Organophosphorus (OP) compounds are found in household pest control products, plastics, and petroleum. Due to the neurotoxic nature of OP compounds, exposure can cause both acute and delayed symptoms, including organophosphate-induced delayed neuropathy (OPIDN). This syndrome is characterized by Wallerian-like degeneration of nerves in the central and peripheral nervous system after exposure to neuropathic OP compounds. There are many questions surrounding the mechanisms of the onset of OPIDN, including possible alterations in proteins associated with neuronal maintenance and repair. This dissertation investigated the changes in levels of neurotrophins in vivo and how in vitro levels of neurotrophin receptors and their downstream signaling cascades are affected after exposure to OP compounds. We also characterized the molecular weight of a soluble factor responsible for inducing neurite outgrowth in vitro after in vivo exposure to a neuropathic OP compound. We evaluated in vivo endpoints using enzyme-linked immunosorbant assays. Results indicated that nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) are found in chicken spinal cord but do not increase as a result of exposure to neuropathic OP compounds. This study also noted that NGF, BDNF, and NT-3 concentrations were not altered after exposure to a non-neuropathic OP compound. We evaluated in vitro endpoints using Western blots, ultrafiltration, and digital morphometry. These studies revealed that activated forms of high-affinity and low-affinity neurotrophin receptors are present after OP compound exposure, that the ratio of these two receptors to each other is stable after OP compound exposure, and that the activated form of the low-affinity receptor, which can lead to apoptosis, was present in greater levels than the activated form of the high-affinity receptor. Furthermore, OP compound exposure resulted in time-dependent changes of protein levels central to the mitogen-activated kinase and phosopholipase C-gamma intracellular pathways. Changes in a third pathway, the protein kinase C pathway, were dependent on the concentration and type of OP compound. Finally, in vitro neurite length was not affected by the type of OP compound administered in vivo or when a whole protein fraction was separated by molecular weight. This research has revealed in vivo consequences and early effects on intracellular protein and activated neurotrophin receptor levels after OP compound exposure. These early effects may contribute to the delayed development of neurotoxic effects associated with OP compound exposure. / Ph. D.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A doenÃa de Parkinson (DP) à a segunda doenÃa neurodegenerativa mais comum afetando cerca de 1% da populaÃÃo mundial. Fatores ambientais e genÃticos poderÃo interagir e contribuir para o desenvolvimento da doenÃa. A 6-hidroxidopamina (6-OHDA) à uma neurotoxina que age em neurÃnios catecolaminÃrgicos, atravÃs da formaÃÃo de espÃcies reativas do oxigÃnio e inibiÃÃo do complexo I da cadeia transportadora de elÃtrons. A berberina à um alcalÃide isoquinolÃnico natural, com atividade antioxidante e aÃÃo na membrana mitocondrial. O presente estudo teve como objetivo investigar a atividade citoprotetora da berberina em modelo de degeneraÃÃo celular induzida pela 6-OHDA em cultura de cÃlulas SH-SY5Y. A berberina (10, 25 e 50Âg/mL) foi adicionada as cÃlulas 15 minutos antes da 6-OHDA 50ÂM, apÃs 24 horas foram feitos os testes para avaliaÃÃo da viabilidade celular (MTT e iodeto de propÃdeo- IP), estresse oxidativo (nitrito, TBARS â quantificaÃÃo de malondialdeÃdo), morfologia/apoptose (hematoxilina/eosina, brometo de etÃdeo/laranja de acridina) e potencial transmembrÃnico mitocondrial (rodamina 123). A 6-OHDA reduziu significativamente a viabilidade celular (Controle: MTT= 99,62%, IP= 98,63%; 6-OHDA: MTT=49,79%, IP= 48,80%), aumentou os nÃveis de nitrito (71,8%) e de malondialdeÃdo (27%). Foi observado fragmentaÃÃo e reduÃÃo do volume celular, perda dos neuritos, grande percentagem de cÃlulas apoptÃticas e necrÃticas (Controle: viÃveis= 95,5%, apoptÃticas= 2,67%, necrÃticas= 1,83%; 6-OHDA:viÃveis= 23,75%, apoptÃticas= 61,92, necrÃticas= 14,34%) e elevou em 56% o nÃmero de cÃlulas que apresentam despolarizaÃÃo mitocondrial. A berberina protegeu significativamente (p<0,05) as cÃlulas dos danos induzidos pela 6-OHDA, elevando a viabilidade celular para (MTT: BERB 25 + 6-OHDA= 68,4; BERB 50 + 6-OHDA= 79%. IP: BERB 25 + 6-OHDA= 61; BERB 50 + 6-OHDA= 58%), reduziu os nÃveis de nitrito (BERB 25+ 6-OHDA= 6,16; BERB 50 + 6-OHDA= 6,20 ÂM) e malondialdeÃdo (BERB 25+ 6-OHDA= 8,53; BERB 50 + 6-OHDA= 6,8 ÂM) AlÃm disso, a berberina reduziu as alteraÃÃes na morfologia celular, na morte por apoptose (BERB 25 + 6-OHDA=apoptose-26,51%; BERB 50 + 6-OHDA= apoptose-30,32%) e o nÃmero de cÃlulas com despolarizaÃÃo mitocondrial (BERB 25+ 6-OHDA= 7,58; BERB 50 + 6-OHDA= 12,9%). Esses resultados mostram a citoproteÃÃo pela berberina, por seu efeito antiapoptÃtico, que pode estar relacionado a uma proteÃÃo mitocondrial e uma aÃÃo antioxidante. Deste modo, podemos sugerir que a berberina pode ser explorada como possÃvel agente neuroprotetor para doenÃa de Parkinson. / Parkinsonâs disease is the second more common neurodegenerative sickness and it affects about 1% of the world population. Environment and genetics factors may interact and contribute to the diseaseâs development. The 6-hydroxydopamine (6-OHDA) is a neurotoxin that acts on catecholaminergic neurons throughthe formation of reactive oxygen species and inhibition of the electron transport chainâs complex I. The berberine is a natural isoquinolinium alkaloid with antioxidant activity and action in the mitochondrial membrane. The present study aimed to investigate the cytoprotective activity of berberine in cell degeneration model induced by 6-OHDA in cultured SH-SY5Y cell. Berberine (10, 25 and 50 Âg/mL) was added to the cells 15 minutes before 6-OHDA 50ÂM and, after 24 hours, the tests were made for evaluation of cellular viability (MTT and propidium iodide-IP), oxidative stress (nitrite, TBARS), morphology/apoptosis (hematoxilin/eosin, ethidium bromide/acridine orange) and mitochondrial transmembrane potencial (rhodamine 123). 6-OHDA reduced significantly the cellular viability (Control: MTT=99,62%, IP=98,63%; 6-OHDA: MTT=49,79%, IP= 48,80%), increased the nitrite (71,8%) and the malondialdehyde levels (27%). It was observed fragmentation and reduction of cell volume, loss of neuritis, large percentage of apoptotic and necrotic cells (Control: viable=23,75%, apoptotic=61,92%, necrotic=1,83%; 6-OHDA: viable= 23,75%, apoptotic= 61,92%, necrotic= 14,34%) and of cells with mitochondrial depolarization 56%. Berberine significantly protected (p<0,05) cells from damage induced by 6-OHDA, increasing cell viability (MTT: BERB 25 + 6-OHDA= 68,10  4,49; BERB 50 + 6-OHDA= 78,81 2,31%. IP: BERB 25 + 6-OHDA= 60,38  0,92; BERB 50 + 6-OHDA= 57,45  1,33%), reduced nitrite (BERB 25 + 6-OHDA= 6,16  0,42; BERB 50 + 6-OHDA= 6,20  0,40ÂM) and malondialdehyde levels (BERB 25+ 6-OHDA= 8,53; BERB 50 + 6-OHDA= 6,8 ÂM). Furthermore, berberine reduced morphology cell alterations, apoptotic death (BERB 25 + 6-OHDA= apoptosis-26,51%; BERB 50 + 6-OHDA= apoptosis-30,32%) and number of cells with mitochondrial depolarization (BERB 25+ 6-OHDA= 7,58; BERB 50 + 6-OHDA= 12,9%). These results show that the cytoprotection of berberine, possibly by its antiapoptotic effects, may be related to a mitochondrial protection and/or an antioxidant action. Thus, we suggest that berberine may be prospected as a possible neuroprotective agent for Parkinsonâs disease.

Certain organophosphorus compounds have the ability to induce a delayed neuropathic condition in sensitive species termed organophosphorus compound-induced delayed neurotoxicity (OPIDN). Esteratic changes associated with OPIDN have been successfully modeled in vitro. The physical characteristics of lesion development in OPIDN including the mode of nerve cell death, cytotoxic initiator and effector molecules, and cytoskeletal involvement have received little in vitro investigation. This dissertation evaluated the mode of cell death (apoptosis versus oncotic-necrosis), and cell cycle, cytoskeletal, nuclear, and mitochondrial alterations induced by OP compounds in SH-SY5Y cultures, an in vitro human neuroblastoma model. The distribution of in vivo neural degeneration in white Leghorn hen models was also assessed as a prelude to validating the mode of OP compound-induced in vivo neural cell death. These endpoints were evaluated by utilizing flow cytometry, spectrophotometry, gel electrophoresis, immunohistochemistry, light, and electron microscopy. Initial data gathered on culture parameters revealed that the mitotic status, basal rates of cell death, and total culture density were dependent on the condition of the media and the initial seeding density. Subsequent in vitro investigations used standardized culture conditions with OP compounds (diisopropylphosphorofluoridate (DFP), paraoxon, parathion, phenyl saligenin phosphate (PSP), tri-ortho-tolyl phosphate (TOTP), and triphenyl phosphite (TPPi); 1uM - 1mM). These studies revealed that OP compounds altered the cell cycle phase, decreased the amount of intracellular f-actin, altered the mitochondrial membrane potential, and induced caspase-3 activation and nuclear partitioning characteristic of apoptosis. The amount of change in these parameters was strongly dependent on the OP compound, the length of incubation time, and the presence of modulators of cytotoxicity such as phenylmethylsulfonyl fluoride (PMSF), carbachol, Ac-DEVD-CHO, Ac-IETD-CHO, and cyclosporin A. Preliminary in vivo experiments designed to validate in vitro results revealed neural degeneration involving fibers, terminals, and cell soma in spinal cord and brain tissue of PSP- and TPPi- exposed hens. The distribution and magnitude of these changes were contingent on the OP compound and length of time post-exposure. Subsequent experiments designed to evaluate the mode of cell death in these tissues revealed little evidence of either necrosis or apoptosis. These results, therefore, did not support or refute in vitro observations. Many OP compound-induced subcellular alterations have been demonstrated in our in vitro SH-SY5Y neuroblastoma model. Even though the mode of cell death observed in SH-SY5Y cells was not validated in in vivo experiments, in vitro observations nonetheless provide stimulating areas to further research the mechanisms of OPIDN and OP compound-induced cell death. / Ph. D.

Le corps humain est composé de plusieurs milliards de cellules neuronales, lesquelles ont un impact primordial sur les systèmes vitaux. En effet, le système nerveux, étant lui-même un système vital du corps humain, effectue de nombreuses fonctions afin de voir au bon fonctionnement des autres systèmes du corps, tels que le système cardiovasculaire, le système digestif, le système musculaire et bien d’autres. Plusieurs maladies neurodégénératives telles que l’Alzheimer et la maladie de Creutzfeldt-Jakob, affectent les cellules nerveuses du corps et occasionnent la perte de différentes fonctions causant une diminution du bien-être et pouvant mener à la mort. La hausse du nombre d’individus atteint de maladies neurodégénératives observée au fils des ans nous montre l’importance de comprendre les phénomènes moléculaires qui se produisent lors des mécanismes de dégénérescence axonale dans les neurones. Lors de ma maîtrise dans le laboratoire du professeur Richard Blouin, j’ai étudié l’impact d’une augmentation intracellulaire de calcium sur la protéine dual leucine zipper kinase (DLK) en utilisant un modèle cellulaire humain, les SH-SY5Y. J’ai pu démontrer que l’entrée de calcium dans la cellule avait un impact sur la quantité de protéine DLK présente dans celles-ci. En effet, j’ai pu observer une diminution de la quantité de DLK dans les cellules suite à une entrée de calcium, un phénomène exclusivement calcium-dépendant. À l’aide d’inhibiteurs pharmacologiques, j’ai pu montrer l’implication, des calpaïnes, des protéases calcium-dépendantes reconnues pour leur rôle dans la dégénérescence axonale. Les essais in vitro montrent que DLK est une cible spécifique des calpaïnes.

Les nanoparticules d'oxyde de fer sont utilisées dans plusieurs domaines notamment en Biomédecine comme agents théranostiques en Cancérologie et aussi comme agents de contraste en Imagerie par Résonance Magnétique. Avec l'augmentation de la production et de l'utilisation de ces NPs de fer, il y a une évidente augmentation de l'exposition humaine et environnementale à ces NPs, et qui peut présenter un risque. Le sujet de ma thèse porte sur l'étude de l’impact physiopathologique et de la toxicité des nanoparticules de fer (NPs de fer) en utilisant un modèle cellulaire de neuroblastome et un modèle d'étude animal, le rat Wistar. L’objectif des travaux de recherche de la partie in vitro est d’évaluer les effets cytotoxiques et génotoxiques ainsi que les effets sur l’expression des protéines cellulaires suite à l’exposition des cellules SH-SY5Y à des concentrations croissantes des NPs de fer. Nous avons montré que les NPs de fer induisent des perturbations cellulaires d’une manière dépendante de la taille et de la concentration. L'analyse protéomique suivie par l'annotation des gènes avec l’ontologie et l'analyse des voies de signalisation a mis en évidence les effets des NPs de fer sur le cytosquelette, l'apoptose et le développement du cancer. Les objectifs de nos travaux de recherche réalisés in vivo consistent à étudier les effets physiopathologiques des NPs de fer administrées par trois voies différentes, intraveineuse, intranasale et orale, à savoir leur impact sur le comportement émotionnel et cognitif ainsi que sur l’homéostasie des neurotransmetteurs et des éléments traces. Les résultats ont montré que les NPs de fer n’induisent pas de modification concernant l’anxiété, la locomotion, l’apprentissage et la mémoire chez le rat quelle que soit la voie d’administration. Cependant, ces NPs provoquent une perturbation des niveaux des catécholamines et des éléments traces au niveau cérébral. Les effets les plus marqués ont été observés suite à l’instillation intranasale des NPs et se manifestent par une diminution de taux du fer sérique, la thrombocytose et la présence des foyers inflammatoires au niveau hépatique. L’analyse comparative des trois voies d’administration a montré que la voie intraveineuse est la moins toxique. Enfin, l’étude protéomique des protéomes du cerveau, du foie et du poumon a permis d’évaluer la toxicité des NPs de fer au niveau protéique et moléculaire. Les résultats obtenus présentent un support important pour l’estimation et la compréhension des effets potentiellement adverses de ces NPs qui présentent une certaine toxicité non négligeable de point de vue moléculaire et physiopathologique. Ainsi, leur utilisation dans le domaine biomédicale doit être prise avec beaucoup de précaution pour éviter au maximum tout risque lié à leur exposition afin d’améliorer leur biocompatibilité et ainsi augmenter leurs avantages. / Iron Oxide Nanoparticles (IONPs) are used in several fields notably in Biomedicine as theranostic agents in Oncology and also as contrast agents in Magnetic Resonance Imaging. With the increase in the production and use of IONPs, there is a clear increase in human and environmental exposure to these NPs, which may pose a risk. The subject of my thesis is the study of the physiopathological impact and toxicity of iron oxide nanoparticles using a cell model of neuroblastoma and an animal study model, the Wistar rat. The aim of the research work of the in vitro part is to evaluate the cytotoxic and genotoxic effects as well as the effects on the expression of cellular proteins following the exposure of SH-SY5Y cells to increasing concentrations of iron NPs. We have shown that iron NPs induce cellular perturbations in a size and concentration dependent manner. Proteomic analysis followed by ontological gene annotation and signaling pathway analysis revealed the effects of IONPs on cytoskeleton, apoptosis and cancer development. The aims of our research carried out in vivo are to investigate the pathophysiological effects of iron NPs administered by three different routes, intravenous, intranasal and oral, also their impact on emotional and cognitive behavior as well as neurotransmitter and trace element homeostasis. The results showed that IONPs do not induce any changes in anxiety, locomotion, learning and memory in rats regardless of the administration route. However, these NPs cause a disruption of catecholamine and trace element levels in the brain. The most marked effects have been observed following intranasal instillation of NPs and are manifested by a decrease in serum iron levels, thrombocytosis and the presence of inflammatory foci in the liver. The comparative analysis of the three routes of administration showed that the intravenous route is the least toxic. Finally, the proteomic study of the proteomes of the brain, liver and lung has made it possible to evaluate the toxicity of the NPs of iron at the protein and molecular level. The obtained results provide an important support for the estimation and understanding of potentially adverse effects of these NPs, which have a certain toxicity that is not negligible from a molecular and physiopathological point of view. Thus, great care must be taken regarding their use in the biomedical field to minimize any risk related to IONP exposure in order to improve their biocompatibility and thus increase their benefits.

Les antipsychotiques sont des antagonistes du récepteur dopaminergique de type 2 (DRD2) et constituent le principal traitement pharmacologique de la schizophrénie. Le traitement à long-terme par les antipsychotiques peut causer la tolérance au traitement et le développement de la dyskinésie tardive, dont le mécanisme est mal compris et le traitement, insatisfaisant. Il a été démontré que le traitement chronique aux antipsychotiques induit la surexpression du récepteur DRD2 et le stress oxydatif, deux mécanismes associés à la dyskinésie tardive. Le lien entre ces deux phénomènes est mal déterminé. Précédemment, il a été rapporté, dans mon laboratoire d'accueil, que le stress oxydatif, induit par le peroxyde d'hydrogène (H[indice inférieur 2]O[indice inférieur 2]), augmente le niveau du récepteur DRD2 sur la lignée cellulaire de neuroblastomes humains SH-SY5Y. L'hypothèse de recherche présentée ici est que le stress oxydatif est impliqué dans la surexpression du récepteur DRD2 induite par les antipsychotiques et que l'administration d'un antioxydant peut atténuer cette surexpression. Le projet présenté rapporte ainsi les effets des antipsychotiques sur les niveaux d'expression du récepteur DRD2 et les effets de l'inhibition du stress oxydatif par le traitement de l'acide lipoïque, un antioxydant, sur la lignée cellulaire de neuroblastomes humains SH-SY5Y. L'halopéridol, un antipsychotique de première génération, induit une augmentation des niveaux d'expression du récepteur DRD2, alors que Pamisulpride, un antipsychotique de deuxième génération, n'a pas d'effet significatif. De plus, l'halopéridol induit une plus importante augmentation des biomarqueurs du stress oxydatif (carbonylation des protéines, peroxydation lipidique et production de l'anion superoxyde) que l'amisulpride. L'acide lipoïque atténue la surexpression du récepteur DRD2 et le stress oxydatif induit par l'antipsychotique. L'inhibition de la synthèse de catécholamine par l'alpha-méthyl-DL-tyrosine (AMPT) élève l'expression du DRD2 et prévient sa surexpression par les antipsychotiques. Les résultats suggèrent que la surexpression du récepteur DRD2 induite par l'halopéridol est liée au stress oxydatif et proposent des mécanismes potentiels par lesquels l'acide lipoïque peut être considéré comme un agent thérapeutique pour la prévention et le traitement des effets secondaires reliés à l'utilisation des antipsychotiques.

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>

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.

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.