藻蓝蛋白复合物对酒精性肝损伤的保护作用

本文对藻蓝蛋白复合物保护化学性肝损伤的功效学进行了研究。以Wistar大鼠建立酒精性肝损伤模型，将藻蓝蛋白复合物分为高、中、低三个剂量组，以联苯双酯作为阳性对照组，灌胃给药42d，分别测定血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、血清丙二醛（MDA）、谷胱甘肽（GSH）、肝匀浆丙二醛（MDA）和谷胱甘肽（GSH）的含量，并对肝脏切片进行病理检查。结果表明：藻蓝蛋白复合物对血清ALT、AST具有显著的抑制作用，显著拮抗肝脏MDA的升高，显著提高肝脏GSH-Px含量。出现肝细胞浊肿、脂肪变性、点状坏死的大鼠数目极少。从而表明藻蓝蛋白复合物具有显著的保护酒精性肝损伤的功效。

高寒草甸植物抗氧化系统对长期增强UV-B辐射的响应

通过研究自然条件下模拟平流层臭氧破坏5%时近地表面增加的太阳UV-B辐射对高寒草甸4种典型植物（矮嵩草Kobresia humilis、垂穗披碱草Elymus nutans、麻花艽Gentiana straminea和鹅绒委陵菜Po-tentilla anserina）的抗氧化系统的影响表明,尽管各植物的抗氧化系统组分变化不同,但4种植物的膜脂过氧化程度没有加剧,长期增强UV-B辐射没有对膜系统造成损伤。在自然长期增强UV-B条件下,4种植物的膜脂过氧化产物——丙二醛（MDA）含量与对照相比无显著差异。垂穗披碱草、鹅绒委陵菜的谷胱甘肽（GSH）含量增加,麻花艽的超氧化物歧化酶（SOD）活性与鹅绒委陵菜的过氧化氢酶（CAT）活性上升,同时麻花艽的类胡萝卜素（Car）含量亦显著增加。可见这些植物已能很好地适应UV-B强辐射,其抗氧化能力除了与抗氧化系统各组分的协同作用有关外,也可能与种的适应性有关。

长期增强UV-B辐射对高寒草甸植物光合速率和抗氧化系统的影响

研究了在野外自然条件下，长期增强UV-B辐射对高寒草甸3种典型植物矮嵩草（Kobresia humilis）、垂穗披碱草（Elymus nutans）和钉柱委陵菜（Potentilla saundersiana）光合放氧速率、光合色素和抗氧化系统的影响。结果表明：长期增强UV-B辐射对3种植物的净光合速率没有明显影响。增强UV-B辐射下，3种植物的叶绿素含量变化不同，Chla／b值，类胡萝卜素（Car）含量，Car／Chl值与对照相比都有升高，说明植物叶片的光合能力、吸收紫外线的能力增强以及忍受逆境能力均有增强，从而产生光保护，有利于光合作用正常进行。由于3种植物膜脂过氧化程度的不同及SOD活性的普遍抑制，植物经受了氧化胁迫。垂穗披碱草叶片GSH含量显著七升，矮嵩草的形态矮小及钉柱委陵菜GSH含量与POD活性显著上升，都能减轻它们所经受的氧化胁迫，使光合器官免受损伤。所以，这些保护性色素的积累和抗氧化系统内部的协同作用可能是高寒草甸植物光合作用正常进行的重要原因。

柴达木盆地唐古特白刺籽油保护肝损伤作用研究

对柴达木盆地唐古特白刺种籽油保护化学性肝损伤作用进行了研究.将小鼠按体重随机分为白刺籽油高、中、低剂量组,另设空白对照组和联苯双酯、藏茵陈阳性对照组.小鼠连续灌胃给药15 d后,用CCl4进行肝损伤,测定小鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST);肝脏过氧化脂质(LPO)降解产物丙二醛(MDA)和谷胱甘肽过氧化物酶(GSH-Px)的含量.结果显示,白刺籽油对血清ALT、AST具有显著的抑制作用,显著拮抗肝脏MDA的升高,显著提高肝脏GSH-Px含量.表明白刺籽油具有明显的保护化学性肝损伤的保健作用.

藏成药安神丸脂溶性提取物对大鼠脂质代谢及抗氧化功能的影响

目的 观察藏成药安神丸饱和脂及不饱和脂提取物对大鼠脂质代谢及抗氧化功能的影响。方法 测定连续ig给药4周、8周及给药8周又停药4周后大鼠血清中丙二醛（MDA）、还原型谷胱甘肽（GSH）及高密谋脂蛋白（HDL）的含量。结果 安神丸的脂溶性提取物可明显增加较大龄鼠血清中的GSH和HDL含量。结论 安神丸脂溶性提取物可改善较大龄鼠脂质代谢功能，提高其抗氧化能力。

高原鼢鼠肌肉脂溶性物质的抗缺氧效果与化学成分

为了确定高在鼢鼠（Myospalax baileyi)体内抗缺氧水成分，通过超临界萃取（SCEF）方法从高原鼢鼠肌肉提取脂溶性部分，我们对其成分通过气相色谱－质谱（GC－MS）联用分析。为了证实高原鼢鼠肌肉脂溶性成分的抗缺氧效果，探讨其抗缺氧机制，我们以小鼠为实验动物，分为5个组，即高剂量实验组（HEG），中剂量实验组（MEG），低剂量实验组（LEG），阳性药物对照组（PEG），空白对照组（CEG），分别以浓度为20％萃取物，10％萃取物，5％萃取物，10％的红景天和蒸馏水连续饲喂10天，然后进行小鼠常压抗缺氧实验，测定小鼠血清超氧化物歧化酶（SOD），谷光甘肽过氧化物酶（GSH－PX），乳酸脱氢酶（LDH）活性及血清丙二(MAD)含量。结果发现，高原鼢鼠肌肉脂溶性物质提取率为11．5％，共确定了17种成分，主要成分是n-十六烷酸（32．293％）,n-十廿烷酸（1．109％）,n-十八烷酸（6．03％），二－羟基－环十五酮（1．198％），顺十六烯酸（6．13％），反十八烯酸（27．3％），和亚油酸（81．4％），小鼠抗缺氧实验和生化指标测试结果表明，高原鼢鼠肌从脂溶性成分在20％，10％，5％时，与空白对照组相比，都不同程度地延长小鼠在缺氧条件下的存活时间，提高小鼠血清SOD和GSH－PX活性，降低血清LDH活性，减少血清MDA含量。

Caratterizzazione proteomica di fluidi e tessuti in diverse condizioni fisio-patologiche

This thesis has been focused on the proteomic characterization of human saliva from donors of different ages, starting from birth up to adult age, and pediatric brain tumor tissues. The first study has been performed in order to compare the acid-insoluble fraction of saliva from preterm with at-term newborns and adults and establish if differences exist. In the second study medulloblastoma and pilocytic astrocytoma pediatric brain tumor extracts have been compared. In both studies 2- DE analysis was coupled with high resolution tandem mass spectrometry (MS/MS). The proteomic characterization of the acid-insoluble fractions of saliva from preterm newborns allowed to integrate data previously obtained on the acid-soluble fraction by HPLC-electrospray ionization (ESI)-mass spectrometry (MS), and to evidence several differences between preterm newborns, at-term newborns and adults. Spots differentially expressed between the three groups, according to image analysis of the gels, were submitted to in-gel tryptic digestion and the peptide mixture analyzed by high performance HPLC-ESI-MS/MS for their characterization. By this strategy, we identified three over-expressed proteins in atterm newborns with respect to preterm newborns and adults (BPI fold-containing family A member 1, two proteoforms of annexin A1, and keratin type 1 cytoskeletal 13), and several over-expressed proteins in adults (fatty acid-binding protein, S100A6, S100A7, two proteoforms of S100A9, several proteoforms of prolactin-inducible protein, Ig kappa chain, two proteoforms of cystatin SN, one proteoform of cystatin S and several proteoforms of α-amylase 1). Moreover, for the first time, it was possible to assign by MS/MS four spots of human saliva 2-DE, already detected by other authors, to different proteoforms of S100A9. The strategy applied used a sequential staining protocol to the 2-DE gels, first with Pro-Q Diamond, that allows specific detection of phosphoproteins, and successively with total protein SYPRO Ruby stain. In the second study, proteomic analysis of two pediatric brain tumor tissues pointed out differences between medulloblastoma, the prevalent malignant tumor in childhood, and pilocytic astrocytoma, the most common, that only rarely shows a malignant progression. Due to the limited availability of bioptic tissue, the study was performed on pooled tumor tissues, and was focused on acid-insoluble fraction to integrate the characterization performed by a group of colleagues in Rome on the acid-soluble fraction by high performance HPLC-ESI-MS/MS. The results indicated that the two tumors exhibit different proteomic profiles and evidenced interesting differential expression of several proteins. Among them, peroxiredoxin- 1, peptidyl-prolyl cis–trans isomerase A, heterogeneous nuclear ribonucleoproteins A2/B1, mitochondrial isoform of malate dehydrogenase, nucleoside diphosphate kinase A, glutathione S-transferase P and fructose bisphosphate aldolase A resulted significantly over-expressed in medulloblastoma while glial fibrillary acidic protein, serotransferrin, α crystallin B chain, ferritin light chain, annexin A5, fatty acid-binding protein (brain), sorcin and apolipoprotein A-I resulted significantly over-expressed in pilocytic astrocytoma. In conclusion, the work done allowed to evidence the usefulness of using an integrated bottom-up/top-down approach, based on 2-DE-MS analysis and high performance MS in order to obtain a complete characterization of the proteome under investigation, revealing and identifying, not only peptides and small proteins, but also proteins with higher MW, that often it is not possible to identify by using exclusively a top-down ESI-MS approach.

Active paraplegics are protected against exercise-induced oxidative damage through the induction of antioxidant enzymes

Exercise improves functional capacity in spinal cord injury (SCI). However, exhaustive exercise, especially when sporadic, is linked to the production of reactive oxygen species that may have a detrimental effect on SCI. We aimed to study the effect of a single bout of exhaustive exercise on systemic oxidative stress parameters and on the expression of antioxidant enzymes in individuals with paraplegia. The study was conducted in the Physical Therapy department and the Physical Education and Sports department of the University of Valencia. Sixteen paraplegic subjects were submitted to a graded exercise test (GET) until volitional exhaustion. They were divided into active or non-active groups. Blood samples were drawn immediately, 1 and 2 h after the GET. We determined plasma malondialdehyde (MDA) and protein carbonylation as markers of oxidative damage. Antioxidant gene expression (catalase and glutathione peroxidase-GPx) was determined in peripheral blood mononuclear cells. We found a significant increase in plasma MDA and protein carbonyls immediately after the GET (P<0.05). This increment correlated significantly with the lactate levels. Active paraplegics showed lower levels of exercise-induced oxidative damage (P<0.05) and higher exercise-induced catalase (P<0.01) and GPx (P<0.05) gene expression after the GET. These results suggest that exercise training may be useful in SCI patients to develop systemic antioxidant defenses that may protect them against exercise-induced oxidative damage.

Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host

Russell M. Morphew, Hazel A. Wright, E. James LaCourse, Debra J. Woods and Peter M. Brophy (2007). Comparative proteomics of excretory-secretory proteins released by the liver fluke Fasciola hepatica in sheep host bile and during in vitro culture ex host. Molecular and Cellular Proteomics, 6 (6), 963-972. Sponsorship: BBSRC / EU RAE2008

Efeitos da tetraciclina em Gambusia holbrooki

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Efeito dos salicilatos em Salmo trutta fario

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Farmacocinética do paracetamol

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Capillary electrophoresis with boron doped diamond electrode for amperometric detection of low molecular weight biological substrate

The research work in this thesis included the sensitive and selective separation of biological substance by capillary electrophoresis with a boron doped diamond electrode for amperometric detection. Chapter 1 introduced the capillary electrophoresis and electrochemical detection. It included the different modes of capillary electrophoresis, polyelectrolyte multilayers coating for open tubular capillary electrochromatography, different modes of electrochemical detection and carbon based electrodes. Chapter 2 showed the synthesized and electropolymerized N-acetyltyramine with a negatively charged sulfobutylether-β-cyclodextrin on a boron doped diamond (BDD) electrode followed by the electropolymerzation of pyrrole to form a stable and permselective film for selective dopamine detection. For comparison, a glassy carbon (GC) electrode with a combined electropolymerized permselective film of polytyramine and polypyrrole-1-propionic acid was used for selective detection of dopamine. The detection limit of dopamine was improved from 100 nM at a GC electrode to 5 nM at a BDD electrode. Chapter 3 showed field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles embedded in poly(diallyldimethylammonium) chloride, which has been investigated for the electrophoretic separation of indoxyl sulphate, homovanillic acid and vanillylmandelic acid. The detection limit of the three analytes obtained by using a boron doped diamond electrode was around 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfereing chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. Chapter 4 showed the selective detection of Pseudomonas Quinolone Signal, PQS for quorum sensing from its precursor HHQ, using a simply boron doped diamond electrode. Furthermore, by combining poly(diallyldimethylammonium) chloride modified fused silica capillary with a BDD electrode for amperometric detection, PQS was separated from HHQ and other analogues. The detection limit of PQS was as low as 65 nM. Different P. aeruginosa mutant strains were studied. Chapter 5 showed the separation of aminothiols by layer-by-layer coating of silica capillary with a boron doped diamond electrode. The capillary was layer-by-layer coated with the polycation poly(diallyldimethylammonium) chloride and negatively charged silica nanoparticles. All the aminothiols was separated and detected using a BDD electrode in an acidic electrolyte. It was a novel scheme for the separation and detection of glutathione reduced and oxidized forms, which is important for estimated overstressed level in the human system.

Mammalian cell cultures as a model system for the determination of cytotoxicity induced by cholesterol oxidation products

Oxysterols are products of cholesterol oxidation, which may be produced endogenously or may be absorbed from the diet where they are commonly found in foods of animal origin. Oxysterols are known to be cyctotoxic to cells in culture and mode of toxicity has been identified as apoptosis in certain cell lines. The cytotoxicity of the oxysterols 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) was examined in two human cell lines, HepG2, a hepatoma cell line, and U937, a monocytic cell line. Both 25-OH and 7β-OH were cytotoxic to the HepG2 cell line but apoptotic cells were not detected and it was concluded that cells underwent necrosis. 25-OH was not cytotoxic to the U937 cell line but it was found to have a cytostatic effect. 7β-OH was shown to induce apoptosis in the U937 line. The mechanism of oxysterol-induced apoptosis has not yet been fully elucidated, however the generation of an oxidative stress and the depletion of glutathione have been associated with the initial stages of the apoptotic process. The concentration of cellular antioxidant enzyme, superoxide dismutase (SOD) was increased in association with 7β-OH induced apoptosis in the U937 cell line. There was no change in the glutathione concentration or the SOD activity of HepG2 cells, which underwent necrosis in the presence of 7β-OH. Many apoptotic pathways center on the activation of caspase-3, which is the key executioner protease of apoptosis. Caspase-3 activity was also shown to increase in association with 7β-OH-induced apoptosis in U937 cells but there was no significant increase in caspase-3 activity in HepG2 cells. DNA fragmentation is regarded as the biochemical hallmark of apoptosis, therefore the comet assay as a measure of DNA fragmentation was assessed as a measure of apoptosis. The level of DNA fragmentation induced by 7β-OH, as measured using the comet assay, was similar for both cell lines. Therefore, it was concluded that the comet assay could not be used to distinguish between 7β-OH-induced apoptosis in U937 cells and 7β-OH-induced necrosis in HepG2 cells. The cytotoxicity and apoptotic potency of oxysterols 25-OH, 7β-OH, cholesterol- 5a,6a-epoxide (a-epoxide), cholesterol-5β,6β-epoxide (β-epoxide), 19-hydroxy-cholesterol (19-OH), and 7-ketocholesterol (7-keto) was compared in the U937 cell line. 7 β-OH, β-epoxide and 7-keto were found to induce apoptosis in U937 cells. 7β-OH-induced apoptosis was associated with a decrease in the cellular glutathione concentration and an increase in SOD activity, 7-keto and β-epoxide did not affect the glutathione concentration or the SOD activity of the cells.a-Epoxide, 19-OH and 25-OH were not cytotoxic to the U937 cell line.

Novel ingredients from brewers' spent grain - bioactivity in cell culture model systems and bioactivity retention in fortified food products

Functional food ingredients, with scientifically proven and validated bioactive effects, present an effective means of inferring physiological health benefits to consumers to reduce the risk of certain diseases. The search for novel bioactive compounds for incorporation into functional foods is particularly active, with brewers’ spent grain (BSG, a brewing industry co-product) representing a unique source of potentially bioactive compounds. The DNA protective, antioxidant and immunomodulatory effects of phenolic extracts from both pale (P1 - P4) and black (B1 – B4) BSG were examined. Black BSG extracts significantly (P < 0.05) protected against DNA damage induced by hydrogen peroxide (H2O2) and extracts with the highest total phenolic content (TPC) protected against 3-morpholinosydnonimine hydrochloride (SIN-1)-induced oxidative DNA damage, measured by the comet assay. Cellular antioxidant activity assays were used to measured antioxidant potential in the U937 cell line. Extracts P1 – P3 and B2 - B4 demonstrated significant (P < 0.05) antioxidant activity, measured by the superoxide dismutase (SOD) activity, catalase (CAT) activity and gluatathione (GSH) content assays. Phenolic extracts P2 and P3 from pale BSG possess anti-inflammatory activity measured in concanavalin-A (conA) stimulated Jurkat T cells by an enzyme-linked immunosorbent assay (ELISA); significantly (P < 0.05) reducing production of interleukin-2 (IL-2), interleukin-4 (IL-4, P2 only), interleukin-10 (IL-10) and interferon-γ (IFN-γ). Black BSG phenolic extracts did not exhibit anti-inflammatory effects in vitro. Hydroxycinnamic acids (HA) have previously been shown to be the phenolic acids present at highest concentration in BSG; therefore the HA profile of the phenolic extracts used in this research, the original barley (before brewing) and whole BSG was characterised and quantified using high performance liquid chromatography (HPLC). The concentration of HA present in the samples was in the order of ferulic acid (FA) > p-coumaric acid (p-CA) derivatives > FA derivatives > p-CA > caffeic acid (CA) > CA derivatives. Results suggested that brewing and roasting decreased the HA content. Protein hydrolysates from BSG were also screened for their antioxidant and anti-inflammatory potential. A total of 34 BSG protein samples were tested. Initial analyses of samples A – J found the protein samples did not exert DNA protective effects (except hydrolysate H) or antioxidant effects by the comet and SOD assays, respectively. Samples D, E, F and J selectively reduced IFN-γ production (P < 0.05) in Jurkat T cells, measured using enzyme linked immunosorbent assay (ELISA). Further testing of hydrolysates K – W, including fractionated hydrolysates with molecular weight < 3, < 5 and > 5 kDa, found that higher molecular weight (> 5 kDa) and unfractionated hydrolysates demonstrate greatest anti-inflammatory effects, while fractionated hydrolysates were also shown to have antioxidant activity, by the SOD activity assay. A commercially available yogurt drink (Actimel) and snack-bar and chocolate-drink formulations were fortified with the most bioactive phenolic and protein samples – P2, B2, W, W < 3 kDa, W < 5 kDa, W > 5 kDa. All fortified foods were subjected to a simulated gastrointestinal in vitro digestion procedure and bioactivity retention in the digestates was determined using the comet and ELISA assays. Yogurt fortified with B2 digestate significantly (P < 0.05) protected against H2O2-induced DNA damage in Caco-2 cells. Greatest immunomodulatory activity was demonstrated by the snack-bar formulation, significantly (P < 0.05) reducing IFN-γ production in con-A stimulated Jurkat T cells. Hydrolysate W significantly (P < 0.05) increased the IFN-γ reducing capacity of the snack-bar. Addition of fractionated hydrolysate W < 3 kDa and W < 5 kDa to yogurt also reduced IL-2 production to a greater extent than the unfortified yogurt (P < 0.05).