There is an increasing concern over the safety of engineered nanoparticles (ENPs) to humans and the environment. It is important that the environmental risks of these particles to be tested under research and regulatory schemes, e.g. Nano Risks to the environment & Human Health (NanoReTox) under Seventh Framework Programme (FP7) in EU. Due to their unique properties and the fact that their detection and characterization in complex matrices is challenging, classic analytical methods and test approaches for assessing environmental risk may not be appropriate for ENPs. In this paper I present the challenges associated with ENPs exposure to the estuarine environment and the testing of a chosen ENPs to generate data on ecotoxicity in the test estuarine organisms for further consideration of risk assessment of marine environment. Careful consideration was given to the selection of the test materials (benthic organisms Nereis diversicolor and Scrobicularia plana), the test system and the test exposure conditions (CdS ENPs and aqueous CdS added to filtered natural seawater with a concentration of 10 µg L-1). Evaluation of the exposure effects was carried out by behavioral tests (burrowing kinetics and feeding rate) and biochemical responses (quantification of biomarkers). Multispecies Freshwater Biomonitor®  (MFB) tests and GST analysis results show significant differences in between control group and CdS NPs exposure one, indicating that CdS NPs are potential to cause sublethal effects in test organisms. Our knowledge in environmental risk assessment of ENPs is still limited. Coordinated research is required to gain a better understanding of the factors and processes affecting ENP fate and effects in the environment as well as to develop more usable, robust and sensitive methods for characterization and detection of ENPs in environmental systems. / NanoReTox, FP7 NMP Work Programme

The physiological responses to stressor exposure can be broadly grouped into the organismal and the cellular stress responses. The organismal stress response involves the release of hormones into general circulation, while the cellular stress response involves the synthesis of proteins, the most important being the heat shock proteins (HSPs), which play a role in maintaining protein homeostasis. Elevated HSP70 expression in response to stressors has been demonstrated in trout (Oncorhynchus mykiss) red blood cells (RBCs). The ease of repeated sampling of blood suggests the possibility of using this tissue as a non-lethal marker of cellular stress in fish. This study tested the hypothesis that stressor exposure will elevate HSP70 expression in trout RBCs and the role of stress hormones in mediating this response. Acute heat shock exposure (+12oC) significantly elevated plasma cortisol, glucose and lactate levels in heat shocked fish over 24 h. A tissue-specific response was seen in HSP70 expression in liver, brain, gill and RBCs. To enable measurement of RBC HSP70 concentrations, an enzyme-linked immunosorbent assay (ELISA) was developed using a commercially available rabbit anti-salmon HSP70 and a recombinant chinook salmon (Oncorhynchus tshawytscha) HSP70. To determine effects of chronic exposure, two studies were conducted exposing trout to either cadmium (0, 0.75 or 2.0 µg/L over 28 d) or municipal wastewater effluent (0, 20 or 90% over 14 d). However, neither exposure elicited a significant HSP70 response. Effects of stress hormones on RBC HSP70 levels were tested by exposing cells in vitro to either cortisol (10 and 100 ng/mL) or epinephrine (10 nM) with or without heat shock. Heat shock elevated HSP70 content in trout RBCs but no modulation by stress hormones was seen. It was shown for the first time that RBCs release HSP70 content into the medium in response to an acute heat shock and this release is attenuated by stress hormones. Overall, HSP70 levels in RBCs have the potential to be a reliable non-lethal marker of acute cellular stress effects in fish. The release of HSP70 from RBCs leads to the hypothesis that HSP70 may also have an extracellular role in fish, and warrants further study.

Intakes of the highly unsaturated fatty acids (HUFA, ≥20 Carbons, ≥3 double bonds) eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3, DHA) greater than 0.25 g/d are currently recommended for health benefits. Targets for omega-3 blood biomarkers have also been proposed based on associations with protection against coronary heart disease mortality. The relationship between diet intakes and blood biomarkers is not well defined, particularly differences between men and women. North American intakes and blood biomarkers of EPA and DHA are typically below recommendations and targets. To address this disparity, adherence to dietary advice strategies to increase EPA + DHA intake was investigated over one year. Adherence was sustained up to 12 weeks and long-term adherence was well characterized by the % of DHA in erythrocytes. For women, n-3 HUFA blood biomarkers increased following nutraceutical or combined strategy dietary advice but not seafood or functional food advice. To assist in the assessment of EPA + DHA intakes, food sources of EPA and DHA in Canada were incorporated into a semi-quantitative, nutrient-specific food frequency questionnaire (FFQ) and validated. The FFQ is an adequate tool for estimating habitual EPA and DHA intake and ranking Canadian adults by their intakes. The blood biomarker response to recommended intakes of 0.25, 0.5 and 1 g/d EPA + DHA was also characterized in adult men and women. Blood n-3 HUFA biomarkers increased in a dose-dependent manner and aligned with blood targets associated with primary cardiac arrest risk reduction. Sex differences in the DHA:EPA ratio in blood observed with low intakes at baseline disappeared following 0.25 g/d EPA + DHA. These findings are applicable towards informing achievable dietary guidelines for EPA + DHA intake and improving measurement of EPA + DHA intake in relation to blood n-3 HUFA biomarkers.

Objective New biomarkers for acute kidney injury are needed and urinary Cystatin C is one alternative. The objective was to validate a urinary Cystatin C method on Mindray BS-380 comparing urine samples from the Uppsala Longitudinal Study of Adult Men (ULSAM-77) and urine samples from a reference group for Cystatin C. A visual control for a relationship between Cystatin C and C-reactive protein (CRP) and interleukin 6 (IL-6) respectively was made. Methods Precision, linearity, recovery, interference, and stability of the urine cystatin C method were investigated. Comparisons were made between ULSAM-77 samples and a reference group samples consisting of ordinary people. Results The highest total imprecision was 10.24 % for the sample with the lowest concentration. The second lowest concentration had 4.21 % total variation coefficient. The linearity equation was y = 0.99x – 0.01 with an R2-value of 0.99. The recovery for all concentrations was always 91 % or more. No interference from hemoglobin at a concentration of 10 g/L was found. The samples were stable at +5°C for seven days. The median for the samples from ULSAM-77 was 0.09 mg/L and the median for the reference samples was 0.06 mg/L. There was no obvious relationship between Cystatin C and CRP/IL-6 from ULSAM-77. Conclusion   Reliable data of urinary Cystatin C can be analyzed on a Mindray BS-380. The level of urinary Cystatin C was higher for people age 77 than for those with a median age of 49. There was no correlation between the concentration of Cystatin C in urine and the levels of CRP and IL-6.

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<p>Diabetic nephropathy is a chronic kidney disease and one of the more severe complications from diabetes mellitus type 2. The glomerular and tubular dysfunctions usually lead to end stage renal disease and the treatments of these patients (dialysis, kidney transplants) are a huge economic burden for the society. Due to an epidemiologic increase of type 2 diabetes, conventional diagnostic markers like creatinine and albumin are not sufficient, since they are only able to identify already existing kidney damage. With targeted metabolomics, the analysis of small molecules produced from metabolism, this project aimed at finding novel and more sensitive metabolic biomarkers from several different classes of metabolites. The different assays were performed with flow injection analysis, high performance liquid chromatography, gas chromatography and mass spectrometry, and with principal component analysis and discriminant analysis, up-and down-regulated metabolites could be identified and their respective biochemical pathways, if possible, explained. In diabetics significantly elevated concentrations of very long chain fatty acids (impaired peroxisomal β-oxidation), urinary sugars and acylcarnitines in plasma could be recognized. Markers indicating kidney damage included significantly increased plasma concentrations of asymmetric dimethylarginine (inhibition of nitric oxide synthase resulting in decreased endothelial functionality) and histamine (indication of uremic pruritus). Oxidative stress was also found to be a potential prognostic marker as indicated by the raised methionine-sulfoxide to methionine ratio in nephrotic patients. To summarize, this project succeeded in identifying metabolic biomarkers both for diabetes type 2 and nephropathy, which in the future might become important tools in slowing down progression or diagnosing these diseases.</p>

The Caspian region and specifically the Apsheron peninsula of Azerbaijan is known to be polluted with a variety of environmental contaminants, making risk assessment difficult. The wetlands of Sumgayit contain particularly complex mixtures of contaminants. Flow cytometry and the micronucleus assay were used to assess chromosomal damage in aquatic turtles and frogs inhabiting contaminated wetlands in Azerbaijan. By evaluating biomarkers that are indicative of somatic effects, elevated chromosomal damage was documented at several sites in Azerbaijan relative to reference sites. Sediment samples were analyzed for polycyclic aromatic hydrocarbons (PAHs), organochlorines (OCs), and mercury to evaluate contaminant associations with genetic damage. Sediment samples revealed heterogeneous patterns of PAH and mercury concentrations throughout Sumgayit. Significant positive correlations were documented between both PAH and mercury sediment concentrations and chromosomal damage. Population genetic methods were employed to study the effects of long-term chronic contaminant exposure in marsh frogs from Sumgayit. The Sumgayit region has reduced levels of genetic diversity, likely due to environmental degradation. One of the most contaminated sites in Sumgayit, WTP, appears to be a source of new mutations as a result of an increased mutation rate. Finally, the Sumgayit region seems to act as an ecological sink, with levels of gene flow into the region exceeding gene flow out of the region. This study provides not only exposure and biomarker data, but also an integrated method for assessing the cumulative population impacts of contaminant exposure by studying both population genetic and evolutionary effects. The results presented here will be used in conjunction with those of ongoing research involving both wildlife and humans to develop comprehensive ecological and human risk assessments.

The human genome contains tens of thousands of gene loci which code for an even greater number of protein and RNA products. The highly complex temporal and spatial expression of these genes makes possible all the biological processes of life. Altered gene expression by mutation or deregulation is fundamental for the development of many human diseases. The ultimate aim of this thesis was to identify gene expression changes relevant to cancer. The advent of genome-wide expression profiling techniques, such as microarrays, has provided powerful new tools to identify such changes and researchers are now faced with an explosion of gene expression data. Processing, comparing and integrating these data present major challenges. I approached these challenges by developing and assessing novel methods for cross-platform analysis of expression data, scalable subspace clustering, and curation of experimental gene regulation data from the published literature. I found that combining results from different expression platforms increases reliability of coexpression predictions. However, I also observed that global correlation between platforms was generally low, and few gene pairs reached reasonable thresholds for high-confidence coexpression. Therefore, I developed a novel subspace clustering algorithm, able to identify coexpressed genes in experimental subsets of very large gene expression datasets. Biological assessment against several metrics indicates that this algorithm performs well. I also developed a novel meta-analysis method to identify consistently reported genes from differential expression studies when raw data are unavailable. This method was applied to thyroid cancer, producing a ranked list of significantly over-represented genes. Tissue microarray analysis of some of these candidates and others identified a number of promising biomarkers for diagnostic and prognostic classification of thyroid cancer. Finally, I present ORegAnno (www.oreganno.org), a resource for the community-driven curation of experimentally verified regulatory sequences. This resource has proven a great success with ~30,000 sequences entered from over 900 publications by ~50 contributing users. These data, methods and resources contribute to our overall understanding of gene regulation, gene expression, and the changes that occur in cancer. Such an understanding should help identify new cancer mechanisms, potential treatment targets, and have significant diagnostic and prognostic implications.

The physiological responses to stressor exposure can be broadly grouped into the organismal and the cellular stress responses. The organismal stress response involves the release of hormones into general circulation, while the cellular stress response involves the synthesis of proteins, the most important being the heat shock proteins (HSPs), which play a role in maintaining protein homeostasis. Elevated HSP70 expression in response to stressors has been demonstrated in trout (Oncorhynchus mykiss) red blood cells (RBCs). The ease of repeated sampling of blood suggests the possibility of using this tissue as a non-lethal marker of cellular stress in fish. This study tested the hypothesis that stressor exposure will elevate HSP70 expression in trout RBCs and the role of stress hormones in mediating this response. Acute heat shock exposure (+12oC) significantly elevated plasma cortisol, glucose and lactate levels in heat shocked fish over 24 h. A tissue-specific response was seen in HSP70 expression in liver, brain, gill and RBCs. To enable measurement of RBC HSP70 concentrations, an enzyme-linked immunosorbent assay (ELISA) was developed using a commercially available rabbit anti-salmon HSP70 and a recombinant chinook salmon (Oncorhynchus tshawytscha) HSP70. To determine effects of chronic exposure, two studies were conducted exposing trout to either cadmium (0, 0.75 or 2.0 µg/L over 28 d) or municipal wastewater effluent (0, 20 or 90% over 14 d). However, neither exposure elicited a significant HSP70 response. Effects of stress hormones on RBC HSP70 levels were tested by exposing cells in vitro to either cortisol (10 and 100 ng/mL) or epinephrine (10 nM) with or without heat shock. Heat shock elevated HSP70 content in trout RBCs but no modulation by stress hormones was seen. It was shown for the first time that RBCs release HSP70 content into the medium in response to an acute heat shock and this release is attenuated by stress hormones. Overall, HSP70 levels in RBCs have the potential to be a reliable non-lethal marker of acute cellular stress effects in fish. The release of HSP70 from RBCs leads to the hypothesis that HSP70 may also have an extracellular role in fish, and warrants further study.

High-throughput mass spectrometry has discovered a plethora of candidates in the biomarker field, however, subsequent verification and validation studies are urgently needed to assess the potential of novel biomarkers in the detection of pancreatic cancer. We have conducted extensive verification and validation studies on two of our most promising biomarkers CUZD1 and LAMC2 with a total of 715 blood samples. In our study, both markers demonstrated consistent diagnostic ability of early- and CA19.9 negative-PDAC cases. When used in combination with CA19.9, CUZD1 and LAMC2 were shown to significantly improve the performance of CA19.9 alone in the diagnosis of PDAC patients. We speculate that CUZD1 and LAMC2 may be good candidates to be used in a panel for monitoring PDAC patients who do not express CA19.9 levels as well as for an aid in screening high risk populations. Further validation of these two proteins is warranted.