The effect of sucrose on protein stability and fibrillation of prion and S6 protein

Aggregation and fibrillation of proteins have a great importance in medicine and industry. Misfolding and aggregation are the basis of many neurodegenerative diseases like Alzheimer and Parkinson. Osmolytes are molecules that can accumulate within cells and act as protective agents and they can inclusively act as protein stabilizers when cells are exposed to stress conditions. Osmolytes can also act as protein stabilizers in vitro. In this work, two different proteins were studied, the ribosomal protein from Thermus thermophilus and the mouse prion protein. The existence of an unstructured N-terminal on the prion protein does not affect its stability. The effect of the osmolyte sucrose on the fibrillation and stabilization of these two proteins was studied through kinectic and equilibrium measurements. It was shown that sucrose is able to compact the native structure of S6 protein in fibrillization conditions. Sucrose affects also folding and unfolding kinetic of S6 protein, delaying unfolding and increasing folding rate constants. The mechanism of stabilization by sucrose is non-specific because it is distributed for all protein structure, as it was demonstrated by a protein engineering approach. Sucrose delays the process of formation and elongation of S6 and prion protein from mouse. This delay is the result of the compaction of the native structure refered above. However, cellular toxicity studies have shown that fibrils formed in the presence of sucrose are more toxic to neuronal cells.

Application of molecular tools for detection of plant viruses

Grapevine leafroll disease (GLRD) is one of the most important virus diseases of grapevines worldwide, causing major economical impact. The disease has a complex aetiology and currently eleven phloem-limited viruses, termed in general Grapevine leafroll-associated virus (GLRaVs), have been identified. Two of the GLRaVs, GLRaV-1 and GLRaV-3, are included in the European certification scheme of propagation material. However, the flawed notion that GLRaV-3 is more frequent than GLRaV-1 and that all other GLRaVs are possibly not as relevant for GLRD, has until now precluded the development of specific serological and molecular detection assays and limited the scope of molecular characterization of the viruses known to be associated with the disease. Hence, few studies have addressed the phylodynamics of GLRaVs or even characterized the genetic structure of their natural populations. This generalized lack of molecular information, in turn underlie the deficient capacity to detect the viruses. The phylogenetic analyses were conducted on the basis of the heat shock protein 70 homologue (HSP70h) and the coat protein (CP) genes for GLRaV-1 and the HSP70h, the heat shock protein 90 homologue (HSP90h) and the CP genes for GLRaV-5. The data obtained for GLRaV-1 contributed 83 new CP sequences. This information was combined with previous analysis by other authors and used for the production of new polyclonal IgG, capable of detecting CP variants from all the phylogroups observed. Successful testing of this new tool included tissue print immunoblotting (TPIB) and in situ immunoassay (ISIA). The data obtained for GLRaV-5, contributed 61 new CP and 28 new HSP90h gene sequences. Eight phylogenetic groups were identified on the basis of the CP. Characterization of the genetic structure of the isolates revealed a higher diversity than previously reported and allowed the identification of dominant virus variants. For both GLRaV-1 and GLRaV-5, the effect of vegetative propagation on the virus transmission dynamics was addressed.

O efeito protector da criatividade face à depressão, à ansiedade e ao stresse em reclusos de estabelecimentos prisionais do Algarve

A criatividade é um potencial que cada indivíduo possui, encontrando-se relacionada com a resolução de problemas, a adaptação e o coping. Esta encontra-se ainda relacionada com indicadores de saúde mental, assim como de psicopatologia. No entanto, a literatura sugere algumas lacunas, havendo a necessidade de compreender melhor as relações entre essas variáveis, assim como de investir em estudos com grupos minoritários. Entre esses grupos, encontram-se a população reclusa, existindo muito pouco investigação nesse âmbito. Esta investigação tem, portanto, como objectivo estudar o possível efeito protector da criatividade face ao mal-estar experienciado durante a reclusão, em termos de depressão, ansiedade e stresse. Os instrumentos utilizados para a avaliação foram a Escala de Personalidade Criativa (EPC) e as Escalas de Ansiedade, Depressão e Stresse de 21 itens (EADS-21). A amostra era constituída por 107 reclusos pertencentes a estabelecimentos prisionais da região do Algarve. Através dos resultados observou-se que a criatividade está relacionada de forma negativa com a depressão e a ansiedade, actuando como um preditor da diminuição dos níveis de depressão, ansiedade e stresse avaliados. Isto sugere que a criatividade poderá ter um efeito protector face a estes indicadores de mal-estar, contribuindo para uma melhor saúde mental dos reclusos.

Searching biocompounds in marine natural extracts with potential use in tumor prevention and treatment

Cancer is a multistage process characterized by three stages: initiation, promotion and progression; and is one of the major killers worldwide. Oxidative stress acts as initiator in tumorigenesis; chronic inflammation promotes cancer; and apoptosis inactivation is an issue in cancer progression. In this study, it was investigated the antioxidant, antiinflammatory and antitumor properties of hexane, ether, chloroform, methanol and water extracts of five species of halophytes: A. macrostachyum, P. coronopus, J. acutus, C. edulis and A. halimus. Antioxidant activity was assessed by DPPH• and ABTS•+ methods, and the total phenolics content (TPC) was evaluated by the Folin-Ciocalteau method. The anti-inflammatory activity of the extracts was determined by the Griess method, and by evaluating the inhibition of NO production in LPS-stimulated RAW- 264.7 macrophages. The cytotoxic activity of the extracts against HepG2 and THP1 cell lines was estimated by the MTT assay, and the results obtained were further compared with the S17 non-tumor cell line. The induction of apoptosis of J. acutus ether extract was assessed by DAPI staining. The highest antioxidant activities was observed in C. edulis methanol and the J. acutus ether extracts against the DPPH• radical; and J. acutus ether and A. halimus ether extracts against the ABTS•+ radical. The methanol extracts of C. edulis and P. coronopus, and the ether extract of J. acutus revealed a high TPC. Generally the antioxidant activity had no correlation with the TPC. The A. halimus chloroform and P. coronopus hexane extracts demonstrated ability to reduce NO production in macrophages (> 50%), revealing their anti-inflammatory capacity. The ether extract of J. acutus showed high cytotoxicity against HepG2 cancer cells, with reduced cellular viability even at the lowest concentrations. This outcome was significantly lower than the obtained with the non-tumor cells (S17). This result was complemented by the induction of apoptosis.

Indução de contaminação mental em vítimas de traição numa amostra de indivíduos não-clínicos

No presente trabalho apresentam-se dois estudos. O primeiro dos estudos teve como objetivo a adaptação ao português do Inventário de Obsessões e Compulsões de Vancouver (VOCI; Thordarson, Radomsky, Rachman, Shafran, Sawchuk & Hakstian, 2004). O segundo dos estudos teve como objetivo induzir, de forma experimental, a sensação de contaminação mental através da imaginação de uma situação de traição. No estudo de adaptação do VOCI participaram 180 indivíduos da população geral não-clínica com idades compreendidas entre os 18 e os 75 anos e 120 estudantes universitários com idades compreendidas entre os 18 e os 25 anos que preencheram além do VOCI, a Escala de sensibilidade de Contaminação (S-CTN; Rachman, 2006 cit in Coughtrey, Shafran, Knibbs & Rachman, 2012). Sessenta dos participantes completaram de novo o VOCI após um intervalo de 8 semanas. A amostra final ficou constituída por 292 participantes. Para conhecer a estrutura interna do VOCI, foi realizada uma análise fatorial exploratória. Obtiveram-se 6 fatores que explicaram 39% da variância total. Os fatores obtidos replicaram a estrutura original do VOCI Contaminação, Verificação, Obsessões, Acumulação, Incerteza, Just Right. Quer o total do VOCI quer as seis subescalas obtidas mostraram excelentes níveis de consistência interna, fiabilidade temporal e validade convergente. De acordo com os nossos resultados, podemos considerar que a versão portuguesa do VOCI apresenta garantias psicométricas suficientes para que possa ser utilizada na população Portuguesa. No segundo estudo, participaram 60 estudantes universitários, 30 do género feminino e 30 do género masculino, com idades compreendidas entre os 18 e os 25 anos. Os participantes foram aleatoriamente divididos em dois grupos: um grupo de controlo (GC) e um grupo experimental (GE). Neste estudo recorreu-se a um desenho experimental 2x4 (2 condições x 4 momentos). Inicialmente os participantes preencheram um conjunto de questionários e de seguida foram instruídos para ouvir uma gravação de voz relativa à sua condição (controlo/experimental). Posteriormente foi-lhes dada uma pausa de 5 minutos e, no quarto e último momento, os participantes foram submetidos a uma pequena entrevista final. Os resultados obtidos após a manipulação experimental revelaram que o GE apresentou valores significativamente mais elevados de desconforto que o GC, ou seja, a manipulação experimental desencadeou emoções negativas (tais como a raiva ou a tristeza). Não se verificou, no entanto, o efeito da manipulação experimental relativamente à capacidade dos participantes para localizar a sujidade em alguma parte do interior ou exterior do corpo. Em suma, no presente estudo não foi possível induzir contaminação mental através da imaginação de uma situação de traição bem como a localização da sensação de sujidade, o aparecimento de emoções/sentimentos negativos, do impulso de se lavar, de evitamento e de estratégias de neutralização e de lavagem.

Study of influence of regulatory polymorphisms of expression in development of breast cancer

The human genome has millions of genetics variants that can affect gene expression. These variants are known as cis-regulatory variants and are responsible for intra-species phenotypic differences and individual susceptibility to disease. One of the diseases affected by cis-regulatory variants is breast cancer. Breast cancer is one of the most common cancers, with approximately 4500 new cases each year in Portugal. Breast cancer has many genes mutated and TP53 has been shown to be relevant for this disease. TP53 is one of the most commonly mutated genes in human cancer and it is involved in cell cycle regulation and apoptosis. Previous work by Maia et al has shown that TP53 has differential allelic expression (DAE), which suggests that this gene may be under the influence of cis-regulatory variants. Also, its DAE pattern is totally altered in breast tumours with normal copy number. We hypothesized that cis-regulatory variants affecting TP53 may have a role in breast cancer development and treatment. The present work aims to identify the cis-regulatory variants playing a role in TP53 expression, using in silico, in vitro and in vivo approaches. By bioinformatic tools we have identified candidate cis-regulatory variants and predicted the possible transcription factor binding sites that they affect. By EMSA we studied DNA-protein interactions in this region of TP53. The in silico analysis allowed us to identified three candidate cis-regulatory SNPs which may affect the binding of seven transcription factors. However, the EMSA experiments have not been conclusive and we have not yet confirmed whether any of the identified SNPs are associated with gene expression control of TP53. We will carry out further experiments to validate our findings.

Molecular structure and functional analysis of runx3 in zebrafish

Tese de doutoramento, Ciências Biomédicas, Universidade do Algarve, Departamento de Ciências Biomédicas e Medicina, 2014

Identification and molecular characterization of bone-related micrornas: functional implications

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2014

Development of non-viral vectors for gene therapy for pathologies of the retina

Tese de doutoramento, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Estudo da atividade protetora de extratos vegetais contra stresse genotóxico

Dissertação de mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Drug repurposing in the treatment of Melanoma Brain Metastasis

Dissertação de Mestrado, Biologia Molecular e Microbiana, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Using genomics for drug discovery in medulloblastoma

Tese de doutoramento, Medicina (Neurocirurgia), Universidade de Lisboa, Faculdade de Medicina, 2014

What is poetic attention

Tese de doutoramento, Estudos de Literatura e de Cultura (Teoria da Literatura), Universidade de Lisboa, Faculdade de Letras, 2014

Human factors required for mycobacterium tuberculosis macrophage infection

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2014

How hydrogen peroxide and CXCL8 modulate neutrophil recruitment "in vivo"

Tese de doutoramento, Ciências Biomédicas (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Medicina, 2014