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Here we use two enriched stable isotopes, 68Znen and 64Znen (>99%), to prepare 68ZnO nanoparticles (NPs) and soluble 64ZnCl2. The standard LUFA 2.2 test soil was dosed with 68ZnO NPs and soluble 64ZnCl2 to 5 mg kg-1 each, plus between 0 and 95 mg kg-1 of soluble ZnCl2 with a natural isotope composition. After 0, 1, 3, 6 and 12 months of soil incubation, earthworms (Eisenia andrei) were introduced for 72-hour exposures. Analyses of soils, pore waters and earthworm tissues using multiple collector ICP-MS allowed the simultaneous measurement of the diagnostic 68Zn/66Zn, 64Zn/66Zn and 68Zn/64Zn ratios, from which the three different isotopic forms of Zn were quantified. Eisenia andrei was able to regulate Zn body concentrations with no difference observed between the different total dosing concentrations. The accumulation of labelled Zn by the earthworms showed a direct relationship with the proportion of labelled to total Zn in the pore water, which increased with longer soil incubation times and decreasing soil pH. The 68Znen/64Znen ratios determined for earthworms (1.09 ± 0.04), soils (1.09 ± 0.02) and pore waters (1.08 ± 0.02) indicate indistinguishable environmental distribution and uptake of the Zn forms, most likely due to rapid dissolution of the ZnO NPs.

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Human neural progenitor cells (hNPC) hold great potential as an ex vivo system for delivery of therapeutic proteins to the central nervous system. When cultured as aggregates, termed neurospheres, hNPC are capable of significant in vitro expansion. In the current study, we present a robust method for lentiviral vector-mediated gene delivery into hNPC that maintains the differentiation and proliferative properties of neurosphere cultures while minimizing the amount of viral vector used and controlling the number of insertion sites per population. This method results in long-term, stable expression even after differentiation of the hNPC to neurons and astrocytes and allows for generation of equivalent transgenic populations of hNPC. In addition, the in vitro analysis presented predicts the behavior of transgenic lines in vivo when transplanted into a rodent model of Parkinson's disease. The methods presented provide a powerful tool for assessing the impact of factors such as promoter systems or different transgenes on the therapeutic utility of these cells.

Vandrammedirektivet foreskriver, at medlemsstaterne skal indsamle oplysninger om type og omfang af de signifikante menneskeskabte belastninger og deres virkninger på kystvandenes tilstand. Forskning har tidligere vurderet, at den mest betydende presfaktor er belastningen med N og P. En række andre presfaktorer kan imidlertid belaste det marine miljø, men der findes ikke en samlet vurdering af betydningen af disse presfaktorer for miljøtilstanden i kystvandene, som den defineres i VRD. For at forbedre plangrundlaget i 3. generations vandplaner har Miljøstyrelsen derfor iværksat indsatser til belysning af andre presfaktorer.I denne analyse har vi på baggrund af strukturerede reviews analyseret presfaktorerne: Råstofindvinding, klapning og graveaktiviter, fysiske konstruktioner, fiskeri, skibstrafik, plastik, miljøfarlige stoffer og invasive arter. De invasive arter er repræsenteret ved 4 karakteristiske arter. Analysen har taget udgangspunkt i, at tilstedeværelse alene ikke er tilstrækkelig, der skal være en påvirkningsmekanisme på et eller flere af kvalitetselementerne (fytoplankton, makroalger, angiospermer og bundfauna) og/eller støtteparametrene (iltforhold og sigtdybde), for at det kan siges, at presfaktorerne har en virkning jf. VRD. Da indsatser for at forbedre vandmiljøet bliver planlagt på niveau af vandområder, skal effekten af presfaktoren endvidere kunne detekteres på dette niveau. For at kunne beskrive og dokumentere en evt. betydende påvirkning af en given presfaktor på kvalitetselementerne i et vandområde er det endvidere nødvendigt, at der er et tilstrækkeligt datagrundlag til at analysere den konkrete sammenhæng mellem presfaktoren og kvalitetselementerne/støtteparametrene.På baggrund af kriterier om dokumenteret påvirkningsmekanisme, potentiel væsentlighed og datagrundlag er de nævnte presfaktorer blevet gennemgået og vi præsenterer her resultater og anbefalinger vedrørende inkludering af presfaktorerne i analyserne i 3. generations vandplaner.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to increase the survival of dopamine neurons in a variety of in vitro and in vivo model systems. Therefore, it constitutes an important therapeutic protein with the potential to ameliorate dopamine neuronal degeneration in Parkinson's disease or to support dopamine neuronal replacement strategies. However, biophysical and practical considerations present obstacles for the direct delivery of the GDNF protein to CNS neurons. Here we show that rodent neural precursor cells isolated and expanded in culture as neurospheres (NS) can be genetically modified to express green fluorescent protein (GFP) or to release GDNF using lentiviral constructs. GDNF-NS increased the fibre outgrowth of primary embryonic dopamine neurons in cocultures, showing that the protein was released in biologically significant quantities. Furthermore, after transplantation into the 6-hydroxydopamine-lesioned rat striatum, GDNF-NS significantly increased the survival of cografted primary dopamine neurons. However, this was not reflected in behavioural recovery in these animals. We found that, by 6 weeks, few cells expressed GDNF or GFP, suggesting either that transgene expression was down-regulated over time or that the cells died. This may explain the initial effects on dopamine neuronal survival within the graft but the lack of long-term effect on subsequent fibre outgrowth and behaviour. Providing sustained levels of neural precursor-mediated transgene expression can be achieved following transplantation in the future; this approach may prove beneficial as an alternative therapeutic strategy in the cell-based management of Parkinson's disease.

Missense mutations and extra copies of the alpha-Synuclein gene result in Parkinson disease (PD). Human stem and progenitor cells can be expanded from embryonic tissues and provide a source of non-transformed neural cells to explore the effects of these pathogenic mutations specifically in human nervous tissue. We over-expressed the wild type, A53T and A30P forms of alpha-synuclein in expanded populations of progenitors derived from the human fetal cortex. The protein localized in the nucleus and around microvesicles. Only the A53T form was acutely toxic, suggesting a unique vulnerability of these progenitors to this mutation. Interestingly, constitutive over-expression of wild-type alpha-synuclein progressively impaired the innate ability of progenitors to switch toward gliogenesis at later passages. To explore the effect of alpha-synuclein on neuronal subtypes selectively affected in PD, such as dopaminergic neurons, alpha-synuclein and its mutations were also over-expressed in terminally differentiating neuroectodermal cultures derived from human embryonic stem cells (hESC). Alpha-synuclein induced acute cytotoxicity and reduced the number of neurons expressing either tyrosine hydroxylase or gamma-aminobutyric acid over time. Consistent with the selective vulnerability of ventral midbrain dopaminergic neurons, alpha-synuclein cytotoxicity appeared most pronounced following FGF8/SHH specification and was decreased by inhibition of dopamine synthesis. Together, these data show that alpha-synuclein over-expressed in human neural embryonic cells results in patterns of degeneration that in some cases match features of Parkinson Disease. Thus, neural cells derived from hESC provide a useful model system to understand the development of alpha-synuclein-related pathologies and allow therapeutic drug screening.

As the number of applications for engineered nanoparticles (ENPs) is increasing, so inevitably is the rate of their release into the environment. The novel properties (size, reactivity) that make their use and the development of applications appealing are also the basis for concerns about their environmental risks. Here the soil nematode Caenorhabditis elegans was used to assess the toxicity of various silver nanoparticle types with respect to gene expression, individual life history traits and population sensitivity. Exposures were carried out reflecting different environmentally relevant conditions that affected ENP fate and bioavailabilty. Characterisation of ENP fate in the media showed that while agglomeration/aggregation and dissolution processes occurred regardless of ENP type and exposure medium, their properties did alter the extent and dynamics of these processes. The toxicity of different Ag-ENPs and ionic silver was investigated and compared. Concentration-dependent decreases in reproduction were found for all silver forms with Ag+ being the most toxic by mass throughout. The effects of environmentally-relevant exposure media on ENP fate and reproductive toxicity to C. elegans was assessed, first by conducting tests in artificial media with varied composition, and second by using pore waters from soils with different properties. In both cases media pH had the greatest impact on observed toxicity. Multigenerational exposure to pristine and aged (sulfidised) Ag-ENPs revealed significant increases in sensitivity towards ionic and pristine Ag-ENP exposures in the second offspring generation (a novel finding), from which populations did not recover; aged ENPs did not induce similar increased sensitivity. Finally, using microarray analysis clear differences in gene expression after exposure to size and surface property-variant ENPs were found, yet changes in neurological functions were identified as the main underlying mode of toxic action regardless of ENP properties. This PhD thus provides a comprehensive assessment of Ag-ENP exposure, mechanisms of toxicity and long-term effects in a soil dwelling species.