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Rey, Stéphanie. "Physiological involvement of presynaptic L-type voltage dependent calcium channels in GABA release of cerebellar molecular layer interneurons". Thesis, Paris 5, 2013. http://www.theses.fr/2013PA05T096/document.
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La libération de neurotransmetteur est provoquée par la dépolarisation de la terminaison présynaptique et l’entrée de calcium à travers les canaux calciques voltagedépendants (VDCCs). Les VDCCs à haut seuil de type-P/Q et de type-N sont classiquement impliqués dans la libération de neurotransmetteurs et sont localisés dans la terminaison axonale près de la zone active. Deux membres de la famille des VDCCs de type-L, Cav1.2 et Cav1.3 sont connus pour être exprimés dans le système nerveux central. Dans le cortex cérébelleux, les propriétés pharmacologiques des VDCCs présynaptiques ont été examinées aux synapses GABAergiques entre les interneurones de la couche moléculaire (MLIs) et entre les MLIs et les cellules de Purkinje. Bien qu’il n’y ait aucun doute que les VDCCs de type- P/Q et de type-N sont les principaux acteurs de l’entrée de calcium présynaptique et de la libération de GABA par les MLIs, l’absence d’effet des dihydropyrines antagonistes a exclut le potentiel rôle des VDCCs de type-L (Forti et al., 2000; Stephens et al., 2001). Il est intéressant de noter que les dihydropyrines antagonistes sont très peu efficaces sur les courants calciques de type-L activés par un potentiel d’action (Helton et al., 2005), ce qui suggère que l’implication des VDCCs de type-L dans la libération de neurotransmetteur a été largement négligée. Dans cette étude, nous avons montré que le BayK8644 (une dihydropyridine agoniste) augmente fortement la fréquence des mIPSCs enregistrés dans les MLIs et dans les cellules de Purkinje suggérant que les VDCCs de type-L peuvent être présents dans les terminaisons axonales des MLIs. Ce résultat a été confirmé par des expériences d’immunohistochimie utilisant la microscopie confocale et électronique ainsi que par des expériences d’imagerie calcique. Nos résultats démontrent que les VDCCs de type-L, souvent négligés dans les terminaisons axonales, ont un rôle crucial dans la libération de GABA par les MLIsPhysiological involvement of presynaptic L-type voltage dependent calcium channels in GABA release of cerebellar molecular layer interneurons
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Zhou, Lei. "Molecular mechanisms regulating dendritic spine morphology". Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=106328.
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In the central nervous system, chemical synapses are highly specialized junctions that are known to be critical for communication between neurons. The ability of synapses to change their physiological and structural properties, known as synaptic plasticity, is important for storing information in neural connections. Dendritic spines are small protrusions on dendrites where the majority of glutamatergic synapses form in the brain. In general, a dendritic spine has an enlarged head region that is connected to the dendritic shaft by a narrow neck. This geometry allows spines to function as individual biochemical compartments and control postsynaptic signaling events. Recent evidence indicates that structural remodeling of spines and the formation of new synaptic contacts may lead to long-term changes in synaptic function including long-term potentiation (LTP) and long-term depression (LTD). These forms of synaptic plasticity are believed to contribute to cognitive processes such as learning and memory. Interestingly, the actin cytoskeleton is enriched in dendritic spines and its turnover contributes to spine shape and motility. A variety of signaling proteins associate with the actin cytoskeleton and are likely critical for controlling the morphological plasticity of spines. However, the molecular mechanisms that regulate actin-based spine dynamics remain unclear. My studies revealed novel pathways downstream of the EphA class of receptor tyrosine kinases that are important for regulating spine plasticity. I showed that PLCγ1 physically interacts with the EphA4 receptor tyrosine kinase and links EphA4 to the downstream actin depolymerizing/severing protein, cofilin. PLCγ1 signaling is critical for maintaining spine morphology and PLC activity is required for spine retraction caused by ephrin ligand binding to EphA4. Remarkably, the amount of cofilin associated with the cell membrane is regulated by PLC and EphA4 activity. Furthermore, I found that ephrin binding to EphA receptors cause the dephosphorylation and activation of cofilin through the phosphatases slingshot (SSH) and calcineurin. Both of the phosphatases are needed for EphA-mediated reorganization of actin filaments and dendritic spine remodeling. These studies contribute new insight into the intricate signaling mechanisms downstream of EphA receptors that control the local remodeling of the actin cytoskeleton in dendritic spines and structural plasticity of excitatory synapses in the central nervous system.Les synapses chimiques sont des jonctions hautement spécialisées du système nerveux central ayant un rôle déterminant pour la communication entre les neurones. Ces dernières sont capables de changer leurs propriétés structurales et physiologiques. Ce phénomène, appelé la plasticité synaptique, est important pour le stockage de l'information. Les épines dendritiques sont de petites saillies sur les dendrites des neurones où la majorité des synapses glutamatergiques du cerveau se forment. De manière générale, une épine dendritique se compose d'une large tête connectée à l'arbre dendritique par une structure plus étroite appelée cou. Cette géométrie permet le fonctionnement des épines comme des compartiments biochimiques indépendant, pouvant ainsi contrôler les événements de transmission synaptique localement, soit au niveau postsynaptique. De récentes évidences expérimentales indiquent que les réarrangements structurels des épines et la formation de nouvelles synapses qui en découle pourraient induire des changements persistant du fonctionnement de la synapse. Ces changements sont de deux types : la potentialisation à long terme (PLT) et la dépression à long terme (DLT). Ce sont deux formes de plasticité synaptique connues pour contribuer aux processus cérébraux de la cognition tels que l'apprentissage et la mémoire. Il est intéressant de noter que le cytosquelette d'actine est très dense dans les épines dendritiques et que son turnover contribue à la morphologie ainsi qu'à la motilité de ces dernières. Une grande variété de protéines de signalisation sont connues pour s'associer avec le cytosquelette d'actine et ont donc probablement un rôle crucial pour le contrôle de la plasticité morphologique des épines. Néanmoins, les mécanismes moléculaires qui régulent la dynamique des épines basée sur le cytosquelette d'actine restent obscurs à ce jour. La présente étude révèle de nouvelles voies de signalisation moléculaire en aval de la classe EphA des récepteurs à la tyrosine kinase ayant un rôle dans la régulation de la plasticité des épines dendritiques. Cette étude montre que la PLCγ1 interagit physiquement avec le récepteur EphA4 tyrosine kinase et relie, en aval, EphA4 à la cofiline, une protéine ayant un pouvoir polymérisant ou dépolymérisant sur les filaments d'actine. De plus, elle démontre que la PLCγ1 est essentielle pour le maintien de la morphologie des épines car la rétraction de celles-ci observée lors de la liaison de l'ephrine à son récepteur EphA4 nécessite une activité des PLC. La quantité de cofiline s'associant à la membrane cellulaire est apparue comme étant régulée de façon remarquable par l'activité de la PLC et du EphA4. Finalement, la démonstration que la liaison de l'ephrine à son récepteur EphA cause la déphosphorylation et l'activation de la cofiline par la phosphatase slingshot (SSH) et la calcineurine a été effectuée. Ces deux phosphatases sont apparues essentielles pour la réorganisation des filaments d'actines et des modifications morphologiques des épines dendritiques induites par EphA. Cette étude contribue donc à la compréhension des mécanismes de signalisation complexes prenant place en aval des récepteurs EphA lors des modifications structurales des épines observées lors des phénomènes de plasticité des synapses excitatrices du système nerveux central.
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Barnabé-Heider, Fanie. "Molecular mechanisms regulating cortical precursor differentiation". Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=100317.
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During development of the mammalian cerebral cortex, precursor cells in the ventricular zone sequentially produce neurons and glial cells. This process clearly involves a complex interplay between intrinsic cellular machinery and extrinsic cues. Remarkably, embryonic cortical precursor cells isolated at the onset of neurogenesis, and cultured under serum-free conditions, will mimic the temporal differentiation pattern observed in vivo, producing neurons first, and then glia. In the studies presented here, I have used this system to identify critical components of the intracellular machinery that co-ordinately control two essential steps in neuronal differentiation, exit from the cell cycle (via the retinoblastoma family) and induction of neuronal gene expression (via the basic helix-loop-helix transcription factors). I then demonstrate that endogenously produced neurotrophins are crucial autocrine/paracrine extracellular factors that regulate the precursor-to-neuron transition by promoting precursor survival (through the PI3-kinase-Akt signaling pathway) and neuronal differentiation (through the MEK-ERK pathway). Finally, I identify the nature of the "cortical timer mechanism" for sequential generation of neurons and glial cells: increasing levels of cytokines are produced by newly born neurons, which act to instruct the remaining precursors to produce glial cells. This neuron-based feedback mechanism was verified by using in utero electroporation to alter levels of cytokine signaling components within the developing cortex, and by analysing mice deficient for the cytokine cardiotrophin-1, which in both cases impaired the onset of gliogenesis. Taken together, our findings have led to a better molecular understanding of cortical precursor biology and cortex development, which could ultimately lead to the design of novel therapeutic strategies for neurodegenerative disorders.
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Nelson, Eric. "Leptin as a molecular modulator of neuroinflammation". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110759.
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The adipocyte derived hormone, leptin, represents an important link betweenmetabolic status and immune function and may be a contributor to the onset ofinflammatory illness. The current project aimed to understand the role of leptin duringboth chronic and acute inflammatory states, and two mouse models were utilized:Experimental autoimmune encephalomyelitis (EAE) and an LPS-induced depression-likesyndrome. Here we have demonstrated that circulating leptin levels peak at early stagesof EAE development, corresponding to elevated SOCS-3 expression in brain, suggestingthe importance of leptin during the pathogenesis of this disease. Furthermore, we showfor the first time, that the LPS-induced recruitment of neutrophils into the brain followsthe time-course of depression-like behaviour after acute sickness responses subside, andthat both neutrophil recruitment and depression-like behaviours are attenuated as a resultof leptin neutralization. Pro-IL-1β expressing, brain invading neutrophils were found tobe most populous at the dissociation point of sickness and depression-like behaviours (48hours after LPS treatment) and leptin neutralization attenuated this recruitment. Finallythe effects of leptin on depression-like behaviour were shown to occur independently ofmicroglial activity or through modulation of hippocampal cell proliferation or survival.La leptine, hormone synthétisée par les adipocytes, est un facteur important aussibien dans la régulation du métabolisme que dans la défense immunitaire. La leptinepourrait jouer un rôle important dans l'apparition des maladies inflammatoires. Notreprojet vise à comprendre le rôle de la leptine dans deux états inflammatoires, l'un étantun état inflammatoire chronique et l'autre une inflammation aigue. L'inflammationchronique a été étudiée en utilisant un modèle expérimental de souris auto-immune(EAE) et l'inflammation aigue a été induite par l'injection de LPS à des souris. Nousavons démontré que la leptine circulante atteint des niveaux maximum à des stadesprécoces du développement des souris EAE, correspondant à un niveau d'expressionélevée de SOCS-3 dans de cerveau, ce qui suggère de rôle important de la leptine aucours de l'apparition de l'inflammation. De plus, nous avons montré pour la premièrefois, que le recrutement des neutrophiles dans le cerveau induit par l'injection de LPS suitl'évolution temporelle des comportements dépressifs qui apparaissent après la disparitiondes premiers symptômes de la maladie. De plus nous avons montré que le recrutementdes neutrophiles et la symptoms de dépression sont atténués suite à la neutralisationleptine. Le Pro-IL-1 ß exprimé par les neutrophiles du cerveau est surtout présent aupoint de dissociation entre les symptômes de la maladie et la dépression (48 heures aprèsle traitement par LPS) et la neutralisation de la leptine atténue ce recrutement. Enfin, leseffets de la leptine sur la dépression sont indépendants de l'activité microgliale ou de lamodulation de la prolifération cellulaire des cellules issues de l'hippocampe ou de leursurvie.
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Shekarabi, Seyed Masoud. "Netrin-1 signaling : cellular consequences and molecular mechanisms". Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=19457.
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During the embryonic development of the nervous System, groups of multipotent cells proliferate, migrate, and differentiate to become neurons. The new born neurons need to make precise connections with their targets. These may be a great distance away from their soma. To make these connections, neurons send their axon through a biochemically complex environment and their growth cones may make many guidance decisions on the ways to their targets. The existence of axon guidance molecules was inferred by the early classical neuroanatomists. We now know of multiple examples of secreted or membrane-bound proteins that either attract or repel axonal growth cônes by causing the growth cône to collapse or extend. The molecular mechanisms that regulate growth cone collapse or extension are closely linked to reorganization of the cytoskeleton. The small Rho GTPases, Cdc42, Rac, and Rho play key roles in neuronal growth cônes by regulating the organization of the cytoskeleton. Netrins are a small family of secreted guidance eues that are implicated in the attraction and repulsion of axons during the development of the nervous System. The netrin-1 receptor deleted in colorectal cancer (DCC) is highly expressed by commissural neurons in the developing spinal cord. The findings described here show that DCC is present at the tips of filopodia and the edges of lamellipodia of HEK293T, NG108-15 neuroblastoma-glioma cells, and the growth cones of embryonic rat spinal commissural neurons. Furthermore, netrin-1 protein causes an increase in filopodia number and surface area of embryonic rat commissural neuron growth cônes and the cell fines when transfected to express DCC. Further experiments indicated that netrin-1 activates the small GTPases Cdc42 and Racl in the cell lines and the embryonic rat commissural spinal cord neurons. The activation of Cdc42 and Racl by netrin-1 requires DCC. Furthermore, netrin-1 causes increased phosphorylation of Pakl, an effector for both Cdc42 and Racl. Expression of a dominant negative form of N-WASP, an effector for Cdc42, blocks the netrin-1 induced morphological changes in the growth cones of commissural neurons. Evidence is presented that netrin-1 induces the formation of a complex of proteins interacting with the intra-cellular domain of DCC that includes Nckl, Cdc42, Racl, Pakl, and N-WASP. The findings described lead to a model whereby netrin-1 binding to DCC triggers the activation of Cdc42 and Racl, which leads to actin based membrane extension and changes in growth cone morphology.
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Mo, Gary. "Molecular physiology of sensory P2X3 ATP receptor channels". Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=107793.
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Purinergic transmission mediated by extracellular release of ATP has been shown to be involved in numerous physiological processes, ranging from bladder function to taste and hearing. The diverse roles of ATP mediated signaling is largely due to widespread distribution of P2 ATP receptors. Since their initial cloning, various P2 receptors have been found to mediate a wide range of cellular processes in different tissues. One P2 receptor in particular, the P2X3 receptor, is almost exclusively expressed on nociceptive dorsal root ganglion (DRG) sensory neurons. Due to its specific distribution, the P2X3 receptor has been a prominent target in pain research, especially in studies of chronic pain conditions. Numerous studies have indicated involvement of P2X3 receptor in mediating increased pain behavior associated with chronic inflammation or neuropathic injury. However, the exact contribution of the P2X3 in chronic pain conditions is still unclear, especially in the case of neuropathic pain. There is inconsistency in reports of changes in P2X3 expression or activity during neuropathic pain. The underlying element of enhanced pain behavior after nerve injury is increased excitability of sensory neurons. The first study of described in this thesis investigates the contribution of the P2X3 receptor to changes in excitability of injured neurons. Activation of protein kinase C (PKC) has been shown to facilitate chronic pain behavior by modulating the activity of various ion channels. Thus, the contribution of PKC to hyperexcitability in injured neurons was also investigated in the first study.Nerve injury induces very dynamic changes in cellular physiology including activation of various intracellular signaling pathways. The activity of the P2X3 receptor may be affected by such cellular processes. Understanding the molecular physiology of the P2X3 receptor may provide additional insight into the specific contribution of the P2X3 receptor in pain physiology. A common component of many cellular signaling pathways is the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) by phospholipase C (PLC), and changes in PIP2 levels modulate the activity of various ion channels, including the P2X2 receptor. The second study in this thesis investigates the modulation of P2X3 by changes in intracellular levels of PIP2. Recent studies have demonstrated possible co-expression of metabotropic P2Y receptors on P2X3 positive sensory neurons. ATP release on sensory neurons that express both P2Y and P2X3 receptors will likely trigger activation of both types of purinergic receptors. The third study in this thesis investigated the modulation of P2X3 receptor activity by metabotropic P2Y receptors. Our understanding of the P2X3 receptor predominantly comes from rodent studies. Because interspecies differences in the functional characteristics of certain P2X receptor subtypes have been reported, there is a pressing need to verify if our understanding of the rodent P2X3 receptor is translatable to species closer to man. To this end, the last study of this thesis compares the pharmacological properties of native P2X3 receptor on rat sensory neurons to native P2X3 receptors on monkey sensory neurons.Il a été montré que la transmission purinergique médiée par la libération extracellulaire d'ATP est impliquée dans de nombreux processus physiologiques, allant du fonctionnement de la vessie aux sens du goût et de l'audition. Les rôles variés de la signalisation par l'ATP sont expliqués principalement par la distribution étendue des récepteurs P2 de l'ATP dans l'organisme. Depuis leur clonage initial dans les années '90, une variété de récepteurs P2 régissant divers mécanismes cellulaires a été découverte dans plusieurs tissus. Un récepteur canal P2 en particulier, P2X3, se trouve exprimé quasi-exclusivement dans les neurones nocicepteurs des ganglions spinaux (DRG). De par cette distribution spécifique, le récepteur P2X3 est une cible importante dans la recherche sur la douleur, principalement dans les études sur la douleur chronique. De nombreuses études indiquent le rôle du récepteur P2X3 dans l'augmentation des réponses à la douleur associée à une inflammation chronique ou une lésion neuropathique. Cependant, la contribution exacte de P2X3 dans la douleur chronique reste incertaine, surtout dans les cas de douleur neuropathqiue. Il existe des contradictions dans les articles sur les changements d'expression ou d'activité de P2X3 en conditions de douleur neuropathique. Un élément clé dans l'exacerbation des comportements douloureux après lésion neuropathique est l'augmentation d'excitabilité des neurones sensoriels. La première étude décrite dans cette thèse explore la contribution de P2X3 dans les changements d'excitabilité des neurones de DRG endommagé. Il a été rapporté que l'activation de la protéine kinase C (PKC) facilite les comportements douloureux en modulant l'activité de certains canaux ioniques. Ainsi, la contribution de PKC à l'hyperexcitabilité des neurones de DRG neuropathique a aussi été étudiée dans ce premier chapitre. Une insulte à un nerf périphérique induit des changements très dynamiques dans sa physiologie cellulaire, incluant l'activation de voies de signalisation intracellulaires. La fonction de P2X3 peut se trouver affectée par ces mécanismes neuronaux. Comprendre la physiologie moléculaire du récepteur P2X3 peut nous éclairer sur sa contribution spécifique dans la douleur. Une étape commune à de nombreuses voies de signalisation cellulaire est le clivage du phosphatidylinositol 4,5-bisphosphate (PIP2) par la phospholipase C (PLC). Les variations de niveaux de PIP2 modulent l'activité de plusieurs canaux ioniques, y compris le récepteur P2X2. Le deuxième chapitre dans cette thèse se concentre sur la modulation de P2X3 par les niveaux intracellulaires de PIP2. Des études récentes ont démontré la co-expression potentielle de récepteurs métabotropiques P2Y et ionotropiques P2X3 sur les neurones sensoriels. L'ATP pouvant activer les deux types de récepteurs, le troisième chapitre se penche sur la modulation de la fonction de P2X3 par les récepteurs P2Y couplés à la phospholipase C.Notre compréhension du récepteur P2X3 provient principalement des données obtenues dans des modèles précliniques de rongeurs. Sachant que des différences interspécifiques marquées dans les propriétés fonctionnelles de certains sous-types de récepteurs P2X ont été documentées, il est urgent et important de vérifier que nos connaissances sur le récepteur P2X3 de rongeur sont transférables aux primates ou à l'homme. À cette fin, dans la dernière étude de cette thèse, nous comparons les propriétés pharmacologiques du récepteur P2X3 natif à la surface des neurones sensoriels de rat avec celles du récepteur P2X3 exprimé dans les neurones sensoriels de singe.
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Fulton, Daniel James. "The molecular basis of long term memory formation". Thesis, University of Sussex, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.288784.
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Strochlic, David E. "Molecular and Genetic Analysis of the Vagus Nerve". Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467391.
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The vagus nerve serves as a primary neural link between the brain and internal organs, detecting a variety of physiological stimuli and controlling a range of autonomic functions essential to homeostatic regulation. However, despite its fundamental importance, little is known about the repertoire of sensory mechanisms residing in vagal afferents, the cellular logic of information coding within the vagus nerve, and the central representation of internal physiological states.
To dissect the neural circuits underlying viscerosensation, we adopted a genome-guided strategy to classify vagal sensory neurons based on G-protein-coupled receptor (GPCR) expression. We identified 5 principal cell types and obtained genetic access to these neurons in vivo using GPCR-ires-cre mouse strains. Using a combination of approaches that support cell-type specific analysis, we investigated the anatomical projections, response profiles, and physiological function of discrete vagal sensory subtypes.
Within the respiratory system, we identified two vagal sensory populations that exert powerful and opposing effects on breathing. P2ry1- and Npy2r-expressing neurons innervate distinct anatomical structures in the lung and send projections to different brainstem targets. Npy2r neurons are largely slow-conducting C fibers while P2ry1 neurons are fast conducting A fibers. Optogenetic activation of Npy2r neurons induces rapid and shallow breathing whereas activating P2ry1 neurons acutely silences respiration, trapping animals in exhalation. Furthermore, activating P2ry1 neurons had no effect on heart rate or gastric pressure, other autonomic functions under vagal control. Thus, the vagus nerve contains intermingled sensory neurons constituting genetically definable labeled lines with different anatomical connections and physiological roles.Medical Sciences
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Alexopoulou, Zoi. "The study of the deubiquitinase USP8 in Parkinson's disease pathogenesis". Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:47c2941b-5232-4bd0-92fa-e59aac16af7c.
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Parkinson's disease is the second commonest neurodegenerative disease currently treated symptomatically. It is a multifactorial disease involving mechanisms ranging from protein aggregation to mitochondrial dysfunction, oxidative stress and dopamine dysregulation. The levels of α-synuclein have been causatively linked to the development and progression of Parkinson's disease. Therefore α-synuclein lowering strategies are valid approaches in Parkinson's disease. Neuropathologically, Lewy Bodies in the vulnerable substantia nigra of Parkinson's disease patients are less ubiquitinated and specifically less K-63 ubiquitinated than Lewy bodies in the cortex, suggesting differential activation or regulation of ubiquitin interactors. A targeted screen for such interactors revealed that the Deubiquitinating enzyme Usp8 is upregulated in the substantia nigra of Parkinson's disease brains and is inversely correlated with the degree of total and K-63 ubiquitination. Using genetic knockdown and overexpression techniques, Usp8 was found to colocalize and directly interact with α-synuclein. It was found to de-ubiquitinate α-synuclein and increase its half-life. Its knockdown increased the total and K-63 α-synuclein ubiquitination and decreased its levels by 35% at least partly by increasing its degradation via the lysosome. In vivo in the Drosophila melanogaster, Usp8 knockdown demonstrated protection against α-synuclein toxicity. It rescued in a specific manner the rough eye phenotype, the age-dependent locomotive defect and the loss of dopaminergic neurons caused by the expression of α-synuclein. Specific and effective pharmacological Usp8 inhibition also has the potential to lower α-synuclein levels. Collectively, the evidence produced in my thesis suggests that Usp8 could be a potential target for the future disease-modifying therapies in Parkinson's disease.
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Ou, Yimiao. "Molecular mechanisms controlling the arborization of dendrites in «Drosophila»". Thesis, McGill University, 2011. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=96940.
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The assembly and function of neural circuits depend on the patterned outgrowth, guidance and targeting of dendrites into appropriate territories during development. As a hallmark of any neuron, cell type-specific dendrite morphology has a crucial role in determining the sensory or synaptic input a neuron receives. Despite recent advances in exploring the molecular and cellular mechanisms that define dendritic architecture, our understanding of dendrite development is still far from complete. In this thesis research, I have taken a genetic screen approach and a candidate molecule approach to discover novel genes and mechanisms that are involved in dendrite morphogenesis, using Drosophila dendritic arborization (da) neurons as a model system. In three independent but related studies, my research led me to focus on 1) the nuclear receptor for the steroid hormone ecdysone (EcR), 2) the transcription factor Longitudinals Lacking (Lola) and 3) the cell surface recognition molecule Turtle (Tutl). I have found that these three different factors each regulate distinct aspects of dendritic arborization including dendrite branching, distribution and self-avoidance. Through their identification, expression patterns, and a characterization of their effects in loss-of-function and gain-of-function experiments, my findings provide novel insight into the regulatory networks that control dendrite morphogenesis.L'élaboration et le fonctionnement harmonieux des circuits nerveux dépendent de la croissance des dendrites et de leur guidage et ciblage vers les territoires appropriés au cours du développement. La morphologie des dendrites sert de signe distinctif pour chaque neurone, et ainsi, joue un rôle crucial dans la détermination des différents influx (synaptiques ou sensoriels) que reçoit un neurone. Malgré de récentes avancées dans la compréhension des mécanismes moléculaires et cellulaires qui contrôlent l'architecture dendritique, notre connaissance du développement des dendrites reste encore incomplète. Mes travaux de recherche se sont attachés à découvrir de nouveaux gènes et mécanismes impliqués dans la morphogenèse dendritique. Dans ce but, j'ai choisi au cours de ma thèse deux méthodes d'étude: une approche par crible génétique et une approche par gènes candidats, que j'ai appliquées aux neurones appelés dendritic arborization (da) de la drosophile, mon modèle d'étude. Mes recherches m'ont permis de me concentrer sur trois molécules: 1) le récepteur nucléaire de l'hormone stéroïde ecdysone (EcR), 2) le facteur de transcription Longitudinals Lacking (Lola) et enfin, 3) la molécule de surface Turtle (Tutl). J'ai pu montrer que chacun de ces facteurs est implique dans des aspects distincts du processus de morphogenèse dendritique incluant le branchement, la distribution et l'auto-répulsion dendritiques. L'identification de ces molécules, la description de leurs patrons d'expression et la caractérisation des phénotypes associés à leurs pertes ou gains de fonctions, m'ont permis d'apporter de nouvelles connaissances des réseaux de régulation contrôlant la morphogenèse dendritique.
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Mandel-Brehm, Caleigh. "A Behavioral and Molecular Approach for Understanding Angelman Syndrome". Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:26718736.
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Autism Spectrum Disorder (ASD) is a set of human developmental disorders that affects ~1 in 68 children. The clinical features of ASD include deficits in social behavior and frequent co-morbidity of motor, emotional and sensory impairment. Currently, there are no effective treatments. A major obstacle for treating ASD is the limited knowledge of the neuronal circuits that drive these complex behaviors.
Several monogenic, or single-gene, disorders that possess similar features to ASD have been identified, implicating a role for molecular pathways in the development of these behavioral circuits. This dissertation focuses on Angelman Syndrome (AS), a neurodevelopmental disorder characterized by social communication deficits, movement disorder and hyper-excitable behavioral traits. The phenotype of AS arises from mutation of the UBE3A gene, encoding an E3 ubiquitin ligase. The overarching goal of this study is to understand how deregulation of UBE3A-dependent pathways contribute to the behavioral phenotype of AS.
Neuronal substrates of UBE3A have been identified and their expression has been shown to be up-regulated in AS neurons. I now test the hypothesis that this deregulation contributes to specific pathology of AS. First, I described clinically relevant behavioral phenotypes in an AS mouse model. Next, I genetically reduced the expression level of two UBE3A substrates, ARC (Activity-Regulated Cytoskeleton-Associated Protein) and EPHEXIN5 (Rho Guanine Nucleotide Exchange Factor 15) in the AS mouse and assayed for reversal of behavioral abnormalities.
I find that the AS mouse model has impaired communication and motor behavior during early postnatal development, enhanced seizure-like activity and an abnormal cortical electroencephalogram (EEG). Reducing the levels of ARC reversed the enhanced seizure-like activity and EEG, but not the communication or motor deficits. The specific rescue of seizure-like activity by reducing ARC, but not EPHEXIN5, reveals a role for molecular diversity in the development of behavioral circuits. Further, these findings suggest that therapeutic interventions that reduce the level of ARC expression have the potential to reverse the seizures associated with AS. Lastly, the identification of aberrant behaviors in AS mice provides clues regarding the neural circuit defects that occur in AS and ultimately allow new approaches for treating this disorder, and broader ASDs.Medical Sciences
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Raouf, Ramin K. "Insights into the molecular physiology of the P2X receptor family". Thesis, McGill University, 2006. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111836.
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Adenosine triphosphate (ATP), the source of metabolic energy for a majority of chemical reactions in the cell, is now considered as an ancient messenger molecule used in intercellular communications. The ionotropic effects of ATP are mediated by P2X receptors which constitute a novel family of ligand-gated ion channels (LGICs). The seven vertebrate subunits, which form homomeric as well as heteromeric channel complexes, show little structural resemblance to other families of LGICs such as the Cys-loop family exemplified by the nicotinic acetylcholine receptors. P2X receptors have been shown to play important physiological roles in processes such as transmission of pain, vasoconstriction, and inflammatory processes involving release of cytokines. The potential importance of these receptors as therapeutic targets in a number of pathophysiological conditions has focused many efforts on the understanding of their molecular physiology and modulation in vivo.This thesis reports the rationale and the results of experiments carried out to advance our knowledge of the molecular physiology of P2X receptors in several fronts: First, development of more selective tools, based on structure-function data, for the knockdown of the receptor function in vivo (manuscripts #1 and #2); second, contributions made to our understanding of the phylogeny and structure-function relationship in this protein family through functional analysis of an invertebrate P2X subunit (manuscripts #3 and #4); and third, advancing our understanding of the modulation of the P2X function through second messenger signalling pathways which would aid the efforts in drug development aimed at this receptor family (manuscript #5).
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Harvey-Girard, Erik. "Molecular and functional characterization of NR2 subunits of teleost NMDA receptors". Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82891.
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Synaptic processing is essential for proper signal integration by sensory neurons. N-methyl-D-aspartate receptors (NMDAR) are ionotropic receptors located at the post-synaptic membrane. NMDARs are considered coincidence detectors because they need postsynaptic depolarisation and neurotransmitter release to open. Several extracellular and intracellular signalling molecules affect NMDARs, which leads to modulation of the synaptic strength. The work presented in this thesis describes sequences of NR2 subunits expressed in pyramidal cells of a sensory system in a gymnotiform fish and analyses the functions of recombinant NMDAR-2B.A rapid amplification cDNA ends (RACE) approach was modified to amplify three NMDAR NR2 subunits expressed in electrosensory lateral line (ELL) pyramidal cells of a weakly electric fish, the gymnotiform Apteronotus leptorhynchus .
The amino acid sequence of the NR2 subunits (aptNR2A/B/C) was determined and compared with the sequence of the murine NR2 subunits. This comparison revealed high levels of sequence conservation throughout the glutamate-binding domain and membrane spanning segments. Lower levels of sequence conservation in the amino- and carboxy-terminal domains (ATD and CTD) were found for the three subunits. However, several known regulation sites in the ATD and several serine/threonine and tyrosine phosphorylation sites in the CTD are conserved.
The functional properties of the apteronotid NMDAR-2B receptor were examined by co-expression of aptNR1 and aptNR2B in HEK293 cells. The recombinant aptNR1/NR2B receptors produced robust currents after stimulation with glycine and glutamate or NMDA. Electrophysiological measurements of the concentration dependencies for these agonists indicated that the agonist-binding sites on the apteronotid receptors are highly conserved, with agonist affinities nearly identical to those of the murine NMDAR-2B. The classical antagonist AP-V and hydrogen ions inhibit the fish NMDAR-2B similarly as they do for mammalian NMDAR-2B. The response kinetics of the fish receptor were also highly conserved, with deactivation rates for the aptNMDAR-2B receptor matching those of its murine homologue. Apteronotid and murine NMDA receptors displayed two functional differences. First, the voltage-dependent Mg++ block is slightly reduced. Second, the affinity of specific non-competitive antagonist ifenprodil for NR2B is also reduced.
These results suggest that the critical features of NMDAR structure and function were established before the evolutionary diversion of fish and higher vertebrates. They suggest also that several signalling mechanisms known to affect synaptic strength in the mammalian CNS are conserved in the apteronotid nervous system and have established the electrosensory system of gymnotiforms as an interesting model to study NMDAR functions in synaptic plasticity of a sensory system.
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Walsh, Gregory S. "Molecular mechanisms regulating survival of peripheral neurons during development and adulthood". Thesis, McGill University, 2005. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86062.
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Developmental sympathetic neuron death is determined by functional interactions between the TrkA/NGF receptor and the p75 neurotrophin receptor (p75NTR). A key question is whether p75NTR promotes apoptosis by directly inhibiting or modulating TrkA activity, or by stimulating cell death independently of TrkA. Here we show that the presence or absence of p75NTR does not alter Trk activity or NGF- and NT-3-mediated downstream survival signaling in primary neurons. Crosses of p75NTR-/- and TrkA-/-mice indicate that the coincident absence of p75NTR substantially rescues TrkA-/-sympathetic neurons from developmental death in vivo. These data support a model where developing sympathetic neurons are "destined to die" by an ongoing p75NTR-mediated apoptotic signal, and one of the major ways that TrkA promotes neuronal survival is by silencing this ongoing death signal. We then examined a role for JNK-p53 apoptotic pathway in NGF-deprived neurons and in nerve injury-induced death. Specifically, inhibition of JNK by overexpression of JIP-1 was sufficient to rescue sympathetic neurons from NGF withdrawal-induced death. In addition, JNK is robustly activated in nerve-injured neonatal facial motoneurons and these neurons are rescued from nerve-injury-induced cell death in p53 null mice. We then investigated the intracellular mechanisms that underlie the relative invulnerability of adult versus developing DRG sensory neurons. In both adult and neonatal neurons, death stimuli induced the apoptotic JNK pathway, but JNK activation only caused death of neonatal neurons, indicating that adult neurons have a downstream block to apoptosis. An essential component of this "block" is the p53 family member, DeltaNp73. Cultured adult p73+/- DRG neurons were more vulnerable to apoptotic stimuli than their p73+/+ counterparts, and invulnerability could be restored to the p73+/- neurons by increased expression of DeltaNp73. Moreover, although DRG neuron development was
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Nudel, Ron. "Molecular genetics of language impairment". Thesis, University of Oxford, 2015. http://ora.ox.ac.uk/objects/uuid:70249129-ef2e-4508-b8f6-50d6eae8e78b.
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Developmental language impairments are neurodevelopmental disorders in which the acquisition of language, a task which children typically perform with ease, is hindered or fraught with difficulty. This work focuses on specific language impairment (SLI), a common and highly heritable language impairment in which language development is abnormal while other developmental domains are normal. Additionally, a case-study of a child with a broader linguistic and behavioural phenotype is also presented. The work described in this thesis includes both genetic and functional investigations which were aimed at identifying candidate genes for language impairment and provide insight into the genetic mechanisms that underlie language development. I performed a genome-wide association study of SLI which included child genotype effects, maternal genotype effects, parent-of-origin effects, and maternal-foetal interaction effects. This study found significant paternal parent-of-origin effects with the gene NOP9 on chromosome 14, and suggestive maternal parent-of-origin effects with a region on chromosome 5 which had previously been implicated in autism and ADHD. Case-control and quantitative association analyses of HLA genes and SLI identified several risk alleles and protective alleles. A case-control association analysis for related individuals which used an isolated population affected by SLI identified a non-synonymous coding variant in the gene NFXL1 which was significantly more frequent in affected individuals than in unaffected individuals. High-throughput sequencing of the coding regions of NFXL1 and LD blocks surrounding associated variants in ATP2C2, CMIP and CNTNAP2 (as reported in previous studies) identified novel or rare non-synonymous coding variants in NFXL1 and ATP2C2 in SLI families as well as intronic variants in all four genes that were significantly more frequent in SLI probands than in population controls. I describe a functional study of NFXL1 examining its expression in various brain regions, the presence of different splice variants across several tissues, its effect on genes it potentially interacts with, and the subcellular localisation of the protein. Finally, I present the case-study of a child with language impairment who had chromosomal rearrangements which spanned the location of FOXP2. I examine the potential influence the chromosomal rearrangements had on FOXP2 expression and describe a lincRNA gene which was disrupted by the chromosomal inversion. In conclusion, this work identified new candidate genes for language impairment, provided further support for the involvement of previously-identified candidate genes in SLI and contributed to the understanding of the molecular function of a newly-identified candidate gene for SLI.
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Nevin, Linda. "Cellular and molecular mechanisms of neurite targeting in the zebrafish visual system". Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3297813.
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Gramolini, Anthony O. "Molecular mechanisms contributing to the expression of utrophin at the mammalian neuromuscular synapse". Thesis, University of Ottawa (Canada), 2000. http://hdl.handle.net/10393/9454.
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Duchenne muscular dystrophy (DMD) is the most severe and prevalent primary myopathy. This disease is characterized by repeated cycles of muscle fiber degeneration and regeneration with an eventual failure to regenerate leading to the progressive replacement of myofibers by adipose and connective tissues. The genetic defects responsible for DMD are mutations in the short arm of the X chromosome which prevent the production of normal size dystrophin, a large cytoskeletal protein of 427 kDa. In contrast to the homogeneous distribution of dystrophin along muscle fibers, utrophin preferentially accumulates at the neuromuscular junction. Due to this sequence similarity between dystrophin and utrophin, it has been suggested that increased expression of utrophin into extrasynaptic regions of dystrophic muscle fibers may represent a therapeutic strategy for DMD. Recently, it has been confirmed that the upregulation of utrophin can, indeed, functionally compensate for the lack of dystrophin and alleviate the muscle pathology. In this context, it thus becomes essential to determine the cellular and molecular mechanisms presiding over utrophin expression in attempts to overexpress the endogenous gene product throughout skeletal muscle fibers. In this Thesis, I explore the mechanisms underlying the selective accumulation of utrophin at the postsynaptic membrane of the neuromuscular synapse. We determined by in situ hybridization that local transcription contributes to the accumulation of utrophin at the neuromuscular junction. Using direct injections of utrophin promoter-reporter constructs into skeletal muscle, we also defined the promoter elements involved in this local transcription and determined that the N-box element is a key consensus sequence that directs transcriptional control of utrophin expression at the neuromuscular junction. Furthermore, additional experiments revealed that utrophin gene transcription is dependent on the extracellular matrix proteins agrin and ARIA/heregulin, and this regulation is dependent upon the N-box element. Indeed, in vitro transfection assays and electromobility shift assays indicated that agrin and ARIA/heregulin may ultimately initiate a cell signaling cascade that activates the ETS-related transcription factor, GA-binding protein (GABP) which binds and activates the N-box element. In these experiments, we determined by RT-PCF, immunoblotting, and nuclear run on assays that, in contrast to the large changes in AChR, utrophin expression was only marginally increased under these conditions. (Abstract shortened by UMI.)
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Bélanger, Jasmin Stéphanie. "Characterization of molecular mechanisms underlying the neurogenic effect of the bHLH protein Hes6". Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=81594.
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Hes1 is a mammalian basic helix-loop-helix (bHLH) transcriptional repressor that inhibits neuronal differentiation and acts together with the corepressor Gro/TLE. Hes1 interacts with Gro/TLE through its WRPW motif and recruits Gro/TLE to specific DNA sites. In contrast, a related Hes family member, Hes6, promotes neuronal differentiation. Little is known about the molecular mechanisms underlying the neurogenic effect of Hes6. To address this question, a structure/function analysis was performed to identify domains of Hes6 important for its biological activity during the differentiation of cortical neurons. It is shown here that the nuclear translocation of Hes6 is required to promote cortical neurogenesis. However, Hes6 neurogenic activity does not appear to be DNA-binding dependent, suggesting that it is mediated by protein-protein interaction mechanisms. Moreover, a conserved potential PEST sequence present at the C-terminus of Hes6, which may regulate protein turnover, is also important for the neurogenic activity of Hes6. It is also shown that the nuclear translocation-incompetent form of Hes6 and, to a lesser extent, the PEST sequence-defective form have a reduced ability to promote a proteolytic degradation of Hes1. This correlation suggests that Hes6 may regulate cortical neurogenesis by a protein-protein interaction mechanism in which Hes6 interacts with Hes1 and promotes a proteolytic degradation of the latter.
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Yao, Jing 1962. "Molecular mechanisms regulating mammalian neurogenesis : roles of GrouchoTLE, BF-1 and HES1 proteins". Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38531.
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Drosophila development has provided a useful model system to study the molecular mechanisms that regulate neurogenesis. Recently much progress has been made in our understanding of how neural progenitor cells undergo commitment and become mature neurons. Studies have shown that a series of transcription factors plays fundamental roles in these neuronal developmental events. From Drosophila to mammals, evolutionarily conserved molecules regulate similar biological processes. The major hypothesis advanced in this thesis is that mammalian homologues of the Drosophila protein Groucho, namely the TLE proteins, share a conserved function in the regulation of mammalian neurogenesis. To investigate the molecular mechanisms regulating mammalian forebrain development, a number of studies were performed focusing on the function of the Groucho/TLE proteins and their partners, Hes1 and BF-1, as transcriptional regulators of mammalian neurogenesis.Groucho regulates neurogenesis by interacting with Drosophila HES proteins genetically and molecularly. Through these interactions, Groucho acts as a transcriptional co-repressor for HES proteins to suppress the transcription of genes promoting neural fate, therefore, inhibiting neurogenesis. Although some of the molecular properties of Groucho/TLE proteins were known, their involvement in mammalian neurogenesis is still poorly understood. In order to define the molecular function of Groucho/TLE1 in these processes, a series of studies were carried out. This thesis comprises three consecutive projects presenting studies of: (1) the expression pattern of TLE proteins during mammalian CNS development and during in vitro neural induction and chondrocyte differentiation, (2) the effect of altered TLE1 expression during in vivo mammalian neurogenesis, and (3) the functional partnerships of Groucho/TLE with other proteins involved in regulating mammalian telencephalon development.
In these studies, we have demonstrated that individual Groucho/TLE proteins are expressed in the developing nervous system in a combinatorial fashion, suggesting a non-redundant function. Furthermore, persistent expression of TLE1 in post-mitotic neurons results in neuronal loss in the developing telencephalon. In addition, TLE proteins physically and functionally interact with HES and BF-1 proteins. Through these associations, Groucho/TLE proteins regulate the transcriptional repressive function of these proteins. These studies provide evidence that Groucho/TLE proteins act as transcriptional co-repressors in controlling neurogenesis in both invertebrate and vertebrate species.
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Turecki, Gustavo. "Molecular and genetic analysis of lithium responsive bipolar disorder". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0011/NQ55387.pdf.
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Godfrey, Grayland W. II. "Characterizing the Role of Key Planar Cell Polarity Pathway Components in Axon Guidance". VCU Scholars Compass, 2017. http://scholarscompass.vcu.edu/etd/4841.
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An essential process to the development of the neural network of the nervous system is axon guidance. The noncanonical Wnt/Planar Cell Polarity pathway has been identified as an integral component in controlling the projection of axons during axon guidance. Prickle, ROR1 and ROR2 are PCP related proteins that do not have clearly defined roles in the process. This study aims to use zebrafish CoPA neurons as a model to study the roles of Prickle, ROR1, and ROR2 in axon guidance. Using in situ hybridization, morpholino knockdown, and CRISPR/Cas9 loss of function experiments were able to identify ror1, ror2 and prickle as potential required components in CoPA neuron axon guidance. Elucidating the role of these protein in axon guidance not only will increase our knowledge of the PCP pathway but it will also increase our understanding of the development of the nervous system.
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Tumova, Katerina. "Uncovering the molecular interplays of dopamine D1-like receptor activation". Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/26347.
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D1-like dopaminergic receptor family consists of two heptahelical G protein-coupled receptors (GPCRs), termed D1A (or D1) and D1B (or D5). In this study, we have used a functional complementation of chimeric D1-like receptors to address the molecular interplay between the third extracellular loop (EL3) and the cytoplasmic tail (CT) in coordinating the functional properties of D1-like receptors expressed in transfected human embryonic kidney 293 (HEK 293) cells. Our results indicate that ED and CT participate in interfering intramolecular interactions that regulate the total functional receptor number and the agonist-mediated G protein coupling properties of the D1-like receptors in HEK 293 cells. In contrast, the agonist-independent activity of the D1-like receptors appears to be regulated solely by the sequences of the CT. In addition, we have identified the proximal region of CT, known as the fourth intracellular loop (IL4), as the pivotal domain underlying the CT-induced properties of the D1-like receptors. Overall, our study provides an insight into the molecular basis of D1-like receptor activation with possible implications for entire field of GPCR signaling.
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Radlinska, Barbara. "«In vivo» imaging of microstructural and molecular neuroplasticity of fibre tracts in human subcortical stroke". Thesis, McGill University, 2013. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=119394.
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Background Studies in acute and chronic post-stroke recovery suggest that both microstructural and molecular changes in stroke-affected fibre tracts are associated with clinical outcome. These processes can be measured noninvasily in the living human brain using Diffusion Tensor Imaging (DTI) and Positron Emission Tomography (PET), and together these neuroimaging techniques provide a detailed multimodal approach to the assessment of post-stroke neuroplasticity. With four controlled prospective studies, this thesis addresses direct and indirect microstructural and neuroinflammatory changes as they progress over time along infarct-affected fibres following subcortical ischaemic stroke of the Pyramidal tract (PT). Methods Patients with subcortical ischaemic stroke either affecting the PT (PT-group) or not (NonPT-group), as well as matched controls with Transient Ischaemic Attack (TIA), underwent both DTI and 11C-[R]-PK11195 PET at 3 weeks and 6 months post-stroke. From the DTI data, the PT (directly affected by the stroke), Callosal Motor Fibres (CMF; indirectly affected by the stroke) and Callosal Occipital Fibres (COF; not affected by stroke) were delineated using deterministic tractography. Fractional Anisotropy (FA) ratios (affected/unaffected hemisphere) were calculated for each tract. FA ratios (rFAPT; rFACMF; rFACOF) were compared within and between groups at both time-points, as well as anterograde and retrograde to the infarct. These ratios were then correlated with clinical outcome measures. From PET data, tracer uptake ratios (URs, affected/unaffected hemisphere) were determined for a set of standardised volumes of interest (VOIs) along the PT. These molecular markers of neuroinflammation were correlated with fibre tract integrity anterograde and retrograde to the infarct and with clinical outcome measures. Results DTI data analyses revealed that mean rFAPT in the PT-group was significantly lower than both NonPT and TIA groups initially and at follow-up, and correlated significantly with clinical outcome measures. PT-group rFACMF decreased over time. At follow-up, PT-group rFACMF was significantly lower than NonPT-group rFACMF and PT-group rFACOF. PT-group rFACMF at follow-up correlated with rFAPT retrograde to the infarct. PET data analyses revealed that PT-group uptake ratios were significantly increased at the level of the infarct and anterograde initially, but only anterograde to the infarct at follow-up. Anterograde uptake ratios were correlated with anterograde rFAPT at the initial time-point, whilst uptake ratios in the infarct were correlated with anterograde rFAPT only at follow-up. After controlling for PT damage, initial brainstem uptake ratios showed a positive correlation with clinical outcome, whereas follow-up uptake ratios in the infarct tended to be negatively correlated. Conclusions Overall, significant progressive changes in both microstructural and molecular neuroimaging parameters can be seen along fibres that have been either directly or indirectly affected by subcortical ischaemic stroke. Changes in fibre integrity, as modelled with DTI, are apparent both anterograde and retrograde to the ischaemic lesion, challenging the notion that decreased FA reflects Wallerian degeneration. This is underscored by the fact that decreases in FA are also apparent in regions with only indirect connections to the infarct. Molecular markers of neuroinflammation are present only in the area of the lesion itself and anterograde to the infarct and are therefore more likely to be associated with Wallerian degeneration. Taken together, DTI and PET provide a clinically meaningful assessment of neuroplasticity in the acute and chronic post-stroke phases.Contexte Les recherches dans le domaine de la récupération suite à un accident vasculaire cérébral (AVC) suggèrent que les changements morphologiques comme moléculaires sont associés à des résultats cliniques. Ces changements peuvent être mesurés de façon non invasive chez l'être humain grâce à l'utilisation de l'Imagerie par Tenseur de Diffusion (ITD) et de la Tomographique par Émission de Positrons (TEP). Au travers de quatre études prospectives contrôlées, cette thèse s'attache à décrire les changements microstructuraux et neuroinflammatoires au cours de leur progression le long des fibres du Faisceau Pyramidal (FP) affectées par un AVC sous-cortical ischémique. Méthodes Des patients présentant un AVC ischémique sous-cortical, qu'il affecte le Faisceau Pyramidal (groupe FP) ou non (groupe NonFP), ainsi que des participants contrôles ayant présenté un Accident Ischémique Transitoire (AIT) se sont vu proposer des examens de ITD et TEP11C-[R]-PK11195 3 semaines et 6 mois après leur AVC. Concernant les données issues de l'examen par ITD, le FP (affecté directement par l'AVC), les Fibres Calleuses Motrices (FCM; affectées indirectement par l'AVC) et les Fibres Calleuses Occipitales (FCO; non affectées par l'AVC) furent délimitées grâce à l'utilisation de la tractographie. Les ratios (l'hémisphère affecté/non affecté) déterminés par l'Anisotropie Fractionnelle (AF) furent calculés. Ces ratios (rAFFP;rAFFCM; rAFFCO) furent comparés à 3 semaines puis 6 mois post-AVC et également analysés selon leur orientation (antérograde ou rétrograde) par rapport à la lésion, et mis en relation avec des résultats cliniques. Concernant les données issues de l'examen de TEP, le ratio d'absorption de la dose traceuse fut déterminé pour un ensemble de zones d'intérêt standardisées le long du FP. Ces marqueurs moléculaires de neuroinflammation furent mis en relation avec l'intégrité des faisceaux, ainsi qu'avec des résultats cliniques. Résultats Les analyses de l'ITD ont révélé que le ratio moyen rAFFP dans le groupe FP était significativement moins élevé que pour les groupes NonFP comme AIT, à 3 semaines et à 6 mois de l'AVC, et que cela était corrélé à des résultats cliniques. Le ratio rAFFCM du groupe FP a décru avec le temps. 6 mois post AVC, le ratio rAFFCM du groupe FP était significativement moins élevé que celui du groupe NonFP comme celui rAFFCO du groupe FP. Le ratio rAFFCM du groupe FP à 6 mois était corrélé à celui rAFFP rétrograde de la zone lésée. Les analyses des données issues de l'examen de TEP ont révélé que les ratios d'absorption du traceurétaient significativement plus élevés au niveau de la zone lésée et dans le sens antérograde 3 semaines post-AVC pour le groupe FP, mais uniquement dans le sens antérograde à la lésion 6 mois post-AVC. Les ratios dans le sens antérograde étaient corrélés avec le ratio rAFFP antérograde 3 semaines post-AVC, alors que les ratios dans la lésion étaient corrélés avec le ratio antérograde rAFFP seulement 6 mois post-AVC. Après avoir contrôlé la présence de dommages sur le faisceau pyramidal, les ratios initiaux d'absorption au niveau du tronc cérébral présentèrent une corrélation positive avec les résultats cliniques, bien que les ratios au niveau de la lésion tendaient à être négativement corrélés. Conclusions Dans l'ensemble, des changements significatifs concernant les fibres affectées directement ou indirectement par un AVC sous-cortical ischémique peuvent être observés au niveau morphologique comme moléculaire grâce à la neuroimagerie. Des changements dans l'intégrité de la fibre, comme démontrés par l'ITD, sont mis en évidence dans les directions antrérograde comme rétrograde à la zone cérébrale lésée, contestant ainsi la notion qu'une faible Anisotropie Fractionnelle reflète une dégénération Wallérienne. Les marqueurs moléculaires de neuroinflammation sont présents uniquement dans la zone lésée elle-même, et dans les fibres antérogrades.
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Savdie, Cheryl. "Characterization of the cellular and molecular determinants for the internalization of the high affinity neurotensin receptor". Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29473.
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Neurotensin is a thirteen amino acid peptide that is found both in the central and peripheral nervous systems. In the CNS, it controls the synthesis and release of dopamine, modulates anterior pituitary hormone secretion, and provides non-opioid-receptor-dependent analgesia. In the gut, it modulates gastric acid secretion, pancreatic secretion, and colonic motility. In the immune system, it modulates IL-1 and mast cell secretion and stimulates macrophage phagocytosis. Neurotensin also acts as a growth factor for normal and cancer cells. Neurotensin mediates its effects through three neurotensin receptors: NTR1, NTR2, and NTR3. NTR1 and NTR2 belong to the G-protein coupled receptor (GPCR) family, while NTR3 is a single transmembrane receptor identical to sortilin. NTR1 has the highest affinity for neurotensin (Kd of 0.1--0.3 nM). Internalization of GPCRs, including NTR1 and NTR2, was shown to play an important role in initiating signaling activity as well as in desensitizing and resensitizing the receptors.In this study, the internalization of the NTR1 was investigated. (Abstract shortened by UMI.)
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Babic, Milos. "Molecular Mechanisms of Mitochondrial Transport in Neurons". Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/556433.
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Dynamic mitochondrial transport into axons and dendrites of neuronal cells is critical for sustaining neuronal excitability, synaptic transmission, and cell survival. Failure of mitochondrial transport is the direct cause of some neurodegenerative diseases, and an aggravating factor for many others. Mitochondrial transport regulation involves many proteins; factoring prominently among them are the atypical mitochondrial GTPase Miro and the Milton/TRAK adaptor proteins, which link microtubule (MT) motors to mitochondria. Motors of the kinesin family mediate mitochondrial transport towards the plus ends of microtubules, while motors of the dynein family mediate mitochondrial transport towards the minus ends. Selective use of these motors determines the ultimate subcellular distribution of mitochondria, but the underlying control mechanisms remain poorly understood. Drosophila Miro (dMiro) is required for kinesin-driven transport of mitochondria, but its role in dynein-driven transport remains controversial. In Chapter 2 of this study, I show that dMiro is also required for the dynein-driven transport of mitochondria. In addition, we used the loss-of-function mutations dMiroT25N and dMiroT460N to analyze the function of dMiro's N- and C-terminal GTPase domains, respectively. We show that dMiroT25N causes lethality and impairs mitochondrial distribution and transport in a manner indistinguishable from dmiro null mutants. Our analysis suggests that both kinesin- and dynein-driven mitochondrial transport require the activity of Miro's N-terminal GTPase domain, which likely controls the transition from a stationary to a motile state irrespective of the transport direction. dMiroT460N reduced only dynein motility during retrograde axonal transport but had no effect on distribution of mitochondria in neurons, indicating that the C-terminal GTPase domain of Miro most likely has only a small modulatory role on transport. Furthermore, we show that commonly used substitutions in Miro's GTPase domains, based on the constitutively active Ras-G12V mutation, appear to cause neomorphic phenotypic effects which are probably unrelated to the normal function of the protein. In mammalian neurons, kinesin and dynein motors are linked to mitochondria via a Miro complex with the adapter proteins TRAK1 and TRAK2, respectively. Differential linkage of TRAK-motor complexes provides a mechanism for determining the direction of transport and controlling mitochondrial distributions within the cell. Drosophila has only one TRAK gene homolog, Milton, which expresses several protein isoform. Milton has been previously been shown to facilitate mitochondrial transport by binding to kinesin and dMiro, a role analogous to TRAK1. However, the question whether Milton might be able mediate dynein-based transport in a manner similar to TRAK2 has remained unknown. In Chapter 3 of this study, I show that protein isoforms A and B of Milton, generated through alternative mRNA splicing, facilitate differential motor activities analogous to mammalian TRAKs. Specifically, overexpression (OE) of Milton-A caused a mitochondrial redistribution and accumulation at axon terminals, which requires kinesin-driven MT plus end directed transport; while OE of Milton-B caused a redistribution of axonal mitochondria into the soma, which requires dynein-driven MT minus end directed transport. I further show that Milton-motor complex binding to mitochondria requires Miro exclusively, and that transport with either of the motor complexes absolutely requires the activity of Miro's N-terminal GTPase domain. Together, these results suggest that Miro controls the transition of mitochondria from a stationary to a motile phase. Thereafter the direction of transport is likely determined by an alternative binding of opposing Milton/TRAK-motor complexes to Miro, a process which appears to be regulated by a Miro-independent mechanism.
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Nanjappa, Purushothama. "Generation and analysis of transgenic zebrafish and mice for the study of Dlx function in the forebrain". Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27277.
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The vertebrate Dlx homeobox genes are organized as three convergently transcribed bigene clusters and show overlapping expression patterns in the forebrain, pharyngeal arches, sensory placodes and limb/fin buds. Little is known about how the Dlx genes are targeted to their sites of expression, or what particular roles individual genes play in development. Cis-acting regulatory elements have been identified in the intergenic and upstream regions of paired Dlx genes and are thought to play a major role in the Dlx regulation in the forebrain. My study was focused on two of the enhancer elements from the Dlxl/Dlx2 locus, I12b and URE2. We used these two elements to target reporter transgene expression to the zebrafish forebrain and analyzed their activity. We also successfully demonstrated the use of Fluorescent Activated Cell Sorting to isolate Dlx-Green Florescent Protein-expressing cells from the zebrafish forebrain. These results contribute to our understanding of Dlx regulation, function and evolution.
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Burton, Lindsay. "Dlx regulation in zebrafish brain development via I56iI56ii and I12aI12b". Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27754.
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Dlx genes are involved in the formation of the forebrain, branchial arches, sensory organs, and limbs. In the forebrain, Dlx genes are expressed in restricted domains in the telencephalon and diencephalon. The telencephalon is one of the most complex structures in the vertebrate central nervous system, but despite variable morphologies of the adult telencephalon, the patterning and basic organization is conserved amongst vertebrates.
Cis-acting regulatory sequences (CREs) that contribute to Dlx expression in the telencephalon were previously identified in the intergenic region between Dlx5 and Dlx6 (I56i and I56ii), between Dlx1 and Dlx2 (I12b), and upstream of Dlx1 (URE2) in zebrafish, mouse, and human. I hypothesize that these CREs contribute to defining distinct subtypes of interneurons during forebrain development. In this study, we have investigated the differential activity of intergenic CREs in the developing zebrafish brain using the established transgenic lines Tg(6kb-dlx1a/dlx2a: EGFP) and Tg(1.4kb-dlx5a/dlx6a:EGFP) in which the green fluorescent protein gene (GFP) was used as a reporter whose expression was controlled by dlx CREs. The two intergenic fragments target reporter transgenes with overlapping patterns of expression in the telencephalon but also show differential activity in the dorsal region of the brain at the level of the pallium. These results suggest that the dlx1a/2a CREs are involved in dlx regulation in a set of cells occupying the subpallium, whereas the dlx5a/6a CREs are involved in dlx regulation in a set of cells that migrate out of the subpallium and travel in a ventral to dorsal manner to occupy the pallium. Co-localization of GFP with GAD65/67 and with molecular markers of the interneuron subtype, calretinin, indicates that the dlx CREs target the reporter constructs to GABAergic interneurons, with at least one subtype expressing calretinin. Results from this study reflect a dynamic regulation of dlx gene expression in the developing zebrafish forebrain through several regulatory elements with distinct and overlapping functions.
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Arauz, Claudia. "Genes required for normal neuronal morphology in Caenorhabditis elegans". Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28627.
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png-1 encodes the Caenorhabditis elegans homolog of Peptide: N-glycanase, a highly conserved cytosolic enzyme that cleaves N-glycans from misfolded glycoproteins. png-1 was found to regulate several aspects of neuronal morphology including axon branching. In this study, we show that mutations in the NDR kinase pathway genes sax-1 and sax-2 result in png-1 like phenotypes, including excessive branching and ectopic neurites. Furthermore, we found that png-1; sax-1; and png-1; sax-2 double mutants display enhanced defects compared to single mutants, suggesting that png-1 and sax-1/sax-2 act in parallel pathways to restrict axon and branch overgrowth. These interactions suggested a sax-1 enhancer screen as a means to identify additional genes in the png-1 pathway as enhancer mutants should phenocopy png-1 and enhance sax-1 branching defects. This approach recovered at least three sax-1 enhancers ( sens) that act like png-1 to limit axon growth and branching. The identification of these genes should provide new insight into how PNG-1 regulates neuronal morphology.
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Mileva, Gerri. "Short Term Synaptic Plasticity Across Multiple Electrosensory Maps in the Weakly Electric Fish Apteronotus leptorhynchus". Thesis, University of Ottawa (Canada), 2010. http://hdl.handle.net/10393/28637.
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The electrosensory lateral line lobe (ELL) is the first order electrosensory processing station in the brain of the electric fish Apteronotus leptorhynchus, and is the only nucleus to receive input from electroreceptors on the skin. The ELL is subdivided into three maps: the centromedial segment (CMS), centrolateral segment (CLS), and lateral segment (LS) which receive input from tuberous electroreceptors processing high frequency signals. Two feedback pathways from higher order nuclei, the nucleus praemenintialis (nP) and the eminentia posterior granularis (EGp) are integral to processing in the ELL and show synaptic plasticity on various time scales. This thesis focuses on characterizing short term plasticity (STP) in nP-ELL synapses between the CMS, CLS and LS, and the development of an in vitro slice containing the entire direct feedback pathway. We find that LS pyramidal cells show greater facilitation in response to high frequency stimulation of direct feedback fibres as compared to CMS.
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Nickerson, James G. "Molecular characterization of the beta-adrenoceptor gene-family of rainbow trout (Oncorhynchus mykiss)". Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/28962.
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beta-Adrenoceptors (beta-ARs) are seven transmembrane domain, G-protein coupled receptors that transduce the cellular effects of the catecholamines, adrenaline (Adr) and noradrenaline (Nadr). Through their interaction with plasma membrane beta-ARs, catecholamines modulate a number of key physiological parameters aimed at allowing an organism to cope with environmental and physiological stressors.
Mammalian species express three distinct beta-AR subtypes (beta 1-, beta2- and beta3-ARs) that exhibit complex modes of regulation and interaction. Relatively few studies have focused on beta-ARs from early branching vertebrates such as fish, particularly at the molecular level. The goal of this study was to characterize the beta-AR gene-family of the rainbow trout (RbT), Oncorhynchus mykiss.
Three putative beta-ARs genes were cloned and a phylogenetic analysis predicted one beta2-AR subtype (RbT beta2-AR) and two beta3-AR subtypes (RbT beta3a- and RbT beta 3b-ARs) relative to the established mammalian beta-AR classification. The RbT beta2-, beta3a- and beta3b-AR genes code for proteins of 409, 427 and 477 amino acids, respectively. Hybridization of gene specific probes to trout tissue RNA indicated that RbT beta 2-AR was highly expressed in the liver, red muscle, and white muscle; RbT beta3a-AR was highly expressed in the gill and heart while RbT beta3b-AR was highly expressed in the blood. Pharmacological analysis indicated RbT beta2-AR binding characteristics consistent with mammalian beta2-ARs while the RbT beta3b-AR showed characteristics that were different from all known mammalian beta-AR subtypes. Differences in the potential regulatory phosphorylation profiles between trout beta-ARs suggests subtype specific sensitivities to the classic mechanisms of beta-AR desensitization.
This molecular characterization of trout beta-ARs is the first study to demonstrate a beta3-AR homolog in fish, it identifies the trout red blood cell beta-AR as a beta3-subtype and provides support for the presence of a complex and unique beta-AR signaling system in the rainbow trout.
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Issa, Amalia Mary. "Effects of the protein phosphatase inhibitors calyculin A and okadaic acid at the cholinergic nerve terminal". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0016/NQ44461.pdf.
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Kirkpatrick, Karen. "Functional role and modulation of a calcium-activated potassium current in rat supraoptic neurons in vitro". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0014/NQ36993.pdf.
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Lazzaro-Albert, Maribeth A. "Characterization of a novel cdc2-related kinase expressed in the nervous system". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0015/NQ36997.pdf.
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Morissette, Nathalie. "Functional studies of laminin-binding integrins and distribution of the laminin alpha2 chain in the developing visual system". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0015/NQ37007.pdf.
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Brake, Wayne Gerard. "Influence of perinatal and early postnatal insults on the adult dopamine stress response in rats". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0016/NQ55304.pdf.
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Causing, Carrie Grace. "Trans-synaptic regulation of neural connections : the role of neuronal activity and brain-derived neurotrophic factor (BDNF)". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0020/NQ55309.pdf.
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Subang, Maria Cristina. "The regulation of ciliary neurotrophic factor, leukemia inhibitory factor and monocyte chemoattractant protein-1 in injured peripheral nervous tissue". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0034/NQ64675.pdf.
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Rashid, Asim J. "Molecular characterization of voltage-gated K+ channels in the weakly electric fish Apteronotusleptorhynchus". Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=20279.
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A PCR approach using degenerate primers specific for the highly conserved pore and S6 transmembrane domains of K+ channels was used to assess the diversity of K+ channels in the electric fish Apteronotus leptorhynchus. DNA sequence analysis has identified nineteen K+ channel genes, each of which can be classified into one of the four major families in a similar distribution to that in mammals. Also, an Apteronotus brain cDNA library was constructed and screened with mammalian K+ channel probes. The first K+ channel clone obtained displays remarkable amino acid sequence homology to rat Kv1.3 and Northern blot analysis and RNase protection assays have suggested that this channel may play similar roles in both fish and mammals. The results indicate that the duplications which gave rise to multiple genes within each of the K+ channel families predate the divergence of the Actinopterygii and Sarcopterygii lineages during early vertebrate evolution and the function of these K+ channel genes has been conserved between the two lineages.
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Roux, Philippe P. "Signaling pathways implicated in p75 neurotrophin receptor-mediated neuronal survival and death". Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38267.
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The neurotrophins are growth factors involved in the development, maintenance, survival, and death of the nervous system. The signal transducing systems that mediate the diverse biological functions of the neurotrophins are initiated by their interactions with two categories of cell surface receptors, the Trk family of tyrosine kinases, and the p75 neurotrophin receptor (p75NTR). In contrast to the rapid progress made in elucidating the mechanism of action of the Trk receptors, the physiological roles of p75NTR are uncertain, but two general functions have been ascribed to p75NTR. First, p75NTR can positively or negatively modulate Trk receptor signaling, and second, p75NTR can autonomously activate signaling cascades that results in cellular apoptosis. The signaling pathways employed by p75NTR to mediate its effects are unclear, but p75NTR was found to activate the NF-kappaB and JNK pathways, and to interact with several adaptor proteins, such as NRAGE, NADE, NRIF, TRAF proteins, and FAP-1.Nervous system injuries represent interesting models to study p75NTR because several types of injury induce p75NTR expression. In the first part of this thesis, we have used the pilocarpine model of seizure in the rat and found that this type of injury induces neuronal apoptosis and p75NTR expression. Apoptosis was tightly linked with p75NTR expression, suggesting that p75NTR may promote apoptosic cell death after seizure, and consistent with this, we have found that p75NTR can promote JNK activation and apoptosis in vitro. In the second study, we discovered that p75NTR can also facilitate survival under some cellular circumstances. The survival-promoting effect of p75NTR was accompanied with PI3-K-dependent Akt activation, and correlated with a reduction in cytosolic protein tyrosine phosphatase activity, suggesting that p75NTR may regulate a tyrosine phosphatase involved in the regulation of Akt and survival. In the last study, we have found that the related neuroprotective compounds, K252a and CEP 1347, are potent. MLK3 inhibitors, yet they simultaneously activated Akt and ERK, and survival through MLK3-independent mechanisms. These findings suggested that K252a and CEP1347 may act on a novel target responsible for their survival-promoting activities.
Taken together, the data in this thesis adds to our understanding of the physiological functions of p75NTR, and contributes to our knowledge of the cellular machinery that control neuronal cell survival and death.
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Zheng, WenHua 1962. "Intracellular signaling of insulin-like growth factor receptors in neuronal cells : activation and regulation of AKT kinase pathway and the forkhead family transcription factor FKHRL1, and their role in cell survival". Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38444.
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Insulin-like growth factor-1 (IGF-1) is a polypeptide trophic factor playing an important role in the survival and differentiation of both neuronal and non-neuronal cells. The biological functions of IGF-1 are mediated by the IGF-1 receptor, a tyrosine kinase receptor expressed in most tissues including the central nervous system. IGF-1 possesses trophic effects in neuronal cells including in hippocampal neurons. However, the intracellular signaling pathways activated by IGF-1 receptors in these cells remain to be fully established. The main objective of the thesis was to investigate the activation and regulation of survival kinases such as Akt and one of its downstream targets the Forkhead family transcription factor FKHRL1 (a proapoptotic protein), as well to establish the role of these events in IGF-1 induced neuroprotection in PC12 cells and hippocampal neurons. Our results show that IGF-1 stimulates tyrosine phosphorylation of the IGF-1 receptor, as well as of IRS-1 and IRS-2, and their association with PI3 kinase, leading to the activation of Akt in PC12 cells and hippocampal neurons. The activation of Akt by IGF-1 is mediated by the PI3 kinase as only PI3 kinase inhibitors such as wortmannin and LY294002 but not the MAP kinase pathway inhibitor PD89059 or the S6p70 kinase pathway inhibitor rapamycin blocked the action of IGF-1. IGF-1 also increases the phosphorylation of FKHRL1 in PC12 cells and hippocampal neurons via the activation of Akt as evidenced by the use of various kinase inhibitors, an in vitro kinase assay and the expression of different forms of Akt. Interestingly, the phosphorylation of Akt and FKHRL1 induced by IGF-1 is attenuated by the stimulation of protein kinase C which modulates IGF-1 receptor signaling at various levels including IRS-1 and Akt in PC12 cells.As in PC12 cells, IGF-1 activates the IGF-1 receptor/IRS-1/PI3K/Akt/FKHRL1 pathway in hippocampal neurons, and the phosphorylation of FKHRL1 is mediated by PI3K/Akt kinase. Functionally, cell survival assay with kinase inhibitors and transfection of different forms of Akt suggest that the PI3K/Akt/FKHRL1 pathway plays a role in IGF-1-promoted survival in neuronal cells. Interestingly, similar results were obtained for neurotrophins such as NGF and BDNF suggesting that neurotrophins induce the activation of this pathway to promote cell survival. Taken together, these finding support the hypothesis that the survival promoting effects of IGF-1 in neuronal cells are mediated, at least in part, by the PI3K/Akt/FKHRL1 pathway.
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Petroulakis, Emmanuel. "Distinct mechanisms of translation of members of the mammalian elongation factor family, eEF1A-1 and eEF1A-2, during neuronal differentiation". Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38510.
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Eukaryotic peptide elongation factor-1 alpha (eEF1A) is an abundantly expressed protein involved in the elongation step of protein synthesis. Its protein expression is regulated at the translational level, as its mRNA shifts from ribonucleoprotein (RNP) complexes to polyribosomes upon growth factor stimulation. The regulation of this shift is mediated by a terminal oligopyrimidine (TOP) element in the 5' untranslated region (UTR), of the eEF1A-1 mRNA. This shifting is also involved during differentiation and mammalian development. During development the expression of eEF1A-1 declines and eEF1A-2, a highly conserved homologue, becomes the dominant eEF1A isoform expressed in brain, heart, and skeletal muscle. In this thesis, three studies were undertaken to examine the regulation and involvement of eEF1A-1 and eEF1A-2 in protein synthesis in neurons. In the first study, the steady-state expression of eEF1A-1 and eEF1A-2 mRNAs and proteins was studied during retinoic acid-induced neuronal differentiation of P19 embryonic teratocarcinoma cells. The decline of eEF1A-1 and the increase in eEF1A-2 expression during neuronal differentiation were found to be regulated primarily at the transcriptional level, since there was a correlation between the levels of mRNA and protein expression. In differentiated P19 neurons translational repression of eEF1A-1 mRNA also contributed to the decrease of eEF1A-1 protein. This indicated that transcriptional and translational repression of eEF1A-1 mRNA occurred during neuronal differentiation. Unlike eEF1A-1, eEF1A-2 mRNA was associated primarily with polyribosomes, suggesting that eEFIA-2 is preferentially translated during P19 neuronal differentiation. In the second study, the eEF1A-1 isoform was shown to be preferentially synthesized in nerve growth factor (NGF) stimulated PC12 cells. The increased synthesis of eEF1A-1 was mediated at the translational level, as shown by the shifting of eEF1A-1 mRNA to polyribosomes after NGF stimula
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Pepio, Anthony M. "The role of the C2 domain of protein kinase C APl II in the nervous system of Aplysia californica /". Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37810.
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In the nervous system of the marine mollusk Aplysia , protein kinase C plays important roles in plasticity and learning. Aplysia PKCs are differentially regulated in sensory neurons of the nervous system. The facilitating neurotransmitter serotonin activates the Ca2+-dependent PKC Apl I but not the Ca2+-independent PKC Apl II. The mechanisms underlying this differential PKC activation still remain unclear. To better understand putative mechanisms regulating PKC activity we sought to characterize Aplysia PKC structural domains. This thesis has explored the characteristics of C1 and C2 domains from Aplysia PKC Apl I and PKC Apl II and whether differences in PKC activation can be attributed to differences in these domains.First, we demonstrate that the presence of the C2 domain of PKC Apl II lowers the affinity of the C1 domain for the protein kinase C activator, phorbol ester. This C2 domain mediated inhibition of activator binding can be overcome by elevating phosphatidylserine concentrations. Further, phosphatidic acid is much more potent than phosphatidylserine in reducing C2 domain mediated inhibition.
Second, we present a comparison of the C1 and C2 domains of PKC Apl I and PKC Apl II. The C2 domain of PKC Apl I binds to lipids constitutively, while the C2 domain of PKC Apl II does not. In contrast, the C1 domains of PKC Apl I and PKC Apl II exhibit only small differences in lipid interactions. This suggests that while the C2 domain of PKC Apl I assists lipid mediated activation that of PKC Apl II hinders activation.
Finally, we show that there are two primary autophosphorylation sites in the C2 domain of PKC Apl II. These sites map to residues serine 2 and serine 36 located in loop 1 of the C2 domain. In vitro, phosphorylation of serine 36 increased binding of the C2 domain to phosphatidylserine membranes. In vivo, the PKC activator phorbol ester stimulated PKC Apl II phosphorylation at serine 36 and PKC phosphorylated at this residue translocated more efficiently to membranes. Moreover, mutation of serine 36 to alanine significantly reduced membrane translocation of PKC Apl II. As a whole, these data suggest a phosphorylation dependent mechanism regulating C2 domain membrane binding of Ca2+-independent PKCs. This mechanism of Ca2+-independent PKC plasma membrane localization may play a role in generating persistent PKC activity and the eventual modulation of synaptic plasticity in the nervous system of Aplysia.
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Hislop, Jonathan. "Cloning of receptor tyrosine kinases from Aplysia californica". Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=21567.
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I have cloned two putative receptor tyrosine kinases (RTKs) from Aplysia californica, designated Aror and Aruf. Aror is homologous to RTKs from the Ror subfamily. Furthermore, Aror contains an extracellular immunoglobulin-like domain, cysteine-rich region, and kringle domain. These domains are all characteristic of Ror receptors. Aruf is homologous to members of the Ret and FGF receptor subfamilies of RTKs. However, Aruf does not have the extracellular domains typical of those receptors. Furthermore the degree of identity between Aruf and either the Ret or FGF receptors is closer to that between members of different RTK subfamilies, than between members of the same subfamily. Therefore, Aruf encodes an RTK belonging to a novel subfamily related to the Ret/FGFR subfamily.
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Greenspun, David. "Distribution of the sst1 and sst2 receptor mRNAs in the hypothalamus and uptake of somatostatin by neurons in the brain of the adult rat". Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=27329.
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The neuropeptide somatostatin is both a hormone and a neuromodulator; It is the major physiological inhibitor of growth hormone secretion. With the aim of identifying the receptor subtypes through which this neuropeptide may be exerting its neuroendocrine actions in the brain, we have examined by in situ hybridization the distribution of the mRNA for the $ rm sst sb1$ and $ rm sst sb2$ receptors in the hypothalamus of adult male and female rats.Several reports focusing on peripheral cells have indicated that somatostatin can be internalized after binding to membrane associated somatostatin receptors. Moreover, it has recently been reported that the neuropeptides neurotensin and substance P, both of which interact with G protein coupled receptors, can be internalized into central neurons by receptor-mediated endocytosis. In light of these reports, we investigated what role the G protein coupled $ rm sst sb1$ or $ rm sst sb2$ receptors might play in mediating the internalization of somatostatin. (Abstract shortened by UMI.)
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Feldstein, Richard 1974. "Construction of a targeting vector for the analysis of the neuroendocrine function of chromogranin A". Thesis, McGill University, 1999. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=29781.
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Parathyroid hormone and Chromogranin A (CgA), are the two major secreted products of the parathyroid gland. CgA is the major member of the granin family of acidic glycoproteins which are expressed in all endocrine and neural cells. Granins are thought to play a role in secretory granule formation and targeting of peptide hormones and neurotransmitters to granules of the secretory pathway.The CgA gene is a single copy gene, that has been characterized in human, bovine, mouse and rat where it was found to be composed of eight exons with conserved exon-intron boundaries. It has been found that several of the peptides encoded within the CgA molecule inhibit hormone and neurotransmitter release in either an autocrine or paracrine fashion. The biosynthesis of CgA is regulated by many different factors, including steroid hormones and a number of intracellular messenger systems, such as intracellular calcium, Protein Kinase A (PKA) and Protein Kinase C (PKC).
Although a number of studies have been conducted on CgA, the precise role that it plays in neuroendocrine function remains unclear. Therefore, to gain insight into the important functions of CgA, this project involved isolation of the CgA gene through screening of a mouse strain genomic DNA library. Restriction fragments of the phage DNA were subcloned into a plasmid vector, sequenced and were confirmed to contain seven of the eight exons of the mouse CgA gene including, eight kilobases of the 5' flanking region. Through the sequence information obtained, a targeting vector was constructed containing approximately nine kilobases of genomic DNA. This vector in turn, will be used for the functional disruption of the CgA gene in mice through the technique of homologous recombination.
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Cohen, Zvi 1967. "Central serotonin (5-HT) neurons in the control of the cerebral circulation : anatomical basis and functional receptors". Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37543.
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Serotonin (5-hydroxytryptamine, 5-HT) is known to influence cerebrovascular functions such as local cerebral blood flow (CBF) and blood brain barrier (BBB) permeability, and has been implicated in cerebrovascular diseases. The present study used a multifaceted approach to determine the distribution, density and origin of the 5-HT innervation of blood vessels at the base of the brain and overlying the cerebral cortex as well as those embedded in the cortical parenchyma. In addition, the type(s) of 5-HT receptor(s) present on intracortical blood vessels as well as their precise cellular localization within the vessel wall was investigated.Firstly, in extracerebral blood vessels, we showed that perivascular serotonergic nerve fibers, immunocytochemically identified for the 5-HT synthesizing enzyme, tryptophan hydroxylase (TPH), are greatly reduced following removal of the superior cervical ganglia, but not after specific lesion of the ascending 5-HT fibers originating from the brainstem raphe nuclei. In addition, we demonstrated that the distribution pattern of TPH-immunolabelled perivascular fibers differed from those containing noradrenaline (identified by dopamine-beta-hydroxylase). These results suggest the existence of a subset of distinct 5-HT nerve fibers in extracerebral arteries and that the serotonergic innervation; most probably, arises from the superior cervical ganglia or a structure closely related to it.
Secondly, in investigating the serotonergic input to the intraparenchymal microcirculation at the ultrastructural level, we found that central TPH-containing nerve terminals are intimately associated with intraparenchymal blood vessels and that these neurovascular associations were closer and/or more frequent in brain regions where manipulations of the brainstem raphe neurons elicited significant changes, as compared to relatively unresponsive cerebral area. These associations frequently involved the perivascular astrocytes, suggesting a possible intermediary role for these non-neuronal cells in the control of microvascular functions. Furthermore, the associations between 5-HT-synthesizing nerve terminals with the microvascular bed appeared relatively selective since neurovascular noradrenaline nerve terminals in the same cortical subdivision did not share the same characteristics in terms of frequency, intimacy or distribution around the vessel walls.
Finally, in an attempt to identify the exact site(s) of action of 5-HT on the blood vessels, we characterized, via reverse transcriptase-polymerase chain reaction and second messenger assays, the 5-HT receptor(s) present on human intracortical blood vessels as well as in cell cultures of human brain astrocytes and of endothelial and smooth muscle cells of micro-vascular origin. We reported the differential expression not only of messages but also of functional proteins for specific 5-HT receptor subtypes in the different cellular compartments of the blood vessel wall; a finding fully compatible with the ability of 5-HT to regulate microvascular perfusion and BBB permeability.
Altogether, the present thesis provides an anatomical substrate for the 5-HT-mediated responses in the microvascular bed. It demonstrates that the indoleamine can affect the microvascular bed by interacting either directly with endothelial and/or smooth muscle cells or indirectly with the perivascular astroglial cells suggesting that the neuronal-glial-vascular triad most likely constitutes the functional unit in the regulation of microvascular related responses. These studies are likely to contribute significantly to our understanding of the relationships between 5-HT and non-neuronal vascular and astroglial cells as they relate to the mechanisms involved in the regulation of CBF and BBB.
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Giasson, Benoit I. "Regulatory and aberrant phosphorylation of neuronal intermediate filament proteins". Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37546.
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The activation of cyclic AMP-dependent protein kinase (PKA) in rat dorsal root ganglion (DRG) cultures, treated concomitantly with low concentrations of okadaic acid that selectively inhibit protein phosphatase-2A, enhanced the disassembly of neuronal intermediate filaments (IFs). The latter disassembly correlated with phosphorylation of the peripherin head domain and a novel, identified PKA site, Ser-2, in the low molecular mass neurofilament (NF) subunit (NFL). On the other hand. insignificant levels of 32P were incorporated into alpha-internxin under control and experimental conditions that promote disassembly. These findings indicate that phosphorylation of the latter protein is not directly involved in the fragmentation of neuronal IFs Phosphopeptide mapping of the mid-size (NF) subunit (NFM) revealed that 32P-labelling of one of its many phosphopeptides is correlated with neuronal IF fragmentation.The expression and Triton X-100 (Triton) solubility of neuronal IF proteins were determined in the developing rat cerebral cortex. The level of expression of alpha-internexin was unchanged from embryonic day 15 (E15) to postnatal day 15 (P15), whereas expression of (NF) subunits increased during this time interval. NFL was largely insoluble in Triton from the time, P5, when there were sufficient amounts for its solubility to be assayed. There was a continual reduction in the Triton solubility of NFM and alpha-internexin during the E15-P15 developmental period. Similar expression patterns and Triton solubility profiles were obtained for neuronal IF proteins in cultured neurons from E15 cerebral cortex. These results suggest that alpha-internexin is expressed earlier than (NF) proteins to provide a more plastic network in the early developing brain.
Correlative studies and direct, in vivo activation of stress-activated protein kinases (SAPKs) were used to demonstrate that SAPKs are involved in aberrant phosphorylation of the perikaryal high molecular mass (NF) subunit (NFH). It was also shown that hyperphosphorylation of perikaryal NFH is a reversible process that does not involve p38 kinases or extracellular signal-regulated kinases (ERKs). The use of defined peptide substrates indicated that SAPKgamma preferentially phosphorylates KSPXE motifs in NFH. SAPKgamma was shown to be located both in the cell body and neurites of cultured DRG neurons, suggesting that it is likely to be involved in the phosphorylation of cytoplasmic proteins. Collectively, these findings strongly support the notion that activation of SAPKs causes the aberrant hyperphosphorylation of perikaryal NFH reported in many neurological diseases.
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Forghani, Reza. "Transcriptional regulatoin of the myelin basic protein gene in Schwann cells". Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37626.
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Myelination of large caliber axons is an essential step in the development of the vertebrate nervous system. In peripheral nerves, axons instruct ensheathing Schwann cells to elaborate and maintain myelin sheaths, and this is achieved in part through the coordinate up-regulation of genes encoding myelin structural proteins. In order to gain insight into the transcriptional regulation of one such gene, Myelin Basic Protein (MBP), I have investigated MBP 5 ' flanking sequence using a combination of sequence analysis, in vitro DNA-protein interaction assays, and functional analysis in transgenic mice including a high-resolution preparation in which a single copy of a reporter construct is inserted into the HPRT locus. These investigations have located a robust 0.6 kb S&barbelow;chwann c&barbelow;ell e&barbelow;nhancer designated SCE1 that corresponds to a highly conserved human regulatory module. A method to prepare transcription factor containing Schwann cell nuclear extracts was developed and using electrophoretic mobility shift assays with such peripheral nerve extracts, multiple candidate regulatory elements were identified. SCE1 contains all the necessary elements for conferring reporter gene expression to myelinating Schwann cells in vivo and is composed of multiple functional sub-domains with independent targeting capabilities and one sub-domain that appears to positively modulate the level of expression. The 3 kb sequence immediately upstream of SCE1 also can target Schwann cell expression and there is indirect evidence for repressor activity in the proximal promoter that serves to limit expression to Schwann cells during embryonic development. In summary, these investigations demonstrate that the expression of MBP in Schwann cells is achieved through the integrated output of multiple interacting regulatory sequences and the techniques established during the course of these investigations will support future higher resolution in vivo studies of myelin gene r
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Couillard-Despres, Sebastien. "Transgenic mouse models to study the role of neurofilaments in motor neuron disease". Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=37882.
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Neurofilaments are the major type of intermediate filaments found in the adult nervous system of mammalians. Abnormalities of the neurofilament network constitute a common finding of many neurological disorders. For instance depositions of neurofilament aggregates in the perikarya and axons of motor neurons are observed in most amyotrophic lateral sclerosis (ALS) cases, sporadic and familial. The impact of such accumulation of neurofilaments on the course of motor neuron disease remains to be fully elucidated.In order to investigate the role of neurofilaments in motor neuron disease, transgenic mice expressing a mutant form of the Cu,Zn superoxide dismutase (SOD1) were used as an animal model of familial ALS. Increasing the perikaryal neurofilament content in these mutant SOD1 mice slowed down the motor neuron disease progression and increased their life span by up to 65%. To date, this approach constitutes the most efficient way to increase the life span of mutant SOD1 mice. Moreover, increasing the axonal neurofilament content in mutant SOD1 mice by human neurofilament-light subunit (hNF-L) overexpression demonstrated that axonal neurofilaments do not constitute an exacerbating factor in the neurodegeneration caused by mutant SOD1.
The pathogenicity of human neurofilament-heavy (hNF-H) proteins expressed in transgenic mice was also investigated. Two alleles of the NF-H gene are present in the normal human population. Expression of both alleles in transgenic mice provoked motor neuron dysfunction. The adverse property of NF-H overexpression is the result of an improper stoichiometry between the NF-L and the NF-H subunits. Restoration of an adequate stoichiometry, via the co-expression of NF-L and NF-H subunits, rescued mice from the motor neuron dysfunction. Finally, expression of the allele called NFH43, bearing less phosphorylation sites than the other allele called NFH44, was shown to be more pathogenic in transgenic mice.
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Bouchard, Pascale. "In vivo interactions between sigma receptors and NPY- & CGRP-relates peptides in the brain". Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41986.
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The existence of sigma ($ sigma$) receptors have been proposed by Martin and co-workers (1976) as a new class of opioid receptors to explain certain "psychotomimetic-like" behaviors induced by some benzomorphans (N-allylnormetazocine, pentazocine) in the chronic spinal dog. Later on, the observation that the typical opioid antagonist, naloxone, failed to antagonize some of these behavioral effects led to a reconsideration of the nature of $ sigma$ receptors. It was determined that although several $ sigma$ ligands displayed reasonable affinity for the phencyclidine (PCP) receptor and that their activation could mimic PCP "psychotomimetic-like" effects in animals, $ sigma$ receptors were a different identity and shared no commonality with opioid or PCP receptors. Since then, $ sigma$ receptors have generated a great deal of interest mostly in relation to their possible implication in psychosis like schizophrenia. More recently, $ sigma$ sites have been associated with various other functions such as the modulation of posture and movement, neuroprotection, anxiety and depression, pain control, drug abuse, regulation of endocrine and immune functions, and cognitive behaviors.Although evidence for the existence of an endogenous $ sigma$ ligand have been reported, the full characterization of such putative endogenous ligand(s) has yet to be achieved. Roman and co-workers (1989) proposed that neuropeptide Y (NPY) and peptide YY (PYY) could act as endogenous $ sigma$ ligands since both NPY and PYY competed with high affinity (nM) for $ rm lbrack sp3 H rbrack ({+})SKF 10,047$ binding sites in rat brain membrane homogenates. However, various laboratories failed to replicate these in vitro findings. In order to clarify this apparent discrepancy, the present thesis investigates the possibility of the existence of in vivo interaction(s) between $ sigma$ receptors and various NPY-related peptides. We first demonstrated that NPY could interact with $ sigma$ sites in vivo, since certain NPY-related peptides could, to some extent, compete for in vivo $ rm lbrack sp3 H rbrack ({+})SKF 10,047$/$ sigma$ binding in the mouse hippocampal formation (Chapter 2). In the course of a specificity study, we found that the calcitonin gene-related peptide (CGRP) also potently competed for in vivo $ rm lbrack sp3 H rbrack ({+})SKF 10,047$/$ sigma$ binding in the mouse hippocampus. We thus included CGRP-related peptides in subsequent studies and found that certain CGRP-related peptides significantly competed for $ rm lbrack sp3 H rbrack ({+})SKF 10,047$/$ sigma$ binding in the mouse hippocampus, in a manner similar to NPY (Chapter 3). Using an ex vivo autoradiographic approach, we further demonstrated that NPY- and CGRP-related peptides could interact with $ sigma$ receptors not only in the hippocampus but in most, if not all, brain areas enriched with $ sigma$ sites (Chapter 4). Finally, the behavioral relevance of these interactions was demonstrated. We found that various NPY- and CGRP-related peptides attenuated learning impairments induced by the systemic administration of the non-competitive NMDA-receptor antagonist MK-801. These effects were blocked by the