Molecular study of cell degeneration and evolution induced by mRNA mistranslation

As proteínas existentes nas células são produzidas pelo mecanismo de tradução do mRNA, no qual a informação genética contida nos genes é descodificada em cadeias polipeptídicas. O código genético, que define as regras de descodificação do genoma, minimiza os erros de tradução do mRNA, garantindo a síntese de proteínas com elevada fidelidade. Esta é essencial para a estabilidade do proteoma e para a manutenção e funcionamento dos processos celulares. Em condições fisiológicas normais, os erros da tradução do mRNA ocorrem com frequências que variam de 10-3 a 10-5 erros por codão descodificado. Situações que aumentam este erro basal geralmente estão associadas ao envelhecimento, stresse e a doenças; no entanto, em certos organismos o código genético é traduzido naturalmente com elevado erro, indicando que a síntese de proteínas aberrantes pode de algum modo ser vantajosa. A fim de estudar a resposta celular aos erros de tradução do mRNA, construímos leveduras que incorporam serina no proteoma em resposta a um codão de leucina, usando a expressão constitutiva de um tRNASer mutante. Este fenómeno genético artificial provocou uma forte diminuição da esporulação, da viabilidade e da eficiência de mating, afectando imensamente a reprodução sexual da levedura. Observou-se também uma grande heterogeneidade no tamanho e na forma das células e elevada instabilidade genómica, com o aparecimento de populações poliplóides e aneuplóides. No sentido de clarificar as bases celulares e moleculares daqueles fenótipos e compreender melhor a biologia do erro de tradução do mRNA, construímos também células de levedura que inserem serina em resposta a um codão de leucina de modo indutível e controlado. Utilizaram-se perfis de mRNA total e de mRNA associado a polissomas para elucidar a resposta celular ao erro de tradução do mRNA. Observou-se a indução de genes envolvidos na resposta ao stresse geral, stresse oxidativo e na unfolded protein response (UPR). Um aumento significativo de espécies reactivas de oxigénio (ROS) e um forte impacto negativo na capacidade das células pós-mitóticas re-iniciarem o crescimento foram também observados. Este fenótipo de perda de viabilidade celular foi resgatado por scavangers de ROS, indicando que o stresse oxidativo é a principal causa de morte celular causada pelos erros de tradução. Este estudo levanta a hipótese de que o stresse oxidativo e a acumulação de ROS, ao invés do colapso súbito do proteoma, são as principais causas da degeneração celular e das doenças humanas associadas aos erros de tradução do genoma. ABSTRACT: Proteins are synthesized through the mechanism of translation, which uses the genetic code to transform the nucleic acids based information of the genome into the amino acids based information of the proteome. The genetic code evolved in such a manner that translational errors are kept to a minimum and even when they occur their impact is minimized by similar chemical properties of the amino acids. Protein synthesis fidelity is essential for proteome stability and for functional maintenance of cellular processes. Indeed, under normal physiological conditions, mistranslation occurs at frequencies that range from 10-3 to 10-5 errors per codon decoded. Situations where this basal error frequency increases are usually associated to aging and disease. However, there are some organisms where genetic code errors occur naturally at high level, suggesting that mRNA mistranslation can somehow be beneficial. In order to study the cellular response to mRNA mistranslation, we have engineered single codon mistranslation in yeast cells, using constitutive expression of mutant tRNASer genes. These mistranslating strains inserted serines at leucine-CUG sites on a proteome wide scale due to competition between the wild type tRNALeu with the mutant tRNASer. Such mistranslation event decreased yeast sporulation, viability and mating efficiencies sharply and affected sexual reproduction strongly. High heterogeneity in cell size and shape and high instability in the genome were also observed, with the appearance of some polyploid or aneuploid cell populations. To further study the cellular and molecular basis of those phenotypes and the biology of mRNA mistranslation, we have also engineered inducible mRNA misreading in yeast and used total mRNA and polysome associated mRNA profiling to determine whether codon misreading affects gene expression. Induced mistranslation up-regulated genes involved in the general stress response, oxidative stress and in the unfolded protein response (UPR). A significant increase in reactive oxygen species (ROS) and a strong negative impact on the capacity of post-mitotic cells to re-initiate growth in fresh media were also observed. This cell viability phenotype was rescued by scavengers of ROS, indicating that oxidative stress is the main cause of cell death caused by mRNA mistranslation. This study provides strong support for the hypothesis that oxidative stress and ROS accumulation, rather than sudden proteome collapse or major proteome disruption, are the main cause of the cellular degeneration observed in human diseases associated mRNA mistranslation.

Evaluation of the matabolic response of osteosarcoma cells to conventional and new anticancer drugs by NMR metabolomics

The main scope of this work was to evaluate the metabolic effects of anticancer agents (three conventional and one new) in osteosarcoma (OS) cells and osteoblasts, by measuring alterations in the metabolic profile of cells by nuclear magnetic resonance (NMR) spectroscopy metabolomics. Chapter 1 gives a theoretical framework of this work, beginning with the main metabolic characteristics that globally describe cancer as well as the families and mechanisms of action of drugs used in chemotherapy. The drugs used nowadays to treat OS are also presented, together with the Palladium(II) complex with spermine, Pd2Spm, potentially active against cancer. Then, the global strategy for cell metabolomics is explained and the state of the art of metabolomic studies that analyze the effect of anticancer agents in cells is presented. In Chapter 2, the fundamentals of the analytical techniques used in this work, namely for biological assays, NMR spectroscopy and multivariate and statistical analysis of the results are described. A detailed description of the experimental procedures adopted throughout this work is given in Chapter 3. The biological and analytical reproducibility of the metabolic profile of MG-63 cells by high resolution magic angle spinning (HRMAS) NMR is evaluated in Chapter 4. The metabolic impact of several factors (cellular integrity, spinning rate, temperature, time and acquisition parameters) on the 1H HRMAS NMR spectral profile and quality is analysed, enabling the definition of the best acquisition parameters for further experiments. The metabolic consequences of increasing number of passages in MG-63 cells as well as the duration of storage are also investigated. Chapter 5 describes the metabolic impact of drugs conventionally used in OS chemotherapy, through NMR metabolomics studies of lysed cells and aqueous extracts analysis. The results show that MG-63 cells treated with cisplatin (cDDP) undergo a strong up-regulation of lipid contents, alterations in phospholipid constituents (choline compounds) and biomarkers of DNA degradation, all associated with cell death by apoptosis. Cells exposed to doxorubicin (DOX) or methotrexate (MTX) showed much slighter metabolic changes, without any relevant alteration in lipid contents. However, metabolic changes associated with altered Krebs cycle, oxidative stress and nucleotides metabolism were detected and were tentatively interpreted at the light of the known mechanisms of action of these drugs. The metabolic impact of the exposure of MG-63 cells and osteoblasts to cDDP and the Pd2Spm complex is described in Chapter 6. Results show that, despite the ability of the two agents to bind DNA, the metabolic consequences that arise from exposure to them are distinct, namely in what concerns to variation in lipid contents (absent for Pd2Spm). Apoptosis detection assays showed that, differently from what was seen for MG-63 cells treated with cDDP, the decreased number of living cells upon exposure to Pd2Spm was not due to cell death by apoptosis or necrosis. Moreover, the latter agent induces more marked alterations in osteoblasts than in cancer cells, while the opposite seemed to occur upon cDDP exposure. Nevertheless, the results from MG-63 cells exposure to combination regimens with cDDP- or Pd2Spm-based cocktails, described in Chapter 7, revealed that, in combination, the two agents induce similar metabolic responses, arising from synergy mechanisms between the tested drugs. Finally, the main conclusions of this thesis are summarized in Chapter 8, and future perspectives in the light of this work are presented.

Effect of photodynamic therapy on the virulence factors of Staphylococcus aureus

Staphylococcus aureus are Gram-positive bacteria who integrate the human microbiota. Nevertheless, these bacteria can be pathogenic to the humans. Due to the increasing occurrence of antibiotic-resistant S. aureus new approaches to control this pathogen are necessary. The antimicrobial photodynamic inactivation process (PDI) is based in the combined use of a light source, an oxidizing agent like oxygen and an intermediary agent (a photosensitizer). These three components interact to form cytotoxic reactive oxygen species that irreversibly damage vital constituents of the microbial cells and ultimately lead to cell death. In fact, PDI is being shown to be a promising alternative to the antibiotic approach in the inactivation of pathogenic microorganisms. However, information on effects of photosensitization on particular virulence factors is strikingly scarce. The objective of this work was to evaluate the effect of PDI on virulence factors of S. aureus. For this, as photosensitizer the 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin tetra-iodide (Tetra-Py+-Me) and six strains of S. aureus (one reference strain, one strain with 1 enterotoxin, two strains with 3 enterotoxins and two strains resistant to methicillin, MRSA – one with 5 enterotoxins and the other without enterotoxins) were used. The effect of photosensitization on catalase activity, beta hemolysis, lipases, thermonuclease, enterotoxins, coagulase production and resistance to methicillin was assessed. The results indicate that the expression of some virulence factors in the cells subjected to this therapy is affected. Additionally the susceptibility of the strains to PDI did not decrease upon successive treatments.

Cannabinoids impact on pregnancy: effects in trophoblast cells

Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.

From genes to radioresistance in head and neck squamous cell carcinoma

Head and Neck Cancers (HNC) are a group of tumours located in the upper aero-digestive tract. Head and Neck Squamous Cell Carcinoma (HNSCC) represent about 90% of all HNC cases. It has been considered the sixth most malignant tumour worldwide and, despite clinical and technological advances, the five-year survival rate has not improved much in the last years. Nowadays, HNSCC is well established as a heterogeneous disease and that its development is due to accumulation of genetic events. Apart from the majority of the patients being diagnosed in an advanced stage, HNSCC is also a disease with poor therapeutic outcome. One of the therapeutic approaches is radiotherapy. However, this approach has different drawbacks like the radioresistance acquired by some tumour cells, leading to a worse prognosis. A major knowledge in radiation biology is imperative to improve this type of treatment and avoid late toxicities, maintaining patient quality of life in the subsequent years after treatment. Then, identification of genetic markers associated to radiotherapy response in patients and possible alterations in cells after radiotherapy are essential steps towards an improved diagnosis, higher survival rate and a better life quality. Not much is known about the radiation effects on cells, so, the principal aim of this study was to contribute to a more extensive knowledge about radiation treatment in HNSCC. For this, two commercial cell lines, HSC-3 and BICR-10, were used and characterized resorting to karyotyping, aCGH and MS-MLPA. These cell lines were submitted to different doses of irradiation and the resulting genetic and methylation alterations were evaluated. Our results showed a great difference in radiation response between the two cell lines, allowing the conclusion that HSC-3 was much more radiosensitive than BICR-10. Bearing this in mind, analysis of cell death, cell cycle and DNA damages was performed to try to elucidate the motifs behind this difference. The characterization of both cell lines allowed the confirmation that HSC-3 was derived from a metastatic tumour and the hypothesis that BICR-10 was derived from a dysplasia. Furthermore, this pilot study enabled the suggestion of some genetic and epigenetic alterations that cells suffer after radiation treatment. Additionally, it also allowed the association of some genetic characteristics that could be related to the differences in radiation response observable in this two cell lines. Taken together all of our results contribute to a better understanding of radiation effects on HNSCC allowing one further step towards the prediction of patients’ outcome, better choice of treatment approaches and ultimately a better quality of life.

O efeito do stress celular na proteína precursora de amilóide de Alzheimer = The effect of cellular stress on the Alzheimer's amyloid precursor protein

A proteína precursora de amilóide de Alzheimer (APP) é um factor chave na doença de Alzheimer (AD). Essencialmente o processamento da APP resulta na produção de Abeta, o peptídeo tóxico depositado nas placas de amilóide dos indivíduos com AD. Ainda permanece por esclarecer se o processamento da APP é afectado sob condições de stress celular, potencialmente aumentando a quantidade de Abeta produzida. Além disso, o stress celular pode induzir alterações moleculares, associadas à AD, que podem representar marcadores moleculares úteis para o diagnóstico da AD. Com estas questões em mente, procurámos identificar alterações, em resposta ao stress celular, no processamento da APP e na expressão de outras proteínas. Nestes estudos de monitorização considerámos que a fosforilação proteica anormal e o stress oxidativo podem contribuir para a condição patológica. Assim, investigámos o processamento da APP dependente da fosforilação durante o stress celular. Os dados obtidos confirmam que a secreção da APP é reduzida em situações de stress, e que o efeito é idêntico em linhas celulares de tipo neuronal e não neuronal. Os resultados obtidos revelam que o PMA, mesmo em situações de stress (azida de sódio 1 mM) pode afectar o processamento da APP, aumentando a produção de sAPP (o fragmento secretado após o processamento de APP) que pode potencialmente reduzir a produção de Abeta. A hipótese de afectar a produção de Abeta dependente da fosforilação, que por sua vez pode ter relevância num quadro clínico, mantém-se mesmo em condições de stress. Os resultados revelaram que a indução de sAPP, após a adição de ésteres de forbol, ou ácido ocadeíco, em condições de stress não é idêntica. Em contraste, sob condições controlo, tanto os ésteres de forbol como o ácido ocadeíco produzem o mesmo efeito em termos da produção de sAPP. Aparentemente estas duas vias podem ser dissociadas em condições de stress, o que de algum modo pode reflectir processamento alterado da APP em condições adversas. Nas experiências em que se analisou a expressão de outros potenciais marcadores moleculares, foram detectadas alterações nos níveis de expressão de várias proteínas. Estes marcadores moleculares representam alvos interessantes para futura validação e potenciais candidatos para um diagnóstico molecular na AD. As proteínas já identificadas são importantes do ponto de vista da transdução de sinais, e incluem a PP1, a HSP70, a PARP e a própria APP.

A morte e o luto em contexto escolar: das vivências na primeira pessoa à (re) significação do conceito de escola acolhedora

A morte é, porventura, o grande tabu da sociedade ocidental contemporânea, fenómeno com clara ressonância na forma como os sobreviventes vivenciam o luto por perda de figura significativa. Numa sociedade que se mantém à margem da morte, as emoções decorrentes do luto são escamoteadas e reprimidas, com sérios riscos para a saúde mental dos enlutados. Essa conspiração do silêncio desagua também nos contextos educativos, onde os valores da juventude, do bem-estar, do prazer e da felicidade, quase não deixam espaço para o sofrimento e a morte. Deste modo, este estudo pretende ser uma contribuição para a compreensão dos efeitos do processo de luto em alunos adolescentes, nomeadamente ao nível do seu desempenho escolar, e, concomitantemente, para a análise do tipo de apoio que a comunidade educativa proporciona a esses alunos, com enfoque no papel dos professores (em geral) e dos diretores de turma (em particular). Pretende ainda apresentar estratégias interventivas, a implementar nas escolas, potenciadoras de uma educação para a vida, mesmo em circunstâncias de morte, e para a gestão do luto, que se revelem promotoras de um lidar pedagógico inclusivo. Sendo o luto um processo que afeta o indivíduo em todas as dimensões que o definem, um paradigma educacional que encontre na complexidade a sua matriz identitária, foi assumido neste estudo como o único capaz de resgatar a importância da gestão equilibrada dos afetos no processo de ensinoaprendizagem. À luz deste paradigma que assume um princípio de totalidade, partindo da totalidade, ou seja, que promove o desenvolvimento do ser humano na sua multidimensionalidade, assumimos também que a missão suprema e última da educação é a construção do sujeito ético. E é nos marcos de uma educação integral, humanista e ética, de responsabilidade pelo Outro, que ganha contornos a figura do professor cuidador, como sendo alguém atento às necessidades emocionais dos seus alunos. Do ponto de vista metodológico, esta investigação desenvolveu-se de acordo com uma abordagem de natureza predominantemente qualitativa, interpretativa e complexa e o estudo realizado centrou-se em três fases, tendo a recolha de dados decorrido entre setembro de 2009 e setembro de 2012: (i) Estudo exploratório, dirigido aos Diretores das 61 escolas secundárias do Distrito do Porto, com base num questionário adaptado, com o objetivo de sustentar a importância e pertinência do estudo principal e recolher indicadores para o orientar; (ii) Estudo de Caso Coletivo, envolvendo três alunas adolescentes em luto por perda de pai. Na primeira parte, e com vista à caracterização do contexto, a escola frequentada pelas três alunas, procedeuse à análise documental, concretamente do Projeto Educativo, mas também ao inquérito por entrevista a vários agentes da comunidade educativa: o Diretor, a Psicóloga, a Coordenadora dos Diretores de Turma do ensino secundário e seis Diretores de Turma. Na segunda parte, procedeu-se a uma abordagem holística e aprofundada da complexidade inerente a cada caso, procurando dar “voz” à forma única como cada uma das alunas vivenciou, significou e enfrentou a sua experiência de luto, tendo-se recorrido ao inquérito por entrevista. De forma a cruzar perspetivas de vários informantes, e identificar significados transversais, complementares ou alternativos, foram também entrevistadas as Encarregadas de Educação e os Diretores de Turma das alunas; (iii) Caraterização das conceções de professores sobre a temática em análise e validação de propostas de intervenção a implementar nas escolas, mobilizando-se, assim, dimensões emergentes das Fases I e II da investigação, mas também do quadro teórico que sustentou o estudo. Para a recolha de dados, foi construído um questionário que se aplicou aos professores do 2º e 3º ciclos do ensino básico e ensino secundário do Agrupamento onde se centrou o estudo de caso desenvolvido na Fase II. Procurando uma leitura global dos resultados obtidos e corroborando o que é amplamente defendido na literatura da especialidade, o estudo demonstra o impacto negativo que o luto tem no desempenho escolar e revela que o padrão afetivo da ambiência escolar não é favorável à expressão emocional de alunos enlutados, prevalecendo uma atitude de evitação por parte dos colegas e professores, o que sugere a necessidade de serem trilhados novos caminhos, por um lado, ao nível da formação de professores e, por outro lado, através da implementação em contexto escolar de uma intervenção pedagógica que eduque para a vida, mas sem descurar os fenómenos de perda afetiva significativa e respetivas vivências de luto. Deste estudo sai também reforçada a responsabilidade acrescida dos Diretores de Turma na constituição de um ethos de suporte envolvendo alunos em luto, cabendo-lhes um importante papel na articulação entre os vários agentes da comunidade educativa. Em suma: para além da necessidade de repensar o perfil de competências dos professores, mais consentâneo com o paradigma de escola acolhedora, deste estudo ressalta também a premência de validar na prática propostas de intervenção articuladas, consistentes e, sobretudo, éticas.

Sinalização celular e a resposta a estrogénios do epitélio mamário

Estrogens, such as 17β-estradiol (E2) are essential for normal growth and differentiation of the mammary gland. There are two estrogen receptors (ERs), ERα and ERβ which are ligand activated transcription factors. ERα stimulates proliferation and is the single most powerful predictor of breast cancer prognosis and since 70% of breast cancers express ERα, strategies to block this receptor are the primary breast cancer treatment. Unlike ERα, the role of ERβ in breast cancer and its potential as alternative therapeutic target remains controversial, mainly due to the lack of correlation between results obtained in vitro and epidemiological studies. The aim of this thesis was to increase our understanding of the molecular and cellular mechanisms of estrogen signaling in normal and cancerous cells, in different cellular contexts and with focus on ERβ. In Paper I we characterized the effect of the flavone PD098059 - which is a commonly used MEK1 inhibitor - on activation of transcription by ERα and ERβ. We found that the estrogenic effect of PD098059 is dose dependent in concentrations ranging from 1 – 10 μM and that activation of transcription by ER is suppressed by the inhibitory effect of PD98059 on MEK1 at concentrations above 50 μM. In agreement with its flavone nature, PD098059 had a much stronger effect on ERβ than on ERα transcriptional activity. Therefore, use of this compound for the study of signalling events in cells expressing ER should be carefully considered. In Paper II we assessed the effect of ERβ agonists in vivo and administered under different conditions in vitro. In basal conditions, ERβ induced apoptosis; however, in vivo ERβ agonists stimulated proliferation and inhibited apoptosis. In vivo effects were reproduced in culture, by activation of MAPK/ERK½ pathway with epidermal growth factor or basement membrane extract. In addition, insulin signalling and PI3-K/AKT activation was necessary for stimulation of proliferation. These results suggest that the cellular context modulates ERβ activity. Manuscript presents preliminary work aimed at the set-up of a methodological strategy to isolate ERs and to identify interacting proteins in different cellular contexts and which could modulate the bi-phased effects of ERβ in cell growth. In conclusion, the studies presented in this thesis contribute to clarify the apparent contradictory information regarding ERβ function in normal and cancerous mammary epithelium and suggest that the cellular context should be considered when ERβ effects are studied.

Functional analysis of String/CDC25 phosphatase in post-mitotic neurons

Cell cycle and differentiation are two highly coordinated processes during organ development. Recent studies have demonstrated that core cell cycle regulators also play cell cycle-independent functions in post-mitotic neurons, and are essential for the maintenance of neuronal homeostasis. CDC25 phosphatases are well-established CDK activators and their activity is mainly associated to proliferating tissues. The expression and activity of mammalian CDC25s has been reported in adult brains. However, their physiological relevance and the potential substrates in a non-proliferative context have never been addressed. string (stg) encodes the Drosophila CDC25 homolog. Previous studies from our group showed that stg is expressed in photoreceptors (PRs) and in lamina neurons, which are two differentiated cell types that compose the fly visual system. The aims of this work are to uncover the function of stg and to identify its potential neuronal substrates, using the Drosophila visual system as a model. To gain insight into the function of stg in a non-dividing context we used the GAL4/UAS system to promote downregulation of stg in PR-neurons, through the use of an RNAi transgene. The defects caused by stg loss-of-function were evaluated in the developing eye imaginal disc by immunofluorescence, and during adult stages by scanning electron microscopy. This genetic approach was combined with a specific proteomic method, two-dimensional difference gel electrophoresis (2D-DIGE), to identify the potential substrates in PR-cells. Our results showed that stg downregulation in PRs affects the well-patterned retina organization, inducing the loss of apical maintenance of PR-nuclei on the eye disc, and ommatidia disorganization. We also detected an abnormal accumulation of cytoskeletal proteins and a disruption of the axon structure. As a consequence, the projection of PR-axons into the lamina and medulla neuropils of the optic lobe was impaired. Upon stg downregulation, we also detected that PR-cells accumulate Cyclin B. Although the rough eye phenotype observed upon stg downregulation suggests neurodegeneration, we did not detect neuronal death during larval stages, suggesting that it likely occurs during pupal stages or during adulthood. By 2D-DIGE, we identified seven proteins which were differentially expressed upon stg downregulation, and are potential neuronal substrates of Stg. Altogether, our observations suggest that Stg phosphatase plays an essential role in the Drosophila visual system neurons, regulating several cell components and processes in order to ensure their homeostasis.

Physiology and metabolic profile of Moringa olifera and Eucalyptus globulus plants subjected to climate changes

As alterações climáticas emergentes têm um grande impacto no crescimento e desenvolvimento de espécies florestais, nomeadamente em espécies de valor industrial e medicinal, como é o caso do eucalipto (Eucalyptus globulus) e da moringa (Moringa oleifera). Assim, é urgente conhecer as respostas fisiológicas e entender as variações que ocorrem nos perfis metabólicos de espécies vegetais. Neste trabalho, plantas jovens de Eucalyptus globulus foram expostas a radiação UVB (12kJ/m2) e foram avaliadas as respostas fisiológicas e o perfil metabólico, um e onze dias após a aplicação da radiação. A dose de UVB usada não afetou as reações fotoquímicas nem as trocas gasosas, contudo ao nível do metabolismo do carbono (AST e amido) e do conteúdo de pigmentos verificaram-se pequenas alterações (AST e pigmentos). Através da análise do perfil metabólico de E. globulus foram encontrados compostos voláteis e semi-voláteis pertencentes às famílias dos terpenos, sesquiterpenos e aldeídos. Em geral, os sesquiterpenos e os álcoois monoterpénicos apresentaram uma tendência para manter e, em alguns casos, diminuir com o stress, enquanto que o grupos dos aldeídos aumentou e os monoterpenos apresentaram um comportamento mais heterogéneo. O E. globulus mostrou ser uma espécie tolerante à aplicação da dose de UVB usada neste trabalho. Por outro lado, plantas jovens de M. oleifera foram expostas a défice hídrico (DH). Um grupo de plantas foi recolhido um dia após o final da exposição e o outro grupo após onze dias do final da exposição. Foi avaliado o perfil metabólico desta espécie através de GC/MS. Os dados cromatográficos indicaram que em condições de stress (DH(1) e DH(11)), as quantidades de compostos associados a vias primárias e secundárias de defesa (como os alcanos, álcoois, ácidos carboxílicos, esteróis, aminoácidos e açucares) sofreram algumas alterações. As plantas analisadas 11 dias após a remoção do stress mostraram maiores variações do perfil de metabolitos. No entanto, tanto um como onze dias após a remoção do stress, as plantas apresentaram a formação de novos rebentos. Apesar do perfil de metabolitos ter sofrido algumas alterações, por não se registarem casos de morte, conclui-se que as plantas de moringa mostraram ser tolerantes aos tratamentos aplicados.