Acute and long-term effects from petroleum discharges on temperate and Arctic Calanus species

Papers 1 and 2 of this thesis are not available in Munin: 1. Jensen, L.K., Carroll, JL., Pedersen, G., Hylland, K., Dahle, S. and Bakke, T.: 'A multigeneration Calanus finmarchicus culturing system for use in long-term oil exposure experiments', Journal of Experimental Marine Biology and Ecology (2006) 333: 71-78. Available at http://dx.doi.org/10.1016/j.jembe.2005.12.001 2. Jensen, L.K. and Carroll, JL.: 'Effects of the volatile petroleum component xylene on Arctic algae and zooplankton' (submitted manuscript to Marine Environmental Research).Summary Petroleum exploitation is emerging in the Arctic. In the Norwegian Arctic the southern Barents Sea is opened for development of oil and gas fields. This same area holds some of the world's largest and well managed commercial fish stocks such as Herring (Clupea harengus), Capelin (Mallotus villosus), Haddock (Melanogrammus aeglefinus), Atlantic Cod (Gadus morhua). For the interest of the fishing industry, as well as for maintaining a healthy ecosystem, petroleum related Environmental Risk Assessments (ERA) for the Barents Sea must be based on sound scientific knowledge of the special features of the Arctic ecosystem. In colder areas the physical behavior of oil is different compared to warmer areas. The lower temperature and lack of sun light during the Arctic winter slows down the natural physical weathering process of oil. In ERA in temperate areas the effects of exposure to the most volatile fraction of the oil are neglected due to short exposure time. However, in the Arctic the exposure to biota of this volatile fraction may be prolonged due to lower evaporation rates and the volatile fraction may be an important contributor to the overall adverse effect of an oil spill. Likewise, the exposure to the heavier oil fractions may be prolonged and knowledge on the long-term effects of exposure is essential. As an adaptation to the Arctic environment species found here have longer life spans, larger body sizes and higher lipid contents compared to temperate equivalent species. Thus Arctic species may accumulate contaminants over longer time and reach higher life time body levels. Meanwhile higher lipid content implies a higher affinity of lipophilic contaminants such as polycyclic aromatic hydrocarbons (PAHs). So, Arctic species may be exposed to oil compounds for a longer time because of the prolonged phycical presence and may, due to special adaptive features, be more efficient at accumulating oil contaminants. Today the knowledge of effects of oil contaminants on Arctic biota is limited, but growing. This thesis addresses a few of the research challenges in the field and provides knowledge on some effects of petroleum exposure to two copepod species; the sub-Arctic Calanus finmarchicus and the Arctic sibling species C. glacialis. First, Calanus was cultured for a generations under sub-Arctic conditions (Paper I). This work establishes that Calanus is suitable for ecological relevant long-term experiments. In an oil spill scenario, Arctic copepods and algae may be exposed to the more volatile fraction of the oil. Paper II provides data showing that the Arctic diatom species, Fragilariopsis oceanica, was the most sensitive species to exposure to the volatile hydrocarbon xylene. Further, the smaller, less lipid rich copepod species C. finmarchicus, was more sensitive compared to the true Arctic C. glacialis. Extrapolating these results to a spill situation, C. finmarchicus and C. glacialis populations would be affected by xylene exposure through both direct exposure (mortality) and indirectly through a decline in prey organisms (algae). Sub-lethal effects after long-term exposures to low concentrations of oil compounds may also be detected in C. finmarchicus and C. glacialis. Feeding was suppressed in C. finmarchicus after exposure to 7.0 µg PAHs l-1 for 11 days (Paper III). No significant effect on feeding was detected in C. glacialis exposed to 10.4 µg PAHs l-1 for 11 days. However, the hatching success of eggs laid by females exposed to 10.4 µg PAHs l-1 for two days was significantly reduced (Paper III). The long term effects of lipophilic PAHs in biota depend on the bioaccumulation capacity and internal concentrations of the various PAHs. Accumulation of lipophilic compounds is particularly important for the lipid rich Arctic species. Paper IV examines the uptake kinetics of two PAHs in C. finmarchicus and show that the accumulation of the lighter and less lipophilic compound, phenanthrene, is faster that the uptake of the heavier and more lipophilic benzo[a]pyrene (B[a]P). Equilibrium was reached for phenanthrene within 192 h exposure while no equilibrium was detected for B[a]P. The bioconcentration factor (BCF) was higher for B[a]P, reflecting the higher affinity for lipids of B[a]P compared to phenanthrene (Paper IV). So the heavier, more lipophilic PAH accumulates slower but to a higher concentration in C. finmarchicus. This thesis shows that not only acute but also long-term exposures may affect Arctic species. In addition, exposure duration may determine which compounds are accumulated by biota. Both indirect effects, such as decrease of food items, and direct effects, such as mortality, reduced feeding and reproduction failure may have implications for population development. These findings should be taken into consideration when developing ERA for the Barents Sea area

Two hundred year old lesson in bullying: building community by producing Honk! Jr., a musical based on The Ugly Duckling

Benjamin Franklin Theatre Project was created in response to recent violence in schools and new anti-bullying legislation. Schools in Washington State are mandated by legislation to adopt anti-bullying policies. After a policy was made official, it was up to schools, particularly educators to guide students to understand, support and integrate the anti-bullying message into the school community. Theatre education, a constructivist, holistic approach to learning, was a successful way to do this at Benjamin Franklin Elementary School. By producing HONK! Jr. a musical play based on Hans Christian Andersen’s story “The Ugly Duckling”, students were able to explore, role-play and recreate a true account of bullying. Andersen’s “The Ugly Duckling”, is based on his life and the bullying he endured as a child. All students deserve a peaceful, safe, positive environment at school. Using theatre education as a bridge for integrated thematic learning provided a safe and pretend environment for students to gain empathy and ultimately understand the dangers of bullying. Producing a musical play was a way to build self-esteem, promote acceptance, and to build strong community bonds. Requiring parent participation and allowing all interested students to join were two key factors that naturally helped detour bullying behavior. This thesis was an exploration of how theatre education, particularly producing a play, can be a tool to take a social message from a policy into the school community value system

Post-Translational Regulation of FAS-Mediated PPARα Activation

The liver is a central organ to whole-body metabolism and mediates many of the adaptive responses to changes in nutrient availability, such that the appropriate energy sources are used and blood glucose levels maintained, whether directly after a meal or after a twelve-hour fast. The adaptive responses to fasting in liver are largely mediated by the nuclear receptor peroxisome proliferator-activated receptor α, or PPARα. PPARα can be activated by a de novo synthesized lipid ligand—16:0/18:1- glycerophosphocholine (16:0/18:1-GPC)—the synthesis of which is dependent on fatty acid synthase (FAS), but little is known about the regulation of this pathway. My thesis focused on post-translational mechanisms controlling endogenous activation of PPARα in the liver and used mouse liver and a hepatocyte cell line as model systems. In addition to its role in PPARα activation during fasting, FAS helps store excess calories as fat during feeding. We demonstrated that this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated FAS, the latter being attached to membranes by a strong peripheral membrane association. To find candidate proteins mediating FAS membrane localization we used a proteomics approach to identify compartment-specific FAS-associated proteins. We identified three proteins—Septin-2, Septin-7, and 40S ribosomal protein S18—that in two different liver model systems associate with FAS exclusively in the membrane fraction. Because the septins are involved in membrane structuring and scaffolding, these proteins may be involved in FAS membrane localization. The ratio of cytoplasmic to membrane FAS specific activity was increased with fasting or in the absence of insulin, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS: cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPAR� target gene expression. mTORC1, a mediator of the feeding/insulin signal to induce lipogenesis, emerged as a mediator of FAS phosphorylation, inhibiting cytoplasmic FAS activity and reducing PPARα target gene expression in a FAS-dependent manner. Next, we investigated the role of ligand transport in FAS-mediated PPARα activation. 16:0/18:1-GPC is synthesized in the cytoplasm and it is not known how it reaches the nuclear PPARα. We identified phosphatidylcholine transfer protein (PCTP) as a possible transport protein for this ligand. PCTP knockdown in Hepa1-6 hepatocytes caused dramatic reductions in expression of PPARα target genes, and PCTP co-immunoprecipitated with PPARα. Immunofluorescent imaging showed that starvation of cells caused an accumulation of PCTP in the nucleus, consistent with a shuttling function controlled by nutrition. Using mass spectrometry, we demonstrated that PCTP binds 16:0/18:1-GPC. We further showed that the binding of this ligand to PCTP is FAS-dependent: in mice with liver-specific knockout of FAS, the amount of 16:0/18:1-GPC bound to PCTP in the nucleus was significantly reduced. Together, these findings suggest that multiple modes of post-translational regulation of FAS combined with regulation of lipid delivery by PCTP control fasting-induced PPARα activation in liver