Characterization of proteotoxic stress in zebrafish

A fidelidade da síntese proteica é fundamental para a estabilidade do proteoma e para a homeostasia celular. Em condições fisiológicas normais as células têm uma taxa de erro basal associada e esta muitas vezes aumenta com o envelhecimento e doença. Problemas na síntese das proteínas estão associados a várias doenças humanas e aos processos de envelhecimento. De facto, a incorporação de erros nas proteínas devido a tRNAs carregados pelas aminoacil-tRNA sintetases com o amino ácido errado causa doenças neurodegenerativas em humanos e ratos. Ainda não é claro como é que estas doenças se desenvolvem e se são uma consequência directa da disrupção do proteoma ou se são o resultado da toxicidade produzida pela acúmulação de proteínas mal traduzidas ao nível do ribossoma. Para elucidar como é que as células eucarióticas lidam com proteínas aberrantes e agregados proteicos (stress proteotóxico) desenvolvemos uma estratégia para destabilizar o proteoma. Para isso estabelecemos um sistema de erros de tradução em embriões de peixe zebra que assenta em tRNAs mutantes capazes de incorporar erradamente serina nas proteínas. As proteínas produzidas neste sistema despoletam as vias de resposta ao stress, nomeadamente a via da ubiquitina-proteassoma (UPP – “ubiquitin protesome pathway”) e a via do retículo endoplasmático (UPR – “unfolded protein response”). O stress proteotóxico gerado pelos erros de tradução altera a expressão génica e perfis de expressão de miRNAs, o desenvolvimento embrionário e viabilidade, aumenta a produção de espécies reactivas de oxigénio (ROS), leva ainda à acumulação de agregados proteicos e à disfunção mitocondrial. As malformações embrionárias e fenótipos de viabilidade que observámos foram revertidos por antioxidantes, o que sugere que os ROS desempenham papéis importantes nos fenótipos degenerativos celulares induzidos pela produção de proteínas aberrantes e agregação proteica. Estabelecemos ainda uma linha de peixe zebra transgénica para o estudo do stress proteotóxico. Este trabalho mostra que a destabilização do proteoma em embriões de peixe zebra com tRNAs mutantes é uma boa metodologia para estudar a biologia do stress proteotóxico visto que permite a agregação controlada do proteoma, mimetizando os processos de agregação de proteínas que ocorrem naturalmente durante o envelhecimento e em doenças conformacionais humanas.

Pregnancy and newborns disorders followed by urine metabolomics

Chapter 1 introduces the scope of the work by identifying the clinically relevant prenatal disorders and presently available diagnostic methods. The methodology followed in this work is presented, along with a brief account of the principles of the analytical and statistical tools employed. A thorough description of the state of the art of metabolomics in prenatal research concludes the chapter, highlighting the merit of this novel strategy to identify robust disease biomarkers. The scarce use of maternal and newborn urine in previous reports enlightens the relevance of this work. Chapter 2 presents a description of all the experimental details involved in the work performed, comprising sampling, sample collection and preparation issues, data acquisition protocols and data analysis procedures. The proton Nuclear Magnetic Resonance (NMR) characterization of maternal urine composition in healthy pregnancies is presented in Chapter 3. The urinary metabolic profile characteristic of each pregnancy trimester was defined and a 21-metabolite signature found descriptive of the metabolic adaptations occurring throughout pregnancy. 8 metabolites were found, for the first time to our knowledge, to vary in connection to pregnancy, while known metabolic effects were confirmed. This chapter includes a study of the effects of non-fasting (used in this work) as a possible confounder. Chapter 4 describes the metabolomic study of 2nd trimester maternal urine for the diagnosis of fetal disorders and prediction of later-developing complications. This was achieved by applying a novel variable selection method developed in the context of this work. It was found that fetal malformations (FM) (and, specifically those of the central nervous system, CNS) and chromosomal disorders (CD) (and, specifically, trisomy 21, T21) are accompanied by changes in energy, amino acids, lipids and nucleotides metabolic pathways, with CD causing a further deregulation in sugars metabolism, urea cycle and/or creatinine biosynthesis. Multivariate analysis models´ validation revealed classification rates (CR) of 84% for FM (87%, CNS) and 85% for CD (94%, T21). For later-diagnosed preterm delivery (PTD), preeclampsia (PE) and intrauterine growth restriction (IUGR), it is found that urinary NMR profiles have early predictive value, with CRs ranging from 84% for PTD (11-20 gestational weeks, g.w., prior to diagnosis), 94% for PE (18-24 g.w. pre-diagnosis) and 94% for IUGR (2-22 g.w. pre-diagnosis). This chapter includes results obtained for an ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) study of pre-PTD samples and correlation with NMR data. One possible marker was detected, although its identification was not possible. Chapter 5 relates to the NMR metabolomic study of gestational diabetes mellitus (GDM), establishing a potentially predictive urinary metabolic profile for GDM, 2-21 g.w. prior to diagnosis (CR 83%). Furthermore, the NMR spectrum was shown to carry information on individual phenotypes, able to predict future insulin treatment requirement (CR 94%). Chapter 6 describes results that demonstrate the impact of delivery mode (CR 88%) and gender (CR 76%) on newborn urinary profile. It was also found that newborn prematurity, respiratory depression, large for gestational age growth and malformations induce relevant metabolic perturbations (CR 82-92%), as well as maternal conditions, namely GDM (CR 82%) and maternal psychiatric disorders (CR 91%). Finally, the main conclusions of this thesis are presented in Chapter 7, highlighting the value of maternal or newborn urine metabolomics for pregnancy monitoring and disease prediction, towards the development of new early and non-invasive diagnostic methods.

Mitochondrial dysfunction in fatty acid β-oxidation disorders

Mitochondria are central organelles for cell survival with particular relevance in energy production and signalling, being mitochondrial fatty acid β–oxidation (FAO) one of the metabolic pathways harboured in this organelle. FAO disorders (FAOD) are among the most well studied inborn errors of metabolism, mainly due to their impact in health. Nevertheless, some questions remain unsolved, as their prevalence in certain European regions and how pathophysiological determinants combine towards the phenotype. Analysis of data from newborn screening programs from Portugal and Spain allowed the estimation of the birth prevalence of FAOD revealing that this group of disorders presents in Iberia (and particularly in Portugal) one of the highest European birth prevalence, mainly due to the high birth prevalence of medium chain acyl-CoA dehydrogenase deficiency. These results highlight the impact of this group of genetic disorders in this European region. The characterization of mitochondrial proteome, from patients fibroblasts with FAOD, namely multiple acyl-CoA dehydrogenase deficiency (MADD) and long chain acyl-CoA dehydrogenase deficiency (LCHADD), provided a global perspective of the mitochondrial proteome plasticity in these disorders and highlights the main molecular pathways involved in their pathogenesis. Severe MADD forms show an overexpression of chaperones, antioxidant enzymes (MnSOD), and apoptotic proteins. An overexpression of glycolytic enzymes, which reflects cellular adaptation to energy deficiency due to FAO blockage, was also observed. When LCHADD fibroblasts were analysed a metabolic switching to glycolysis was also observed with overexpression of apoptotic proteins and modulation of the antioxidant defence system. Severe LCHADD present increased ROS alongside with up regulation of MnSOD while moderate forms have lower ROS and down-regulation of MnSOD. This probably reflects the role of MnSOD in buffering cellular ROS, maintain them at levels that allow cells to avoid damage and start a cellular response towards survival. When ROS levels are very high cells have to overexpress MnSOD for detoxifying proposes. When severe forms of MADD were compared to moderate forms no major differences were noticed, most probably because ROS levels in moderate MADD are high enough to trigger a response similar to that observed in severe forms. Our data highlights, for the first time, the differences in the modulation of antioxidant defence among FAOD spectrum. Overall, the data reveals the main pathways modulated in FAOD and the importance of ROS levels and antioxidant defence system modulation for disease severity. These results highlight the complex interaction between phenotypic determinants in FAOD that include genetic, epigenetic and environmental factors. The development of future better treatment approaches is dependent on the knowledge on how all these determinants interact towards phenotype.!

Lipidomic analysis of mesenchymal cells candidates for cell therapy

Mesenchymal stromal cells are adult stem cells found mostly in the bone marrow. They have immunosuppressive properties and they have been successfully applied as biological therapy in several clinical trials regarding autoimmune diseases. Despite the great number of clinical trials, MSCs’ action is not fully understand and there are no identified markers that correlate themselves with the immunomodulatory power. A lipidomic approach can solve some of these problems once lipids are one of the major cells’ components. Therefore, in this study cells’ lipidome was analysed and its deviations were evaluated according to the medium of culture and to the presence of pro-inflammatory stimuli, mimicking physiological conditions in which these cells are used. This was the first study ever made that aimed to analyse the differences in the phospholipid profile between mesenchymal stromal cells non-stimulated and stimulated with proinflammatory stimulus. This analysis was conducted in both cells cultured in medium supplemented with animal serum and in cells cultured in a synthetic medium. In cells cultured in the standard medium the levels of phosphatidylcholine (PC) species with shorter fatty acids (FAs) acyl chains decreased under pro-inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of lysoPC (LPC)(18:0) - an anti-inflammatory LPC - observed in cells subjected to TNF-α and IFN-γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF-α and IFN- γ also enhanced the levels of phosphatidylethanolamine PE(40:6) and decreased the levels of PE(38:6). Higher expression of phosphatidylserine PS(36:1) and sphingomyelin SM(34:0) along with a decrease in PS(38:6) levels were observed. However, in cells cultured in a synthetic medium, TNF-α and IFN-γ only enhanced the levels of PS(36:1). These results indicate that lipid metabolism and signaling is modulated during mesenchymal stromal cells action.

Development of infrared spectroscopy for assessing bacterial quality in foods

Rapid and specific detection of foodborne bacteria that can cause food spoilage or illness associated to its consumption is an increasingly important task in food industry. Bacterial detection, identification, and classification are generally performed using traditional methods based on biochemical or serological tests and the molecular methods based on DNA or RNA fingerprints. However, these methodologies are expensive, time consuming and laborious. Infrared spectroscopy is a reliable, rapid, and economic technique which could be explored as a tool for bacterial analysis in the food industry. In this thesis it was evaluated the potential of IR spectroscopy to study the bacterial quality of foods. In Chapter 2, it was developed a calibration model that successfully allowed to predict the bacterial concentration of naturally contaminated cooked ham samples kept at refrigeration temperature during 8 days. In this part, it was developed the methodology that allowed the best reproducibility of spectra from bacteria colonies with minimal sample preparation, which was used in the subsequent work. Several attempts trying different resolutions and number of scans in the IR were made. A spectral resolution of 4 cm-1, with 32 scans were the settings that allowed the best results. Subsequently, in Chapter 3, it was made an attempt to identify 22 different foodborne bacterial genera/species using IR spectroscopy coupled with multivariate analysis. The principal component analysis, used as an exploratory technique, allowed to form distinct groups, each one corresponding to a different genus, in most of the cases. Then, a hierarchical cluster analysis was performed to further analyse the group formation and the possibility of distinction between species of the same bacterial genus. It was observed that IR spectroscopy not only is suitable to the distinction of the different genera, but also to differentiate species of the same genus, with the simultaneous use of principal component analysis and cluster analysis techniques. The utilization of IR spectroscopy and multivariate statistical analysis were also investigated in Chapter 4, in order to confirm the presence of Listeria monocytogenes and Salmonella spp. isolated from contaminated foods, after growth in selective medium. This would allow to substitute the traditional biochemical and serological methods that are used to confirm these pathogens and that delay the obtainment of the results up to 2 days. The obtained results allowed the distinction of 3 different Listeria species and the distinction of Salmonella spp. from other bacteria that can be mistaken with them. Finally, in chapter 5, high pressure processing, an emerging methodology that permits to produce microbiologically safe foods and extend their shelf-life, was applied to 12 foodborne bacteria to determine their resistance and the effects of pressure in cells. A treatment of 300 MPa, during 15 minutes at room temperature was applied. Gram-negative bacteria were inactivated to undetectable levels and Gram-positive showed different resistances. Bacillus cereus and Staphylococcus aureus decreased only 2 logs and Listeria innocua decreased about 5 logs. IR spectroscopy was performed in bacterial colonies before and after HPP in order to investigate the alterations of the cellular compounds. It was found that high pressure alters bands assigned to some cellular components as proteins, lipids, oligopolysaccharides, phosphate groups from the cell wall and nucleic acids, suggesting disruption of the cell envelopes. In this work, bacterial quantification and classification, as well as assessment of cellular compounds modification with high pressure processing were successfully performed. Taking this into account, it was showed that IR spectroscopy is a very promising technique to analyse bacteria in a simple and inexpensive manner.

Aplicação da terapia fotodinâmica no processo de esterilização de sangue

A importância médica do sangue associada ao risco de doenças infeciosas levou a um melhoramento das técnicas de rastreio de patogénicos no sangue doado. No entanto, devido aos períodos de "janela", durante o qual os agentes infeciosos não podem ser detetados, a desinfeção de sangue e seus derivados assume uma importância vital. Considerando que as técnicas convencionais de desinfeção (tratamento com solvente-detergente ou irradiação com UV ou radiação gama) pode ser empregue em concentrados de plasma ou de proteínas, o efeito colateral associado aos respetivos tratamentos não permite a sua utilização em frações celulares. Consequentemente, é necessário o desenvolvimento de uma nova alternativa eficaz para inativar microrganismos em sangue. Uma boa estratégia que merece ser considerada baseia-se na terapia fotodinâmica antimicrobiana (aPDT). aPDT envolve a interação entre a luz e um fotossensibilizador (PS) na presença de oxigénio molecular. Esta interação produz espécies reativas de oxigénio (ROS), que causam danos oxidativos às moléculas microbianas necessárias à sobrevivência do microrganismo. Em alguns países, esta metodologia já está aprovada para descontaminação de plasma, utilizando azul de metileno ou psoraleno como PSs. O objetivo deste estudo foi avaliar a adequação de de estrutura do tipo ftalocianina (Pc) e porfirina (Por) para desinfeção fotodinâmica de hemoderivados. Plasma e sangue total foram infetados com 108 unidades formadoras de colónias (CFU) / mL de Escherichia coli e após incubação com os derivados Pc e Por em estudo, expostos respetivamente a luz vermelha ou a luz branca com uma irradiância de 150 W/m2durante 270 min. As concentrações de E. coli viáveis foram determinadas a 0, 30, 60, 90, 180 e 270 min e comparadas com as obtidas nos controlos claro (amostras irradiadas na ausência de PS) e controlos escuro (amostras incubadas com PS mas não irradiadas). O efeito do tratamento aPDT nas células do sangue (glóbulos vermelhos e brancos) também foi avaliado. Os resultados obtidos mostram que, em todos os componentes do sangue, a Por em estudo é mais eficaz na inativação de E. coli que o derivado Pc. Após o tratamento aPDT, o número de células vermelhas e brancas no sangue é semelhante aos valores observados nas amostras de controlo. A eficiente inativação de células de E. coli e a ausência de efeito sobre as células de sangue transformam os derivados porfirínicos e ftalocianinas potenciais candidatos a serem utilizados com fotossensibilizadores na desinfeção fotodinâmica de produtos derivados do sangue.

Infection mechanism of Diplodia corticola

Diplodia corticola is regarded as the most virulent fungus involved in cork oak decline, being able to infect not only Quercus species (mainly Q. suber and Q. ilex), but also grapevines (Vitis vinifera) and eucalypts (Eucalyptus sp.). This endophytic fungus is also a pathogen whose virulence usually manifests with the onset of plant stress. Considering that the infection normally culminates in host death, there is a growing ecologic and socio-economic concern about D. corticola propagation. The molecular mechanisms of infection are hitherto largely unknown. Accordingly, the aim of this study was to unveil potential virulence effectors implicated in D. corticola infection. This knowledge is fundamental to outline the molecular framework that permits the fungal invasion and proliferation in plant hosts, causing disease. Since the effectors deployed are mostly proteins, we adopted a proteomic approach. We performed in planta pathogenicity tests to select two D. corticola strains with distinct virulence degrees for our studies. Like other filamentous fungi D. corticola secretes protein at low concentrations in vitro in the presence of high levels of polysaccharides, two characteristics that hamper the fungal secretome analysis. Therefore, we first compared several methods of extracellular protein extraction to assess their performance and compatibility with 1D and 2D electrophoretic separation. TCA-Acetone and TCA-phenol protein precipitation were the most efficient methods and the former was adopted for further studies. The proteins were extracted and separated by 2D-PAGE, proteins were digested with trypsin and the resulting peptides were further analysed by MS/MS. Their identification was performed by de novo sequencing and/or MASCOT search. We were able to identify 80 extracellular and 162 intracellular proteins, a milestone for the Botryosphaeriaceae family that contains only one member with the proteome characterized. We also performed an extensive comparative 2D gel analysis to highlight the differentially expressed proteins during the host mimicry. Moreover, we compared the protein profiles of the two strains with different degrees of virulence. In short, we characterized for the first time the secretome and proteome of D. corticola. The obtained results contribute to the elucidation of some aspects of the biology of the fungus. The avirulent strain contains an assortment of proteins that facilitate the adaptation to diverse substrates and the identified proteins suggest that the fungus degrades the host tissues through Fenton reactions. On the other hand, the virulent strain seems to have adapted its secretome to the host characteristics. Furthermore, the results indicate that this strain metabolizes aminobutyric acid, a molecule that might be the triggering factor of the transition from a latent to a pathogenic state. Lastly, the secretome includes potential pathogenicity effectors, such as deuterolysin (peptidase M35) and cerato-platanin, proteins that might play an active role in the phytopathogenic lifestyle of the fungus. Overall, our results suggest that D. corticola has a hemibiotrophic lifestyle, switching from a biotrophic to a necrotrophic interaction after plant physiologic disturbances.This understanding is essential for further development of effective plant protection measures.

Impact of exercise training on cancer-induced cardiac dysfunction

Cachexia is a complex syndrome characterized by severe weight loss frequently observed in cancer patients and associated with poor prognosis. Cancer cachexia is also related to modifications in cardiac muscle structure and metabolism leading to cardiac dysfunction. In order to better understand the cardiac remodeling induced by bladder cancer and the impact of exercise training after diagnosis on its regulation, we used an animal model of bladder cancer induced by exposition to N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in the drinking water. Healthy animals and previously BBN exposed animals were submitted to a training program in a treadmill at a speed of 20m/min, 60 min/day, 5 days/week during 13 weeks. At the end of the protocol, animals exposed to BBN presented a significant decrease of body weight, in comparison with control groups, supporting the presence of cancer cachexia. Morphological analysis of the cardiac muscle sections revealed the presence of fibrosis and a significant decrease of cardiomyocyte’s cross-sectional area, suggesting the occurrence of cardiac dysfunction associated with bladder cancer. These modifications were accompanied by heart metabolic remodeling characterized by a decreased fatty acid oxidation given by diminished levels of ETFDH and of complex II subunit  from the respiratory chain. Exercise training promoted an increment of connexin 43, a protein involved in cardioprotection, and of c-kit, a protein present in cardiac stem cells. These results suggest an improved heart regenerative capacity induced by exercise training. In conclusion, endurance training seems an attractive non-pharmacological therapeutic option for the management of cardiac dysfunction in cancer cachexia.

Identification of molecules and pathways regulating mistranslation in Candida albicans

Candida albicans is the major fungal pathogen in humans, causing diseases ranging from mild skin infections to severe systemic infections in immunocompromised individuals. The pathogenic nature of this organism is mostly due to its capacity to proliferate in numerous body sites and to its ability to adapt to drastic changes in the environment. Candida albicans exhibit a unique translational system, decoding the leucine-CUG codon ambiguously as leucine (3% of codons) and serine (97%) using a hybrid serine tRNA (tRNACAGSer). This tRNACAGSer is aminoacylated by two aminoacyl tRNA synthetases (aaRSs): leucyl-tRNA synthetase (LeuRS) and seryl-tRNA synthetase (SerRS). Previous studies showed that exposure of C. albicans to macrophages, oxidative, pH stress and antifungals increases Leu misincorporation levels from 3% to 15%, suggesting that C. albicans has the ability to regulate mistranslation levels in response to host defenses, antifungals and environmental stresses. Therefore, the hypothesis tested in this work is that Leu and Ser misincorporation at CUG codons is dependent upon competition between the LeuRS and SerRS for the tRNACAGSer. To test this hypothesis, levels of the SerRS and LeuRS were indirectly quantified under different physiological conditions, using a fluorescent reporter system that measures the activity of the respective promoters. Results suggest that an increase in Leu misincorporation at CUG codons is associated with an increase in LeuRS expression, with levels of SerRS being maintained. In the second part of the work, the objective was to identify putative regulators of SerRS and LeuRS expression. To accomplish this goal, C. albicans strains from a transcription factor knock-out collection were transformed with the fluorescent reporter system and expression of both aaRSs was quantified. Alterations in the LeuRS/SerRS expression of mutant strains compared to wild type strain allowed the identification of 5 transcription factors as possible regulators of expression of LeuRS and SerRS: ASH1, HAP2, HAP3, RTG3 and STB5. Globally, this work provides the first step to elucidate the molecular mechanism of regulation of mistranslation in C. albicans.

Uma abordagem de matabolómica no estudo comparativo do metabolismo de glucose e galactose no patogénico humano Streptococcus pneumoniae

Dissertação mest., Ciências Biomédicas, Universidade do Algarve, 2010

Optimisation of an aptamer-biosensor to detect C-reactive protein

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009