Inanga (Galaxias maculatus) are a culturally and economically important fish species in New Zealand and abroad. However, very little is known about their ability to deal with trace element contamination. As a scaleless fish with the ability to survive in relatively extreme environments, they may not fit toxicity models (such as the biotic ligand model; BLM) based on other fish species. The aim of this study was to determine how this fish responds to elevated trace elements in both the laboratory and field in order to determine the applicability of these toxicity models. In order to determine the impacts of stress on ion transport and subsequent metal toxicity, inanga were exposed to handling stress and measures of ion uptake were collected. Handling stress was shown to result in increased ventilation rates, resulting in stimulated sodium (Na+) efflux. A compensatory increase in Na+ influx was also measured as a result of this stress. Inanga largely recovered from this ionoregulatory stress within 2 hours, with full recovery after 24 hours. This was indicative of a rapid homeostatic response for maintaining ion balance. Enhanced Na+ uptake in response to this stress resulted in increased copper (Cu) uptake in Cu-contaminated water, suggesting stressed fish will accumulate more Cu (and likely other Na+ mimics) than an unstressed fish. These results suggest a heightened vulnerability of inanga to this type of contaminant as a result of exercise stress during migrations. A combination of field and laboratory studies was used in order to measure trace element accumulation in inanga. In situ field studies showed changes to aluminum (Al) and iron (Fe) body burdens when inanga were placed in streams of varying trace element concentrations along the West Coast of the South Island. However, other trace elements measured did not alter over the period of exposure (9-10 days). Biochemical biomarker analysis showed no changes in the activity of Na+/K+-ATPase (NKA), but a marker of lipid peroxidation (thiobarbituric acid reactive substances; TBARS) was elevated in one stream. Analysis suggested that stream pH was the major driver of this effect, whether directly or via changes to metal bioavailability. Subsequent laboratory exposures (96 h) of inanga to 1.2, 2.7, 10.8, and 44 µg L-1 dissolved Fe and 5.6, 23.3, 60.7, and 128.7 µg L-1 dissolved zinc (Zn) showed no difference in whole body trace element accumulation, ammonia excretion, ion influx (Ca2+ and Na+), and TBARS. There were significant differences in oxygen consumption (MO2) after Fe exposures, with increases in the 2.7 and 44 µg L-1 dissolved Fe exposures. Laboratory exposure results suggest inanga are relatively insensitive to short-term Fe and Zn exposures. Both in vivo (whole body partitioning) and in vitro (Ussing chamber) techniques were used to determine the influence of cutaneous ion transport on preventing trace element accumulation. Results suggest inanga use their skin as an additional site of calcium (Ca2+) and Na+ uptake. This is the first study to confirm these ion transport capabilities in inanga, and revealed that up to 48% of Na+ uptake may occur across the skin. Pharmacological inhibition of Ca2+ uptake was achieved by known Ca2+ channel blockers (verapamil and lanthanum). Furthermore Fe and Zn impaired cutaneous Ca2+ transport, indicating that ion transport pathways in the skin modulate in response to these metals.

Solid cancers often shed (sub)cellular materials into the circulation, such as circulating tumor cells and extracellular microvesicles. Mounting evidence supports that these circulating materials could serve as surrogate cancer markers for classifying primary tumors, stratifying patients for targeted therapies, assessing treatment efficacy, and achieving clinical benefits. A sensor platform capable of sensitive and portable detection of circulating cancer markers would thus be an invaluable tool, that will advance our understanding of tumor biology as well as clinical outcomes. This dissertation describes various systems that we have developed for quantitative analyses of circulating cancer biomarkers. Firstly, we have developed a novel magnetic resonance sensing platform for microvesicle analyses. By using a chip-based platform that combines microfiltration and bioorthogonal nanoparticle targeting, we demonstrate for the first time that magnetic biosensing can be applied for clinical evaluation of circulating microvesicles in blood samples to monitor cancer therapy. Secondly, we have advanced a new plasmonic sensor to achieve label-free detection of microvesicles. Based on periodic nanohole arrays, this platform has been applied for high-throughput protein profiling of microvesicles in native ascites. Finally, we have implemented microfluidic devices to effectively enrich circulating tumor cells from peripheral whole blood, and to enable comprehensive molecular analyses of isolated tumor cells at a single cell resolution. By enabling rapid, sensitive and cost-effective detection of circulating cancer markers, these developed platforms could significantly expand the reach of preclinical and clinical cancer research, in informing therapy selection, rationally directing trials, and improving sequential monitoring to achieve better clinical outcomes.

Innovation is not always enough. In the beginning of the 2000s established pharmaceutical firms had developed several drugs, yet these new products were far too few. Patents of many blockbuster drugs were to soon expire and substantial profit would then be lost. A potential solution emerged: implementing new biomarker technologies in drug development. Biomarkers are required for knowledge creation about the drug effect on underlying causes of a disease. The problem is this: although academia, industry, and policy makers have deemed biomarkers as necessary for successful drug development, pharmaceutical firms have not used them in actual drug development projects.  Since the 1990s, established pharmaceutical firms have invested financially and restructured organizationally in order to implement biomarkers. Still, cases show that more than 50% of project termination in Clinical Phase 2 (the bottle neck of drug development) can be attributed to the lack of implementing biomarkers.   Challenges of established firms transforming in the face of technology change is a commonly studied phenomenon within innovation management literature. Several explanations have attempted to determine why established firms fail in following technology change. However, most of this literature has been based upon an empirical context where technology change is conceptualized as an innovation of the dominant product design in the industry. Consequently, the challenge is to develop or adapt a discontinuous product innovation. Conversely, implementing biomarkers is a case of technology change that impacts R&amp;D. Since drugs lose their value when the patent protection expires, the established pharmaceutical firms need to continuously develop new block buster drugs – not just one product. More research is needed to fill this gap in the literature in order to develop an understanding of the established firm challenge in implementing biomarkers. This thesis builds upon a longitudinal case study of AstraZeneca. Using multiple data sources, the findings show that the dominant architecture of the drug development process during the 2000s impeded the implementation of biomarkers. AstraZeneca required an “architectural process innovation” in order to complete this implementation. The company’s process-based management structures distorted it from recognizing the need for process change. This thesis has three contributions: First, it describes the process change and the firm’s managerial challenges associated with biomarker implementation; Second, it contributes to the literature on the established firm challenge by developing an understanding of the phenomenon of architectural process innovation; Third, it develops a process-based framework for studying technology change that affects R&amp;D. / <p>QC 20151106</p>

We exposed a native endangered species, bonytail chub (Gila elegans), to graded concentrations of secondarily-treated effluent. At the end of each treatment period, we collected water from all raceways and samples were analyzed for the presence of 83 organic wastewater compounds. We extracted blood plasma from control and treatment fish in each raceway and samples were analyzed for hormones, 17β-estradiol and 11-ketotestosterone, and the egg yolk protein, vitellogenin.17β-estradiol concentrations were consistently lower in treatment females than those detected in control females. The 17β-estradiol and vitellogenin concentrations were always higher in treatment males than those found in control males. Concentrations of 11-ketotestosterone were consistently lower in treatment males than those detected in control males.Endocrine disrupting effects occurred in male and female fish exposed to low concentrations of effluent. Changes in hormone levels suggested a feminizing effect in treatment male fish and an androgenizing effect in treatment female fish.

Protein biomarkers circulating in blood hold the promise of improved diagnosis, prognosis and follow-up of treatment of disease via minimally invasive procedures. For the discovery and validation of such biomarkers, methods are needed that can facilitate parallel, highly specific and in-depth analysis of the blood proteome. The work presented in this thesis intends to develop and apply such assays, building on the concept of the proximity ligation assay (PLA). In paper I, I present an easy and non-expensive alternative for the conjugation of oligonucleotides to antibodies via biotin-streptavidin-biotin interaction. This approach can be used when large sets of antibodies and/or oligos need to be validated for their performance as probes in PLA reactions. In paper II, a solid-phase variant of PLA (SP-PLA) for the detection and quantification of proteins in blood is presented. SP-PLA exhibited an improved limit of detection compared to commercial ELISA assays by two orders of magnitude. In addition SP-PLA exhibited a broader dynamic range by at least one order of magnitude and required only 5 μl of sample, rendering the method very well suited for analyses of precious bio-banked material. Last but not least, SP-PLA was used to validate the diagnostic potential of GDF-15 as a biomarker for cardiovascular disease in a set of cardiovascular disease patients and healthy controls. Paper III discusses the development of a multiplex SP-PLA (MultiPLAy) for the simultaneous detection of 36 proteins in just 5 μl of sample. MultiPLAy exhibited an improved LOD when compared to state-of-the-art bead-based sandwich assays. Most importantly, we observed only a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that much higher levels of multiplexing will be possible. The assay was used to identify putative biomarkers in sample cohorts of colorectal cancer (CRC) and cardiovascular disease (CVD). Subsequent multivariate analysis revealed previously known diagnostic biomarkers. Furthermore, we successfully applied next-generation sequencing as a readout for the protein assays, allowing for the first time digital recording of protein profiles in blood. In paper IV, we investigated the suitability of prostasomes as blood biomarkers in patients with prostate cancer using a newly developed PLA assay (4PLA) that utilizes five binders for the detection of complex target molecules. The assay successfully detected significantly elevated levels of prostasomes in blood samples from prostate cancer patients prior to radical prostatectomy, compared to controls and men with benign biopsy results.

No description available.

The goal of this thesis was to test the applicability of biomarker analyses to better understand the glacial stratigraphic record of the Hudson Bay Lowlands and Oak Ridges Moraine. A biomarker analysis conducted on three geologic deposits from the Hudson Bay Lowlands showed that they can be differentiated based on organic matter (OM) inputs and stage of diagenesis, relating to paleoclimate and depositional environments. In the second study, a biomarker analysis was applied to samples from ten deposits in the Oak Ridges Moraine. These deposits were differentiated based on OM inputs relating to paleovegetation. Additionally, reincorporation and post-deposition alteration led to sample heterogeneity confirming the current understanding of glacial depositional processes and environments. This thesis shows that biomarker analyses can effectively differentiate and contextualize geologic deposits based on OM inputs and stage of diagenesis. This in turn will provide a more robust understanding of the stratigraphic record.

The goal of this thesis was to test the applicability of biomarker analyses to better understand the glacial stratigraphic record of the Hudson Bay Lowlands and Oak Ridges Moraine. A biomarker analysis conducted on three geologic deposits from the Hudson Bay Lowlands showed that they can be differentiated based on organic matter (OM) inputs and stage of diagenesis, relating to paleoclimate and depositional environments. In the second study, a biomarker analysis was applied to samples from ten deposits in the Oak Ridges Moraine. These deposits were differentiated based on OM inputs relating to paleovegetation. Additionally, reincorporation and post-deposition alteration led to sample heterogeneity confirming the current understanding of glacial depositional processes and environments. This thesis shows that biomarker analyses can effectively differentiate and contextualize geologic deposits based on OM inputs and stage of diagenesis. This in turn will provide a more robust understanding of the stratigraphic record.

Two popular workflows exist for quantitative proteome analysis: two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), with staining to visualize proteins, and multidimensional solution phase separations of isotopically labelled peptides coupled to mass spectrometry (MS). However, the development of an alternative strategy, which combines easy-to-read differential profiling as seen in 2D-PAGE, with the sensitivity of MS for detection and identification, is needed. This thesis presents work towards the development of a workflow for high-throughput protein biomarker discovery. Multidimensional separations are vital to obtain sufficient fractionation of complex proteome mixtures. As a first dimension of separation, ion exchange chromatography (IEC) is a common choice, though it has yet to be thoroughly evaluated in terms of its effectiveness as a proteome prefractionation tool. This study used a defined set of protein standards to establish the resolution and proteome yield obtained through IEC. The evaluation uncovered significant bias in terms of protein recovery and separation. To improve throughput of a multidimensional separation strategy, a multiplexed (8-column) reversed phase liquid chromatography (RPLC) platform was constructed. The system design allowed for even distribution of flow across all columns with limited cross-loading during sample loading. This system was directly coupled to matrix-assisted laser desorption/ionization (MALDI) through a novel well plate device. The Teflon wells allowed for high recovery and no cross-contamination during collection/spotting, improved throughput, and greatly reduced the number of sample manipulation steps. An evaluation of MALDI MS, using the ThermoFisher vMALDI LTQ, for quantitative profiling was performed, employing the multiplexed LC-MALDI platform. The use of MALDI MS allowed for fast (< 5.5 hours) acquisition of quantitative data from isotopically differentiated samples partitioned over 640 fractions from two-dimensional LC. Proteins comprising 0.1% of the proteome were detected and quantified using this method. Finally, the effects of varying concentrations of acetonitrile (ACN) upon the products generated from tryptic digestions were explored. Poor enzymatic efficiency in 80% ACN was found to be responsible for an increased concentration of peptides containing missed cleavage sites. These peptides often contained unique amino acid sequences, which were not detected from complete digestions, resulting in improved protein sequence coverage following MS analysis.

Traumatic brain injury (TBI) is a relatively unknown disease considering the tens of millions of people affected around the world each year. Many TBI patients die from their injuries and survivors often suffer from life-long disabilities. The primary injury initiates a variety of cellular and molecular processes that are both beneficial and detrimental for the brain, but that are not fully understood. The focus of this thesis has been to study the role of astrocytes in clearance of dead cells after TBI and to identify injury specific proteins that may function as biomarkers, by using cell cultures, animal models and in cerebrospinal fluid (CSF) from TBI patients. The result demonstrates a new function in that astrocytes, the most numerous cell type in the brain, engulf dead cells after injury both in cell cultures and in adult mice and thereby save neurons from contact-induced apoptosis. Astrocytes are effective phagocytes, but degrade the ingested dead cells very slowly. Moreover, astrocytes express the lysosome-alkalizing proteins Rab27a and Nox2 as well as major histocompatibility complex class II, the receptors on which antigens are being presented. By lowering the pH of the lysosomes with acidic nanoparticles, the degradation increases, but the astrocytes still remained less effective than macrophages. Taken together, the data indicates that the low acidification in astrocytes can preserve antigens and that astrocytes may be able to activate T cells. The expression and secretion of injury-specific proteins was studied in a cell culture model of TBI by separate mass spectrometry analysis of cells and medium. Interestingly, close to 30 % of the injury-specific proteins in medium are linked to actin, for example ezrin of the ezrin/radixin/moesin (ERM) protein family. Ezrin, but none of the other ERM proteins or actin, is actively secreted after injury. Extracellular ezrin also increases in CSF in response to experimental TBI in rats and is present in CSF from TBI patients, indicating that ezrin is a potential biomarker for TBI.