In the studies underlying this thesis, various source tracing techniques were applied to environmental samples from the Baltic region. Comprehensive sampling and analysis of polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in surface sediments in Swedish coastal and offshore areas resulted in a unique data set for this region. Nearly 150 samples of surface sediments were analyzed for all tetra- to octa-chlorinated PCDD/Fs. The levels showed large spatial variability with hotspots in several coastal regions. Neither Sweden nor the EU has introduced guideline values for PCDD/Fs in sediment, but comparisons to available guidelines and quality standards from other countries indicate that large areas of primarily coastal sediments may constitute a risk to marine organisms. Multivariate pattern analysis techniques and receptor models, such as Principal Component Analysis (PCA) and Positive Matrix Factorization (PMF), were used to trace sources. These analyses suggested that three to six source types can explain most of the observed pattern variations found in the sediment samples. Atmospheric deposition was suggested as the most important source to offshore areas, thus confirming earlier estimates. However, spatial differences indicated a larger fraction of local/regional atmospheric sources, characterized by PCDFs, in the south. This was indicated by the identification of several patterns of atmospheric origin. In coastal areas, the influence of direct emission sources was larger, and among these, chlorophenol used for wood preservation and emissions from pulp/paper production and other wood related industry appeared to be most important. The historic emissions connected to processes involving chemical reactions with chlorine (e.g. pulp bleaching) were found to be of less importance except at some coastal sites. The analysis of PCDD/Fs in Baltic herring also revealed spatial variations in the levels and pollution patterns along the coast. The geographical match against areas with elevated sediment levels indicated that transfer from sediments via water to organisms was one possible explanation. Fugacity, a concept used to predict the net transport direction between environmental matrices, was used to explore the gas exchange of hexachlorocyclohexanes (HCHs) and polychlorinated biphenyls (PCBs) between air and water. These estimates suggested that, in the Kattegat Sea, the gaseous exchange of HCHs primarily resulted in net deposition while PCBs were net volatilized under certain environmental conditions. The study also indicated that, while the air concentrations of both PCBs and γ-HCH are mostly dependent upon the origin of the air mass, the fluctuations in α-HCH were primarily influenced by seasonal changes.

La formation de la gaine de myéline est un processus complexe et finement régulé. Une altération de l’expression des gènes codant pour les protéines structurales de cette gaine entraine de graves neuropathies démyélinisantes. Notre objectif est d’identifier de nouvelles voies de signalisation capables de moduler l’expression de ces gènes. Les cellules de Schwann et les oligodendrocytes contiennent et synthétisent de grande quantité de dérivés oxydés du cholestérol : les oxystérols. Ces molécules sont connues pour leurs rôles dans le maintien de l’homéostasie du cholestérol et dans la progression des maladies neurodégénératives. Les oxystérols peuvent être classés en deux groupes : ceux dont l’oxydation a lieu sur la chaine carbonée latérale (25OH) et ceux qui portent une oxydation sur l’un des cycles du cholestérol (7KC). Nous nous sommes tout d’abord intéressés à la première catégorie d’oxystérols. Nous avons montré que le 25OH, réprimait l’expression des gènes de la myéline périphérique P0 et PMP22. Cette activité répressive était le fruit d’un mécanisme direct conduisant à une augmentation de la quantité des LXRs liés à leurs éléments de réponse sur les promoteurs des gènes de la myéline, et d’un mécanisme indirect provoquant une diminution de l’activité de la voie Wnt/β-caténine. En revanche, dans le SNC, nos résultats indiquent que le 25OH active l’expression des gènes de la myéline PLP et MBP. Le traitement, par ces oxystérols, de cultures organotypiques de cervelet démyélinisées par la lysolécithine permet une remyélinisation des axones des cellules de Purkinje. Nous nous sommes ensuite penchés sur le rôle du corégulateur transcriptionnel RIP140. Ce dernier peut soit agir comme un corépresseur soit comme un coactivateur. Il peut interagir avec le LXR. L’invalidation de RIP140 dans le poisson zèbre altère les gaines de myéline. Nous avons montré que RIP140 possédait des rôles bivalents dans la régulation de la myélinisation. En effet, il est capable d’activer mais aussi de réprimer l’activité transcriptionnelle de P0 et de PMP22. Enfin, nous nous sommes intéressés à la seconde catégorie d’oxystérols. Le 7KC est l’oxystérol majoritairement présent dans le SNP et la CS. Il est connu pour moduler l’action du récepteur aux dioxines : le AhR. Ce récepteur a été très largement étudié dans un cadre toxicologique. Cependant ses rôles et ses ligands endogènes restent à ce jour encore assez méconnus. Nos résultats indiquent que le AhR est impliqué dans le contrôle de l’expression des gènes de la myéline périphérique. L’invalidation du AhR, chez la souris, provoque des anomalies structurales de la gaine de myéline conduisant à des déficits moteurs. Cette étude a permis de mieux comprendre les dialogues entre les voies de signalisation gouvernant le processus de myélinisation. Ce travail apporte également de nouvelles perspectives thérapeutiques des maladies neurodégénératives comme la CMT1A ou la sclérose en plaques. / The myelination of axons is a complex process performed by Schwann cells (SC) and by oligodendrocytes (OL) respectively in the peripheral nervous system (PNS) and in the central nervous system (CNS). A slight change in expression of myelin structural proteins has a deep impact on the development and preservation of nerve fibers and their myelin sheaths, as observed for example in Charcot-Marie-Tooth disease or in Pelizaeus-Merzbacher disease. Our aim is to identify new signaling pathways able to control the expression of these structural proteins. SC and OL contain and synthesize high amount of reactive molecules generated from the oxidation of cholesterol: the oxysterols. Their implication in cholesterol homeostasis and in the progression of neurodegenerative disorders is well known but few data are available for their functions in myelination of PNS and CNS. Firstly, we demonstrate that oxysterols inhibit peripheral myelin gene expression: MPZ and PMP22. This downregulation is mediated by two mechanisms: by increasing the binding of LXRs to myelin genes promoters and by inhibiting the Wnt/β-catenin pathway leading to a decrease of b-catenin recruitment at the levels of the MPZ and PMP22 promoters. However, in the CNS, our data demonstrate that activation of LXRS by oxysterols stimulate myelin genes expression (PLP and MBP). Interestingly, by using demyelinated organotipc culture of cerebellum, we show that oxysterols enhance OL differentiation and promote remyelination, via LXRs. Then, we studied the role of the transcriptional coregulatory, RIP140, in myelination. RIP140 is able to act as a corepressor or as a coactivator and can interact with LXRs. In Zebrafish, the knocked down of the orthologue of RIP140 led to a decrease of peripheral and central myelin gene expression and to a defect in myelin sheath ultrastructure. Finally, we focused on impact of AhR in myelination process. AhR is a ligand activated transcription factor mostly known to interact with environmental pollutant like dioxins to mediate their toxic and carcinogenic effect. However, its detoxifying activity is posterior to the apparition of the gene and its physiological roles and endogenous ligands remain elusive. We show that the main oxysterol in the nervous system is 7-ketocholesterol which is an endogenous modulator of AhR. We report that the constitutive absence of AhR in mice leads to defects in locomotion behaviors. We studied the impact of this invalidation on the myelin of sciatic nerve. We observed a severe demyelinating phenotype and deregulation of myelin genes expression. Moreover, we demonstrated a cross-talk between AhR and Wnt/β-catenin pathways. Our data reveal a new endogenous role of AhR in myelination process.

High levels of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) in edible fish in the Baltic Sea have raised health concerns in the Baltic region and the rest of Europe. Thus, there are urgent needs to characterize sources in order to formulate effective mitigation strategies. The aim of this thesis is to contribute to a better understanding of past and present sources of PCDD/Fs in the Baltic Sea environment by exploring chemical fingerprints in sediments, air, and biota. The spatial and temporal patterns of PCDD/F distributions in the Baltic Sea during the 20th century were studied in Swedish coastal and offshore sediment cores. The results showed that PCDD/F levels peaked in 1975 (± 7 years) in coastal and 1991 (± 5 years) in offshore areas. The time trends of PCDD/Fs in the sediment cores also showed that environmental half-lives of these pollutants have been shorter in coastal than in offshore areas (15 ± 5 and 29 ± 14 years, respectively). Consequently, there have been remarkable recoveries in coastal areas, but slower recovery in offshore areas with 81 ± 12% and 38 ± 11% reductions from peak levels, respectively. Source-to-receptor multivariate modeling by Positive Matrix Factorization (PMF) showed that six types of PCDD/F sources are and have been important for the Baltic Sea environment: PCDD/Fs related to i) atmospheric background, ii) thermal processes, iii) manufacture and use of tetra-chlorophenol (TCP) and iv) penta-chlorophenol (PCP), v) industrial use of elementary chlo- rine and the chloralkali-process (Chl), and vi) hexa-CDD sources. The results showed that diffuse sources (i and ii) have consistently contributed >80% of the total amounts in the Southern Baltic Sea. In the Northern Baltic Sea, where the biota is most heavily contaminated, impacts of local sources (TCP, PCP and Chl) have been higher, contributing ca. 50% of total amounts. Among the six sources, only Thermal and chlorophenols (ii-iv) have had major impacts on biota. The impact of thermal sources has, however, been declining as shown from source apportioned time-trend data of PCDD/Fs in Baltic herring. In contrast, impacts of chlorophenol-associated sources generally increased, remained at steady-state or slowly decreased during 1990-2010, suggesting that these sources have substantially contributed to the persistently high levels of PCDD/Fs in Baltic biota. Atmospheric sources of PCDD/Fs for the Baltic region (Northern Europe) were also investigated, and specifically whether the inclusion of parallel measurements of metals in the analysis of air would help back-tracking sources. PCDD/Fs and metals in high-volume air samples from a rural field station near the shore of the central Baltic Sea were measured. The study focused on the winter season and air from the S and E sectors, as these samples showed elevated levels of PCDD/Fs, particularly PCDFs. Several metals were found to correlate significantly with the PCDFs. The wide range of candidate metals as source markers for PCDD/F emissions, and the lack of an up-to-date extensive compilation of source characteristics for metal emission from vari- ous sources, limited the use of the metals as source markers. The study was not able to pin-point primary PCDD/F sources for Baltic air, but it demonstrated a new promising approach for source tracing of air emissions. The best leads for back-tracking primary sources of atmospheric PCDD/Fs in Baltic air were seasonal trends and PCDD/F congener patterns, pointing at non-industrial related thermal sources related to heating. The non-localized natures of the sources raise challenges for managing the emissions and thus societal efforts are required to better control atmospheric emissions of PCDD/Fs. / EcoChange / BalticPOPs

The aryl hydrocarbon receptor (AhR) heterodimerizes with the aryl hydrocarbon receptor nuclear translocator (Arnt) for transcriptional regulation. We generated three N-terminal deletion constructs of the human AhR of 12-24 KDa in size—namely D1 (aa 84-295), D2 (aa 84-192) and D3 (aa 191-295)—to suppress the Arnt function. We observed that all three constructs interact with the human Arnt with similar affinities. D2, which contains part of the AhR PAS-A domain and interacts with the PAS-A domain of Arnt, inhibits the formation of the AhR gel shift complex. D2 suppresses the 3-methylcholanthrene-induced, dioxin response element (DRE)-driven luciferase activity in Hep3B cells and exogenous Arnt reverses this D2 suppression. D2 suppresses the induction of CYP1A1 at both the message and protein levels in Hep3B cells; however, the CYP1B1 induction is not affected. D2 suppresses the recruitment of Arnt to the cyp1a1 promoter but not to the cyp1b1 promoter, partly because the AhR/Arnt heterodimer binds better to the cyp1b1 DRE than to the cyp1a1 DRE. Interestingly, D2 has no effect on the cobalt chloride-induced, hypoxia inducible factor-1 (HIF-1)-dependent expression of vegf, aldolase c, and ldh-a messages. Our data reveal that the flanking sequences of the DRE contribute to the binding affinity of the AhR/Arnt heterodimer to its endogenous enhancers and the function of AhR and HIF-1 can be differentially suppressed by the D2 inhibitory molecule. In chapter 2, a Pichia Pastoris expression system was constructed expressing codon optimized human full length AhR. This codon optimization is necessary for overexpression of huAhR. RT-PCR data showed that the codon optimized mRNA was more stably expressed than wild types. Overexpressed huAhR protein was degraded by proteinase when using a regular P. Pastoris strain yJC100 whereas the proteinase deficient ySMD1163 maintained a much higher level of huAhR. P. Pastoris expressed huAhR was natively purified and analyzed. Coimmunopricipitation assay shows its interaction with endogenous Arnt. A ligand-dependent gel shift was also observed. In addition, we performed an in vitro coprecipitation assay to study its binding to endogenous cyp1b1 DREs. The result shows that the DRE3, known as a critical DRE for cyp1b1 transcriptional activity, has the highest binding affinity to AhR/Arnt complex. Taking together, we constructed a novel P. Pastoris expression system to overexpress human full length AhR. Purified huAhR is a good reagent for studing its ligand and DNA binding. In chapter 3, an adeno-associated virus (AAV) expression system was constructed to express an AhR deletion contruct CΔ553 (aa1-295) for tumor injection. Western blot shows the expression of CΔ553 (aa1-295) in hela cells infected by AAV-553, but the low yield of AAV-553 limited its application on tumor treatment. Possible solutions were discussed for future work.

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