This dissertation investigated locational risks to ecosystem health associated with proximity to industrial complexes. The study was performed at the behest of ranchers and citizens living and working down-prevailing wind from the Formosa Plastics, Inc. and ALCOA facilities located in Calhoun County, Texas. Concerns expressed were for potential genotoxicity resulting from exposure to complex chemical mixtures released by the facilities. Exposure assessment of the marine environment was performed with sediments and oysters from Lavaca Bay being analyzed. Numerous chemicals were found to be present at concentrations considered likely to result in adverse responses in exposed populations. Bayesian geostatistical analysis was performed to determine if the concentrations were affected by a spatial process. Mercury and polycyclic aromatic hydrocarbons were the most notable of the chemicals found to be present at elevated concentrations and affected by a spatial process. Evaluation of maps generated from spatial modeling revealed that proximity to ALCOA resulted in elevated risks for exposure to harmful concentrations of pollutants. Genotoxicity was measured in two sentinel species. Oysters (Crassostrea virginica) were utilized for evaluation of the marine environment and cattle (Bos taurus and Bos taurus crossbred cattle) were chosen for evaluation of the terrestrial environment. Chromosomal aberration analysis was performed on oyster hematocytes. Analysis of the results failed to demonstrate the presence of an important generalized spatial process but some specific locations close to the ALCOA plant had elevations in this measure of genotoxicity. Stress as measured by the lysosomal destabilization assay was also performed on oyster hematocytes. These results were found to be affected by a significant spatial process with the highest degree of destabilization occurring in close proximity to ALCOA. Genotoxicity in cattle was evaluated with the single cell gel electrophoresis assay and chromosomal aberration analysis. Bayesian geostatistical analyis revealed the presence of important spatial processes. DNA-protein cross-linkage was the most notable with a strong indication of increased damage down-prevailing wind from the industrial complexes. Results indicated that proximity to industrial facilities increased the risk for harmful exposures, genotoxicity, and lysosomal destabilization.

Long-term dynamics of lake-water total organic carbon (LW-TOC) concentrations in freshwater lakes provide an important perspective on the recent increases in LW-TOC observed in many of these systems and may assist with the identification of natural and anthropogenic drivers of change. This study examines how LW-TOC in Dragsjön, a lake situated in an area with a long history of anthropogenic land use, has changed in response to natural and anthropogenic perturbations throughout the Holocene. To provide a better understanding of the processes involved, a multi-proxy study was conducted and included multi-element geochemistry (17 major and trace elements), biogenic silica, organic matter (OM) content and composition, and the faecal biomarker “coprostanol”. The direct biomarker for anthropogenic presence, “coprostanol”, and a detailed characterisation of OM composition are for the first time applied for tracing changes in LW-TOC. Natural processes contributed to stable LW-TOC concentrations in Dragsjön for most part of the Holocene. Humans were present in the catchment from AD 100 as indicated by coprostanol, but did not begin to affect LW-TOC until c. AD 1500. In the last 500 years LW-TOC steadily declined from 17 to 10 mg L-1 in response to anthropogenic alterations to the terrestrial biomass balance. The increase in LW-TOC during the last 70 years likely represents a recovery from anthropogenic disturbance rather than a baseline shift in response to any of the number of proposed recent stressors. The faecal biomarker coprostanol and OM composition provided information essential for identifying and characterising the effects of anthropogenic disturbance.

Human disease processes are often characterized by a deviation from the normal physiological concentration of critical biomarkers. The detection of disease biomarkers requires the development of novel sensing methods which are sensitive, specific, efficient and low-cost. To address this need, a novel conductive and recognitive hydrogel composite material has been developed. This work investigated the fabrication methods, the chemical and physical composition, the sensing capabilities, and the biocompatibility of the proposed conductive and recognitive hydrogel composite materials. The conductive polymer was found to respond by changing conductivity in the presence of biomolecules. Specificity can then be incorporated into the system by integrating the conductive polymer with a molecularly imprinted hydrogel. The demonstration of a conductive and recognitive hydrogel composite is a step towards the integration of these materials into close-loop sensing and drug delivery systems. / text

The use of biomarkers is considered a promising alternative, or complement, to traditional ecotoxicological assays. Toxic effects are often initially manifested at the molecular or biochemical level, biomarkers are therefore used as sensitive indicators of toxic exposure. Ideally, biomarkers would also indicate reduced fitness and possible later effects at the individual or population levels. However, implementing biomarkers in ecotoxicology is challenging and few biomarkers have an established connection to reduced individual fitness. The aim of this thesis was to increase the value and improve the interpretation of biomarker responses in ecotoxicological studies by examining the impact of confounding factors and the relationship between oxidative biomarkers and reproductive effects in crustaceans. The sensitivity of biomarkers was confirmed in paper I as toxic effects of pharmaceuticals with conserved drug target orthologs were observed at the molecular and biochemical levels both earlier and at lower concentrations than effects on mortality and reproduction. No toxic effects were observed for the pharmaceutical without identified drug target orthologs, thus stressing the importance of considering toxic mechanisms and being aware of the most likely target when evaluating toxic effects also in non-target species. Many xenobiotics and environmental stressors interfere with oxidative processes, making oxidative biomarkers interesting to study in ecotoxicology and stress ecology. Still, feeding rate was identified as a confounding factor for antioxidant capacity (assayed as oxygen radical absorbance capacity, ORAC) and lipid peroxidation in ecotoxicological studies (paper II). However, ORAC normalized to protein was independent of altered feeding rates, hence it can be applied as a suitable exposure biomarker without considering alterations and effects of feeding rate. The connection between reproduction and oxidative stress is dual, as reproduction both can be inhibited by oxidative stress and induce pro-oxidative processes. Further, a positive association was found between ORAC and the occurrence of embryo aberrations in the benthic amphipod Monoporeia affinis (paper III). An association between antioxidant defense and reproduction was also observed for Daphnia magna (paper IV). Threshold values for identification of exposed individuals and prediction of possible later reproductive effects were established for ORAC. This thesis has contributed to diminishing some of the knowledge gaps limiting the use of oxidative biomarkers in ecotoxicology, by contributing to increased understanding of how oxidative biomarkers relate to important life-traits. Moreover, ORAC has been identified as a suitable biomarker of not only exposure, but also reproductive effects. Future research should continue to establish connections between biomarker responses and effects at higher levels, and focus on providing defined threshold values to enable predictions about later effects. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Coastal metal pollution is a major concern to the health and well-being of aquatic organisms. Bioindicator organisms such as mussels have the potential to monitor coastal metal pollution. In New Zealand, the feasibility of employing green-lipped mussels, Perna canaliculus as a bioindicator species is yet to be investigated. This thesis focuses on applying a suite of biomarkers on green-lipped mussels exposed to cadmium (Cd) to evaluate the utility of the biomarkers and investigate the utility of the mussels in assessment of metal pollution. Cd is a non-essential metal and is known to be highly toxic to many aquatic organisms. This research consisted of a laboratory study to understand the mechanistic effects of Cd toxicity in green-lipped mussels. Physiological, biochemical, immunocytotoxic and cytogenotoxic biomarker responses were measured in mussels exposed to acute (96 h; 2000 µg L⁻¹ and 4000 µg L⁻¹) and subchronic (28 d; 200 µg L⁻¹ and 2000 µg L⁻¹) Cd treatments. The 96 h LC₅₀ value for P. canaliculus was 8160 µg L⁻¹, indicating that the green-lipped mussels were relatively tolerant to Cd exposure. Results from the Cd exposures, indicated that Cd had a negative impact on physiological processes such as feeding and oxygen consumption. Cd-induced physiological impairments caused an imbalance between energy gain and energy loss in the mussels that led to negative scope for growth. Detoxification (metallothionein-like protein) and defence mechanisms (catalase) were induced in the mussels to provide protection against the toxic effects of Cd. However, the defence mechanisms were not sufficient to protect the mussels from damage due to lipid peroxidation. DNA damage was also observed in the haemocytes of mussels as a result of Cd exposure. Cellular homeostasis (alkaline phosphatase) mechanisms were also perturbed. The immunocytotoxic endpoints reflected differences in haemocyte proportions in the haemolymph of Cd-exposed mussels. Exposure to Cd also led to the formation of several nuclear aberrations in the gill cells of mussels. Overall the laboratory study highlighted toxic effects of Cd on green-lipped mussels that were dependent on the dose and/or the duration of exposure to Cd. Among the biomarkers tested, clearance rate, metallothionein-like protein induction and the formation of nuclear aberrations in mussel gill cells correlated strongly to Cd accumulation levels and reflected Cd exposure effects. The feasibility of employing green-lipped mussels as bioindicators was tested during the field study. Green-lipped mussels were collected from different coastal sites along the South Island in NZ. Metal concentrations in the sediment and in four different mussel tissues were analysed. The findings indicated a significant geographical difference in metal concentration in the environment and in the metal accumulation levels in the mussels. Overall, the field study indicated that the green-lipped mussel, Perna canaliculus has the potential to be used as a bioindicator species for assessment of coastal metal pollution levels in NZ.

Ecotoxicology studies attempt to evaluate the consequences of exposure to environmental contaminants by defining exposure and effects parameters across multiple levels of biological organization. Genetic markers are well-suited for these studies as they can track both somatic and evolutionary effects. In the studies reported here, connections among contaminant levels in environmental media and biota, in vitro bioassay results, and changes in individual- and population-level biomarkers were explored. Sediment and/or wildlife samples were collected from each of three sites of documented environmental contamination, Sumgayit and Baku in the Republic of Azerbaijan and East Fork Poplar Creek in Tennessee. Sumgayit and Baku are heavily contaminated with petroleum and petrochemical wastes. Sediments from several areas and tissues from turtles inhabiting a contaminated wetland contained high levels of several compounds, including mercury, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organochlorine pesticides. Sediment extracts produced variable responses in the Salmonella/microsome mutagenicity assay and did not necessarily reflect contaminant burden. Micronucleus counts in European pond turtles were not statistically different from counts in the same species from an uncontaminated reference site. The counts were statistically correlated with tissue levels of mercury, heptachlor, DDD, hexachlorobenzene, and trans-nonachlor. These results confirmed that Sumgayit and Baku are heavily contaminated with a complex mixture of pollutants and demonstrated that genotoxic effects from exposure to contaminated sediments appear to be slight. East Fork Poplar Creek is a stream that receives contaminant influx from a former Department of Energy nuclear weapons production facility and several point and non-point sources around the city of Oak Ridge, Tennessee. In this study, coefficient of variation in cellular DNA content and randomly amplified polymorphic DNA markers were examined in central stonerollers and compared to previous studies in which the same markers were evaluated in red-breasted sunfish from the same sites. While assay responses were attenuated in stonerollers compared to the sunfish, there is some evidence of genetic damage in both species at the most contaminated sampling site. A common problem in the wildlife studies was high within sample variability combined with small sample size, which most likely masked potential contaminant-induced differences in markers used in these studies.

Proteins are the effector molecules of life. They are encoded in DNA that is inherited from generation to generation, but most cellular functions are executed by proteins. Proteins rarely act on their own – most actions are carried out through an interplay of tens of proteins and other biomolecules. Here I describe how synthetic DNA can be used to study proteins and protein complexes. Variants of proximity ligation assays (PLA) are used to generate DNA reporter molecules upon proximal binding by pairs of DNA oligonucleotide-modified affinity reagents. In Paper I, a robust protocol was set up for PLA on paramagnetic microparticles, and we demonstrated that this solid phase PLA had superior performance for detecting nine candidate cancer biomarkers compared to other immunoassays. Based on the protocol described in Paper I I then developed further variants of PLA that allows detection of protein aggregates and protein interactions. I sensitively detected aggregated amyloid protofibrils of prion proteins in paper II, and in paper III I studied binary interactions between several proteins of the NFκB family. For all immunoassays the selection of high quality affinity binders represents a major challenge. I have therefore established a protocol where a large set of protein binders can be simultaneously validated to identify optimal pairs for dual recognition immunoassays (Paper IV).

Background: Vitamin D may have beneficial effects on cardiometabolic disease, but the evidence is equivocal. This may be due to unaccounted confounders, such as lifestyle factors and genetic variation. We examined the association between circulating 25-hydroxyvitamin D [25(OH)D] and biomarkers of cardiometabolic disease risk, including biomarkers of inflammation, glycemic dysregulation and lipid metabolism, and a panel of 54 plasma proteomic biomarkers, and determined whether lifestyle variables and genetic variation modified these associations. Methods: Participants were from the Toronto Nutrigenomics and Health Study, an ethnically diverse population of individuals aged 20-29 years. Anthropometric measurements were obtained. Participants answered general health and lifestyle and food frequency questionnaires and provided a fasting blood sample for biochemical measurements and genotyping. Results: Across ethnic groups, women who used hormonal contraceptives (HC) had higher 25(OH)D and C-reactive protein (CRP) than women HC non-users and men. Circulating 25(OH)D was positively associated with CRP in the entire population in models not accounting for HC use. However, there was no association after accounting for HC use. 25(OH)D was also not associated with inflammatory cytokines after adjusting for HC use. 25(OH)D was inversely associated with insulin, HOMA-IR, and HOMA-Beta among Caucasians and East Asians and among men and women HC non-users. No biomarkers were associated with 25(OH)D among South Asians and women HC users, although non-significant inverse trends were observed for markers of glycemic dysregulation. Only two of the 54 plasma proteomic biomarkers were associated with 25(OH)D in women HC non-users, and none were associated in men. Among women HC users, after accounting for hormone dose, only three proteins were associated with 25(OH)D. Finally, 25(OH)D affected the association between rs2239182, a variant in the vitamin D receptor (VDR) and the pro-inflammatory cytokine interferon gamma-induced protein 10 (IP-10). However, the association was suggestive of heterosis and may have been due to chance. Conclusions: We identified a confounding effect of HC use on the association between 25(OH)D, biomarkers of inflammation and plasma proteomic biomarkers. In addition, HC use might also affect the association between 25(OH)D and biomarkers of glycemic dysregulation. Genetic variation in VDR did not modify any associations.

Background: Vitamin D may have beneficial effects on cardiometabolic disease, but the evidence is equivocal. This may be due to unaccounted confounders, such as lifestyle factors and genetic variation. We examined the association between circulating 25-hydroxyvitamin D [25(OH)D] and biomarkers of cardiometabolic disease risk, including biomarkers of inflammation, glycemic dysregulation and lipid metabolism, and a panel of 54 plasma proteomic biomarkers, and determined whether lifestyle variables and genetic variation modified these associations. Methods: Participants were from the Toronto Nutrigenomics and Health Study, an ethnically diverse population of individuals aged 20-29 years. Anthropometric measurements were obtained. Participants answered general health and lifestyle and food frequency questionnaires and provided a fasting blood sample for biochemical measurements and genotyping. Results: Across ethnic groups, women who used hormonal contraceptives (HC) had higher 25(OH)D and C-reactive protein (CRP) than women HC non-users and men. Circulating 25(OH)D was positively associated with CRP in the entire population in models not accounting for HC use. However, there was no association after accounting for HC use. 25(OH)D was also not associated with inflammatory cytokines after adjusting for HC use. 25(OH)D was inversely associated with insulin, HOMA-IR, and HOMA-Beta among Caucasians and East Asians and among men and women HC non-users. No biomarkers were associated with 25(OH)D among South Asians and women HC users, although non-significant inverse trends were observed for markers of glycemic dysregulation. Only two of the 54 plasma proteomic biomarkers were associated with 25(OH)D in women HC non-users, and none were associated in men. Among women HC users, after accounting for hormone dose, only three proteins were associated with 25(OH)D. Finally, 25(OH)D affected the association between rs2239182, a variant in the vitamin D receptor (VDR) and the pro-inflammatory cytokine interferon gamma-induced protein 10 (IP-10). However, the association was suggestive of heterosis and may have been due to chance. Conclusions: We identified a confounding effect of HC use on the association between 25(OH)D, biomarkers of inflammation and plasma proteomic biomarkers. In addition, HC use might also affect the association between 25(OH)D and biomarkers of glycemic dysregulation. Genetic variation in VDR did not modify any associations.

The Arctic is considered as a focal region in the ongoing climate change debate. The currently observed and predicted climate warming is particularly pronounced in the high northern latitudes. Rising temperatures in the Arctic cause progressive deepening and duration of permafrost thawing during the arctic summer, creating an ‘active layer’ with high bioavailability of nutrients and labile carbon for microbial consumption. The microbial mineralization of permafrost carbon creates large amounts of greenhouse gases, including carbon dioxide and methane, which can be released to the atmosphere, creating a positive feedback to global warming. However, to date, the microbial communities that drive the overall carbon cycle and specifically methane production in the Arctic are poorly constrained. To assess how these microbial communities will respond to the predicted climate changes, such as an increase in atmospheric and soil temperatures causing increased bioavailability of organic carbon, it is necessary to investigate the current status of this environment, but also how these microbial communities reacted to climate changes in the past. This PhD thesis investigated three records from two different study sites in the Russian Arctic, including permafrost, lake shore and lake deposits from Siberia and Chukotka. A combined stratigraphic approach of microbial and molecular organic geochemical techniques were used to identify and quantify characteristic microbial gene and lipid biomarkers. Based on this data it was possible to characterize and identify the climate response of microbial communities involved in past carbon cycling during the Middle Pleistocene and the Late Pleistocene to Holocene. It is shown that previous warmer periods were associated with an expansion of bacterial and archaeal communities throughout the Russian Arctic, similar to present day conditions. Different from this situation, past glacial and stadial periods experienced a substantial decrease in the abundance of Bacteria and Archaea. This trend can also be confirmed for the community of methanogenic archaea that were highly abundant and diverse during warm and particularly wet conditions. For the terrestrial permafrost, a direct effect of the temperature on the microbial communities is likely. In contrast, it is suggested that the temperature rise in scope of the glacial-interglacial climate variations led to an increase of the primary production in the Arctic lake setting, as can be seen in the corresponding biogenic silica distribution. The availability of this algae-derived carbon is suggested to be a driver for the observed pattern in the microbial abundance. This work demonstrates the effect of climate changes on the community composition of methanogenic archae. Methanosarcina-related species were abundant throughout the Russian Arctic and were able to adapt to changing environmental conditions. In contrast, members of Methanocellales and Methanomicrobiales were not able to adapt to past climate changes. This PhD thesis provides first evidence that past climatic warming led to an increased abundance of microbial communities in the Arctic, closely linked to the cycling of carbon and methane production. With the predicted climate warming, it may, therefore, be anticipated that extensive amounts of microbial communities will develop. Increasing temperatures in the Arctic will affect the temperature sensitive parts of the current microbiological communities, possibly leading to a suppression of cold adapted species and the prevalence of methanogenic archaea that tolerate or adapt to increasing temperatures. These changes in the composition of methanogenic archaea will likely increase the methane production potential of high latitude terrestrial regions, changing the Arctic from a carbon sink to a source. / Die Arktis ist in den gegenwärtigen Diskussionen zum Klimawandel von besonderem Interesse. Die derzeitig beobachtete globale Erwärmung ist in den hohen nördlichen Breiten besonders ausgeprägt. Dies führt dazu, dass ehemals gefrorene Böden zunehmend tiefer auftauen und daher im Boden enthaltene Kohlenstoffquellen für die mikrobielle Umsetzung und Mineralisierung zur Verfügung stehen. Aufgrund dieser Prozesse entstehen klimarelevant Gase, darunter Kohlendioxid und Methan, die aus den Böden und Sedimenten freigesetzt werden können. Wenn man bedenkt, dass in den nördlichen Permafrostgebieten die Hälfte des weltweit unter der Bodenoberfläche gelagerten Kohlenstoffs gelagert ist, wird die Bedeutung dieser Region für das Verständnis des globalen Kohlenstoffkreislaufes und der möglichen Treibhaus-gasemissionen sichtbar. Trotz dieser Relevanz, sind die am Kohlenstoffumsatz beteiligten Mikroorganismen in der Arktis wenig untersucht und ihre Anpassungsfähigkeit an die gegenwärtigen Klimaveränderungen unbekannt. Die vorliegende Arbeit untersucht daher, wie sich Klimaveränderungen in der Vergangenheit auf die Anzahl und Zusammensetzung der mikrobiellen Gemeinschaften ausgewirkt haben. Dabei liegt ein besonderer Fokus auf die methanbildenden Archaeen, um das Verständnis der mikrobiellen Methandynamik zu vertiefen. Im Rahmen dieser Arbeit wurden drei Bohrkerne aus zwei verschiedenen Standorten in der russischen Arktis untersucht, darunter terrestrischer Permafrost und Seesedimente aus Sibirien und Chukotka, Russland. Mittels der Identifikation und Quantifizierung von mikrobiellen Genen und charakteristischen Bestandteilen der mikrobiellen Zellmembran war es möglich, fossile mikrobielle Gemeinschaften in Seesedimenten mit einem Alter von bis zu 480 000 Jahren und in Permafrostablagerungen mit einem Alter von bis zu 42 000 Jahren zu rekonstruieren. Es wurde gezeigt, dass es während vergangener warmen Perioden zu einem Wachstum von Bakterien und Archaeen in allen untersuchten Standorten gekommen ist. Dieser Trend konnte auch für die Gemeinschaft der methanogenen Archaeen gezeigt werden, die während warmen und insbesondere feuchten Klimabedingungen in großer Anzahl und Diversität vorhanden waren, was wiederrum Rückschlüsse auf mögliche Methanemissionen erlaubt. In den terrestrischen Permafroststandorten wird der Temperaturanstieg als direkte Ursache für die gefundene Reaktion der mikrobiellen Gemeinschaft vermutet. Im Gegenzug dazu, führte der Temperaturanstieg im untersuchten arktischen See wahrscheinlich zu einer erhöhten Primärproduktion von organischem Kohlenstoff, die wiederum das Wachstum der Mikroorganismen antrieb. Weiterhin konnte im Rahmen dieser Arbeit gezeigt werden, dass Methanosarcina-verwandte Spezies in der Russischen Arktis weit verbreitet sind und sich an veränderte Umweltbedingungen gut anpassen können. Im Gegensatz dazu stehen Vertreter von Methanocellales und Methano-microbiales, die nicht in der Lage sich an veränderte Lebensbedingungen anzupassen. Im Rahmen dieser Arbeit konnte erstmalig gezeigt werden, dass es in früheren Warmphasen zu einem vermehrten Wachstum der an der Umsetzung des organischen Kohlenstoffs beteiligten Mikroorganismen in der Russischen Arktis gekommen ist. Im Zusammenhang mit der zukünftigen Erwärmung der Arktis kann also von einer Veränderung der am Kohlenstoffkreislauf beteiligten Mikroorganismen ausgegangen werden kann. Mit den steigenden Temperaturen werden sich einige Vertreter der methanproduzierenden Mikroorganismen an die veränderten Bedingungen anpassen können, während Temperatur-empfindliche Vertreter aus dem Habitat verdrängt werden. Diese Veränderungen in der mikrobiellen Gemeinschaft können die Methanproduktion der hohen noerdlichen Breiten erhoehen und dazu beitragen, dass aus der Arktis als eine Kohlenstoffsenke eine Kohlenstoffquelle wird.