As taxas de produção embrionária tanto da FIV (30-40%) como da TN (23%) ainda estão aquém do esperado. Desta forma, a pré-maturação com inibidores do ciclo celular é uma das alternativas que vem sendo estudada para aumentar a competência dos oócitos utilizados na PIV, na tentativa de otimizar o sucesso das biotécnicas. Sabe-se que neurotrofinas desempenham funções no sistema reprodutor. O BDNF é um exemplo de neurotrofina que parece estar relacionada com a maturação dos oócitos. Desta forma, o objetivo deste trabalho foi de aperfeiçoar a pré-maturação e a maturação in vitro de oócitos bovinos submetidos posteriormente à ativação partenogenética, visando seu uso na biotécnica de transferência de núcleo. Oócitos bovinos foram submetidos à maturação na presença (MIV/BD) ou ausência (MIV) de BDNF ou pré-maturados com BLI e suplementados (BL/BD) ou não (BL) com a neurotrofina. Posteriormente foram avaliados quanto à taxa de maturação (metáfase II), ativação (formação de prónúcleo) e desenvolvimento embrionário (produção e qualidade dos blastocistos). Não houve diferença (P>0,05) entre taxas de MII após a maturação com ou sem BDNF. Porém, o grupo pré-maturado e suplementado (BL/BD n=73; 91,2%) apresentou maior taxa de MII (P<0,05) em relação ao grupo não suplementado (BL n=66; 76,7%). Oócitos maturados nas mesmas condições foram ativados quimicamente para análise do desenvolvimento embrionário. Os grupos MIV/BD (n=30; 71,4%) e MIV (n=41; 91.1%) apresentaram diferença (P<0,05) em relação à taxa de ativação. Porém, não foi observada diferença quanto aos outros parâmetros do desenvolvimento. Quando os oócitos foram pré-maturados a taxa de clivagem do grupo suplementado (BL/BD: n=227; 65,2%) foi superior (P<0,05) ao grupo não suplementado (BL: n=187; 57,7%), mas não foram observadas diferenças (P>0,05) para os outros parâmetros de desenvolvimento. A qualidade dos embriões ativados também não foi afetada pelos tratamentos. Os grupos submetidos à TN (MIV e BL/BD) apresentaram diferenças (P<0,05) para extrusão do 1°CP (n=639; 63,5% e n=693; 69,5%, respectivamente) e para taxa de fusão (n=345; 72,9% e n=397; 79,2%, respectivamente) não havendo diferença para nenhum outro parâmetro avaliado. A qualidade dos embriões clonados também foi avaliada e não foi observada diferença. Após a transferência dos embriões dos grupos MIV (n=28) e BL/BD (n=26) para as receptoras, os grupos foram capazes de produzir gestação avançadas (10,7 e 11,5%, respectivamente) de forma similar (P>0,05). Com base nestes resultados podemos concluir que a suplementação, tanto da maturação como a pré-maturação não causa prejuízo no posterior desenvolvimento embrionário. Ainda, embriões clonados produzidos a partir de oócitos bloqueados são capazes de estabelecer gestações avançadas em bovinos. / Embryo production rates obtained from both IVF (30-40%) and NT (23%) are still below the expected values. Therefore, oocyte pre-maturation using cell cycle inhibitors is one of the alternatives which has been studied to increase the competence of oocytes used for IVP, as an attempt to optimize the success rates of these biotechniques. Neurotrophins are known to play several roles in the reproductive system. BDNF is an example of a neurotrophin that seems to be related to oocyte maturation. Therefore, the objective of this study was to improve techniques of pre-maturation and maturation of bovine oocytes submitted to parthenogenetic activation, aiming for its use on cloning by nuclear transfer. Bovine oocytes were submitted to maturation either in presence (IVM/BD) or absence (IVM) of BDNF or pre-matured with BLI and supplemented (BL/BD) or not (BL) with the neurotrophin. Groups were evaluated for maturation rates (metaphase II), activation (pro-nucleus formation) and embryo development (blastocyst formation rate and quality). There was no difference (P>0.05) in MII rate after the maturation with or without BDNF. However, pre-maturation in the supplemented group (BL/BD, n=73; 91.2%) resulted in higher MII rate (P<0.05) when compared with the nonsupplemented group (BL, n=66; 76.7%). Oocytes which were matured under the same conditions were also activated chemically for embryonic development analysis. Activation rates were different (P<0.05) from IVM/BD groups (n=30; 71.4%) and IVM (n=41; 91.1%). However, no difference were observed for development parameters. When the oocytes were prematured, cleavage rates in the supplemented group were superior (P<0.05) than non supplemented group (BL/BD: n=227; 65.2%) and (BL: n=187; 57.7%), but no difference was observed for other developmental parameters. Embryo quality was also evaluated and no difference was observed between treatments. Groups submitted to NT (IVM and BL/BD) differed regarding (P<0.05) the 1stPB extrusion (n=639; 63.5% and n=693; 69.5%, respectively) and fusion rate (n=345; 72.9% and n=397; 79.2%, respectively), but did not present differences for other evaluated parameters. Embryo quality was evaluated again and no differences were observed. After the embryos were transferred to recipient cows, groups IVM (n=3; 10.7%) and BL/BD (n=3; 11.5%) were capable of producing advanced gestations at similar rates (P>0.05). Based on these results, it may be concluded that supplementation of both maturation and pre-maturation does not impair embryonic development. Additionally, cloned embryos produced from blocked oocytes are able to establish advanced gestation in cattle.

A adolescência é caracterizada como um período de grande estresse na vida humana, sendo o bullying um dos principais estressores desencadeantes de distúrbios psiquiátricos. Modelos animais de depressão usam o estresse social prolongado como indutores de depressão. Utilizamos o modelo de subjugação (ou derrota) social prolongada em camundongos machos adolescentes para estudar a regulação do BDNF neste contexto. Os animais submetidos ao estresse psicossocial apresentaram anedonia no teste de preferência por sacarose e esquiva social no teste de interação social. Explorando a variabilidade comportamental, identificamos grupos suscetíveis e resilientes ao estresse. Animais suscetíveis apresentaram uma redução na expressão do transcrito Bdnf4 e dos níveis proteicos de BDNF total e sua isoforma truncada somente no estriado dorsal, área ainda pouco relacionada à depressão, enquanto que não ocorreram alterações no córtex pré-frontal e hipocampo, áreas comumente afetadas durante a depressão. / Adolescence is characterized by a period of life with great amount of stress, being the bullying one of the main stressors leading to psychiatry disorders. Animal models of depression use prolonged social stress to model depression. We used the prolonged social defeat model in adolescent male mice to study BDNF regulation in this context. Defeated mice showed anhedonia in the sucrose preference test, and social avoidance in the social interaction test. We took advantage of the behavioral outcome variability to identify susceptible and resilient groups to the stress. The susceptible mice showed a reduction in Bdnf4 transcripts, and in total BDNF protein levels, as well as its truncated form in the dorsal striatum, a brain area not much related to depression. However, BDNF gene or protein expression did not differ in the prefrontal cortex and hippocampus, areas commonly associated with depression.

A adolescência é caracterizada como um período de grande estresse na vida humana, sendo o bullying um dos principais estressores desencadeantes de distúrbios psiquiátricos. Modelos animais de depressão usam o estresse social prolongado como indutores de depressão. Utilizamos o modelo de subjugação (ou derrota) social prolongada em camundongos machos adolescentes para estudar a regulação do BDNF neste contexto. Os animais submetidos ao estresse psicossocial apresentaram anedonia no teste de preferência por sacarose e esquiva social no teste de interação social. Explorando a variabilidade comportamental, identificamos grupos suscetíveis e resilientes ao estresse. Animais suscetíveis apresentaram uma redução na expressão do transcrito Bdnf4 e dos níveis proteicos de BDNF total e sua isoforma truncada somente no estriado dorsal, área ainda pouco relacionada à depressão, enquanto que não ocorreram alterações no córtex pré-frontal e hipocampo, áreas comumente afetadas durante a depressão. / Adolescence is characterized by a period of life with great amount of stress, being the bullying one of the main stressors leading to psychiatry disorders. Animal models of depression use prolonged social stress to model depression. We used the prolonged social defeat model in adolescent male mice to study BDNF regulation in this context. Defeated mice showed anhedonia in the sucrose preference test, and social avoidance in the social interaction test. We took advantage of the behavioral outcome variability to identify susceptible and resilient groups to the stress. The susceptible mice showed a reduction in Bdnf4 transcripts, and in total BDNF protein levels, as well as its truncated form in the dorsal striatum, a brain area not much related to depression. However, BDNF gene or protein expression did not differ in the prefrontal cortex and hippocampus, areas commonly associated with depression.

La maladie de Huntington (HD) est une maladie génétique neurodégénérative qui touche environ 6000 personnes en France. Les manifestations psychiatriques sont une des composantes majeures des symptômes précoces de la pathologie. Ainsi, des épisodes dépressifs parfois associés à de l’anxiété généralisée sont communément observés au cours des stades pré-symptomatiques de la maladie. On connaît mal à l’heure actuelle les raisons de cette prévalence élevée. L'allèle responsable de la maladie code une protéine appelée huntingtine (HTT) dont l'expansion polyglutaminique (polyQ) en N-terminal est plus longue que dans la HTT non pathogénique. La huntingtine est impliquée dans diverses fonctions cellulaires et notamment dans le transport et l’expression d’un facteur neurotrophique, le Brain-Derived Neurotrophic Factor (BDNF). Celui-ci est d’ailleurs connu pour son rôle dans la régulation des troubles de l’humeur, de la neurogénèse hippocampique chez l’adulte, ainsi que dans la réponse thérapeutique aux antidépresseurs. Nous avons émis l'hypothèse que la huntingtine, en plus de ses rôles connus dans le cortex et le striatum, puisse jouer également un rôle dans l'hippocampe. Ainsi, une altération du transport de BDNF dans l’hippocampe pourrait en partie expliquer les troubles de l’humeur observés chez les patients HD.Par une approche in vivo, en utilisant différents modèles de souris, nous avons ainsi démontré que la huntingtine stimule le trafic vésiculaire et la sécrétion de BDNF dans les neurones hippocampiques et que cette action peut être modulée par la mutation polyQ ou par le statut de phosphorylation de la protéine sur les sérines 1181 et 1201. Cela aboutit à des modifications des voies de signalisation (Akt, ERK, CREB) activées par le BDNF. Nous mettons également en évidence que la huntingtine sauvage est impliquée dans le soutien exercé par les neurones matures sur les nouveaux neurones, nécessaire à leur survie à long terme et à la formation d’une arborisation dendritique complexe. Le BDNF est l’intermédiaire idéal grâce à ses effets sur la neurogenèse hippocampique. Enfin, la huntingtine sauvage et ses formes mutées (polyQ et phosphorylation sur les sérines 1181 et 1201) sont impliquées dans le comportement anxio-dépressif des souris. / Huntington disease (HD) is a genetic neurodegenerative disorder that affects about 6,000 people in France. Psychiatric manifestations are an important component of the early symptoms of the disease. Indeed, depressive episodes sometimes associated with generalized anxiety are commonly observed during the pre-symptomatic stages of disease. Few information is available about the reasons for this high prevalence.The allele responsible for the disease encodes a protein called huntingtin (HTT) whose polyglutamine expansion (polyQ) in the N-terminal region is longer than in the non-pathogenic HTT. Huntingtin is involved in various cellular functions including the transport and the expression of a neurotrophic factor, the Brain-Derived Neurotrophic Factor (BDNF). This factor is also known for its role in the regulation of mood, adult hippocampal neurogenesis, and in the therapeutic response to antidepressants.We hypothesized that huntingtin, in addition to its known roles in the cortex and striatum, may play a role in the hippocampus. Thus, an impaired transport of BDNF in the hippocampus could partly explain the mood disorders observed in HD patients.By an in vivo approach using different mouse models, we demonstrated that huntingtin stimulates vesicular trafficking and secretion of BDNF in hippocampal neurons and that this action may be modulated by the polyQ mutation or by the phosphorylation status of the protein on serines 1181 and 1201. These lead to changes in signaling pathways (Akt, ERK, CREB) activated by BDNF.We also demonstrate that normal huntingtin is involved in the support provided by mature neurons to new neurons for their long-term survival and the formation of a complex dendritic arborization. BDNF is the ideal candidate to mediate these effects on hippocampal neurogenesis. Finally, normal huntingtin and its mutated forms (polyQ and phosphorylated on serines 1181 and 1201) are involved in anxiety and depressive-like phenotype in mice.

O fator neurotrófico derivado do cérebro (BDNF) tem sido associado com a angiogênese por meio do seu receptor específico tropomiosina quinase B (TrkB). Uma vez que ambos são expressos em células endoteliais vasculares e o treinamento físico aeróbio (TF) pode aumentar os níveis séricos de BDNF, este estudo teve como objetivo testar as hipóteses de que: 1) Os níveis séricos de BDNF modulam o fluxo de sangue periférico; e 2) A presença do alelo Met no polimorfismo BDNF Val66Met prejudica o ganho de vasodilatação por TF. Foram genotipados para o polimorfismo do gene BDNF 304 voluntários saudáveis do sexo masculino (Val66Val, n = 221; Val66Met, n = 83) que foram submetidos a intenso TF em uma pista de corrida 3 vezes/semana durante 4 meses. Foram avaliados pré e pós-TF as concentrações circulantes de BDNF e pró-BDNF (ELISA), frequência cardíaca (FC), pressão arterial média (PAM), o fluxo de sangue do antebraço (FSA) e resistência vascular periférica (RVP). No período pré-TF, BDNF, pró-BDNF, a razão BDNF/pró-BDNF, FSA e RVP foram semelhantes entre todos os genótipos. Depois do TF, a capacidade funcional (VO2pico) aumentou e houve a diminuição da FC de repouso de forma semelhante em ambos os grupos. O grupo Val66Val, mas não o Val66Met, aumentou os níveis de BDNF (interação, p=0,04) e aumentou a razão BDNF/pró-BDNF (interação, p < 0,001). Curiosamente, as respostas do FSA (Interação, p=0,04) e da RVP (Interação, p=0,01), durante o exercício handgrip, do grupo Val66Val, apresentou melhoras quando comparado com o grupo Val66Met, mesmo com respostas similares de FC e PAM. Outras análises mostraram associação entre a razão BDNF/pró-BDNF e FSA (R=0,64; p < 0,001) e RVP (R=-0,58; p < 0,001). Estes resultados mostram que, em resposta ao TF, tanto a reatividade vascular periférica quanto o BDNF circulante são prejudicados pela presença do polimorfismo Val66Met do gene BDNF e esta responsividade está associada às concentrações de BDNF sérico em indivíduos saudáveis / The neurotrophin Brain-derived neurotrophic factor (BDNF) has been associated with angiogenesis through its specific receptor tropomyosin-related kinase B (TrkB). Since both are expressed in vascular endothelial cells and aerobic training (ET) can increase serum BDNF levels, this study aimed to test the hypotheses that: 1) Serum BDNF levels modulate peripheral blood flow; and 2) The presence of the allele Met in the BDNF Val66Met polymorphism impairs vasodilation gain by ET. We genotyped for BDNF polymorphism 304 healthy male volunteers (Val66Val, n= 221; Val66Met, n=83) which underwent to intense aerobic ET on a running track 3 times/week for 4 months. We evaluated pre and post ET serum BDNF and proBDNF concentration (ELISA), heart rate (HR), mean blood pressure (MBP), forearm blood flow (FBF) and forearm vascular resistance (FVR). In the pre ET, BDNF, proBDNF, BDNF/proBDNF ratio, FBF and FVR were similar between all genotypes. After ET, functional capacity (VO2peak) increased and HR decreased similarly in both groups. Val66Val, but not Val66Met, increased BDNF (Interaction, p= 0.04) and BDNF/proBDNF ratio (Interaction, p < 0.001). Interestingly, FBF (Interaction, p=0.04) and the FVR (Interaction, p=0.01) responses during handgrip exercise improved in Val66Val compared with Val66Met, even with similar responses of HR and MBP. Further analyses showed association between BDNF/proBDNF ratio and FBF (R=0.64, p < 0.001) and FVR (R=-0.56, p < 0001). These results show that peripheral vascular reactivity and serum BDNF responses to ET are impaired by the BDNF Val66Met polymorphism and such responsiveness is associated to the serum BDNF concentrations in healthy subjects

Introdução: Penfigoide bolhoso (PB) resulta da produção de autoanticorpos contra proteínas hemidesmossomais BP (Bullous Pemphigoid) 180 e/ou 230, acomete os idosos, e está associado com doenças neurológicas (DN), especialmente com a demência (DEM). BP180/230 foram identificadas no Sistema Nervoso Central (SNC), aventando-se possível mimetismo antigênico entre moléculas da pele e do SNC. O fator neurotrófico derivado do cérebro (BDNF) participa da neurogênese, sinaptogênese e sobrevivência neuronal, e a sua diminuição sérica tem sido relacionada com DN. Objetivo: Quantificar o peptídeo BDNF e os anticorpos anti-BP180/230 na relação PB com DEM. Material e Métodos: Em estudo comparativo, 50 pacientes com PB, 50 com demência e 50 controles foram avaliados. A detecção dos anticorpos anti-BP180/P230 e do peptídeo BDNF foi determinada por ensaios ELISA. Imunofluorescência indireta (IFI) foi conduzida nas amostras de soro dos pacientes do grupo DEM e dos controles que apresentaram positividade para anti-BP180/230. Resultados: No grupo PB, a frequência de DN foi de 26%: DEM 16%, acidente vascular cerebral 6%, e epilepsia 4% - 5/8 (63%) pacientes apresentaram demência vascular e 3/8 (38%) demência por doença de Alzheimer. Positividade para anti-BP180/230 foi observada no grupo PB (74% e 40%, respectivamente), no grupo DEM (10% e 10%) e nos controles (14% e 0%). No grupo DEM, em 2/10 pacientes que apresentaram positividade para antiBP180/230, a IFI evidenciou depósito de IgG e C3 no lado epidérmico da clivagem, configurando quadro subclínico de PB. A mediana do BDNF resultou menor no grupo DEM (25,41 pg/ml) comparado aos controles (38,21 pg/mL), e o grupo PB apresentou os menores valores de BDNF (16,88 pg/mL). Não houve correlação dos títulos de anticorpos antiPB180/230 com a concentração do peptídeo BDNF no grupo PB. Os pacientes do grupo DEM foram alocados de acordo com a escala de demência - CDR1, CDR2 e CDR3; a mediana de BDNF do subgrupo CDR3 (23,37 pg/mL) foi inferior ao CDR1 (30,17 pg/ mL). Não houve diferença na concentração do BDNF segundo o tipo de demência. O grupo PB, quando estratificado em - com DEM, outras DN e sem DN, aqueles com associação com DEM apresentaram menores níveis de BDNF (9,1 pg/mL), comparados ao grupo sem DN e ao subgrupo CDR3 do grupo DEM. Conclusão: Marcadores para PB não são úteis para o diagnóstico de DEM. Valores sorológicos baixos de BDNF no grupo PB podem sugerir associação com DEM. BDNF pode ser utilizado como biomarcador de gravidade da DEM. / Introduction: Bullous pemphigoid (BP) is characterized by autoantibodies against the hemidesmossomal proteins BP180 and/or BP230, affects the elderly people and has been strongly associated with neurological disorders (ND), especially dementia. A possible antigenic mimicry hypothesis between the skin and the nervous system molecules is strong reasonable because BP peptides have also been identified in the central nervous system (CNS). Brain-derived neurotrophic factor (BDNF) plays a role in the synaptogenesis, neurogenesis, and neuronal survival, and some studies have been correlated the decreased serum BDNF levels with ND. The aim of this study was to quantify the BDNF peptide and the anti-BP180 and anti-BP230 antibodies in the BP and DEM relationship. Methods: AntiBP180/230 and BDNF quantification were analyzed in three groups: 50 patients with BP, 50 patients with dementia and 50 elderly individuals comprised a case-control study. Serum IgG anti-180/230, and the BDNF peptide were evaluated by using ELISA commercial kits; and immunofluorescence allied to salt split skin technique (SSS) was conducted in serum samples from patients of the dementia group and from controls who showed positive anti-BP180/230. Results: In BP group, 26% were associated with ND, and dementia was the most frequent (16%), followed by stroke (6%) and epilepsy (4%) - 5/8 (63%) patients showed vascular dementia and 3/8 (38%) patients presented dementia due to Alzheimer\'s disease. AntiBP180/230 positivity was observed in BP group (74%, 40%, respectively), in dementia group (10%, 10%) and in controls (14%, 0%). In 2/10 patients of the dementia group with positive anti-BP180/230, IIF showed IgG and C3 deposition in the epidermal side of cleavage, configuring a subclinical BP dermatosis. The medians of BDNF resulted lower in the patients of the dementia group (25.41 pg/mL) compared with controls (38.21 pg/mL), and the BP group presented the lowest BDNF values (16.88 pg/mL). There was no correlation between the anti-BP180/230 antibodies titres and the BDNF levels in BP group. There was no difference of BDNF levels accordingly with the clinical type of dementia in the dementia group. Patients of the dementia group were sub grouped accordingly with the clinical dementia severity - CDR1, CDR2 and CDR3 - being the median of BDNF in CDR3 (23.37 pg/mL) lower than in CDR1 (30.17 pg/mL) subgroup. BP group had lower levels of BDNF compared to CDR3 subgroup. BP patients when stratified with dementia, other ND and without ND, those with association with dementia presented the lowest levels of BDNF (9.1 pg/mL) compared to the PB patients without DN and to the CDR3 subgroup. Conclusion: BP biomarkers are not useful for the diagnosis of dementia. Low BDNF levels seen in BP patients may suggest an association with dementia. BDNF may be used as a biomarker of severity of dementia.

La pratique régulière d’une activité physique (AP) est un important message de santé publique tant pour ces bénéfices cardiovasculaires que cérébraux. Par ailleurs, la rééducation par le mouvement et notamment l’exercice sur tapis roulant est de plus en plus utilisée pour accélérer la récupération après un accident vasculaire cérébral (AVC). Il est admis que l’AP impacte positivement le fonctionnement cérébral via l’augmentation des taux de BDNF (brain-derived neurotrophic factor) dans le cerveau et que son bénéfice cardiovasculaire est à relier à une modification du phénotype endothélial. Dans un premier temps, nous avons mis au point le dosage par Western blotting du proBDNF et du BDNF mature (BDNFm) ainsi que les techniques immunohistochimiques permettant de localiser le BDNF à l’étage cellulaire. Dans un second temps, nous avons comparé l’effet d’une AP (tapis roulant, 30min/j, 18m/min, 7 jours consécutifs) sur le métabolisme et la localisation du BDNF chez des rats sains versus soumis à une ischémie cérébrale focale. Dans un dernier temps, nous nous sommes intéressés à l’expression du BDNF cardiovasculaire chez des rats normo- ou hypertendus, sédentaires ou soumis au modèle d’AP précédent. Les principaux résultats montrent que 1) l’AP augmente les taux de proBDNF et de BDNFm aussi bien dans le cerveau (neurones et cellules endothéliales) que dans le système cardiovasculaire (endothélium vasculaire et cardiaque), 2) l’ischémie cérébrale n’entrave pas les effets cérébraux de l’AP sur le BDNFm, 3) l’expression endothéliale (cœur, aorte, artère coronaire) du BDNF est moindre en cas d’hypertension, 4) la synthèse et la sécrétion de BDNF par des cellules endothéliales en culture augmentent lorsque les cellules sont soumises à des contraintes de cisaillement, 5) le BDNF exerce un effet vasodilatateur sur le modèle d’aorte isolée. En conclusion, notre travail montre qu’il est possible de sélectionner, chez des rats sains, des protocoles d’AP capables d’augmenter la neuroplasticité dépendante du BDNF chez des rats ischémiés. Il identifie également le BDNF d’origine endothéliale comme un marqueur potentiel de la fonction endothéliale et un acteur jusque-là ignoré des changements neuroplastiques induits par l’AP. / The regular practice of physical activity is an important message for public health as it has both cardiovascular and brain benefits. Furthermore, rehabilitation programs involving movement especially the treadmill exercise are being used increasingly to accelerate post stroke recovery. It is recognised that the physical activity has a positive impact on brain function through increased levels of BDNF (brain-derived neurotrophic factor) in the brain and that its cardiovascular benefit is connected to a change in endothelial phenotype. As a first step, we developped the dosage by Western blotting of proBDNF and mature BDNF (mBDNF) and the immunohistochemical techniques in order to localise BDNF in the cell floor. As a second step, we compared the effect of physical activity (treadmill exercise, 30min/d, 18m/min, 7 consecutive days) on the metabolism and localisation of BDNF in healthy rats versus rats subjected to focal cerebral ischemia. As a final step, we looked into the expression of cardiovascular BDNF in normotensive or hypertensive rats, sedentary or subjected to the same physical activity. The main results show that 1) physical activity increases the levels of proBDNF and mBDNF in both brain (neurons and endothelial cells) and cardiovascular system (heart and vascular endothelium), 2) cerebral ischemia does not change the cerebral effects of physical activity on mBDNF, 3) endothelial expression (heart, aorta, coronary artery) of BDNF is reduced in the presence of hypertension, 4) the synthesis and secretion of BDNF by endothelial cells in culture increase when the cells are subjected to shear stress, 5) BDNF has a vasodilatator effect on the isolated aorta model. In conclusion, our work shows that it is possible to select, in healthy rats, protocols of physical activity that are able to increase the BDNF-dependent neuroplasticity in ischemic rats. It also identifies endothelial BDNF as a potential marker of endothelial function as well as a potential contributor of physical activity-induced neuroplasticity.

As taxas de produção embrionária tanto da FIV (30-40%) como da TN (23%) ainda estão aquém do esperado. Desta forma, a pré-maturação com inibidores do ciclo celular é uma das alternativas que vem sendo estudada para aumentar a competência dos oócitos utilizados na PIV, na tentativa de otimizar o sucesso das biotécnicas. Sabe-se que neurotrofinas desempenham funções no sistema reprodutor. O BDNF é um exemplo de neurotrofina que parece estar relacionada com a maturação dos oócitos. Desta forma, o objetivo deste trabalho foi de aperfeiçoar a pré-maturação e a maturação in vitro de oócitos bovinos submetidos posteriormente à ativação partenogenética, visando seu uso na biotécnica de transferência de núcleo. Oócitos bovinos foram submetidos à maturação na presença (MIV/BD) ou ausência (MIV) de BDNF ou pré-maturados com BLI e suplementados (BL/BD) ou não (BL) com a neurotrofina. Posteriormente foram avaliados quanto à taxa de maturação (metáfase II), ativação (formação de prónúcleo) e desenvolvimento embrionário (produção e qualidade dos blastocistos). Não houve diferença (P>0,05) entre taxas de MII após a maturação com ou sem BDNF. Porém, o grupo pré-maturado e suplementado (BL/BD n=73; 91,2%) apresentou maior taxa de MII (P<0,05) em relação ao grupo não suplementado (BL n=66; 76,7%). Oócitos maturados nas mesmas condições foram ativados quimicamente para análise do desenvolvimento embrionário. Os grupos MIV/BD (n=30; 71,4%) e MIV (n=41; 91.1%) apresentaram diferença (P<0,05) em relação à taxa de ativação. Porém, não foi observada diferença quanto aos outros parâmetros do desenvolvimento. Quando os oócitos foram pré-maturados a taxa de clivagem do grupo suplementado (BL/BD: n=227; 65,2%) foi superior (P<0,05) ao grupo não suplementado (BL: n=187; 57,7%), mas não foram observadas diferenças (P>0,05) para os outros parâmetros de desenvolvimento. A qualidade dos embriões ativados também não foi afetada pelos tratamentos. Os grupos submetidos à TN (MIV e BL/BD) apresentaram diferenças (P<0,05) para extrusão do 1°CP (n=639; 63,5% e n=693; 69,5%, respectivamente) e para taxa de fusão (n=345; 72,9% e n=397; 79,2%, respectivamente) não havendo diferença para nenhum outro parâmetro avaliado. A qualidade dos embriões clonados também foi avaliada e não foi observada diferença. Após a transferência dos embriões dos grupos MIV (n=28) e BL/BD (n=26) para as receptoras, os grupos foram capazes de produzir gestação avançadas (10,7 e 11,5%, respectivamente) de forma similar (P>0,05). Com base nestes resultados podemos concluir que a suplementação, tanto da maturação como a pré-maturação não causa prejuízo no posterior desenvolvimento embrionário. Ainda, embriões clonados produzidos a partir de oócitos bloqueados são capazes de estabelecer gestações avançadas em bovinos. / Embryo production rates obtained from both IVF (30-40%) and NT (23%) are still below the expected values. Therefore, oocyte pre-maturation using cell cycle inhibitors is one of the alternatives which has been studied to increase the competence of oocytes used for IVP, as an attempt to optimize the success rates of these biotechniques. Neurotrophins are known to play several roles in the reproductive system. BDNF is an example of a neurotrophin that seems to be related to oocyte maturation. Therefore, the objective of this study was to improve techniques of pre-maturation and maturation of bovine oocytes submitted to parthenogenetic activation, aiming for its use on cloning by nuclear transfer. Bovine oocytes were submitted to maturation either in presence (IVM/BD) or absence (IVM) of BDNF or pre-matured with BLI and supplemented (BL/BD) or not (BL) with the neurotrophin. Groups were evaluated for maturation rates (metaphase II), activation (pro-nucleus formation) and embryo development (blastocyst formation rate and quality). There was no difference (P>0.05) in MII rate after the maturation with or without BDNF. However, pre-maturation in the supplemented group (BL/BD, n=73; 91.2%) resulted in higher MII rate (P<0.05) when compared with the nonsupplemented group (BL, n=66; 76.7%). Oocytes which were matured under the same conditions were also activated chemically for embryonic development analysis. Activation rates were different (P<0.05) from IVM/BD groups (n=30; 71.4%) and IVM (n=41; 91.1%). However, no difference were observed for development parameters. When the oocytes were prematured, cleavage rates in the supplemented group were superior (P<0.05) than non supplemented group (BL/BD: n=227; 65.2%) and (BL: n=187; 57.7%), but no difference was observed for other developmental parameters. Embryo quality was also evaluated and no difference was observed between treatments. Groups submitted to NT (IVM and BL/BD) differed regarding (P<0.05) the 1stPB extrusion (n=639; 63.5% and n=693; 69.5%, respectively) and fusion rate (n=345; 72.9% and n=397; 79.2%, respectively), but did not present differences for other evaluated parameters. Embryo quality was evaluated again and no differences were observed. After the embryos were transferred to recipient cows, groups IVM (n=3; 10.7%) and BL/BD (n=3; 11.5%) were capable of producing advanced gestations at similar rates (P>0.05). Based on these results, it may be concluded that supplementation of both maturation and pre-maturation does not impair embryonic development. Additionally, cloned embryos produced from blocked oocytes are able to establish advanced gestation in cattle.

Les troubles de l’humeur font partie des problèmes de santé majeurs dans le monde, du fait de leur forte incidence et récurrence dans la population générale, de la nuisance pour la qualité de vie des patients ainsi que la répercussion majeure sur les systèmes de santé. A ce jour, l’étiologie ainsi que les mécanismes biologiques sous-jacents les troubles de l’humeur sous encore très mal connus. Un nombre grandissant de preuves suggère qu’une interaction complexe entre les gènes et l’environnement serait a l’origine de la mise en place et évolution des épisodes dépressifs majeurs – un des troubles de l’humeur les plus répandus. Par conséquent, des régulations épigénétiques complexes, qui consistent en des mécanismes clefs par lesquels l’environnement induit des changements persistant sur l’expression des gènes (sans modifier le code génétique), joueraient un rôle prépondérant dans la pathophysiologie de la dépression. De manière plus spécifique, la répression épigénétique du gène codant pour le brain-derived neurotrophic factor (BDNF) – un facteur de croissance impliqué dans la plasticité neuronale et développement du système nerveux central – serait un mécanisme clef dans la mise en place de la dépression et autres troubles de l’humeur. Dans ce contexte, les travaux de recherche présentés dans cette thèse visent à explorer le rôle des régulations épigénétiques au niveau de la signalisation BDNF/TrkB dans la physiopathologie et traitement des troubles de l’humeur. Les résultats montrent que les régulations épigénétiques au niveau de la signalisation BDNF/TrkB sont fortement impliquées dans la mise en place et maintenance de la plasticité neuronale. De plus, les variations environnementales, particulièrement au cours du développement, sont capables d’induire une reprogrammation épigénétique stable et persistante au niveau du complexe BDNF/TrkB ainsi qu’une altération de la neuroplasticité, conduisant à une augmentation de la vulnérabilité au stress et troubles de l’humeur. De manière intéressante, la signalisation du récepteur TrkB est nécessaire pour les effets neurobiologiques et comportementaux des antidépresseurs. De ce fait, une approche pharmocologique ciblée sur le complexe BDNF/TrkB et ses régulations épigénétiques sous-jacentes apparaît comme stratégie thérapeutique prometteuse pour le traitement des troubles de l’humeur tel que la dépression. / Mood disorders are among the major health problems worldwide due to the high prevalence and recurrence in the general population, and the significant burden for individual life quality and the repercussion on healthcare systems and society. Up to date, the etiology and biological mechanisms underlying mood disorders are still poorly understood. Mounting evidences suggest that a complex interaction between genes and environment might account in the development and course of major depression i.e. one of the most prevalent affective disorders. Accordingly, complex epigenetic regulations - consisting of key mechanisms by which environmental factors induce enduring changes in gene expression without altering the DNA code - have been suspected to plays a pivotal role in the pathophysiology of depression. More specifically, epigenetic repression of the gene encoding for brain-derived neurotrophic factor (BDNF) - a small-secreted growth factor implicated in brain development and neuronal plasticity - may have a preponderant role in the onset of depression and other mood disorders. In this context, the research presented in this thesis aimed at exploring the role of BDNF signaling and its downstream epigenetic regulations in the pathophysiology and treatment of mood disorders. Our findings indicate that epigenetic regulation at BDNF/TrkB signaling is critically important in the establishment and maintenance of neuronal plasticity. Moreover, environmental variations, especially when occurring in development, are able to induce stable and enduring epigenetic reprogramming involving aberrant BDNF/TrkB signaling and impaired neuroplasticity, thereby increasing vulnerability to stress and mood disorders. Interestingly, antidepressants require TrkB to exert some of their neurochemical and behavioral effects. Hence, targeting the BDNF receptor TrkB to restore a normal epigenetic regulation and neuronal functioning appears to be a promising strategy for the treatment of mood disorders.

Le facteur neurotrophique dérivé du cerveau (BDNF) synthétisé sous la forme d'un précurseur (proBDNF) qui peut être clivé pour donner sa forme mature (mBDNF). Le mBDNF et le proBDNF produisent des réponses physiologiques opposées par l'activation de deux classes distinctes de récepteurs transmembranaires : respectivement, le récepteur TrkB et p75NTR. Le ratio mature/pro-BDNF est un élément important impliqué dans la plasticité synaptique, la formation des circuits neuronaux et in fine les fonctions cognitives. Les altérations dans ce clivage peuvent ainsi expliquer l’émergence de conditions pathologiques post-lésionnelles, comme la mort cellulaire induite par un état de mal épileptique. Au cours de ma thèse, j'ai montré que l'altération de la maturation du BDNF in vitro, provoquait, via le récepteur p75NTR, une altération de l’activité GABAergique. Par ailleurs, au cours des crises d'épilepsies, les réponses dépolarisantes et excitatrices du GABA, soutenus par la baisse d’expression et d’activité du co-transporteur KCC2, ont été rapportées. Ainsi, in vivo, j’ai montré que la voie proBDNF/p75NTR module l'homéostasie chlore au cours du développement et dans des processus d’épileptogenèse. Pendant le développement, l’activation de la voie proBDNF/p75NTR contrôle le passage d’un GABA immature dépolarisant à un GABA mature hyperpolarisant via KCC2. Pendant l’épileptogenèse, le proBDNF via p75NTR contribuerait à l’hyperexcitabilité des réseaux neuronaux. De plus, le blocage de p75NTR permet de réduire le nombre de crises épileptiques. En conclusion, proBDNF/p75NTR est un facteur clé dans la séquence maturative du GABA et dans la mise en place de l’épilepsie du lobe temporal. / The brain-derived neurotophic factor (BDNF) is synthesized as a precursor (proBDNF) that can be processed intracellularly to the mature form (mBDNF). mBDNF and proBDNF are assumed to produce opposing physiological responses mediated by the activation of two distinct classes of transmembrane receptors, the TrkB and the p75NTR respectively. The proteolysis of proBDNF is crucial for cognitive functions; its impairment may account for the emergence of brain disorders such as epilepsy. During my thesis, I showed that alteration in BDNF maturation in vitro triggers an up-regulation of p75NTR, inducing a disruption of GABAergic transmission. Moreover, in epilepsy, depolarizing and excitatory GABAergic responses, due to alteration of KCC2, have been reported. Interestingly, I described novel insights into the proBDNF/p75NTR mechanisms and function in vivo in modulating chloride homeostasis during the development of neuronal networks and in the pathogenesis of epilepsy. In physiological conditions, p75NTR activation by proBDNF may be a key regulator in shaping neural circuitry and synaptic plasticity. Moreover, I have shown that proBDNF/p75NTR to mBDNF/TrkB ratio may control the timing of the developmental shift of GABA depolarizing to hyperpolarizing. During epileptogenesis, proBDNF via p75NTR alters the excitatory/inhibitory equilibrium thereby enhancing neuronal activity through the inhibition of KCC2 function. Hence, blockade of p75NTR can prevent some of the epileptogenic mechanisms. Altogether, these data provide the first compelling evidence that proBDNF disrupts the GABA excitatory/inhibitory developmental sequence, which then favors the emergence of epileptic disorders.