Ngai Lei-Ka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 113-125). / Abstracts in English and Chinese. / List of Abbreviations --- p.ix / List of Tables --- p.x / List of Figures --- p.xii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Flavonoids --- p.1 / Chapter 1.1.1 --- The six major classes of flavonoids --- p.1 / Chapter 1.1.1.1 --- Flavonones --- p.1 / Chapter 1.1.1.2 --- Flavones --- p.2 / Chapter 1.1.1.3 --- Flavonols --- p.2 / Chapter 1.1.1.4 --- Isoflavonoids --- p.2 / Chapter 1.1.1.5 --- Anthocyanidins --- p.3 / Chapter 1.1.1.6 --- Flavans --- p.3 / Chapter 1.1.2 --- Structural variation of flavonoids --- p.3 / Chapter 1.1.3 --- The roles of flavonoids --- p.6 / Chapter 1.2 --- "Free radicals, oxidative stress and antioxidants" --- p.7 / Chapter 1.2.1 --- Oxidants and free radicals --- p.7 / Chapter 1.2.2 --- Lipid peroxidation (LPO) --- p.8 / Chapter 1.2.3 --- Oxidative stress and human diseases --- p.9 / Chapter 1.2.4 --- Role of food antioxidants --- p.10 / Chapter 1.2.5 --- Synthetic and natural food antioxidants --- p.11 / Chapter 1.3 --- Antioxidant activity of flavonoids --- p.12 / Chapter 1.4 --- Determination of antioxidant activity --- p.13 / Chapter 1.4.1 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.13 / Chapter 1.4.2 --- DPPH radical scavenging assay --- p.14 / Chapter 1.4.3 --- β-carotene bleaching assay --- p.15 / Chapter 1.5 --- Single cell gel electrophoresis assay (Comet assay) --- p.16 / Chapter 1.6 --- Determination of the genotoxicity --- p.18 / Chapter 1.7 --- Research objectives --- p.19 / Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Standards and reagents --- p.26 / Chapter 2.2 --- Sample Preparation --- p.26 / Chapter 2.3 --- Trolox equivalent antioxidant capacity (TEAC) assay --- p.27 / Chapter 2.4 --- DPPH´Ø radical scavenging assay --- p.28 / Chapter 2.5 --- β-carotene bleaching assay --- p.29 / Chapter 2.6 --- The comet assay --- p.30 / Chapter 2.6.1 --- Preparation of reagents --- p.31 / Chapter 2.6.2 --- Blood sample --- p.32 / Chapter 2.6.3 --- Optimal condition of comet assay --- p.32 / Chapter 2.6.3.1 --- Induction of DNA damage --- p.32 / Chapter 2.6.3.2 --- Antioxidant pre-treatment --- p.32 / Chapter 2.6.4 --- Hydrogen peroxide (H2O2) treatment --- p.32 / Chapter 2.6.4.1 --- Pre-incubation system --- p.32 / Chapter 2.6.4.2 --- Co-incubation system --- p.33 / Chapter 2.6.5 --- Slide preparation --- p.33 / Chapter 2.6.6 --- Cell lysis --- p.33 / Chapter 2.6.7 --- Alkaline treatment and electrophoresis --- p.34 / Chapter 2.6.8 --- Neutralization --- p.34 / Chapter 2.6.9 --- Quantification of DNA damage --- p.34 / Chapter 2.6.10 --- Cell viability analysis --- p.35 / Chapter 2.6.11 --- Statistical Analysis --- p.35 / Chapter 2.7 --- Mutatox® test --- p.36 / Chapter 2.8 --- Statistical analysis --- p.36 / Chapter 3 --- Result --- p.45 / Chapter 3.1 --- Determination of antioxidant activity using Trolox equivalent antioxidant capacity (TEAC) assay --- p.45 / Chapter 3.1.1 --- Trolox standard reference --- p.45 / Chapter 3.1.2 --- Antioxidant activity: ABTS´Ø+ scavenging capacity --- p.45 / Chapter 3.2 --- Evaluation of antioxidant activity using free radical scavenging assay --- p.46 / Chapter 3.2.1 --- Free radical scavenging abilities at 5 minutes --- p.46 / Chapter 3.2.2 --- Antiradical efficiency --- p.47 / Chapter 3.3 --- Evaluation of antioxidant activity using β-carotene bleaching assay --- p.47 / Chapter 3.3.1 --- Optimal incubation time --- p.47 / Chapter 3.2.2 --- Antioxidant activities on β-carotene bleaching assay --- p.47 / Chapter 3.4 --- Structure and antioxidant activities relationship (SAR) of flavonoids --- p.48 / Chapter 3.4.1 --- Effect of hydroxylation on the antioxidant activities of flavonoids --- p.48 / Chapter 3.4.1.1 --- Hydroxylation positions --- p.48 / Chapter 3.4.1.2 --- Polyhydroxylation --- p.48 / Chapter 3.4.2 --- Importance of B ring structure --- p.49 / Chapter 3.4.2.1 --- Increase hydroxylation in B ring --- p.49 / Chapter 3.4.2.2 --- The othro-dihydroxyl arranagement in B ring --- p.49 / Chapter 3.4.3 --- Importance of C ring structure --- p.50 / Chapter 3.4.3.1 --- The presence of a hydroxyl group at C3 --- p.50 / Chapter 3.4.3.2 --- The blockage of hydroxylation at C3 --- p.50 / Chapter 3.4.3.3 --- The presence of a double bond between C2 to C3 --- p.50 / Chapter 3.4.3.4 --- The presence of a carbonyl group at C4 --- p.50 / Chapter 3.4.3.5 --- The conjugation of a carbonyl at with a double bond between C2 to C3 --- p.51 / Chapter 3.4.4 --- Effect of glycosylation --- p.51 / Chapter 3.5 --- Evaluation of protective effects on DNA damage using comet assay --- p.51 / Chapter 3.5.1 --- Optimization of conditions fro the determination of H2O2-induced DNA damage --- p.52 / Chapter 3.5.1.1 --- H2O2 concentration & treatment temperature --- p.52 / Chapter 3.5.1.2 --- H2O2 treatment time --- p.52 / Chapter 3.5.1.3 --- Sample volume --- p.52 / Chapter 3.5.2 --- Protective effect of vitamin C on DNA damage --- p.53 / Chapter 3.5.3 --- Protective effect of selected flavonoids on DNA damage --- p.53 / Chapter 3.5.4 --- Structure and antioxidant activities relationship (SAR) of flavonoids in comet assay --- p.54 / Chapter 3.5.4.1 --- The importance of B ring structures --- p.54 / Chapter 3.5.4.2 --- The importance of C ring structures --- p.54 / Chapter 3.6 --- Evaluation of genotoxicity of flavonoids using Mutatox test --- p.55 / Chapter 4 --- Discussion --- p.96 / Chapter 4.1 --- Comparison of antioxidant activities between hydrophilic and lipophilic assays --- p.97 / Chapter 4.2 --- Structure and antioxidant activity relationship of flavonoids --- p.98 / Chapter 4.3 --- The comet assay --- p.103 / Chapter 4.4 --- Comparison of protective effect on DNA damage between pre-incubation and co-incubation systems --- p.105 / Chapter 4.5 --- Structure and protective effect of flavonoids in the comet assay --- p.106 / Chapter 4.6 --- Genotoxicity of flavonoids --- p.108 / Chapter 4.7 --- Significance and future works --- p.108 / Chapter 5 --- Conclusion --- p.111 / References --- p.113

Dietary phosphate is essential for normal fracture healing and bone growth. Previous studies have established that mice given a phosphate deficient diet after a fracture demonstrate delayed cartilage maturation and callus mineralization, as well as changes in gene expression consistent with oxidative phosphorylation dysfunction. This study was undertaken in order to examine the role of inorganic and organic phosphate availability on chondrocyte differentiation and mineralization, and to define the relationship between these processes and changes in chondrocyte metabolic function. ATDC5 murine chondroprogenitor cell line, which has been shown to undergo in vitro differentiation and extracellular matrix mineralization, was cultured under both differentiating and non-differentiating media conditions under conditions in 1mM -0.25mM sodium phosphate monobasic (inorganic phosphate) in the presence or absence of 4mM β-glycerol phosphate (organic phosphate). In the first series of studies, overall cell growth (total DNA and protein contents), mineralization (calcium accumulation), and cell-normalized oxidative metabolism (basal respiration, maximal respiration, ATP turnover, spare capacity, proton leak, and non-mitochondrial respiration rates) were measured over a 28 day time course in cultures grown in differentiating (ascorbic acid, insulin-transferrin-selenium, and β-glycerol phosphate) conditions in 1mM phosphate. These studies found that when the cells were induced to differentiate, there was a measurable increase in protein content while DNA content decreased by 30%, indicating a fraction of the cells underwent cell death. Differentiation was further associated with an overall two-fold increase in oxidative respiration. Next we assessed how differentiation, the promotion of matrix mineralization, and inorganic phosphate availability affected oxidative respiration. When differentiation was not induced with ascorbic acid and β-glycerol phosphate, there was no over growth in the cultures nor any change in total extracellular matrix mineralization or oxidative respiration. In the absence only of β-glycerol phosphate, differentiation proceeded but matrix mineralization did not occur. However, overall protein content and oxidative respiration were statistically two- and 1.5-fold higher, respectively, independent of the inorganic phosphate contents of the growth media. These results suggest that both differentiation and overall protein accumulation are strongly associated with increased oxidative metabolism while mineralization of the matrix decreased oxidative function. Only at the lowest phosphate levels were changes in basal oxidative function observed. These results are consistent with previous in vivo findings suggesting that diminished expression of mitochondrial associated genes in callus tissues from hypophosphatemic mice were associated with an overall decrease in chondrocyte differentiation.

Catalyst-Controlled Divergent C–H Functionalization of Unsymmetrical 2-Aryl Cyclic 1,3-Dicarbonyl Compounds with Alkynes and Alkenes A problem faced within the area of C–H functionalization is achieving siteselectivity when several similar C–H bonds are present within a given compound. One solution to this problem is the development of reactions that employ different catalytic systems to control the required selectivity. Herein, it is shown that such catalyst-controlled selectivity could be achieved on 2-aryl cyclic 1,3-dicarbonyl compounds where there exist two potential, non-adjacent sites for C–H functionalization. Examples demonstrate the palladium- and ruthenium-catalysed oxidative annulations of the 2-aryl cyclic 1,3-dicarbonyl substrates with alkynes, as well as with alkenes, where initial C–H bond cleavage occurs at one of two potential sites, depending on the catalyst used, which give unique products. 1,4-Rhodium(III) Migration in the One-Carbon Oxidative Annulations of 2-Arylphenols, Benzamides, and Benzoic Acids with 1,3-Enynes Oxidative annulations of 2-arylphenols, benzamides, and benzoic acids with alkynes and enynes are precedented and provide a range of heterocyclic products. However, in these examples, either the alkyne or enyne acts as a two-carbon annulation partner, reacting only across the alkynyl moiety. Herein, a more expansive scope of a previously published process in which 1,3-enynes, possessing allylic hydrogen atoms cis to the alkyne, undergo oxidative annulations with the three aforementioned classes of substrates as a one-carbon annulation partner is described. Proposed to occur via the 1,4-migration of a rhodium(III) species, annulated products were formed from a range of 1,3-enynes and substrates possessing a variety of functional groups.

Orientador: Roberto Calderon Gonçalves / Banca: Simone Biagio Chiacchio / Banca: Raimundo de Souza Lopes / Banca: Fernando José Benesi / Banca: Débora Cristina Damasceno / Resumo: Nas espécies domésticas as pneumonias cursam com intensa resposta inflamatória e acúmulo de células fagocíticas nos pulmões, levando a danos expressivos das estruturas do trato respiratório e à função pulmonar devido ao estresse oxidativo decorrente da liberação de grandes quantidades de Espécies Reativas do Oxigênio (ERO) durante a explosão respiratória. O objetivo deste estudo foi analisar o status oxidativo, a resposta inflamatória e a gasometria arterial, de ovinos sadios (n=20) e com diagnóstico clínico de pneumonia (n=20). Inicialmente os animais foram submetidos ao exame clínico e divididos em dois grupos: I) G1/controle, composto pelos animais clinicamente sadios e II) G2, composto pelos animais portadores de pneumonia. O status oxidativo foi avaliado por determinação indireta da atividade enzimática da Superóxido Dismutase (SOD) e Glutationa Peroxidase (GSH-Px) e das concentrações de Glutationa total (GSH-t) e Substâncias Reativas ao Ácido Tiobarbitúrico (TBARS) no sangue periférico por método colorimétrico. A resposta inflamatória foi avaliada pelo hemograma e proteína total e fibrinogênio plasmáticos e a função pulmonar pela determinação das variáveis hemogasométricas Pressão Arterial de Oxigênio (PaO2), Pressão Arterial de Gás Carbônico (PaCO2), Hidrogeniônico (pH), Saturação de Oxigênio (SO2), Bicarbonato (HCO3¯), Dióxido de Carbono Total (TCO2) e Excesso de Bases (EB), avaliados em sangue arterial. O leucograma revelou leucocitose com neutrofilia, eosinofilia, monocitose e linfopenia nos animais doentes (p<0,05). Com relação aos parâmetros bioquímicos, os ovinos portadores de pneumonia apresentaram aumento significativo (p>0,05) da concentração de fibrinogênio e proteína plasmática total. Os animais portadores de pneumonia apresentaram diminuição estatisticamente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In domestic species, pneumonia is accompanied by intense inflammatory response and accumulation of phagocytic cells in the lungs, causing structural damage of the respiratory tract due to oxidative stress resulting from the release of large amounts of Reactive Oxygen Species (ROS) during the respiratory burst. The aim of this study was to analyze the oxidative status, inflammatory response and arterial blood gases values in healthy sheep (n=20) and animals with a clinically diagnosed pneumonia (n = 20). After physical examination the animals were divided into two groups: i) G1/control, composed of clinically healthy animals and ii) G2, composed of animals with pneumonia. The oxidative status was assessed by indirect determinations of enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and concentrations of total glutathione (GSH-t) and thiobarbituric acid reactive substances (TBARS) in peripheral blood by a colorimetric method. The inflammatory response was evaluated by complete blood count and total protein and plasma fibrinogen. The lung function was evaluated by determinations of blood gas parameters in arterial blood: Oxygen Pressure (PaO2) Pressure of Carbon Dioxide (PaCO2), Pressure Hydrogen (pH), Oxygen Saturation (SO2), Bicarbonate (HCO3¯), Total Carbon Dioxide (TCO2) and Base Excess (EB). The leucogram results showed Leukocytosis with neutrophilia, eosinophilia, monocytosis and lymphopenia in sick animals (p<0,05). With regard to biochemical parameters, sheep with pneumonia showed a significant increase (p<0,05) of fibrinogen and total plasma protein concentrations. The animals from group G2 had a statistically significant reduction (p<0,05) in SOD and GSH-Px enzymatic activity and GSH-t concentration, while TBARS concentration was significantly higher (p<0,05). Arterial blood... (Complete abstract click electronic access below) / Doutor

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / In last years, considerable efforts have been made to identify neuroactive and neuroprotective peptides derived from the venom of different natural species. In this work, were studied the activity of Dinoponera quadriceps native (DqV) and denatured (DqDV) venom on chemically induced seizures models in vivo and on in vitro cortical astrocytes viability. Male Swiss mice (28- 33g) were pretreated with DqV (0.1 or 0.5 mg/kg, e.v., n = 6-8), DqDV (0.5 or 2.0 mg/kg, i.p., n = 6-8) or DqDV (0.1 or 0.5 mg/kg, e.v., n = 6-8). 30 minutes after the intraperitoneal pretreatment or ten minutes after intravenous pretreatment with the venom was induced seizures in all animals by the administration of pentylenetetrazole (80 mg/kg) pilocarpine (400 mg/kg) or strychnine (3.0 mg/kg). In behavioral analysis, we recorded the time to the first seizure and to death and the percentage of survival. To determine the parameters of oxidative stress was dissected three brain areas (prefrontal cortex, hippocampus and striatum) of animals used in behavioral analysis, in order to determine the degree of lipid peroxidation, by measuring the levels of malondialdehyde (MDA), and the content of nitrite. In in vitro assay, cell viability of cortical astrocytes was determined after treatment with different concentrations of DqV, PTZ and DqV + PTZ. The data were analyzed by ANOVA followed by a Student Newman-Keuls (for in vivo tests) or Bonferroni (for in vitro experiments) as post hoc tests. It was observed that the DqV had effect only on the PTZ model, both in behavioral analysis as for the determination of the oxidative parameters. Pretreatment with DqV significantly reduced the time until the occurrence of first seizure (0.1 mg/kg: 77.83 Â 5.27 compared to the 101.0 Â 3.31 seconds in the control group; 0.5 mg/kg: 74.43 Â 3.94 compared to the 101.0 Â 3.31 seconds in the control group), while DqDV caused an increase in the percentage of survival when pretreated by i.p. (0.5 mg/kg: 25% of survival compared with 0% in the control group, 2.0 mg/kg: 62.5% of survival compared with 0% in control group) and e.v (0.5 mg/kg: 28.57% of survival compared with 0% in control group). routes. In relation to the oxidative stress parameters, both pretreatments with DqV and with DqDV caused increases of MDA levels in all three brain areas analyzed. The nitrite content also increased after pretreatment with DqV in the three areas of the brain and after pretreatment with DqDV via e.v., only in the hippocampus. About cell viability assays, were observed that DqV was not able to change this parameter. The PTZ reduced the cell viability of astrocytes in a dose-dependent way, with an IC 50 (cytotoxicity index to 50% of the cell population under study) corresponding to 33.12 mM. The combined treatment of DqV (100 Âg/mL) and PTZ (IC 50) also caused a reduction in cell viability. The results suggest that the DqV, probably, has both neurotoxic and neuroprotective components, and that the astrocytes should be the cells involved in the venomâs neurotoxicity. / Nos Ãltimos anos, esforÃos considerÃveis tÃm sido feitos no sentido de identificar peptÃdeos naturais neuroativos e neuroprotetores derivados do veneno de diferentes espÃcies animais. Nesse trabalho foi estudada a atividade do veneno de Dinoponera quadriceps nativo (vDq) e desnaturado (vdDq) em modelos animais de convulsÃo quimicamente induzidos in vivo e sobre a viabilidade de astrÃcitos corticais in vitro. Camundongos Swiss machos (28-33g) foram prÃ-tratados com o vDq (0,1 ou 0,5 mg/kg, e.v, n= 6-8), vdDq (0,5 ou 2,0 mg/kg, i.p., n= 6-8) ou com vdDq (0,1 ou 0,5 mg/kg, e.v., n= 6-8). Meia hora apÃs o prÃ-tratamento intraperitoneal ou dez minutos apÃs o prÃ-tratamento endovenoso com o veneno foi induzida a convulsÃo em todos os animais atravÃs da administraÃÃo de pentilenotetrazol (80 mg/kg), pilocarpina (400 mg/kg,) ou estricnina (3,0 mg/kg). Na anÃlise comportamental, foram registrados os tempos para ocorrÃncia da primeira convulsÃo e morte e o percentual de sobrevida. Para determinaÃÃo dos parÃmetros de stress oxidativo foram utilizadas trÃs Ãreas cerebrais (cÃrtex prÃ-frontal, hipocampo e corpo estriado) de animais utilizados na anÃlise comportamental, a fim de se determinar o grau de peroxidaÃÃo lipÃdica, pela mensuraÃÃo dos nÃveis de malondialdeÃdo (MDA), e o conteÃdo de nitrito. No ensaio in vitro, foi determinada a viabilidade celular de astrÃcitos corticais apÃs o tratamento com diferentes concentraÃÃes de vDq, PTZ e vDq + PTZ. Os dados foram analisados por ANOVA e Student-Newman-Keuls como pÃs-teste, para os ensaios in vivo, e ANOVA seguido pelo pÃs-teste de Bonferroni, para as experimentaÃÃes in vitro. Foi observado que o vDq apresentou efeito apenas no modelo de PTZ, tanto na anÃlise comportamental quanto na determinaÃÃo dos parÃmetros oxidativos. O prÃ-tratamento com vDq reduziu significativamente o tempo para ocorrÃncia da primeira convulsÃo (0,1 mg/kg: 77,83 Â 5,27 comparado com 101,0 Â 3,31 segundos no grupo controle; 0,5 mg/kg: 74,43 Â 3,94 comparado com 101,0 Â 3,31 segundos no grupo controle), enquanto que o vdDq causou aumento do percentual de sobrevida quando prÃ-tratado por via i.p. (0,5 mg/kg: 25% de sobrevida comparado com 0% no grupo controle; 2,0 mg/kg: 62,5% de sobrevida comparado com 0% no grupo controle) e e.v (0,5 mg/kg: 28,57% de sobrevida comparado com 0% no grupo controle). Em relaÃÃo aos parÃmetros de estresse oxidativo, tanto o prÃ-tratamento com o vDq quanto com o vdDq causaram aumentos dos nÃveis de MDA nas trÃs Ãreas cerebrais analisadas. O conteÃdo de nitrito tambÃm sofreu elevaÃÃo apÃs prÃ-tratamento com vDq, nas trÃs Ãreas cerebrais, e apÃs o prÃ-tratamento com vdDq, via e.v., apenas no hipocampo. Quanto aos ensaios de viabilidade celular, foi observado que o vDq, isoladamente, nÃo foi capaz de alterar esse parÃmetro. O PTZ causou reduÃÃo da viabilidade de astrÃcitos de modo dose-dependente e com uma IC 50 (Ãndice de citotoxicidade para 50% da populaÃÃo celular em estudo) correspondente a 33,12 mM. O tratamento combinado entre vDq (100 Âg/mL) e PTZ (IC 50) tambÃm causou reduÃÃo da viabilidade das cÃlulas. Os resultados sugerem que o vDq possivelmente apresenta ambos componentes neurotÃxicos e neuroprotetores, e os astrÃcitos devem ser uma das cÃlulas envolvidas na neurotoxicidade do veneno.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-04-04T14:38:17Z No. of bitstreams: 1 Alessandra Melise Golke.pdf: 1584787 bytes, checksum: c4cea85302181139401e8596c156ca0a (MD5) / Made available in DSpace on 2016-04-04T14:38:18Z (GMT). No. of bitstreams: 1 Alessandra Melise Golke.pdf: 1584787 bytes, checksum: c4cea85302181139401e8596c156ca0a (MD5) Previous issue date: 2015-07-27 / A malária é causada por protozoários do gênero Plasmodium, sendo que o P. falciparum é responsável pela maioria das mortes. O quadro clínico dessa parasitose inclui febre, calafrios, cefaleia, vômito, diarreia, anemia, coma e morte. O mais antigo antimalárico é a quinina, um alcaloide obtido a partir da casca da árvore de espécies de Cinchona. Devido a sua toxicidade e ao surgimento de novos fármacos, seu uso foi limitado, porém voltou a ter maior importância em virtude da resistência do plasmódio a outros medicamentos. Uma alternativa para minimizar a toxicidade é o desenvolvimento de uma forma farmacêutica capaz de tornar esse fármaco menos tóxico, como os sistemas nanoencapsulados. O presente trabalho teve como objetivo verificar parâmetros hematológicos, bioquímicos e de estresse oxidativo em ratos Wistar tratados com nanocápsulas contendo quinina. Os animais foram divididos em quatro grupos, os quais receberam os seguintes tratamentos: controle (solução salina - NaCl 0,9%); quinina livre (75mg/Kg/dia); nanocápsulas brancas; nanocápsulas contendo quinina (75mg/Kg/dia). As formulações foram administradas três vezes ao dia por via intraperitoneal durante sete dias consecutivos. No oitavo dia os ratos foram eutanasiados e o sangue venoso foi coletado para análises posteriores. O tratamento com nanocápsulas contendo quinina foi capaz de manter os níveis hematológicos dentro dos limites normais. Os marcadores das funções cardíaca, hepática e renal apresentaram níveis sanguíneos significativamente diminuídos no grupo tratado com nanocápsulas contendo quinina em relação ao grupo tratado com quinina livre. As enzimas antioxidantes catalase, superóxido dismutase e glutationa peroxidase apresentaram uma atividade significativamente mais elevada no grupo tratado com nanocápsulas contendo quinina comparado ao grupo quinina livre, assim como o nível de glutationa reduzida. A quinina foi capaz de induzir dano lipídico, proteico e genético, e sua nanoencapsulação atenuou o dano. Assim, estes resultados demonstraram que a nanoencapsulação pode ser eficaz na redução dos efeitos adversos causados pela quinina, tornando este medicamento mais seguro para o tratamento da malária. / Malaria is caused by protozoa of the genus Plasmodium, which P. falciparum is responsible for the most deaths. The clinical picture this parasitosis include fever, chills, headache, vomit, diarrhea, anaemia, coma and death. The oldest antimalarial is quinine, an alkaloid obtained bark species Cinchona. Due to their toxicity and the appearance of new drugs, its use was limited, but again had greater importance because to Plasmodium resistance to other drugs. An alternative to minimize toxicity is the development of a pharmaceutical form capable of less toxic drug, as nanoencapsulated systems. This present work had objective to verify hematological, biochemical and oxidative stress parameters in rats Wistar treated with nanocapsules containing quinine. The animals were divided into four groups which received the following treatments: control (solution saline - 0.9% NaCl); free quinine (75mg/kg/day); blank nanocapsules; quinine-loaded nanocapsules (75 mg/kg/day). The formulations were administered three times a day intraperitoneally during seven consecutive days. On the eighth day the rats were euthanized and venous blood was collected for followed analysis. Treatment with quinine-loaded nanocapsules was able to maintain the blood levels with normal limits. The markers of cardiac, hepatic and renal function showed blood levels significantly decreased in the group treated with quinine-loaded nanocapsules compared to the group treated with free quinine. The antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase were significantly higher activity in the group treated with quinine-loaded nanocapsules compared to free quinine group, as well as the level of reduced glutathione. Quinine was able to induce lipid, protein and genetic damage, and its nanoencapsulation attenuate the damage. Then, these results demonstrate that nanoencapsulation can be effective in reducing the adverse effects caused by quinine, making this a safer medicament for treatment malaria.

"Oxidative stress is a type of cellular stress that can damage and kill cells. While it is naturally occurring, many non-natural substances found in our environment can also induce the formation of reactive oxygen species (ROS), which then cause oxidative stress within the cell. Oxidative stress has been shown to be involved in the death of neurons in a number of neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis. The primary causes for these diseases are still unknown; however, we do know oxidative stress plays a primary role in their development. In conditions where oxidative stress is present, adenosine receptor expression has been upregulated and has played a cytoprotective role, but the specific mechanism of action is unknown. In this thesis, oxidative stress was studied in a model eukaryote, Saccharomyces cerevisiae, and the effects of the expression of the human A1 and A2A receptors upon stress response was examined. Oxidative stress was induced by the addition of hydrogen peroxide at concentrations of .5 mM, 1 mM, and 2 mM. The growth of cells expressing either A1-GFP R or A2A-GFP R at the varying hydrogen peroxide concentrations were compared to the parental cells. Confocal microscopy was performed to determine the receptor expression levels, and to confirm the expression of the receptors via their GFP tag. Immunoblots were also performed to assess the receptor expression level at the differing hydrogen peroxide concentrations. A ROS assay was also performed to show the presence of ROS and oxidative stress in the cells. No significant increase in receptor level expression or localization for either A1 R or A2A R at the varying hydrogen peroxide concentrations was found. The data did show trends indicating that A2A receptors may help process the oxidative stress better than A1 receptors and that A2A receptor containing cells had a shorter doubling time. However, more research on this subject should be performed in the future. However, the concentration of hydrogen peroxide should be greatly increased for further experiments in S. cerevisiae in order to better differentiate the trends observed." / 1 / Bryan Goldman

Human hydroxysteroid sulfotransferase (hSULT2A1) catalyzes the sulfation of a broad range of endogenous (e.g., hormones, neurotransmitters, bile acids) as well as xenobiotic (e.g, drugs, environmental pollutants) compounds. Alteration in the catalytic activity of hSULT2A1 can lead to outcomes like endocrine disruptions or aberrant drug metabolism and xenobiotic toxicity. Oxidative and electrophilic stresses are known to cause physiological damage and be implicated as possible underlying pathologic mechanisms of a wide range of diseases. To examine the oxidative as well as electrophilic regulation of hSULT2A1, model oxidants (glutathione disulfide (GSSG), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), diamide, tert-butyl hydroperoxide (TBHP)) and electrophiles such as quinone metabolites of polychlorinated biphenyls (PCB-quinones) and phenyl-p- benzoquinone were chosen for this study. Mechanistic studies correlating the enzyme structural modifications with alteration in the catalytic properties were performed to elucidate the catalytic regulative mechanism of an individual oxidant or electrophile. Thiol oxidants including GSSG, DTNB, and diamide showed catalytic regulation of hSULT2A1. Changes in protein intrinsic fluorescence indicated conformational alterations in hSULT2A1 following the reaction with diamide. Binding properties of hSULT2A1 for its substrates were also altered after reaction with these thiol oxidants, which could be one major reason for the kinetic alteration due to oxidative modification. Formation of mixed disulfides with cysteines in hSULT2A1 was also identified as a result of reaction with GSSG and DTNB. TBHP was chosen as a model for lipid peroxides, and reaction with this hydroperoxide decreased the catalytic function of hSULT2A1. The dissociation constant for binding of dehydroepiandrosterone (DHEA) was significantly altered with TBHP-pretreatment, but this did not affect the binding of 3',5'-adenosine diphosphate (PAP) to the enzyme. Structural analysis identified cysteine sulfonic acids and methionine sulfoxide formation after reaction of hSULT2A1 with TBHP, which could account for the alterations in the binding properties and the catalytic activity. Both PCB-quinones and PBQ could regulate the catalytic activity of hSULT2A1. Although PCB-quinones only caused decreases in the catalytic activity at all concentrations tested, pretreatment with PBQ indicated that lower concentrations resulted in an increase in the catalytic activity of hSULT2A1 that was followed by a decrease in the catalytic activity of hSULT2A1 upon increasing the concentration of PBQ in the pretreatment. Differences in the dissociation constants of PAP after PBQ-pretreatment were also observed, indicating the key role played by these PCB-quinones in altering the binding of either PAP or the sulfuryl donors, PAPS. Adducts at cysteines in hSULT2A1 were formed following reactions with PCB-quinones and PBQ. Small amounts of cysteine sulfonic acids and methionine sulfoxides were also formed following reaction of the protein with PCB-quinones and PBQ. Therefore, alterations in both the catalytic function as well as the structural properties of hSULT2A1 by interaction with oxidants and electrophiles may lead to changes in the metabolism of xenobiotics, as well as alterations in the endogenous balance of various steroid hormones. Such changes may be an important component in physiological damage that occurs under oxidative and electrophilic stress.

There is increasing evidence that hydrogen peroxide (H2O2) can act as a signalling molecule capable of modulating a variety of biochemical and genetic systems. Using Jurkat T-lymphocytes, this study initially investigated the involvement of H2O2 in the activation of a specific signalling protein extracellular signal-regulated protein kinase (ERK). It was found that as a result of H2O2 treatment, mitochondrial complex activities decreased which led to subsequent increase of mitochondrial reactive oxygen species (ROS) production. The increase of ROS resulted in higher cellular H2O2 as well as increased ERK activation. This study demonstrated that in an oxidative stress setting, H2O2 production from the mitochondria was an essential component in maintaining the activation of a signalling protein. One way in which H2O2 could influence protein function is by the oxidation of susceptible thiol groups of cysteine residues. To further understand the variety of signalling pathways that H2O2 may be involved in, an improved proteomics technique was developed to globally identify proteins with susceptible thiol groups. The

Thesis (M.Sc.)--York University, 2004. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 100-121). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL:http://gateway.proquest.com/openurl?url%5Fver=Z39.88-2004&res%5Fdat=xri:pqdiss&rft%5Fval%5Ffmt=info:ofi/fmt:kev:mtx:dissertation&rft%5Fdat=xri:pqdiss:MQ99388