Browsing by Author "Bravo, Joana"
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- 3D vs 2D Cell Cultures in the Evaluation of Radiobiological Effects of Exposition to Low Doses - Medical Imaging Levels - of Ionizing RadiationPublication . Costa, Pedro; Caires, Hugo; Lemos, Joana; Cunha, Lídia; Bravo, Joana; Bravo, Isabel; Silva, Regina; Summavielle, Teresa; Metello, Luís F.Pretending to develop advanced biological models to study biological effects of low doses of ionizing radiation and following the actual policies on Animal Sciences, based on 3 R’s Rule (to Reduce, Refine and Replace) – that limits as much as possible the application of animal models – scientific research using cellular models is constantly increasing. Nevertheless, the intrinsic limitations of actual cellular models quite often had been recognized on a significant number of papers pointing a significant number of non-concordances between results obtained using in vitro and in vivo studies. Actually, an increasing number of authors admit that three-dimensional cell culture (and spheroid cell culture in particular) could represent an interesting solution and a step further on use of cellular models. The work here to be presented reflects the first phase on the use of this methodology on the study, evaluation and quantification of cellular effects of low doses – starting on medical imaging level - of exposition to ionizing radiation.
- Acetyl-L-Carnitine Improves Cell BioenergeticsPublication . Cunha, Lídia; Bravo, Joana; Costa, Pedro; Fernandes, Sílvia; Oliveira, Marta; Castro, Rosa; Metello, Luís F.; Summavielle, TeresaIntroduction: Acetyl-L-Carnitine (ALC), a natural occurring compound in all mammalian species, plays a variety of vital functions in the body. The most important are related to mitochondria, namely the transport of fatty acids for energy production through β oxidation and the control of acyl-CoA/CoA ratio. Due to this close interaction with cell bioenergetics, it plays a role in many diseases, especially those related to the mitochondria. We propose to characterize the action of ALC in mitochondrial bioenergetics and functional integrity.
- ALC Neuroprotection through autophagy and ups acitivityPublication . Bravo, Joana; Cunha, Lídia; Fernandes, Sílvia; Binienda, Zbigniew; Summavielle, TeresaAcetyl–L-carnitine (ALC) has beneficial effects in neurodegenerative diseases and was shown to be protective against exposure to methamphetamine (METH) reducing mitochondrial dysfunction and oxidative stress. However, the mechanisms underlying ALC action are still unknown, limiting its putative therapeutic use.
- Astrocyte-derived TNF and glutamate critically modulate microglia activation by methamphetaminePublication . Canedo, Teresa; Portugal, Camila Cabral; Socodato, Renato; Almeida, Tiago Oliveira; Terceiro, Ana Filipa; Bravo, Joana; Silva, Ana Isabel; Magalhães, João Duarte; Guerra-Gomes, Sónia; Oliveira, João Filipe; Sousa, Nuno; Magalhães, Ana; Relvas, João Bettencourt; Summavielle, TeresaMethamphetamine (Meth) is a powerful illicit psychostimulant, widely used for recreational purposes. Besides disrupting the monoaminergic system and promoting oxidative brain damage, Meth also causes neuroinflammation, contributing to synaptic dysfunction and behavioral deficits. Aberrant activation of microglia, the largest myeloid cell population in the brain, is a common feature in neurological disorders triggered by neuroinflammation. In this study, we investigated the mechanisms underlying the aberrant activation of microglia elicited by Meth in the adult mouse brain. We found that binge Meth exposure caused microgliosis and disrupted risk assessment behavior (a feature that usually occurs in individuals who abuse Meth), both of which required astrocyte-to-microglia crosstalk. Mechanistically, Meth triggered a detrimental increase of glutamate exocytosis from astrocytes (in a process dependent on TNF production and calcium mobilization), promoting microglial expansion and reactivity. Ablating TNF production, or suppressing astrocytic calcium mobilization, prevented Meth-elicited microglia reactivity and re-established risk assessment behavior as tested by elevated plus maze (EPM). Overall, our data indicate that glial crosstalk is critical to relay alterations caused by acute Meth exposure.
- Evaluation of 99mTc-Sestamibi as a potential tool to investigate PgP activity in inflammationPublication . Costa, Pedro; Cunha, Lídia; Bravo, Joana; Alves, Cecília J.; Summavielle, Teresa; Metello, Luís F.In the XXI Century’s Society the scientific investigation process has been rowing steadily, and the field of the pharmaceutical research is one of the most enthusiastic and relevant. Here, it is very important to correlate bserved functional alterations with possibly modified drug bio distribution patterns. Cancer, inflammation and infection are processes that induce many olecular intermediates like cytokines, chemokines and other chemical complexes that an alter the pharmacokinetics of many drugs. One cause of such changes is hought to be the modulator action of these complexes in the P-Glycoprotein activity, because they can act like inducers/inhibitors of MDR-1 expression. This protein results from the expression of MDR-1 gene, and acts as an ATP energy-dependent efflux pump, withtheir substrates including many drugs, like antiretrovirals, anticancers, anti-infectives, immunosuppressants, steroids or opioids. Because of the lack of methods to provide helpful information in he investigation of in vivo molecular changes in Pgp activity during fection/infl ammation processes, and its value in the explanation of the altered drug harmacokinetic, this paper want to evaluate the potential utility of 99m Tc-Sestamibi scintigraphy during this kind of health sciences investigation. Although the aim is indeed to create a technique to the in vivo study of Pgp activity, this preliminary Project only reaches the in vitro study phase, assumed as the first step in a n evaluation period for a new tool development. Materials and Methods: For that reason , we are performing in vitro studies of influx and efflux of 99m Tc - Sestamibi ( that is a substrate of Pgp) in hepatocytes cell line (HepG2). We are interested in clarify the cellular behavior of this radiopharmaceutical in Lipopolysaccharide(LPS) stimulated cells ( well known in vitro model of inflammation) to possibly approve this methodology. To validate the results, the Pgp expression will be finally evaluated using Western Blot technique. Results: Up to this moment , we still don’t have the final results, but we have already enough data to let us believe that LPS stimulation induce a downregulation of MDR - 1, and consequently Pgp, which could conduce to a prolonged retention of 99m Tc - Sestamibi in the inflamed cells . Conclusions: If and when this methodology demonstrate the promising results we expect, one will be able to con clude that Nuclear Medicine is an important tool to help evidence based research also on this specific field .
- Exploring cinnamic acid scaffold: development of promising neuroprotective lipophilic antioxidantsPublication . Chavarria, Daniel; Silva, Tiago; Martins, Daniel; Bravo, Joana; Summavielle, Teresa; Garrido, Jorge; Borges, FernandaNew lipophilic hydroxycinnamic acid based derivatives were designed and synthesized and their antioxidant and neuroprotective activities evaluated. The chemical modification introduced in the cinnamic acid scaffold leads to compounds with amplified lipophilicity and in general with increased antioxidant activity when compared to natural models (caffeic and ferulic acids). The compounds did not display cytotoxicity and present a significant neuroprotective effect against 6-OH-DA induced damage to SH-SY5Y cells. Compound 6 stands out as an efficient radical scavenger and iron(II) chelator that ensures drug-like properties. Moreover, neuroprotection against oxidative damage was observed even at low concentration (1 μM). Therefore, compound 6 developed by a biology-oriented approach displays a combination of important features for a further optimization process that will generate a new effective antioxidant with therapeutic application for oxidative-stress-related events, namely neurodegenerative diseases.
- Mitochondrial function and acetyl-L-carnitine neuroprotectionPublication . Cunha, Lídia; Bravo, Joana; Gonçalves, Raquel; Metello, Luís F.; Rodrigues, Adriano; Summavielle, TeresaMitochondrial dysfunction is involved in a number of chronic neurodegenerative disorders. Acetyl-L-carnitine (ALC) has been proposed to confer effective neuroprotection by increasing mitochondria viability, but little is known regarding the molecular mechanisms involved in its action
- A mouse model reproducing the pathophysiology of neonatal group B streptococcal infectionPublication . Bonifácio Andrade, Elva; Magalhães, Ana; Puga, Ana; Costa, Madalena; Bravo, Joana; Portugal, Camila Cabral; Ribeiro, Adília; Correia-Neves, Margarida; Faustino, Augusto; Firon, Arnaud; Trieu-Cuot, Patrick; Summavielle, Teresa; Ferreira, PaulaGroup B streptococcal (GBS) meningitis remains a devastating disease. The absence of an animal model reproducing the natural infectious process has limited our understanding of the disease and, consequently, delayed the development of effective treatments. We describe here a mouse model in which bacteria are transmitted to the offspring from vaginally colonised pregnant females, the natural route of infection. We show that GBS strain BM110, belonging to the CC17 clonal complex, is more virulent in this vertical transmission model than the isogenic mutant BM110∆cylE, which is deprived of hemolysin/cytolysin. Pups exposed to the more virulent strain exhibit higher mortality rates and lung inflammation than those exposed to the attenuated strain. Moreover, pups that survive to BM110 infection present neurological developmental disability, revealed by impaired learning performance and memory in adulthood. The use of this new mouse model, that reproduces key steps of GBS infection in newborns, will promote a better understanding of the physiopathology of GBS-induced meningitis.
- Neuron–microglia contact-dependent mechanisms attenuate methamphetamine-induced microglia reactivity and enhance neuronal plasticityPublication . Bravo, Joana; Ribeiro, Inês; Terceiro, Ana Filipa; Andrade, Elva B.; Portugal, Camila Cabral; Lopes, Igor M.; Azevedo, Maria M.; Sousa, Mafalda; Lopes, Cátia D. F.; Lobo, Andrea C.; Canedo, Teresa; Relvas, João Bettencourt; Summavielle, TeresaExposure to methamphetamine (Meth) has been classically associated with damage to neuronal terminals. However, it is now becoming clear that addiction may also result from the interplay between glial cells and neurons. Recently, we demonstrated that binge Meth administration promotes microgliosis and microglia pro-inflammation via astrocytic glutamate release in a TNF/IP3R2-Ca2+-dependent manner. Here, we investigated the contribution of neuronal cells to this process. As the crosstalk between microglia and neurons may occur by contact-dependent and/or contact-independent mechanisms, we developed co-cultures of primary neurons and microglia in microfluidic devices to investigate how their interaction affects Meth-induced microglia activation. Our results show that neurons exposed to Meth do not activate microglia in a cell-autonomous way but require astrocyte mediation. Importantly, we found that neurons can partially prevent Meth-induced microglia activation via astrocytes, which seems to be achieved by increasing arginase 1 expression and strengthening the CD200/CD200r pathway. We also observed an increase in synaptic individual area, as determined by co-localization of pre- and post-synaptic markers. The present study provides evidence that contact-dependent mechanisms between neurons and microglia can attenuate pro-inflammatory events such as Meth-induced microglia activation.
- Regulation of CD47 expression by interferon-gamma under chronic Methamphetamine exposure TitlePublication . Rodrigues, João; Bravo, Joana; Bonifácio Andrade, Elva; Canedo, Teresa; Azevedo, Maria; Summavielle, TeresaExposure to methamphetamine (Meth), a highly addictive widely used psychostimulant, is classically associated with damage to neuronal terminals, but its neurotoxicity can also be mediated via activation of the neuroinflammatory response. Microglia, the resident immune cells of the brain, become highly activated and increase the release of proinflammatory mediators upon exposure to Meth. However, their role in Meth-associated neurotoxicity is still not sufficiently understood. Data from our lab shows that, in the hippocampus, chronic Meth administration leads to microglia homeostasis dysregulation, synapse dysregulation, and downregulation of cluster-differentiation 47 protein (CD47). The crosstalk between CD47 and its receptor, signal regulatory protein α (SIRPα), is an important “don’t eat me signal” that inhibits phagocytosis. CD47 has been shown to protect synapses from excessive microglia-mediated pruning during development and neurodegeneration. Of note, in cancer cells CD47 expression is modulated by interferon-gamma (IFN-γ). Consistently, after chronic Meth, we observed a significant decrease of meningeal T cells, and a decrease in the production of IFN-y by these cells. Here we aim to clarify if IFNγ is regulating CD47 in the brain after chronic Meth administration, and consequently regulating synaptic pruning, using IFNyKO mice and wild-type mice injected with recombinant IFNy via stereotaxic surgery. Preliminary results indicate that IFNy/CD47 does not modulate microglia morphology and number after chronic Meth in the hippocampus. Currently we are evaluating synapses and phagocytosis, and we further expect to clarify the impact of IFNγ /CD47 in the chronic Meth conditioning and in memory.