L'épilepsie affecte des millions de patients dans le monde. Les traitements disponibles sont symptomatiques, ils per récepteur mGlu7 du glutamate dans la modulation non seulement de l’excitabilité, mais également de l'hypersynchronisation des réseaux de neurones, deux facteurs cruciaux dans les crises d'épilepsie. J'ai parachevé ces découvertes qui ont été à l’origine d’une première publication (Tassin, Girard et al. 2016).A l’aide d’un nouvel agoniste du récepteur mGlu7, le LSP2-9166, lors de ma thèse j'ai ensuite étudié l’impact de ce récepteur dans différents modèles d’épilepsie chez la souris. Deux modèles complémentaires ont été utilisés : le kindling (embrasement), modèle chimique induit par le pentylènetétrazol (PTZ) qui sensibilise le cerveau jusqu’à générer des crises tonico-cloniques généralisées, et l’injection intra-hippocampique de kainate, mimant l’épilepsie mésiale du lobe temporal chez l'homme.Dans un premier temps j'ai observé une atténuation de la progression de la sévérité des crises dans le modèle de kindling au PTZ, sous l’activation du récepteur mGlu7. Cet effet a été corrélé à une inflammation, et une activation microgliale et astrocytaire plus faibles. Dans le modèle d’injection intra-hippocampique de KA, considéré comme pharmaco-résistant, l’activation du récepteur mGlu7 pendant la période d’épileptogenèse a augmenté la durée des périodes interictales et diminué la durée des crises ainsi que la réorganisation neuronale. Une fois les crises chroniques installées, l’activation aigu du récepteur mGlu7 a diminué le nombre de crises aussi fortement que le diazépam couramment utilisé dans le cadre clinique. Pour finir, des injections chroniques de LSP2-9166 chez des animaux naïfs (non épileptiques) n’engendrent pas de déficit cognitif ou comportemental détectables, ni de modification du niveau d’ARNm du récepteur mGlu7. L’activation du récepteur mGlu7 offre donc une cible stratégique dans nos deux modèles.Ces travaux permettent une meilleurmettent de traiter les crises sans pour autant éviter la progression de la maladie et présentent de lourds effets secondaires. La découverte de nouvelles cibles thérapeutiques et de nouveaux composés reste primordiale pour dépasser les limites des stratégies thérapeutiques actuelles. Au début de ma thèse, des études précédentes avaient montré une implication due compréhension du rôle du récepteur mGlu7 dans l’épileptogenèse. Ils participent ainsi à la recherche de futurs traitements. antiépileptiques adéquats. / Epilepsy affects millions of patients worldwide. The available treatments are symptomatic, they treat seizures without preventing the progression of the disease and have heavy side effects. The discovery of new therapeutic targets and new compounds is therefore essential to overcome the limitations of current therapeutic strategies. Previous studies have demonstrated substantial involvement of the mGlu7 receptor in modulating not only excitability but also hypersynchronization of neural networks, two crucial factors affecting epileptic seizures. These discoveries were at the origin of a first publication that I completed at the beginning of my thesis (Tassin, Girard et al., 2016).Using a new mGlu7 receptor agonist, LSP2-9166, in my thesis I then studied the impact of this receptor in different epilepsy models in mice. Two complementary models were used: kindling, a chemical model induced by pentylenetetrazol (PTZ) which sensitizes the brain to induce generalized tonic-clonic seizures, and intra-hippocampal injection of kainate, mimicking mesial temporal lobe epilepsy in humans.At first, I observed an attenuation of the progression of the seizures severity in the PTZ kindling model, under the activation of the mGlu7 receptor. This effect was correlated with weaker inflammation, and microglial and astrocytic activation. In the intra-hippocampal injection model of KA, considered as drug-resistant, activation of the mGlu7 receptor during the epileptogenesis period increased the duration of interictal periods and decreased the duration of seizures as well as neuronal reorganization. Once chronic seizures were established, acute activation of the mGlu7 receptor decreased the number of seizures as strongly as diazepam, commonly used in clinical settings. Finally, chronic injections of LSP2-9166 into naive (non epileptic) animals do not generate any detectable cognitive or behavioral deficits or changes in mGlu7 receptor mRNA level. The activation of the mGlu7 receptor thus presents a strategic target in our two models.This work provides a better understanding of the role of the mGlu7 receptor in epileptogenesis. It participates in the search for future more adequate treatments.

Orientador: Mario Sergio Palma / Banca: Zila Luz Paulino Simões / Banca: Norberto Peporine Lopes / Resumo: As abelhas têm sido utilizadas como modelos robustos e influentes para o estudo de memória e aprendizagem, contribuindo para o melhor entendimento das bases da cognição. Nesse contexto, diferentes metabólitos foram caracterizados por desempenharem funções distintas no processo de aprendizagem e formação de memória em insetos. Considerando que pouco se sabe sobre os metabólitos em relação ao desenvolvimento das habilidades cognitivas em A. mellifera, ou mesmo em relação aos comportamentos reflexos (condicionados e/ou não condicionados), o presente estudo teve como objetivo a análise metabolômica do cérebro de abelhas submetidas ao ensaio comportamental de reflexo de extensão de probóscide (REP). Para isto, foi padronizada a técnica de análise metabolômica com o uso do sistema LC-ESI- MS e MSn, construindo-se inicialmente uma biblioteca de compostos característicos de cérebro de abelhas (neurotransmissores, aminoácidos livres, poliaminas, nucleotídeos, nucleosídeos, ácidos orgânicos, etc). Nesta primeira abordagem, dentre os 112 compostos da biblioteca, 48 foram identificados e quantificados; alguns destes compostos foram únicos para o grupo controle (cadaverina, espermina, glicose, uracila e n-acetil-L-glutamato 5-semialdeido), enquanto que outros foram únicos para o grupo REP (fenilalanina, betaína, espermidina, serina e creatina). Dentre os compostos identificados em ambos os grupos, apenas 5 compostos apresentaram diferenças quantitativas estatisticamente significantes (arginina, asparagina, guanosina monofosfato, putrescina e 4-guanitinobutanoato). Visando o estudo dos perfis metabólicos regionais (em cada região do cérebro), foi também padronizado o protocolo experimental utilizando-se a estratégia MALDI Spectral Imaging e o desenvolvimento de um método semi-quantitativo de metabólitos ... (Resumo completo, clicar acesso eletrônico) / Abstract: The honeybee Apis mellifera has long served as an invertebrate model organism for learning and memory research, contributing to a better understanding of cognition bases. In this context, different metabolites (especially neurotransmitters) were characterized by play distinct roles in learning process and in memory formation in insects. Whereas little is known about the metabolites in relation to the development of cognitive skills in A. mellifera, the present study aims to perform a metabolomic analysis of the honeybee brains submitted to the behavioral test of proboscis extension reflex (PER). For this, has been standardized a metabolomic analysis technique through the use of LC-ESI-MS and MSn system. Initially a low molecular weight compounds library was created, containing characteristic compounds of bee brain (neurotransmitters, free amino acids, polyamines, nucleotides, nucleosides, organic acids, etc.). In this first approach, from the library of 112 compounds, 48 compounds were identified and quantified; some of these compounds were only identified in the control group (cadaverine, spermine, glucose, uracil and N-acetyl-L-glutamate 5- semialdehyde), while others were only identified in PER group (phenylalanine, betaine, spermidine, serine and creatine). Among the compounds identified in both groups, only five compounds showed statistically significant differences in quantitative results (arginine, asparagine, guanosine monophosphate, putrescine and 4-guanidinebutanoate). In order to study the metabolic profiles by regions (within each brain region) it was also standardized an experimental protocol using a novel semi-quantitative method of MALDI Spectral Imaging strategy. This strategy allowed the study and the mapping of the spatial distribution of metabolites identified in honeybee brain sections, as well as a better understanding ... (Complete abstract click electronic access below) / Mestre

A wide range of conditions are characterised by focal neurological symptoms, yet the pathophysiolgy often remains poorly understood. This thesis has focussed on applying functional neuroimaging in clinical groups. Migraines with aura are amongst the most common conditions posing a significant burden to sufferers. Elevated connectivity was detected in the visual cortex of migraine with aura patients, potentially complementing one of the leading proposed mechanisms for attacks. Minor strokes patients are also affected by focal symptoms after events which in some cases can be prolonged. Altered connectivity was observed in a number of regions reflecting previous findings for acute stroke. A group of transient ischaemic attack patients were also analysed, revealing subtle differences necessitating further study. Lastly disorders of consciousness pose acute challenges for treatment and ongoing care. Task based imaging was applied to form a more accurate picture of residual cognition. Additionally the correlation between measures derived from resting state data and cerebral glucose consumption was explored.

A Neurobiologia Vegetal é um recente ramo das ciências vegetais que objetiva esclarecer os complexos padrões de comportamento vegetal, no que se refere à percepção, processamento, armazenamento e transmissão de sinais na planta e entre plantas. A detecção de vizinhos, é uma capacidade que implica em auto reconhecimento, uma vez que um organismo só terá sucesso em interações competitivas se for capaz de auto/não-auto discriminação. Assim, objetivou-se com este trabalho verificar se raízes de ervilha (Pisum sativum) cv. Mikado apresentam crescimento diferenciado quando na presença de raízes da mesma planta, e de raízes de outras plantas, mas pertencentes ao mesmo genótipo, para que se possa averiguar sua capacidade de auto/não-auto discriminação. Além disso, avaliou-se também o crescimento da parte aérea para observar em que grau a presença de plantas vizinhas pode influenciar o desenvolvimento vegetativo de plantas de ervilha. Quatro dias após a germinação, plântulas de Pisum sativum cv. Mikado tiveram a raiz principal cortada 5 mm abaixo do hipocótilo. Passados sete dias, foram retiradas as raízes secundárias, deixando-se apenas duas raízes, de igual tamanho, por planta (split-root). Plantas com duas raízes iguais foram replantadas, com cada vaso contendo duas raízes da mesma planta (tratamento Auto) ou duas raízes de plantas diferentes (Tratamento Não-auto). Os vasos foram agrupados em tríades. O experimento foi mantido em estufa incubadora sob condições de temperatura e fotoperíodo controladas e após 18 dias foram feitas avaliações do crescimento da parte aérea e das raízes, através das medições de: altura da planta (cm), peso fresco de parte aérea e de raiz (g), peso seco de parte aérea e de raiz (g), área foliar (cm2), área radicular (cm2), comprimento total de raiz (cm) e diâmetro médio de raiz (cm). A análise dos dados considerando os valores médios de cada tríade revelou não haver diferença significativa entre os tratamentos Auto e Não-auto com relação ao crescimento de parte aérea. No que se refere ao crescimento da raiz, com exceção do diâmetro médio, as demais variáveis diferiram significativamente, sendo que as plantas pertencentes ao tratamento Auto apresentaram valores de peso seco, área superficial e comprimento total 36,71%, 27,84% e 23,18%, respectivamente, maiores do que as plantas do tratamento Não-auto. Ou seja, as plantas que não estavam sob competição apresentaram maior crescimento de raiz. No entanto, quando se observou o comportamento das plantas entre si, em cada tríade, verificou-se, no tratamento não-auto, diferenças visíveis de crescimento tanto em parte aérea como na raiz entre as três plantas que constituía cada tríade. Verificou-se também que a raiz de uma mesma planta cresceu diferentemente de acordo com a identidade da raiz vizinha. Enquanto que no tratamento auto as três plantas que constituíam uma tríade tinham aproximadamente o mesmo tamanho de parte aérea e raiz. Assim, podemos afirmar que o crescimento das plantas no tratamento não-auto foi influenciado pelas interações entre as raízes e mais que isto, foi dependente da identidade da raiz vizinha implicando em auto/não-auto discriminação e reconhecimento parental. / The Plant Neurobiology is a recent branch of plant science that aims to clarify the complex patterns of behavior vegetable, with respect to perception, processing, storage and transmission of signals in plant and between plants. The detection of neighbors, is a capacity that involves self-recognition and an individual will only be successful in competitive interactions if it is capable of self/non-self discrimination. Thus, the objective was to determine whether roots of pea (Pisum sativum) cv. Mikado grow differently in the presence of the same plant roots, and roots of other plants, but within the same genotype, so that we can determine its capacity for self/non-self discrimination. In addition, we assessed also the growth of the shoot to see to what degree the presence of neighboring plants can influence the vegetative growth of pea plants. Five days from germination, seedlings of Pisum sativum cv. Mikado had the seminal root severed 5 mm below the hypocotyl. After seven days, all but two of these roots were removed, leaving only two roots of equal size per plant (split-root). Plants with two equal roots were replanted, with each pot containing two roots of the same plant (treatment self) or two roots of different plants (Treatment non-self). Pots were grouped in triplets. The experiment was kept in an incubator camera under controlled conditions of temperature and photoperiod and after 18 days were evaluated for growth of shoots and roots. It was measure plant height (cm), fresh weight of shoot and root (g), dry weight of shoot and root (g), leaf area (cm2), root area (cm2), total length of root (cm) and average root diameter (cm). The analysis of data considering the average values of each triplets showed no significant difference between treatments self and non-self in relation to the growth of shoots. With respect to root growth, except for the diameter, the other variables differed significantly, and plants belonging to treatment self had values of dry weight, surface area and total length of 36.71%, 27.84 % and 23.18%, respectively, higher than the treatment plants non-self. That is, plants that were not under competition had higher root growth. However, when we observe the behavior of plants in each triplet, it was found that the treatment non-self, the plants had sizes of shoot and root differ. It was also found that the root of the same plant grew differently depending on the identity of neighboring roots. While in treatment self, the three plants that constituted a triplet had, approximately, the same size of shoot and root. Thus, we can say that the growth of plants to treatment non-self was influenced by the interactions between roots and more that this was dependent on the identity of neighboring roots implying self/non-self discrimination and kin recognition.

Encontramos no bulbo diversos núcleos, assim como diversos neurotransmissores, relacionados com a manutenção da pressão arterial. Dentre os núcleos, o núcleo do trato solitário se destaca por ser um dos principais moduladores do sistema nervoso autônomo, sendo o primeiro a receber aferências dos barorreceptores e encaminhá-los para diversos outros núcleos. Dentre estes neurotransmissores, encontramos o glutamato e as catecolaminas, sendo ambos essenciais para a manutenção da pressão arterial. É sabido que a atuação de transmissores em células do sistema nervoso pode levar a alterações em outras vias de neurotransmissão, alterando assim a resposta das células a estímulos. Levando em consideração a importância do glutamato e das catecolaminas na modulação da pressão arterial, e que tanto os receptores glutamatérgicos quanto catecolaminérgicos podem interferir no metabolismo celular e gerar mudanças estruturais nos neurônios, cogitamos que a atuação do sistema glutamatérgico poderia modular o sistema catecolaminérgico. Neste trabalho, avaliamos se o sistema glutamatérgico e catecolaminérgico podem interagir em culturas de células do bulbo de ratos neonatos, a partir de tratamentos das culturas com glutamato ou noradrenalina. Observamos que o tratamento destas culturas com glutamato leva a uma redução nos níveis de proteína e de mRNA da enzima tirosina hidroxilase e do receptor _2 adrenérgico. A modulação do sistema glutamatérgico a partir de tratamentos com noradrenalina não mostrou variações significativas. Concluímos que o sistema glutamatérgico pode modular o sistema catecolaminérgico em células do bulbo de ratos neonatos, e que esta modulação pode ser importante na regulação da pressão arterial pelos núcleos bulbares. / It is found in the medulla oblongata several nuclei, as well as several neurotransmitters, related with the maintenance of the arterial pressure. Among these nuclei, the nucleus of the solitary tract stands aside for being one of the main modulators of the autonomic nervous system, being the first to receive afferences from baroreceptors and to send their stimuli to other nuclei. Among these neurotransmitters, glutamate and the catecholamines are both essentials to the maintenance of the arterial pressure. It is known that the stimulation of brain cells by neurotransmitters can result in changes in other neurotransmitter pathways, changing the cell response to certain stimuli. Taking in consideration the importance of glutamate and the catecholamines in the modulation of the arterial pressure, and that both of them can interfere in the cellular metabolism and create structural changes in neurons, we have speculated that the stimulation of the glutamatergic system could modulate the catecholaminergic system. In this work, it was evaluated if the glutamatergic and catecholaminergic systems could interact in cell cultures of the medulla oblongata of newborn rats, from treatments of the cultures with glutamate or noradrenaline. It was found that the treatment of these cultures with glutamate leads to a reduction in the protein and mRNA levels of the enzyme tyrosine hydroxylase and the receptor _2 adrenergic. The modulation of the glutamatergic system from treatments with noradrenaline did not show significative variation. We concluded that the glutamatergic system can modulate the catecholaminergic system in medulla oblongata cell cultures, and that this modulation can be important in the regulation of the arterial pressure by nuclei present in the medulla oblongata.

L’addiction se caractérise par une recherche et une consommation pathologiques de la drogue, maintenues malgré leurs conséquences néfastes. C’est une pathologie chronique car, le plus souvent, les tentatives de sevrage se soldent par une rechute. L’addiction à la cocaïne se développe chez 15 à 20 % des usagers, après un usage plus ou moins prolongé. Les solutions thérapeutiques font gravement défaut et c’est un enjeu que de comprendre les mécanismes neurobiologiques qui sous-tendent cette addiction. Les études cliniques et précliniques proposent que l’addiction résulte d’un déséquilibre entre les circuits cortico-subcorticaux qui gèrent la valeur motivationnelle de la drogue et ceux qui sont impliqués dans le contrôle cognitif inhibiteur. Des changements séquentiels dans des circuits interconnectés qui incluent notamment le noyau basolatéral de l’amygdale, le noyau accumbens et le cortex préfrontal seraient au cœur de processus motivationnels pathologiques et d’une difficulté à inhiber le craving et la consommation. L’étude de l’addiction, à l’échelle des circuits neuronaux, fait face à plusieurs défis. Techniquement limitée chez l’homme, elle peut bénéficier des modèles animaux, mais seulement s’ils capturent des dimensions de la pathologie. Au cours des dix dernières années, de tels modèles ont été mis en œuvre, mais exclusivement chez le rat. Or, les outils pour l’exploration fonctionnelle fine des circuits neuronaux ont été majoritairement développés chez la souris. Un autre défi consiste à pouvoir questionner la fonctionnalité des circuits, en temps réel, sur l’individu se comportant. Mes travaux de thèse ont eu pour objectif : 1. L’étude de marqueurs de connectivité fonctionnelle chez des rats Addict et des rats Non-addict à la cocaïne. Notre modèle d’addiction à la cocaïne permet d’identifier 15 à 20 % de rats qui, après une période prolongée d’autoadministration intraveineuse de cocaïne, et bien qu’ils aient consommé la même quantité de cocaïne que les autres, montrent une très forte motivation pour la substance, une difficulté à limiter la recherche de drogue, et maintiennent la prise de cocaïne malgré ses conséquences néfastes. L’électrophysiologie in vivo, multi-site, au moyen d’enregistrements unitaires ou de potentiels de champs locaux est un outil de choix pour l’exploration de la connectivité fonctionnelle chez le rongeur. Un défi technique a été de l’adapter pour la coupler à notre modèle d’addiction à la cocaïne chez le rat. Nous avons montré des différences significatives de connectivité fonctionnelle entre rats Addict et Non-addict, suggérant un défaut de fonctionnalité du cortex préfrontal médian (PFM) chez les Addict. 2. L’étude du rôle du cortex prélimbique (PL) dans le contrôle du comportement d’autoadministration de cocaïne chez le rat. Des données récentes de la littérature remettent en cause le dogme selon lequel le PL exerce exclusivement un rôle facilitateur sur les propriétés motivationnelles de la cocaïne. Nous avons cherché à clarifier le rôle du PL dans le comportement d’autoadministration de cocaïne avant que ne se développe une addiction : comprendre son rôle dans l’usage précoce de cocaïne pour, à terme, étudier l’évolution de son implication selon que l’individu développe ou non une addiction. Nous avons montré que l’inactivation du PL peut s’accompagner, chez le même individu, d’une diminution ou d’une exacerbation du comportement de recherche de cocaïne selon les contingences expérimentales. Les neurones du PL émettent des projections vers plusieurs structures. Pour étudier leur rôle dans les effets comportementaux observés, nous avons travaillé à la mise au point d’outils optogénétiques pour la manipulation de l’activité de voies neuronales spécifiques, chez le rat, pour lequel ils sont encore très peu développés. Mes travaux de thèse contribuent tant sur le plan théorique que technique à la compréhension des mécanismes psychobiologiques de l’addiction à la cocaïne. / Drug addiction is characterized by pathological drug seeking and taking, maintained despite their negative consequences. This is a chronic pathology, withdrawal attempts being unsuccessful in most cases. Cocaine addiction develops in about 15 to 20 % of habitual users. For cocaine, therapeutic options are lacking, which could be explained by the relatively poor understanding of the neurobiological mechanisms underlying cocaine addiction to date. Clinical and preclinical studies propose that addiction results from an imbalance between the cortical-subcortical circuits that process motivational value of drug-related stimuli versus those involved in cognitive inhibitory control. Hierarchical sequential changes in distinct, but interconnected circuits, including the basolateral amygdala, the nucleus accumbens and the prefrontal cortex could be at the core of pathological incentive processes and difficulty to control craving and drug taking. Studying addiction at the neuronal circuit level faces many challenges. Technically limited in humans, it can benefit from animal models, but only if they properly capture dimensions of the pathology. Over the last ten years, such models have been developed, but exclusively in rats. However, tools for a refined functional exploration of neuronal circuits have been established mostly in mice, and until recently they have begun to be explored in rats. In addition, another main challenge is the ability to investigate functional connectivity in real time in behaving animals. My thesis work had two objectives: 1. Studying markers of functional connectivity in rats showing a cocaine addiction-like behavior (Addict) or not (Non-addict). Our model of cocaine addiction allows identifying 15-20% of rats that show a high motivation for cocaine, a difficulty to limit drug seeking and that maintain drug taking despite negative consequences. These extreme behaviors occur after prolonged cocaine self-administration and despite that these rats have used a comparable amount of cocaine as compared to the others. In vivo, multi-site electrophysiology recordings, applied to single units or local field potentials, is a tool of choice for studying functional connectivity in rodents. A technical challenge has been to adapt and couple it to our model of cocaine addiction in the rat. We have evidenced significant differences in connectivity between Addict and Non-addict rats, which suggest a default of functionality of the medial prefrontal cortex in the Addict rats. 2. Studying the role of the prelimbic cortex (PL) in cocaine self-administration behavior in the rat. The canonical role of the PL in exclusively promoting drug seeking was recently questioned, with studies involving it also in inhibition of drug seeking. Our first goal was to clarify this role of the PL in early cocaine self-administration, i.e. before addiction-like behavior develops: understanding its early role to eventually compare it to its late role and whether an addiction-like behavior develops or not. We have shown that optogenetic PL inactivation can decrease or increase cocaine seeking in the same individual, according to experimental contingencies. PL neurons project to several remote structures. To study the role of these different neuronal pathways, we have worked in establishing optogenetic tools for the manipulation of specific neuronal pathways, in the rat, for which they are still poorly developed. My thesis work contribute, both theoretically and technically, to the understanding of the psychobiology of cocaine addiction.

Chronic traumatic encephalopathy is the neurodegenerative disease that is ascribed to the long term development of cognitive, behavioral, emotional, and motor deficits as a result of the exposure to high amounts of sub concussive traumatic brain injuries. The disease has gained recent popularity in the media for its prevalence in American football as a response to recent research that has suggested the prominence of the disease in nearly every NFL player that is examined post mortem. This has produced a growing concern for the consequences of head impact and participation in contact sports. Despite media attention, little is currently known about the specific causes of the disease and an in life diagnosis is still nonexistent. The present study proposes that the chemokine, CCL11, could prove to be a viable biomarker for recognizing the onset and progression of chronic traumatic encephalopathy. The results of our study suggest that football players who are clinically suspicious of CTE show significantly higher levels of CCL11 in their cerebrospinal fluid than do sedentary controls and noncontact athletes. Our results demonstrate that this increase in CCL11 is correlated with the number of years that a football player had participated in. We also suggest that this increase in CCL11 is associated with a unique immune response through results showing that the CCL11 expression increase is correlated with an increase in the expression of the cytokine IL-4 and substantial decrease in IFN-gamma. The analysis of CCL11 expression levels in the cerebrospinal fluid may prove to be a viable method of diagnosing and providing treatment for patients who may be at risk of chronic traumatic encephalopathy.

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Understanding pathways of neurological disorders requires extensive research on both functional and structural characteristics of the brain. This dissertation introduced two interrelated research endeavors, describing (1) a novel integrated approach for constructing functional connectivity networks (FCNs) of brain using non-invasive scalp EEG recordings; and (2) a decision aid for estimating intracranial volume (ICV). The approach in (1) was developed to study the alterations of networks in patients with pediatric epilepsy. Results demonstrated the existence of statistically significant (p

No description available.