Tesis sobre el tema "GLRA2"

Des variants pathogènes du gène GLRA2 codant pour la sous-unité α2 du récepteur glycinergique ont été récemment impliqués dans le trouble du spectre autistique (TSA) et la déficience intellectuelle. Notre groupe a précédemment montré que les souris mâles déficientes en Glra2 (Glra2–/Y) présentent un déficit de mémoire lors du test de reconnaissance d’objets (TRO) et une altération de la plasticité synaptique du cortex préfrontal (CPF), une région fortement impliquée dans le TSA. Par ailleurs, des études développementales des souris Glra2–/Y ont décrit un défaut de migration des interneurones et une perte de neurones de projection corticaux associée à une microcéphalie. Dans ce projet, nous avons recherché les altérations cellulaires et fonctionnelles qui sous-tendent les défauts comportementaux et synaptiques des souris Glra2–/Y, en se focalisant sur le CPF. Contrairement à ce qui a été rapporté précédemment, les souris Glra2–/Y ne sont pas microcéphales et ne présentent ni perte globale de neurones excitateurs ou inhibiteurs, ni perte de sous-populations d’interneurones à parvalbumine, calrétinine ou cholécystokinine dans le CPF ou le cortex somatosensoriel. Cependant, le nombre d'interneurones corticaux à somatostatine est augmenté chez les souris Glra2–/Y. Ces résultats démontrent un rôle plus subtil de Glra2 dans le développement du cortex que ce qui avait été suggéré auparavant, cohérent avec le phénotype des patients mâles porteurs de mutations dans GLRA2. Au moyen d’approches anatomiques et électrophysiologiques, nous avons également mis en évidence chez les souris Glra2–/Y des altérations caractéristiques des troubles neurodéveloppementaux, retrouvées dans d'autres modèles murins du TSA. Dans le CPF, ces souris mutantes présentent une diminution du nombre de synapses inhibitrices, une augmentation de la densité des épines et de la complexité dendritique des neurones pyramidaux, et une augmentation de l’activité synaptique excitatrice des neurones pyramidaux, sans effet sur la transmission synaptique inhibitrice. L'ensemble de ces résultats révèle l’existence d’un déséquilibre de la balance excitation/inhibition dans le cortex des souris Glra2–/Y. Afin d'identifier les régions cérébrales impliquées dans le déficit cognitif des souris Glra2–/Y, nous avons quantifié les neurones exprimant c-Fos, un marqueur d’activation neurale, après le TRO. Nous avons mis en évidence une hypoactivation sélective du CPF infralimbique rostral chez les souris Glra2–/Y après cette tache. Par colocalisation de c-Fos avec des marqueurs neuronaux, nous avons montré que cette hypoactivation était due à un déficit d’activation des neurones glutamatergiques. Pour évaluer plus finement l'activité neuronale dans le CPF des souris Glra2–/Y lors de l'apprentissage, nous avons mesuré l’activité des neurones glutamatergiques du cortex infralimbique par photométrie de fibre à l’aide d’un senseur calcique, pendant le TRO. Chez les souris sauvages, l'exposition répétée à des objets pendant la phase d'entraînement induit une réduction progressive de l'activité calcique, alors que cette atténuation est absente chez les souris Glra2–/Y, renforçant l’implication d’une altération de l'activité des neurones excitateurs dans le déficit cognitif de ces souris. En outre, malgré l'absence de déficits sociaux apparents, la réponse calcique des neurones glutamatergiques à la nouveauté sociale est atténuée chez les souris Glra2–/Y. Dans leur ensemble, ces résultats montrent que des altérations subtiles des circuits préfrontaux chez les souris Glra2–/Y engendrent une altération de la balance excitation/inhibition et un dysfonctionnement des neurones glutamatergiques dans le CPF lors du TRO, entraînant un déficit de mémoire. Ils suggèrent que la sous-unité α2 est cruciale pour le développement normal du CPF, et que des défauts des circuits préfrontaux pourraient sous-tendre le dysfonctionnement neurocognitif observé chez les patients présentant une perte de GLRA2
Pathogenic variants in the GLRA2 gene, which encodes the glycine receptor α2 subunit, have been recently implicated as a novel cause of autism spectrum disorder (ASD) and intellectual disability. Our group previously showed that Glra2-deficient male (Glra2 /Y) mice display impaired learning and memory in the novel object recognition (NOR) task and altered synaptic plasticity in the prefrontal cortex (PFC), a region consistently implicated in ASD. In addition, developmental studies in mice expressing the same Glra2 mutation reported deficits in interneuron migration and loss of cortical projection neurons associated with microcephaly. In this project, we investigated the cellular and functional alterations underlying the behavioural and synaptic defects of Glra2 /Y mice, focusing on the PFC. In contrast with previous reports, Glra2 /Y mice were not microcephalic and neuronal quantification showed no loss of either glutamatergic neurons or interneurons, including parvalbumin, calretinin and cholecystokinin interneuron subpopulations in the PFC or the somatosensory cortex. However, the number of cortical somatostatin interneurons was increased in these regions in mutant mice. These findings imply that Glra2 plays a more subtle role in neocortical development and assembly than previously suggested and are consistent with the phenotype of male patients with pathogenic GLRA2 variants, who are not microcephalic and have normal brain imaging. We also show that Glra2 /Y mice exhibit many of the hallmarks of neurodevelopmental brain dysfunction observed in other rodent models of ASD. In the adult PFC, Glra2 /Y mice show a decreased number of inhibitory synapses and increased spine density and dendritic complexity of pyramidal neurons, whilst young mice (P14-P21) have increased excitatory synaptic inputs to prefrontal pyramidal neurons, with no effect on inhibitory synaptic transmission. Taken together, these findings point to excitatory hyperconnectivity in the PFC of Glra2 /Y mice, and suggest an imbalance of excitatory and inhibitory neurotransmission in these mutant mice. To identify which brain regions are associated with the recognition memory deficit observed in Glra2 /Y mice, we quantified c-Fos expression as a marker of neuronal activation following NOR. We found that the rostral infralimbic PFC was hypoactivated in Glra2 /Y mice following this task, whilst other brain regions quantified showed similar levels of c-Fos expression compared to wild-type mice. c-Fos colocalization with neuronal markers revealed that the hypoactivation of the PFC was driven by impaired activation of glutamatergic neurons following the task. To further assess neuronal activity in the PFC in Glra2 /Y mice during cognition, we recorded calcium transients from infralimbic glutamatergic neurons using in vivo fiber photometry during NOR, and compared them with the calcium response induced by social interaction with a novel mouse. In wild-type animals, repeated exposure to objects during the training phase of the NOR task caused a progressive reduction in calcium-dependent neuronal activity during exploration. This attenuation of the calcium signals was absent from Glra2 /Y mice, further implicating an impairment of prefrontal glutamatergic activity in the NOR deficit observed in this model. In addition, despite a lack of apparent social deficits, Glra2 /Y mice exhibited an attenuated glutamatergic calcium response to novel social stimuli in the PFC. Overall, these findings show that subtle alterations in prefrontal circuit organization and physiology in Glra2 /Y mice result in altered inhibitory/excitatory balance and an aberrant response of prefrontal glutamatergic neurons during recognition memory leading to impaired task performance. These results suggest that the glycine receptor α2 subunit is crucial for normal PFC development, and that defects in prefrontal circuits may underlie the neurocognitive dysfunction observed in patients lacking GLRA2

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Glutamate receptors are the mediators of most excitatory neurotransmission processes in the central nervous system, acting as prominent targets for the treatment of several neurological disorders such as Epilepsy, Amyotrophic Lateral Sclerosis, Parkinson’s disease and Alzheimer’s disease. Hence an improved understanding of how glutamate and other ligands interact with the binding domain, of these receptors, can bring relevant insights to the development of new ligands. Therefore, this work aims to study the GluA2–ligand interaction using the structure of GluA2 co-crystallized with the ligands glutamate, AMPA, kainate and DNQX applying a method based on the Density Functional Theory combined with the molecular fractionation with conjugate caps scheme. To address that the dielectric constant of the GluA2 receptor is not homogeneous, a novel molecular approach was proposed and it was applied to study the interaction between the GluA2 and the ligands glutamate, AMPA, kainate and DNQX. The results obtained, considering the inhomogeneous model, were compared with those obtained using an uniform dielectric function for the GluA2 receptor and with data published in the literature establishing a more detailed description of the relevant amino acid residues for the protein-ligand binding interaction. Molecular dynamics studies and protein DFT calculations usually consider a fixed value for the protein dielectric function. In this work when ε = 1 is considered, many amino acid residues seem important, but when the dielectric constant shield was considered, they lost their relevance. The results for the GluA2-ligand total interaction energy and the D1-ligand and D2-ligand total interaction energy also shed some light on the differentiation between full and partial agonists, and between agonists and antagonists. Additionally, the results allow a hypothesis on the correlation between the Glu705-ligand interaction energy and the ligand action, paving the way for the use of the inhomogeneous dielectric function to study glutamate receptors and other protein-ligand systems. Finally, the results also suggests that for different ligands, different homogeneous dielectric constant will be able to well represent the system GluA2-ligand, making it necessary the previous analyses with the inhomogeneous dielectric constant approach.
Os receptores de glutamato são os mediadores da maioria dos processos de neurotransmissão excitatória no sistema nervoso central, atuando como alvos proeminentes para o tratamento de vários distúrbios neurológicos, como Epilepsia, Esclerose Lateral Amiotrófica, Doença de Parkinson e Doença de Alzheimer. Assim, uma compreensão aprimorada de como o glutamato e outros ligantes interagem com o domínio de interação, desses receptores, pode trazer informações relevantes para o desenvolvimento de novos ligantes. Portanto, este trabalho teve por objetivo estudar a interação GluA2-ligante utilizando a estrutura de GluA2 co-cristalizada com os ligantes Glutamato, AMPA, Cainato e DNQX utilizando método baseado na Teoria do Funcional da Densidade combinado com o esquema de fracionamento molecular com capas conjugadas. Para abordar que a constante dielétrica do receptor GluA2 não é homogênea, foi proposta uma nova abordagem molecular, que foi aplicada para estudar a interação entre a GluA2 e os ligantes Glutamato, AMPA, Cainato e DNQX. Os resultados obtidos, considerando o modelo não-homogêneo, foram comparados com aqueles obtidos usando uma função dielétrica uniforme para o receptor GluA2 e com dados publicados na literatura, estabelecendo uma descrição mais detalhada dos resíduos de aminoácido mais relevantes para a interação proteína-ligante. Estudos de dinâmica molecular e cálculos DFT de sistemas proteicos normalmente consideram um valor fixo para a função dielétrica proteica. Nesse trabalho quando ε = 1 é considerado, muitos resíduos de aminoácido parecem relevantes, mas quando a blindagem da constante dielétrica foi considerada, eles perderam sua relevância. Os resultados apresentados para a energia de interação total GluA2-ligante e a energia de interação total D1-ligante e D2-ligante contribuiu com a diferenciação entre agonistas totais e agonistas parciais e entre agonistas e antagonistas. Além disso, os resultados permitem que seja feita hipótese sobre a correlação entre a energia de interação Glu705-ligante e a ação do ligante, abrindo caminho para o uso da função dielétrica não-homogênea para estudar receptores de glutamato e outros sistemas proteína-ligante. Por fim, os resultados também sugerem que para diferentes ligantes, diferentes constantes dielétricas homogêneas serão capazes de representar bem o sistema GluA2-ligante, tornando necessária a análise prévia com a abordagem da constante dielétrica não-homogênea.

Grapevine leafroll-associated virus-2 (GLRaV-2), GLRaV-3, and grapevine fleck virus (GFkV) are widespread in grapes around the world. These viruses can cause significant crop loss and affect wine quality by reducing sugar accumulation and compromising skin color. Mealybugs are vectors of grapevine leafroll-associated viruses (GLRaVs). A statewide survey of commercial and wild grapevines in Virginia was conducted during 2009 through 2011. Also, vector management options were tested in two field studies. GLRaV-2, GLRaV-3, and GFkV were detected in 8%, 25%, and 1%, respectively, of over 1,200 vine samples (41 wine grape varieties) from 77 locations, and 64% of vineyards were positive for at least one of the tested viruses. All 100 wild grapevines tested were free of these three viruses, indicating that they are not alternative hosts. The majority of infected vines from commercial vineyards were planted prior to the 1990\'s; however, some new plantings were also found to be positive, indicating movement of the viruses among vineyards and also potential infection prior to planting. The high frequency of virus-infected vines emphasizes the importance of clean plant materials, as well as management of vector insects. The insecticide trials resulted in promising vector control with dinotefuran and spirotetramat; however, acetamiprid and pryrethroid resulted in an increase in mealybug population. This study is the first to examine multiple grape viruses in VA. It will aid in developing better strategies aimed at controlling mealybugs to restrict the movement of viral diseases.
Master of Science in Life Sciences

Le synaptic scaling est une forme de plasticité homéostatique par lequel les synapses ajustent leur efficacité pour compenser des variations normales ou pathologiques de l'activité neuronale notamment lors des maladies neurodégeneratives ou suite à la perte d’afférences sensorielles après une lésion. Dans un modèle expérimental classique, le traitement chronique des neurones primaires avec la tétrodotoxine (TTX) pour bloquer la propagation des potentiels d'action présynaptiques induit une augmentation significative de l'amplitude des courants miniatures excitateurs transmis par les récepteurs du glutamate AMPA postsynaptiques. Plusieurs voies de signalisation ont été proposées, dont celle impliquant les microARNs (miRs), de petits ARN non-codants qui inhibent la traduction des protéines en se liant aux ARN messagers cibles. Dans ce contexte, nous avons exploré l'hypothèse que le microARN, miR-124, fortement exprimé dans le cerveau, pourrait être un régulateur important de l'homéostasie synaptique en contrôlant l'expression de la protéine synaptopodine, une protéine structurante des épines dendritiques et indispensable à l'expression du synaptic scaling.En combinant des approches de RTq-PCR, d'immunocytochimie et d'électrophysiologie in vitro, nous avons montré dans un premier temps que la privation globale de l'activité des neurones primaires d’hippocampe diminuait le niveau d'expression de miR-124 et augmentait celui de la synaptopodine et des récepteurs AMPA dont la sous-unité GluA2 est une autre cible de miR-124. Par ailleurs, en rendant des synapses individuelles inactives via l’expression présynaptique de la toxine tétanique, nous avons observé que le recrutement synaptique des récepteurs AMPA et de la synaptopodine était spécifique de ces synapses, suggérant une régulation homéostatique locale. Dans un deuxième temps, nous avons trouvé que la surexpression de miR-124 ou l’inhibition de son interaction avec l’ARNm de la synaptopodine ou de GluA2 bloquaient la réponse synaptique homéostatique induite par le traitement TTX. Enfin, des expériences de FRAP ont suggéré que la synaptopodine influençait le trafic des récepteurs AMPA à la membrane probablement en les stabilisant à la synapse, ce qui expliquerait ainsi son rôle pendant la plasticité homéostatique
Synaptic scaling is a form of homeostatic plasticity where synapses adjust their own efficacy to compensate for normal or pathological variations in neuronal activity such as neurodegenerative disorders or sensory deprivation after a lesion. In a well-established paradigm, the chronic application of tetrodotoxin (TTX) in primary neurons, to block presynaptic action potential propagation, induces a significant upscaling of miniature excitatory postsynaptic currents mediated-AMPA receptors. Numerous regulators of this plasticity have been identified including microRNAs (miR), which are small endogenous non-coding RNAs, inhibiting protein translation by binding to mRNA targets. This led us to hypothesize that the most highly expressed microRNA in the brain, miR-124, could be an important regulator of homeostatic scaling by controlling the expression of synaptopodin, a structural protein of dendritic spines playing a crucial role in homeostatic plasticity.By combining qRT-PCR, immunocytochemistry and in vitro electrophysiology approaches, first we showed that a global 48hrs TTX treatment in hippocampal primary neurons led to a decrease in miR-124 level and an increase in the expression of synaptopodin and synaptic AMPA receptors containing the GluA2 subunit which is another miR-124 target. Moreover, we observed that the synaptic accumulation of AMPA receptors and synaptopodin could be synapse-specific by expressing the tetanus toxin to block the activity of individual presynapses, which suggested a local homeostatic regulation. Importantly, we found that overexpressing miR-124 or inhibiting its interaction with synaptopodin or GluA2 mRNAs blocked the synaptic homeostatic response. In addition, FRAP experiments suggested that synaptopodin controlled AMPA receptor trafficking at the membrane by probably retaining them in dendritic spines, which could explain its role during homeostatic plasticity

L’enroulement de la vigne cause des pertes de rendement et de la qualité des vins au niveau mondial. Il est causé par quatre espèces de Grapevine leafroll-associated virus(GLRaVs) ; GLRaV-1, -2, -3, -4-like. Si le GLRaV-2 (genre Closterovirus) ne possède pas de vecteur connu, les trois autres espèces (genre Ampelovirus) sont transmises par cochenilles(Coccoidea) qui contribuent à leur dispersion dans et entre les vignobles. Les vignobles de la France septentrionale (Alsace, Bourgogne, Champagne) sont les plus impactés par l’enroulement viral. Ce travail a porté sur le rôle vecteur de Phenacoccus aceris, espèce connue pour son efficacité de transmission et de dispersion des ampélovirus, ainsi que de vitivirus souvent associés. Les interactions cellulaires et moléculaires entre virus et cochenille restent peu connues. Une approche pluridisciplinaire combinant entomologie,virologie, biologie cellulaire et moléculaire a été développée pour étudier la biologie de la vection du GLRaV-1 et du Grapevine virus A (GVA) par P. aceris. Des expériences de transmission ont montré que ces virus sont transmis selon le mode semi-persistant non circulant. L’étude préliminaire du comportement alimentaire de P. aceris sur vigne par électropénétrographie a révélé une activité similaire à celle d'autres espèces de cochenilles déjà décrites et suggère un effet de l’infection sur le comportement alimentaire. L’anatomie des pièces buccales de P. aceris, organes directement impliqués dans la transmission et la rétention de virus non-circulants a été décrite et une méthode basée sur l’acquisition de virus purifié sur membrane a été développée pour rechercher les sites de rétention virale dans le vecteur
Grapevine leafroll disease affects grape yield and wine quality worldwide. It is caused by four species of Grapevine leafroll-associated virus (GLRaVs) (GLRaV-1, -2, -3, -4-like).While GLRaV-2 (genus Closterovirus) has no known vector, the other three (genus Ampelovirus) are transmitted by mealybugs (Coccoidea) and thus prone to be dispersed within and between vineyards. In north-eastern France (Alsace, Bourgogne et Champagne),vineyards are more impacted by Grapevine leafroll disease. This thesis focusses on the vector role of the species Phenacoccus aceris, known for its efficiency in transmission and dissemination of ampeloviruses, as well as often associated vitiviruses. Molecular and cellular interactions between viruses and mealybugs remain poorly known. A multidisciplinary approach, combining entomology, virology, molecular and cellular biology, was developed to analyse the vector biology of GLRaV-1 and Grapevine virus A (GVA) by P. aceris.Transmission experiments showed that GLRaV-1 and GVA transmission follows the semipersistent non-circulative mode. A preliminary study of P. aceris feeding behavior on grape using electropenetrography revealed an activity similar to that of other mealybug species already described and suggested a potential effect of infection on Ph. aceris feeding behavior. The anatomy of mouth parts, directely implied in transmission and retention of non circulative viruses was described and a method for membrane acquisition of purified virus was developed to search for virus retention sites within the vector

Nrf2 project: The protein nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that provides protection against oxidative stress and the dysfunction of this pathway has been suggested to be implicated in many neurodegenerative diseases. The aim of this thesis was to identify novel Nrf2 activators that disrupt the protein-protein interaction between Nrf2 and Keap1 and thereby induce increased expression of antioxidant enzymes and protective genes. The crystal structure of the Keap1-Nrf2 interface was used to perform a virtual screen and compounds from the screen were assayed using a cellular nuclear complementation assay that measures the nuclear translocation of Nrf2 from the cytosol. Although two novel compounds were found to increase the Nrf2 nuclear translocation, they had low activity and further characterisation did not provide sufficient evidence of a Nrf2-Keap1 robust interaction. iGluRs project: AMPA and kainate receptors are ionotropic glutamate receptors (iGluRs) that are important for excitatory transmission and synaptic plasticity and are linked to several neurological disorders such as epilepsy, schizophrenia and autism. This project aimed to find novel allosteric modulators binding in the ligand-binding domain (LBD) of the GluA2 and GluK1 and GluK2 subtypes of AMPA and kainate receptors, respectively, using protein purification and X-ray crystallography methodologies. Fragment screening for GluA2 identified eight novel fragments, five of which were located at the dimer interface and three located in a novel site near the glycine-threonine dipeptide linker. As regards kainate receptors, structural information on the Gluk1 and GluK2 LBD was obtained, both proteins were soaked with in-house fragments with one compound displaying 20% occupancy in the GluK2 dimer interface. These data form the basis of future studies in the search for novel drugs for the treatment of epilepsy and schizophrenia.

Les virus de l'enroulement de la vigne (Grapevine leafroll-associated virus, GLRaV) sont répandus mondialement et transmis à la vigne uniquement par cochenilles (Coccoidea). En France, l'enroulement viral affecte particulièrement les vignobles des régions septentrionales.L'approche biologique de la vection a montré la capacité de Phenacoccus aceris à transmettre à la vigne les GLRaV-1, -3, -4, -5, -6, -9 et ceux du bois strié Grapevine virus A et B. Cette étude est la première démonstration de la transmission du GLRaV-6 et confirme l'absence de spécificité des cochenilles dans la transmission des Ampelovirus. Les larves néonates de P. aceris et de Neopulvinaria innumerabilis représentent un stade de développement efficace pour la transmission de ces virus. En conséquence, leurs capacités vectrices, associées à leur fort potentiel de dissémination anémophile, impliquent un risque important de dispersion naturelle de ces virus dans un vignoble infesté. Les relevés sur quatre parcelles distinctes montrent que Parthenolecanium corni, Pulvinaria vitis, Heliococcus bohemicus et P. aceris sont communes, chaque vignoble différant par la diversité spécifique, le taux de ceps infestés et l'abondance des cochenilles. L'étude épidémiologique prouve le rôle des cochenilles dans la dispersion de l'enroulement viral dans les vignobles septentrionaux. A Bonzon, la responsabilité de P. aceris dans la diffusion rapide du GLRaV-1 est mise en évidence. Cette découverte représente la première preuve en Europe d'une dispersion naturelle du GLRaV-1. A Marsannayla-Côte, l'incidence du GLRaV-1 reste faible, la colonie de P. aceris ne semblant avoir qu'un rôle très limité dans la diffusion de la maladie. L'épidémiologie moléculaire à Bonzon révèle une diversité génétique importante du GLRaV-1 à l'échelle parcellaire et fournit pour la première fois des données sur le polymorphisme génétique d'une population de GLRaV-1 ayant été dispersée par des cochenilles.

Thesis (PhD (Genetics))--University of Stellenbosch, 2011.
Includes bibliography
ENGLISH ABSTRACT: Grapevine diseases, in particular virus and virus-like diseases, are threatening grapevine industries worldwide; also in South Africa. Grapevine leafroll (GLR) is one of the most important diseases of grapevines, occurring in all grape-producing countries worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is known to be closely associated with GLR disease and occurs commonly in South African vineyards. In this study three genetic variants of GLRaV-3 were identified in vineyards of the Western Cape, South Africaby single strand conformation polymorphism (SSCP) profiles generated from a region amplified in ORF5. A specific SSCP profile could be assigned to each variant group and these wereconfirmed by sequencing of the ORF5 regions.These results demonstrated that SSCP analysis on this region in ORF5 provides a fast and reliable indication of the GLRaV-3 variant status of a plant, which in many instances showed mixed infections. The full genome sequence of one representative of each variant group i.e. isolates 621 (group I), 623 (group II) and PL-20 (group III), was determined by sequencing overlapping cloned fragments of these isolates. The sequences of genomic 5’ ends of these isolates were determined by RLM-RACE. Sequence alignment of the 5’UTRs indicated significant sequence and length variation in this region, between the three South African variant groups. Nucleotide sequence alignment of the Hsp70h and CP gene regions of these isolates with those of isolates from elsewhere in the world, followed by phylogenetic analysis, further supported the presence of three GLRaV-3 variants in South Africa, and that two or three additional variant groups occurs elsewhere in the world. We further investigated the prevalence of these three GLRaV-3 variants in mother blocksof different cultivars and from different vine growing regions, using SSCP analysis. The majority of the plants studied, were infected with the group II variant, similar to isolates 623 and GP18. The distribution of the three GLRaV-3 variants within a spatio-temporally recorded cluster of diseased plants was studied by means of SSCP profile analysis. We showed that different GLRaV-3 variants are transmitted to adjacent plants in an infection cluster. Results showed that, in some leafroll disease clusters, the variant that was present in the original GLRaV-3 infected plant of a cluster was transmitted to adjacent plants in a row and across rows. Some plants in the cluster were also infected with variants not present in the original plant. These infections could have been caused by mealybug vectors feeding on plants from surrounding areas and then infecting these plants. The scientific information generated on GLRaV-3 variants in this project contributed to the advancement of our knowledge of genetic variability and provides a basis of further epidemiology and vector-virus studies. The study showed for the first time that different GLRaV-3 variants were transmitted to adjacent plants in a row and across rows in a GLR disease cluster. The diversity detected in the 5’UTR between variants from the three genetic groups provides a platform for the further study of the biological characteristics of GLRaV-3 variants.
AFRIKAANSE OPSOMMING: Wingerdsiektes, veral virus siektes, bedreig wingerd industrieë wêreldwyd, asook die Suid Afrikaanse wingerdbedryf. Rolbladsiekte is een van die belangrikste siektes op wingerd en kom wêreldwyd voor. Die virus, grapevine leafroll-associated virus 3 (GLRaV-3), word sterk geassosieer met Rolbladsiekte en kom wydverspreid voor in Suid Afrikaanse wingerde. Tydens hierdie studie is drie genetiese variante van GLRaV-3 geïdentifiseer in wingerd moederblokke in die Wes-Kaap. Die GLRaV-3 variante is geïdentifiseer met ‘n tegniek wat ‘single-strand conformation polymorphism (SSCP)’ genoem word. Die SSCP profiele was gegenereer vanaf PKR produkte van die ORF5 area op die genoom van GLRaV-3. Die geamplifiseerde produk van die ORF5 gebied is gebruik om die SSCP profiele te verkry en DNA-volgorde data in die gebied het die drie SSCP profiele gestaaf. Hierdie metode om virus variasie te bestudeer in plante is vinnig en betroubare resultate is verkry. Gemengde infeksies, wat gereeld in wingerd voorkom, kon ook met die tegniek opgespoor word. Die volledige nukleotied-volgorde van elkeen van die drie GLRaV-3 genome is volledig bepaal. Die isolate wat die drie variant groepe verteenwoordig is isolaat 621 (groep I), 623 (groep II) en PL-20 (groep III). Die nukleotiedvolgorde in die 5’UTR is bepaal met die RLM-RACE tegniek. Wanneer die 5’UTRs van die drie variante vergelyk is, het dit getoon dat daar verskille is in die volgordes en lengtes voorgekom het. Ander dele van die genoom, o.a. die dopproteïen (CP) en Hsp70 areas, is filogeneties vergelyk met isolate van regoor die wêreld. In die filogenetiese analise is bevind dat die drie GLRaV-3 variante saamgegroepeer het met ander isolate in die wêreld en dat daar elders ook twee to drie addisionele variant groepe van GLRaV-3 voorkom. Die verspreiding van die drie GLRaV-3 variante in wingerde is bestudeer in verskillende kultivars en in verskillende verbouingsgebiede. Die meerderheid van die plante in die studie was geïnfekteer met die groep II variant wat dieselfde is as isolate 623 en GP18. Die voorkoms van die drie variante in ‘n siekte cluster is bestudeer d.m.v SSCP. Die studie het gewys dat verskillende GLRaV-3 variante versprei word na aangrensende plante in ‘n ry en tussen rye. In sommige gevalle is die variant wat in die oorspronklik geïnfekteerde plant voorkom, oorgedra na naasliggende plante. Sommige van die plante in the infeksie area was ook met ander GLRaV-3 variante geïnfekteer wat moontlik deur wolluise oorgedra is vanaf naburige geïnfekteerde plante. Die wetenskaplike inligting wat tydens hierdie studie beskryf word aangaande die identifikasie van GLRaV-3 variante, dra by tot die molekulêre kennis van GLRaV-3 en verskaf ‘n basis vir verdure epidemiologiese -en insek oordragingstudies. Die studie het vir die eerste keer bewys dat verskillende GLRaV-3 variante na aanliggende plante in ‘n ry asook oor rye oorgedra word. Die diversiteit tussen die GLRaV-3 variant groepe in die 5’UTR moet verder ondersoek word en die deel van die genoom kan ‘n belangrike rol speel in die biologiese eienskappe van die variante.

Early-life stress is associated with maladaptive long-lasting brain effects. Such alterations increase the likelihood of developing several psychiatric disorders. However, the molecular consequences of early-life stress are poorly understood. Here, we evaluated the impact of maternal separation with early weaning (MSEW) at different phases of cocaine self-administration (SA) and the subsequent molecular alterations in brain reward regions of male and female mice. Our findings show that MSEW affects males while females appear to be resilient to this kind of stress. Maternally separated mice evidence higher percentage of acquisition, more cocaine intake and decrease capacity to extinguish the SA behaviour. Moreover, molecular analyses of the brain areas studied reveal sex-induced alterations in the AMPA receptor composition and MSEW-induced changes in the expression of GluA1, GluA2, pCREB and CREB. Cocaine also alters the expression of these molecules. Together, results suggest that MSEW induces molecular alterations in areas related to reward processing, potentiating the vulnerability to cocaine-seeking behaviour and depression.
El estrés en la etapa temprana de la vida se asocial con efectos cerebrales maladaptativos y duraderos. Dichas alteraciones pueden aumentar la probabilidad de desarrollar diversos trastornos psiquiátricos. Sin embargo, las consecuencias moleculares del estrés en la vida temprana son poco conocidas. En este trabajo evaluamos el impacto de la separación maternal con destete temprano (MSEW, por sus siglas en inglés) en diferentes fases de la autoadministración de cocaína, así como las posteriores alteraciones moleculares en regiones cerebrales asociadas al sistema de recompensa, en ratones machos y hembras. Nuestros resultados muestran que la MSEW afecta a los machos, mientras que las hembras parecen ser resistentes a este tipo de estrés. Los ratones separados maternalmente muestran un mayor porcentaje de adquisición, más consumo de cocaína y una menor capacidad para extinguir el comportamiento de autoadministración. Además, los análisis moleculares de las áreas cerebrales estudiadas revelan alteraciones inducidas por el sexo en la composición del receptor AMPA y cambios inducidos por la MSEW en la expresión de GluA1, GluA2, pCREB y CREB. La cocaína también altera la expresión de estas moléculas. Los resultados en conjunto sugieren que la MSEW induce alteraciones moleculares en áreas relacionadas con el procesamiento de la recompensa, potenciando la vulnerabilidad al comportamiento de búsqueda de cocaína y la depresión.

Worldwide, agriculture production continues to be constrained by a number of abiotic and biotic stresses. In areas with Mediterranean climate, drought events are expected to strengthen in terms of intensity, frequency and duration in the near future. Thus, in this area, soil water deficit is considered the main environmental factor influencing grapevine growth. At the same time, grapevines are highly subjected to different viruses from which Grapevine leafroll associated virus-3 (GLRaV-3) is one of the most widespread ones, provoking considerable economic losses in many vineyards worldwide. The identification of how different stress responses are integrated and how they affects plant growth and physiological traits, is therefore very important to ensure the continuity of vine growing. The first main objective of the present thesis was to study the sanitary status of Majorcan minority grapevines cultivars and to highlight the prevalence of GLRaV-3 in local cultivars. In this sense, the results showed that virus incidence is very high and can rise up (91.75 %) in the local grapevine cultivars conserved in the germplasm collection of the Balearic Islands. Remarkably, GLRaV-3 is the most prevalent virus in this collection (82 %). This situation urges a speedy implementation of the sanitation techniques in order to obtain virus-free certified plants. Hence, two sanitation techniques ―shoot tips culture and thermotherapy in combination with shoot tips culture‖ have been optimized for double and triple viruses‘ eradication from two local cultivars with high enological potential, Argamussa and Gorgollassa. The second main objective was to dissect the effects of virus infection, moderate water stress and its combination on main physiological processes in two local grapevine cultivars, Malvasia de Banyalbufar and Giró Ros. Each of the two studied stresses resulted in a negative effect on leaf gas-exchange parameters. Water stress in non-infected plants (WS-NI) significantly reduced plant growth and net CO2 assimilation (AN) but had only small effect on metabolic changes, indicating that AN changes were mainly constrained by diffusive parameters (stomatal (gs) and mesophyll (gm) conductance‘s). With regard to virus infection under well-watered conditions (WW-VI), the results also revealed that GLRaV-3 impaired AN through diffusional limitations rather than biochemical ones. In this case, stomatal conductance was mainly regulated by changes in hydraulic conductance at leaf level, namely leaf (Kleaf) and petiole (Kpetiole) hydraulic conductance, in which Kpetiole resulted to be the most relevant parameter to be checked when studding the effect of phloemetic viruses in plant hydraulics. Remarkably, the results of this thesis indicate that the combined stress resulted in a detrimental effect on plant growth and physiology; but any additive effect between WS and VI has been highlighted. Moreover, the combination of stresses triggers a specific response at the metabolic level, which was not quantitatively predicted by the sum of each individual stress. The observed changes in primary metabolites were closely associated with respiration metabolisms. Indeed, the specific adjustment of respiratory metabolism (i.e; cysteine, threonine, fumarate and erythronate 1,4-lactone) could potentially explain the maintenance of leaf carbon balance and growth in cultivars studies under combined stress conditions. In the present thesis, we move a step further, by revealing the importance of studding grapevine-virus infection-water stress interactions under field conditions, particularly, in white cultivars where viruses are asymptomatic and difficult to identify. Further research with differents virus concentration and high number of cultivars are needed to confirm if virus effect is concentration dependent and if there is a cultivar-susceptibility difference.
A escala mundial, la producción agrícola sigue estando limitada por una serie de estreses abióticos y bióticos. En las zonas de clima mediterráneo, es muy probable que, en un futuro próximo, la duración, la frecuencia y/o la intensidad de la sequía aumenten. De esta forma, en el mediterráneo, el déficit hídrico se considera el principal factor ambiental limitante del crecimiento de la vid. Al mismo tiempo, el cultivo de la vid está expuesto a diferentes enfermedades provocadas por virus, de los cuales el virus del enrollado 3 (GLRaV-3) es uno de los más extendidos, provocando importantes pérdidas económicas en muchos viñedos de grandes regiones vitícolas de todo el mundo. La identificación de cómo se integran las respuestas de diferentes estreses en la viña y también de cómo están afectando el crecimiento y la fisiología de las plantas es, por tanto, de gran interés para garantizar la continuidad del cultivo de la vid. El primer objetivo de esta tesis es estudiar el estado sanitario de las variedades minoritarios de la vid y destacar la incidencia del GLRaV-3 en las variedades locales de vid. Los resultados obtenidos han puesto de manifiesto que la incidencia de los virus es muy alta (91,75%) en las variedades locales de vid conservadas en la colección de germoplasma de las Islas Baleares. Sorprendentemente, en esta colección, GLRaV-3 es el virus que mostró una mayor incidencia (82%). En este sentido, instamos a la rápida aplicación de técnicas adecuadas de saneamiento para obtener plantas certificadas libres de virus. Por ello, se han optimizado dos técnicas de saneamiento " cultivo in vitro de ápices caulinares y la combinación de termoterapia y cultivo in vitro de ápices caulinares‖ para la erradicación de las infecciones víricas dobles y triples de dos cultivares locales de gran potencial enológico, Argamussa y Gorgollassa. El segundo objetivo principal de esta tesis es explorar los efectos del estrés hídrico, la infección vírica y la combinación de ambos sobre los principales procesos fisiológicos de dos variedades locales de vid, Malvasia de Banyalbufar y Giró Ros. Cada uno de los dos estreses estudiados resultó en un efecto negativo sobre los parámetros de intercambio gaseoso foliar. El estrés hídrico in plantas sanas (WS-NI) redujo significativamente el crecimiento de las plantas y la asimilación neta de CO2 (AN), pero solo demostró tener un efecto reducido sobre los niveles metabólicos, indicando en este caso, que los cambios de AN durante el WS se ven limitados por factores difusivos (la conductancia estomática (gs) y del mesofilo (gm)). Respecto a las plants infectadas en condiciones de riego (WW-VI) las correlaciones obtenidas entre los parámetros fisiológicos (AN, gs y gm), la concentración de virus y los metabolitos sugiere firmemente que el GLRaV-3 afecta a la AN mediante limitaciones difusivas más que bioquímicas. En este caso, la conductancia estomática fue regulada principalmente por la conductancia hidráulica a nivel foliar, concretamente conductancia hidráulica de hoja (Kleaf) y pecíolo (Kpetiole). En este trabajo, Kpetiole resultó ser el parámetro más relevante para estudiar el efecto de los virus sistémicos de tipo floemático sobre la hidráulica de la planta. Notablemente, los resultados de esta tesis indican que el estrés combinado tuvo un efecto negativo sobre el crecimiento y la fisiología de la vid; pero no se destacó ninguna interacción entre WS y VI. Por el contrario, la combinación de los dos estreses subrayó una respuesta específica a nivel metabólico, no pudiéndose predecir cuantitativamente este efecto en base a la suma de cada uno de ellos. Los cambios observados en los metabolitos primarios se asociaron estrechamente con el metabolismo respiratorio. De hecho, el ajuste específico del metabolismo respiratorio (-i.e, cisteína, treonina, fumarato y eritronato 1,4-lactona) podría explicar potencialmente el mantenimiento del balance de carbono foliar y el crecimiento de las variedades estudias bajo condiciones de estrés combinado. En la presente tesis, hemos revelado la importancia de estudiar las interacciones vid-virus-estrés hídrico en condiciones de campo, particularmente en las variedades blancas donde las infecciones son asintomáticas y difíciles de identificar. Para completar este trabajo, sería interesante realizar un experimento con diferentes concentraciones de virus en un mayor número de cultivares y así confirmar si el efecto del virus depende de la concentración y/o si hay una diferencia en la susceptibilidad entre los cultivares.
Actualment, la producció agrícola mundial es troba limitada per una sèrie d‘estressos abiòtics i biòtics. A les zones de clima Mediterrani, s'espera que en un futur pròxim els esdeveniments de sequera siguin encara de major importància en termes d'intensitat, freqüència i durada. Per tant, en aquestes zones, el dèficit hídric és considerat com el principal factor ambiental capaç d‘afectar el creixement de la vinya. A més, la vinya es troba altament sotmesa a diferents virus, entre els quals, Grapevine leafroll-associated virus-3 (GLRaV-3) és un dels més estesos, provocant pèrdues econòmiques considerables a moltes vinyes d‘arreu del món. La identificació de com les diferents respostes a l‘estrès es troben integrades a la vinya i de com aquestes afecten el creixement i la fisiologia de les plantes és, per tant, de gran interès per a garantir la continuïtat del cultiu de la vinya. El primer objectiu d‘aquesta tesi tracta d‘estudiar l'estat sanitari dels cultivars minoritaris de vinya a Mallorca i destacar la prevalença a la vinya del virus GLRaV-3. Aquest estudi demostra una incidència d'infeccions víriques simples i múltiples (91,75%) molt alta en els cultivars locals de vinya, actualment conservats a la col·lecció de germoplasma de les Illes Balears. Sorprenentment, GLRaV-3 és el virus que més predomina en aquesta col·lecció (82%). Aquest resultat impulsa la necessitat d'una aplicació dràstica de tècniques de sanejament per tal d'obtenir plantes certificades lliures de virus. En aquest treball, s'han optimitzat dues tècniques de sanejament ―shoot tips culture and thermotherapy in combination with shoot tips culture‖ per a l'eradicació de virus dobles i triples a dos cultivars locals amb alt potencial enològic, Argamussa i Gorgollassa. El segon objectiu principal d‘aquesta tesi és estudiar els efectes de la infecció vírica, l'estrès hídric moderat i la seva combinació sobre els principals paràmetres fisiològics a dues varietats locals de vinya, Malvasia de Banyalbufar i Giró Ros. Cada un dels estressos estudiats, va resultar en un efecte negatiu sobre l'intercanvi de gasos a nivell foliar. L'estrès hídric a plantes sanes (WS-NI) va reduir de manera significativa el creixement de les plantes i l'assimilació neta al CO2 (AN), en canvi, sols va afectar lleugerament els canvis metabòlics, el que indicà que canvis sobre la AN durant l‘estrès hídric foren principalment deguts a les limitacions sofertes sobre determinats paràmetres difusius, com son la conductància estomàtica (gs) i la conductància del mesòfil (gm). A les plantes infectades per virus i cultivades sota condicions de reg (WW-VI), s‘obtingueren correlacions significatives entre els paràmetres fisiològics (AN, gs i gm), la concentració de virus i la presència de metabòlits. Aquestes correlacions varen desvelar que la presència de GLRaV-3 afectà la AN a través de limitacions difusives i no bioquímiques. En aquest cas, la conductància estomàtica es regí principalment pels canvis en la conductància hidràulica a nivell de la fulla (Kleaf) i el pecíol (Kpetiole), essent Kpetiole el paràmetre més rellevant alhora d‘estudiar l'efecte de virus floemàtics sobre el sistema hidràulic de la planta. Cal remarcar que els resultats obtinguts en aquest treball indiquen que la presència d‘un estrès combinat tingué un efecte negatiu sobre el creixement i la fisiologia de la vinya; però en cap cas es destacà una interacció entre WS i VI. Per contra, la combinació d‘ambdós estressos desencadenà una resposta específica a nivell metabòlic, que no va ser quantitativament predita per la suma dels dos. Els canvis observats en els metabòlits primaris varen resultar estar estretament associats amb el metabolisme respiratori. De fet, l'ajust específic del metabolisme respiratori (és a dir, cisteïna, treonina, fumarat i eritronat 1,4-lactona) podria explicar el manteniment de l'equilibri entre el balanç de carboni i el creixement en els dos cultivars estudiats. En el present treball, s‘ha avançat en quant a revelar quant d‘important és l‘estudi de les interaccions virus-estrès hídric a la vinya en condicions de camp, en particular, en els cultivars de raïm blancs on els efectes del virus són asimptomàtics i més difícils d'identificar. En un futur, es necessitaria aprofundir en aquest camp mitjançant la realització d‘estudis addicionals amb diferents concentracions víriques i un major nombre de cultivars per a ser capaços de confirmar si l'efecte del virus sobre la vinya depèn de la seva concentració i si cada cultivar presenta diferent susceptibilitat a la presència del virus.

Les premières étapes de développement du cerveau dépendent fortement de programmes génétiques. Après la naissance, les connexions neuronales deviennent susceptibles d’être remodelées en réponse à des changements de l’environnement. Cette plasticité culmine au cours de fenêtres temporelles appelées périodes critiques. Les mécanismes sous-jacents sont encore mal connus. Dans le cadre de ma thèse, j’ai étudié les mécanismes qui sous-tendent la plasticité des réseaux au cours de deux périodes critiques chez la souris : la période critique de plasticité de dominance oculaire dans le cortex visuel et la période critique de l’élagage synaptique dans le cortex préfrontal. Dans une première étude, je me suis intéressée aux synapses excitatrices dans les interneurones PV du cortex visuel au cours du développement postnatal. Mes résultats suggèrent que la régulation développementale de l’expression de la protéine GRIP1 dans les interneurones PV gouverne la composition des récepteurs AMPA et la plasticité homéostatique des synapses, et pourrait jouer un rôle dans l’ouverture de la période critique du cortex visuel. Dans une deuxième étude, j’ai étudié les conséquences d’une expression élevée du gène du complément C4, qui chez l’homme prédispose à la schizophrénie, sur la maturation corticale. J’ai montré que la surexpression de C4 dans le cortex préfrontal cause plusieurs phénotypes qui, chez l’homme, ont été associés à la schizophrénie : perte d’épines dendritiques, modifications de la transmission portée par les récepteurs NMDA, altérations de la transmission GABAergique et déficits de la mémoire de travail
The early stages of brain development are largely determined by genetic programs. After birth, neuronal connections become susceptible to remodeling in response to changes in the environment. This plasticity culminates during specific time windows termed critical periods. The underlying mechanisms are still poorly understood. In my thesis, I studied the mechanisms underlying network plasticity during two critical periods in mice: the critical period of ocular dominance plasticity in the visual cortex and the critical period of synaptic pruning in the prefrontal cortex. In a first study, I studied the properties of excitatory synapses in PV interneurons of the visual cortex during postnatal development. My results suggest that the developmental regulation of GRIP1 protein expression in PV interneurons governs the composition of AMPA receptors and homeostatic synaptic plasticity, and may play a role in the opening of the critical period in the visual cortex. In a second study, I investigated the consequences of high expression of the C4 complement gene, which in humans predisposes to schizophrenia, on cortical maturation. I showed that overexpression of C4 in the prefrontal cortex causes several phenotypes that in humans have been associated with schizophrenia: loss of dendritic spines, changes in transmission carried by NMDA receptors, alterations in GABAergic transmission and deficits in working memory

La viticoltura si trova ad affrontare il problema dalla lotta contro i patogeni che comporta costi ingenti dovuti anche alla sostituzione di piante infette. Le patologie più gravi sono l’Infezione degenerativa, l’Accartocciamento fogliare e il Complesso del legno riccio, tutti causati da infezioni virali. L’impresa cilena Concha y Toro ha deciso di studiare la presenza dei virus all’interno dei propri vigneti in modo da evitarne la diffusione, applicando la tecnica analitica della Real Time PCR, strategia SYBR® Green e TaqMan®. L’obiettivo di questa ricerca è di mettere a punto un protocollo che permetta di rilevare la presenza dei virus applicando una strategia multiplex, al fine di ridurre i costi e i tempi di analisi. Inoltre è possibile inserire un controllo interno che assicuri la veridicità di una risposta negativa alla presenza del virus. Sono state costruite curve standard per le analisi in singleplex dei virus testati dall’azienda e del gene actina di vite, allo scopo di ottimizzare l’analisi. È seguito lo studio delle analisi in duplex prima con l’introduzione del controllo interno e in seguito con test per analizzare combinazioni di virus. Sono state confrontate le efficienze delle curve standard verificando che il valore rimanesse all’interno di un range stabilito e che non ci fosse differenza significativa tra l’analisi in singleplex e la corrispondente in duplex. Lo stesso è stato ripetuto per un protocollo in triplex. Sono state studiate la sensibilità e la specificità di ogni analisi in modo da validare il metodo. I risultati ottenuti hanno dato la possibilità di ottimizzare i protocolli singleplex e confermato la possibilità di introdurre il gene actina come controllo interno nelle analisi. Sono stati raggiunti ottimi risultati anche in molte delle combinazioni in duplex tra virus. In futuro sarà necessario approfondire lo studio delle sensibilità e specificità d’analisi in modo da validare il metodo evitando falsi negativi o falsi positivi.

Dans cette these sont presentes les travaux visant a ameliorer les conditions de detection de deux closterovirus de la vigne, le virus a (gva) et le virus 1 associe a l'enroulement de la vigne (glrav 1), a demontrer le role du gva dans l'etiologie d'un syndrome de cannelures du tronc, le kober stem grooving (ksg), et a voir des relations entre la multiplication des virus et le niveau d'expression des symptomes de cannelures. Une technique de tres haute sensibilite, basee sur l'amplification genique (pcr) d'un fragment de genome du gva precedee d'une etape d'immunocapture du virus, a ete adaptee a la detection du virus dans la vigne. Pour le glrav 1, la sequence primaire de l'extremite n-terminale de sa coque proteique a ete determinee pour la production d'amorces specifiques pour la synthese et le clonage d'adn complementaire au rna viral, necessaires a sa detection par pcr. La recherche par pcr du gva dans des vignes malades et/ou traitees par thermotherapie a demontre l'etroite correlation entre la presence du gva et le ksg ; l'inoculation du gva a la vigne par heterogreffage in vitro sur n. Benthamiana infecte a entraine le developpement de symptomes sur le porte-greffe sensible kober 5bb, ce qui a confirme l'implication du gva dans l'etiologie du ksg et a montre que le ksg restait latent sans greffage. La detection in situ du gva par immunomarquage de coupes de tiges de kober 5bb infecte n'a revele aucune difference quantitative dans sa multiplication avant et apres greffage, mais que la surinfection par le glrav 1, accentue la severite des symptomes de ksg

A dependência de drogas é um transtorno multifatorial complexo que se desenvolve em uma minoria de indivíduos que fazem uso dessas substâncias. Memórias associativas entre a droga e o contexto funcionam como gatilho para disparar comportamentos não adaptativos de busca e consumo, além de recaídas após períodos de abstinência. Subjacentes a essas mudanças comportamentais, existem modificações nas subunidades de receptores glutamatérgicos do tipo AMPA em estruturas envolvidas com memória (Hipocampo) e recompensa (Núcleo Accumbens). Por isso, estratégias que enfraqueçam a associação do contexto com a droga e que aprofundem o conhecimento dos circuitos envolvidos nesses comportamentos são de extrema relevância terapêutica. A memória quando evocada pode passar por dois processos pós-evocação: a extinção, em que uma nova memória é formada inibindo uma prévia associação, e a reconsolidação, em que a memória original entra em um estado lábil e suscetível a modificações, em que é possível enfraquecê-la através da inibição de sua reconsolidação. A reconsolidação da memória mostra-se uma estratégica mais eficaz e duradoura em relação à extinção, já que a memória original é modificada. Como modelo animal para o estudo da memória na dependência de drogas, o condicionamento de preferência por local (CPL) é bastante utilizado e sabe-se que é possível enfraquecer a preferência através do bloqueio da reconsolidação. Porém, são escassos os estudos que investigaram a existência da reconsolidação no modelo de sensibilização locomotora, que parece ocorrer, na maioria dos casos, em condição dependente do contexto de aquisição do comportamento, embora existam exemplos que demonstrem sua independência. As questões a serem respondidas neste trabalho são (a) se é possível reverter conjuntamente a preferência por local e a sensibilização locomotora à morfina (5 mg/kg) em ratos Wistar adultos machos, inibindo-se a síntese proteica com cicloheximida (CHX) i.p. logo após uma sessão de reativação contextual da memória no CPL, (b) se a reversão dos comportamentos reflete alterações (já descritas por outros autores) em GluA1, GluA1p (Ser845) e GluA2, no Hipocampo dorsal (HPCd) e no Núcleo Accumbens (NAc), e (c) se o mesmo tratamento em ambas estruturas reverte os dois parâmetros avaliados – comportamental e neuroquímico – de forma diferente ou igual. Nossos resultados mostraram ser possível reverter a preferência por local e a sensibilização locomotora por inibição sistêmica de síntese proteica, e que o condicionamento com exposição à morfina induz alterações nas subunidades analisadas de AMPA, conforme verificado no HPCd e NAc, embora a CHX não tenha produzido um efeito tão bem definido. Os animais que receberam infusões centrais no HPCd e NAc (central) não exibiram preferência por local, nem sensibilização. Em conjunto, nossos resultados mostraram, pela primeira vez em um mesmo desenho experimental, que é possível reverter diferentes aspectos da memória de recompensa (preferência e sensibilização) por meio do bloqueio da reconsolidação.
Drug addiction is a complex and multifactorial disorder that develops in a few people who use these substances. Associative memories between the drug and context of use act as a trigger for maladaptive behavior such as drug seeking and drug use, in addition to relapse after an extended period of withdrawal. Underlying these behavioral changes are modifications in glutamatergic reception (AMPA) in structures involved with memory (Hippocampus) and reward (Nucleus Accumbens). Therefore, strategies that weaken the drug and context association and deepen knowledge of circuits involved in these behaviors are extremely relevant therapeutically. When retrieved, a memory can undergo two distinct processes post-retrieval: extinction, in which a new memory inhibiting a previous association is generated, and reconsolidation, in which the original memory can enter a labile state and is susceptible to modifications, when it can be weakened by inhibition of its reconsolidation. Reconsolidation of memory has been shown to be a more effective and long lasting strategy in relation to extinction, since the original memory is modified. An animal model for studying drug addiction, conditioned place preference (CPP) is largely used and it is well known that it is possible to weaken preference by disrupting reconsolidation. However, there are few studies that investigate the existence of reconsolidation in a locomotor sensitization paradigm, which seems to occur in a condition dependent on context of acquisition, although some works report its independence. The questions answered in this work were (a) if it is possible to reverse both, context preference and locomotor sensitization to morphine (5mg/kg) by protein synthesis inhibition (CHX) after a contextual memory reactivation session in CPP, (b) if the disruption of behaviors reflects a reversal of changes of GluA1, GluA1p (Ser845) e GluA2 in dorsal Hippocampus (dHPC) and Nucleus Accumbens (NAc) and (c) if the same treatment in these structures differentially reverts the two parameters assessed. Our results indicate that it is possible to revert context preference and locomotor sensitization via systemic disruption of protein synthesis and that morphine conditioning induces changes in AMPA subunits in dHPC and NAc, although CHX did not have an evident effect on molecular reversal. Animals cannulated in dHPC and NAc core did not induce preference or sensitization. Taken together, our results demonstrated, for the first time, using the same experimental design that is possible to revert different aspects of reward memory (preference and sensitization) by disrupting the reconsolidation process.

Dissertação de Mestrado em Engenharia Agronómica
A vinha pode ser alvo de várias pragas e doenças parasitárias, entre elas diversas viroses. Atualmente, estão identificados vários vírus responsáveis por algumas dessas patologias da videira embora apenas alguns afetem a sua produtividade, maturação e longevidade. A doença do enrolamento foliar da videira é a virose que está mais expandida pelas vinhas portuguesas, sendo Grapevine leafroll-associated virus 3 (GLRaV3) o mais difundido. Em Pegões está o Pólo de Conservação da Diversidade da Videira da Associação Portuguesa para a Diversidade da Videira (PORVID), associação esta responsável pela seleção clonal das videiras em Portugal. Alguns clones localizados nesse Pólo Experimental foram testados por ELISA em 2013 para o GLRaV-3 tendo resultados positivos. Nos anos seguintes os mesmos clones não manifestaram sintomas, pelo que com este estudo pretende-se analisar se essas videiras continuam infetadas por GLRaV3 de modo a verificar se as elevadas temperaturas de verão na região influenciam a presença do vírus na planta e respetiva manifestação dos sintomas da doença. No verão de 2017 fez-se acompanhamento visual dos sintomas das plantas de vários clones em seis castas e, em Setembro, fez-se a recolha das amostras e os testes ELISA de modo a analisar a concentração de GLRaV-3. Após a análise dos resultados mostrou-se conveniente fazer uma nova visita ao campo experimental em Pegões e tiraram-se novas fotografias a alguns clones estudados. Foi feito um estudo comparativo entre os resultados obtidos nos testes ELISA realizados em 2013 e em 2017, bem como entre os resultados de 2017 e a situação sintomática observada. Os resultados evidenciaram a existência de plantas isentas de GLRaV-3 em 2017 que, no entanto, estavam infetadas por este vírus em 2013. Nas plantas que continuaram positivas também houve várias situações em que os sintomas não se manifestaram, apesar da infeção pelo GLRaV-3 permanecer. Estes parâmetros sugerem que as temperaturas elevadas durante o verão em Pegões possam inativar ou até eliminar o vírus.
Vineyards are the target to many pests and parasitic diseases, among them several viruses. There are currently identified several viruses responsible for some of these grapevine pathologies, although only a few affect their productivity, maturation, and longevity. The grapevine leafroll disease is the most expanded virus in the portuguese vines, being Grapevine leafroll-associated virus 3 (GLRaV-3) the most widespread. In Pegões is located the Pole of Conservation of the Vineyard of the “Associação Portuguesa para a Diversidade da Videira” (PORVID), which is responsible for the clonal selection of grapevine in Portugal. Some of the clones in this experimental pole were submitted to ELISA in 2013 for the GLRaV-3 with positive results for the virus. During the following years the same clones showed no symptoms, so this study intends to analyze if these vines are still infected with GLRaV-3 in order to verify if the high summer temperatures in the region influence the presence of the virus in the plant and the manifestation of the disease symptoms. In the summer of 2017, plant symptoms were visually monitored in several clones from six grapevine varieties and, in September, the samples collected and ELISA tests for GLRaV-3 were carried out. After the analysis of the results, it was convenient to make a new visit to the experimental field in Pegões and new photos were taken to some studied clones. A comparative study was carried out between the results obtained in ELISA tests performed in 2013 and in 2017, as well as, between the results of 2017 and the symptomatic situation observed. The results showed the existence of GLRaV-3-free plants in 2017 that, however, were infected by this virus in 2013. In the plants that remained positive there were also several cases in which the symptoms were not present, although the GLRaV-3 infection still remains. These parameters suggest that summer temperatures in Pegões can inactivate or even eliminate the virus.

"April, 2004"
"List of figures" - inside back cover.
Bibliography: leaves 83-93
93, [8] leaves : ill., plates (some col.), photos ; 30 cm.
The specific objectives of this work include: 1. Completion of the GLRaV-1 genome sequence and bioinformatic analysis of the viral open reading frames ; 2. Production of an appropriate GLRaV-1 certification protocol addressing the shortcomings of the current tests for leafroll detection ; 3. Intracellular localisation of the GLRaV-1 gene products via generation of green fluorescent protein (GFP)-fusion constructs, in an attempt to further characterise the function of these proteins.
Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, Discipline of Wine and Horticulture, 2004

Substance IEM 1460 is an antagonist of AMPA receptors in the brain. It is a derivative of adamantine, which has already been tested in several studies as a potential age-related anticonvulsant. In the research part of the thesis there is a summary of the knowledge about receptors, focusing on AMPA receptors, IEM 1460, epilepsy and ontogenetic development of laboratory rat. The research section evaluates the effect of IEM 1460 on motor skills of a rat in a few postnatal days of its life. To evaluate spontaneous animal motor skills, Open-Field tracking was used, and several specific tests were used to evaluate provoked motor skills. A total of 30 animals were included in the research. Substances (IEM 1460 at 3 mg / kg, IEM 1460 at 10 mg / kg or 2 ml / kg saline) were infused intraperitoneally on five consecutive days (ages P7-P11) and the animals were repeatedly tested at age 12, 15, 18, 21, 25, 31 and 60 days. In this study, IEM 1460 has not been shown to have significant effect on the gross rat motor skills and therefore the substance remains as a serious candidate for age-specific antiepileptic drugs.

Tese de doutoramento em Biologia Experimental e Biomedicina, no ramo de Neurociências e Doença, apresentada ao Instituto de Investigação Interdisciplinar da Universidade de Coimbra
As redes neuronais estão constantemente a adaptar a densidade sináptica e a eficiência da transmissão sináptica em resposta ao normal desenvolvimento neuronal e a processos de aprendizagem. Formas de plasticidade Hebbiana, co mo a potenciação de longo termo ( LTP ) e depressão de longo termo ( LTD ) , são consideradas as fundações celulares da aprendizagem e memória. Estas formas de plasticidade têm como base um mecanismo de feedback positivo que facilita o reforço das conexões sinápticas específicas. Contudo, se não forem regulados, estes mecanismos podem promover uma diminuição do limiar de atividade necessário para indução de LTP/LTD, conduzindo a eventos extremos de hiperexcitabilidade ou eliminação sináptica. Mecanismos homeostáticos, como o escalamento sináptico, previnem esse tipo de situações, atuando como uma resposta compensatória de feedback negativo que mantém a estabilidade das redes neuronais. No escalamento sináptico, os neurónio s detetam níveis de atividade globais, através da despolarização da membrana e do influxo de cálcio, promovendo o escalamento da sua força sináptica ao ajustarem os níveis sinápticos de recetores AMPA. Muitos dos componentes associados à maquinaria que pro move o escalamento das sinapses estão identificados, contudo ainda falta compreender de que forma estes mecanismos promovem a regulação da resposta neuronal. A janela temporal do escalamento das sinapses sugere a implicação de alterações nos programas de t ranscrição e pós - transcrição nestes processos; porém, estes programas estão pouco caracterizados. De facto, vários paradigmas de escalamento sináptico requerem síntese proteica local para promoverem rapidamente alterações funcionais necessárias para a indução de escalamento sináptico. Os microRNAs (miRNAs) desempenham um papel fundamental como reguladores pós - transcrição da síntese proteica local: um miRNA pode regular múltiplos transcritos e a expressão dos miRNAs é rapidamente alterada por atividade n euronal. Até agora, os miRNAs têm sido implicados em vários processos fisiológicos, incluindo desenvolvimento neuronal e plasticidade sináptica. Contudo, poucos miRNAs têm sido associados com mecanismos de escalamento sináptico. No presente estuo, observám os que o bloqueio crónico de recetores AMPA e NMDA induz escalamento sináptico positivo das correntes pós - sinápticas excitatórias mediadas por recetores AMPA e aumenta o conteúdo sináptico de GluA1 e GluA2. Além disso, este paradigma de bloqueio de ativida de afeta o transcriptoma neuronal, afetando particularmente os níveis de transcritos associados com processos de desenvolvimento neuronal e plasticidade sináptica, incluindo vários transcritos associados previamente com o escalamento sináptico, como Arc , B dnf , CamKII β , Plk2 , Rps6ka1 , entre outros. O perfil neuronal de miRNAs também é regulado por mecanismos de escalamento sináptico . A expressão do miR - 186 - 5p é muito afetada, estando consideravelmente diminuída de forma persistente após 2h de bloqueio iv crónic o de atividade sináptica. Adicionalmente, inibição basal do miR - 186 - 5p impede o escalamento sináptico da amplitude das correntes mediadas por recetores AMPA. O miR - 186 - 5p regula a expressão endógena de GluA2 em condições fisiológicas, através de um local - a lvo localizado na 3’UTR do Gria2 . Para além disso, supressão da atividade sináptica leva ao aumento da expressão de GluA2 através de mecanismos dependentes da região 3’UTR do Gria2 . Inibição do miR - 186 - 5p impede o escalamento positivo dos níveis totais e s inápticos de GluA2 em condições de bloqueio de atividade, de acordo com os estudos de electrofisiologia que mostram que inibição do miR - 186 - 5p impede o escalamento sináptico. Tendo em conta padrão de expressão do miR - 186 - 5p ao longo do desenvolvimento, e a previsão de alvos do miR - 186 - 5p que codificam proteínas com uma função no desenvolvimento dendrítico e sináptico, avaliámos o seu envolvimento no desenvolvimento neuronal. Reportamos que o miR - 186 - 5p não está envolvido na fase final do desenvolvimento de ndrítico mas promove a maturação das espículas dendríticas. Além disso, testámos o papel do mirR - 186 - 5p na formação e/ou manutenção das sinapses excitatórias e inibitórias através da análise das respetivas proteínas pós - sinápticas, PSD95 e gefirina. Demons trámos que a expressão do miR - 186 - 5p promove um aumento da densidade de sinapses excitatórias e no conteúdo sináptico de PSD95, enquanto reduz os níveis de gefirina. Estes resultados indicam que o miR - 186 - 5p regula a balanço sináptico entre excitação e ini bição (E/I) hipocampo. Globalmente, os nosso resultados implicam o miR - 186 - 5p em processos homeostáticos de escalamento sináptico, na regulação da expressão de GluA2 e na coordenação do rácio neuronal de E/I, sugerindo que o miR - 186 - 5p desempenha um impor tante papel no desenvolvimento cerebral e em respostas homeostáticas cerebrais a estímulos internos e externos.
Neuronal networks are constantly adapting their synapse number and strength in response to developmental or learning-related processes. Hebbian forms of plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), are thought to be the cellular counterpart of learning and memory. These forms of plasticity rely on positive feedback mechanisms to facilitate the reinforcement of specific synaptic connections. However, if left unchecked, these mechanisms could decrease the threshold for LTP/LTD induction, leading to runaway excitation or synapse elimination. Homeostatic mechanisms, such as synaptic scaling, prevent extreme activity levels, acting as a compensatory negative-feedback response to maintain the stability of neuronal function. In synaptic scaling, neurons perceive their overall levels of activity through depolarization and calcium influx, and scale their synaptic strength accordingly by adjusting synaptic AMPA receptor levels. Many components of the synaptic scaling apparatus have been identified but full understanding of how synaptic scaling mechanisms work to regulate neuronal response is still lacking. The time window of synaptic scaling suggests that transcriptional and post-transcriptional programs may be activated upon chronic changes in neuronal activity, but these programs are poorly characterized. In fact several synaptic scaling paradigms require local protein synthesis to rapidly promote the functional modifications needed for synaptic scaling induction. microRNAs (miRNAs) play a pivotal role as posttranscriptional regulators of local protein synthesis: a single miRNA can target multiple transcripts and their expression is rapidly regulated by neuronal activity. So far, miRNAs have been implicated in several physiological processes, including neuronal development and synaptic plasticity. However, few miRNAs have been implicated in synaptic scaling mechanisms. In this present work, we report that chronic blockade of excitatory synaptic activity induces synaptic upscaling of AMPA receptor-mediated mini excitatory postsynaptic currents (mEPSCs) and increases the synaptic content of GluA1 and GluA2 AMPA receptor subunits. Moreover, this paradigm of activity blockade affects the neuronal transcriptome, impacting on the levels of transcripts associated with neuronal development and synaptic plasticity processes, including several transcripts previously associated with synaptic scaling, such as Arc, Bdnf, CamKIIβ, Plk2, Rps6ka1, among others. The neuronal miRNA profile is also regulated by synaptic scaling mechanisms. The expression of miR-186-5p is highly affected, being persistently downregulated starting at 2h of blockade of synaptic activity. Additionally, inhibition of miR-186-5p hampers upscaling of the amplitude of AMPA receptor-mediated mEPSCs upon synaptic activity suppression. Notably, we found that miR-186-5p is a regulator of endogenous GluA2 expression under physiological conditions, through a targeting site located in the Gria2 3’UTR. In fact, increased GluA2 expression is triggered by prolonged suppression of synaptic activity, in a manner dependent on regulatory elements located on the Gria2 3’ untranslated region (UTR). Long-term inhibition of miR-186-5p blocked upscaling of total and synaptic GluA2 content upon synaptic activity blockade, in agreement with electrophysiological studies showing that inhibition of miR-186-5p hampers synaptic scaling. Given the developmental expression pattern of miR-186-5p, and predictions suggesting that it targets transcripts encoding proteins with a role in dendritic and synaptic development, we tested its implication in neuronal development and synapse formation/maintenance. We report that miR-186-5p is not involved in late dendritic development but it promotes dendritic spine maturation. Further, we evaluated how miR-186-5p impacts on excitatory and inhibitory synapses by analysing their respective postsynaptic proteins, PSD95 and gephyrin. We show that expression of miR-186-5p promotes an increase in excitatory synapse density and synaptic PSD95 content, while reducing the levels of gephyrin. These results indicate that miR-186-5p regulates the excitatory/inhibitory (E/I) synaptic balance in the hippocampus. Overall, our results implicate miR-186-5p in homeostatic synaptic scaling processes, in the regulation of GluA2 expression and in the coordination of the neuronal E/I ratio, suggesting that miR-186-5p has an important role in brain development and in brain homeostatic responses to internal and external stimuli.