviors. Students participate in social skills training sessions on the stigma of mental disorders, adolescence, self-esteem, problem-solving skills, and wellbeing. Problem statement: To identify the qualitative impact of the +Contigo Project. Objectives: To identify the importance of the project for students, the problem situations which it helped to solve, and its impact on an individual level. Research methods: Content analysis (Bardin, 2009) with a posteriori categorical identification. Semi-structured interview guide with 5 questions. A convenience sample of 16 students, from three schools of one school cluster in the Center Region of Portugal, was used. The ethical aspects of parental consent, voluntary participation and data confidentiality were addressed. Findings: Fourteen categories emerged from the data analysis. The answers to Question 4 'How was this project important to you?' were particularly relevant. Students mentioned that it improved their self-esteem by increasing self-confidence and interpersonal skills, and that it increased their reflection and awareness on problems with which they learned how to cope during the sessions. Conclusions: All interviewees considered that the +Contigo project helped them to solve problems, improve their interpersonal skills and cope with the adolescence period. Students improved their self-esteem and self-knowledge, and were able to apply what they had learned in the sessions in their daily lives. We concluded that the project had a positive impact on the students' mental health.

Saúde mental em adolescentes portugueses: contributos do + Contigo

Conclusões: Cerca de um quarto dos adolescentes apresentam níveis de sintomatologia depressiva, em que 14,4% apresentam níveis moderados ou severos, sendo estes mais evidentes nas raparigas e agravando-se ao longo do sua progressão académica entre o 7º e o 10ºano. As adolescentes apresentam maiores níveis de vulnerabilidade em saúde mental. Estes achados reforçam a necessidade de privilegiar a promoção da saúde mental junto da comunidade educativa e, particularmente, junto dos adolescentes. Tendo em conta os dados recolhidos, a supervisão, monitorização e acompanhamento de comportamentos de risco assume particular importância na promoção de saúde mental, evidenciando assim a relevância do projeto + Contigo.