As the world energy demand increases, drilling deeper wells is inevitable. Deeper wells have abnormal pressure zones where the difference between pore pressure and fracture pressure gradient, is very small. Smaller drilling margins make it harder to drill the well and result in high operation costs due to the increase of non-productive time. One of the major factors influence non-productive time in drilling operations is lost circulation due to drilling induced fractures. The most common approach is still plugging the fractures by using various loss circulation materials and there are several wellbore strengthening techniques present in the literature to explain the physics behind this treatment. This thesis focuses on development of a rock mechanics/hydraulic model for quantifying the stress distribution around the wellbore and fracture geometry after fracture initiation, propagation and plugging the fracture with loss circulation materials. In addition, fracture behavior is investigated in different stress states, for different permeability values and in the presence of multiple fractures. The following chapters contain detailed description of this model, and analysis results. / text

Judicious addition of photolabile caging groups (PCGs) to protect biologically im- portant molecules, has enabled the development of many powerful chemical tools for the study of biological processes. These tools have the potential to be activated in a cellular setting by irradiation with light of appropriate wavelengths, restoring functionality, with excellent spatial and temporal control. This D. Phil. dissertation highlights two biologically relevant examples where PCGs can be applied: (i) 4,5-dimethoxynitrobenzyl (DMNB) caged derivatives of GSH and ESG, were synthesised and uncaging of the DMNB group at 350 nm to reveal the free α-carboxylic acid of the glycine residue was demonstrated. These molecules have the potential to probe the binding mode within the KefC KTN binding domain, a ligand-gated K+ efflux system, critical for bacterial response to electrophilic assault. The molecules are currently with collaborators awaiting further evaluation. (ii) The wavelength-dependent application of PCGs toward the study of protein post- translational modifications (PTMs) was developed. PTMs modulate protein function and have a ubiquitous role in a diverse range of cellular functions. A wavelength selective sequential pair of PCGs was developed and demonstrated in a tripeptide, using the diethylamino coumarin (DEACM), which was cleaved at 420 nm, and the DMNB caging group, which was subsequently cleaved at 350 nm. A chromatically orthogonal pair of PCGs, with possible applications in both organic synthesis and biological systems, incorporating the BODIPY-based and DEACM caging groups, which could be cleaved at 530 nm and 420 nm, respectively, was also developed. Photolysis was conducted on a tripeptide and hexapeptide and an in vitro application was demonstrated where when caged, the peptide was not susceptible to peptidolysis when incubated with enzymes. Following irradiation at 420 nm, the DEACM group was uncaged and the peptide underwent peptidolysis in the presence of Endoproteinase AspN, whereas following irradiation at 530 nm to uncage the BODIPY group, the peptide underwent peptidolysis in the presence of Endoproteinase LysC. These results not only provide conclusive evidence of the ability of this pair of caging groups to act in a wavelength orthogonal sense but also demonstrates the potential applicability of the pair to biochemical studies. This pair of PCGs represents the first example of an orthogonal pair of caging groups where both PCGs could be cleaved, at wavelengths longer than 400 nm, in a sequence independent manner.

The main objective of this study was to investigate small-bodied fish caging as an approach to evaluate selenium (Se) bioaccumulation and speciation in native fish species inhabiting lakes influenced by uranium (U) milling effluent in northern Saskatchewan, Canada. In contaminated environments freshwater fish show a high propensity to accumulate Se beyond levels needed for normal physiological function. Maternal transfer of elevated Se concentrations to offspring can cause deformities and reduced survival in fry, and in certain cases negatively impact the sustainability of native fish populations. This research included a caging validation study using wild, naïve (i.e., collected from a reference lake) lake chub (Couesius plumbeus) and spottail shiner (Notropis hudsonius), and three field based 21-day caging studies to investigate the dominance of the feeding pathway with respect to Se uptake and speciation in wild populations of northern small-bodied fish exposed to a gradient of Se. Three feeding regimes were used: an in situ benthic diet, a basal Se diet of Chironomus dilutus (1.5 µg Se/g dry weight) and a Se-spiked diet of C. dilutus (5.5 µg Se/g dry weight). Lake chub were identified as more suitable candidates for caging due to higher survival and condition factor at the completion of the in situ 21-day trial. The resulting Se bioaccumulation was compared among treatments as well as to wild small-bodied fish populations from the study area. Results from the caging experiments showed that caged lake chub exposed to natural and controlled diets with elevated Se had significantly greater whole-body Se concentrations after 21 days compared to fish caged in the reference lake. The results also showed that whole-body Se concentrations exceeded conservative Se thresholds, and approached the currently proposed USEPA regulatory threshold (7.91 µg/g dry weight) designed to protect fish species in only three weeks. The use of stable carbon (C), nitrogen (N), and sulphur (S) isotope ratios indicated that alternate benthic food sources native to the exposure lake were consumed in conjunction with the controlled diets. Stable isotope analysis of both wild and caged lake chub indicated that the N and S isotopic signatures decreased with increasing Se exposure, representing differences in isotopic signatures of the food sources. Speciation results from caged lake chub indicated that Se substituted for S in methionine (i.e. selenomethionine) was the dominant Se species found in caged lake chub exposed to dietary sources of elevated Se. Overall, this research demonstrates that using caged native lake chub represents a useful biomonitoring approach to investigate patterns of Se bioaccumulation and speciation in fish.

The main objective of this study was to investigate small-bodied fish caging as an approach to evaluate selenium (Se) bioaccumulation and speciation in native fish species inhabiting lakes influenced by uranium (U) milling effluent in northern Saskatchewan, Canada. In contaminated environments freshwater fish show a high propensity to accumulate Se beyond levels needed for normal physiological function. Maternal transfer of elevated Se concentrations to offspring can cause deformities and reduced survival in fry, and in certain cases negatively impact the sustainability of native fish populations. This research included a caging validation study using wild, naïve (i.e., collected from a reference lake) lake chub (Couesius plumbeus) and spottail shiner (Notropis hudsonius), and three field based 21-day caging studies to investigate the dominance of the feeding pathway with respect to Se uptake and speciation in wild populations of northern small-bodied fish exposed to a gradient of Se. Three feeding regimes were used: an in situ benthic diet, a basal Se diet of Chironomus dilutus (1.5 µg Se/g dry weight) and a Se-spiked diet of C. dilutus (5.5 µg Se/g dry weight). Lake chub were identified as more suitable candidates for caging due to higher survival and condition factor at the completion of the in situ 21-day trial. The resulting Se bioaccumulation was compared among treatments as well as to wild small-bodied fish populations from the study area. Results from the caging experiments showed that caged lake chub exposed to natural and controlled diets with elevated Se had significantly greater whole-body Se concentrations after 21 days compared to fish caged in the reference lake. The results also showed that whole-body Se concentrations exceeded conservative Se thresholds, and approached the currently proposed USEPA regulatory threshold (7.91 µg/g dry weight) designed to protect fish species in only three weeks. The use of stable carbon (C), nitrogen (N), and sulphur (S) isotope ratios indicated that alternate benthic food sources native to the exposure lake were consumed in conjunction with the controlled diets. Stable isotope analysis of both wild and caged lake chub indicated that the N and S isotopic signatures decreased with increasing Se exposure, representing differences in isotopic signatures of the food sources. Speciation results from caged lake chub indicated that Se substituted for S in methionine (i.e. selenomethionine) was the dominant Se species found in caged lake chub exposed to dietary sources of elevated Se. Overall, this research demonstrates that using caged native lake chub represents a useful biomonitoring approach to investigate patterns of Se bioaccumulation and speciation in fish.

Spinal cord injuries (SCI) are characterized by the inability of mature neurons to regenerate or repair by themselves. In an attempt to overcome the SCI, a novel photo-sensitive cyclic Arg-Gly-Asp-Ser (RGDS) peptide was synthesized using solid phase peptide synthesis (SPSS) to control 3T3 fibroblast cell attachment on hyaluronic acid (HA) hydrogel. The circular RGDS peptide was designed using RGDS sequence labeled with Anp group (3-Na-fmoc-amino-3-(2-nitrophenyl) propionic acid) at the N terminus. The peptide was photo-labile cyclic caged to shelter its bioactivity and UV light was used to make the peptide uncaged. Accuracy of the cyclic caged RGDS peptide was confirmed by high-performance liquid chromatography (HPLC) and mass spectrum (MS). The molecular weight of cyclic caged RGDS peptide was confirmed as 881 by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrum. Stability of the cyclic caged RGDS peptide under various pH conditions was verified by circular dichroism spectroscopy. The bioactivity of cyclic caged and uncaged RGDS peptide was tested by photo-controllable directing cell growth based on cell attachment study, cell counting study, and cell morphology study. Three dimensional model structures of cyclic caged and uncaged RGDS peptides were computed by Hyperchem program. The first order reaction theory of Anp uncaging reaction was confirmed by kinetic study. Bioactivity caging and uncaging property of the peptide was also fully confirmed by cell attachment study. This cyclic caged RGDS peptide would be a promising tool in cell patterning for repairing of SCI.

Spinal cord injuries (SCI) are characterized by the inability of mature neurons to regenerate or repair by themselves. In an attempt to overcome the SCI, a novel photo-sensitive cyclic Arg-Gly-Asp-Ser (RGDS) peptide was synthesized using solid phase peptide synthesis (SPSS) to control 3T3 fibroblast cell attachment on hyaluronic acid (HA) hydrogel. The circular RGDS peptide was designed using RGDS sequence labeled with Anp group (3-Na-fmoc-amino-3-(2-nitrophenyl) propionic acid) at the N terminus. The peptide was photo-labile cyclic caged to shelter its bioactivity and UV light was used to make the peptide uncaged. Accuracy of the cyclic caged RGDS peptide was confirmed by high-performance liquid chromatography (HPLC) and mass spectrum (MS). The molecular weight of cyclic caged RGDS peptide was confirmed as 881 by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrum. Stability of the cyclic caged RGDS peptide under various pH conditions was verified by circular dichroism spectroscopy. The bioactivity of cyclic caged and uncaged RGDS peptide was tested by photo-controllable directing cell growth based on cell attachment study, cell counting study, and cell morphology study. Three dimensional model structures of cyclic caged and uncaged RGDS peptides were computed by Hyperchem program. The first order reaction theory of Anp uncaging reaction was confirmed by kinetic study. Bioactivity caging and uncaging property of the peptide was also fully confirmed by cell attachment study. This cyclic caged RGDS peptide would be a promising tool in cell patterning for repairing of SCI.

L’évaluation de l’impact des polluants dans l’environnement est une des préoccupations majeures qui s’inscrit dans la Directive Européenne Cadre Eau 2000. Les réglementations préconisées ont notamment pour objectif de parvenir au bon état chimique et écologique des masses d’eau. Dans ce contexte, notre étude a consisté à développer une approche multibiomarqueur sur des juvéniles de poisson afin d’évaluer les effets biologiques de la pollution chimique en milieu littoral. Des paramètres moléculaires de détoxification (EROD, GST) et une enzyme antioxydante (CAT) ont été utilisés en tant que «système d’alarme » susceptibles de détecter une perturbation avant l’apparition de signes pathologiques irréversibles. En parallèle, différents biomarqueurs physiologiques (croissance somatique et récente, rapport ARN/ADN, indices morphométrique et lipidique) ont été analysés en considérant que ceux-ci pourraient révéler les dommages induit par les polluants sur l’état de santé des juvéniles. La sensibilité et la pertinence des biomarqueurs moléculaires et physiologiques ont été testés expérimentalement sur des juvéniles : i) de bar exposés à une pollution aigüe de pétrole, ii) de bar et de turbot soumis à des mélanges de contaminants en concentrations environnementales en conditions contrôlée et semi-contrôlée (« caging »). Nos résultats montrent la capacité de l’EROD, et à un degré moindre de la GST, à détecter une exposition courte (2 et 4 jours) des organismes au pétrole et à refléter ses effets délétères sur leur état de santé. Cette relation entre biomarqueurs moléculaires et physiologiques a par contre été plus difficilement établie dans un contexte de pollution multiple. Les indices de croissance et de condition utilisés se sont avérés plus sensibles aux différents niveaux de contamination analysés (métaux et HAPs). Leur utilisation a permis d’évaluer la condition affaiblie des organismes mis en cage en milieu portuaire pendant 38 jours. Cette expérience de « caging » s’est révélée concluante, notamment pour les juvéniles de bar, sur lesquels aucun stress physiologique de la mise en cage n’a été détecté dans la station de référence. Les effets délétères des contaminants chimiques sur l’état de santé des juvéniles de turbot ont également été observés en condition contrôlée après exposition de 21 jours aux mêmes sédiments portuaires et à un sédiment estuarien. En complément de ces expériences, une étude de terrain a été réalisée sur des juvéniles de flet prélevés dans des estuaires le long de la côte française et belge. Une diminution des indices morphométrique et lipidique des juvéniles de flet, issu des trois estuaires anthropisés, a été observée en relation avec des bioconcentrations en métaux plus élevées que l’estuaire de référence. Les résultats issus de ces différentes études montrent la potentialité des indices de croissance et de condition à révéler les effets biologiques des contaminants chimiques sur les juvéniles de poissons marins. Cependant, leur spécificité vis à vis des polluants étant plus faible que les paramètres de détoxification, leur utilisation peut être limitée. Ces travaux montrent ainsi le besoin d’utiliser des biomarqueurs à différents niveaux d’organisation biologique dans les programmes de biosurveillance. / Evaluation of pollutant effects in environment is one of the major issues of the European Water Framework Directive 2000. Regulations have particularly the objective to reach to a good chemical and ecological status of water bodies. In this context, the aim of our study was to develop a multibiomarker approach on juvenile marine fish in order to evaluate the biological effects of chemical pollution in coastal areas. Molecular detoxification parameters (EROD, GST) and an antioxidant enzyme (CAT) were used as early warning tools of toxicity allowing the prevention of irreversible damages. In parallel, different physiological biomarkers (somatic and recent growth, RNA:DNA ratio, morphometric and lipidic indices) were analysed as reflecting damages on juveniles health. Sensitivity and relevance of molecular and physiological biomarkers were tested experimentally on juvenile : i) sea bass exposed to acute petroleum pollution, ii) sea bass and turbot submitted to a mix of contaminants in environmental concentrations during controlled and semi-controlled (caging) conditions. Our results show the ability of EROD, and in lower degree the GST, to detect short exposure (2 and 4 days) of organisms to petroleum and to reflect their deleterious effects on fish health. This relationship between molecular and physiological biomarkers was more difficultly established under multiple pollutions. Growth and condition indices were found to be more sensitive to the different levels of chemical contamination analysed (metal, PAHs). Their analyses allowed us to evaluate the weakened condition of organisms caged in the harbour area during 38 days. This caging experiment was relevant especially for juvenile sea bass in which no physiological stress was detected in the reference station. Deleterious effects of chemical contaminant on turbot juvenile health were also observed in controlled condition after 21 days exposure to the same harbour sediments and to an estuarine sediment. In complement to these experiments, a field study was realized on juvenile flounders sampled in some estuaries along the French and Belgium coast. A decrease of morphometric and lipidic indices were found in juvenile flounders from the three anthropogenic estuaries showing the highest metal bioconcentrations compared to the reference estuary. Results from these different studies showed the potentiality of growth and condition indices to reflect biological effects of chemical contaminants on juvenile marine fish. However, their use could be limited by their lower specificity to pollutant than parameters involved in detoxification. These works show therefore the need to use biomarkers at different level of biological organization in biomonitoring programs.

Caged compounds are biologically active molecules that are rendered inert by masking an important functionality with a photolabile protecting, ‘caging’, group. The caging group can be removed by irradiation with light to reveal the active compound with restored pharmacological activity with high spatial and temporal control. This technology provides an ideal tool for the study of many chemical, physiological and biological systems. This DPhil dissertation highlights several projects in which caging technology has been employed to address biological problems and questions. The first example of spatially controlled mitochondrial inactivation is reported - a tool for the study of the role of mitochondria in Ca2+ signalling. Caged TRPV1 agonists and antagonists have been developed to probe TRPV1, specifically the location of the agonist-binding site. T cell activation has been controlled with light as a tool to gain insight into the adaptive immune response. Caged sodium channel blockers have been investigated. Wavelength-orthogonal photolysis in a neuronal system has been demonstrated using the neurotransmitters glutamate and GABA - this represents a significant advancement in caging technology. This dissertation also includes investigations into the development of novel caging groups.

Biocide-based antifouling (AF) paints are the most common method for preventing biofouling, i.e. the growth of algae, barnacles and other organisms on boat hulls. AF paints for leisure boats are predominantly based on copper (Cu) as the main biocide, with zinc (Zn) present as a pigment and stabilizer. Both metals are released from the paint matrix into the water column, leading to contamination of marinas which typically have only limited water exchange. Thus, the aim of this PhD thesis was to describe the use of AF paints in different regions in Sweden, as well as the associated environmental consequences with regard to contamination of the environment and toxicity to non-target aquatic snails. Using a recently developed X-ray fluorescence application, high levels of Cu were detected on boats moored in freshwaters, despite a more than 20-year-old ban, as well as high levels of tin (Sn) on 10 % of the boats, indicating the presence of (old) tributyltin paints (TBT), which might pose an environmental risk and a health hazard for people performing the paint scraping (paper 1). In addition, very high levels of Cu and Zn were measured in the biofouling material collected from the boat hulls, and this is problematic because the biofouling is commonly disposed of on the soil in boatyards at the end of each season. No difference was found in the amount of biofouling on boats coated with Cu or biocide-free paints, which implies that Cu might be currently overused in areas of low salinity and low barnacle density (paper 2). This work also introduces the use of a new species for ecotoxicological field experiments, the snail Theodoxus fluviatilis. Chronic field experiments (paper 3) revealed 6-fold increases in snail mortality, negative growth and up to 67-fold decreased reproduction in marinas, compared to areas not impacted by boating (‘reference areas’). Moreover, a higher prevalence of snails with histopathological alterations (e.g. necrosis of gills, gonads, midgut gland and parasite infestation, among others) was observed in the marinas, compared to the reference areas (paper 4). Statistical modelling indicated that the majority of the toxic effects were best predicted by the metals, most likely originating from AF paints. The results presented in this thesis depict some important aspects of AF paint use in brackish water and highlight the necessity of implementing a suitable management practice for the heavily contaminated biofouling waste in order to minimize the risk to soils. In addition, the evidence of toxicity to snails in marinas can be used as a basis to increase the public understanding of the impact of recreational boating and encourage people to choose less toxic alternatives to AF paints. / <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>

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.