Research in the field of molecular neuroscience contributes a better perception of the events that trigger neurodegeneration. At the forefront of this work is the study of intracellular calcium as a consequence of mitochondrial dumping and NMDA receptor activation by glutamate. Increased intracellular calcium presages excitotoxicity with ultimate apoptosis of the cell. Among the many disorders involving this sequence is depression, a disorder that in and of itself is a risk factor for neurodegenerative disorders such as Alzheimer disease. The successful use of anti-depressants to alleviate the depressive state leads to the question about whether these pharmacological agents, as part of their effect to ameliorate depression, might have an effect on intracellular calcium. Until now, this has not been explored directly but such exploration was initiated with this thesis. As a model for astrocytes and neurons, two cell lines, C6 and SH-SY5Y were used. These were differentiated with all-trans retinoic acid into astrocyte-like and neuron-like cells. Graphic imaging of intracellular calcium by ratiometrics is not new, but what is new is using this technique to evaluate the effect of the antidepressants mirtazapine and citalopram on intracellular calcium fluxes induced by glutamate. Furthermore, comparing the ratiometric intracellular calcium flux in the presence of mirtazapine and citalopram to that of known NMDA blockers was also done for the first time. Also studied were the acute and chronic effects of mirtazapine and citalopram on cell viability. The antidepressant agents, mirtazapine and citalopram, were chosen for this study. Mirtazapine blocks the adrenergic and serotonergic inhibitory autoreceptors which results in the increased release of these neurotransmitters and increases their concentration in the synapse. And it also has been shown to have an anti-oxidant and a calcium modulatory effect. Citalopram has the highest degree of serotonin reuptake selectivity of all the selective serotonin reuptake inhibitors. The ratiometric studies found that mirtazapine and citalopram reduce the effect of glutamate-induced increase in relative [Ca2+]i by either a direct or indirect action on NMDA receptors. This effect is not similar to the NMDA blockers memantine and AP5. The supporting evidence is that CCCP, which normally releases calcium from mitochondria, has no effect in cells treated with acute mirtazapine or citalopram. This indicates that no calcium entered the cell – and subsequently none was taken up by mitochondria – in response to glutamate. However, these observations were with a limited number of cells and, therefore, these results will have to be verified by different techniques by different laboratories. In microplate studies, all drugs studied reduced cell viability but the mechanism behind this reduced viability remains to be determined. This may be due to mutations in enzymatic expression, uptake of drug through the cell membrane, or other perturbations. The reduction in cell viability induced by acute glutamate was attenuated by pretreatment with mirtazapine or citalopram. Moreover, chronic treatment of the cells with mirtazapine or citalopram for 10 weeks before acute treatment with glutamate either attenuated the effect on viability or reversed it. Based on this present study, mirtazapine and citalopram may be useful as neuroprotective agents to alleviate not only depression but also to reduce cell death in neurodegenerative diseases, trauma and stroke.

<p dir="ltr">Mycobacterium abscessus (Mab) is a type of mycobacterium that is known for its remarkable resistance to a variety of antibiotics. This pathogen poses a significant risk for individuals with weakened immune systems as it can cause skin and soft tissue infections, pulmonary disease and disseminated infections. Mab's ability to expel antibiotics through efflux pumps and form strong biofilms makes it even more challenging to treat infections. Lipids form a major part of the extracellular matrix of Mab biofilms. Efflux pumps have been shown to export lipids to the cell surface. Despite ongoing research into Mab's antibiotic tolerance, there is still much to learn about the impact of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis during biofilm development in Mab. In this study, we investigated the impact of the EPIs; CCCP (carbonyl cyanide m-chlorophenyl hydrazone), piperine (PIP), reserpine (RES), berberine (BER), and verapamil (VER) on efflux activity, biofilm formation, antibiotic resistance, and lipid biosynthesis in Mab during planktonic and biofilm growth conditions. We found that Mab cells had a higher tolerance to EPIs in biofilm-stimulating medium and that the presence of EPIs led to a decrease in minimum inhibitory concentrations of frontline antibiotics, reduced efflux activity within Mab cells, and significantly inhibited biofilm formation. We examined the effects of EPIs that inhibited biofilm formation on lipid metabolism in Mab using radiolabeling with 14C?palmitic acid and 14C-acetic acid which are precursors of lipid biosynthesis. We observed that the EPI berberine inhibited the incorporation of 14C-palmitic acid into glycopeptidolipids in the surface lipids of planktonic cells and increased cellular glycopeptidolipid (GPL) in biofilm cells. Verapamil-treated cells showed a 55 % increase in cellular trehalose monomycolate (TMM) compared to controls. Piperine-treated cells exhibited a 50 % increase in cardiolipin. The incorporation of 14C-acetate into biofilm cells showed that piperine-treated biofilm cells showed a 146 % increase in surface glycopeptidolipids. Overall, our study enhances our understanding of lipid biosynthesis in Mab, the effects of EPIs on Mab biofilms, efflux mechanisms, and antibiotic resistance and offers insights for combating Mab-related infections.</p>

O processo de isquemia-reperfusão é responsável pela geração de um dano agudo em uma série de órgãos do corpo humano, e, no coração, é o principal causador da doença crônica conhecida por insuficiências cardíaca. Atualmente não existe nenhuma opção terapêutica disponível na prática clínica contra esta injúria. Com o objetivo de desenvolver uma nova estratégia de combate a este dano, no presente trabalho investigamos a promessa da aplicação da recém-descoberta via UPRam num modelo de hipóxia reoxigenação in-vitro, sem obter sucesso. Contudo, os resultados gerados nestes experimentos forneceram pistas de que o uso do desacoplador CCCP é capaz de reduzir o dano deste insulto, porém o mecanismo celular responsável por esta proteção permanece desconhecido. Tentativas de desvendar este mecanismo utilizando a via lisossomal-autofágica ou a clivagem de OPA-1 falharam, mas produziram importantes insights a respeito do papel da protease mitocondrial OMA-1 no processo de hipóxia-reoxigenação, abrindo caminho para novos estudos subsequentes. / Ischemia-reperfusion injury is a process that occurs in many human organs including the heart, where it is the main trigger to heart failure, a chronic disease that kills over 40% patients only five years following the first diagnosis. Despite the bulky research on the subject, there is no available therapy on clinical practice against this insult. Attempting to develop a novel strategy to mitigate this damage, we investigated if the pro-survival effect of the recently discovered UPRam pathway could be protective in an in-vitro model of ischemia reperfusion. Despite the negative results regarding its conservation on mammalian cells, treatment with the mitochondrial uncoupler CCCP was proven to reduce cell death under this process, but the cellular mechanism responsible for this protection remained elusive. Aspiring to unravel this cellular response, we tested whether autophagy or OPA-1 cleavage was capable of abrogating the verified protection, but the results came back negative. Regardless of that, the behavior of OMA-/- cells over H/R stress has given new insights on novel strategies comprising I/R injury abrogation.

The master's thesis is focused on the study of response of the intracellular pH of the yeast cells on various external environments, primarily in a relation to the protonophore carbonyl cyanide m-chlorophenylhydrazone, CCCP. To measure the intracellular pH of the yeast cells we used a genetically coded fluorescent probe the ratiometric pHluorin. Using the method of synchronously scanned fluorescent spectra we were able to measure the intracellular pH of the cells with high precision. As a part of these experiments we also studied the influence of ionic strength of the cell suspensions buffers on the surface potential as well as the influence of the mineral salt KCl on the depolarization of the yeast membranes and cytosolic acidification induced by the protonophore CCCP. We examined the changes of cytosolic pH as such but we also used the measured pH as an indicator of the processes and the state of environment outside the cell. One of the most notable outcomes of this thesis is a new method of monitoring the value of the surface potential of the yeast cells by measuring the titration curves of cytosolic acidification induced by the protonophore CCCP.