Antioxidant Activity of Sesame (Seasamum indicum L.) Cake Extract for the Stabilization of Olein Based Butter

This study investigated the effect of ethanolic sesame cake extract on oxidative stabilization of olein based butter. Fractionation of cream was performed by the dry fractionation technique at 10 °C, ethanolic sesame cake extract (SCE) was incorporated into olein butter at three different concentrations; 50, 100, 150 ppm (T1, T2, T3) and compared with a control. The total phenolic content of SCE was 1.72 (mg gallic acid equivalent g−1 dry weight). The HPLC characterization of ethanolic sesame cake revealed the presence of antioxidant substances viz. sesamol, sesamin and sesamolin in higher extents. The DPPH free radical scavenging activity of SCE was 83 % as compared to 64 and 75 % in BHA and BHT. Fractionation of milk fat at 10 °C significantly (p < 0.05) influenced the fatty acid profile of olein and stearin fractions from the parent milk fat. Concentration of oleic acid and linoleic acid in olein fraction was 29.62 and 33.46 % greater than the parent milk fat. The loss of C18:1 in 90 days stored control and T3 was 24.37 and 3.58 %, respectively, 58 % C18:2 was broken down into oxidation products over 8.55 % loss in T3. The peroxide value of control, T1, T2, BHT and T3 in the Schaal oven test was 8.59, 8.12, 5.34, 4.52 and 2.49 (mequiv O2/kg). The peroxide value and anisidine value of 3 months stored control and T3 were 1.21, 0.42 (mequiv O2/kg) and 27.25, 13.25, respectively. The concentration of conjugated dienes in T3 was substantially less than the control. The induction period of T3 was considerably higher than BHT with no difference in sensory characteristics (p > 0.05). Ethanolic SCE can be used for the long-term preservation of olein butter, with acceptable sensory characteristics.

Purple sweet potato colour - a potential therapy for galactosemia?

Galactosemia is an inherited metabolic disease in which galactose is not properly metabolised. There are various theories to explain the molecular pathology, and recent experimental evidence strongly suggests that oxidative stress plays a key role. High galactose diets are damaging to experimental animals and oxidative stress also plays a role in this toxicity which can be alleviated by purple sweet potato colour (PSPC). This plant extract is rich in acetylated anthocyanins which have been shown to quench free radical production. The objective of this Commentary is to advance the hypothesis that PSPC, or compounds therefrom, may be a viable basis for a novel therapy for galactosemia.

Systemic oxidative-nitrosative-inflammatory stress during acute exercise in hypoxia; implications for microvascular oxygenation and aerobic capacity

New Findings

What is the central question of this study?Exercise performance is limited during hypoxia by a critical reduction in cerebral and skeletal tissue oxygenation. To what extent an elevation in systemic free radical accumulation contributes to microvascular deoxygenation and the corresponding reduction in maximal aerobic capacity remains unknown.What is the main finding and its importance?We show that altered free radical metabolism is not a limiting factor for exercise performance in hypoxia, providing important insight into the fundamental mechanisms involved in the control of vascular oxygen transport.

Exercise performance in hypoxia may be limited by a critical reduction in cerebral and skeletal tissue oxygenation, although the underlying mechanisms remain unclear. We examined whether increased systemic free radical accumulation during hypoxia would be associated with elevated microvascular deoxygenation and reduced maximal aerobic capacity (). Eleven healthy men were randomly assigned single-blind to an incremental semi-recumbent cycling test to determine  in both normoxia (21% O_{2) and hypoxia (12% O2) separated by a week. Continuous-wave near-infrared spectroscopy was employed to monitor concentration changes in oxy- and deoxyhaemoglobin in the left vastus lateralis muscle and frontal cerebral cortex. Antecubital venous blood samples were obtained at rest and at  to determine oxidative (ascorbate radical by electron paramagnetic resonance spectroscopy), nitrosative (nitric oxide metabolites by ozone-based chemiluminescence and 3-nitrotyrosine by enzyme-linked immunosorbent assay) and inflammatory stress biomarkers (soluble intercellular/vascular cell adhesion 1 molecules by enzyme-linked immunosorbent assay). Hypoxia was associated with increased cerebral and muscle tissue deoxygenation and lower  (P < 0.05 versus normoxia). Despite an exercise-induced increase in oxidative–nitrosative–inflammatory stress, hypoxia per se did not have an additive effect (P > 0.05 versus normoxia). Consequently, we failed to observe correlations between any metabolic, haemodynamic and cardiorespiratory parameters (P > 0.05). Collectively, these findings suggest that altered free radical metabolism cannot explain the elevated microvascular deoxygenation and corresponding lower  in hypoxia. Further research is required to determine whether free radicals when present in excess do indeed contribute to the premature termination of exercise in hypoxia.}

Conduit artery structure and function in lowlanders and native highlanders: relationships with oxidative stress and role of sympathoexcitation

Research detailing the normal vascular adaptions to high altitude is minimal and often confounded by pathology (e.g. chronic mountain sickness) and methodological issues. We examined vascular function and structure in: (1) healthy lowlanders during acute hypoxia and prolonged (∼2 weeks) exposure to high altitude, and (2) high-altitude natives at 5050 m (highlanders). In 12 healthy lowlanders (aged 32 ± 7 years) and 12 highlanders (Sherpa; 33 ± 14 years) we assessed brachial endothelium-dependent flow-mediated dilatation (FMD), endothelium-independent dilatation (via glyceryl trinitrate; GTN), common carotid intima–media thickness (CIMT) and diameter (ultrasound), and arterial stiffness via pulse wave velocity (PWV; applanation tonometry). Cephalic venous biomarkers of free radical-mediated lipid peroxidation (lipid hydroperoxides, LOOH), nitrite (NO₂^–) and lipid soluble antioxidants were also obtained at rest. In lowlanders, measurements were performed at sea level (334 m) and between days 3–4 (acute high altitude) and 12–14 (chronic high altitude) following arrival to 5050 m. Highlanders were assessed once at 5050 m. Compared with sea level, acute high altitude reduced lowlanders’ FMD (7.9 ± 0.4 vs. 6.8 ± 0.4%; P = 0.004) and GTN-induced dilatation (16.6 ± 0.9 vs. 14.5 ± 0.8%; P = 0.006), and raised central PWV (6.0 ± 0.2vs. 6.6 ± 0.3 m s⁻¹; P = 0.001). These changes persisted at days 12–14, and after allometrically scaling FMD to adjust for altered baseline diameter. Compared to lowlanders at sea level and high altitude, highlanders had a lower carotid wall:lumen ratio (∼19%, P ≤ 0.04), attributable to a narrower CIMT and wider lumen. Although both LOOH and NO₂^– increased with high altitude in lowlanders, only LOOH correlated with the reduction in GTN-induced dilatation evident during acute (n = 11, r = −0.53) and chronic (n = 7, r = −0.69; P ≤ 0.01) exposure to 5050 m. In a follow-up, placebo-controlled experiment (n = 11 healthy lowlanders) conducted in a normobaric hypoxic chamber (inspired O₂ fraction () = 0.11; 6 h), a sustained reduction in FMD was evident within 1 h of hypoxic exposure when compared to normoxic baseline (5.7 ± 1.6 vs. 8.0 ±1.3%; P < 0.01); this decline in FMD was largely reversed following α₁-adrenoreceptor blockade. In conclusion, high-altitude exposure in lowlanders caused persistent impairment in vascular function, which was mediated partially via oxidative stress and sympathoexcitation. Although a lifetime of high-altitude exposure neither intensifies nor attenuates the impairments seen with short-term exposure, chronic high-altitude exposure appears to be associated with arterial remodelling.

Hydrogel antimicrobial capture coatings for endotracheal tubes; a pharmaceutical strategy designed to prevent ventilator-associated pneumonia.

This paper presents a novel strategy for the prevention of ventilator-associatedpneumonia that involves coating poly(vinyl chloride, PVC) endotracheal tubes (ET) withhydrogels that may be subsequently used to entrap nebulized antimicrobial solutions. Candidatehydrogels were prepared containing a range of ratios of hydroxyethyl methacrylate (HEMA) andmethacrylic acid (MAA) from 100:0 to 70:30 using free radical polymerization and, whenrequired, simultaneous attachment to PVC was performed. The mechanical properties, glasstransition temperatures, swelling kinetics, uptake of gentamicin from an aqueous medium, andgentamicin release were characterized. Increasing the MAA content of the hydrogels significantlydecreased the ultimate tensile strength, % elongation at break, Young’s modulus, and increasedthe glass transition temperature, the swelling ratio, and gentamicin uptake. Microbial(Staphylococcus aureus and Pseudomonas aeruginosa) adherence to control (drug-free) hydrogelswas observed; however, while adherence to gentamicin-containing p(HEMA) occurred, noadherence occurred to gentamicin-containing HEMA:MAA copolymers. Antimicrobialpersistence of gentamicin-containing hydrogels was examined by determining the zone ofinhibition against each microorganism on successive days. Hydrogel composition affected the observed antimicrobial persistence,with the hydrogel composed of 70:30 HEMA:MAA exhibiting >20 days persistence against S. aureus and P. aeruginosa,respectively. To simulate clinical use, the hydrogels (coated onto PVC) were first exposed to a nebulized solution of gentamicin(4 mL, 80 mg for 20 min), and then to nebulized bacteria (4 mL ca. 1 × 109 colony forming units mL−1, 30 min). Viable bacteriawere not observed on the gentamicin-treated p(HEMA: MAA) copolymers, whereas growth was observed on gentamicin-treatedp(HEMA). In light of the excellent antimicrobial activity and physicochemical properties, p(HEMA: MAA) copolymerscomposed of ratios of 80:20 or 70:30 HEMA: MAA were identified as potentially useful coatings of endotracheal tubes to be usedin conjunction with the clinical nebulization of gentamicin and designed for the prevention of ventilator-associated pneumonia

The role lipid aldehydes and ALEs in the pathogenesis of diabetic retinopathy

Antioxidant,antimicrobial and toxicological properties of Schinus molle L. essential oils

Ethnopharmacological relevance: Schinus molle L. has been used in folk medicine as antibacterial, antiviral, topical antiseptic, antifungal, antioxidant, anti-inflammatory, anti-tumoural as well as antispasmodic and analgesic; however, there are few studies of pharmacological and toxicological properties of S. molle essential oils. Aim of the study: The aim of this study was to evaluate the antioxidant and antimicrobial activities of S. molle leaf and fruit essential oils, correlated with their chemical composition and evaluate their acute toxicity. Materials and methods: The chemical composition of S. molle leaf and fruit essential oils were evaluated by GC-FID and GC-MS. Antioxidant properties were determined using the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) free radical and β-carotene/linoleic acid methods. Antimicrobial properties were evaluated by the agar disc diffusion method and minimal inhibitory concentration assay. Toxicity in Artemia salina and acute toxicity with behavioural screening in mice were evaluated. Results: The dominant compounds found in leaf and fruit essential oils (EOs) were monoterpene hydrocarbons, namely -phellandrene, β-phellandrene, β-myrcene, limonene and α-pinene. EOs showed low scavenging antioxidant activity by the DPPH free radical method and a higher activity by the β-carotene/linoleic acid method. Antimicrobial activity of EOs was observed for Gram+, Gram– pathogenic bacteria and food spoilage fungi. EOs showed cytotoxicity for Artemia salina and lower toxicity in Swiss mice. Conclusions: The result showed that EOs of leaves and fruits of S. molle demonstrated antioxidant and antimicrobial properties, suggesting their potential use in food or pharmaceutical industries.

ANTIOXIDANT, ANTIMICROBIAL AND TOXICOLOGICAL PROPERTIES OF SCHINUS MOLLE L. ESSENTIAL OILS

Schinus molle L. is commonly known as pink pepper or American pepper, of Anacardiaceae family, from subtropical regions of South America, introduced and naturalized in South Europe, including Portugal. In folk medicine, plant extracts and essential oil has related as having antibacterial, antiviral, antifungal, anti-inflammatory, antitumoral, antispasmodic, analgesic and antidepressive properties. The aim of present study was to evaluate the chemical composition and biological activities of essential oil extracted from leaves and fruits of S. molle. For this purpose, the essential oils were analyzed by gas chromatography (GC/FID) and antioxidant properties were evaluated by the free radical DPPH and by system β-carotene/linoleic acid methods. The antimicrobial activities were screened against pathogenic bacteria and fungi and food spoiling fungi by the disc diffusion assay and minimal inhibitory concentration (MIC) was determined for sensitive strains. Toxicity of essential oils were carried out by the brine shrimp mortality test (EC50) and acute lethal dose (DL50) determination after oral administration in Swiss mice The major components in leaf essential oil were α-phellandrene, β-phellandrene and limonene, while myrcene, α-phellandrene and 1,8-cineole are the main components in the fruit essential oil. The essential oils of leaf and fruit of S. molle showed antioxidant activity through the two mechanisms: the ability to capture free radicals and protection of lipid peroxidation. These oils exhibited also a broad microbial activity spectrum, against pathogenic bacteria Gram-positive and Gram-negative and Candida spp. The fruit essential oil showed high cytotoxicity against Artemia salina. Essential oils of leaves and fruits of S. molle showed significant antioxidant and microbial properties, so the studies continue to clarify more in deep its toxicity, including hepatotoxicity and nephrotoxicity, and to evaluate its medicinal or nutraceutical potential.

Synthesis of new vegetable oil-based polymeric materials

A crescente procura de recursos fosseis a que se tem assistido nos ultimos anos, tem resultado num crescimento sem precedentes dos precos, com consequencias imprevisiveis e que levara, no espaco de decadas, ao seu inevitavel esgotamento. A procura de um modelo de desenvolvimento sustentavel, baseado em recursos renovaveis e o grande desafio que se coloca a civilizacao no seculo XXI. A biomassa vegetal, atraves das designadas gBio-refinarias h, e uma alternativa logica para a producao de produtos quimicos e de materiais mas tambem de combustiveis e energia. Os oleos vegetais constituem uma das fracoes da biomassa vegetal, cuja exploracao tem merecido redobrada atencao nos ultimos anos, como fonte de materiais e de combustiveis. Assim, a presente dissertacao tem por objetivo o desenvolvimento de novos materiais polimericos derivados de oleos vegetais, seguindo duas abordagens distintas, nomeadamente a preparacao de polimeros atraves de polimerizacao por etapas e polimerizacao em cadeia (Parte B e C, respetivamente). Em primeiro lugar, foram sintetizados poliesteres alifaticos de cadeia longa a partir de monomeros provenientes do oleo de colza (Capitulo III). A auto-metatese do acido erucico com catalisadores de rutenio, seguida de hidrogenacao da ligacao dupla, originou o acido 1,26-hexacosanodioico, que por sua vez foi convertido em hexacosano-1,26-diol. Subsequentemente, a policondensacao do acido ƒ¿,ƒÖ-dicarboxilico de cadeia longa com o hexacosano-1,26-diol originou o poliester 26,26. O diacido C26 foi tambem polimerizado com outros alcano-diois de cadeia curta, nomeadamente o dodecano-1,12-diol e o butano-1,2-diol, produzindo, respetivamente, os poliesteres 12,26 e 4,26. Estes poliesteres de fontes 100% renovaveis possuem valores de Mn na ordem dos 8-14 kDa e valores de PDI entre 2.1 e 2.7. As propriedades destes poliesteres alifaticos foram avaliadas atraves de varias tecnicas, revelando elevada cristalinidade (com uma estrutura cristalina como a do polietileno) e elevadas temperaturas de fusao (74-104 ‹C), cristalizacao (68-92 ‹C) e degradacao (323-386 ‹C). Em segundo lugar, foram sintetizados polimeros lineares termo-reversiveis a partir de derivados do oleo de ricinio (Capitulo IV). Para tal foram preparados monomeros que incorporam aneis furanicos inseridos atraves do acoplamento tiol-eno, e que posteriormente foram polimerizados pela reacao de Diels-Alder (DA) entre os grupos furano (dieno A) e estruturas complementares do tipo maleimida (dienofilo B). Para as polimerizacoes DA foram consideradas duas abordagens diferentes, nomeadamente (i) o uso de monomeros com dois aneis furanicos terminais em conjunto com uma bismaleimida (sistemas AA+BB) e (ii) a utilizacao de um monomero que incorpora ambos os grupos reativos, furano e maleimida, na sua estrutura (sistema AB). Este estudo demonstrou claramente que ambas as estratégias foram bem sucedidas embora com diferentes resultados em termos da natureza dos produtos obtidos. Estes polímeros lineares apresentam valores relativamente baixos de Tg (-40 to -2 °C) devido à natureza flexível dos grupos separadores das funções reativas, e de Mn (4.5-9.0 kDa) dada a observada tendência de ciclização associada a concentrações baixas de monómero. A aplicação da reação de retro-DA aos polímeros em causa confirmou o seu caráter reversível, ou seja, a possibilidade de promover, em condições controladas, a despolimerização com recuperação dos monómeros de partida. Esta particularidade abre caminhos para materiais macromoleculares originais com aplicações promissoras tais como auto-reparação e reciclabilidade. Em terceiro lugar, sintetizaram-se polímeros não-lineares termo-reversíveis a partir de derivados do óleo de ricínio (Capítulo V). Para tal foram preparados monómeros trifuncionais e posteriormente polimerizados através da reação de DA entre os grupos reativos complementares furano/maleimida. Foram consideradas três abordagens distintas para preparar estes polímeros não-lineares, nomeadamente através da utilização de (i) um monómero bisfurânico em combinação com uma trismaleimida (sistema A2+B3) e (ii) um monómero trisfurânico em conjunto com uma bismaleimida (sistema A3+B2) que originaram materiais ramificados ou reticulados, e ainda (iii) a utilização de monómeros assimetricamente substituídos do tipo A2B ou AB2 capazes de originar estruturas macromoleculares hiper-ramificadas. Todos os sistemas apresentaram valores de Tg perto de 0 °C, o que era de esperar para estes materiais não-lineares. A aplicação da reação de retro-DA comprovou mais uma vez o caráter termo-reversível das polimerizações em causa. Em quarto lugar e último lugar, foram preparados copolímeros de acetato de vinilo (VAc) com monómeros derivados de óleo de girassol (Capítulo VI). Ésteres vinílicos de ácidos gordos (FAVE) foram sintetizados por transvinilação dos ácidos oleico e linoleico com VAc catalisada por um complexo de irídio. Os monómeros vinílicos preparados foram caracterizados e posteriormente homopolimerizados e copolimerizados com VAc através do uso dos grupos vinílicos terminais como função inicial de polimerização. A variação do tipo e quantidade de monómero FAVE e da quantidade de iniciador radicalar originou copolímeros de VAc com valores de Mn na gama de 1.2-3.0 kDa e valores de Tg de -5 a 16 °C. Os copolímeros foram avaliados em testes de cura oxidativa através das insaturações nas suas cadeias alifáticas para formar materiais reticulados, e os resultados sugerem que eles podem ser sistemas efetivos de cura para aplicações como tintas, vernizes e outros tipos de revestimento. Todos os materiais poliméricos preparados ao longo deste trabalho constituem contribuições atrativas para a área dos polímeros oriundos de recursos renováveis e representam uma prova indiscutível de que os óleos vegetais são percursores promissores de materiais macromoleculares com potenciais aplicações.

Synthesis and biological activities of polyphenolic hybrids - evaluation of anti-cancer, anti-inflammatory and antioxidant activities

Os compostos polifenólicos constituem uma classe de metabolitos secundários de plantas, mas existe também uma enorme quantidade de derivados sintéticos ou semi-sintéticos contendo múltiplas unidades fenólicas. Estes compostos apresentam importantes características biológicas, que dependem das suas estruturas básicas. Certos derivados desta família de compostos, tais como flavonoides, cromonas e cumarinas contribuem para os benefícios da dieta humana, e partilham o núcleo de benzopiran-(2 e 4)-ona ou benzofuran-3-ona. A presente dissertação inclui uma introdução geral e três capítulos que descrevem as novas rotas sintéticas estabelecidas para a preparação de novos híbridos de diversos compostos polifenólicos, assim como a sua elucidação estrutural e termina com a presentação dos resultados da avaliação biológica desses mesmos compostos. No segundo capítulo discute-se a preparação de híbridos de pirimidina- e imidazolidina-polifenóis, especialmente a síntese diastereoseletiva de novos híbridos benzofuran-3-ona-hidantoína e derivados de uracilo. A rota sintética envolve a ação de carbodiimidas sobre os ácidos cromona-(2- e 3)-carboxílicos num só passo ou em dois passos sequenciais, catalisada por uma base orgânica ou inorgânica. O terceiro capítulo descreve reações do tipo adições conjugadas 1,4 - hetero-ciclisações em cascata de compostos 1,3-dicarbonílicos em ácido cromona-3-carboxílico catalisadas por uma base orgânica, que originaram novas cromonas, cromanonas e flavonas polissubstituídas. As bispiranonas [bispiran-2 e 4)-onas] foram elaboradas numa reacção de acoplamento da 4-hidroxicumarina ou da lactona do ácido triacético com o ácido cromona-3-carboxílico ou precursores formil-funcionalizados (ω-formil-2’-hydroxy acetofenonas e cromona-3-carbaldeídos) utilizando organocatálise básica. Finalmente, alargou-se o estudo das adições conjugadas 1,4 para uma variedade de 4-hidroxipiran-2-onas e cetonas α,β-insaturadas para originar novos análogos de warfarina. Obteve-se uma variedade de estruturas complexas por hibridação das unidades de 4-hidroxicumarina ou da lactona do ácido triacético com os novos derivados de cromonas polissubstituídas. Todos as reações foram executadas em condições suaves e ambientalmente favoráveis, utilizando a 4-pirrolidinopiridina como organocatalisador básico. As estruturas dos novos híbridos polifenólicos foram caracterizados por técnicas espectroscópicas de alta resolução, incluindo espectroscopia de ressonância magnética nuclear (1D e 2D) e por difractometria de raios-X, que nos permitiram resolver o complexidade estrutural dos compostos sintetizados. O quarto capítulo apresenta os resultados da avaliação biológica obtidos com os híbridos polifenólicos sintetizados neste trabalho, mostrando a possibilidade de seu envolvimento na terapia do cancro. A maioria dos compostos foram avaliados quanto ao seu efeito sobre a citotoxicidade e proliferação de células leucémicas e ao seu envolvimento na regulação de via pró-inflamatória NF-kB, na qual, os híbridos de biscumarinas exibiram actividades elevadas (IC50 = 6-19 μM para inibição de NF-kB depois de 8 horas de incubação e IC50 = 15-39 μM para efeitos citotóxicos em células cancerosas, após 24 horas de incubação). Uma inibição moderada das enzimas HDAC e Cdc25 foi induzida pelos derivados de benzofuran-3-ona-hidantoína. Catorze dos novos derivados polifenólicos polissubstituídos, tendo como estrutura básica a benzopiran-4-ona, foram avaliados pela sua actividade quimiopreventiva do cancro mediada pela indução de sinalização citoprotectora Nrf2 (fator 2 relacionado com o fator nuclear da proteína E2) e capacidade para inibir a proliferação das células de cancro da mama. Os derivados da classe das cromanonas foram identificados como os indutores mais potentes da actividade Nrf2. As concentrações necessárias para aumentar a actividade de luciferase em 10 vezes (C10) foram de 2,8-21,3 μM. Todos os novos híbridos polifenólicos que apresentam atividade citotóxica e anti-proliferativa não afectam o crescimento de células saudáveis periféricas do sangue (PBMC) (IC50 > 50 μM), indicando a sua seletividade para as células cancerosas e sugerindo que alguns deles são estruturalmente interessantes para posteriores análises. A avaliação da atividade antioxidante utilizando os testes do radical livre DPPH e o poder redutor do ião férrico FRAP foram realizados em algumas estruturas híbridas polifenólicas.

Development of 2-oxazoline-based hydrogels and porous polyester microparticles in scCO2

Thesis submitted to Faculdade de Ciências e Tecnologia from Universidade Nova de Lisboa in partial fulfillment of the requirements for the obtention of the degree of Master of Science in Biotechnology

Desenvolvimento e aplicação de um biossensor para monitorização de creatinina, um biomarcador associado a doenças emergentes

Este trabalho descreve o desenvolvimento de um material sensor para creatinina por impressão molecular em estrutura polimérica (MIP) e a sua aplicação no desenvolvimento de um dispositivo de natureza potenciométrica para a determinação da molécula alvo em fluidos biológicos. A creatinina é um dos biomarcadores mais utilizados no acompanhamento da doença renal, já que é um bom indicador da taxa de filtração glomerular (TFG). Os materiais biomiméticos desenhados para interação com a creatinina foram obtidos por polimerização radicalar, recorrendo a monómeros de ácido metacríclico ou de vinilpiridina e a um agente de reticulação apropriado. De modo a aferir o efeito da impressão da creatinina na resposta dos materiais MIP à sua presença, foram também preparados e avaliados materiais de controlo, obtidos sem impressão molecular (NIP). O controlo da constituição química destes materiais, incluindo a extração da molécula impressa, foi realizado por Espectroscopia de Raman e de Infravermelho com Transformada de Fourrier. A afinidade de ligação entre estes materiais e a creatinina foi também avaliada com base em estudos cinéticos. Todos os materiais descritos foram integrados em membranas selectivas de elétrodos seletivos de ião, preparadas sem ou com aditivo iónico lipófilo, de carga negativa ou positiva. A avaliação das características gerais de funcionamento destes elétrodos, em meios de composição e pH diferentes, indicaram que as membranas com materiais impressos e aditivo aniónico eram as únicas com utilidade analítica. Os melhores resultados foram obtidos em solução tampão Piperazine-N,N′-bis(2- ethanesulfonic acid), PIPES, de pH 2,8, condição que permitiu obter uma resposta quasi-Nernstiana, a partir de 1,6×10-5 mol L-1. Estes elétrodos demonstraram ainda uma boa selectividade ao apresentaram uma resposta preferencial para a creatinina quando na presença de ureia, carnitina, glucose, ácido ascórbico, albumina, cloreto de cálcio, cloreto de potássio, cloreto de sódio e sulfato de magnésio. Os elétrodos foram ainda aplicados com sucesso na análise de amostras sintéticas de urina, quando os materiais sensores eram baseados em ácido metacrilico, e soro, quando os materiais sensores utilizados eram baseados em vinilpiridina.

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: Therapeutic potential of mitochondrially targeted antioxidants.

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.

Mitochondrial uncoupling as a regulator of life-history trajectories in birds: an experimental study in the zebra finch.

Mitochondria have a fundamental role in the transduction of energy from food into ATP. The coupling between food oxidation and ATP production is never perfect, but may nevertheless be of evolutionary significance. The 'uncoupling to survive' hypothesis suggests that 'mild' mitochondrial uncoupling evolved as a protective mechanism against the excessive production of damaging reactive oxygen species (ROS). Because resource allocation and ROS production are thought to shape animal life histories, alternative life-history trajectories might be driven by individual variation in the degree of mitochondrial uncoupling. We tested this hypothesis in a small bird species, the zebra finch (Taeniopygia guttata), by treating adults with the artificial mitochondrial uncoupler 2,4-dinitrophenol (DNP) over a 32-month period. In agreement with our expectations, the uncoupling treatment increased metabolic rate. However, we found no evidence that treated birds enjoyed lower oxidative stress levels or greater survival rates, in contrast to previous results in other taxa. In vitro experiments revealed lower sensitivity of ROS production to DNP in mitochondria isolated from skeletal muscles of zebra finch than mouse. In addition, we found significant reductions in the number of eggs laid and in the inflammatory immune response in treated birds. Altogether, our data suggest that the 'uncoupling to survive' hypothesis may not be applicable for zebra finches, presumably because of lower effects of mitochondrial uncoupling on mitochondrial ROS production in birds than in mammals. Nevertheless, mitochondrial uncoupling appeared to be a potential life-history regulator of traits such as fecundity and immunity at adulthood, even with food supplied ad libitum.

Vieillissement vasculaire chez des patients athérosclérotiques: Sénescence prématurée des cellules endothéliales?

La dysfonction de l’endothélium vasculaire, associée à une diminution de ses propriétés vasorelaxantes et anti-thrombogéniques, survient avec le vieillissement mais également chez de plus jeunes patients athérosclérotiques présentant plusieurs facteurs de risque cardiovasculaire. Au niveau cellulaire, le vieillissement des cellules endothéliales (CE) mène à un état irréversible de non division cellulaire appelé sénescence. Ces cellules sénescentes présentent des changements spécifiques au niveau de leur morphologie et de l’expression génique, menant à leur dysfonction. La sénescence dite réplicative est déclenchée par le raccourcissement des télomères survenant à chaque division cellulaire, mais peut également être induite prématurément par le stress oxydant (SIPS). L’objectif principal de cette étude est de caractériser la sénescence de CE vasculaires isolées à partir de patients athérosclérotiques, et d’observer l’impact des facteurs de risque sur cette sénescence. Afin de confirmer la contribution des deux principales voies de la sénescence, nous avons par la suite étudié conjointement ou séparément, l’impact d’un traitement chronique avec un antioxydant sur la sénescence de CE, et d’une surexpression de la sous-unité catalytique de la télomérase (hTERT), une enzyme responsable de l’allongement des télomères. Nous avons isolé et cultivé des CE provenant d’artères mammaires internes prélevées lors de pontages coronariens. Selon les études, les cellules ont été infectées ou non avec un lentivirus surexprimant la hTERT, et cultivées in vitro jusqu’à sénescence, en présence ou en absence de l’antioxydant N-acétyl-L-cystéine (NAC). Différents marqueurs des deux principales voies de la sénescence (réplicative ou SIPS) ont été quantifiés. La sénescence cellulaire se développe exponentiellement avec le temps et est associée à une réduction de la viabilité et de la prolifération cellulaires. Chez les patients athérosclérotiques, le vieillissement des CE passe par les deux principales voies de la sénescence : des télomères courts initialement en culture et la durée d’exposition in vivo aux facteurs de risque cardiovasculaire prédisent une apparition prématurée de la sénescence. Toutefois, chez les fumeurs, la sénescence est exclusivement du type SIPS. Ces facteurs de risque cardiovasculaire et principalement l’hypertension, semblent accélèrer le vieillissement biologique et favoriser la dysfonction des CE. Lorsque traitées chroniquement avec le NAC, les CE présentant initialement de moindres dommages cellulaires et moléculaires ainsi qu’une meilleure défense antioxydante développent une sénescence retardée. Lorsque le NAC est combiné à une surexpression de la hTERT, les deux voies de la sénescence sont bloquées et une immortalisation cellulaire est observée. À l’inverse, dans les CE les plus endommagées par les ROS in vivo, le NAC n’a aucun effet sur le développement de la sénescence, la hTERT, seule ou en combinaison avec le NAC, retarde légèrement la sénescence mais aucune immortalisation n’est observée lorsque ces traitements sont combinés. En conclusion, nos études démontrent que l’exposition chronique au stress oxydant associé aux facteurs de risque cardiovasculaire accélère le développement de la sénescence de CE vasculaires, contribuant potentiellement à l’athérogénèse. Dans les cellules de patients athérosclérotiques, il semble exister un seuil de dommages cellulaires et moléculaires subis in vivo au-delà duquel, aucun traitement (antioxydant ou hTERT) ne peut être bénéfique.