Browsing by Author "Fonseca, Beatriz Marques da"
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- Bioanalytical tools for unraveling part of the puzzle of the biochemical phenomena in epilepsy: neuromediators and antiepileptic drugsPublication . Fonseca, Beatriz Marques da; Alves, Gilberto LourençoThe earliest descriptions of epilepsy date back to 3000 years B.C., which was, throughout history, linked to divine factors, and only in the mid-nineteenth century it was widely accepted as a pathology originating in the brain. Thus, summarily, epilepsy is a disease of the brain involving recurrent unprovoked seizures, which arise due to an abnormal excessive or synchronous neuronal activity. Although the scientific knowledge has evolved tremendously over the last decades, particularly in the field of neurosciences, it is believed that we are still far from knowing in detail the neurobiochemical and molecular phenomena intrinsic to the complexity of the processes underlying this disease. In fact, a deeper understanding of the mechanisms that lead to the development of epilepsy (epileptogenesis) and those underlying the ictogenesis will be certainly favorable to find novel therapeutic approaches, capable of improving the suppression of seizures, particularly in patients who develop phenotypes of drug-resistant epilepsy, but also capable of modifying, and if possible interrupting, the cascade of molecular, structural and functional pathophysiologic changes that occur in brain during epileptogenesis. Given the scarcity of information on the neurobiological phenomena underlying the pharmacological response, as well as on the biochemical and molecular processes responsible for drug resistance and epileptogenesis, new studies are imperative to contribute to increase knowledge about these topics. The involvement of neuromediators in the initiation and spread of epileptic seizures is an increasingly recognized reality and the quantitative determination of these endogenous substances will be fundamental to improve our knowledge at neurobiochemical level, thus enabling a better understanding of the etiopathogenesis of epilepsy and the mechanisms of action of antiepileptic drugs as well. Therefore, in this context, bioanalysis will play a vital role in supporting and helping to interpret the results obtained from many research studies developed in this field. Consequently, the global aim of this doctoral project was to developed simple and reliable bioanalytical tools for the determination of a series of key neuromediators involved in the neurotransmission and neuronal excitability. In addition, the development of a novel bioanalytical tool to simultaneously quantify important antiepileptic drugs and some widely used chemoconvulsant agents was also considered. Thus, the research work supporting this doctoral thesis began with the development and full validation of an analytical technique of high-performance liquid chromatography coupled to fluorescence detection (HPLC-FLD) for the simultaneous quantification of several catecholamines and related endogenous compounds (i.e., dopamine, norepinephrine, epinephrine, homovanillic acid, levodopa and 3-O-methyldopa) in rat brain tissue. Posteriorly, another methodology using the same analytical system (HPLC-FLD) was also developed for the determination of five neuroactive amino acids (i.e., glutamate, aspartate, taurine, glutamine and gamma-aminobutyric acid) in rat brain tissue; however, in this case, a precolumn derivatization step was required because these amino acids do not have native fluorescence. Moreover, to perform more integrated analyses of the anticonvulsant and convulsant effects in nonclinical studies it will be essential the availability of bioanalytical tools that enable the simultaneous determination of the target antiepileptic drugs and convulsant agents. Therefore, within the scope of this thesis, it was also developed a liquid chromatography method coupled to diode array detection for the simultaneous determination, in plasma and rat brain samples, three established antiepileptics (levetiracetam, zonisamide and lamotrigine) and two important chemoconvulsant agents (pentylenetetrazole and pilocarpine) frequently used to experimentally induce acute seizures and/or chronic epilepsy in whole-animal models. This set of bioanalytical tools may allow the study of neurochemical changes that occur in several brain regions associated with epilepsy and/or epileptic seizures, and thus can help to understand multiple aspects still unclear in the field of neurosciences. Hence, the achievements obtained with the work presented in this doctoral thesis may provide quantitative support for future nonclinical studies aimed at deepening the knowledge about the neurobiochemical mechanisms underlying the epileptogenesis and ictogenesis phenomena, as well as the action of antiepileptic drugs and chemoconvulsant agents. Finally, this work represents a small but useful contribution to unravel part of the puzzle of the complex neurobiochemical mechanisms involved in epilepsy.
- Determinação de marcadores de exposição ao tabaco em amostras de fluido oral de indivíduos não fumadoresPublication . Fonseca, Beatriz Marques da; Alba, Maria Eugénia Gallardo; Calheiros, José Manuel Lage CampeloA exposição à poluição tabágica ambiental constitui um sério problema de saúde pública, já que os fumadores passivos estão expostos a mais de quatro mil substâncias tóxicas, entre as quais dezenas de cancerígenos e nicotina, principal agente responsável pelo desenvolvimento da dependência ao tabaco. Este alcalóide de ocorrência natural deriva das folhas de tabaco e é rápida e extensivamente metabolizado no fígado para formar uma larga variedade de metabolitos. A exposição crónica à nicotina, a droga mais consumida em todo o planeta, é responsável pela aparição de problemas de saúde como cancro, doenças do foro respiratório e neurológico, entre outros. Como problema de saúde pública, é uma prioridade conhecer a verdadeira prevalência de indivíduos expostos a este tipo de poluição. Assim o principal objectivo do presente estudo é a determinação de nicotina e os seus principais metabolitos, cotinina e trans-3- hidroxicotinina em amostras de fluido oral por cromatografia gasosa acoplada a espectrometria de massa em tandem, de forma a avaliar a exposição de não fumadores a estas substâncias. A metodologia proposta foi validada de acordo com critérios internacionalmente aceites para a validação de métodos bioanalíticos da Food and Drug Administration (FDA). Utilizando apenas 200 μL de amostra, o método demonstrou ser linear no intervalo de concentrações de 0,5 a 1000 ng/mL, exacto e preciso. Os limites de detecção e quantificação foram de 0,5 ng/mL e a eficiência de extracção foi superior a 85% para todos os compostos de estudo. Tendo em conta os resultados obtidos, podemos afirmar que a cromatografia gasosa acoplada a espectrometria de massa em tandem é uma poderosa e indispensável ferramenta que providencia uma elevada sensibilidade para a quantificação simultânea de biomarcadores de exposição ao fumo de tabaco num volume reduzido de fluido oral.