Les dommages tissulaires associés à l'ischémie/reperfusion ont été largement étudiés. Plusieurs études ont montré que des dysfonctionnements mitochondriaux sont responsables de la survenue de ces dommages, et que la transition de perméabilité pourrait y être impliquée. Cette transition de perméabilité est médiée par l'ouverture du pore de transition de perméabilité (PTP). Cette ouverture du PTP pourrait survenir pendant la phase d'ischémie ou pendant la phase de reperfusion. L'objectif de ce travail était de visualiser l'ouverture du PTP dans des conditions d'ischémie/reperfusion sur cellules intactes (HMEC-1 et INS-1) et d'étudier son implication dans ce phénomène. Nous avons pu visualiser pour la première fois l'ouverture du PTP par le suivi des dommages qu'elle engendre (sortie du NADH et la chute du ΔΨ) induits par une ischémie/reperfusion. Nous avons constaté que l'activation du PTP a lieu pendant la phase d'ischémie tant dans les cellules HMEC-1 que dans les cellules INS-1. Cette induction a été prévenue dans les deux modèles cellulaires par la cyclosporine A. Nos résultats suggèrent également que le complexe I pourrait être impliqué dans la prévention de la chute du ΔΨ et de la sortie du NADH. Nous avons aussi montré que la capacité de rétention calcique des cellules perméabilisées diminue à l'ischémie et que cette diminution disparait après 60 minutes de reperfusion. Ainsi, la visualisation de l'ouverture du PTP dans un modèle d'ischémie/reperfusion constituerait une piste intéressante qui apporterait plus de certitude quant à l'implication du PTP dans ce phénomène. De plus, l'étude du phénomène d'ischémie in vitro, apporterait plus de réponses quant à l'implication des modifications du fonctionnement cellulaire dans les dommages tissulaires. / Several studies have shown that ischemia/reperfusion injury is strongly related to mitochondrial dysfunction. These studies have mostly focused on the involvement of the permeability transition in this phenomenon. The permeability transition is mediated by the opening of the mitochondrial permeability transition pore (mPTP). PTP activation may occur during ischemia or reperfusion. The aim of this work was to visualize mPTP opening during ischemia/reperfusion conditions on intact cells (HMEC-1 and INS-1) and to study its involvement in this phenomenon. For the first time, we observed the opening of the mPTP mediated by ischemia/reperfusion by monitoring the damages caused by its activation (NADH efflux and ΔΨ decrease). This mPTP activation occurred during ischemia in HMEC-1 and INS-1 cells. However, mPTP induction was prevented by cyclosporine A in both cell models. Furthermore, our results showed the involvement of complex I in the prevention of NADH efflux and ΔΨ decrease. A decrease in the mitochondrial calcium retention capacity was also shown in permeabilised cells during ischemia, which disappear after 60 minutes of reperfusion. Thus, the visualisation of the mPTP opening in a model of ischemia / reperfusion is an interesting observation indicating the involvement of PTP in this phenomenon. Furthermore, the study of ischemia in vitro would provide answers regarding the involvement of cellular function changes in tissue damage.

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A subset of cyclophilins have been implicated in mechanisms of neuroinflammation and neurodegeneration that contributes to the pathogenesis of Multiple Sclerosis. Mitochondrial dysfunction leading to mitochondrial permeability transition plays a pivotal role in axonal damage and disease progression in Multiple Sclerosis. Cyclophilin D (CypD) is a crucial regulatory component of the mitochondrial permeability transition pore and it was demonstrated that the cyclophilin D knockout animals showed reduced experimental allergic encephalomyelitis (EAE) clinical disease severity and axonal injury. We investigated the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive and non-selective cyclophilin inhibitor, on the course and severity of EAE. EAE mice treated with NIM811 showed a significant reduction in clinical disease severity compared to vehicle treated mice. FACS analysis performed with the dissociated thoracolumbar spine showed that NIM811 treatment was associated with a reduction in CNS macrophages but does not alter T-helper lineage frequencies. In addition, we demonstrated NIM811’s effect on crude mitochondrial fraction obtained from brain and liver homogenates of both wild type and CypD knockout mice in order to determine drug specificity. Benefits observed from the pharmacological inhibition of cyclophilin may lead to a novel MS therapy but NIM811’s exact mechanism of action has yet to be elucidated.

Phenylarsine oxide (PAO) is a membrane-permeable trivalent arsenic compounds, which interfere the biochemical activity of intracellular enzymes or proteins through reacting specifically with sulfhydryl and vicinal dithiol groups in the protein structure. Although the deleterious effects of arsenic compounds in bioorganisms have been extensively studied, however its role in the synaptogenesis is still obscure. Here we test the role of PAO on the synaptic activity at developing Xenopus neuromuscular synapse by using whole-cell patch clamp recording. Bath application of PAO dose-dependently increases the frequency of spontaneous synaptic currents (SSC frequency) and reaches its maximal effect at 10 £gM. The SSC frequency is robustly facilitated in 10~15 minutes after PAO application and then the release of neurotransmitter were abruptly ceased due to the degenerative collapse of the presynaptic motoneuron. Pretreatment of the culture with Ca2+ chelator BAPTA-AM significantly blunted the SSC frequency facilitation induced by PAO, suggesting a rise in Ca2+ in presynaptic motoneuron is a prerequisite. The PAO-induced SSC frequency facilitation is unaffected even that Ca2+ is eliminated from culture medium or addition of pharmacological Ca2+ channel inhibitor cadmium, indicating the influx of extracellular Ca2+ is not needed for the rise of [Ca2+]i. Depletion of endoplasmic reticulum Ca2+ pool with thapsigargin effectively hampered the PAO-induced SSC frequency facilitation. Pretreatment of ryanodine receptor inhibitor TMB-8 but not IP3 receptor inhibitor XeC significantly occluded the increase of SSC frequency elicited by PAO. Furthermore, bath application of the culture with either mitochondria oxidative phosphorylation uncoupler FCCP or mitochondrial permeability transition pore inhibitor cyclosporin A significantly abolished the SSC facilitating effect of PAO. Pretreatment the culture with TMB-8 and cyclosporin A have no addictive effects on the occlusion of PAO-induced SSC frequency facilitation, suggesting a consecutively released Ca2+ from internal store through ryanodine receptor and mitochondria is responsible for PAO-induced SSC frequency facilitation. The synaptic facilitating effect of PAO is eliminated while incubated with free radical scavenger n-acetylcysteine. Furthermore, treating cultures with complex III of electron transport chain (ETC) inhibitor antimycin A, but not complex I inhibitor rotenone, abolished PAO-induced facilitation of synaptic transmission. PAO elicited no facilitation effects on SSC frequency when pretreatment the culture with either thiol-modifying agent NEM or thiol-reducing agent DTT. Overall, results from our current study provide evidences that reactive oxygen species derived from PAO inhibition on complex III of ETC induce the open of MPT pore in mitochondria, the accompanied Ca2+ leak from mitochondria and Ca2+-induced Ca2+ release from endoplasmic reticulum resulted in a robustly release of neurotransmitter and a destructive damage on the neuron.

Hypochlorous acid (HOCl) is a powerful oxidant produced by activated phagocytes at sites of inflammation to kill a wide range of pathogens. Yet, it may also damage and kill the neighbouring host cells. The abundance of dead macrophages in atherosclerotic plaques and their colocalization with HOCl-modified proteins implicate HOCl may play a role in killing macrophages, contributing to disease progression. The first part of this research was to investigate the cytotoxic effect and cell death mechanism(s) of HOCl on macrophages. Macrophages require efficient defense mechanism(s) against HOCl to function properly at inflammatory sites. The second part of the thesis was to examine the antioxidative effects of glutathione (GSH) and 7,8-dihydroneopterin (7,8-NP) on HOCl-induced cellular damage in macrophages. GSH is an efficient scavenger of HOCl and a major intracellular antioxidant against oxidative stress, whereas 7,8-NP is secreted by human macrophages upon interferon-γ (IFN-γ) induction during inflammation and can also scavenge HOCl. HOCl caused concentration-dependent cell viability loss in human monocyte derived macrophage (HMDM) cells above a specific concentration threshold. HOCl reacted with HMDMs to cause viability loss within the first 10 minutes of treatment, and it posed no latent effect on the cells afterwards regardless of the HOCl concentrations. The lack of caspase-3 activation, rapid influx of propidium iodide (PI) dye, rapid loss of intracellular ATP and cell morphological changes (cell swelling, cell membrane integrity loss and rupture) were observed in HMDM cells treated with HOCl. These results indicate that HOCl caused HMDM cells to undergo necrotic cell death. In addition to the loss of intracellular ATP, HOCl also caused rapid loss of GAPDH enzymatic activity and mitochondrial membrane potential, indicating impairment of the metabolic energy production. Loss of the mitochondrial membrane potential was mediated by mitochondrial permeability transition (MPT), as blocking MPT pore formation using cyclosporin A (CSA) prevented mitochondrial membrane potential loss. HOCl caused an increase in cytosolic calcium ion (Ca2+) level, which was due to both intra- and extra-cellular sources. However, extracellular sources only contributed significantly above a certain HOCl concentration. Preventing cytosolic Ca2+ increase significantly inhibited HOCl-induced cell viability loss. This suggests that cytosolic Ca2+ increase was associated with HOCl-induced necrotic cell death in HMDM cells, possibly via the activation of Ca2+-dependent calpain cysteine proteases. Calpain inhibitors prevented HOCl-induced lysosomal destabilisation and cell viability loss in HMDM cells. Calpains induced HOCl-induced necrotic cell death possibly by degrading cytoskeletal and other cellular proteins, or causing the release of cathepsin proteases from ruptured lysosomes that also degraded cellular components. The HOCl-induced cytosolic Ca2+ increase also caused mitochondrial Ca2+ accumulation and MPT activation-mediated mitochondrial membrane potential loss. MPT activation, like calpain activation, was also associated with the HOCl-induced necrotic cell death, as preventing MPT activation completely inhibited HOCl-induced cell viability loss. The involvement of both calpain activation and MPT activation in HOCl-induced necrotic cell death in HMDM cells implies a cause and effect relationship between these two events. HMDM cells depleted of intracellular GSH using diethyl maleate showed increased susceptibility towards HOCl insult compared to HMDM cells with intact intracellular GSH levels, indicating that intracellular GSH played an important role in protecting HMDM cells against HOCl exposure. Intracellular GSH level in each HMDM cell preparation directly correlated with HOCl concentration required to kill 50% of population for each cell preparation, indicating intracellular GSH concentrations determine the efficiency of GSH in preventing HOCl-induced damage to HMDM cells. Intracellular GSH and cell viability loss induced by 400 μM HOCl were significantly prevented by 300 μM extracellular 7,8-NP, indicating that added 7,8-NP is an efficient scavenger of HOCl and out-competed intracellular GSH for HOCl. The amount of 7,8-NP synthesized by HMDM cells upon IFN-γ induction was too low to efficiently prevent HOCl-mediated intracellular GSH and cell viability loss. HOCl clearly causes HMDM cells to undergo necrosis when the concentration exceeds the intracellular GSH concentrations. Above this concentration HOCl causes oxidative damage to the Ca2+ ion channels on cell and ER membranes, resulting in an influx of Ca2+ ions into the cytosol and possibly the mitochondria. The rise in Ca2+ ions triggers calpain activation, resulting in the MPT-mediated loss of mitochondrial membrane potential, lysosomal instability and cellular necrosis.

There is a large body of clinical and experimental evidence linking heart disease with impairment of myocardial energetics, particularly the creatine kinase (CK) system. The goal of the experiments described in this thesis was to develop and study models of increased CK phosphotransfer, by overexpressing the CK isoenzymes and/or augmenting intracellular creatine stores. Pilot experiments were performed in cultured cells, which were used to (a) study the effects of CK overexpression in vitro, and (b) validate constructs prior to generation of transgenic mice. Expression was verified at the protein level for all constructs in HL-1 and HEK293 cells, and enzymatic activity was confirmed. Mitochondrial CK (CKmt) was expressed in the mitochondria, as expected, and CKmt overexpression was associated with a significant reduction in cell death in a model of ischemia/reperfusion injury (68.1 ± 7.1% of control, p≤0.05). Transgenic mice overexpressing CKmt in the heart were generated by a targeted approach, using PhiC31 integration at the ROSA26 locus. Transgene expression was confirmed in vitro in embryonic stem cells, and in vivo at the mRNA and protein levels. There was only a modest increase in CKmt activity; therefore, homozygous transgenic mice were generated to increase expression levels, and had 27% higher CKmt activity than wild-types (p≤0.01). Mitochondrial localization of CKmt was confirmed by electron microscopy. Citrate synthase activity, a marker of mitochondrial volume, was ~10% lower in transgenic mice (p≤0.05). Baseline phenotyping studies found that CKmt-overexpressing mice have normal cardiac structure and function. These mice are currently being backcrossed onto a pure C57BL/6 background for further studies in models of heart disease. In addition to CKmt, transgenic mice overexpressing the cytosolic CK isoenzymes, CK-M and CK-B, were generated. Due to the modest level of expression observed at ROSA26, random-integration transgenesis was used, and multiple lines were generated for each construct (carrying 2 or 6 transgene copies in the CK-M line; 2, 3, or ~30 in CK-B). Transgene expression was validated at the mRNA, protein, and activity levels. These lines are currently being expanded for further validation and phenotyping studies. Previous experiments in our group have demonstrated that increasing intracellular creatine (Cr) reduces ischemia/reperfusion injury, and a series of in vitro experiments was performed to determine whether this effect may be mediated by inhibition of the mitochondrial permeability transition pore (mPTP). The mPTP plays a significant role in ischemia/reperfusion, and there is evidence linking the CK system to regulation of the mPTP. Therefore, a model was developed to test whether Cr affects mPTP opening in cardiac-derived HL-1 cells, as this mechanism may contribute to the protective effect observed in vivo. Cr incubation conditions were determined empirically, and 24-hour incubation with 5mM or 10mM Cr was found to significantly delay mPTP opening, to a similar degree to the established mPTP inhibitor, cyclosporin A. This provides evidence that Cr may exert protective effects in the heart by a variety of mechanisms, in addition to its traditional role in energy metabolism. In summary, the experiments conducted in this thesis have produced a range of tools for studying augmentation of the creatine kinase system as a therapeutic target in heart disease. The results of in vitro assays indicate that mitochondrial CK may be a particularly promising target, and that inhibition of the mitochondrial permeability transition pore may contribute to the cardioprotective effect of creatine. Finally, the transgenic models generated and validated over the course of this project will allow for a wide range of future studies into the potential benefits of CK overexpression in the mammalian heart.

Orientador: Aníbal Eugênio Vercesi / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T00:12:21Z (GMT). No. of bitstreams: 1 Ferreira_ViniciusVercesiAlmadaNogueira_M.pdf: 1353533 bytes, checksum: b60bd544902662a56e45ea9cf1e14b01 (MD5) Previous issue date: 2013 / Resumo: Além de essencial na respiração celular, a mitocôndria é considerada uma organela de papel fundamental em processos de sinalização e morte celular. A geração de espécies reativas de oxigênio (EROs) e de espécies reativas de nitrogênio (ERNs) pela mitocôndria podem ser consequência destes processos de sinalização tanto em condições fisiológicas quanto em patologias, tais como a dislipidemia e câncer. A transição de permeabilidade mitocondrial (TPM) é um tipo de permeabilização não-seletiva da membrana mitocôndrial interna que permite a passagem de moléculas de até 1,5 KDa, causando dissipação do potencial eletroquímico de H+ e inchamento da organela. O termo transição é usado porque a permeabilização pode ser parcialmente revertida, logo após o inicio do processo, pela adição de quelantes de Ca2+ como o EGTA, por exemplo, ou redutores tiólicos. A oxidação de grupamentos tiólicos promove a TPM. Os experimentos apresentados nesta tese indicam o composto 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO) caracteriza-se como um inibidor da TPM. As pequenas alterações observadas em relação à respiração e fosforilação oxidativa não se correlacionam com a abertura do PTP, pelo menos com base na literatura corrente. Em outras palavras, a presença de cPTIO diminuiu os níveis de NOo mitocondrial e desviou a reação deste composto com o ânion superóxido diminuindo a produção de peroxinitrito, provável indutor de abertura do PTP nestas condições experimentais / Abstract: In addition to be the site of cellular respiration, the mitochondrion has important role in cell signaling for cell physiology and death. The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by the mitochondrion can participate in cellular signaling both in physiological and pathological conditions, such as dyslipidemia and cancer. The Mitochondrial Permeability Transition (MPT) is a non-selective inner membrane permeabilization that enables free passage of molecules up to 1.5 kDa, dissipating the H+ electrochemical gradient and the organelle's swelling. The term transition is applied because the permeabilization can be partially reverted, right after the beginning of the process, by chelating extramitochondrial Ca2+ with EGTA, for example, or by using thiol reducers. The oxidation of membrane protein thiols leads to MPT. The experiments shown in this thesis indicate that the compound 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO) has proven to be a strong inhibitor agent of the MPT. The small changes observed in relation to respiration and oxidative phosphorylation did not correlate with the opening of PTP, at least on the basis of the current literature. In other words, the presence of cPTIO decreased levels of NOo and mitochondrial diverted reaction of this compound with superoxide anion decreasing production of peroxynitrite, likely inducing PTP opening under these experimental conditions / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

Orientadores: Anibal Eugênio Vercesi, Helena Coutinho Franco de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T11:59:13Z (GMT). No. of bitstreams: 1 Marques_AnaCarolina_M.pdf: 1109688 bytes, checksum: 76a66df390130e764ca54b415b0756b4 (MD5) Previous issue date: 2014 / Resumo: Os camundongos hipercolesterolêmicos (LDLr-/-) provenientes do Jackson Laboratory são modelos experimentais valiosos para o estudo da aterosclerose. Estes animais apresentam elevadas taxas de lipogênese, processo que consome grandes quantidades de NADPH. Pesquisas recentes revelaram que esta linhagem possui, além da deleção do gene do receptor de LDL, uma mutação no gene da NADP- transidrogenase (NNT). A falta da NNT pode gerar estresse oxidativo devido ao fornecimento deficiente de NADPH mitocondrial. O objetivo deste trabalho foi investigar a participação da elevação da lipogênese e da deficiência de NNT sobre o estado redox mitocondrial e suscetibilidade à transição de permeabilidade mitocondrial (TPM). Para tanto foram comparadas três linhagens de camundongos: LDLr-/- (deficiente do receptor de LDL e da NNT), C57BL6/J (deficiente apenas da NNT) e C57BL6/JUnib (controle). Foram avaliados: o controle respiratório mitocondrial (consumo de oxigênio), o estado redox de NAD(P) (fluorimetria), a susceptibilidade à transição de permeabilidade mitocondrial induzida por cálcio (inchamento e dissipação do potencial elétrico de membrana (??) sensíveis à ciclosprina A e a geração de peróxido de hidrogênio (H2O2) (Amplex red®) em mitocôndrias isoladas de coração e fígado. Observamos que não houve diferenças significativas nos parâmetros respiratórios mitocondriais nos dois tecidos das três linhagens estudadas. Como esperado, as mitocôndrias dos camundongos LDLr-/- e C57BL6/J não podem sustentar o estado reduzido de NADPH in vitro, uma vez que são deficientes de NNT. Observamos que houve diferenças significativas entre as 3 linhagens quanto à TPM da seguinte maneira: LDLr-/- > C57BL6/J > C57BL6/JUnib (controle) em mitocôndrias isoladas de fígado (inchamento e dissipação de ??) e em mitocôndrias de coração (??). Além disso, a produção de H2O2 por mitocôndrias hepáticas seguiu o mesmo padrão, sendo LDLr-/- > C57BL6/J > C57BL6/JUnib (controle). Em conjunto, estes resultados indicam que a maior suscetibilidade à TPM das mitocôndrias de camundongos LDLr-/- está correlacionada com diminuição de NADPH, tanto por aumento de consumo (devido a elevada lipogênese) quanto por diminuição de sua produção (deficiência em NNT) / Abstract: Hypercholesterolemic LDL receptor knockout mice (LDLr-/-) from Jackson Laboratory are valuable experimental models to study atherosclerosis development. These mice exhibit high rates of lipogenesis, a process that consumes large amounts of NADPH. It was recently discovered that the mice strain used to produce the LDLr-/- also carries a homozygous NADP-transhydrogenase (NNT) mutation. Loss of NNT may cause oxidative stress due to a poor supply of mitochondrial NADPH. The objective of this study was to investigate the role of elevated lipogenesis and NNT deficiency on the mitochondrial redox status and susceptibility to mitochondrial permeability transition (MPT). Three mice strains were compared: LDLr-/- mice (deficient of both LDL receptor and NTT), C57BL6/J (deficient in NNT only) and the wild type control mice C57BL6/JUnib. We evaluated the mitochondrial respiratory control (oxygen consumption), the NAD(P) redox status (fluorimetry), the susceptibility to calcium induced mitochondrial permeability transition (swelling and dissipation of membrane potential (??) sensitive to cyclosporin A) and the generation of H2O2 (Amplex red®) in isolated heart and liver mitochondria. We observed no significant differences in mitochondrial respiratory parameters in both tissues of the three mice strains studied. As expected, the mitochondria of LDLr-/- and C57BL6/J mice cannot maintain the NADP in the reduced state in vitro, since they are deficient in NNT. Regarding the susceptibility to MPT, we observed significant differences among mitochondria from the 3 strains, as follows: LDLr-/- > C57BL6/J > C57BL6/JUnib (control) in isolated liver mitochondria (swelling and potential dissipation) and in heart mitochondria potential dissipation). Furthermore, the production of H2O2 by the liver mitochondria followed the same MPT pattern: LDLr-/- > C57BL6/J > C57BL6/JUnib (control). Together, these results indicated that the greater susceptibility of LDLr-/- mitochondria to MPT is correlated with decreased NADPH which is explained by both increased consumption (due to high lipogenesis) and decreased production (deficiency NNT) / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestra em Ciências

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