Aß biology and its contribution to Alzheimer's disease

A doença de Alzheimer (DA) é uma desordem neurodegenerativa progressiva patologicamente caracterizada pela presença de placas de amilóide (placas senis) insolúveis e também pela presença de tranças neurofibrilhares,formadas pela proteína Tau hiperfosforiladada. O principal constituinte das placas senis é o peptídeo beta-amilóide (Ab), que deriva do processamento proteolítico da proteína precursora de amilóide de Alzheimer (APP). Embora Ab exista como um agregado pouco solúvel nas placas senis, ele é secretado pelas células como uma molécula solúvel. O Ab “per se” pode afectar o metabolismo da APP. Alguns autores sugerem que o Ab exerce o seu efeito alterando o processamento ou catabolismo da APP, outros sugerem que ele também induz a transcrição da APP, onde aumentando os níveis da APP pode estar a contribuir para a sua própria produção (mecanismo de “feedback” positivo). Assim sendo, torna-se difícil consolidar todas estas observações e identificar as potenciais funções fisiológicas do Ab “in vivo”, ou as consequências da sua produção. Neste trabalho caracterizaram-se os efeitos do Ab no metabolismo da APP. Os nossos estudos revelaram que um dos mecanismos induzidos pelo Ab é a acumulação intracelular do fragmento neuroprotector sAPP (isAPPa) em estruturas com características vesiculares associadas ao citosqueleto. Estudos adicionais em culturas primárias revelaram que o Ab estava a exercer o seu efeito ao nível da secreção vesicular, provavelmente interferindo com o transporte de APP/sAPP ao longo da rede do citosqueleto. Esta hipótese é sustentada pelo facto do Ab estar a afectar a estabilidade e a polimerização de proteínas envolvidas na dinâmica do citosqueleto. Contrariamente a publicações anteriores o Ab não induziu a transcrição da APP, na verdade em culturas primárias neuronais foi observado uma diminuição nos níveis de expressão da APP. Isto foi acompanhado por um aumento nos fragmentos C-terminais da APP (CTFs) e uma diminuição na localização nuclear do seu domínio intracelular (AICD), sugerindo alterações na sinalização nuclear da APP. O Ab pode afectar outras vias de sinalização, particularmente alterando o balanço entre as actividades das proteínas cinases e fosfatases, o que pode ter consequências para o desenvolvimento da doença. Os dados obtidos indicam que o Ab é capaz de inibir a actividade da proteína fosfatase1, a sua importância numa perspectiva de futuras terapias é discutida. Devido à relevância da agregação do Ab para a sua toxicidade, a formação de complexos com proteínas que promovem a sua desagregação/degradação e o seu efeito no processamento da APP foi avaliado. Na presença destes complexos observou-se uma reversão da acumulação isAPP, demonstrando o potencial terapêutico destas proteínas como moduladores do metabolismo da APP. Este trabalho permitiu compreender melhor os mecanismos envolvidos nos efeitos do Ab no processamento da APP e descobrir algumas moléculas que podem ser relevantes numa perspectiva de diagnóstico e terapia na DA.

PP1 interactomes as a means of characterizing protein functions

A maioria das funções celulares, incluindo expressão de genes, crescimento e proliferação celulares, metabolismo, morfologia, motilidade, comunicação intercelular e apoptose, é regulada por interações proteína-proteína (IPP). A célula responde a uma variedade de estímulos, como tal a expressão de proteínas é um processo dinâmico e os complexos formados são constituídos transitoriamente mudando de acordo com o seu ciclo funcional, adicionalmente, muitas proteínas são expressas de uma forma dependente do tipo de célula. Em qualquer instante a célula pode conter cerca de centenas de milhares de IPPs binárias, e encontrar os companheiros de interação de uma proteína é um meio de inferir a sua função. Alterações em redes de IPP podem também fornecer informações acerca de mecanismos de doença. O método de identificação binário mais frequentemente usado é o sistema Dois Hibrido de Levedura, adaptado para rastreio em larga escala. Esta metodologia foi aqui usada para identificar os interactomas específicos de isoforma da Proteína Fosfatase 1 (PP1), em cérebro humano. A PP1 é uma proteína fosfatase de Ser/Thr envolvida numa grande variedade de vias e eventos celulares. É uma proteína conservada codificada por três genes, que originam as isoformas α, β, e γ, com a última a originar γ1 e γ2 por splicing alternativo. As diferentes isoformas da PP1 são reguladas pelos companheiros de interação – proteínas que interagem com a PP1 (PIPs). A natureza modular dos complexos da PP1, bem como a sua associação combinacional, gera um largo reportório de complexos reguladores e papéis em circuitos de sinalização celular. Os interactomas da PP1 específicos de isofoma, em cérebro, foram aqui descritos, com um total de 263 interações identificadas e integradas com os dados recolhidos de várias bases de dados de IPPs. Adicionalmente, duas PIPs foram selecionadas para uma caracterização mais aprofundada da interação: Taperina e Sinfilina-1A. A Taperina é uma proteína ainda pouco descrita, descoberta recentemente como sendo uma PIP. A sua interação com as diferentes isoformas da PP1 e localização celulares foram analisadas. Foi descoberto que a Taperina é clivada e que está presente no citoplasma, membrana e núcleo e que aumenta os níveis de PP1, em células HeLa. Na membrana ela co-localiza com a PP1 e a actina e uma forma mutada da Taperina, no motivo de ligação à PP1, está enriquecida no núcleo, juntamente com a actina. Mais, foi descoberto que a Taperina é expressa em testículo e localiza-se na região acrossómica da cabeça do espermatozoide, uma estrutura onde a PP1 e a actina estão também presentes. A Sinfilina-1A, uma isoforma da Sinfilina-1, é uma proteína com tendência para agregar e tóxica, envolvida na doença de Parkinson. Foi mostrado que a Sinfilina-1A liga às isoformas da PP1, por co-transformação em levedura, e que mutação do seu motivo de ligação à PP1 diminuiu significativamente a interação, num ensaio de overlay. Quando sobre-expressa em células Cos-7, a Sinfilina-1A formou corpos de inclusão onde a PP1 estava presente, no entanto a forma mutada da Sinfilina-1A também foi capaz de agregar, indicando que a formação de inclusões não foi dependente de ligação à PP1. Este trabalho dá uma nova perspetiva dos interactomas da PP1, incluindo a identificação de dezenas de companheiros de ligação específicos de isoforma, e enfatiza a importância das PIPs, não apenas na compreensão das funções celulares da PP1 mas também, como alvos de intervenção terapêutica.

Identification of reliable reference genes for quantitative gene expression in the Mozambique tilapia, Oreochromis mossambicus

Dissertação de mest., Biologia Marinha (Aquacultura), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2010

Interacção do vanádio com a miosina de músculo esquelético de coelho

Relatório de projecto de licenciatura, Bioquímica, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2009

Identification of the cellular and molecular mechanism by which CD2AP, a regulator of neuronal intracellular transport contributes to the development of Alzheimer’s disease

Tese de mestrado. Biologia (Biologia Molecular e Genética). Universidade de Lisboa, Faculdade de Ciências,2014

The role of AmotL2 in organ formation and tumorigenesis

Tese de mestrado, Oncobiologia, Faculdade de Medicina, Universidade de Lisboa, 2014

Development of a new cellular model for the visualization and study of GFAP oligomerization in living cells

Tese de mestrado, Neurociências, Faculdade de Medicina, Universidade de Lisboa, 2016

Cancer-associated fibroblasts in brain metastases

Tese de mestrado. Oncobiologia, Faculdade de Medicina, Universidade de Lisboa, 2015

Cellular polarity in aging: role of redox regulation and nutrition

Cellular polarity concerns the spatial asymmetric organization of cellular components and structures. Such organization is important not only for biological behavior at the individual cell level, but also for the 3D organization of tissues and organs in living organisms. Processes like cell migration and motility, asymmetric inheritance, and spatial organization of daughter cells in tissues are all dependent of cell polarity. Many of these processes are compromised during aging and cellular senescence. For example, permeability epithelium barriers are leakier during aging; elderly people have impaired vascular function and increased frequency of cancer, and asymmetrical inheritance is compromised in senescent cells, including stem cells. Here, we review the cellular regulation of polarity, as well as the signaling mechanisms and respective redox regulation of the pathways involved in defining cellular polarity. Emphasis will be put on the role of cytoskeleton and the AMP-activated protein kinase pathway. We also discuss how nutrients can affect polarity-dependent processes, both by direct exposure of the gastrointestinal epithelium to nutrients and by indirect effects elicited by the metabolism of nutrients, such as activation of antioxidant response and phase-II detoxification enzymes through the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In summary, cellular polarity emerges as a key process whose redox deregulation is hypothesized to have a central role in aging and cellular senescence.

Characterization of the effects of antiepileptic drugs on bone metabolism: in vitro studies with human osteoblastic and osteoclastic cells

Bone is constantly being molded and shaped by the action of osteoclasts and osteoblasts. A proper equilibrium between both cell types metabolic activities is required to ensure an adequate skeletal tissue structure, and it involves resorption of old bone and formation of new bone tissue. It is reported that treatment with antiepileptic drugs (AEDs) can elicit alterations in skeletal structure, in particular in bone mineral density. Nevertheless, the knowledge regarding the effects of AEDs on bone cells are still scarce. In this context, the aim of this study was to investigate the effects of five different AEDs on human osteoclastic, osteoblastic and co-cultured cells. Osteoclastic cell cultures were established from precursor cells isolated from human peripheral blood and were characterized for tartrate-resistant acid phosphatase (TRAP) activity, number of TRAP+ multinucleated cells, presence of cells with actin rings and expressing vitronectin and calcitonin receptors and apoptosis rate. Also, the involvement of several signaling pathways on the cellular response was addressed. Osteoblastic cell cultures were obtained from femur heads of patients (25-45 years old) undergoing orthopaedic surgery procedures and were then studied for cellular proliferation/viability, ALP activity, histochemical staining of ALP and apoptosis rate. Also the expression of osteoblast-related genes and the involvement of some osteoblastogenesis-related signalling pathways on cellular response were addressed. For co-cultured cells, osteoblastic cells were firstly seeded and cultured. After that, PBMC were added to the osteoblastic cells and co-cultures were evaluated using the same osteoclast and osteoblast parameters mentioned above for the corresponding isolated cell. Cell-cultures were maintained in the absence (control) or in the presence of different AEDs (carbamazepine, gabapentin, lamotrigine, topiramate and valproic acid). All the tested drugs were able to affect osteoclastic and osteoblastic cells development, although with different profiles on their osteoclastogenic and osteoblastogenic modulation properties. Globally, the tendency was to inhibit the process. Furthermore, the signaling pathways involved in the process also seemed to be differently affected by the AEDs, suggesting that the different drugs may affect osteoclastogenesis and/or osteoblastogenesis through different mechanisms. In conclusion, the present study showed that the different AEDs had the ability to directly and indirectly modulate bone cells differentiation, shedding new light towards a better understanding of how these drugs can affect bone tissue.

Besnoitia besnoiti and Toxoplasma gondii: two apicomplexan strategies to manipulate the host cell centrosome and Golgi apparatus

Besnoitia besnoiti and Toxoplasma gondii are two closely related parasites that interact with the host cell microtubule cytoskeleton during host cell invasion. Here we studied the relationship between the ability of these parasites to invade and to recruit the host cell centrosome and the Golgi apparatus. We observed that T. gondii recruits the host cell centrosome towards the parasitophorous vacuole (PV), whereas B. besnoiti does not. Notably, both parasites recruit the host Golgi apparatus to the PV but its organization is affected in different ways. We also investigated the impact of depleting and over-expressing the host centrosomal protein TBCCD1, involved in centrosome positioning and Golgi apparatus integrity, on the ability of these parasites to invade and replicate. Toxoplasma gondii replication rate decreases in cells over-expressing TBCCD1 but not in TBCCD1-depleted cells; while for B. besnoiti no differences were found. However, B. besnoiti promotes a reorganization of the Golgi ribbon previously fragmented by TBCCD1 depletion. These results suggest that successful establishment of PVs in the host cell requires modulation of the Golgi apparatus which probably involves modifications in microtubule cytoskeleton organization and dynamics. These differences in how T. gondii and B. besnoiti interact with their host cells may indicate different evolutionary paths.

Methamphetamine promotes a-tubulin deacetylation in endothelial cells: The protective role of acetyl-L-carnitine

Methamphetamine (METH) is a powerful psychostimulant drug used worldwide for its reinforcing properties. In addition to the classic long-lasting monoaminergic-disrupting effects extensively described in the literature, METH has been consistently reported to increase blood brain barrier (BBB) permeability, both in vivo and in vitro, as a result of tight junction and cytoskeleton disarrangement. Microtubules play a critical role in cell stability, which relies on post-translational modifications such as a-tubulin acetylation. As there is evidence that psychostimulants drugs modulate the expression of histone deacetylases (HDACs), we hypothesized that in endothelial cells METH-mediation of cytoplasmatic HDAC6 activity could affect tubulin acetylation and further contribute to BBB dysfunction. To validate our hypothesis, we exposed the bEnd.3 endothelial cells to increasing doses of METH and verified that itleads to an extensivea-tubulin deacetylation mediated by HDACs activation. Furthermore, since we recently reported that acetyl-L-carnitine (ALC), a natural occurring compound, prevents BBB structural loss in a context of METH exposure, we reasoned that ALC could also preserve the acetylation of microtubules under METH action. The present results confirm that ALC is able to prevent METH-induced deacetylation providing effective protection on microtubule acetylation. Although further investigation is still needed, HDACs regulation may become a new therapeutic target for ALC.

Acetyl-L-Carnitine Prevents Methamphetamine-Induced Structural Damage on Endothelial Cells via ILK-Related MMP-9 Activity

Methamphetamine (METH) is a potent psychostimulant highly used worldwide. Recent studies evidenced the involvement of METH in the breakdown of the blood-brain-barrier (BBB) integrity leading to compromised function. The involvement of the matrix metalloproteinases (MMPs) in the degradation of the neurovascular matrix components and tight junctions (TJs) is one of the most recent findings in METH-induced toxicity. As BBB dysfunction is a pathological feature of many neurological conditions, unveiling new protective agents in this field is of major relevance. AcetylL-carnitine (ALC) has been described to protect the BBB function in different paradigms, but the mechanisms underling its action remain mostly unknown. Here, the immortalized bEnd.3 cell line was used to evaluate the neuroprotective features of ALC in METH-induced damage. Cells were exposed to ranging concentrations of METH, and the protective effect of ALC 1 mM was assessed 24 h after treatment. F-actin rearrangement, TJ expression and distribution, and MMPs activity were evaluated. Integrin-linked kinase (ILK) knockdown cells were used to assess role of ALC in ILK mediated METHtriggered MMPs’ activity. Our results show that METH led to disruption of the actin filaments concomitant with claudin-5 translocation to the cytoplasm. These events were mediated by MMP-9 activation in association with ILK overexpression. Pretreatment with ALC prevented METH-induced activation of MMP-9, preserving claudin-5 location and the structural arrangement of the actin filaments. The present results support the potential of ALC in preserving BBB integrity, highlighting ILK as a new target for the ALC therapeutic use.

Role of oxidative stress-induced systemic and cavernosal molecular alterations in the progression of diabetic erectile dysfunction

Background Erectile dysfunction (ED) is a prevalent complication of diabetes, and oxidative stress is an important feature of diabetic ED. Oxidative stress-induced damage plays a pivotal role in the development of tissue alterations. However, the deleterious effects of oxidative stress in the corpus cavernosum with the progression of diabetes remain unclear. The aim of this study was to evaluate systemic and penile oxidative stress status in the early and late stages of diabetes. Methods Male Wistar streptozotocin-diabetic rats (and age-matched controls) were examined 2 (early) and 8 weeks (late) after the induction of diabetes. Systemic oxidative stress was evaluated by urinary H2O2 and the ratio of circulating reduced/oxidized glutathione (GSH/GSSG). Penile oxidative status was assessed by H2O2 production and 3-nitrotyrosine (3-NT) formation. Cavernosal endothelial nitric oxide synthase (eNOS) was analyzed by quantitative immunohistochemistry. Dual immunofluorescence was also performed for 3-NT and α-smooth muscle actin (α-SMA) and eNOS–α-SMA. Results There was a significant increase in urinary H2O2 levels in both diabetic groups. The plasma GSH/GSSG ratio was significantly augmented in late diabetes. In cavernosal tissue, H2O2 production was significantly increased in late diabetes. Reactivity for 3-NT was located predominantly in cavernosal smooth muscle (SM) and was significantly reduced in late diabetes. Quantitative immunohistochemistry revealed a significant decrease in eNOS levels in cavernosal SM and endothelium in late diabetes. Conclusions The findings indicate that the noxious effects of oxidative stress are more prominent in late diabetes. Increased penile protein oxidative modifications and decreased eNOS expression may be responsible for structural and/or functional deregulation, contributing to the progression of diabetes-associated ED.

Spatiotemporal mechanisms for actomyosin ring assembly and contraction in budding yeast cell division

Dissertation presented to obtain the Ph.D degree in Molecular Medicine