Oxidation and glycoxidation of phosphatidylserine and their effect in immune response : a lipidomic approach

Phosphatidylserine (PS) is a member of the class of phospholipids, and is distributed among all cells of mammalians, playing important roles in diverse biological processes, including blood clotting and apoptosis. When externalized, PS is a ligand that is recognized on apoptotic cells. It has been considered that before externalization PS is oxidized and oxPS enhance the recognition by macrophages receptors, however the knowledge about oxidation of PS is still limited. PS, like others phospholipids, has two fatty acyl chains and one polar head group, in this case is the amino acid serine. The modifications in PS structure can occur by oxidation of the unsaturated fatty acyl chains and by glycation of the polar head group, due to free amine group, thus increasing the susceptibility to oxidative events. The main goal of this work was to characterize and identify oxidized and glycoxidized PS, contributing to the knowledge of the biological role of oxidation products of PS, as well as of glycated PS, in immune and inflammatory processes. To achieve this goal, PS standards (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho- L-serine (POPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (DPPS), 1- palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (PLPS) and 1-palmitoyl-2- arachidonoyl-sn-glycero-3-phospho-L-serine (PAPS)) and glycated PS (PAPS and POPS) were induced to oxidize in model systems, using different oxidant reagents: HO��� and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) . The detailed structural characterization of the oxidative products was performed by ESI-MS and MS/MS coupled to separation techniques such as off line TLC-MS and on line LC-MS, in order to obtained better characterization of the larger number of PS and glycated PS oxidation products. The results obtained in this work allowed to identify several oxidation products of PS and glycated PS with modifications in unsaturated fatty acyl chain. Also, oxidation products formed due to structural changes in the serine polar head with formation of terminal acetamide, terminal hydroperoxyacetaldehyde.and terminal acetic acid (glycerophosphacetic acid, GPAA) were identified. The mass spectrometric specific fragmentation pathway of each type of oxidation product was determined using different mass spectrometry approaches. Based on the identified fragmentation pathways, targeted lipidomic analysis was performed to detect oxidation products modified in serine polar head in HaCaT cell line treated with AAPH. The GPAA was detected in HaCaT cells treated with AAPH to induce oxidative stress, thus confirming that modifications in PS polar head is possible to occur in biological systems. Furthermore, it was found that glycated PS species are more prone to oxidative modifications when compared with non glycated PS. During oxidation of glycated PS, besides the oxidation in acyl chains, new oxidation products due to oxidation of the glucose moiety were identified, including PS advanced glycation end products (PSAGES). To investigate if UVA oxidative stress exerted changes in the lipidome of melanoma cell lines, particularly in PS profile, a lipidomic analysis was performed. The lipid profile was obtained using HILIC-LC-MS and GC-MS analysis of the total lipid extracts obtained from human melanoma cell line (SKMEL- 28) after UVA irradiation at 0, 2 and 24 hours. The results did not showed significant differences in PS content. At molecular level, only PS (18:0:18:1) decreased at the moment of irradiation. The most significant changes in phospholipids content occurred in phosphatidylcholines (PC) and phosphatidylinositol (PI) classes, with an increase of mono-unsaturated fatty acid (MUFA), similarly as observed for the fatty acid analysis. Overall, these data indicate that the observed membrane lipid changes associated with lipogenesis after UVA exposure may be correlated with malignant transformations associated with cancer development and progression. Despite of UVA radiation is associated with oxidative damage, in this work was not possible observe oxidation phospholipids. The anti/pro-inflammatory properties of the oxidized PLPS (oxPLPS) versus non-oxidized PLPS were tested on LPS stimulated RAW 264.7 macrophages. The modulation of intracellular signaling pathways such as NF-kB and MAPK cascades by oxPLPS and PS was also examined in this study. The results obtained from evaluation of anti/pro-inflammatory properties showed that neither PLPS or oxPLPS species activated the macrophages. Moreover only oxidized PLS were found to significantly inhibit NO production and iNOS and il1�� gene transcription induced by LPS. The analysis at molecular level showed that this was the result of the attenuation of LPS-induced c-Jun-N-terminal kinase (JNK) and p65 NF-kB nuclear translocation. Overall these data suggest that oxPLPS, but not native PLPS, mitigates pro-inflammatory signaling in macrophages, contributing to containment of inflammation during apoptotic cell engulfment. The results obtained in this work provides new information on the modifications of PS, facilitating the identification of oxidized species in complex samples, namely under physiopathologic conditions and also contributes to a better understanding of the role of oxPS and PS in the inflammatory response, in the apoptotic process and other biological functions.

Development of polymeric materials to inhibit bacterial quorum sensing

Bacterial infections are an increasing problem for human health. In fact, an increasing number of infections are caused by bacteria that are resistant to most antibiotics and their combinations. Therefore, the scientific community is currently searching for new solutions to fight bacteria and infectious diseases, without promoting antimicrobial resistance. One of the most promising strategies is the disruption or attenuation of bacterial Quorum Sensing (QS), a refined system that bacteria use to communicate. In a QS event, bacteria produce and release specific small chemicals, signal molecules - autoinducers (AIs) - into the environment. At the same time that bacterial population grows, the concentration of AIs in the bacterial environment increases. When a threshold concentration of AIs is reached, bacterial cells respond to it by altering their gene expression profile. AIs regulate gene expression as a function of cell population density. Phenotypes mediated by QS (QSphenotypes) include virulence factors, toxin production, antibiotic resistance and biofilm formation. In this work, two polymeric materials (linear polymers and molecularly imprinted nanoparticles) were developed and their ability to attenuate QS was evaluated. Both types of polymers should to be able to adsorb bacterial signal molecules, limiting their availability in the extracellular environment, with expected disruption of QS. Linear polymers were composed by one of two monomers (itaconic acid and methacrylic acid), which are known to possess strong interactions with the bacterial signal molecules. Molecularly imprinted polymer nanoparticles (MIP NPs) are particles with recognition capabilities for the analyte of interest. This ability is attained by including the target analyte at the synthesis stage. Vibrio fischeri and Aeromonas hydrophila were used as model species for the study. Both the linear polymers and MIP NPs, tested free in solutions and coated to surfaces, showed ability to disrupt QS by decreasing bioluminescence of V. fischeri and biofilm formation of A. hydrophila. No significant effect on bacterial growth was detected. The cytotoxicity of the two types of polymers to a fibroblast-like cell line (Vero cells) was also tested in order to evaluate their safety. The results showed that both the linear polymers and MIP NPs were not cytotoxic in the testing conditions. In conclusion, the results reported in this thesis, show that the polymers developed are a promising strategy to disrupt QS and reduce bacterial infection and resistance. In addition, due to their low toxicity, solubility and easy integration by surface coating, the polymers have potential for applications in scenarios where bacterial infection is a problem: medicine, pharmaceutical, food industry and in agriculture or aquaculture.

Transforma����es organocatal��ticas e mediadas por metais de transi����o na s��ntese de novos heterociclos nitrogenados e ciclo-hexanos

Nas ��ltimas d��cadas a quiralidade tornou-se essencial na conce����o, descoberta, desenvolvimento e comercializa����o de novos medicamentos. A import��ncia da quiralidade na efic��cia e seguran��a dos f��rmacos tem sido globalmente reconhecida tanto pelas ind��strias farmac��uticas como pelas ag��ncias reguladoras de todo o mundo. De forma a produzir eficazmente medicamentos seguros e dar resposta �� demanda da ind��stria de compostos enantiomericamente puros, a pesquisa de novos m��todos de s��ntese assim��trica, assim como o desenvolvimento estrat��gico dos m��todos j�� dispon��veis tem sido um dos principais objetos de estudo de diversos grupos de investiga����o tanto na academia como na ind��stria farmac��utica No primeiro cap��tulo desta disserta����o s��o introduzidos alguns dos conceitos fundamentais associados �� s��ntese de mol��culas quirais e descritas algumas das estrat��gias que podem ser utilizadas na sua s��ntese. Apresenta-se ainda uma breve revis��o bibliogr��fica acerca dos antecedentes do grupo de investiga����o e sobre a ocorr��ncia natural, atividade biol��gica e m��todos de s��ntese e transforma����es de compostos do tipo (E,E)-cinamilidenoacetofenona. O segundo cap��tulo centra-se na adi����o de Michael enantiosseletiva de diversos nucle��filos a derivados de (E,E)-cinamilidenoacetofenona. Inicialmente descreve-se a s��ntese de derivados de (E,E)-cinamilidenoacetofenona atrav��s de uma condensa����o ald��lica de acetofenonas e cinamalde��dos apropriadamente substitu��dos. Estes derivados s��o posteriormente utilizados como substratos na adi����o de Michael enantiosseletiva de tr��s diferentes nucle��filos: nitrometano, malononitrilo e 2-[(difenilmetileno)amino]acetato de metilo. Nestas rea����es s��o utilizados diferentes organocatalisadores de forma a induzir enantiosseletividade nos aductos de Michael para serem utilizados na s��ntese de compostos com potencial interesse terap��utico. �� descrita ainda uma nova metodologia de s��ntese de ��1-pirrolinas atrav��s de um procedimento one-pot de redu����o/cicliza����o/desidrata����o mediada por ferro na presen��a de ��cido ac��tico de (R,E)-1,5-diaril-3-(nitrometil)pent-4-en-1-onas com bons rendimentos e excelentes excessos enantiom��ricos. O terceiro cap��tulo centra-se no estabelecimento de novas rotas de s��ntese e transforma����o de derivados do ciclo-hexano. Ap��s uma breve revis��o bibliogr��fica, s��o descritas tr��s metodologias enantiosseletivas distintas, sendo que a primeira envolve a utiliza����o de organocatalisadores e catalisadores de transfer��ncia de fase derivados de alcaloides cinchona. Os derivados do ciclo-hexano foram obtidos a partir da rea����o entre as (E,E)-cinamilidenoacetofenonas e o malononitrilo com bons rendimentos, mas baixas enantiosseletividades independentemente do catalisador utilizado. De forma a contornar este problema e uma vez que a forma����o do derivado do ciclo-hexano envolve inicialmente a forma����o in-situ do aducto de Michael, a segunda e terceira metodologias de s��ntese envolvem a utiliza����o dos aductos de Michael enantiomericamente puros preparados no segundo cap��tulo. Assim, a rea����o do (S,E)-2-(1,5-diaril-1-oxopent-4-en-3-il)malononitrilo com os derivados de (E,E)-cinamilidenoacetofenona organocatalisada pela hidroquinina permitiu obter os compostos pretendidos com excelentes excessos enantiom��ricos. A utiliza����o de um catalisador de transfer��ncia de fase n��o foi t��o eficiente em termos de enantiosseletividades obtidas na rea����o entre as (R,E)-1,5-diaril-3-(nitrometil)pent-4-en-1-onas e os derivados de (E,E)-cinamilidenoacetofenona, apesar de estes terem sido obtidos em bons rendimentos. A prepara����o destes derivados levou ainda �� idealiza����o de uma nova metodologia de s��ntese de an��logos do ��cido ��-aminobut��rico (GABA) devido �� presen��a de um grupo nitro em posi����o gama relativamente a um grupo carbox��lico. No entanto, apesar de terem sido testadas v��rias metodologias, n��o foi poss��vel obter os compostos pretendidos. No quarto cap��tulo apresenta-se uma breve revis��o bibliogr��fica acerca da ocorr��ncia natural, atividade biol��gica e m��todos de s��ntese de derivados de di-hidro- e tetra-hidropiridinas, assim como um enquadramento te��rico acerca das rea����es peric��clicas utilizadas na s��ntese dos compostos pretendidos. Inicialmente �� descrita a prepara����o de N-sulfonilazatrienos substitu��dos atrav��s da condensa����o direta de derivados de (E,E)-cinamilidenoacetofenona e sulfonamidas. Estes compostos s��o posteriormente utilizados na s��ntese de derivados de 1,2-di-hidropiridinas atrav��s de uma aza-eletrocicliza����o-6�� por duas metodologias distintas: utiliza����o de organocatalisadores quirais e utiliza����o de complexos met��licos de bisoxazolinas. Na s��ntese das tetra-hidropiridinas os N-sulfonilazatrienos s��o utilizados como dienos e o ��toxi-eteno como dien��filo numa rea����o hetero-Diels-Alder inversa utilizando tamb��m os complexos met��licos de bisoxazolinas como catalisadores. Todos os novos compostos sintetizados foram caracterizados estruturalmente recorrendo a estudos de espetroscopia de resson��ncia magn��tica nuclear (RMN), incluindo espetros de 1H e 13C e estudos bidimensionais de correla����o espetrosc��pica homonuclear e heteronuclear e de efeito nuclear de Overhauser (NOESY). Foram tamb��m efetuados, sempre que poss��vel, espetros de massa (EM) e an��lises elementares ou espetros de massa de alta resolu����o (EMAR) para todos os novos compostos sintetizados.

Effects of environmental factors on the toxicity of pesticides to zebrafish embryos

During the last century mean global temperatures have been increasing. According to the predictions, the temperature change is expected to exceed 1.5��C in this century and the warming is likely to continue. Freshwater ecosystems are among the most sensitive mainly due to changes in the hydrologic cycle and consequently changes in several physico-chemical parameters (e.g. pH, dissolved oxygen). Alterations in environmental parameters of freshwater systems are likely to affect distribution, morphology, physiology and richness of a wide range of species leading to important changes in ecosystem biodiversity and function. Moreover, they can also work as co-stressors in environments where organisms have already to cope with chemical contamination (such as pesticides), increasing the environmental risk due to potential interactions. Therefore, the objective of this work was to evaluate the effects of climate change related environmental parameters on the toxicity of pesticides to zebrafish embryos. The following environmental factors were studied: pH (3.0-12.0), dissolved oxygen level (0-8 mg/L) and UV radiation (0-500 mW/m2). The pesticides studied were the carbamate insecticide carbaryl and the benzimidazole fungicide carbendazim. Stressors were firstly tested separately in order to derive concentration- or intensity-response curves to further study the effects of binary combinations (environmental factors x pesticides) by applying mixture models. Characterization of zebrafish embryos response to environmental stress revealed that pH effects were fully established after 24 h of exposure and survival was only affected at pH values below 5 and above 10. Low oxygen levels also affected embryos development at concentrations below 4 mg/L (delay, heart rate decrease and edema), and at concentrations below 0.5 mg/L the survival was drastically reduced. Continuous exposure to UV radiation showed a strong time-dependent impact on embryos survival leading to 100% of mortality after 72 hours of exposure. The toxicity of pesticides carbaryl and carbendazim was characterized at several levels of biological organization including developmental, biochemical and behavioural allowing a mechanistic understanding of the effects and highlighting the usefulness of behavioural responses (locomotion) as a sensitive endpoint in ecotoxicology. Once the individual concentration response relationship of each stressor was established, a combined toxicity study was conducted to evaluate the effects of pH on the toxicity of carbaryl. We have shown that pH can modify the toxicity of the pesticide carbaryl. The conceptual model concentration addition allowed a precise prediction of the toxicity of the jointeffects of acid pH and carbaryl. Nevertheless, for alkaline condition both concepts failed in predicting the effects. Deviations to the model were however easy to explain as high pH values favour the hydrolysis of carbaryl with the consequent formation of the more toxic degradation product 1- naphtol. Although in the present study such explanatory process was easy to establish, for many other combinations the ���interactive��� nature is not so evident. In the context of the climate change few scenarios predict such increase in the pH of aquatic systems, however this was a first approach focused in the lethal effects only. In a second tier assessment effects at sublethal level would be sought and it is expectable that more subtle pH changes (more realistic in terms of climate changes scenarios) may have an effect at physiological and biochemical levels with possible long term consequences for the population fitness.

Codon ambiguities as a mechanism to alter the genetic code in Saccharomyces cerevisiae

Although the genetic code is generally viewed as immutable, alterations to its standard form occur in the three domains of life. A remarkable alteration to the standard genetic code occurs in many fungi of the Saccharomycotina CTG clade where the Leucine CUG codon has been reassigned to Serine by a novel transfer RNA (Ser-tRNACAG). The host laboratory made a major breakthrough by reversing this atypical genetic code alteration in the human pathogen Candida albicans using a combination of tRNA engineering, gene recombination and forced evolution. These results raised the hypothesis that synthetic codon ambiguities combined with experimental evolution may release codons from their frozen state. In this thesis we tested this hypothesis using S. cerevisiae as a model system. We generated ambiguity at specific codons in a two-step approach, involving deletion of tRNA genes followed by expression of non-cognate tRNAs that are able to compensate the deleted tRNA. Driven by the notion that rare codons are more susceptible to reassignment than those that are frequently used, we used two deletion strains where there is no cognate tRNA to decode the rare CUC-Leu codon and AGG-Arg codon. We exploited the vulnerability of the latter by engineering mutant tRNAs that misincorporate Ser at these sites. These recombinant strains were evolved over time using experimental evolution. Although there was a strong negative impact on the growth rate of strains expressing mutant tRNAs at high level, such expression at low level had little effect on cell fitness. We found that not only codon ambiguity, but also destabilization of the endogenous tRNA pool has a strong negative impact in growth rate. After evolution, strains expressing the mutant tRNA at high level recovered significantly in several growth parameters, showing that these strains adapt and exhibit higher tolerance to codon ambiguity. A fluorescent reporter system allowing the monitoring of Ser misincorporation showed that serine was indeed incorporated and possibly codon reassignment was achieved. Beside the overall negative consequences of codon ambiguity, we demonstrated that codons that tolerate the loss of their cognate tRNA can also tolerate high Ser misincorporation. This raises the hypothesis that these codons can be reassigned to standard and eventually to new amino acids for the production of proteins with novel properties, contributing to the field of synthetic biology and biotechnology.

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.

Post-synthetic modification of metal���organic frameworks

Post-synthetic modification (PSM) of metal-organic frameworks encompassing the chemical transformation of the linker present is a promising new route for engineering optical centres and tuning the light emission properties of materials, both in the visible and in the near infrared (NIR) spectral regions. Here, PSM of isoreticular metal-organic framework-3 (IRMOF-3) with ethyl oxalyl monochloride, ethyl acetoacetate, pentane-2,4-dione, 3-(2- hydroxyphenyl)-3-oxopropanal, 2-chloroacetic acid, glyoxylic acid, methyl vinyl ketone and diethyl (ethoxymethylene)malonate followed by chelation of trivalent lanthanide ions afforded intriguing near infrared (Nd3+) and visible (Eu3+, Tb3+) light emitters. IRMOF-3 was used as a case in point due to both its highly porous crystalline structure and the presence of non-coordinating amino groups on the benzenedicarboxylate (bdc) linker amenable to modification. The materials were characterised by elemental analysis, powder X-ray diffraction, optical, scanning and transmission electron microscopy, Fourier transform infrared spectroscopy, and liquid and solid-state nuclear magnetic resonance. The solid-state luminescence properties of Ln-modified-IRMOF-3 were investigated at room temperature. The presence of the bdc aromatic ring, ����� diketonate and oxalate enhanced the Ln3+ sensitization via ligand-to-metal energy transfer (anthena effect). As far as photocalysis is concerned, we have synthesized metal���organic frameworks (Cr-MIL-125-AC, Ag-MIL-125-AC) by a green method (solid���vapors reactions). The resulting functionalized materials show a photocatalytic activity for methylene blue degradation up to 6.52 times larger than that of the commercial photocatalyst hombikat UV-100. These findings open the door for further research for improving the photocatalytic performance of metal-organic frameworks.

Uma abordagem de matabol��mica no estudo comparativo do metabolismo de glucose e galactose no patog��nico humano Streptococcus pneumoniae

Disserta����o mest., Ci��ncias Biom��dicas, Universidade do Algarve, 2010

Estudo da recupera����o da defici��ncia de ferro em plantas de morangueiro (Fragaria x ananassa Duch.) numa perspectiva sustent��vel

Consulta interdita at�� nova indica����o

A palinologia ao acesso de todos?: estudo comparativo de duas t��cnicas de an��lise de sedimentos

Disserta����o mest., Biologia e Geologia, Universidade do Algarve, 2006

Perfil de ��cidos gordos de larvas rec��m eclodidas produzidas em cativeiro de camar��es marinhos ornamentais candidatos a aquacultura

Disserta����o de Mestrado, Biologia Marinha, especializa����o em Pescas e Aquacultura, Faculdade de Ci��ncias do Mar e do Ambiente, Universidade do Algarve, 2009

Sub-produtos de desinfec����o da ��gua: s��ntese e caracteriza����o espectrosc��pica, implementa����o de metodologias anal��ticas e estudo da actividade genot��xica

Disserta����o de mest., Engenharia Biol��gica, Faculdade de Engenharia e de Recursos Naturais, Univ. do Algarve, 2008

Growth conditions, omega - 3 fatty acid concentrations and gene expression of fatty acid desaturases in Portulaca oleracea leaves

Tese de dout., Ci��ncias Biotecnol��gicas (Biotecnologia Vegetal), Univ. do Algarve, 2009

Estudo da evolu����o da microbiota em fermenta����es naturais de azeitona verde do Algarve

Dissserta����o de mest., Tecnologia de Alimentos, Instituto Superior de Engenharia, Univ. do Algarve, 2011

Conserva����o dos frutos de duas cultivares de framboesa (Sevillana e Maravilla) em fresco e em doce

Disserta����o de mest., Tecnologia de Alimentos, Instituto Superior de Engenharia, Univ. do Algarve, 2012