Attenuation of muscle atrophy in a murine model of cachexia by inhibition of the dsRNA-dependent protein kinase

Atrophy of skeletal muscle is due to a depression in protein synthesis and an increase in degradation. Studies in vitro have suggested that activation of the dsRNA-dependent protein kinase (PKR) may be responsible for these changes in protein synthesis and degradation. In order to evaluate whether this is also applicable to cancer cachexia the action of a PKR inhibitor on the development of cachexia has been studied in mice bearing the MAC16 tumour. Treatment of animals with the PKR inhibitor (5 mg kg-1) significantly reduced levels of phospho-PKR in muscle down to that found in non-tumour-bearing mice, and effectively attenuated the depression of body weight, with increased muscle mass, and also inhibited tumour growth. There was an increase in protein synthesis in skeletal muscle, which paralleled a decrease in eukaryotic initiation factor 2α phosphorylation. Protein degradation rates in skeletal muscle were also significantly decreased, as was proteasome activity levels and expression. Myosin levels were increased up to values found in non-tumour-bearing animals. Proteasome expression correlated with a decreased nuclear accumulation of nuclear factor-κB (NF-κB). The PKR inhibitor also significantly inhibited tumour growth, although this appeared to be a separate event from the effect on muscle wasting. These results suggest that inhibition of the autophosphorylation of PKR may represent an appropriate target for the attenuation of muscle atrophy in cancer cachexia. © 2007 Cancer Research UK.

Induction of proteasome expression in skeletal muscle is attenuated by inhibitors of NF-κB activation

The potential for inhibitors of nuclear factor-κB (NF-κB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-κB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-κB inhibitor SN50 (18 μM) attenuated the expression of 205 proteasome α-subunits, two subunits of the 195 regulator MSSI and p42, and the ubiquitin-conjugating enzyme, E214k, as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-κB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 μM) and resveratrol (30 μM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2 14k. However, curcumin (150 and 300 mg kg-1) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg-1) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-κB DNA-binding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-κB may prove useful for the treatment of muscle wasting in cancer cachexia.

Angiotensin II directly induces muscle protein catabolism through the ubiquitin-proteasome proteolytic pathway and may play a role in cancer cachexia

The ability of angiotensin I (Ang I) and II (Ang II) to induce directly protein degradation in skeletal muscle has been studied in murine myotubes. Angiotensin I stimulated protein degradation with a parabolic dose-response curve and with a maximal effect between 0.05 and 0.1 μM. The effect was attenuated by coincubation with the angiotensin-converting enzyme (ACE) inhibitor imidaprilat, suggesting that angiotensin I stimulated protein degradation through conversion to Ang II. Angiotensin II also stimulated protein breakdown with a similar dose-response curve, and with a maximal effect between 1 and 2.5 μM. Total protein degradation, induced by both Ang I and Ang II, was attenuated by the proteasome inhibitors lactacystin (5 μM) and MG132 (10 μM), suggesting that the effect was mediated through upregulation of the ubiquitin-proteasome proteolytic pathway. Both Ang I and Ang II stimulated an increased proteasome 'chymotrypsin-like' enzyme activity as well as an increase in protein expression of 20S proteasome α-subunits, the 19S subunits MSSI and p42, at the same concentrations as those inducing protein degradation. The effect of Ang I was attenuated by imidaprilat, confirming that it arose from conversion to Ang II. These results suggest that Ang II stimulates protein degradation in myotubes through induction of the ubiquitin-proteasome pathway. Protein degradation induced by Ang II was inhibited by insulin-like growth factor and by the polyunsaturated fatty acid, eicosapentaenoic acid. These results suggest that Ang II has the potential to cause muscle atrophy through an increase in protein degradation. The highly lipophilic ACE inhibitor imidapril (Vitor™) (30 mg kg-1) attenuated the development of weight loss in mice bearing the MAC16 tumour, suggesting that Ang II may play a role in the development of cachexia in this model. © 2005 Cancer Research.

Problematising L2 listening pedagogy:the potential of process-based listening strategy instruction in the L2 classroom

Listening is typically the first language skill to develop in first language (L1) users and has been recognized as a basic and fundamental tool for communication. Despite the importance of listening, aural abilities are often taken for granted, and many people overlook their dependency on listening and the complexities that combine to enable this multi-faceted skill. When second language (L2) students are learning their new language, listening is crucial, as it provides access to oral input and facilitates social interaction. Yet L2 students find listening challenging, and L2 teachers often lack sufficient pedagogy to help learners develop listening abilities that they can use in and beyond the classroom. In an effort to provide a pedagogic alternative to more traditional and limited L2 listening instruction, this thesis investigated the viability of listening strategy instruction (LSI) over three semesters at a private university in Japan through a qualitative action research (AR) intervention. An LSI program was planned and implemented with six classes over the course of three AR phases. Two teachers used the LSI with 121 learners throughout the project. Following each AR phase, student and teacher perceptions of the methodology were investigated via questionnaires and interviews, which were primary data collection methods. Secondary research methods (class observations, pre/post-semester test scores, and a research journal) supplemented the primary methods. Data were analyzed and triangulated for emerging themes related to participants’ perceptions of LSI and the viability thereof. These data showed consistent positive perceptions of LSI on the parts of both learners and teachers, although some aspects of LSI required additional refinement. This project provided insights on LSI specific to the university context in Japan and also produced principles for LSI program planning and implementation that can inform the broader L2 education community.

Glucosamine-induced insulin resistance in L6 muscle cells

Background: Glucosamine increases flux through the hexosamine pathway, causing insulin resistance and disturbances similar to diabetic glucose toxicity. Aim: This study examines the effect of glucosamine on glucose uptake by cultured L6 muscle cells as a model of insulin resistance. Methods: Glucose uptake by L6 myotubes was measured using the non-metabolized glucose analogue 2-deoxy-D-glucose after incubation with glucosamine for 4 and 24 h, with and without insulin and several other agents (metformin, peroxovanadium and D-pinitol) that improve glucose uptake in diabetic states. Results: After 4 h, high concentrations of glucosamine (5 × 10-3 and 10-2 M) reduced basal and insulin-stimulated glucose uptake by up to 50%. After 24 h, the effect of insulin was completely abolished by 10-2 M glucosamine and reduced over 50% by 5 × 10-3 M glucosamine. Lower concentrations of glucosamine did not significantly alter glucose uptake. The effect of glucosamine could not be attributed to cytotoxicity assessed by the Trypan Blue test. Metformin, peroxovanadium and D-pinitol, each of which increased glucose uptake by L6 cells, did not prevent the decrease in glucose uptake with glucosamine. Conclusion: Glucosamine decreased insulin-stimulated glucose uptake by L6 muscle cells, providing a potential model of insulin resistance with similarities to glucose toxicity. Insulin resistance induced by glucosamine was not reversed by three agents (metformin, peroxovanadium and D-pinitol) known to enhance or partially mimic the effects of insulin. © 2004 Blackwell Publishing Ltd.

Novel dipeptidyl peptidase IV resistant analogues of glucagon-like peptide-1(7-36)amide have preserved biological activities in vitro conferring improved glucose-lowering action in vivo

Although the incretin hormone glucagon-like peptide-1 (GLP-1) is a potent stimulator of insulin release, its rapid degradation in vivo by the enzyme dipeptidyl peptidase IV (DPP IV) greatly limits its potential for treatment of type 2 diabetes. Here, we report two novel Ala8-substituted analogues of GLP-1, (Abu8)GLP-1 and (Val8)GLP-1 which were completely resistant to inactivation by DPP IV or human plasma. (Abu8)GLP-1 and (Val8)GLP-1 exhibited moderate affinities (IC50: 4.76 and 81.1 nM, respectively) for the human GLP-1 receptor compared with native GLP-1 (IC50: 0.37 nM). (Abu8)GLP-1 and (Val8)GLP-1 dose-dependently stimulated cAMP in insulin-secreting BRIN BD11 cells with reduced potency compared with native GLP-1 (1.5- and 3.5-fold, respectively). Consistent with other mechanisms of action, the analogues showed similar, or in the case of (Val8)GLP-1 slightly impaired insulin releasing activity in BRIN BD11 cells. Using adult obese (ob/ob) mice, (Abu8 )GLP-1 had similar glucose-lowering potency to native GLP-1 whereas the action of (Val8)GLP-1 was enhanced by 37%. The in vivo insulin-releasing activities were similar. These data indicate that substitution of Ala8 in GLP-1 with Abu or Val confers resistance to DPP IV inactivation and that (Val8)GLP-1 is a particularly potent N-terminally modified GLP-1 analogue of possible use in type 2 diabetes.

Time for action? Elementary engineering education - Challenging teachers, policy makers and parents

Grounded in the findings of a three year exploratory student whereby teachers' and policy makers' perceptions of elementary level engineering education were analysed, this paper focuses upon three strands of engineering education activity: Pedagogy: Practice, and: Policy. Taking into account the challenges associated with introducing engineering education at an elementary level across the UK, the paper critiques the role played by the 'competition model' in promoting engineering to children and 4 to 11 years. In considering the 'added value' that appropriately developed engineering education activities can offer in the classroom the discussion argues that elementary level engineering has the potential to reach across the curriculum, offering context and depth in many different areas. The paper concludes by arguing that by introducing the discipline to children at a foundational level, switching on their 'Engineering Imaginations' and getting them to experience the value and excitement of engineering, maths and applied science a new "Educational Frontier" will be forged. © American Society for Engineering Education, 2014.

Attenuation of muscle wasting in murine C2C12myotubes by epigallocatechin-3-gallate

Background: Loss of muscle protein is a common feature of wasting diseases where currently treatment is limited. This study investigates the potential of epigallocatechin-3-gallate (EGCg), the most abundant catechin in green tea, to reverse the increased protein degradation and rescue the decreased protein synthesis which leads to muscle atrophy. Methods: Studies were conducted in vitro using murine C2C12myotubes. Increased protein degradation and reduced rates of protein synthesis were induced by serum starvation and tumour necrosis factor-α (TNF-α). Results: EGCg effectively attenuated the depression of protein synthesis and increase in protein degradation in murine myotubes at concentrations as low as 10 μM. Serum starvation increased expression of the proteasome 20S and 19S subunits, as well as the proteasome ‘chymotrypsin-like’ enzyme activity, and these were all attenuated down to basal values in the presence of EGCg. Serum starvation did not increase expression of the ubiquitin ligases MuRF1 and MAFbx, but EGCg reduced their expression below basal levels, possibly due to an increased expression of phospho Akt (pAkt) and phospho forkhead box O3a (pFoxO3a). Attenuation of protein degradation by EGCg was increased in the presence of ZnSO4, suggesting an EGCg-Zn2+complex may be the active species. Conclusion: The ability of EGCg to attenuate depressed protein synthesis and increase protein degradation in the myotubule model system suggests that it may be effective in preserving skeletal muscle mass in catabolic conditions.

Muscle movement and electrodes motion artifact during vibration treatment

Vibration treatment by oscillating platforms is more and more employed in the fields of exercise physiology and bone research. The rationale of this treatment is based on the neuromuscular system response elicited by vibration loads. surface Electromyography (EMG) is largely utilized to assess muscular response elicited by vibrations and Root Mean Square of the electromyography signals is often used as a concise quantitative index of muscle activity; in general, EMG envelope or RMS is expected to increase during vibration. However, it is well known that during surface bio-potential recording, motion artifacts may arise from relative motion between electrodes and skin and between skin layers. Also the only skin stretch, modifying the internal charge distribution, results in a variation of electrode potential. The aim of this study is to highlight the movements of muscles, and the succeeding relevance of motion artifacts on electrodes, in subjects undergoing vibration treatments. EMGs from quadriceps of fifteen subjects were recorded during vibration at different frequencies (15-40 Hz); Triaxial accelerometers were placed onto quadriceps, as close as possible to muscle belly, to monitor motion. The computed muscle belly displacements showed a peculiar behavior reflecting the mechanical properties of the structures involved. Motion artifact related to the impressed vibration have been recognized and related to movement of the soft tissues. In fact large artifacts are visible on EMGs and patellar electrodes recordings during vibration. Signals spectra also revealed sharp peaks corresponding to vibration frequency and its harmonics, in accordance with accelerometers data. © 2008 Springer-Verlag.

Fatty acids and stable isotopes as indicators of early-life feeding and potential maternal resource dependency in the bull shark Carcharhinus leucas

The degree of reliance of newborn sharks on energy reserves from maternal resource allocation and the timescales over which these animals develop foraging skills are critical factors towards understanding the ecological role of top predators in marine ecosystems. We used muscle tissue stable carbon isotopic composition and fatty acid analysis of bull sharks Carcharhinus leucas to investigate early-life feeding ecology in conjunction with maternal resource dependency. Values of δ13C of some young-of-the-year sharks were highly enriched, reflecting inputs from the marine-based diet and foraging locations of their mothers. This group of sharks also contained high levels of the 20:3ω9 fatty acid, which accumulates during periods of essential fatty acid deficiency, suggesting inadequate or undeveloped foraging skills and possible reliance on maternal provisioning. A loss of maternal signal in δ13C values occurred at a length of approximately 100 cm, with muscle tissue δ13C values reflecting a transition from more freshwater/estuarine-based diets to marine-based diets with increasing length. Similarly, fatty acids from sharks >100 cm indicated no signs of essential fatty acid deficiency, implying adequate foraging. By combining stable carbon isotopes and fatty acids, our results provided important constraints on the timing of the loss of maternal isotopic signal and the development of foraging skills in relation to shark size and imply that molecular markers such as fatty acids are useful for the determination of maternal resource dependency.

Comparison of the stable carbon and nitrogen isotopic values of gill and white muscle tissue of fish

The potential use of stable carbon and nitrogen isotope ratios (d13C, d15N) of fish gills for studies on fish feeding ecology was evaluated by comparing the d13C and d15N of gill tissue with the more commonly used white muscle tissue. To account for the effect of lipid content on the d13C signatures, a study-specific lipid correction model based on C:N ratios was developed and applied to the bulk d13C data. For the majority of species in the study, we found no significant difference in d13C values between gill and muscle tissue after correction, but several species showed a small (0.3-1.4 per mil) depletion in 13C in white muscle compared to gill tissue. The average species difference in d15N between muscle and gill tissue ranged from -0.2 to 1.6 per mil for the different fish species with muscle tissue generally more enriched in 15N. The d13C values of muscle and gill were strongly linearly correlated (R**2 = 0.85) over a large isotopic range (13 per mil), suggesting that both tissues can be used to determine long-term feeding or migratory habits of fish. Muscle and gill tissue bulk d15N values were also strongly positively correlated (R**2= 0.76) but with a small difference between muscle and gill tissue. This difference indicates that the bulk d15N of the two tissue types may be influenced by different isotopic turnover rates or a different composition of amino acids.

Análise fitoquímica e avaliação das atividades anti-inflamatória, antipeçonhenta e citotóxica de extratos aquosos de Aspidosperma pyrifolium e Ipomoea asarifolia

Envenomation caused by venomous animals, mainly scorpions and snakes, are a serious matter of public health. Tityus serrulatus is considered the most venomous scorpion in South America because of the high level of toxicity of its venom. It is responsible for causing serious accidents, mainly with kids. The species Bothrops jararaca is a serpent that has in its venom a complex mixture of enzyme, peptides and other molecules. The toxins of the venom of B. jararaca induce local and systemic inflammatory responses. The treatment chosen to serious cases of envenomation is the intravenous administration of the specific antivenom. However, the treatment is not always accessible to those residents in rural areas, so that they use medicinal plant extracts as the treatment. In this context, aqueous extracts, fractions and isolated compounds of Aspidosperma pyrifolium (pereiro) and Ipomoea asarifolia (salsa, salsa-brava), used in popular medicine, were studied in this research to evaluate the anti-inflammatory activity in the peritonitis models induced by carrageenan and peritonitis induced by the venom of the T. serrulatus (VTs), and in the local oedema model and inflammatory infiltrate induced by the venom of the B. jararaca, administrated intravenously. The results of the assays of cytotoxicity, using the MTT, showed that the aqueous extracts from the plant species presented low toxicity to the cells that came from the fibroblast of the mouse embryo (3T3).The chemical analysis of the extracts by High Performance Liquid Chromatography revealed the presence of the rutin flavonoid, in A. pyrifoliu, and rutin, clorogenic acid and caffeic acid, in I. asarifolia. Concerning the pharmacological evaluation, the results showed that the pre-treatment using aqueous extracts and fractions reduced the total leukocyte migration to the abdominal cavity in the peritonitis model caused by the carrageenan and in the peritonitis model induced by the T. serulatus venom. Yet, these groups presented anti-oedematous activity, in the local oedema model caused by the venom of the B. jararaca, and reduced the inflammatory infiltrate to the muscle. The serum (anti-arachnid and anti-bothropic) specific to each venom acted inhibiting the inflammatory action of the venoms and were used as control. The compounds identified in the extracts were also tested and, similar to the plant extracts, showed meaningful anti-inflammatory effects, in the tested doses. Thus, these results are indicating the potential anti-inflammatory activity of the plants studied. This is the first research that evaluated the possible biological effects of the A. pyrifolium and I. asarifolia, showing the biological potential that these species have.

Engineering Highly-functional, Self-regenerative Skeletal Muscle Tissues with Enhanced Vascularization and Survival in Vivo

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

Investigating the Role of PDE4D1/2 in Vascular Smooth Muscle Cell Desensitization

Vascular smooth muscle cell (VSMC) behaviour and phenotypic modulation is critical to vessel repair following damage, and the progression of various cardiovascular diseases. The second messenger cyclic adenosine monophosphosphate (cAMP) plays a key role in VSMC function under the synthetic/activated phenotype, which is typically associated with unhealthy cell behaviour. Consequently, cAMP signaling is often targeted in attempts to impact several pathological diseases, including atherosclerosis, restenosis, and pulmonary arterial hypertension (PAH). The cyclic nucleotide phosphodiesterases (PDEs) catalyze hydrolysis of cAMP to an inactive form, and therefore directly regulate cAMP signaling. The PDE4D family dominates in synthetic VSMCs, and there is considerable interest in determining how distinct PDE4D isoforms affect cell function. Specifically, we are interested in the potential link between short isoforms of PDE4D and VSMC desensitization to pharmacological agents that impact cardiovascular disease via cAMP signaling. This study extends on previous work that assessed the expression of PDE4D splice variants in rat aortic VSMCs following prolonged challenge with cAMP-elevating agents. It was determined that PDE4D1 and PDE4D2 were uniquely expressed in synthetic VSMCs incubated with these agents, and that this upregulation impacted PDE activity and cAMP accumulation in these cells. Here, we report that PDE4D1 and PDE4D2 are markedly upregulated in synthetic human aortic smooth muscle cells (HASMCs) following prolonged challenge with cAMP-elevating agents. Using a combination of RNAi-based and pharmacological approaches, we establish that this upregulation is reflected in levels of cAMP PDE activity, and restricted to the cytosolic sub-cellular compartment. Our results suggest a role for localized PDE4D1 and PDE4D2 activity in regulating cAMP-mediated desensitization in HASMCs, and highlight their therapeutic potential in treating various cardiovascular diseases.

Molecular docking and geographical information systems as tools to assess the potential impact of veterinary medicines on non-target organisms and the environment

Veterinary medicines (VMs) from agricultural industry can enter the environment in a number of ways. This includes direct exposure through aquaculture, accidental spillage and disposal, and indirect entry by leaching from manure or runoff after treatment. Many compounds used in animal treatments have ecotoxic properties that may have chronic or sometimes lethal effects when they come into contact with non-target organisms. VMs enter the environment in mixtures, potentially having additive effects. Traditional ecotoxicology tests are used to determine the lethal and sometimes reproductive effects on freshwater and terrestrial organisms. However, organisms used in ecotoxicology tests can be unrepresentative of the populations that are likely to be exposed to the compound in the environment. Most often the tests are on single compound toxicity but mixture effects may be significant and should be included in ecotoxicology testing. This work investigates the use, measured environmental concentrations (MECs) and potential impact of sea lice treatments on salmon farms in Scotland. Alternative methods for ecotoxicology testing including mixture toxicity, and the use of in silico techniques to predict the chronic impact of VMs on different species of aquatic organisms were also investigated. The Scottish Environmental Protection Agency (SEPA) provided information on the use of five sea lice treatments from 2008-2011 on Scottish salmon farms. This information was combined with the recently available data on sediment MECs for the years 2009-2012 provided by SEPA using ArcGIS 10.1. In depth analysis of this data showed that from a total of 55 sites, 30 sites had a MEC higher than the maximum allowable concentration (MAC) as set out by SEPA for emamectin benzoate and 7 sites had a higher MEC than MAC for teflubenzuron. A number of sites that were up to 16 km away from the nearest salmon farm reported as using either emamectin benzoate or teflubenzuron measured positive for the two treatments. There was no relationship between current direction and the distribution of the sea lice treatments, nor was there any evidence for alternative sources of the compounds e.g. land treatments. The sites that had MECs higher than the MAC could pose a risk to non-target organisms and disrupt the species dynamics of the area. There was evidence that some marine protected sites might be at risk of exposure to these compounds. To complement this work, effects on acute mixture toxicity of the 5 sea lice treatments, plus one major metabolite 3-phenoxybenzoic acid (3PBA), were measured using an assay using the bioluminescent bacteria Aliivibrio fischeri. When exposed to the 5 sea lice treatments and 3PBA A. fischeri showed a response to 3PBA, emamectin benzoate and azamethiphos as well as combinations of the three. In order to establish any additive effect of the sea lice treatments, the efficacy of two mixture prediction equations, concentration addition (CA) and independent action ii(IA) were tested using the results from single compound dose response curves. In this instance IA was the more effective prediction method with a linear regression confidence interval of 82.6% compared with 22.6% of CA. In silico molecular docking was carried out to predict the chronic effects of 15 VMs (including the five used as sea lice control). Molecular docking has been proposed as an alternative screening method for the chronic effects of large animal treatments on non-target organisms. Oestrogen receptor alpha (ERα) of 7 non-target bony fish and the African clawed frog Xenopus laevis were modelled using SwissModel. These models were then ‘docked’ to oestradiol, the synthetic oestrogen ethinylestradiol, two known xenoestrogens dichlorodiphenyltrichloroethane (DDT) and bisphenol A (BPA), the antioestrogen breast cancer treatment tamoxifen and 15 VMs using Auto Dock 4. Based on the results of this work, four VMs were identified as being possible xenoestrogens or anti-oestrogens; these were cypermethrin, deltamethrin, fenbendazole and teflubenzuron. Further investigation, using in vitro assays, into these four VMs has been suggested as future work. A modified recombinant yeast oestrogen screen (YES) was attempted using the cDNA of the ERα of the zebrafish Danio rerio and the rainbow trout Oncorhynchus mykiss. Due to time and difficulties in cloning protocols this work was unable to be completed. Use of such in vitro assays would allow for further investigation of the highlighted VMs into their oestrogenic potential. In conclusion, VMs used as sea lice treatments, such as teflubenzuron and emamectin benzoate may be more persistent and have a wider range in the environment than previously thought. Mixtures of sea lice treatments have been found to persist together in the environment, and effects of these mixtures on the bacteria A. fischeri can be predicted using the IA equation. Finally, molecular docking may be a suitable tool to predict chronic endocrine disrupting effects and identify varying degrees of impact on the ERα of nine species of aquatic organisms.