Pesticides including organophosphates (OPs) and carbamates (CBs) are widelyused to control undesirable pests. These compounds are neurotoxic and inhibithydrolysis of the neurotransmitter acetylcholine by acetylcholinesterase. Public healthconcerns have increased with the escalating usage of pesticides. Reliable monitoringprograms are required to detect and quantify pesticide exposure, as well as to promotean understanding of their neurotoxic properties. In this dissertation, both theanticholinergic (Part I) toxicity and neurotoxicity in neuroblastoma cells (Part II) ofpesticides were explored using mass spectrometry (MS). The high sensitivity andhigh-throughput of this technique renders it well-suited for proteomics analysis.Part I describes the study of butyrylcholinesterase (BChE) inhibition resultingfrom OP and CB exposure. The main hypothesis of Part I is that the specialmodification of BChE can provide the origin and extent of pesticide exposure. A novelmethod for detection and quantification of pesticide exposure was designed using aproteomics approach and equine BChE (eBChE) as a model system. The methodologyfeatured detection and analysis of phosphorylated or carbamylated peptides at theactive site serine residue. The developed technique was successfully applied towardsthe study of human BChE (hBChE) inhibition in vitro and in serum samples. Aspecially designed affinity column enabled an isolation of BChE from serum. EnrichedBChE was subjected to enzymatic digestion by a novel on-bead double digestionprotocol. LC/MS/MS was employed to produce a calibration system for the analysis ofhBChE inhibition, which was then applied towards quantification of the enzyme.Part II describes a proteomic study of the neurotoxicity in neuroblastoma cellscaused from chlorpyrifos (CPF), an organophosphate pesticide. The concerns of CPFexposure to pregnant women, infants and children are increasing due todevelopmentally neurotoxic effects of this chemical. The main hypothesis of Part II isthat CPF can cause protein alterations and these altered proteins can be detected usingproteomics. Systematic studies at subcellular levels evaluated proteome changes inSH-SY5Y cells exposed to CPF. Two-dimensional gel electrophoresis (2DE) wasapplied with MALDI-TOF-MS to analyze differential protein expression. Thirty sevencommon unique altered proteins were identified, which play important roles inmetabolic pathway.

<p>Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1). I have also investigated the possible involvement of UCP3 in IGF-1-mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an <i>in vitro</i> cell model. The major findings were as follows:</p><p>i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4. </p><p>ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF-1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways. </p><p>iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1-mediated neuroprotection.</p><p>iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3 </p><p>v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection.</p>

Studies of the phenotypes of animals at different parts of their geographic range often reveal striking variability. It is of considerable fundamental and applied interest to discover the extent to which such variation depends on genetic as opposed to environmental differences. A first step towards disentangling these effects is to use an empirical approach known as the common environment method in which wild-caught juveniles from different regions are reared under common laboratory conditions. I used this approach to determine the population and thermal responses of Atlantic cod, a species with a wide distribution and geographic variation in life history traits. Life history traits were investigated in cod from three areas around the British Isles of differing thermal regimes, namely St Andrews Bay on the Scottish east coast, the Clyde Sea on the Scottish west coast, and from near Lowestoft in the southern North Sea. Concurrently haemoglobin genotype and behaviour were also studied. Spatially significant differences in life history traits and thermal responses were revealed in juvenile and adult growth rate, gonadal investment and behaviour, suggestive of population differentiation. Behavioural differences between cod of differing haemoglobin genotypes were also demonstrated. Results suggested that juvenile growth rates may be modified by competitive interactions. At a group level, growth rate of cod from the Clyde Sea was suppressed in the presence of cod from St Andrews Bay. Pairwise trials demonstrated that cod from the Clyde Sea consumed a higher prey share than those from St Andrews Bay but that those from St Andrews Bay were more aggressive and thus could potentially restrict feeding of cod from the Clyde Sea, resulting in a reduced growth rate. There were no population differences in the distribution of haemoglobin genotype, but haemoglobin genotype did have a strong influence on behaviour in pairwise contests. Cod of the HbI-2*2 genotype displayed a higher level of aggression than other genotypes and this effect was stronger than the population difference. Juvenile cod from the Clyde Sea exhibited a growth rate 24 % higher than those from St Andrews Bay. Cod from the Clyde Sea and from Lowestoft expressed higher growth rates as adults than those from St Andrews Bay. Body size and thus growth appeared to be the main driver of fecundity in the females and body size and liver were the main influences on gonadosomatic index (GSI) in the males. Females from the Clyde Sea invested more into fecundity than those from St Andrews Bay and males from St Andrews Bay had a higher testis investment than those from the Clyde Sea and Lowestoft. Temperature had a large influence on both the juvenile growth and egg development. Growth rate increased linearly and in parallel over the experimental temperatures, within their normal range. Egg development was strongly affected by temperature, resulting in a decrease in hatch time and an increase in embryonic cardiac rate, and a smaller larval size at hatch for a given temperature at higher temperatures. Temperature did not directly influence fecundity or GSI in males but warmer temperatures resulted in higher growth rates and thus a larger body size, which in turn resulted in a greater fecundity or GSI. These differences in life history traits, demonstrated under controlled environment conditions, raises the possibility that there may be a genetic basis to the variation and that cod may be locally adapted to their thermal environments in areas around the British Isles. However, effects of environmental differences prior to capture, including maternal effects, cannot be ruled out. This greater understanding of life history variation in cod will be important in the conservation of phenotypic diversity, vital for the long-term persistence of the species, while the findings of plasticity in response to temperature will enhance predictions of responses to sea temperature rise.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Marine Protected Areas (MPAs) have been suggested as one of the solutions to coral reef fisheries management. It is thought that their effects on biomass, species diversity and habitat could improve surrounding fisheries yields through fish migration and increased recruitment. However, MPAs' benefits on surrounding fisheries are difficult to establish due to the lack of historical data. Furthermore, the lack of involvement of stakeholders have led to a number of failures. MPAs in the form of No Take Zones (NTZ), which aim to promote the sustainable use of fisheries resources, might contribute to the successful management of coral reef fisheries. However, few studies have considered their benefits from the point of view of surrounding fishing communities. It is evident that if stakeholders are to be further involved in MPA management, they need to perceive the benefits. The hypothesis tested was that if NTZs are of benefit to surrounding communities, their food security situation would be improved. The fieldwork was carried out in Southern Kenya with five fishing communities located around a long established MPA. The study showed that a range of food security indicators gave good information on MPAs' benefits to the surrounding communities. It was found that these benefits were highly affected by distances. Thus, although fishing households were the least food secure, they were better off if they fished nearer the protected reefs. Households dependent on MPA-related tourism were the most food secure but this dependency decreased with the communities' distance from the main tour operators. The results showed that MPAs' benefits were not equally shared by the communities bearing most of the costs. In addition, it was also found that tourism seasonality does not always compensate for the seasonality of other activities. Furthermore, tourism could not be assumed to develop around MPAs and provide reliable alternative employment.

This thesis describes the use of in vivo and in vitro models to better understand the molecular correlates of sleep and sleep deprivation. Unlike previous studies, we utilise a timecourse based experimental design throughout, which has the advantage of identifying how the abundance of molecules return to baseline following sleep deprivation. Chapter 3 outlines the transcriptome of mouse cortex collected over 54 hours from mice subjected to varied durations of sleep deprivation. The timecourse experimental design aids in the identification of genes that are induced during both spontaneous and enforced wakefulness, and facilitates the dissociation of genes whose expression is tightly linked to the current wake state of the animal from those whose expression is linked to the total amount of wakefulness recently experienced by the animal. Like previous studies, we identify several genes involved in the unfolded protein response and synaptic function that are upregulated by sleep deprivation. We also find that increasing durations of sleep deprivation progressively reduces the total number of rhythmically expressed genes in mouse cortex, with only a handful of transcripts identified as diurnal following 12 hour sleep deprivation. Chapter 4 outlines the proteomic and metabolomic effects of 12 hour sleep deprivation. Proteomic analyses indicate that the abundance of ribosomal and nucleosomal proteins is suppressed for at least 24 hours following sleep deprivation, whilst the abundance of several phosphodiesterases are acutely increased following sleep deprivation. Metabolomic analyses of sleep deprived mouse cortex identified 3 molecular species whose abundance profile implicate them as sleep homeostats. Finally, we also set out to develop an in vitro model of sleep deprivation based on the optogenetic activation of a neuroblastoma cell line, which is outlined in Chapter 5. Following several rounds of optimisation, the stable expression of an opsin was found to induce intracellular calcium spikes and immediate early gene expression during illumination. Transcriptomic profiling of illuminated SH-SY5Y cells induced large scale transcriptomic changes, and modulated the expression of genes involved in synapses, cholesterol synthesis, the molecular clock and the unfolded protein response. Although these functional classes are reminiscent of those modulated by in vivo sleep deprivation, there was only a slight enrichment of individual genes modulated by in vivo sleep deprivation amongst the blue light sensitive genes, indicating further work is required to more closely model in vivo sleep deprivation.

Background: For people with type 1 diabetes, there is no alternative to treatment with insulin. The major side effect of insulin is severe hypoglycaemia (SH), when the patient needs help or even becomes unconscious. Material: We have studied a geographic population of yearly 130-140 unselected type 1 diabetes patients aged 1-18 years during 1992-1999. They were intensively treated with 87-96% on 4-7 daily insulin doses, combined with active self-control, psychosocial support and problem-based education from onset. Average HbA1c was 6.5 with Mono-S standard (1.15% beow DCCT level). Methods: We evaluated use of a prospective patient questionnaire for continuous long-term registration of treatment and outcome data and analysed HbA1c, SH and other variables. Over years, 95-100% response rate was achieved. We used also temporary questionnaries. Results: We found SH with unconsciousness reported from on average 11% of patients yearly, SH without unconsciousness but needing assistance from on average 36% yearly and weak associations to HbA1c, such as reletive risk of SH 1.24 for yearly mean HbA1c &lt;7.0% compared to ≥7.0% There was a seasonal variation in HbA1c (p=0.023) and incidence of SH. The strongest predictor for SH was SH during the previous year (r=9.38, p&lt;0.0001). The impact from SH showed great variation, and 20-30% of events led to practical disturbancies for parents and/or other people. Hospital visits took place only at 5% and hospitalisations at 3% of events. Social activities for patients were cancelled after 10% of events. Increased worry for patients was reported after 8% of events, bad sleep after 7%. We estimated the average socio-economic cost for SH at EURO 239 per event of SH with unconsciousness, and EURO 63 per event of SH without unconsciousness but needing assistance. Mass-distributed self-study material (brochures and videos) aimed at the prevention of SH without compromising metabolic control reached high dissemination and was widely appreciated by patients. The material copy cost was only EURO 7 per patient. It also seems to have contributed to a decrease in SH with unconsciousness from yearly 13% of patients before to 9% after intervention (3-years average), but controlled studies are needed. Conclusions: We conclude that SH remains a very serious problem of multifactorial aethiology. It causes considerable discomfort and costs. Systematic patient education mgiht reduce the incidence. Interventions using mass-distribution of high quality self-study material such as videos and brochures seem to have a potential to be cost-effective. There is a great patient/consumer interest in high quality- and advanced information/education materials.

Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1). I have also investigated the possible involvement of UCP3 in IGF-1-mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an in vitro cell model. The major findings were as follows: i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4. ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF-1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways. iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1-mediated neuroprotection. iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3 v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection.

The thesis examines the possible causes of decline of Spartina anglica marshes along the south coast of Britain with emphasis on the Solent marshes. The study shows that although there may be some genotypic differences between S. anglica gathered from sites in Britain. The disparities are not large enough to explain the significant differences in morphological vigour of S. anglica observed in the field. It concludes that the discrepancies observed in the field are the result of phenotypic differences resulting from environmental factors. The work shows that in the S. anglica marshes of the south coast, anaerobic soil conditions prevail with impeded drainage being the most likely cause of the dwarf growth forms and lack of re-colonisation of pans and mudflats observed in the field. The thesis concludes that the ultimate demise of the S. anglica marshes of the south coast of England is the result of frontal and creek erosion of the mature marsh and the failure of S. anglica to establish itself on the newly exposed sediments of the foreshore. When S. anglica establishes itself in a flood /ebb neutral zone of an estuary, it changes the bathymetry to that of ebb dominant morphology. As a result eroded sediment is swept away with the outgoing tide. Furthermore, S. anglica is then unable to recolonise the exposed foreshore sediments because of its low redox potential resulting from poor permeability which is the consequence of the of historic overburden pressure of a once colonising marsh.

Poor salmonid spawning habitat due to excessive fine sediment inputs has been identified as a major factor limiting survival in chalk rivers. A lack of knowledge about the complex processes and factors affecting survival was the driver for this study and gaps in the research were identified concerning the sources of fine sediment and the impact organic material had on salmonid survival in chalk streams. Consequently the main objectives of this study were to characterise spawning habitat quality of a chalk catchment, assess the sources of sediments accumulating within artificial redds, describe the composition of organic sediments using emerging technology and to create a novel method to assess the sediment oxygen consumption of those sediments. Methods were based around a catchment wide field based monitoring programme, consisting of artificially constructed spawning gravels which allowed hyporheic measurements to be taken, and sediment analysis and sediment oxygen consumption methods were carried out using different laboratory methods. Spawning habitat characteristics of the chalk catchment were found to exhibit; low sediment accumulation rates although original levels of fine sediment were high, high organic matter content, variable intra-gravel flow and intra-gravel oxygen concentrations and groundwater influences. Primary sources of fine sediment accumulating in spawning gravels and suspended sediments were found to be attributed to catchment surface sources, namely pasture (50-68%) and arable (32-50%) using inorganic and organic parameters. Organic composition of redd gravels was found to be dominated by protein material rather than humic substances, the more commonly found fluorescent compound in freshwater systems and the sediment oxygen consumption of sediments varied throughout the catchment and was found to consume the greatest oxygen in <63μm size fraction. Application of sediment oxygen consumption rates to existing parameter based models that predict salmonid survival, highlighted the need to address the sensitivity of current models to rivers experiencing low sediment accumulation rates. Outcomes of this study further the knowledge of the sources, organic composition and sediment oxygen consumption capacity of fine sediments accumulating in spawning gravels which can lead to appropriate mitigation on chalk rivers to improve salmonid spawning habitat.