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Computational biophysics evaluation of promising smart metallodrug delivery systems
No full textTese de mestrado, Bioquímica e Biomedicina , 2023, Universidade de Lisboa, Faculdade de CiênciasTriple-negative breast cancer is an aggressive subtype of breast cancer, lacking hormonal and human epidermal growth factor receptors, that render current targeted therapies used for other subtypes
ineffective. Thus, treatment options are currently limited to classical chemotherapy drugs, such as cisplatin, that have known devastating side effects, while lacking selectivity. The current work focuses on
the study of ruthenium-based compounds, that have shown promising anti-tumor capabilities, with increased selectivity for the tumor microenvironment. We studied three derivatives of TM34, substituted
with a pH-sensitive linker based on hydrazone, and a peptide targeting triple-negative breast cancer cells.
Their active species are substituted with a hydrazide group in the cyclopentadienyl coligand, or an acetyl
group in the cyclopentadienyl or bipyridine coligands. The main goal of this work is to evaluate the
impact of said derivations on the compound’s biophysical properties when interacting with a membrane
model.
By performing unrestrained Molecular Dynamics simulations, we studied the preferential partitioning region and orientation of the TM34 derivatives into the membrane phase, identifying their respective
preferred insertion depth (∼1.3 nm from the membrane center), and their preference towards having the
triphenylphosphine group oriented along the membrane normal vector and facing the membrane interior.
Additionally, we performed Umbrella Sampling simulations, which allowed us to study the insertion
profile of the compounds across the membrane, as well as their permeability coefficients. Despite being
cations, all compounds had a strong preference towards the membrane phase, with an energy minimum
below the average phosphate group position of the lipids, and permeability coefficient values comparable to those of other drugs currently used. Overall, we concluded that the acetyl substitution in the
cyclopentadienyl ligands has significantly less impact on the compound’s membrane partitioning than
the substitution with a hydrazine group, which we show introduces a higher desolvation penalty upon
insertion
Development of a new hybrid therapeutic agent against malaria by targeting the Aquaglyceroporin from plasmodium spp
No full textTese de mestrado, Bioinformática e Biologia Computacional , 2023, Universidade de Lisboa, Faculdade de CiênciasDespite the availability of antimalarial drugs, malaria is one of the largest public health problems. Severe malaria, mostly caused by Plasmodium (P.) falciparum, can lead to anemia, end-organ damage,
pulmonary issues, and hypoglycemia. To tackle resistant parasite strains, hybrid antimalarial agents are
being pursued. The aquaporin of P. falciparum (PfAQP), a water and glycerol membrane protein channel, is a promising therapeutic target for new antimalarial therapies due to its role in the fast reproduction
of the parasite. In this thesis, we have developed a bioinformatics approach focused on the identification
of PfAQP structural features regulating the permeation of water, glycerol, erythritol, and xylitol, to boost
the development of hybrid therapeutical agents that block or transport available antimalarial drugs to P.
falciparum medium. Using Molecular Dynamics simulations, we have started by identifying the most
adequate simulation model based on the analysis the stability of the protein using different membrane
sizes. We then performed Umbrella Sampling simulations and used the Inhomogeneous Solubility Diffusion Model to calculate the free energy profile and the permeability coefficients of water and glycerol,
along with erythritol, xylitol, and glycerol derivatives. Our results are in agreement with the available
experimental results that indicate that PfAQP, as a bi-functional aquaglyceroporin, shows similar permeability coefficients for both water and glycerol. Additionally, erythritol showed the highest energy
barriers close to the selectivity filter, and consequently, the lowest permeability coefficients, making this
compound a promising blocker of PfAQP. Regarding xylitol, it showed the higher permeability coefficient, indicating its promising carrier characteristics for an antimalarial drug. In conclusion, our results
demonstrate a high correlation between the permeation barriers at the selectivity filter and the permeability coefficients, which can be taken into account in the future development of a hybrid new antimalarial
therapeutics
Regulation of adenosine levels as a new therapeutic strategy for Rett Syndrome
No full textRett Syndrome (RTT) is a rare, genetically caused neurodevelopmental disorder that affects approximately 1:10000-15000 girls worldwide. It is characterized by an apparently normal development up to 6 to 18 months of age, followed by a regression phase, in which there is a loss of acquired abilities. During the progression of this disease, which takes place over four distinct stages, the following stand out: appearance of stereotyped and repetitive hand movements with progressive loss of functionality, cognitive and motor dysfunction and epilepsy. Currently, this disease has no cure and there are few therapeutic options for symptomatic control, which makes this disease devastating for both patients and caregivers.
Genetic studies carried out in recent years have established that this syndrome is mainly due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene, located on the X chromosome. This gene encodes the MeCP2 protein, which performs multiple functions where its role stands out as an epigenetic modulator and regulator of the structure of chromatin, controlling the expression of several other genes, making it a key protein in the development and maturation of the central nervous system (CNS). One of the proteins whose expression is controlled by MeCP2 is the brain-derived neurotrophic factor (BDNF), a neurotrophin with essential functions in cell maturation and differentiation, synaptic plasticity and neuronal survival. Consequently, alterations in MeCP2 compromise BDNF expression levels and function, and this evidence has already been demonstrated in multiple studies in RTT animal models.
Those studies have also shown that the increase in BDNF expression can reverse some of the dysfunctions and symptoms present in RTT animal models. However, the therapeutic use of BDNF is not yet applicable since the blood-brain barrier (BBB) is impervious to this neurotrophic factor, preventing it from reaching the brain and performing its functions properly. In an attempt to facilitate the effects of BDNF, new strategies have been developed involving, for example, the use of molecules that cross the BBB and potentiate the neuroprotective action of BDNF. One of the molecules that has deserved particular attention is adenosine. Adenosine is a CNS neuromodulator that exerts its functions through the activation of four receptors, A1, A2A, A3 and A2B (AR). In particular, the activation of A2AR is crucial for the maintenance of BDNF and its receptor, TrkB-FL (full-length tropomyosin-related kinase B), levels as well as for its synaptic effects. It is noteworthy that the adenosinergic system, in addition to be crucial in BDNF-mediated signaling, also has a prominent role in the control of synaptic excitability through the activation of inhibitory A1 receptors, recognized as potential therapeutic targets in the control of epilepsy. These actions of adenosine, as well as the presence of some symptoms in patients with RTT overlapping with diseases with adenosinergic dysfunction already described, suggest the possibility that this neuromodulator is also affected in RTT.
Thus, this project aimed to: 1) characterize in detail the adenosinergic system and BDNF-mediated signaling, through the use of models with distinct phenotypes: 1.1) animal model with an severe phenotype, Mecp2-null mutant male mice (Mecp2-/ y); 1.2) animal model with a moderate phenotype, female mice heterozygous for Mecp2 (Mecp2+/-); and 2) explore the augmentation of adenosine levels as a possible therapeutic strategy.
Through Western-Blot (WB) assays it was possible to detect decreased BDNF protein levels in hippocampus, cortex, brainstem, and cerebellum homogenates obtained from Mecp2-/y symptomatic animals. The changes detected in the pre-symptomatic stage were less marked, with only a decrease in BDNF levels observed in the striatum of 3-week-old Mecp2-/y animals. Also, in Mecp2+/- animals, decreases in BDNF levels were detected both in the cortex and in the hippocampus in the symptomatic stage. Regarding protein levels of BDNF receptors, decreases in TrkB-FL were observed in the symptomatic stage in cortical and hippocampal homogenates, and in the pre-symptomatic phase in the cortex and striatum. In this brain area, an increase in TrkB-FL levels was also observed in the symptomatic phase. Analyzing the protein levels of truncated isoforms of the TrkB receptor (TrkB-Tc), negative modulators of BDNF action, an increase were detected in brainstem homogenates from 1-week and 6-week-old animals. In Mecp2+/- animals, no alterations were detected in any of the studied BDNF receptors.
Electrophysiological recordings performed in the hippocampus allowed the study of synaptic plasticity, namely through the study of long-term potentiation (LTP). In Mecp2-/y animals there was a decrease in the magnitude of LTP and an absence of the facilitatory effect of BDNF upon LTP. In Mecp2+/- animals, although the basal magnitude of LTP was not affected, BDNF also lost its ability to potentiate this phenomenon associated with synaptic plasticity.
The study of the adenosinergic system, analyzed by high performance liquid chromatography (HPLC), allowed to detect a decrease in the levels of adenosine and its precursor adenosine monophosphate (AMP), both in hippocampal and cortical homogenates of Mecp2-/y animals in the symptomatic stage. Simultaneously, increases in protein levels of A1R in the cortex and hippocampus and a decrease in A2AR in the cortex were found. The same changes in adenosine receptors were found in Mecp2+/- animals, but also a decrease in A2AR levels in the hippocampus. Despite the decrease in BDNF levels, in both models, it was possible to recover the facilitatory effect of BDNF upon the magnitude of hippocampal LTP, through the activation of A2AR by the selective agonist CGS21680.
These data supported the hypothesis that a therapy targeting the adenosinergic system could be beneficial. Thus, 5-6 weeks old Mecp2-/y animals were administered intraperitoneally with an adenosine kinase (ADK) inhibitor drug, 5-iodotubercidin (ITU). This drug allows an increase in adenosine levels by inhibiting its metabolism. In addition to the efficacy of ADK inhibition already demonstrated in other diseases, such as epilepsy models, in Mecp2-/y animals, by WB analysis, increased ADK protein levels were detected in cortex homogenates during the pre-symptomatic stage, supporting the study of ADK as a potential therapeutic target in RTT.
Through the study of ITU administration, carried out in vivo, it was possible to observe a recovery of the effect of BDNF upon LTP potentiation, in electrophysiological recordings performed in hippocampal slices, as well as a recovery of protein levels of TrkB-FL receptors in hippocampal homogenates from ITU-treated Mecp2-/y animals.
Overall, the results point to a dysfunction either in signaling mediated by BDNF and in adenosinergic system, in two different phenotypes of the disease, suggesting a possible involvement of both in the pathophysiology of the disease. The positive data obtained regarding the reversal of some deficits present in the animal models studied, through the pharmacological inhibition of ADK, reinforces the importance of adenosinergic system involvement while suggesting the increase of adenosine levels as a strategy to be explored in RTT.Universidade de Lisboa (BD2016
Evaluation of the morphological and functional properties of erythrocytes in patients with amyotrophic lateral sclerosis
No full textTese de mestrado, Neurociências, Universidade de Lisboa, Faculdade de Medicina, 2022A Esclerose Lateral Amiotrófica (ELA) é uma doença neurodegenerativa, fatal e devastadora que se caracteriza pela degeneração progressiva dos neurónios motores situados na espinhal medula, tronco cerebral e córtex motor. A maioria dos doentes com ELA tem entre os 40 e os 60 anos de idade à data do seu diagnóstico. Após o diagnóstico da doença, a esperança média de vida de um doente com ELA é de aproximadamente 3 a 5 anos. A ELA é uma doença complexa devido à sua heterogeneidade e à sua fisiopatologia podendo ser alterada por fatores biológicos. Cerca de 90% dos casos de ELA são considerados esporádicos podendo ter origem, por exemplo, em fatores ambientais ou em disfunções relacionadas com o envelhecimento. Os restantes 10% dos casos de ELA têm um histórico familiar. Relativamente ao fenótipo da doença, a maioria dos casos diagnosticados são considerados de origem espinhal (cerca de 70%). Porém, também existem mais dois fenótipos de ELA menos comuns, o bulbar (representando cerca de 25% dos casos) e o respiratório (cerca de 3%). Atualmente, o diagnóstico é efetuado com base nos critérios El Escorial Revistos, em conjunto com a eletromiografia (Critérios Awaji) para confirmar a extensão da desenervação. Também são realizados testes laboratoriais e exames de imagem para distinguir os doentes ELA de doentes com outras doenças que se possam assemelhar a ELA. Existe também um questionário utilizado a nível mundial, onde a soma das pontuações das respostas origina o valor da escala revista de avaliação funcional da doença (ALSFRS-R). Esta escala visa avaliar o estado funcional global e respiratório do doente e o declínio da doença ao longo do tempo. A escala ALSFRS-R global avalia 12 parâmetros no doente, como por exemplo, a sua função física, a capacidade de engolir, de utilizar utensílios, de subir escadas e a sua função respiratória. O questionário também permite a avaliação da dispneia, ortopneia e a necessidade de suporte ventilatório no doente.
A disfunção respiratória é um fator determinante do comprometimento funcional e morte na ELA. A hipoxia aparece após o enfraquecimento dos músculos respiratórios.
Alterações na estrutura e função dos eritrócitos no fluxo sanguíneo nunca foram descritos na ELA, embora sejam relevantes para uma adequada oxigenação dos tecidos. Existem evidências de que a inflamação está associada à ELA, possivelmente aumentada pelo desconforto respiratório nos doentes. Este fato poderá estar relacionado com o aumento do fibrinogénio plasmático na circulação sanguínea. Níveis aumentados de fibrinogénio plasmático têm sido associados ao aumento da agregação eritrocitária, podendo ter um papel central nos mecanismos que originam eventos trombóticos. A inter-relação entre insuficiência respiratória, inflamação, alterações do fibrinogénio plasmático e da morfologia do eritrócito, com implicações na perfusão periférica, nunca foi explorada na ELA.
O tromboembolismo venoso (TEV) é uma condição médica responsável pela formação de coágulos sanguíneos nas veias. Eventos de TEV foram reportados em alguns casos de doentes com ELA, geralmente associados a fatores como a falta de mobilidade, o envelhecimento e também a insuficiência respiratória. No entanto, alguns fatores de risco associados a TEV ainda não foram devidamente explorados nesta doença.
A formação de coágulos sanguíneos pode dever-se a alterações que ocorrem ao nível da membrana dos eritrócitos (p.ex. alterações da sua elasticidade) que dependem (entre outros fatores) do seu conteúdo lipídico. Em condições inflamatórias, o fibrinogénio ’ (uma variante in vivo do fibrinogénio) está presente no plasma em elevadas concentrações, o que representa um aumento da sua percentagem em relação aos valores totais de fibrinogénio plasmático. Esta variante do fibrinogénio, o fibrinogénio ’, conduz a alterações na arquitetura e na formação de coágulos sanguíneos sendo estes mais resistentes à fibrinólise. Assim, os principais objetivos desta tese consistem em avaliar as alterações morfológicas, biomecânicas e as propriedades biofísicas dos eritrócitos em doentes com ELA. Adicionalmente pretende-se quantificar os níveis de fibrinogénio ’ no plasma sanguíneo, com o intuito de perceber se este marcador de risco inflamatório está alterado na ELA.
No desenvolvimento do trabalho experimental, foram colhidas amostras humanas de sangue provenientes de doentes com ELA que foram analisadas e posteriormente comparadas com um grupo de dadores saudáveis e dadores não saudáveis com outras doenças neurológicas. Numa primeira fase, foram obtidos os hemogramas de todos os pacientes e dadores que participaram neste estudo, e foi efetuada uma avaliação clínica dos dadores não saudáveis e dos doentes com ELA.
As amostras de sangue colhidas foram posteriormente analisadas recorrendo à microscopia de força atómica (AFM) onde foram efetuadas imagens dos eritrócitos, obtidas curvas de elasticidade destas células e foi ainda medida a profundidade a que a ponta do AFM penetra no eritrócito. Com as imagens de AFM foi possível estudar individualmente as características morfológicas do eritrócito nos diferentes grupos de estudo, tais como área, volume, altura, percentagem de eritrócitos sem concavidade, assim como a sua rugosidade de membrana.
Para complementar a análise dos dados obtidos no AFM, foram efetuadas medições do potencial zeta da membrana dos eritrócitos na ausência e na presença de diferentes concentrações de fibrinogénio (0, 0.4, 1.0, 2.0 mg/ml). Foi também realizada a quantificação dos níveis de fibrinogénio ’ no plasma sanguíneo dos diferentes grupos de estudo através de um ensaio imunoenzimático.
Os resultados obtidos sugerem que os eritrócitos de doentes com ELA têm uma área maior e apresentam menor concavidade por comparação com os eritrócitos de dadores saudáveis. A partir das medições do potencial zeta, foi possível verificar que as membranas dos eritrócitos de doentes com ELA apresentam uma carga menos negativa do que o respetivo controlo saudável na presença de elevadas concentrações de fibrinogénio. Para além disso, as membranas dos eritrócitos de doentes com ELA apresentaram um perfil menos rugoso que as dos eritrócitos de controlos saudáveis. Adicionalmente, a profundidade a que a ponta do AFM consegue penetrar é menor em células provenientes de doentes com ELA comparativamente com controlos, podendo se concluir que os eritrócitos de doentes com ELA têm maior rigidez do que os controlos. Por fim, os pacientes com ELA apresentaram concentrações mais elevadas da variante fibrinogénio ’ plasmático em comparação a controlos saudáveis e não saudáveis.
Ao longo deste estudo foram também efetuadas análises de regressão múltipla e análise longitudinal aos parâmetros estudados. A análise de regressão múltipla permite comparar os três grupos para diferentes variáveis de estudo e perceber se os resultados obtidos são ou não influenciados por outras variáveis independentes, tal como a idade e o género. Por outro lado, a análise longitudinal, permitiu fornecer informação relativamente à progressão e agravamento da doença em pacientes com ELA. Os resultados obtidos após a realização da análise longitudinal, revelaram que existem correlações negativas entre a escala ALSFRS-R global e/ou respiratória com a rugosidade dos eritrócitos, a profundidade de penetração nestas células e uma correlação positiva com os níveis plasmáticos de fibrinogénio ’.
Os resultados sugerem que os eritrócitos de pacientes com ELA apresentam alterações eletrostáticas, biomecânicas e morfológicas quando comparados com os controlos saudáveis. Os resultados apontam para um maior risco de inflamação na ELA. Este processo inflamatório parece estar associado a um efeito retardador na progressão da doença.
Estes resultados podem ainda ajudar a compreender, não só o papel do fibrinogénio na agregação eritrocitária, como também as alterações podem ocorrer na membrana dos eritrócitos que comprometem o seu normal funcionamento em doentes com ELA. As alterações
morfológicas dos eritrócitos na ELA poderão contribuir para o início ou agravamento de eventos trombóticos e inflamatórios. O risco de tais eventos trombóticos representa um agravamento do estado de saúde dos indivíduos com ELA que poderá ter impacto na sua mortalidade.
Este estudo foi realizado com um número restrito de doentes. Novos estudos deverão ser efetuados com um grupo mais alargado de doentes de modo a validar os resultados obtidos ao longo desta tese. No entanto, tanto a concentração de fibrinogénio ' plasmático como a rugosidade da superfície da membrana eritrocitária representam dois possíveis biomarcadores da progressão da doença de ELA. Níveis elevados de fibrinogénio ' podem estar associados ao desenvolvimento de processos inflamatórios na ELA. Consequentemente, este processo inflamatório parece estar associado a um processo de diminuição da progressão da doença de ELA (diminuição do declínio da escala funcional global e respiratória).Amyotrophic Lateral Sclerosis (ALS) is a rapidly progressive and fatal neurological disease. Erythrocyte structure and function abnormalities and their role on blood flow never have been described in ALS, although relevant for proper tissue oxygenation. There is large evidence that inflammation is associated with ALS, possibly increased by respiratory distress. This is potentially related to fibrinogen increase in blood circulation. Increased levels of plasma fibrinogen have been associated with the increase of erythrocyte aggregation which may have a central role on the mechanism of thrombosis. The complex interplay between respiratory insufficiency, inflammation, fibrinogen changes and abnormal erythrocyte structure, with implications in peripheral perfusion, has been never explored in ALS.
The main purpose of our study was to evaluate changes in morphological, biomechanical, and biophysical properties of erythrocytes from ALS patients and to quantify ’ fibrinogen plasma levels to evaluate if this marker of inflammation is altered in ALS.
Blood samples from ALS patients were analysed and compared with healthy donors and non-healthy donors with other neurological diseases. Samples were examined by Atomic Force Microscopy (AFM) to evaluate the changes in morphology and elasticity of erythrocytes, zeta-potential analysis, and quantification of ’ fibrinogen plasma levels.
Results showed that erythrocytes from ALS patients have higher area, are less negatively charged and smoother than healthy controls. Also, they presented higher ’ fibrinogen concentrations than controls. A multiple-regression analysis and longitudinal assessment showed negative correlations between ALSFRS global and/or respiratory scores and erythrocyte roughness, erythrocyte penetration depth; and positive correlation with ́ ́fibrinogen plasma levels.
The results suggest that erythrocytes from ALS patients present electrostatic, biomechanical, and morphologic changes when compared with healthy control. The results also pointed to a higher risk of inflammation in ALS. This inflammation process seems to be associated to a retarding process in the progression of ALS disease
The works of homemaking: migration, domestic materiality, and everyday life
No full textWhat are the potentialities and limitations of an ethnographic approach to the material aspects
of contemporary migrations? What does the study of domestic materiality and homemaking
in contexts of migration reveal about movement and its outcomes? By addressing these two
questions, this chapter aims to contribute to further consolidating the study of material culture
and consumption practices in migration contexts and discuss the empirical advantages of tackling
contemporary circulations of people through a focus on things, in general, and on things
from home, in particular.info:eu-repo/semantics/publishedVersio
Characterization of the metabolic profile of differentiated cells derived from subventricular zone neural stem cells
No full textTese de mestrado, Investigação Biomédica, Universidade de Lisboa, Faculdade de Medicina, 2022As células estaminais neurais (CENs) são células auto-renováveis e multipotentes, tendo deste modo a capacidade de proliferarem e de se diferenciarem em astrócitos, oligodendrócitos e neurónios. No cérebro adulto de mamíferos existem locais específicos onde as CENs se localizam, chamados, nichos neurogénicos. Estes nichos são, a zona subgranular no giro denteado do hipocampo e a zona subventricular (ZSV) na parede dos Ventrículos Laterais. A formação de novos neurónios (neurogénese) na fase adulta está bem estabelecida e caracterizada em modelos animais como em roedores. No entanto, embora a existência de neurogénese adulta em humanos já tenha sido demonstrada em diversos estudos, este tema ainda é alvo de grande debate na comunidade científica.
A mitocôndria é um organelo que tem várias propriedades, tais como: bioenergética, responsável pela produção de ATP; dinâmica mitocondrial, que reflete os ciclos de fusão e fissão da mitocôndria; biogénese, que é a capacidade de formar novas mitocôndrias; e mitofagia, que mantém a qualidade mitocondrial por um processo de eliminação de mitocôndrias danificadas. Apesar da mitocôndria ser maioritariamente conhecida pelas suas capacidades energéticas, também é muito relevante na regulação de vias de sinalização celulares. Este organelo, tem vindo a revelar-se como fundamental para processos de regulação das CENs, tanto para a proliferação, como para a diferenciação. Vários estudos têm vindo a realçar a importância da mitocôndria para a regulação do processo de decisão da linhagem em que as CENs se vão comprometer. Mais concretamente, resultados previamente obtidos no laboratório mostraram que não só a morfologia mitocondrial é diferente após a diferenciação, como também varia entre linhagens, sugerindo uma adaptação mitocondrial dependente da linhagem celular. Deste modo, foi colocada a hipótese de que outras propriedades mitocondriais, tais como a bioenergética, poderiam alterar aquando da diferenciação e de forma especifica à linhagem. Embora ainda se desconheça o potencial papel da bioenergética na decisão da linhagem das CENs, estudos já reportaram que CENs são inicialmente mais dependentes da glicólise, e com a diferenciação em neurónios, as células ficam mais dependentes da fosforilação oxidativa. No entanto, os astrócitos e os oligodendrócitos não têm sido alvo de muito estudos, faltando informação relevante, particularmente alusiva a estes tipos celulares. Assim, o objetivo deste projeto foi caracterizar o perfil metabólico das diferentes células diferenciadas a partir das CENs da ZSV. Para atingir este objetivo, foram utilizadas culturas primárias de neuroesferas da ZSV de murganhos C57BL/6 ao dia pós-natal 1-3. Foram efetuadas duas passagens às neuroesferas, para aumentar o rendimento celular. Para promover a diferenciação das células das neuroesferas terciárias, estas foram dissociadas e plaqueadas num substrato e foi usado um meio com metade da concentração dos fatores de crescimento. Após 96h em meio com baixa concentração de fatores de crescimento, as células diferenciadas foram separadas através de Magnetic-Activated Cell Sorting, obtendo frações enriquecidas em astrócitos, oligodendrócitos e neurónios separadamente. Vários controlos com as células não separadas foram considerados, tais como, um controlo de células em meio de baixa concentração de fatores de crescimento durante 24h, que foi o controlo da diferenciação; um controlo com células em meio de baixa concentração de fatores de crescimento durante 96h, que representa a mistura heterogénia de células quando estas já se encontram diferenciadas, e outro controlo nos mesmos tempos nos quais as células separadas foram analisadas (96h+3/4days). Todas as condições foram analisadas por imunocitoquímica e por testes respiratórios e de conteúdo de ATP. A imunocitoquímica foi utilizada para confirmar se cada condição continha os tipos celulares pretendidos. Por exemplo, foi usada para verificar se a separação magnética dos astrócitos, oligodendrócitos e neurónios correu como planeado, portanto se cada fração de células separadas contém a linhagem pretendida, sem contaminações significativas de outras linhagens.
Nos testes respiratórios foi analisada a Taxa de Consumo de Oxigénio. Para tal foram adicionados vários moduladores da cadeia transportadora de eletrões: Oligomicína inibe o complexo V, impedindo a produção de ATP pela respiração mitocondrial; o FCCP é um desacoplador, que interrompe o gradiente de protões e permite que a cadeia transportadora de eletrões trabalhe na sua capacidade máxima; por fim a Rotenona e Antimicina A são inibidores dos complexos I e III, respetivamente. Esta experiência permitiu obter parâmetros, tais como, Respiração Basal, ATP ligado à Respiração Mitocondrial, Fuga de protões, Respiração Máxima, Capacidade Respiratória Sobresselente e Consumo de Oxigénio não Mitocondrial.
O ensaio de conteúdo de ATP baseia-se num protocolo de luciferina, sendo que, a reação da luciferina com o ATP, catalisada pela luciferase, produz luz. Através da análise da luminescência é possível calcular a quantidade de ATP nas nossas amostras utilizando uma curva padrão com determinados valores de ATP.
Relativamente aos resultados, começámos por caracterizar as culturas de neuroesferas, observando o seu tamanho e efetuando um estudo ao nível da proliferação das células. Neste estudo foram utilizados dois marcadores de proliferação diferentes: o BrdU que é um análogo da timidina que se incorpora no ADN durante a fase S da mitose; e o Ki-67 que é expresso durante o ciclo celular ativo (fases G1, S, G2 e M). Esta análise permitiu-nos concluir que embora o meio de baixa concentração de fatores de crescimento promova a diferenciação das CENs, este também tem a capacidade de favorecer a auto-renovação das mesmas.
Observámos através de imunocitoquímica que as frações astróciticas, oligodendrociticas, e neuronais continham maioritariamente o tipo celular pretendido e sem contaminações significativas de outros tipos celulares.
Através dos testes respiratórios observámos que o controlo das 96h+4days tem uma produção de ATP ligado à Respiração Mitocondrial significativamente superior ao controlo das 96h. O que sugere que com a diferenciação e maturação das células, estas ficam mais dependentes da fosforilação oxidativa. Seria interessante observar futuramente se alterações metabólicas mais mínimas acontecem aquando da decisão de futuro das células, utilizando por exemplo, ensaios de atividade dos complexos da cadeia transportadora de eletrões.
Também observámos que com a diferenciação astrocítica, a respiração basal das células aumenta e ocorre uma tendência para a redução da flexibilidade energética. Enquanto que com a diferenciação oligodendrocítica ocorre uma diminuição estatisticamente significativa da respiração máxima e da capacidade respiratória sobresselente. Embora os neurónios tenham a respiração basal inferior comparativamente às outras linhagens, estes também são a linhagem com maior capacidade respiratória sobresselente, o que reflete uma maior capacidade para responder em ambientes mais energeticamente exigentes. Estes dados estão de acordo com a função deste tipo celular, como a transmissão sináptica, que é extremamente exigente energeticamente.
Relativamente ao conteúdo de ATP não foram observadas diferenças entre as diferentes linhagens. Sabendo que o controlo das 96h+4days tem uma produção de ATP ligado à Respiração significativamente superior ao controlo das 96h, e observando que o nível de ATP é equivalente, isto sugere que ou as células às 96h+4days consomem mais ATP do que as das 96h, ou que as das 96h produzem mais ATP através de outras vias metabólicas que não a fosforilação oxidativa.
Portanto, embora ainda muito trabalho se tenha de efetuar de forma a determinar mais concretamente o perfil bioenergético das diferentes linhagens das CENs da ZSV, estes resultados reforçam as diferenças metabólicas ao longo da diferenciação e entre as três linhagens. Deste modo, estes resultados apoiam a hipótese de que a bioenergética mitocondrial muda ao longo da diferenciação e de forma dependente da linhagem celular.
Este projeto poderá ser importante para futuramente estudar se será possível modular a decisão de destino das CENs através da modulação das propriedades mitocondriais. Esta possível modulação poderá ter relevância ao nível de tratamento de doenças do sistema nervoso central, como por exemplo, promovendo a diferenciação de oligodendrócitos em pacientes com Esclerose Múltipla, uma doença caracterizada pela perda de oligodendrócitos.Neural stem cells (NSCs) are self-renewing and multipotent cells that can differentiate into astrocytes, oligodendrocytes, and neurons. There are two main niches where NSCs reside in the adult mammalian brain, the hippocampal dentate gyrus, and the subventricular zone (SVZ). The molecular cues associated with the decision of the NSCs to commit to either the astroglial, oligodendroglial, or neuronal lineages have been a research focus of the field. Importantly, mitochondria have been associated with the regulation of both proliferation and differentiation of NSCs. Particularly, results from the Host Laboratory show that mitochondrial morphology changes significantly not only with differentiation but also in a lineage specific way. Therefore, we hypothesized that other mitochondrial properties, such as bioenergetics, may also contribute to the decision-making process, and that changes in mitochondrial properties may be involved in determining the fate of NSCs.
This project aimed to characterize the metabolic profile of the differentiated cells derived from the SVZ NSCs. Towards this aim, we used neurospheres primary cultures from the SVZ of P1-3 C57BL/6 mice, as an in vitro model to study SVZ NSCs. We sorted the differentiated astrocytes, oligodendrocytes, and neurons derived from SVZ neurospheres with a Magnetic-Activated Cell Sorting protocol and performed Respiratory Assays, ATP content assays, and Immunocytochemistry.
We observed that with differentiation and maturation, the SVZ-derived cells become more reliant on Oxidative Phosphorylation (OXPHOS). Furthermore, the basal respiration is higher during astrocytic differentiation, while with oligodendrogenesis, cells became more reliant on OXPHOS and less energetically flexible. Finally, although neurons present the lowest basal respiration compared with the astrocytes and oligodendrocytes, they are highly energetically flexible.
Although additional assays to fully comprehend the bioenergetic profile of the different lineages from the SVZ NSCs is still required, our results already propose the existence of metabolic differences throughout differentiation within each cell lineage
The role of moral disengagement in cyberbullying
No full textCyberbullying is a complex phenomenon with multiple factors involved, both individual as well as contextual, therefore a multiplicity of interventions are possible. Nonetheless, there are specific individual factors, such as moral disengagement (MD), which have already been investigated previously in relation to bullying, that are considered risk factors for cyberbullying involvement. Thus, this investigation focused specifically on MD and how it is related to cyberbullying from the perspective of adolescents. The processes involved in cyberbullying can be seen as the two sides from the same coin. On one side, there are the protective factors, and on the other side, there are the risk factors. Hence, considering the fact that cyberbullying is a complex phenomenon, which can be explained by several factors, we also aimed to understand the relation between MD and empathy. This provided evidence on how to include these two constructs together in order to develop more effective anti-cyberbullying interventions. Hence, in a first study, we proposed to examine how students belonging to different cyberbullying roles, perceived beliefs related to cyberbullying, both at the individual level and concerning the peer group. Specifically, we aimed to understand how adolescents (N=404) perceived their personal and normative beliefs about cyberbullying, considering their specific role in this type of aggressive behavior. For this purpose, students answered to the Inventory of Observed Cyberbullying Incidents. To this objective, 34 adolescents participated in semi-structured interviews with scenarios, and content analysis was used with a mixed approach, based on the Social Cognitive Theory (SCT). Results from hierarchical regressions followed by Post Hoc Tukey test revealed that those who were involved as bystanders, victims and aggressors presented the lowest scores on all four types of beliefs. Specifically, this group believed that cyberbullying was less severe and less unfair, and thought that their peer group believed cyberbullying was less severe and unfair than others (i.e., bystanders, bystanders-victims and those who were not involved at all). Moreover, the most used MD mechanisms were blaming the victim and euphemistic labeling (regarding the seriousness of the situation). Therefore, we concluded that those who were involved as bystanders, victims and aggressors that would most benefit from cyberbullying interventions, specifically targeted at clarifying beliefs about the fairness and severity of these types of behavior. Furthermore, this study enabled us to understand the role of MD, considering that specific mechanisms are more related to the aggressors’ behavior and others are more related to bystanders’ aggressive behavior. Furthermore, the adolescents’ perspectives of cyberbullying led to a cyclical model, where some mechanisms were related to the antecedents, others to the behavior, and finally, others mechanisms were related to the consequents.
In a second study, we investigated how adolescents reported empathy in online contexts and MD in cyberbullying incidents. To accomplish this goal, we had to adapt and develop two instruments, the short version of the Empathy Quotient, to Portuguese and to online contexts, which originated the Empathy Quotient in Virtual Contexts (EQVC), and develop the Process Moral Disengagement in Cyberbullying Situations Questionnaire (PMDCSQ), based on the content analysis from the previous study and on the SCT. Exploratory factor analyses (N=234) and Confirmatory factor analyses (N=345) to assess both instruments revealed empathy as a bi-dimensional structure including difficulty and self-efficacy in empathizing (Cronbach's α = .44, .83, respectively), and process MD was assessed with four unidimensional questionnaires including locus of behavior, agency, outcome and recipient (Cronbach's α = .76, .65, .77, .69, respectively). Results of a correlational study showed that difficulty in empathizing was negatively associated with sex, meaning that girls revealed more difficulty than boys. Difficulty in empathizing was also negatively associated with all MD loci, with exception for behavior. Self-efficacy in empathizing was not associated with any variable. Lastly, MD was positively correlated with sex, suggesting that boys morally disengaged more from cyberbullying than girls.
Lastly, we analyzed how MD and empathy could be related, longitudinally. Specifically, two gamified tasks (one for empathy and other for MD) were analyzed. These tasks were developed attending to the specificities of the cyberbullying scenarios presented in a serious game. To accomplish this goal, data from gamified tasks (N=208), from 4 different moments, were analyzed through multilevel linear modeling. Results suggest that there was a change in adolescents’ MD over time. Participants with greater empathy revealed lower MD overall. Over time, adolescents with greater empathy revealed lower MD within their own growth rate. Overall, our results provide important information about the dynamic relationship between MD, empathy and cyberbullying, which informs future studies and interventions
Prediction of Antimicrobial Resistance for Personalized Prevention and Clinical Management of Infectious Diseases
No full textTese de mestrado, Ciência de Dados, 2023, Universidade de Lisboa, Faculdade de CiênciasAntibiotics are a very important class of drugs in modern Medicine; its discovery and introduction constituted a major revolution in Medicine. As such, the decrease of their effectiveness due to the rising
levels of antibiotic resistance is a great concern to our society.
Hospital-Acquired Infections (HAI), also known as nosocomial infections, are infections that a patient acquires in the context of medical treatments. Nosocomial infections are closely related to the
problem of resistance to antibiotics, because a large part of these infections are caused by antibiotic
resistant bacteria. This aspect motivates the tackling of these two problems jointly.
This work was performed in the frame of the RESISTIR project, that aims to develop a Decision
Support System for intelligent control of infection and personalized antibiotherapy. The data was collected by a portuguese software company, Maxdata Software, S.A.; then, in the scope of this project, it
was pre-processed and inserted into a database. The database contains information generated by clinical
episodes with origin in three portuguese hospitals, dated from 2013 to 2016.
The available data allowed us to develop predictive models of risk of antibiotic resistance (AMR
models) and risk of nosocomial infection (HAI model). For the AMR models, we aggregated the antibiotics into the level 4 of the ATC classification system, and focused on four classes of antibiotics:
J01MA (fluoroquinolones), J01CA (penicillins with extended spectrum), J01DC (second-generation
cephalosporins) and J01DH (carbapenems). In all predictive models developed, the features generated
related to the clinical history of the patients and the health units involved in the episodes were found to
be the most significant.
We concluded that the main goals of this project were achieved, with the development of prototype
predictive models of risk of antibiotic resistance and risk of nosocomial infection. The predictive power
of the models, as measured by the ROC-AUC, ranged from 0.720 to 0.857 for the AMR models, and was
0.915 for the HAI model. These prototype predictive models that we were able to develop show that it is
possible to deploy in healthcare units a Decision Support System that can help to monitor and reduce the
problem of resistance to antibiotics and the nosocomial infections.
For the HAI predictive model developed, we made an estimate of its benefits if it would be deployed
in production in the three hospitals involved in this project. We drew the conclusion that the following
savings would be obtained, in 2007 euros and considering only the hospitalizations: about 20 million
euros and a reduction of approximately 60 000 days in the hospital stays, per year
Contrasting capture methods and health indicators among juvenile sharks in the nursery area of Boa Vista, Cabo Verde
No full textTese de mestrado, Ecologia Marinha, 2025, Universidade de Lisboa, Faculdade de CiênciasStress levels in neonate and juvenile sharks can influence the health and survival of the individual, as
well as the overall population resilience. This thesis explores interspecific differences in health and
stress indicators in juvenile sharks caught in Sal Rei Bay (SRB), a multi-specific shark nursery area in
Boa Vista Island, Cabo Verde. Body condition, capture resilience, and a range of biochemical
parameters (cholesterol, glucose, phosphate, creatine, alanine aminotransferase, alkaline phosphatase,
potassium, total protein) were assessed in four shark species frequenting SRB — milk shark
(Rhizoprionodon acutus), blacktip shark (Carcharhinus limbatus), scalloped hammerhead shark
(Sphyrna lewini), and Atlantic weasel shark (Paragaleus pectoralis) — caught using artisanal fishing
methods traditionally employed in the region (gillnets and angling). Recuperation scores varied among
species and generally indicated faster recovery in individuals captured by angling compared to gillnets.
Milk sharks, however, tended to recover quicker after gillnet capture. Hammerhead sharks showed the
overall slowest recovery. Significant species-specific differences were revealed in nearly all analysed
indicators, with a frequent influence of the umbilical scar. A significant influence of gear type was only
detected for the two biomarkers alanine aminotransferase and alkaline phosphatase. The present results
showcase the influence of species, development, and fishing-method on physiological indicators in
juvenile sharks in SRB, discussing how species-specific ecological and behavioural characteristics
might have influenced the results obtained. The influence of environmental and other anthropogenic
pressures should be considered more thoroughly in future studies. By yielding the first fundamental
insights into the interspecific differences in stress levels among juvenile sharks in the SRB nursery area,
the present dissertation contributes to the design of future and ongoing monitoring efforts. Further, it
enhances the comprehension of local interactions between sharks and the local artisanal fishery,
addressing a knowledge gap for formulating effective, locally-tailored conservation strategies