L'insuffisance cardiaque (IC) touche 1-2% des adultes dans les pays développés. On distingue classiquement 2 formes d’IC : l’IC chronique, dont la mise en place est lente, et l’IC aigue dont l’apparition est rapide ou due à l'aggravation des symptômes et/ou des signes de l’IC. Quelles soient chroniques ou aigues, certaines formes d'IC n'ont pas de traitement efficace. L'insuffisance cardiaque à fraction d'éjection préservée (ICFEp) devient la forme prédominante d'IC chronique mais la compréhension de sa physiopathologie reste encore incomplète. Un modèle de rat transgénique sur-exprimant le récepteur β3- adrénergique humain au niveau endothélial a été développé dans l'équipe. Comme les patients, ce modèle développe une ICFEp avec l’âge qui se manifeste notamment par une dysfonction diastolique et une intolérance au stress. Dans le cadre de ma thèse et sur la base d'une étude transcriptomique, j'ai évalué les altérations du cycle du calcium, du métabolisme ainsi que l'apparition de fibrose et d'inflammation. Le choc septique est considéré comme une IC aigue particulièrement violente. Le deuxième axe de ma thèse a été de développer des modèles pertinents de choc septique et d’évaluer le potentiel thérapeutique d’une modification post-traductionnelle : la O-GlcNAcylation, déjà connue pour ses effets bénéfiques dans des formes d’IC aigues. Ainsi, une augmentation des niveaux de l'O-GlcNAc, grâce à un agent pharmacologique, le NButGT, améliore la fonction cardiovasculaire et la survie des animaux en choc septique. / Heart failure (HF) affects 1-2% of adults in developed countries. There are two distinct forms of HF: chronic HF, with a slow development, and acute HF that appears rapidly or is due to an aggravation of the symptoms and/or signs of HF. For some forms of HF, acute or chronic, no effective treatment exists. Heart failure with preserved ejection fraction (HFpEF) is becoming the predominant form of chronic HF but the understanding of its pathophysiology remains incomplete. A transgenic rat model over-expressing the human endothelial β3-adrenergic receptor has been developed in the team. As in patients, this model develops HFpEF with age, and is characterized by diastolic dysfunction and stress intolerance. During my thesis and based on transcriptome data, I evaluated alterations of the calcium cycle, metabolism as well as the appearance of fibrosis and inflammation. Septic shock is considered to be a form of severe acute HF. The second axis of my thesis was to develop relevant models of septic shock and to evaluate the therapeutic potential of a post-translational modification: O-GlcNAcylation, already known for its beneficial effects in acute HF forms. Indeed, an increase in levels of O-GlcNAc, through a pharmacological agent, NButGT, improved cardiovascular function and survival of animals in septic shock.

Thesis advisor: Jia Niu / O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification plays important roles in cellular network/diseases such as autophagy, transcriptional activity, protein stability, phosphorylation modification competition, cancers, neurodegenerative disorders, etc. However, there are still lots of unknown roles of it in gene regulation especially while there is existence of more than 1000 transcription factors and many other coactivators/repressors which might have influence in expression level with or without O-GlcNAc modification. In this project, we use CRISPR technology with catalytically inactive Cas9 from Streptococcus pyogenes fused with the only writer and eraser of O-GlcNAc to selectively target to the DNA sequence of interest by guide RNA to see what the result of additional modification or cleavage of O-GlcNAc on proximal proteins in gene regulation is. A cost and time efficient way for guide RNA construction is developed in the project with one-piece PCR and Gibson assembly for 2 different backbones of guide RNA: 11 guide RNA for each. Other different cloning methods have also been used for the future work. For the future work, second reporter will be introduced to normalize the luciferase signal. In addition, new metabolic O-GlcNAc reporter and Y289L GalT/ UDP-GalNAz could be used to find out proteins which might be modify by dCas9-OGT/OGA with click reaction. Furthermore, split OGA/Turbo ID system could also be used to reduce the background and find out proteins/modification sites which might be important to the gene regulation for DNA-protein or protein-protein interaction. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Les Oligosaccharides soluble (OS) sont essentiellement générés durant le processus de dégradation des N-glycoprotéines nouvellement synthétisées et mal conformées (ERAD) et durant la voie « turn-over » des glycoprotéines matures. Dans le but de déterminer si la modification post-traductionnelle O-GlcNAc est effectivement impliquée dans le processus de dégradation des N-glycoprotéines, nous avons analysé par spectrométrie de masse les OS provenant des cellules CHO après traitement par l’inhibiteur PUGNAc. Le PUGNAc ou « O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbanate » est un inhibiteur puissant de l’O-GlcNAcase (OGA) qui catalyse l’hydrolyse du résidu O-GlcNAc des résidus sérine et thréonine des protéines O-GlcNAcylées.L’analyse par spectrométrie de masse révèle l’apparition d’une population d’OS de structures anormaux dans les cellules CHO suite au traitement par le PUGNAc. Cette population a été identifiée comme ayant des structures possédant des résidus GlcNAc au niveau de leur extrémité non-réductrice issues d’une dégradation lysosomale incomplète des glycoprotéines. Contrairement au PUGNAc, le NButGT, un autre inhibiteur de l’OGA, n’aboutit pas à l’apparition de cette population. Ainsi, Nous avons démontré que ces structures s’accumulent exclusivement dans la fraction membranaire conséquence de l’inhibition des β-hexosaminidases lysosomaux par le PUGNAc. Notre étude avait permis, d’un part, de mettre en évidence la capacité du PUGNAc de mimer une maladie de surcharge lysosomale et, d’autre part, de montrer un autre aspect des effets indésirables induits par le PUGNAc et qui nécessite d’être pris en considération lors de l’utilisation de cet inhibiteur. / Free oligosaccharides (fOS) are generated as a result of glycoproteins catabolism that occurs in two principal distinct pathways: the endoplasmic reticulum-associated degradation (ERAD) of misfolded newly synthesized N-glycoproteins and the mature N-glycoproteins turnover pathway. We analyzed fOS by Mass spectrometry in PUGNAc CHO treated cells in order to investigate whether O-GlcNAc modified proteins were involved in N-glycoprotein degradation process. The O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbanate (PUGNAc) is a potent inhibitor of the O-GlcNAcase (OGA) catalyzing the cleavage of β-O-linked 2-acetamido-2-deoxy-beta-D-glucopyranoside (O-GlcNAc) from serine and threonine residues of post-translationally modified proteins. Mass spectrometry (MS) analysis revealed the appearance of an unusual population of fOS after PUGNAc treatment. The structures representing this population have been identified as containing non-reducing end GlcNAc residues resulting from incomplete lysosomal fOS degradation. Only observed after PUGNAc treatment, the NButGt, another OGA inhibitor, did not lead to the appearance of the population. These structures have clearly been shown to accumulate in membrane fractions as the consequence of lysosomal β-hexosaminidases inhibition by PUGNAc. As Lysosomal Storage Disorders (LSD) are characterized by the accumulation of fOS in various tissues, our study evokes that PUGNAc mimics a LSD and shows another off target effects that needs to be taken into account in the use of this drug.

Continual intake of high-fat foods, coupled with limited physical activity, can lead to metabolic inflexibility. Eventually, this may lead to significant health issues such as obesity, insulin resistance, cardiovascular disease, and other chronic diseases. Metabolic flexibility of human skeletal muscles is influenced by changes to mitochondrial, nuclear, and cytosolic proteins, in part as a result of posttranslational modifications (PTMs). O-linked B-D-N-acetylglucosamine, known as O-GlcNAc, has recently been identified as an important posttranslational modification that responds to nutrient sensing and cellular stress. Unlike other PTMs, O-GlcNAc has only two cycling enzymes. Because of its novelty, little research has been performed on the role of O-GlcNAc in human skeletal muscle and metabolic flexibility. The purpose of the current study was to establish the effects of a 5-day high-fat diet on skeletal muscle O-GlcNAcylation. In the proposed study, 13 non-obese, sedentary, college-aged males consumed a controlled diet for two weeks followed by a high-fat diet composed of 55% fat, 30% carbohydrate, and 15% protein. Muscle biopsies were taken from the vastus lateralis both fasted and four hours after a high-fat meal, following both the control diet and the high-fat diet. Western blot analysis was used to assess global O-GlcNAc and protein concentrations of O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) in whole-homogenates and isolated mitochondria from skeletal muscle. Results were analyzed using independent, two-tailed t-tests and 2-way ANOVA analysis with repeated measures and Bonferroni corrections; a p-value was set to α less than or equal to 0.05. It was found that O-GlcNAc and OGT levels remained stable, although fasting levels of OGA significantly decreased after the 5-day high-fat diet. It is possible that healthy individuals are capable of maintaining normal levels of O-GlcNAc and its cycling enzymes, but there is still more to learn about O-GlcNAc and its role in metabolic flexibility. / Master of Science

Vários mecanismos controlam os processos de contração e relaxamento das células musculares lisas vasculares (CMLVs). Receptores e moléculas sinalizadoras intracelulares, os quais controlam os processos de contração e relaxamento das CMLVs, são alvo de modificações pós-traducionais, como a O-GlcNAcilação que modula respostas vasculares. O aumento de proteínas modificadas por O-GlcNAc apresenta efeito ambíguo sobre as CMLVs, sendo protetor em situações de aumento agudo, mas lesivo quando mantido cronicamente. O aumento crônico de O-GlcNAc em animais está associado a repostas contráteis mais intensas e redução do relaxamento vascular, assim como o aumento da produção de espécies reativas de oxigênio (EROs), denominado estresse oxidativo, alterações constantemente descritas em doenças crônicas como diabetes e hipertensão arterial. Considerando que algumas proteínas que controlam a contratilidade vascular são modificadas por O-GlcNAc e que pouco se sabe a respeito da via de Biossíntese das Hexosaminas (VBH) e sua relação com o sistema vascular em humanos, nós investigamos a hipótese de que modificações de proteínas por O-GlcNAc estão relacionadas a alterações vasculares na hipertensão arterial em humanos. Durante a realização de nossos experimentos, demonstramos que os principais componentes da VBH estão presentes em CMLVs humanas. O tratamento com Thiamet G (TMG) por 24 h aumentou os níveis de proteínas modificadas por O-GlcNAc nas CMLVs pela redução da atividade de OGA, assim como induziu efeito compensatório de aumento da expressão dessa enzima. TMG reduziu a atividade de OGA em CMLVs no grupo Controle, mas não promoveu alteração na geração de EROs. Após tratamento com TMG, artérias de grupo Controle apresentaram maior sensibilidade à noradrenalina (NA) e maior relaxamento ao nitroprussiato de sódio (NPS); enquanto o grupo Hipertenso não apresentou alterações na contratilidade ou no relaxamento arterial. Artérias do grupo Hipertenso apresentaram maior sensibilidade à NA que o grupo Controle antes de qualquer tratamento, além de deficiência no relaxamento, com menor sensibilidade e menor resposta máxima ao NPS em comparação ao grupo Controle. O grupo Hipertenso apresentou aumento da pressão arterial média de internação (PAMi), assim como da idade, quando comparado com o grupo Controle. Em conclusão, a VBH está presente nas CMLVs humanas. A inibição da atividade da OGA por TMG aumenta os níveis de proteínas modificadas por O-GlcNAc, a expressão de OGA e modula a reatividade vascular no grupo Controle, mas não no grupo Hipertenso. Os resultados demonstram que pacientes hipertensos apresentam respostas vasculares a drogas vasoativas diferentes daquelas observadas em pacientes controle, antes e após o aumento dos níveis de proteínas modificadas por O-GlcNAc nas CMLVs. Novos estudos serão necessários para determinar se as alterações observadas são decorrentes da hipertensão arterial e/ou do tratamento farmacológico aos quais os pacientes estão submetidos. / Several mechanisms control the contraction and relaxation processes in the vascular smooth muscles cells (VSMC). Intracellular receptors and signaling molecules involved in contraction and relaxation mechanisms are targets of post-translational modifications (PTM), such as O-GlcNAcylation, which modulates vascular responses. Augmented levels of O-GlcNAc-modified proteins show a dual effect in VSMC, being protective during acute stressful events and deleterious when O-GlcNAc is chronically augmented. In animals, chronic increases in O-GlcNAc-modified proteins are linked to increased vascular responses to constrictor agents, to reduced vascular relaxation in response to dilator drugs, and to increased production of reactive oxygen species (ROS), named oxidative stress. All these changes are frequently described in chronic diseases such as diabetes and arterial hypertension. Since proteins involved in vascular contractility are modified by O-GlcNAc, and our knowledge on the influence of the hexosamine biosynthesis pathway (HBP) in the human vascular system is limited, we tested the hypothesis that proteins modified by O-GlcNAc contribute to vascular changes in hypertensive patients. Our data show that human VSMC express the main components of the HBP; the treatment of human VSMC with Thiamet G (TMG) for 24 h augmented O-GlcNAc levels, decreased OGA activity and induced a compensatory increase in OGA\'s expression. TMG reduced OGA activity, increased levels of O-GlcNAc-modified proteins but did not change ROS generation in human arteries from the Control group. After treatment with TMG, arteries from the Control group exhibited increased sensitivity to norepinephrine (NE) and to sodium nitroprusside (SNP), as well as increased maximum relaxation to SNP. Augmented O-GlcNAc levels produced no changes in contractile or relaxation responses in the Hypertensive group. Arteries from the Hypertensive group exhibited an increased sensitivity to NE as well as decreased sensitivity and maximum relaxation to SNP when compared to arteries from the Control group. Mean arterial blood pressure (hMABP) and the average age were increased in patients from the Hypertensive group. In conclusion, the HBP is present in human VSMC and the inhibition of OGA activity with TMG increases O-GlcNAc levels, increases OGA expression and modifies vascular responses to constrictor and dilator stimuli in human arteries from the Control group, but not from the Hypertensive group. These results indicate that hypertensive patients have altered vascular responses to vasoactive drugs both in the absence and in the presence of augmented O-GlcNAc levels. Further research is needed to explain whether these differences are due to the hypertensive disease and/or to the patient\'s medical treatment.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação póstraducional altamente dinâmica, controlada por duas enzimas: a OGT, responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina; e a OGA, que catalisa a remoção de O-GlcNAc das proteínas modificadas. Condições crônicas de aumento de proteínas O-GlcNAc estão associadas a disfunções vasculares e a efeitos negativos no coração. No entanto, os efeitos agudos produzidos pelo aumento de O-GlcNAc estão associados a uma melhora na função cardíaca, redução dos níveis vasculares de mediadores pró-inflamatórios. Neste estudo testamos a hipótese que o aumento agudo da modificação por O-GlcNAc reduz os níveis sistêmicos de citocinas pró-inflamatórias, como IL-1?, IL-6 e TNF-?, bem como a ativação do fator de transcrição NF-?B, reduzindo o processo inflamatório e disfunção vascular em animais com endotoxemia. Foram utilizados camundongos C57BL6/J que receberam lipopolissacarídeo (LPS) para o desenvolvimento de endotoxemia severa (LPS sev, 20 mg/Kg, i.p.) ou veículo (controle). Os animais também receberam glucosamina (GlcN; 300 mg/Kg; i.v.; 30 min antes do LPS), ThG (150 ?g/Kg, i.v.; 12 h antes do LPS) ou veículo e foram sacrificados 6 horas após a indução da endotoxemia com LPS. Os tratamentos prévios com GlcN e ThG aumentaram a sobrevida dos animais (50 % e 60 % na endotoxemia sev). A determinação dos níveis sistêmicos de IL-1?, IL-6 e TNF-? e da expressão gênica vascular destas citocinas [mRNA (2-??CT) em aorta e leito mesentérico] demonstrou que os tratamentos prévios com GlcN e ThG reduziram a expressão de IL- 1?, IL-6 e TNF-? em animais com endotoxemia. O tratamento prévio com GlcN atenuou, mas não normalizou a hipotensão arterial causada pela endotoxemia por LPS. Não foram observadas alterações de reatividade na artéria aorta de animais que receberam LPS, ou LPS + GlcN. Entretanto, os tratamentos prévios com GlcN e ThG atenuaram a refratariedade a vasoconstrictores observada em artéria mesentérica de resistência de animais que receberam somente LPS. O aumento dos níveis de proteínas glicosiladas, com GlcN e ThG, em cultura de macrófagos provenientes da medula óssea de camundongos C57BL6/J corroboram os resultados descritos acima. Os níveis de citocinas no sobrenadante da cultura de macrófagos, bem como a expressão gênica destas citocinas, e a produção de nitrito por estas células foram menores em células submetidas ao tratamento com GlcN e ThG. Além da endotoxemia por LPS, avaliamos os efeitos do tratamento com GlcN em um modelo de ligadura e perfuração do ceco (CLP) e os resultados corroboram os dados obtidos nos animais com endotoxemia e na cultura de macrófagos. Portanto, o aumento agudo da modificação por O-GlcNAc em animais com endotoxemia por LPS promove aumento da sobrevida dos animais, redução nos níveis de citocinas pró-inflamatórias, como IL-1?, IL-6 e TNF-? e redução da disfunção vascular e hipotensão arterial. Conclusão: Aumento agudo de O-GlcNAc reduz o processo inflamatório e atenua eventos cardiovasculares associados a endotoxemia por LPS, sugerindo que essa via representa um possível alvo em condições de resposta inflamatória sistêmica. / The post-translational modification of proteins by O-GlcNAcylation (O-GlcNAc) is highly dynamic and modulates cell-signaling processes. Chronic conditions that increase the levels of O-GlcNAc-modified proteins are associated with vascular disorders. However, acute increases in O-GlcNAc levels reduce the release of proinflammatory mediators and negatively regulate inflammatory processes by decreasing e.g. NF-?B activation. This study tested the hypothesis that acute increases of O-GlcNAc levels reduce mortality and inflammatory processes in experimental models of sepsis. Male C57BL6/J mice received lipopolysaccharide (LPS) for the development of severe (LPS sev, 20 mg / kg, i.p.) endotoxemia or vehicle (control). Mice also received glucosamine (GlcN; 300 mg / kg, iv; 30 min) or ThG (150 ?g / kg, iv; 12 h) or vehicle before LPS administration. Mice were killed 6 hours after LPS. The acute increase of OGlcNAc modification in animals with LPS-induced endotoxemia increased survival, reduced levels of proinflammatory cytokines such as IL-1?, IL-6 and TNF-? and attenuated vascular dysfunction and arterial hypotension. Treatments with GlcN and ThG reduced IL-1?, IL-6 and TNF-? [plasma levels and vascular gene expression [mRNA] (2- ??CT) in the aorta and mesenteric bed] in animals with endotoxemia. GlcN and ThG also attenuated the decreased vasoconstrictor responses and hypotension induced by LPS. Increased levels of glycosylated proteins decreased levels (in the supernatant) and expression of cytokines in cultured bone marrow-derived macrophages from C57BL6/J mice. GlcN treatment produced similar effects in an experimental model that closely resembles human sepsis, the cecum ligation and perforation (CLP) model. Conclusion: Acute increase of O-GlcNAc reduces the inflammatory process and attenuates cardiovascular events associated with LPS endotoxemia, suggesting that this pathway represents a possible target under conditions of systemic inflammatory response.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação póstraducional altamente dinâmica, controlada por duas enzimas: a OGT, responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina; e a OGA, que catalisa a remoção de O-GlcNAc das proteínas modificadas. Condições crônicas de aumento de proteínas O-GlcNAc estão associadas a disfunções vasculares e a efeitos negativos no coração. No entanto, os efeitos agudos produzidos pelo aumento de O-GlcNAc estão associados a uma melhora na função cardíaca, redução dos níveis vasculares de mediadores pró-inflamatórios. Neste estudo testamos a hipótese que o aumento agudo da modificação por O-GlcNAc reduz os níveis sistêmicos de citocinas pró-inflamatórias, como IL-1?, IL-6 e TNF-?, bem como a ativação do fator de transcrição NF-?B, reduzindo o processo inflamatório e disfunção vascular em animais com endotoxemia. Foram utilizados camundongos C57BL6/J que receberam lipopolissacarídeo (LPS) para o desenvolvimento de endotoxemia severa (LPS sev, 20 mg/Kg, i.p.) ou veículo (controle). Os animais também receberam glucosamina (GlcN; 300 mg/Kg; i.v.; 30 min antes do LPS), ThG (150 ?g/Kg, i.v.; 12 h antes do LPS) ou veículo e foram sacrificados 6 horas após a indução da endotoxemia com LPS. Os tratamentos prévios com GlcN e ThG aumentaram a sobrevida dos animais (50 % e 60 % na endotoxemia sev). A determinação dos níveis sistêmicos de IL-1?, IL-6 e TNF-? e da expressão gênica vascular destas citocinas [mRNA (2-??CT) em aorta e leito mesentérico] demonstrou que os tratamentos prévios com GlcN e ThG reduziram a expressão de IL- 1?, IL-6 e TNF-? em animais com endotoxemia. O tratamento prévio com GlcN atenuou, mas não normalizou a hipotensão arterial causada pela endotoxemia por LPS. Não foram observadas alterações de reatividade na artéria aorta de animais que receberam LPS, ou LPS + GlcN. Entretanto, os tratamentos prévios com GlcN e ThG atenuaram a refratariedade a vasoconstrictores observada em artéria mesentérica de resistência de animais que receberam somente LPS. O aumento dos níveis de proteínas glicosiladas, com GlcN e ThG, em cultura de macrófagos provenientes da medula óssea de camundongos C57BL6/J corroboram os resultados descritos acima. Os níveis de citocinas no sobrenadante da cultura de macrófagos, bem como a expressão gênica destas citocinas, e a produção de nitrito por estas células foram menores em células submetidas ao tratamento com GlcN e ThG. Além da endotoxemia por LPS, avaliamos os efeitos do tratamento com GlcN em um modelo de ligadura e perfuração do ceco (CLP) e os resultados corroboram os dados obtidos nos animais com endotoxemia e na cultura de macrófagos. Portanto, o aumento agudo da modificação por O-GlcNAc em animais com endotoxemia por LPS promove aumento da sobrevida dos animais, redução nos níveis de citocinas pró-inflamatórias, como IL-1?, IL-6 e TNF-? e redução da disfunção vascular e hipotensão arterial. Conclusão: Aumento agudo de O-GlcNAc reduz o processo inflamatório e atenua eventos cardiovasculares associados a endotoxemia por LPS, sugerindo que essa via representa um possível alvo em condições de resposta inflamatória sistêmica. / The post-translational modification of proteins by O-GlcNAcylation (O-GlcNAc) is highly dynamic and modulates cell-signaling processes. Chronic conditions that increase the levels of O-GlcNAc-modified proteins are associated with vascular disorders. However, acute increases in O-GlcNAc levels reduce the release of proinflammatory mediators and negatively regulate inflammatory processes by decreasing e.g. NF-?B activation. This study tested the hypothesis that acute increases of O-GlcNAc levels reduce mortality and inflammatory processes in experimental models of sepsis. Male C57BL6/J mice received lipopolysaccharide (LPS) for the development of severe (LPS sev, 20 mg / kg, i.p.) endotoxemia or vehicle (control). Mice also received glucosamine (GlcN; 300 mg / kg, iv; 30 min) or ThG (150 ?g / kg, iv; 12 h) or vehicle before LPS administration. Mice were killed 6 hours after LPS. The acute increase of OGlcNAc modification in animals with LPS-induced endotoxemia increased survival, reduced levels of proinflammatory cytokines such as IL-1?, IL-6 and TNF-? and attenuated vascular dysfunction and arterial hypotension. Treatments with GlcN and ThG reduced IL-1?, IL-6 and TNF-? [plasma levels and vascular gene expression [mRNA] (2- ??CT) in the aorta and mesenteric bed] in animals with endotoxemia. GlcN and ThG also attenuated the decreased vasoconstrictor responses and hypotension induced by LPS. Increased levels of glycosylated proteins decreased levels (in the supernatant) and expression of cytokines in cultured bone marrow-derived macrophages from C57BL6/J mice. GlcN treatment produced similar effects in an experimental model that closely resembles human sepsis, the cecum ligation and perforation (CLP) model. Conclusion: Acute increase of O-GlcNAc reduces the inflammatory process and attenuates cardiovascular events associated with LPS endotoxemia, suggesting that this pathway represents a possible target under conditions of systemic inflammatory response.

Vários mecanismos controlam os processos de contração e relaxamento das células musculares lisas vasculares (CMLVs). Receptores e moléculas sinalizadoras intracelulares, os quais controlam os processos de contração e relaxamento das CMLVs, são alvo de modificações pós-traducionais, como a O-GlcNAcilação que modula respostas vasculares. O aumento de proteínas modificadas por O-GlcNAc apresenta efeito ambíguo sobre as CMLVs, sendo protetor em situações de aumento agudo, mas lesivo quando mantido cronicamente. O aumento crônico de O-GlcNAc em animais está associado a repostas contráteis mais intensas e redução do relaxamento vascular, assim como o aumento da produção de espécies reativas de oxigênio (EROs), denominado estresse oxidativo, alterações constantemente descritas em doenças crônicas como diabetes e hipertensão arterial. Considerando que algumas proteínas que controlam a contratilidade vascular são modificadas por O-GlcNAc e que pouco se sabe a respeito da via de Biossíntese das Hexosaminas (VBH) e sua relação com o sistema vascular em humanos, nós investigamos a hipótese de que modificações de proteínas por O-GlcNAc estão relacionadas a alterações vasculares na hipertensão arterial em humanos. Durante a realização de nossos experimentos, demonstramos que os principais componentes da VBH estão presentes em CMLVs humanas. O tratamento com Thiamet G (TMG) por 24 h aumentou os níveis de proteínas modificadas por O-GlcNAc nas CMLVs pela redução da atividade de OGA, assim como induziu efeito compensatório de aumento da expressão dessa enzima. TMG reduziu a atividade de OGA em CMLVs no grupo Controle, mas não promoveu alteração na geração de EROs. Após tratamento com TMG, artérias de grupo Controle apresentaram maior sensibilidade à noradrenalina (NA) e maior relaxamento ao nitroprussiato de sódio (NPS); enquanto o grupo Hipertenso não apresentou alterações na contratilidade ou no relaxamento arterial. Artérias do grupo Hipertenso apresentaram maior sensibilidade à NA que o grupo Controle antes de qualquer tratamento, além de deficiência no relaxamento, com menor sensibilidade e menor resposta máxima ao NPS em comparação ao grupo Controle. O grupo Hipertenso apresentou aumento da pressão arterial média de internação (PAMi), assim como da idade, quando comparado com o grupo Controle. Em conclusão, a VBH está presente nas CMLVs humanas. A inibição da atividade da OGA por TMG aumenta os níveis de proteínas modificadas por O-GlcNAc, a expressão de OGA e modula a reatividade vascular no grupo Controle, mas não no grupo Hipertenso. Os resultados demonstram que pacientes hipertensos apresentam respostas vasculares a drogas vasoativas diferentes daquelas observadas em pacientes controle, antes e após o aumento dos níveis de proteínas modificadas por O-GlcNAc nas CMLVs. Novos estudos serão necessários para determinar se as alterações observadas são decorrentes da hipertensão arterial e/ou do tratamento farmacológico aos quais os pacientes estão submetidos. / Several mechanisms control the contraction and relaxation processes in the vascular smooth muscles cells (VSMC). Intracellular receptors and signaling molecules involved in contraction and relaxation mechanisms are targets of post-translational modifications (PTM), such as O-GlcNAcylation, which modulates vascular responses. Augmented levels of O-GlcNAc-modified proteins show a dual effect in VSMC, being protective during acute stressful events and deleterious when O-GlcNAc is chronically augmented. In animals, chronic increases in O-GlcNAc-modified proteins are linked to increased vascular responses to constrictor agents, to reduced vascular relaxation in response to dilator drugs, and to increased production of reactive oxygen species (ROS), named oxidative stress. All these changes are frequently described in chronic diseases such as diabetes and arterial hypertension. Since proteins involved in vascular contractility are modified by O-GlcNAc, and our knowledge on the influence of the hexosamine biosynthesis pathway (HBP) in the human vascular system is limited, we tested the hypothesis that proteins modified by O-GlcNAc contribute to vascular changes in hypertensive patients. Our data show that human VSMC express the main components of the HBP; the treatment of human VSMC with Thiamet G (TMG) for 24 h augmented O-GlcNAc levels, decreased OGA activity and induced a compensatory increase in OGA\'s expression. TMG reduced OGA activity, increased levels of O-GlcNAc-modified proteins but did not change ROS generation in human arteries from the Control group. After treatment with TMG, arteries from the Control group exhibited increased sensitivity to norepinephrine (NE) and to sodium nitroprusside (SNP), as well as increased maximum relaxation to SNP. Augmented O-GlcNAc levels produced no changes in contractile or relaxation responses in the Hypertensive group. Arteries from the Hypertensive group exhibited an increased sensitivity to NE as well as decreased sensitivity and maximum relaxation to SNP when compared to arteries from the Control group. Mean arterial blood pressure (hMABP) and the average age were increased in patients from the Hypertensive group. In conclusion, the HBP is present in human VSMC and the inhibition of OGA activity with TMG increases O-GlcNAc levels, increases OGA expression and modifies vascular responses to constrictor and dilator stimuli in human arteries from the Control group, but not from the Hypertensive group. These results indicate that hypertensive patients have altered vascular responses to vasoactive drugs both in the absence and in the presence of augmented O-GlcNAc levels. Further research is needed to explain whether these differences are due to the hypertensive disease and/or to the patient\'s medical treatment.

La O-GlcNAc est une glycosylation dynamique, résidente du cytosol et du noyau, participant à la régulation de processus biologiques tels que le cycle cellulaire et l'embryogenèse. Nos travaux ont porté dans un premier temps sur le contrôle par la O-GlcNAc de la reprise méiotique de l'ovocyte deXenopus laevis, processus analogue à la transition G2/M du cycle cellulaire. Cette transition G2/M est caractérisée par l'activation simultanée du M-phase Promoting Factor, facteur universel d'entrée en phase M et de la voie MAPK-Erk2, et par une augmentation du niveau de O-GlcNAc. Nous avons démontré que cette augmentation de O-GlcNAc était primordiale pour la reprise méiotique ovocytaire puisque l'inhibition de l'OGT, l'enzyme transférant le résidu de GlcNAc, empêche la transition G2/M de l'ovocyte alors que sa surexpression accélère ce phénomène. Nous avons identifié 24 protéines dont le niveau de O-GlcNAc augmente au cours de la reprise méiotique dont des protéines du cytosquelette, la kinase erk2, la phosphatase PP2A, des enzymes de la glycolyse et des protéines ribosomales. Nous avons également entrepris l'étude des variations de O-GlcNAc, d'OGT et d'UDP-GlcNAc au cours de l'ovogenèse et de l'embryogenèse précoce chez Xenopus laevis et avons montré que la dynamique de la O-GlcNAc était complexe tout au long de ces deux processus. Notamment, nous avons observé une diminution drastique et transitoire de la O-GlcNAc au début de la gastrulation suggérant une implication de la glycosylation dans les phénomènes de migration cellulaire caractéristiques de cette étape du développement mettant en place les trois feuillets embryonnaires à l'origine de tous les tissus de l'adulte. / O-GlcNAc is a dynamic and reversible post-translational modification found within the cytosol and the nucleus that take part in the regulation of many cellular processes among which cell cycle and embryogenesis. First, our works have focused on the study of O-GlcNAc implication in the control of Xenopus laevis oocyte meiotic resumption, a process analogous to the G2/M transition of the cell cycle. This G2/M transition is characterized by the simultaneous activation of the M-phase Promoting Factor, the universal regulator of the M-Phase entry and of the MAPK-Erk2 pathway but also by a sudden increase in the oocyte O-GlcNAc content. We have demonstrated that this O-GlcNAc increase was essential for meiotic resumption since the inhibition of OGT, the enzyme transferring the O-GlcNAc, prevents the oocyte G2/M transition whereas OGT overexpression accelerates this process. We identified 24 proteins that O-GlcNAc modification increases during meiotic resumption among which cytoskeletal proteins, the kinase erk2, the phosphatase PP2A, several glycolysis enzymes and sorne ribosomal proteins. Second, we have undertaken the study of O-GlcNAc, OGT and UDP-GlcNAc variations during the oogenesis and the early development of Xenopus laevis and we showed that the O-GlcNAc dynamism is intricate from the Xenopus oogenesis to embryogenesis. ln particular, we observed a drastic and transitory O-GlcNAc decrease at the onset of gastrulation, suggesting a role for O-GlcNAc in the regulation of cell migration characteristic of this stage of development since it permits the generation of the three germ layers, precursors ofthe whole adult tissues.

Axonal regeneration within the mammalian central nervous system following traumatic damage is limited and interventions to enable regrowth is a crucial goal in regenerative medicine. The nematode Caenorhabditis elegans is an excellent model to identify the intrinsic genetic programs that govern axonal regrowth. Here we demonstrate that alterations in O-linked N- beta-acetylglucosamine (O-GlcNAc) post-translational modifications of proteins can increase the regenerative potential of individual neurons. O-GlcNAc are single monosaccharide protein modifications that occur on serines/threonines in nucleocytoplasmic compartments. Changes in O-GlcNAc levels serve as a sensor of cellular nutrients and acts in part through the insulin-signaling pathway. Loss of O-GlcNAc via mutation of the O-GlcNAc Transferase (OGT), the enzyme that adds O-GlcNAc onto target proteins, enhances regeneration by 70%. Remarkably, hyper-O-GlcNAcyation via mutation of the O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from target proteins, also enhances regeneration by 40%. Our results shed light on this apparent contradiction by demonstrating that O-GlcNAc enzyme mutants differentially modulate the insulin-signaling pathway. OGT mutants act through AKT1 to modulate glycolysis. In contrast, OGA mutants act through the FOXO/DAF-16 transcription factor to improve the mitochondrial stress response. These findings reveal for the first time the importance of O-GlcNAc post-translational modifications in axon regeneration and provide evidence that regulation of metabolic programs can dictate the regenerative capacity of a neuron. / 2021-02-20T00:00:00Z