6 research outputs found
First steps in using machine learning on fMRI data to predict intrusive memories of traumatic film footage
No full textAfter psychological trauma, why do some only some parts of the traumatic event return as intrusive memories while others do not? Intrusive memories are key to cognitive behavioural treatment for post-traumatic stress disorder, and an aetiological understanding is warranted. We present here analyses using multivariate pattern analysis (MVPA) and a machine learning classifier to investigate whether peri-traumatic brain activation was able to predict later intrusive memories (i.e. before they had happened). To provide a methodological basis for understanding the context of the current results, we first show how functional magnetic resonance imaging (fMRI) during an experimental analogue of trauma (a trauma film) via a prospective event-related design was able to capture an individual's later intrusive memories. Results showed widespread increases in brain activation at encoding when viewing a scene in the scanner that would later return as an intrusive memory in the real world. These fMRI results were replicated in a second study. While traditional mass univariate regression analysis highlighted an association between brain processing and symptomatology, this is not the same as prediction. Using MVPA and a machine learning classifier, it was possible to predict later intrusive memories across participants with 68% accuracy, and within a participant with 97% accuracy; i.e. the classifier could identify out of multiple scenes those that would later return as an intrusive memory. We also report here brain networks key in intrusive memory prediction. MVPA opens the possibility of decoding brain activity to reconstruct idiosyncratic cognitive events with relevance to understanding and predicting mental health symptoms
Avaliação do estresse oxidativo na cardiopatia chagásica crônica
No full textDissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-graduação em Farmácia.O desequilíbrio entre os mecanismos que causam condições oxidativas e as defesas antioxidantes provoca uma variedade de mudanças fisiológicas, chamadas coletivamente de estresse oxidativo. A cardiopatia chagásica é uma patologia com características de inflamação crônica muito próxima a uma doença auto-imune que tem mecanismo de ação ainda obscuro. O objetivo deste trabalho foi analisar as enzimas antioxidantes nos eritrócitos de pacientes cardiopatas chagásicos crônicos puros, classificados em 4 grupos nomeados de I à IV (cada grupo contendo n=10), variando entre estes grupos o grau de comprometimento cardíaco, segundo classificação de Los Andes modificada. Cada um dos quatro grupos chagásicos foi igualado a indivíduos saudáveis pela idade, formando 4 grupos-controle, e ainda, um grupo V formado por 10 pacientes não chagásicos com insuficiência cardíaca de etiologia orovalvar com sobrecarga de pressão e volume, perfazendo um total de 90 pacientes. Foram examinadas as enzimas antioxidantes catalase (CAT), superóxido dismutase (SOD), glutationa peroxidase (GPx), glutationa redutase (GR), glutationa S-transferase (GST) e ainda os tióis não proteicos (GSH), (GT) e (GSSG). As atividades da SOD, CAT, GR e os níveis de GT e GSSG essencialmente não apresentaram diferenças entre os grupos chagásicos. A concentração de GSH diminui com a progressão da doença e apresentou aumento em relação aos controles nos grupos I, II e III. As atividades da GST e GPx estiveram aumentadas no grupo III e diminuídas no grupo II e IV, indicando uma diminuição destes antioxidantes com a progressão da doença. O grupo V, exceção às atividades da SOD e da CAT que foram semelhantes, mostrou aumentos das enzimas em relação ao grupo IV, constituindo um comportamento diferenciado neste sentido. Esses resultados sugerem a existência de um quadro de estresse oxidativo com o aumento das condições oxidativas paralelamente à evolução da doença, ou seja, pacientes com grau mais elevado de acometimento cardíaco têm uma capacidade antioxidante diminuída, o que sugere uma perda grande das defesas antioxidantes. Os dados do presente trabalho têm grande importância quanto ao sistema antioxidante relacionado com os mecanismos de agressão do parasita nesta patologia crônica
Ação da intervenção antioxidante na síndrome de Down
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2015.A Síndrome de Down (SD) é a mais frequente desordem genética humana, e é causada, na sua quase totalidade, pela trissomia do cromossomo 21. A geração excessiva de espécies reativas de oxigênio (EROs) está envolvida na patogenia da SD. O objetivo deste estudo foi (I) avaliar o status antioxidante no sangue de crianças e adolescentes SD, antes e após a suplementação com vitaminas E e C, (II) bem como o efeito da administração de melatonina (MEL) sobre os biomarcadores de estresse oxidativo (EO) e de neurogênese, em um modelo animal de SD (camundongos Ts65Dn). Biomarcadores de EO e níveis de citocinas inflamatórias foram avaliados em pacientes com SD (n=21), antes e após suplementação diária (vitamina E 400 mg, C 500 mg) durante 6 meses, seguida de interrupção da terapia (por 6 meses) e posteriormente submetidos a uma nova intervenção antioxidante de 6 meses. Crianças saudáveis (n=18) sem SD foram recrutadas para constituir o grupo controle. As atividades da superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx), glutationa redutase (GR), glutationa S-transferase (GST), gama-glutamiltransferase (GGT), glicose-6-fosfato desidrogenase (G6PD) e mieloperoxidase (MPO), assim como os conteúdos de glutationa reduzida (GSH), ácido úrico (AU), vitamina E, substâncias que reagem com o ácido tiobarbitúrico (TBARS), proteína carbonilada (PC), transferrina, TNF-a e IL-1ß, foram mensuradas no sangue destes indivíduos. Antes da suplementação, os indivíduos SD, apresentaram aumento da atividade enzimática da SOD, CAT, GR, GGT e MPO, assim como dos níveis séricos de AU, transferrina, TNF-a e IL-1ß enquanto que a atividade da GST e os níveis de GSH e PC mostraram valores diminuídos. No entanto, as atividades da GPx e G6PD, assim como os níveis plasmáticos de vitamina E e TBARS, não apresentaram diferenças significativas em comparação aos controles. Após a suplementação antioxidante, as atividades das enzimas SOD, CAT, GPx, GR, GGT e MPO, bem como o conteúdo de TBARS foram diminuídos, as atividades da G6PD e GST, e os níveis de AU, PC, transferrina, TNF-a e IL-1ß permaneceram inalterados, enquanto que os conteúdos de GSH e vitamina E mostraram significativo aumento. Após a interrupção da suplementação, houve aumento das atividades da GPx e GGT nos indivíduos SD, assim como nos níveis de AU e TBARS. Nenhuma mudança foi observada nas atividades da SOD, CAT, GR, GST, G6PD e MPO, bem como nos níveis de GSH, vitamina E, PC,transferrina, TNF-a e IL-1ß. Após nova suplementação, houve aumento dos níveis plasmáticos de vitamina E e diminuição da atividade da GGT nenhuma alteração nas atividades da SOD, CAT, GPx, GR, G6PD, GST e MPO, assim como nos conteúdos de GSH, AU, transferrina, TBARS, PC, TNF-a e IL-1ß. Para o estudo da MEL foram utilizados animais jovens e adultos. Para ambas as idades, foram utilizados dois grupos de camundongos com genótipos diferentes, Controles (Dissômicos) e Ts65Dn (Trissômicos), os quais foram subdividos e tratados com veículo (água contendo 0,06% etanol) e/ou MEL (100 mg/L), formando 4 grupos experimentais: CO+Veículo, CO+Mel, TS+Veículo e TS+Mel. Para os jovens, o tratamento ocorreu desde a fase pré-natal com as fêmeas prenhas e se estenderam até a idade de experimento (5,5 meses de idade). Já para os adultos o tratamento iniciou com 5,5 e perdurou até 10,5 meses de idade. Tanto para jovens e adultos, foram avaliados biomarcadores de EO no córtex e hipocampo do cérebro desses animais. A neurogênese dos animais jovens (em adultos são dados já publicados) foi avaliada através de imunohistoquímica para Ki-67 (proteína nuclear, expressa em células em mitose) e DAPI (marcador nuclear) no hipocampo desses animais. A administração de MEL diminuiu a atividade da SOD e CAT enquanto não houve mudanças na atividade da GPx e GR e nos níveis de TBARS e PC no córtex e hipocampo. Nos animais adultos, o tratamento com a MEL diminuiu a atividade da SOD apenas no córtex, assim como os níveis de TBARS e PC no hipocampo desses animais, enquanto não houve mudanças na atividade da CAT, GPx e GR no córtex e hipocampo desses animais. Não houve mudanças na densidade de células Ki-67 e DAPI após tratamento com MEL nos animais jovens. Conclusão: (I) A presença da trissomia 21 em crianças e adolescentes resultou em alterações bioquímicas que contribuem para o EO sistêmico e exacerbado nesses pacientes. (II) A terapia antioxidante com vitaminas E e C após 6 meses atenuou o EO. Adicionalmente, (III) o efeito da intervenção antioxidante persistiu significativamente após 6 meses de interrupção da suplementação. (IV). No estudo com camundongos TS também foi observado aumento do EO nos camundongos TS. A MEL foi capaz de diminuir o EO causado pela TS, especialmente nos animais adultos. Além disso, (V) o tratamento com a MEL não modificou a neurogênese nos animais jovens.Abstract : Down syndrome (DS), the most frequent genetic disorder, is almost entirely caused by trisomy of human chromosome 21. The overgeneration of reactive oxygen species (ROS) is involved in DS pathogenesis. The aim of this study was (I) to evaluate biomarkers of oxidative stress (OS) in the blood of DS children and adolescents before and after an antioxidant supplementation with vitamins E and C, as well as (II) the effect of administration of melatonin (MEL) on OS biomarkers and neurogenesis in an SD animal model (Ts65Dn mice). Biomarkers of OS and contents of inflammatory cytokines were evaluated in the blood of DS patients (n=21) before and after a daily antioxidant intervention (vitamin E 400 mg, vitamin C 500 mg) during 6 months followed by an interruption of the supplementation (also 6 months), followed by a new supplementation (6 months). Healthy children (n=18) without DS were recruited to constitute the control group. The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), gamma-glutamyltransferase (GGT), glucose-6-phosphate dehydrogenase (G6PD) and myeloperoxidase (MPO), as well as the contents of reduced glutathione (GSH), uric acid (UA), vitamin E, thiobarbituric acid reactive substances (TBARS), protein carbonyls (PC), transferrin, TNF-a and IL-1ß, were measured. Before the antioxidant therapy DS patients showed elevated SOD, CAT, GR, GGT and MPO activity and also elevated UA, transferrin, TNF-a and IL-1ß levels. The GST activity, GSH and PC levels were decreased, while GPx and G6PD activity and also plasma levels of vitamin E and TBARS showed no significant differences compared to controls. After the antioxidant supplementation, the activity of SOD, CAT, GPx, GR, GGT and MPO were downregulated, while TBARS contents were strongly decreased. No changes in G6PD and GST activities as well as in UA, PC, transferrin, TNF-a and IL-1ß levels were detected, while the contents of GSH and vitamin E were significantly increased. After interruption of the antioxidant therapy, DS patients showed elevated GPx and GGT activities as well as elevated UA and TBARS levels, while no changes in SOD, CAT, GR, GST, G6PD and MPO activities as well as in GSH, vitamin E, PC, transferrin, TNF-a and IL-1ß levels were detected. After the new period of supplementation there was an increase in plasma levels of vitamin E and decreased GGT activity, while nochanges in SOD, CAT, GPx, GR, G6PD, GST and MPO activity as well as in the contents of GSH, UA, transferrin, TBARS, PC, TNF-a and IL-1ß. The MEL intervention in young and adult animals used two groups of mice with different genotypes, controls (euploid littermates) and Ts65Dn (with trisomy), which were subdivided and treated with vehicle (water containing 0.06% ethanol) and/or MEL (100 mg/L), forming four groups: CO+Vehicle, CO+MEL, TS+Vehicle and TS+MEL. For young animals treatment occurred from the pre-natal stage with pregnant females and extended until the age of experiment (5.5 months), while for adults the treatment began and lasted 5.5 to 10.5 months of age. Both young and adults were assessed through OS biomarkers in the cortex and hippocampus. Neurogenesis of young animals (in adults data were already published) was assessed by immunohistochemistry for Ki-67 (nuclear protein expressed in cells in mitosis) and DAPI (nuclear marker) in the hippocampus. MEL administration decreased SOD and CAT activity while no changes in the activity of GPx and GR, as well as in levels of TBARS and PC in the cortex and hippocampus were detected. In adults MEL treatment promoted decreased SOD activity only in the cortex, as well as TBARS and PC levels in the hippocampus, whereas no changes in the CAT, GPx and GR activity in the cortex and hippocampus were detected. No changes in the density of Ki-67 and DAPI cells after treatment with MEL in young animals. Conclusions: (I) The presence of trisomy 21 in children and adolescents results in biochemical changes that strongly contributes to a systemic and exacerbated oxidative stress in these patients. (II) The antioxidant intervention with vitamins E and C for 6 months consistently attenuated such oxidative insult. Furthermore, (III) the effect of the antioxidant intervention persisted after 6 months of withdrawal of the antioxidant supplementation. (IV) In the study with TS mice, increased OS in brain was also observed. (V) MEL was able to decrease OS caused by TS, especially in adult animals, while it did not alter neurogenesis in young animals
Endoplasmic reticulum H₂O₂ : Ero1-driven generation and GPx-mediated detoxification
No full textEndoplasmic reticulum (ER) oxidoreductin 1 alpha (Ero1alpha) is an ER-resident oxidase, which utilizes molecular oxygen (O2) as terminal electron acceptor to produce disulfide bonds and hydrogen peroxide (H2O2). The major target for Ero1alpha-derived disulfides is protein disulfide isomerase (PDI), which transfers them onto substrate proteins and plays an additional role as homeostatic regulator of Ero1alpha.
In this thesis, I demonstrated that PDI-mediated activation of Ero1alpha extends beyond the reduction of the known inhibitory disulfides Cys94-Cys131 and Cys99-Cys104 and involves an additional disulfide, Cys208-Cys241. Opening of this disulfide by PDI apparently enables diffusion of O2 towards and of H2O2 away from the catalytic flavin cofactor in Ero1alpha. Expression of a constitutively active Ero1alpha mutant, which is devoid of all three regulatory disulfides, compromises cell viability. Hence, redox regulation of the O2/H2O2 diffusion pathway in Ero1alpha emerges as critical determinant of ER homeostasis, in which PDI takes center stage by directly regulating O2 consumption.
I also elucidated the molecular basis for the specificity of glutathione peroxidase 8 (GPx8) to detoxify Ero1alpha-derived H2O2, as this enzyme binds to the site of H2O2 release in Ero1alpha. Only depletion of GPx8 but not of the abundant ER peroxidase peroxiredoxin IV (PrxIV) exhibited an additive effect with deregulated Ero1alpha on ER hyperoxidation and induction of unfolded protein response and antioxidant response target genes. Furthermore, only upon GPx8 knockdown I was able to detect leakage of Ero1alpha-derived H2O2 from ER to cytosol. Therefore, GPx8 acts as a specific molecular gatekeeper to protect the cytosol from Ero1alpha-derived H2O2. The exclusion of PrxIV from this process revealed a previously unappreciated compartmentalization of electron transport pathways in the ER.
Moreover, I successfully isolated mixed-disulfide interaction partners of the ER-resident peroxidase GPx7 and of PDI. The interactome of the latter was analyzed and found to be mainly comprised of other members of the PDI family, which, in conjunction with its function as Ero1alpha activator, places PDI as central regulator of ER disulfide homeostasis. With regard to GPx7 interaction partners I am confident that their identification will serve as basis for future elucidation of novel cellular functions of this peroxidase
Investigations into effects of 12-lipoxygenase and NADPH oxidase on platelet activity, and influences of dietary dark chocolate
No full textPhDPlatelets play a pivotal role in both normal hemostasis and pathological bleeding and importantly also contribute to the development of atherothrombosis. Even though platelet function tests traditionally are utilised mainly for the diagnosis and management of patients presenting with bleeding problems rather than thrombosis, new and improved platelet function tests are now increasingly used to monitor anti-platelet therapy in patients and to identify patients at risk of arterial disease. Based on light transmission traditional aggregometry, this thesis reports data from a new model of platelet aggregation using a modified 96-well plate format. This method allows examination of many agonists at a range of concentrations at the same time. Thus, more information can be collated about different aspects of platelet function and smaller assay volumes can be used while still obtaining reliable results. To further utilise this method, agonist combinations were used in the 96-well plate approach that resemble the actions of machines such as the PFA-100, which uses combined agonists within a cartridge, but at much lower cost. Platelet cyclooxygenase has been widely studied; however, the functions of platelet 12-lipoxygenase and NADPH oxidase in platelets are still generally not understood. Data presented here demonstrate that both pathways are partly essential in platelet activation following exposure to stimulatory agonists. To further explore the relationship between dietary intake and the risk of atherothrombosis, an in vivo study was performed to observe the antiplatelet effects following from consumption of dark chocolate in baseline hypertensive patients. Based on findings in this thesis, it can be
4
concluded that this new method of evaluating platelet aggregation and adhesion in a 96-well plate format is very useful, and that new observations into influences on platelets of pathways other than cyclooxygenase may be beneficial in the development of new antiplatelet drugs.funded by Academician Training Scheme for Public Institute of Higher Education, Ministry of Higher Education of Malaysia
Effect of protein glycation by methylglyoxal on pancreatic beta cell function
No full textMethylglyoxal is a physiological dicarbonyl metabolite and potent argininedirected glycating agent. It often modifies proteins at functional sites producing loss of positive charge, structural distortion and inactivation. Plasma methylglyoxal is increased in hyperglycaemia associated with diabetes and is linked to the development of vascular complications of diabetes – particularly nephropathy, retinopathy and neuropathy. The effects of dicarbonyl glycation on beta cells and involvement in early stage dysfunction and development of type 2 diabetes mellitus are not known. The aim of this project was to investigate the effect of dicarbonyl protein glycation on beta cell function and related involvement in the development of diabetes. Studies were performed in an in vitro model of beta cell dysfunction - MIN6 insulinoma cells incubated under low and high glucose concentrations, and in a pre-clinical in vivo model of decline of glucose tolerance preceding development of type 2 diabetes - high fat diet-induced insulin resistant mice. Dicarbonyl metabolism and protein damage by glycation and oxidation were studied by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry. Localisation of methylglyoxal glycation adducts within the pancreas were visualised by immunostaining. Interactions between the extracellular matrix protein, collagen IV, and MIN6 cells in vitro were investigated and impairments in adhesion were assessed following glycation with methylglyoxal. Impairments in adhesion of MIN6 cells to methylglyoxal-glycated collagen IV were assessed using atomic force microscopy force spectroscopy.
The results show that MIN6 cells were resistant to accumulation of methylglyoxal when incubated in high glucose concentration although the flux of methylglyoxal was increased 41%. Glycation of collagen IV by methylglyoxal impairs binding to MIN6 cells in vitro resulting in a 91% decrease in the energy necessary to detach cells from the extracellular matrix protein. In high fat diet fed mice the concentration of methylglyoxal in the pancreas was increased. Visualisation of MG-H1 adduct residues in the pancreas showed they were predominantly on the extracellular matrix.
In conclusion, protein glycation by methylglyoxal occurs in MIN6 cells in vitro and in the mouse pancreas in vivo. Although the methylglyoxal concentration in the pancreas of high fat diet fed, insulin resistant mice was increased, the lack of a concurrent increase in methylglyoxal protein glycation adducts suggests there may be increased turnover of methylglyoxal-modified proteins. Impairment of beta cell attachment to the extracellular matrix protein, collagen IV, by methylglyoxal and increased protein turnover stimulated by an increased rate of methylglyoxal
glycation may impair beta cell function in pre-diabetes in vivo. Glycation by methylglyoxal may contribute to beta cell glucotoxicity and dysfunction with progression to type 2 diabetes mellitus
