Optimisation of chromatographic separation of oxidised phospholipids and detection with targeted approaches provides greater coverage of the oxidised lipidome

Phospholipid oxidation by adventitious damage generates a wide variety of products with potentially novel biological activities that can modulate inflammatory processes associated with various diseases. To understand the biological importance of oxidised phospholipids (OxPL) and their potential role as disease biomarkers requires precise information about the abundance of these compounds in cells and tissues. There are many chemiluminescence and spectrophotometric assays available for detecting oxidised phospholipids, but they all have some limitations. Mass spectrometry coupled with liquid chromatography is a powerful and sensitive approach that can provide detailed information about the oxidative lipidome, but challenges still remain. The aim of this work is to develop improved methods for detection of OxPLs by optimisation of chromatographic separation through testing several reverse phase columns and solvent systems, and using targeted mass spectrometry approaches. Initial experiments were carried out using oxidation products generated in vitro to optimise the chromatography separation parameters and mass spectrometry parameters. We have evaluated the chromatographic separation of oxidised phosphatidylcholines (OxPCs) and oxidised phosphatidylethanolamines (OXPEs) using C8, C18 and C30 reverse phase, polystyrene – divinylbenzene based monolithic and mixed – mode hydrophilic interaction (HILIC) columns, interfaced with mass spectrometry. Our results suggest that the monolithic column was best able to separate short chain OxPCs and OxPEs from long chain oxidised and native PCs and PEs. However, variation in charge of polar head groups and extreme diversity of oxidised species make analysis of several classes of OxPLs within one analytical run impractical. We evaluated and optimised the chromatographic separation of OxPLs by serially coupling two columns: HILIC and monolith column that provided us the larger coverage of OxPL species in a single analytical run.

Serum α-tocopherol has a nonlinear inverse association with periodontitis among US adults

Previous experimental models suggest that vitamin E may ameliorate periodontitis. However, epidemiologic studies show inconsistent evidence in supporting this plausible association. We aimed to investigate the association between serum α-tocopherol (αT) and γ-tocopherol (γT) and periodontitis in a large cross-sectional US population. This study included 4708 participants in the 1999–2001 NHANES. Serum tocopherols were measured by HPLC and values were adjusted by total cholesterol (TC). Periodontal status was assessed by mean clinical attachment loss (CAL) and probing pocket depth (PPD). Total periodontitis (TPD) was defined as the sum of mild, moderate, and severe periodontitis. All measurements were performed by NHANES. Means ± SDs of serum αT:TC ratio from low to high quartiles were 4.0 ± 0.4, 4.8 ± 0.2, 5.7 ± 0.4, and 9.1 ± 2.7 μmol/mmol. In multivariate regression models, αT:TC quartiles were inversely associated with mean CAL (P-trend = 0.06), mean PPD (P-trend < 0.001), and TPD (P-trend < 0.001) overall. Adjusted mean differences (95% CIs) between the first and fourth quartile of αT:TC were 0.12 mm (0.03, 0.20; P-difference = 0.005) for mean CAL and 0.12 mm (0.06, 0.17; P < 0.001) for mean PPD, whereas corresponding OR for TPD was 1.65 (95% CI: 1.26, 2.16; P-difference = 0.001). In a dose-response analysis, a clear inverse association between αT:TC and mean CAL, mean PPD, and TPD was observed among participants with relatively low αT:TC. No differences were seen in participants with higher αT:TC ratios. Participants with γT:TC ratio in the interquartile range showed a significantly lower mean PPD than those in the highest quartile. A nonlinear inverse association was observed between serum αT and severity of periodontitis, which was restricted to adults with normal but relatively low αT status. These findings warrant further confirmation in longitudinal or intervention settings.

Synthesis of azulenylsilane nitrones as diagnostic tools for superoxide detection

The superoxide radical is considered to play important roles in physiological processes as well as in the genesis of diverse cytotoxic conditions such as cancer, various cardiovascular disorders and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson’s disease (PD) and Alzheimer’s disease (AD). The detection and quantification of superoxide within cells is of critical importance to understand biological roles of superoxide and to develop preventive strategies against free radical-mediated diseases. Cyclic nitrone spin traps such as DMPO, EMPO, DEPMPO, BMPO and their derivatives have been widely used in conjunction with ESR spectroscopy to detect cellular superoxide with some success. However, the formation of unstable superoxide adducts from the reaction of cyclic nitrones with superoxide is a stumbling block in detecting superoxide by using electron spin resonance (ESR). A chemiluminescent probe, lucigenin, and fluorogenic probes, hydroethidium and MitoSox, are the other frequently used methods in detecting superoxide. However, luceginen undergoes redox-cycling producing superoxide by itself, and hydroethidium and MitoSox react with other oxidants apart from superoxide forming red fluorescent products contributing to artefacts in these assays. Hence, both methods were deemed to be inappropriate for superoxide detection. In this study, an effective approach, a selective mechanism-based colorimetric detection of superoxide anion has been developed by using silylated azulenyl nitrones spin traps. Since a nitrone moiety and an adjacent silyl group react readily with radicals and oxygen anions respectively, such nitrones can trap superoxide efficiently because superoxide is both a radical and an oxygen anion. Moreover, the synthesized nitrone is designed to be triggered solely by superoxide and not by other commonly observed oxygen radicals such as hydroxyl radical, alkoxyl radicals and peroxyl radical. In vitro studies have shown that these synthesized silylated azylenyl nitrones and the mitochondrial-targeted guanylhydrazone analog can trap superoxide efficiently yielding UV-vis identifiable and even potentially fluorescence-detectable orange products. Therefore, the chromotropic detection of superoxide using these nitrones can be a promising method in contrast to other available methods.

The role of protein oxidation in ageing & disease

Ageing is a natural phenomenon of the human lifecycle, yet it is still not understood what causes the deterioration of the human body near the end of the lifespan. One popular theory is the Free Radical Theory of Ageing, which proposes that oxidative damage to biomolecules causes ageing of tissues. The ageing population is affected by many chronic diseases. This study focused on sarcopenia (muscle loss in ageing) and obesity as two models for comparison of oxidative damage in muscle proteins in mice. The aim of the study was to develop advanced mass spectrometry methods to detect specific oxidative modifications to mouse muscle proteins, including oxidation, nitration, chlorination, and carbonyl group formation, but western blotting was also used to provide complementary information on the oxidative state of proteins from aged and obese muscle. Mass spectrometry proved to be a powerful tool, enabling identification of the types of modifications present, the sites at which they were present and percentage of the peptide populations that were modified. Targeted and semi-targeted mass spectrometry methods were optimised for the identification and quantitation of the oxidised residues in muscle proteins. The development of the quantitative methods enabled comparisons of mass spectrometry instruments. Both the Time of Flight and QTRAP systems showed advantages of using the different mass analysers to quantify oxidative modifications. Several oxidised residues were characterised and quantified in both the obese and sarcopenic models, and higher levels of oxidation were found compared to their control counterparts. Residues found to be oxidised were oxidation of proline, tyrosine and tryptophan, dioxidation of methionine, allysine and nitration of tyrosine. However quantification was performed on methionine dioxidation and cysteine trioxidation containing residues in SERCA. The combination of measuring residue susceptibility and functional studies could contribute to understanding the overall role of oxidation in ageing and obesity.

Prospecção química e avaliação biológica de Banisteriopsis laevifolia (Malpighiaceae)

The Banisteriopsis genus is widespread in traditional medicine. This work aims to contribute with information about the chemical composition and on the evaluation of the biological activity of the essential oil, the ethanol extract of the leaves and partitions of the Banisteriopsis laevifolia. The phytochemical screeningtest of ethanol extract and partitions of leaves indicated the presence of flavonoids, terpenoids, saponins, phenols and steroids compounds. Nitrogenous compounds, characteristic of some species of this family, were not detected. Flavonoids were the predominant metabolite, with the highest concentrations on the partitions ethyl acetate and n-butanol. The antibacterial activity, antifungal and cytotoxicity of the essetial oil, ethanol extract and partitions were assyed by microdilution broth method (MBM), where the minimum inhibitory concentrations (MIC) were calculated. The ethanol extract and partitions did not inhibit growth against to Gram positive bacteria tested, with MIC less than 400 mg L-1. For the Gram negative bacteria tested, the hexane and hydroethanol partitios were more effective against F. nucleatum bacteria (MIC 100 ug mL-1). The ethanol extract showed antifungal activity with MIC of 31.2 mg L-1. Ethyl acetate and n-butanol partitions showed MIC 187.5 mg L-1 and 93.7 mg L-1, respectively, arousing interest for isolation studies. The antioxidant activity was evaluated by the DPPH free radical method. The ethanolic extract, ethyl acetate and n-butanol partitions were active, since they showed EC50 values (4.53 ug mL-1, 4.07 and 8.39 ug mL-1, respectively), values equivalent to the BHT (7.3 mg L-1). The analysis by HPLC-MS/MS of the most active fractions (ethyl acetate and n-butanol) identified phenolic compounds (flavonols and phenolic acids) which exert recognized biological activity. The GC-MS analysis of the essential oils from leaves collected in two periods studied (dry and wet), showed a small variation in the number of compounds. The major classes identified for the oil collected in the dry period were aliphatic alcohols (23,4%), terpenoids (18.7%), sterols (10.4%) and long-chain alkanes (9.2%) compounds. Terpenoids (26.8%) were the major class for the rain season. The major compounds (3Z) -hexenol, phytol and untriacontano are present in the two seasons but in different amounts (19.4%, 9.8% and 7.5% during the dry season, and 17.0 %, 14.9% and 15.3% in the rainy season, respectively). The essential oil from rainy season was not effective against to the oral bacteria Gram positive and Gram negative tested. However, showed significant antifungal activity with MIC 1000 mg L-1 against Candidas. Thus, the promising results with respect to biological assays of ethanolic extract and partitions from B. laevifolia contributed to the chemical and biological knowledge of the species B. laevifolia.

Estudo fitoquímico e biológico das folhas de Banisteriopsis argyrophylla (A. Juss.) B. Gates (Malpighiaceae)

With the increasing fungi resistance compared with existing drugs on the market and the side effects reported by some compounds with antioxidant properties and enzymatic inhibitors, in particular against α-amylase and α-glucosidase, the discovery of new compounds with biological potential, becomes a need. In this context, natural products can be an important source for the discovery of new active molecular architectures. Then, this study aimed to evaluate the antioxidant activity, the enzymatic inhibitory activity of α-amylase and α-glucosidase, the antifungal and cytotoxic activities of ethanolic extract (EE) the leaves of Banisteriopsis argyrophylla (Malpighiaceae) and their fractions, obtained by liquid-liquid extraction using solvents of increasing polarity. The antioxidant activity was evaluated by the free radical DPPH scavenging method (2,2-diphenyl-1-picrylhydrazyl) and the ethyl acetate fractions (FAE) and n-butanol (FB) were the most active, confirmed by the peak current and the oxidation potential obtained by differential pulse voltammetry (DPV). The inhibitory activity of the α-amylase and α-glucosidase was analyzed considering the reactions between substrates α-(2-chloro-4-nitrophenyl)-β-1,4-galactopiranosilmaltoside (Gal-α-G2-CNP) and 4-nitrophenyl-α-D-glucopyranoside (p-NPG), respectively. Initially, it was found that the EE showed considerable activity against α-amylase (EC50 = 2.89±0.1 μg m L–1) compared to the acarbose used as positive control (EC50 = 0.08±0.1 μg mL–1) and that did not showed promising activity against the α-glucosidase. After this observation we evaluated the inhibitory activity of α-amylase fractions, with FAE (EC50 = 2.33±0.1 μg mL–1) and FB (EC50 = 2.57 ± 0.1 μg mL–1) showing the best inhibitions. The antifungal activity was evaluated against Candida species, and the FAE had better antifungal potential (MIC's between 93.75 and 11.72 μg mL–1) compared with amphotericin as positive standard (MIC = 1.00 and 2.00 μg L–1 for C. parapsilosis and C. krusei used as controls, respectively). The EE (CC50 = 360.00 ± 12 μg mL–1) and fractions (CC50's> 270.00 μg mL–1) were considerably less toxic to Vero cells than the cisplatin used as positive control (CC50 = 7.01 ± 0 6 μg mL–1). The FAE showed the best results for the activities studied, this fraction was submitted to ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS)), and the following flavonoids have been identified: (±)-catechin, quercetin-3-O-β-D-Glc/ quercetin-3-O-β-D-Gal, quercetin-3-O-β-L-Ara, quercetin-3-O-β-D-Xyl, quercetin-3-O-α-L-Rha, kaempferol-3-O-α-L-Rha, quercetin-3-O-(2''-galoil)-α-L-Rha, quercetin-3-O-(3''-galoil)-α-L-Rha and kaempferol-3-O-(3''-galoil)-α-L-Rha,. FAE was submitted to column chromatography using C18 phase, and (±)-catechin was isolated (FAE-A1, 73 mg) and three fractions consisting of a mixture of flavonoids were obtained (FAE-A2, FAE-A3 and FAE-A4). These compounds were identified by thin layer chromatography (TLC) and (–)-ESI-MS. The (±)-catechin fraction showed an MIC = 2.83 μg ml–1 in assay using C. glabrata, with amphotericin as positive control. The fractions FAE-A2, FAE-A3, FAE-A4, showed less antifungal potential in tested concentrations. The identified flavonoids are described in the literature, regarding their antioxidant capacity and (±)-catechin, quercetin-3-O-Rha and kaempferol-3-O-Rha are described as α-amylase inhibitors. Thus, B. argyrophylla is an important species that produces compounds with antioxidant potential that can be related to the traditional use as anti-inflammatory and also has antifungal compounds and inhibitors of α-amylase. Therefore, these leaves are promising resources for the production of new drugs.

Active Surface Deformation Technology for Management of Marine Biofouling

Biofouling, the accumulation of biomolecules, cells, organisms and their deposits on submerged and implanted surfaces, is a ubiquitous problem across various human endeavors including maritime operations, medicine, food industries and biotechnology. Since several decades, there have been substantial research efforts towards developing various types of antifouling and fouling release approaches to control bioaccumulation on man-made surfaces. In this work we hypothesized, investigated and developed dynamic change of the surface area and topology of elastomers as a general approach for biofouling management. Further, we combined dynamic surface deformation of elastomers with other existing antifouling and fouling-release approaches to develop multifunctional, pro-active biofouling control strategies.

This research work was focused on developing fundamental, new and environment-friendly approaches for biofouling management with emphasis on marine model systems and applications, but which also provided fundamental insights into the control of infectious biofilms on biomedical devices. We used different methods (mechanical stretching, electrical-actuation and pneumatic-actuation) to generate dynamic deformation of elastomer surfaces. Our initial studies showed that dynamic surface deformation methods are effective in detaching laboratory grown bacterial biofilms and barnacles. Further systematic studies revealed that a threshold critical surface strain is required to debond a biofilm from the surface, and this critical strain is dependent on the biofilm mechanical properties including adhesion energy, thickness and modulus. To test the dynamic surface deformation approach in natural environment, we conducted field studies (at Beaufort, NC) in natural seawater using pneumatic-actuation of silicone elastomer. The field studies also confirmed that a critical substrate strain is needed to detach natural biofilm accumulated in seawater. Additionally, the results from the field studies suggested that substrate modulus also affect the critical strain needed to debond biofilms. To sum up, both the laboratory and the field studies proved that dynamic surface deformation approach can effectively detach various biofilms and barnacles, and therefore offers a non-toxic and environmental friendly approach for biofouling management.

Deformable elastomer systems used in our studies are easy to fabricate and can be used as complementary approach for existing commercial strategies for biofouling control. To this end, we aimed towards developed proactive multifunctional surfaces and proposed two different approaches: (i) modification of elastomers with antifouling polymers to produce multifunctional, and (ii) incorporation of silicone-oil additives into the elastomer to enhance fouling-release performance.

In approach (i), we modified poly(vinylmethylsiloxane) elastomer surfaces with zwitterionic polymers using thiol-ene click chemistry and controlled free radical polymerization. These surfaces exhibited both fouling resistance and triggered fouling-release functionalities. The zwitterionic polymers exhibited fouling resistance over short-term (∼hours) exposure to bacteria and barnacle cyprids. The biofilms that eventually accumulated over prolonged-exposure (∼days) were easily detached by applying mechanical strain to the elastomer substrate. In approach (ii), we incorporated silicone-oil additives in deformable elastomer and studied synergistic effect of silicone-oils and surface strain on barnacle detachment. We hypothesized that incorporation of silicone-oil additive reduces the amount of surface strain needed to detach barnacles. Our experimental results supported the above hypothesis and suggested that surface-action of silicone-oils plays a major role in decreasing the strain needed to detach barnacles. Further, we also examined the effect of change in substrate modulus and showed that stiffer substrates require lower amount of strain to detach barnacles.

In summary, this study shows that (1) dynamic surface deformation can be used as an effective, environmental friendly approach for biofouling control (2) stretchable elastomer surfaces modified with anti-fouling polymers provides a pro-active, dual-mode approach for biofouling control, and (3) incorporation of silicone-oils additives into stretchable elastomers improves the fouling-release performance of dynamic surface deformation technology. Dynamic surface deformation by itself and as a supplementary approach can be utilized biofouling management in biomedical, industrial and marine applications.

MEDIUM-CHAIN-LENGTH POLY(3-HYDROXYALKANOATE) NANOPARTICLE SUSPENSIONS

The main goal of this thesis was to prepare medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA) nanoparticle suspensions at high solids content (≥ 10 % w/v). A two-stage emulsification-solvent evaporation process was employed to produce poly-3-hydroxydecanoate (PHD) suspensions. The formulation and processing conditions including ultrasonication time and amplitude, selection of solvent, and selection of surfactants and their concentrations were investigated to make concentrated suspensions (10 and 30 % (w/v)) of PHD with particles less than 300 nm. Among the ionic surfactants tested to stabilize the suspension, the anionic, sodium dodecyl sulphate (SDS), and the cationic, dodecyltrimethylammonium bromide (DTAB) surfactants produced the smallest particle sizes (~100 nm). However, more stabilized nanoparticles were obtained when the ionic surfactant, SDS, was combined with any of the non-ionic surfactants tested, with polyoxyethylene octyl phenyl ether (Triton X-100) or polyoxyethylene (20) sorbitan monooleate (Tween 80) resulting in a slight increase in zeta potential over 30 days while the zeta potential with other non-ionic surfactants decreased. Mcl-PHA containing 11 and 18 % of carboxyl groups was synthesized via free radical addition reaction of 11-mercaptoundecanoic acid to the pendant double bonds of unsaturated poly-3-hydroxynonanoate (PHNU). Colloidal suspensions prepared by ultrasonication needed a surfactant to maintain stability, even at 0.4 % solids of mcl-PHA containing 11 % carboxylation (PHNC-1) unlike the stable suspensions prepared without surfactants by the titration method. Similar particle sizes (155.6 ± 8.4 to 163.4 ± 11.3 nm) and polydispersity indices (0.42 ± 0.03 to 0.49 ± 0.04) were obtained when several non-ionic surfactants were tested to minimize particle agglomeration, with the smallest particles obtained with Triton X-100. When Triton X-100 was combined with a variety of ionic surfactants, smaller nanoparticles (97.1 ± 1.1 to 121.7 ± 5.7 nm) with a narrower particle size distribution (0.21 ± 0.001 to 0.25 ± 0.003) were produced. The SDS and Triton X-100 combination was chosen to evaluate other mcl-PHAs at 10 % (w/v) solids content. Slightly smaller nanoparticles were formed with carboxylated mcl-PHAs compared to mcl-PHAs having aliphatic pendant side chains. Mcl-PHA consisting of 18 % carboxylation (PHNC-2) formed a much smaller nanoparticles and higher zeta potential.

Contribution to the ethnobotanical study of antidiabetic medicinal plants of the Central Middle Atlas region (Morocco)

An ethnobotanical survey of medicinal plants was carried out in the Central Middle Atlas in the years 2013 and 2014 to establish the catalog of medicinal plants used in traditional medicine in the treatment of diabetes. Thus, 1560 people were interviewed, using questionnaires. The latter enabled us to gather information on traditional healing practices of the local population including scientific name, French name, vernacular name, plant parts used , therapeutic indications , revenues and mode of administration. The results show that 76 medicinal species were inventoried in the study area. These plant species are included into 67 genus and 40 families. The most represented families are: Lamiaceae (12 species), Asteraceae and Brassicaceae species with 14 each. Of 76 medicinal species found in the region, four species are reported for the first time in the traditional treatment of diabetes in Morocco. They are Pistacia atlantica, Ptychotis verticillata, Anacyclus pyrethrum, Alyssum spinosum, Cistus albidus, Juniperus thurifera, Ephedra nebrodensis, Thymus algeriensis, Th. munbyanus, Th. zygis, Abelmoschus esculentus, Fraxinus augustifolia, Sorghum vulgare and, Eriobotrya japonica. The leaves are the most used organs (38%). The decoction is the dominant mode of preparation (50%) and administration is mostly for oral use (97%).

Chemical characterization and evaluation of biological activity of Cynara cardunculus extractable compounds

The Mediterranean species Cynara cardunculus L. is recognized in the traditional medicine, for their hepatoprotective and choleretic effects. Biomass of C. cardunculus L. var. altilis (DC), or cultivated cardoon, may be explored not only for the production of energy and pulp fibers, but also for the extraction of bioactive compounds. The chemical characterization of extractable components, namely terpenic and phenolic compounds, may valorize the cultivated cardoon plantation, due to their antioxidant, antitumoral and antimicrobial activities. In this study, the chemical composition of lipophilic and phenolic fractions of C. cardunculus L. var. altilis (DC), cultivated in the south of Portugal (Baixo Alentejo region) was characterized in detail, intending the integral valorization of its biomass. The biological activity of cultivated cardoon extracts was evaluated in terms of antioxidant, human tumor cell antiproliferative and antibacterial effects. Gas chromatography-mass spectrometry (GC-MS) was used for the chemical analysis of lipophilic compounds. Sixty-five lipophilic compounds were identified, from which 1 sesquiterpene lactone and 4 pentacyclic triterpenes were described, for the first time, as cultivated cardoon components, such as: deacylcynaropicrin, acetates of β- and α-amyrin, lupenyl acetate and ψ-taraxasteryl acetate. Sesquiterpene lactones were the major family of lipophilic components of leaves (≈94.5 g/kg), mostly represented by cynaropicrin (≈87.4 g/kg). Pentacyclic triterpenes were also detected, in considerably high contents, in the remaining parts of cultivated cardoon, especially in the florets (≈27.5 g/kg). Taraxasteryl acetate was the main pentacyclic triterpene (≈8.9 g/kg in florets). High pressure liquid chromatography-mass spectrometry (HPLC-MS) was utilized for the chemical analysis of phenolic compounds. Among the identified 28 phenolic compounds, eriodictyol hexoside was reported for the first time as C. cardunculus L. component, and 6 as cultivated cardoon components, namely 1,4-di-O-caffeoylquinic acid, naringenin 7-O-glucoside, naringenin rutinoside, naringenin, luteolin acetylhexoside and apigenin acetylhexoside. The highest content of the identified phenolic compounds was observed in the florets (≈12.6 g/kg). Stalks outer part contained the highest hydroxycinnamic acids abundance (≈10.3 g/kg), and florets presented the highest flavonoids content (≈10.3 g/kg). The antioxidant activity of phenolic fraction was examined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Stalks outer part, and receptacles and bracts extracts demonstrated the highest antioxidant effect on DPPH (IC50 of 34.35 μg/mL and 35.25 μg/mL, respectively). (cont.) abstract (cont.) The DPPH scavenging effect was linearly correlated with the total contents of hydroxycinnamic acids (r = -0.990). The in vitro antiproliferative activity of cultivated cardoon lipophilic and phenolic extracts was evaluated on a human tumor cells line of triple-negative breast cancer (MDA-MB-231), one of the most refractory human cancers to conventional therapeutics. After 48 h of exposition, leaves lipophilic extract showed higher inhibitory effect (IC50 = 10.39 μg/mL) than florets lipophilic extract (IC50 = 315.22 μg/mL), upon MDA-MB-231 cellular viability. Pure compound of cynaropicrin, representative of the main compound identified in leaves lipophilic extract, also prevented the cell proliferation of MDA-MB-231 (IC50 = 17.86 μM). MDA-MB-231 cells were much more resistant to the 48 h- treatment with phenolic extracts of stalks outer part (IC50 = 3341.20 μg/mL) and florets (IC50 > 4500 μg/mL), and also with the pure compound of 1,5-di-O-caffeoylquinic acid (IC50 = 1741.69 μM). MDA-MB-231 cells were exposed, for 48 h, to the respective IC50 concentrations of leaves lipophilic extract and pure compound of cynaropicrin, in order to understand their ability in modelling cellular responses, and consequently important potentially signaling pathways for the cellular viability decrease. Leaves lipophilic extract increased the caspase-3 enzymatic activity, contrarily to pure compound of cynaropicrin. Additionally, leaves lipophilic extract and pure compound of cynaropicrin caused G2 cell cycle arrest, possibly by upregulating the p21Waf1/Cip1 and the accumulation of phospho-Tyr15-CDK1 and cyclin B1. The inhibitory effects of leaves lipophilic extract and cynaropicrin pure compound, against the MDA-MB-231 cell proliferation, may also be related to the downregulation of phospho-Ser473-Akt. The antibacterial activity of cultivated cardoon lipophilic and phenolic extracts was assessed, for the first time, on two multidrug-resistant bacteria, such as the Gram-negative Pseudomonas aeruginosa PAO1 and the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), two of the main bacteria responsible for health care-associated infections. Accordingly, the minimum inhibitory concentrations (MIC) were determined. Lipophilic and phenolic extracts of florets did not have antibacterial activity on P. aeruginosa PAO1 and MRSA (MIC > 2048 μg/mL). Leaves lipophilic extract did not prevent the P. aeruginosa PAO1 growth, but pure compound of cynaropicrin was slightly active (MIC = 2048 μg/mL). Leaves lipophilic extract and pure compound of cynaropicrin blocked MRSA growth (MIC of 1024 and 256 μg/mL, respectively). The scientific knowledge revealed in this thesis, either by the chemical viewpoint, or by the biological viewpoint, contributes for the valorization of C. cardunculus L. var. altilis (DC) biomass. Cultivated cardoon has potential to be exploited as source of bioactive compounds, in conciliation with other valorization pathways, and Portuguese traditional cheeses manufacturing.

Identification of Genes Modulating Mitochondrial Biogenesis in Caenorhabditis elegans

Since Altmann recognized ubiquitously distributed "bioblasts" in 1890, understanding of mitochondria has evolved from "elementary organisms" living inside cells and carrying out vital functions, over the Harman's "free radical theory" in 1956, to one of the driving forces of aging and cause of multiple associated diseases impacting society today. While a tremendous amount of work has contributed to the understanding of mitochondrial biology in different model organisms, the precise molecular mechanisms of basic mitochondrial function have yet to be deciphered. By employing an RNA interference mediated screen in Caenorhabditis elegans, we identified two transcription factors: SPTF-3, a member of Sp1 family, and an uncharacterized, nematode specific W04D2.4. We propose that both proteins modulate expression of many genes with regard to mitochondrial function including mitochondrial single-stranded binding protein encoded by mtss-1, whose promoter was used as transcriptional reporter in the screen. Further, RNA sequencing data indicate that W04D2.4 indirectly regulates expression of mitochondrial DNA via control of genes functionally related to mitochondrial replication and translation machineries. We also demonstrate that from all interventions targeting cytosolic translation, MTSS-1 levels are elevated only upon knockdown of genes encoding cytosolic ribosomal proteins. Reduction of ribosomes leads to increased sptf-3 translation, most likely in an internal ribosome entry side (IRES) mediated manner, eventually inducing mtss-1 expression. Moreover, we identify a novel role for SPTF-3 in the regulation of mitochondrial unfolded stress response (UPRmt) activation, but not endoplasmatic reticulum or oxidative stress responses. Taken together, this study identifies two transcription factors previously not associated with mitochondrial biogenesis and UPRmt in C. elegans, establishing a basis for further investigation of mito-nuclear interactions.

Interference lithography for optical devices and coatings

Interference lithography can create large-area, defect-free nanostructures with unique optical properties. In this thesis, interference lithography will be utilized to create photonic crystals for functional devices or coatings. For instance, typical lithographic processing techniques were used to create 1, 2 and 3 dimensional photonic crystals in SU8 photoresist. These structures were in-filled with birefringent liquid crystal to make active devices, and the orientation of the liquid crystal directors within the SU8 matrix was studied. Most of this thesis will be focused on utilizing polymerization induced phase separation as a single-step method for fabrication by interference lithography. For example, layered polymer/nanoparticle composites have been created through the one-step two-beam interference lithographic exposure of a dispersion of 25 and 50 nm silica particles within a photopolymerizable mixture at a wavelength of 532 nm. In the areas of constructive interference, the monomer begins to polymerize via a free-radical process and concurrently the nanoparticles move into the regions of destructive interference. The holographic exposure of the particles within the monomer resin offers a single-step method to anisotropically structure the nanoconstituents within a composite. A one-step holographic exposure was also used to fabricate self- healing coatings that use water from the environment to catalyze polymerization. Polymerization induced phase separation was used to sequester an isocyanate monomer within an acrylate matrix. Due to the periodic modulation of the index of refraction between the monomer and polymer, the coating can reflect a desired wavelength, allowing for tunable coloration. When the coating is scratched, polymerization of the liquid isocyanate is catalyzed by moisture in air; if the indices of the two polymers are matched, the coatings turn transparent after healing. Interference lithography offers a method of creating multifunctional self-healing coatings that readout when damage has occurred.

Obtenção de biopeptídeos com atividade antioxidante a partir de proteínas de origem animal

Pele, ossos, espinhas, entre outros, separados durante o processamento de produtos cárneos podem ser uma boa fonte de proteína, especialmente de colágeno. Para obtenção de colágeno nativo a partir de ossos é necessário um tratamento prévio de desproteinização e desmineralização. Portanto, o objetivo deste trabalho foi determinar os melhores parâmetros para a desmineralização de ossos de pescado e frango utilizando soluções de HCl e EDTA um complexante de íons metálicos. O melhor efeito da desmineralização foi obtido com solução de HCl 1,0 mol/L. Após 48 h de extração, 99,4 e 95,4% das substâncias minerais foram solubilizadas para os ossos de pescado e para ossos de frango, respectivamente. Paralelamente, a menor perda de colágeno também foi observada nessas condições. O processo realizado empregando soluções de EDTA foi menos eficaz do que com solução de HCl. Após 48 h de extração com EDTA 0,1 mol/L, 37,5 e 32,4% dos compostos minerais foram removidos dos ossos de pescado e dos ossos de frango, respectivamente. Uma maior eficiência foi alcançada com solução de EDTA 0,5 mol/L. O rendimento do processo foi de cerca de 66,6% a partir dos ossos de pescado e 70,6% a partir os ossos de frango. A desmineralização com EDTA não provocou perda de colágeno.

Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective- This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods- Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results- Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 �g/ml) nor native LDL (100 �g/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CA VSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 �M), and MnTBAP (a free radical scavenger, 50��M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogen-induced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and O2 .- levels were determined in IMA versus CA VSMC. Conclusions- Enhanced intrinsic antioxidant capacity may confer on IMA VSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.