Bupivacaine is one of the amide type local anesthetics and is widely used for epidural anesthesia and blockade of nerves. Bupivacaine administration locally could result in neuron injury showing transient neurologic symptoms. Dexamethasone is a synthetic glucocorticoid and may exert cytoprotective properties against damage induced by some stimuli. In the present study, we evaluated the effects of dexamethasone on bupivacaine-induced toxicity in mouse neuroblastoma N2a cells. N2a cells were exposed to bupivacaine in the presence or absence of dexamethasone. After treatment, the cell viability, nuclear condensation, and lactate dehydrogenase levels were evaluated. Mitochondrial potential and Akt (threonine-serine protein kinase B) activation were also examined. In a separate experiment, we examined the effect of Akt inhibition by triciribine on cell viability following dexamethasone treatment. We also investigated whether dexamethasone could prevent lidocaine-induced neurotoxicity. Treatment of N2a cells with bupivacaine resulted in significant cell injury as evidenced by morphological changes, LDH leakage, and nuclear condensation. Pretreatment of the cells with dexamethasone significantly attenuated bupivacaine- and lidocaine-induced cell injury. Dexamethasone treatment prevented the decline of mitochondrial potential caused by bupivacaine and increased the levels of Akt phosphorylation. Importantly, pharmacological inhibition of Akt abolished the protective effect of dexamethasone against bupivacaine-induced cell injury. Our data suggest that pretreatment of neuroblastoma cells with dexamethasone exerts a protective effect on bupivacaine-induced neuronal cell injury. The mechanisms involve activating the Akt signaling pathway.

>Magister Scientiae - MSc / Silver nanoparticles (AgNPs) apart from being chemically significant, have shown a lot of health benefits, the most studied being their anti-bacterial and anti-inflammatory properties. These biological properties can be further enhanced by adding compounds with known medical properties giving rise to even more desired potent materials. Anti-bacterial and cytotoxicity studies show that these AgNPs can kill bacteria, prevent infections and regenerate skin cells. On the other hand, previous studies have reported dimethyl sulfoxide (DMSO) with attractive wound healing abilities specifically cell growth promotion. It was then envisaged that the combination of DMSO and AgNPs could lead to a potent wound healing agent. It is a well-known fact that non-healing wounds pose a socioeconomic threat to a large population worldwide. / 2023

<b>Français : </b> La mitochondrie est un organite dont les fonctions dépassent largement le rôle bioénergétique. De ce fait, il apparaît de plus en plus clairement qu’un grand nombre de pathologies sont liées à un dysfonctionnement mitochondrial. Au cours de ces dernières années l’existence d’une communication moléculaire rétrograde entre la mitochondrie non fonctionnelle et le noyau a été mise en évidence dans les cellules eucaryotes de mammifères. Les voies de signalisation moléculaire menant à l’expression différentielle de gènes nucléaires en réponse à un dysfonctionnement mitochondrial sont néanmoins encore peu connues. Dans ce domaine, l’utilisation de lignées cellulaire totalement (r0) ou partiellement (r-) déplétées en ADN mitochondrial (ADNmt) s’est révélée essentielle dans l’étude de la réponse cellulaire induite par un dysfonctionnement mitochondrial. L’objectif de ce travail était de mieux comprendre les mécanismes moléculaires impliqués dans la réponse cellulaire à un dysfonctionnement mitochondrial 1) en recherchant comment les cellules déplétées en ADNmt maintiennent un potentiel de membrane mitochondrial en étudiant les mécanismes impliqués dans le maintien de la biogenèse mitochondriale et 3) en caractérisant la sensibilité des cellules déplétées en ADNmt à l’apoptose. Au cours de la première partie de ce travail, nous avons mis en évidence le rôle de la protéine mtCLIC dans le maintien du Dym des cellules déplétées en ADNmt. Nous avons ainsi démontré que le gène codant cette protéine est surexprimé dans les cellules présentant un dysfonctionnement mitochondrial, et que l’activité de canal à chlore pouvait rendre compte du maintien du Dym dans ces cellules. Dans la deuxième partie de ce travail, nous avons caractérisé et comparé les populations mitochondriales des cellules parentales et déplétées en ADNmt (143B r0, ostéosarcome humain). L’activité de certains facteurs de transcription décrits pour jouer un rôle dans le processus de biogenèse mitochondriale a été recherchée ainsi que le niveau d’expression de certaines protéines marqueurs de la biogenèse mitochondriale. Le rôle de la voie calcium-CaMKIV-CREB dans le maintien de la biogenèse mitochondriale des cellules r0 a ainsi pu être mis en évidence. Nous avons également mis en évidence une diminution de l’activité d’importation de protéines chimériques matricielles dans les mitochondries des cellules déplétées en ADNmt. Cette diminution peut s’expliquer par la réduction du Dym et de la charge en ATP dans ces cellules mais n’est pas généralisable à l’ensemble des protéines mitochondriales. En effet, l’importation du cytochrome c est augmentée et celle de la sous-unité ß de la F1-ATPase est inchangée dans des cellules 143B r0. La dernière partie de ce travail a été consacrée à la caractérisation et à la comparaison de la réponse des cellules 143B et 143B r0 à un stimulus pro-apoptotique. Après avoir clairement établi que les cellules r0 présentent une sensibilité accrue à la staurosporine, nous avons recherché les mécanismes moléculaires pouvant expliquer cette réponse différentielle. Nous proposons que la sensibilité accrue des cellules r0 peut s’expliquer par la sous-expression constitutive des protéines anti-apoptotiques Bcl-2 et Bcl-XL. De plus, nous montrons également que les mécanismes impliqués pourraient faire intervenir la cathepsine B, libérée du lysosome par un mécanisme non encore élucidé. Nous montrons également que l’activation spécifique de l’autophagie dans les cellules 143B r0 en réponse à la staurosporine pourrait également contribuer à la plus grande sensibilité à l’apoptose des cellules présentant un dysfonctionnement mitochondrial. Les résultats obtenus au cours de ce travail ont permis d’identifier certains mécanismes d’adaptation mis en place dans des cellules de mammifères soumises à un stress énergétique chronique, et donc de mieux comprendre les implications d’un dysfonctionnement mitochondrial, une situation associée à ou responsable de nombreuses pathologies mitochondriales. <b>English : </b> Mitochondria are involved in numerous cell processes, such as ATP production, calcium homeostasis, fatty acid metabolism, heme synthesis, urea cycle, redox cell status, autophagy and apoptosis. Impairment of its bioenergetic activity is thus obviously associated with numerous pathologies. However, while various origins and symptoms have been described for mitochondrial diseases over the past 10 years, only very few retrograde signalling pathways (that could be defined as communication between impaired mitochondria and nucleus) have been identified. In addition, little is still known about the molecular mechanisms leading to differential gene expression in response to chronic or acute mitochondrial dysfunction. In that research field, the generation of cells totally (r0) or partially (r-) depleted in mtDNA has been very useful to study the response of cells to a chronic energetic stress. The major aim of this work was to get a better understanding of the molecular mechanisms involved in the retrograde communication between impaired mitochondria and the nucleus that participate to the maintenance of 1) the mitochondrial membrane potential (Dym), 2) the mitochondrial biogenesis and 3) the apoptotic response to staurosporine, an alkaloïd that inhibits numerous kinases. In the first part of this work, we highlighted the role of the protein mtCLIC/CLIC4 in the maintenance of the Dym in mtDNA-depleted cells. Using a “mRNA RT-PCR differential display” approach, we first identified that the gene was over-expressed in mtDNA-depleted cells. We also show that modifications of its abundance (over expresion and silencing by siRNA) were able to modify the Dym. Finally, we evidenced that mtCLIC allows the importation of chlorine into mitochondria of r-L929 (murine fibrosarcoma cells). In the second part of this work, we characterized and compared mitochondrial populations between 143B (osteosarcoma cell line) and 143B r0 cells. We monitored the activity status of several key transcription factors known to be involved in the control of mitochondrial biogenesis and we determined the expression level of several mitochondrial proteins used as common markers of mitochondrial biogenesis. We also clearly demonstrated the role for calcium-CaMKIV-CREB pathway in the maintenance of mitochondrial biogenesis in mtDNA-depleted cells. Indeed, we show that the over-expression of cytochrome c and the higher mitochondrial NAO (Nonyl Acridine Orange) staining (two indicators for a higher abundance of mitochondrial mass) observed in mtDNA-depleted cells could be reduced in r0 cells that over-express either a dominant negative forms of CREB or CaMKIV. Moreover, we show that the importation of matrix-targeted proteins is reduced in mtDNAdepleted cells, a feature that can be explained by the lower Dym and reduced ATP content in these cells. As several evidence were reported to link mitochondrial dysfunction and apoptosis in vivo, the last part of this work has been dedicated to the characterization of the apoptotic response of mtDNAdepleted cells to staurosporine. Indeed, the higher or lower sensitivity of mtDNA-depleted cells to apoptotic stimuli is still a debated question in the literature. We first show that r0 143B cells are hypersensitive to staurosporine-induced apoptosis, a phenomenon that could most likely be explained by the constitutive down-regulation of anti-apoptotic proteins such as Bcl-2 an Bcl-XL in r0 cells. Moreover, we show that the mechanisms of r0 cells response to staurosporine seems to be different from those triggered in parental cells. Indeed, we show that cathepsin B might play a role in staurosporine-induced mtDNA-depleted cell apoptosis, despite the activation of many caspases. Finally, we show that autopahgy is also triggered by staurosporine in r0 143B cells, an upstream event of caspase activation as 3-methyladenine (3-MA) strongly reduces caspase activation. In conclusion, our results bring new information in the understanding of mechanisms and cell signalling activated in mammalian cells facing a chronic energetic stress, and thus bring new insights into the cellular consequences of mitochondrial impairment, a feature found in numerous mitochondrial diseases and pathologies associated with aging.

Orientador: Prof. Dr. Daniel Carneiro Carrettiero / Tese (doutorado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2017. / A estreptozotocina (STZ) e uma toxina derivada da bacteria Streptomyces acromogenes. A administracao cerebral de STZ promove alteracoes metabolicas que mimetizam as encontradas em pacientes com doenca de Alzheimer (DA) esporadica. Por este motivo, a STZ vem, sendo utilizada intracerebroventricularmente na criacao de modelos animais para o estudo de DA. A DA possui duas importantes caracteristicas histopatologicas, o acumulo de peptideo ¿À amiloide, levando a formacao das placas senis, e o aumento da fosforilacao da proteina tau, que culmina com a formacao dos emaranhados neurofibrilares, que possuem papel eminente no processo neurodegenerativo da DA. Alguns fatores estao relacionados com o surgimento dessas caracteristicas no paciente, como por exemplo, a geracao de estresse oxidativo, ativacao de Caspase e morte neuronal. A co chaperona BAG2 e fundamental para degradacao da proteina Tau fosforilada inibindo sua ubiquitinacao e favorecendo sua degradacao pela via proteossomica independente de ubiquitina. O objetivo do estudo foi avaliar os efeitos da STZ nos parametros relacionadas a citotoxicidade (MTT, Senescencia, Caspasee potencial mitocondrial), na geracao de estresse oxidativo (Diclorofluorisceina e citometria), dosagem de oxido nitrico (Griess), bem como estudar sua acao nos niveis das proteinas oxido nitrico sintase (nNOS e iNOS), Tau fosforilada e BAG2. Para o estudo, foram utilizadas cultura primaria de celulas do hipocampo. As celulas foram tratadas com STZ (0,05; 0,5 e 5 mM) pelos periodos de 6, 12 e 24 horas. A STZ apresentou citotoxicidade na concentracao de 5 mM por 24 horas, no ensaio de MTT e pelo teste de Senescencia celular nas concentracoes de 0,05 e 5 mM em 12 e 24 horas, promovendo aumento na morte celular e niveis de caspase total ativa em celulas tratadas 0,05; 0,5 e 5 mM por 12 e 24 horas. Apesar disso, nao houve alteracao no potencial mitocondrial em nenhuma das concentracoes e tratamentos com STZ. O tratamento promoveu aumento tanto na quantidade de especies reativas de oxigenio avaliado pela Diclorofluorisceina e estresse oxidativo por citometria de fluxo em celulas submetidas as concentracoes de 0,5 e 5 mM por 12 e 24 horas. Os niveis de Nitrito apresentaram uma diminuicao nas concentracoes de 0,5 e 5 mM em 12 horas de tratamento e um aumento em celulas tratadas com 5 mM por 24 horas. Em relacao aos niveis de nNOS houve uma reducao em seus niveis em celulas tratadas na concentracao de 5 mM por 24 horas. Ja a iNOS nao teve seus niveis alterados em nenhum dos tratamentos. Apos o tratamento com STZ foi observado a diminuicao nas formas fosforiladas de proteina Tau, nas concentracoes de 0,05; 0,5 e 5 mM por 12 e 24 horas. O tratamento com STZ 14 promoveu uma reducao nos niveis de Tau total nas concentracoes de 0,5 e 5 mM em 12 horas e 0,05; 0,5 e 5 mM por 24 horas. A razao entre a proteina Tau fosforilada e total nao apresentou alteracao significativa. A proteina BAG2 apresentou diminuicao em seus niveis em celulas tratadas com 0,5 e 5 mM por 12 horas de STZ e 5 mM por 24 horas. Desse modo, conclui-se que a STZ, em celulas de cultura primaria de hipocampo, mostrou ser uma ferramenta interessante de estresse oxidativo, caracteristica da DA, alem de apresentar morte celular tambem um fator envolvido com a doenca. / Cancer is the leading cause of death worldwide and it is considered a public health problem. Tumor cells exhibit a variety of features that enable tumor growth and dissemination, like resistance to cell death mechanisms. Phenothiazines is a group of drugs that have been used in the treatment of psychiatric disorders for a long time. Literature data demonstrate that these compounds exhibit relevant biological effects including antitumor activity. Publications from our group have evidenced extremely important effects of phenotiazines and analogues on mitochondria related to the increase of calcium cytosolic concentration promoted by this drug. In our earlier work with isolated mitochondria, it was demonstrated that these drugs promote the mitochondrial membrane permeability due to the opening of permeability transition pore complex with consequent dissipation of mitochondrial transmembrane potential, calcium efflux and cytochrome c release. Additionally, in our latest publication, we demonstrated the cytotoxicity of phenothiazines in hepatoma cells, accompanied by cellular morphological alterations, plasma membrane permeability and immediate dissipation of mitochondrial transmembrane potential. Among the studied phenothiazines, the most potent was thioridazine, which was chosen for the present work. The goal of this work was to underlie the molecular mechanisms of thioridazine-­induced cell death in leukemic cells, evaluating the role of BCL-­2 family proteins, as well as changes in signaling pathways associated to cell death, including endoplasmic reticulum (ER) stress. This compound was able to induce apoptosis in a concentration and time-­dependent manner, and also inhibit the cell cycle progression in K562 cells. Furthermore, tioridazine-­induced cell death was accompanied by dissipation of mitochondrial transmembrane potential, alterations in BCL-­2 proteins expression as well as activation of the kinases JNK, ERK1/2 and p38. Our results also demonstrated that thioridazine was able to activate the pro ­apoptotic protein BAX and the release of the mitochondrial protein Omi, resulting in mitochondrial outer membrane permeabilization. In addition, we observed that thioridazine was able to induce a severe ER stress, promoting an increase in the expression of the major sensor proteins in this signaling pathway, leading to cell death. We can conclude that both mitochondrial and ER stress contributes to thioridazine-­induced cell death in K562 cells. Besides the importance of our results to the elucidation of phenothiazines-­induced tumor cells death, it also confirms the promising therapeutic potential of this class of drugs as antitumor agents.