Orientador: Cristina Antoniali Silva / Banca: Fernando Silva Carneiro / Banca: Carlos Alan Candido Dias Junior / Banca: Graziela Scalianti Ceravolo / Banca: Angela de Castro Resende / Resumo: A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas en... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Reduction of vascular reactivity to phenylephrine (PE) in aortaof spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higherproduction and/or higerbioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation,by PI3K/Akt.Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc)is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOSon serine siteleads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this studywas to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteriesof pregnant rats. The O-GlcNAc-protein content and also the enzymesexpressionthat participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2ndor 3rdbranch) of non-pregnant (NP) and pregnant (P), normotensiverats(Wistar) and SHR.Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolaein the aorta and the mesenteric artery in the presence or absence of methyl-β-cyclodextrin (dextrin, 10 mmol/L) by electronicmicroscopy.In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 μmol/L) and of the caveolae, using a caveolae disassembler, (Complete abstract electronic access below) / Doutor

O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença. / Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease.

O câncer de próstata é a segunda causa de morte entre homens no Brasil. É tipo um câncer de crescimento lento, podendo levar anos para o tumor atingir 1 cm3, porém, em alguns casos ele pode se espalhar pelo corpo, sendo o osso o principal sítio de metástase. No estágio de desenvolvimento do câncer conhecido como metástase, o principal tratamento consiste em terapia de restrição andrógena, levando as células prostáticas a pararem de proliferar, uma vez que elas crescem em resposta a presença de hormônios andrógenos, como a diidrotestosterona e testosterona. Porém, em alguns casos, as células proliferam mesmo na ausência de andrógenos e isto se deve a diversos fatores que, em geral, estão associados a mutações no receptor andrógeno e/ou alterações no metabolismo andrógeno. Quando isto acontece, os tratamentos disponíveis são menos efetivos e costumam falhar. Porém, estudos sugerem que o γ-orizanol, um fitoesterol extraído do óleo do farelo do arroz; e extratos amplamente utilizados na medicina popular, como o extrato hidroalcólico de Thuya occidentalis, poderiam atuar inibindo o desenvolvimento e progressão do câncer de próstata. Neste estudo, com o uso de abordagens bioquímicas e de biologia molecular, foi demonstrado que o tratamento com γ-orizanol diminui a viabilidade e biomassa celular em cultura, associado ao aumento da morte celular por apoptose e/ou necrose, em linhagens celulares responsivas (LNCaP) e não-responsivas a andrógenos (PC3 e DU145), além de aumentar a pERK1/2 em células LNCaP e DU145. O γ-orizanol também foi capaz de bloquear o ciclo celular em G2/M nas células PC3 e LNCaP e em G0/G1 nas células DU145. Estes efeitos foram ainda acompanhados por uma redução da expressão do gene e proteína caveolina-1- uma importante molécula envolvida no aumento da agressividade do câncer de próstata, e também, na progressão da doença para o fenótipo andrógeno resistente - nas células não-responsivas a andrógenos, e do gene PCGEM1 - gene específico da próstata regulado por andrógeno - nas células LNCaP e DU145. Ainda, γ-orizanol também mostrou capacidade de regular vários miRNAs - pequenas moléculas de RNA não codificantes de proteínas - envolvidos no controle de funções associadas ao desenvolvimento, progressão e invasão no câncer de próstata, como o miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 e miR222. O extrato hidroalcólico de Thuya occidentalis reduziu a viabilidade e biomassa celular nas linhagens responsiva (LNCaP) e não-responsivas (DU145 e PC3) a andrógenos, além de induzir parada do ciclo celular na fase G0/G1 nas células DU145 e aumentar a morte celular por apoptose e/ou necrose em todas as linhagens. Da mesma forma que o γ-orizanol, este extrato reduziu a expressão da caveolina-1 nas linhagens não-responsivas a andrógenos. Trabalhos anteriores mostram que o monoterpeno α-tujona é o principal composto ativo do extrato de Thuya occidentalis. Por cromatografia gasosa acoplada a detector de massas foi mostrada a existência de 0,0016 μg de α-tujona na dose de extrato usada neste estudo. No entanto, o tratamento com 0,0016μg de α-tujona foi efetivo somente sobre linhagem LNCaP, não tendo efeito sobre as outras linhagens estudadas, reforçando a hipótese da diferença de sensibilidade entre as linhagens responsivas e não responsivas a andrógeno e mostrando a contribuição de outros componentes do extrato nos efeitos observados neste estudo. Concluindo, estes resultados demonstram que tanto γ-orizanol como o extrato de Thuya occidentalis podem vir a ser agentes terapêuticos promissores no tratamento de câncer de próstata, não só por inibirem o crescimento celular, mas também e principalmente pela possibilidade de induzirem a recuperação da sensibilidade a andrógenos, aumentando as possibilidades de tratamento da doença. / Prostate cancer is the second cause of death among men in Brazil. It is a slowgrowing cancer and it may take years for tumor to reach 1 cm3, but in some cases it can spread throughout the body and the bone is the main site of metastasis. At this cancer stage known as metastasis, the principal treatment involves antiandrogen therapy, leading to prostate cells stop proliferating, because they grow in response to presence of androgens such as testosterone and dihydrotestosterone. However, in some cases, the cells can proliferate even in the absence of androgens and this fact occurs due to many factors and they are generally associated with mutations in the androgen receptor and/or alterations in androgen metabolism. In this stage, the treatments available are less effective and usually fail. However, studies suggest that γ-oryzanol, a phytosterol extracted of rice bran oil; and extracts widely used in folk medicine, as Thuya occidentalis hidroalcolic extract, could act inhibiting the development and progression of prostate cancer. In this study, using molecular biology and biochemical approaches we showed that γ- oryzanol treatment was able to decrease cell viability and biomass in culture, and this fact was linked to increased cell death by apoptosis and/or necrosis in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) prostate cancer cell lines, besides increasing pERK1/2 in LNCaP and DU145 cells. γ- oryzanol was also able to cause cell cycle arrest at G2/M phase in LNCaP and PC3 cells and at G0/G1 phase in DU145 cells. These effects were also accompanied by a reduction in caveolin-1 gene and protein expression - an important molecule related to high aggressiveness in prostate cancer and also in the progression of the disease to androgen resistant phenotype - in androgen unresponsive cells, and also PCGEM1 gene - a prostate specific gene regulated by androgens - in LNCaP and DU145 cells. γ-oryzanol also showed ability to regulate several miRNAs - small non-coding RNA molecules - involved in the control of many functions associated with the development, progression and invasion of prostate cancer, such as miR16-1, miR19b-2, miR24b-1, miR24b-2, miR99a, miR133a-5p, miR182-5p, miR198 and miR222. Thuya occidentalis hidroalcolic extract also reduce cell viability and biomass in androgen responsive (LNCaP) and unresponsive (DU145 and PC3) cells, in addition to inducing cell cycle arrest at G0/G1 phase in DU145 cells and to increase apoptosis and/or necrosis cell death in all cell lines. The same way that γ-oryzanol, this extract reduced the caveolin-1 expression in androgen unresponsive prostate cancer cells. Prior studies showed that the monoterpene α-thujone is the main active compound in the T. occidentalis extract. By gas chromatography coupled to mass detector it was showed the existence of 0.0016 μg of α-thujone in extract dose used in this study. However, the treatment with 0.0016 μg of α-thujone was effective only on LNCaP cell line, having no effect on the other studied lines, supporting the hypothesis of difference in sensitivity between responsive and unresponsive cell lines and showing the contribution of other components in the effects caused by the extract, observed it this study. In conclusion, these results demonstrate that both γ-oryzanol as T. occidentalis extract may become promising therapeutic agents in treatment of prostate cancer, not only inhibit cell growth but also and manly by the possibility of inducing the recovery of androgen sensitivity, increasing the treatment chances of treatment this disease.

No description available.

Le syndrome de Wernicke-Korsakoff (SWK) est un désordre neuropsychiatrique causé par la déficience en thiamine (DT). Dans la DT expérimentale comme dans le SWK, on observe une mort neuronale et des hémorragies dans certaines régions précises du diencéphale et du tronc cérébral. Les lésions diencéphaliques du SWK sont particulièrement sévères et entraînent souvent des séquelles amnésiques permanentes. Le lien entre la dysfonction métabolique induite par la DT et la mort neuronale n’est pas connu. Des rapports précédents ont démontré que la perméabilité de la barrière hémato-encéphalique (BHE) était altérée et ce, précédant l’apparition du dommage neuronal, suggérant un rôle critique de la dysfonction vasculaire. Les jonctions serrées (JS) interendothéliales, la base anatomique de la BHE, constituent un réseau moléculaire incluant l’occludin et les zonula occludens (ZOs). Cette thèse démontre une perte d’expression et une altération de la morphologie de ces protéines en relation avec la dysfonction de la BHE dans le thalamus de souris déficientes en thiamine, fournissant une explication pour la présence d’hémorragies. Le stress oxydatif peut entraîner des dommages directs aux protéines des JS et interférer avec leurs mécanismes de régulation. De plus, l’oxyde nitrique (NO) peut induire la métalloprotéinase matricielle-9 (MMP-9) impliquée dans la dégradation de ces protéines. L’endothélium vasculaire cérébral (EVC) semble être une source importante de NO dans la DT, l’expression de l’oxyde nitrique synthase endothéliale (eNOS) étant sélectivement induite dans les régions vulnérables. Le NO peut réagir avec les espèces réactives oxygénées et former du peroxynitrite, entraînant un stress oxydatif/nitrosatif endothélial. Les résultats présentés démontrent que la délétion du gène de eNOS prévient le stress oxydatif/nitrosatif cérébrovasculaire, l’extravasation des immunoglobulins G (IgGs) et l’altération de l’occludin et des ZOs dans le thalamus de souris déficientes en thiamine. De plus, cette délétion prévient l’induction de l’expression de MMP-9 dans l’EVC. Des résultats similaires ont été obtenus avec l’antioxydant N-acétylcystéine (NAC). Les mécanismes précis par lesquels les espèces réactives altèrent les protéines des JS sont inconnus. Caveolin-1, une composante majeure du caveolæ de l’EVC, est impliquée dans la régulation de l’expression des protéines des JS, et celle-ci est modulée par le stress oxydatif/nitrosatif; l’altération de l’expression de caveolin-1 a été récemment associée à la rupture de la BHE. Les résultats présentés démontrent que l’expression de caveolin-1 est sélectivement altérée dans l’EVC du thalamus de souris déficientes en thiamine, coïcidant avec la rupture de la BHE, et démontrent que la normalisation de l’expression de caveolin-1 par le NAC est associée avec l’atténuation du dommage à la BHE. Pris ensemble, ces résultats démontrent un rôle central du stress oxydatif/nitrosatif cérébrovasculaire, particulièrement celui provenant de eNOS, dans l’altération des JS de la BHE via des dommages directs et via l’induction de MMP-9 et de caveolin-1. Cette rupture de la BHE contribue par conséquent à la mort neuronale dans le thalamus, puisque la prévention des altérations cérébrovasculaires par la délétion du gène de eNOS et le NAC atténue significativement la mort neuronale. L’administration précoce d’antioxydants en combinaison avec la thiamine devrait donc être une considération importante pour le traitement du SWK. / Wernicke-Korsakoff syndrome (WKS) is a neuropsychiatric disorder caused by thiamine deficiency (TD). In experimental TD as in WKS, neuronal cell death and hemorrhages are observed in specific diencephalic and brainstem areas. Diencephalic lesions in WKS are especially severe and often lead to permanent amnesic symptoms. The link between TD-induced metabolic dysfunction and neuronal cell death is unknown. Previous reports have shown that blood-brain barrier (BBB) permeability was impaired and that this occurred prior to the onset of neuronal damage, suggesting a critical role for vascular dysfunction. Interendothelial tight junctions (TJs), the anatomical basis of the BBB, constitute a molecular network comprising occludin and zonula occludens (ZOs). This thesis shows a loss of expression and alterations in the morphology of these proteins in relation to BBB dysfunction in the thalamus of thiamine-deficient mice, providing an explanation for the presence of hemorrhages. Oxidative stress can lead to direct oxidative damage to TJ proteins and interfere with their regulation mechanisms. Also, nitric oxide (NO) can induce matrix metalloproteinase-9 (MMP-9) involved in the degradation of these proteins. Cerebral vascular endothelium (CVE) seems to be an important source of NO in TD, since endothelial nitric oxide synthase (eNOS) expression is selectively induced in vulnerable areas. NO can react with reactive oxygen species and form peroxynitrite, leading to endothelial oxidative/nitrosative stress. Results have show that eNOS gene deletion prevents cerebrovascular oxidative/nitrosative stress, immunoglobulins G (IgGs) extravasation and occludin and ZOs alterations in the thalamus of thiamine-deficient mice. Also, eNOS gene deletion prevents the induction of MMP-9 in CVE. Similar results have been obtained with the antioxidant N-acetylcysteine (NAC). Precise mechanisms by which reactive species alter TJ proteins are unknown. Caveolin-1, a major component of CVE caveolæ, is involved in the regulation of TJ protein expression, and is modulated by oxidative/nitrosative stress; alteration in caveolin-1 expression has been recently associated with BBB breakdown. The present results show that caveolin-1 expression is selectively altered in CVE of the thalamus of thiamine-deficient mice, and show that normalization of caveolin-1 expression by NAC is associated with the attenuation of BBB damage. Taken together, these results demonstrate a central role for cerebrovascular oxidative/nitrosative stress, especially coming from eNOS, in BBB TJ protein alterations via direct damage and via induction of MMP-9 and caveolin-1. As a result, BBB breakdown contributes to neuronal cell death in the thalamus, since prevention of cerebrovascular alterations by eNOS gene deletion and NAC significantly attenuates neuronal cell death. Early administration of antioxidants combined with thiamine should therefore be an important consideration for the treatment of WKS.

Le syndrome de Wernicke-Korsakoff (SWK) est un désordre neuropsychiatrique causé par la déficience en thiamine (DT). Dans la DT expérimentale comme dans le SWK, on observe une mort neuronale et des hémorragies dans certaines régions précises du diencéphale et du tronc cérébral. Les lésions diencéphaliques du SWK sont particulièrement sévères et entraînent souvent des séquelles amnésiques permanentes. Le lien entre la dysfonction métabolique induite par la DT et la mort neuronale n’est pas connu. Des rapports précédents ont démontré que la perméabilité de la barrière hémato-encéphalique (BHE) était altérée et ce, précédant l’apparition du dommage neuronal, suggérant un rôle critique de la dysfonction vasculaire. Les jonctions serrées (JS) interendothéliales, la base anatomique de la BHE, constituent un réseau moléculaire incluant l’occludin et les zonula occludens (ZOs). Cette thèse démontre une perte d’expression et une altération de la morphologie de ces protéines en relation avec la dysfonction de la BHE dans le thalamus de souris déficientes en thiamine, fournissant une explication pour la présence d’hémorragies. Le stress oxydatif peut entraîner des dommages directs aux protéines des JS et interférer avec leurs mécanismes de régulation. De plus, l’oxyde nitrique (NO) peut induire la métalloprotéinase matricielle-9 (MMP-9) impliquée dans la dégradation de ces protéines. L’endothélium vasculaire cérébral (EVC) semble être une source importante de NO dans la DT, l’expression de l’oxyde nitrique synthase endothéliale (eNOS) étant sélectivement induite dans les régions vulnérables. Le NO peut réagir avec les espèces réactives oxygénées et former du peroxynitrite, entraînant un stress oxydatif/nitrosatif endothélial. Les résultats présentés démontrent que la délétion du gène de eNOS prévient le stress oxydatif/nitrosatif cérébrovasculaire, l’extravasation des immunoglobulins G (IgGs) et l’altération de l’occludin et des ZOs dans le thalamus de souris déficientes en thiamine. De plus, cette délétion prévient l’induction de l’expression de MMP-9 dans l’EVC. Des résultats similaires ont été obtenus avec l’antioxydant N-acétylcystéine (NAC). Les mécanismes précis par lesquels les espèces réactives altèrent les protéines des JS sont inconnus. Caveolin-1, une composante majeure du caveolæ de l’EVC, est impliquée dans la régulation de l’expression des protéines des JS, et celle-ci est modulée par le stress oxydatif/nitrosatif; l’altération de l’expression de caveolin-1 a été récemment associée à la rupture de la BHE. Les résultats présentés démontrent que l’expression de caveolin-1 est sélectivement altérée dans l’EVC du thalamus de souris déficientes en thiamine, coïcidant avec la rupture de la BHE, et démontrent que la normalisation de l’expression de caveolin-1 par le NAC est associée avec l’atténuation du dommage à la BHE. Pris ensemble, ces résultats démontrent un rôle central du stress oxydatif/nitrosatif cérébrovasculaire, particulièrement celui provenant de eNOS, dans l’altération des JS de la BHE via des dommages directs et via l’induction de MMP-9 et de caveolin-1. Cette rupture de la BHE contribue par conséquent à la mort neuronale dans le thalamus, puisque la prévention des altérations cérébrovasculaires par la délétion du gène de eNOS et le NAC atténue significativement la mort neuronale. L’administration précoce d’antioxydants en combinaison avec la thiamine devrait donc être une considération importante pour le traitement du SWK. / Wernicke-Korsakoff syndrome (WKS) is a neuropsychiatric disorder caused by thiamine deficiency (TD). In experimental TD as in WKS, neuronal cell death and hemorrhages are observed in specific diencephalic and brainstem areas. Diencephalic lesions in WKS are especially severe and often lead to permanent amnesic symptoms. The link between TD-induced metabolic dysfunction and neuronal cell death is unknown. Previous reports have shown that blood-brain barrier (BBB) permeability was impaired and that this occurred prior to the onset of neuronal damage, suggesting a critical role for vascular dysfunction. Interendothelial tight junctions (TJs), the anatomical basis of the BBB, constitute a molecular network comprising occludin and zonula occludens (ZOs). This thesis shows a loss of expression and alterations in the morphology of these proteins in relation to BBB dysfunction in the thalamus of thiamine-deficient mice, providing an explanation for the presence of hemorrhages. Oxidative stress can lead to direct oxidative damage to TJ proteins and interfere with their regulation mechanisms. Also, nitric oxide (NO) can induce matrix metalloproteinase-9 (MMP-9) involved in the degradation of these proteins. Cerebral vascular endothelium (CVE) seems to be an important source of NO in TD, since endothelial nitric oxide synthase (eNOS) expression is selectively induced in vulnerable areas. NO can react with reactive oxygen species and form peroxynitrite, leading to endothelial oxidative/nitrosative stress. Results have show that eNOS gene deletion prevents cerebrovascular oxidative/nitrosative stress, immunoglobulins G (IgGs) extravasation and occludin and ZOs alterations in the thalamus of thiamine-deficient mice. Also, eNOS gene deletion prevents the induction of MMP-9 in CVE. Similar results have been obtained with the antioxidant N-acetylcysteine (NAC). Precise mechanisms by which reactive species alter TJ proteins are unknown. Caveolin-1, a major component of CVE caveolæ, is involved in the regulation of TJ protein expression, and is modulated by oxidative/nitrosative stress; alteration in caveolin-1 expression has been recently associated with BBB breakdown. The present results show that caveolin-1 expression is selectively altered in CVE of the thalamus of thiamine-deficient mice, and show that normalization of caveolin-1 expression by NAC is associated with the attenuation of BBB damage. Taken together, these results demonstrate a central role for cerebrovascular oxidative/nitrosative stress, especially coming from eNOS, in BBB TJ protein alterations via direct damage and via induction of MMP-9 and caveolin-1. As a result, BBB breakdown contributes to neuronal cell death in the thalamus, since prevention of cerebrovascular alterations by eNOS gene deletion and NAC significantly attenuates neuronal cell death. Early administration of antioxidants combined with thiamine should therefore be an important consideration for the treatment of WKS.