Дисертації з теми "Tyrosine oxidation"

Le but de mon travail de thèse était de caractériser les produits de l'oxydation du peptide beta-amyloïde (Abeta) impliqué dans le développement de la maladie d'Alzheimer. Pour étudier l'effet de la structure de peptide sur les processus redox, les propriétés du peptide Abeta (1-40) ont été comparées au peptide de séquence inverse, Abeta (40-1). Les radicaux libres choisis ont été produits en radiolyse gamma. Les produits finaux ont été caractérisés par des techniques d'analyse diverses (HPLC, GC, MALDI-TOF MS, spectrométrie de fluorescence, spectrométrie raman). Pour établir l'effet de l'environnement sur le processus d'oxydation, peptides ont été oxydés dans trois systèmes différents: solution aqueuse homogène, milieu micellaire (SDS) et système des vésicules phospholipidiques (POPC). En solution aqueuse homogène, les produits d'oxydation sont différents pour les deux peptides. Les résidus facilement oxydables sont Met35 pour Abeta(1-40) et Tyr10 pour Abeta(40-1). La présence des micelles ainsi que des vésicules phospholipidiques change profondément le cours de l'oxydation. Une étude structurale en dichroïsme circulaire nous permet d'avancer des hypothèses pour interpréter ces résultats. Nous avons montré que des produits de dégradation des peptides Abeta peuvent induire d'une manière catalytique l'altération des phospholipides. Cette propriété est attribuée à l'action des atomes d'hydrogène sur le peptide. Nos résultats sont intéressants dans le contexte du développement de la maladie d'Alzheimer, car ils peuvent aider à éclairer le rôle de l'interaction de peptide Abeta(1-40) avec des membranes phospholipidique dans les propriétés redox du peptide
The goal of my thesis work was to characterize oxidation products of beta-amyloid peptide (Abeta), which is implied in the development of Alzheimer's disease. To study the effect of peptide structure on the redox processes, oxidation properties of Abeta(1-40) were compared to the peptide with reverse sequence, Abeta(40-1). Azide and hydroxyl radicals used for oxidation were produced by gamma radiolysis. Final products were characterized by a variety of analytical techniques (HPLC, GC, MALDI-TOF MS, fluorescence and raman spectrometry). To establish the role of peptide environment on its redox properties, oxidation was carried out in three different systems: homogeneous aqueous solution, micellar system (SDS) and in the presence of phospholipids vesicles (POPC). In homogeneous aqueous solution, oxidation products are different for both peptides. The main oxidation targets are Met35 for Abeta(1-40) and Tyr10 for Abeta(40-1). The presence of micelles and phospholipid vesicles has an important impact on the oxidation pathways. These changes could be related to changes in peptide conformations studied by circular dichroism. We have also shown that Abeta degradation products may catalytically induce alternation of phospholipids. This process is initiated by reaction of hydrogen radicals with the peptide. Our results are interesting in the context of the development of Alzheimer's disease as they may bring an insight into the role of Abeta(1-40) interaction with phospholipids membrane for the redox properties of the peptide

The photosynthetic reaction centre (RC) is central to conversion of solar energy into chemical energy. In this thesis, in order to introduce the redox-active cofactors similar to that of the Photosystem II RC, a non-photosynthetic protein scaffold was used as an in vitro model. The protein tasked for this purpose was the heme containing bacterioferritin (BFR) protein found in E. coli. The BFR protein naturally expresses as a homodimer based on a 4-helix bundle monomer. Desirable properties included: (i) a promiscuos di-nuclear metal binding site which provides ligands for class II metals such as Mn (ii) a hydrophobic pocket at the dimer interface which can bind a photosensitive porphyrin, in this case a chlorin (Ce6), and (iii) presence of tyrosine residues proximal to the bound cofactors, which can be utilised as efficient electron-tunnelling intermediates. The work in this thesis extends earlier work in the group by refining and improving the BFR system. Several mutants were made and an improved protein expression system was developed. For these samples experiments demonstrated ligation of weakly coupled equivalent Mn2II,II at the di-nuclear binding site of apo-BFR, and binding of the photo-active pigment ZnCe6 in hydrophobic pocket of the protein. Light-induced electron transfer from proximal tyrosine residue(s) to the photo-oxidised ZnCe6+, in the modified BFR reconsitituted with both ZnCe6 and MnII is presented. Three site-specific tyrosine mutants (Y25F, Y58F and Y45F) were made to localise the redox-active tyrosine in this engineered system. The results indicate that: (i) presence of bound MnII is necessary to observe tyrosine oxidation in all BFR variants, (ii) Y45 (within van Der Waals network of ZnCe6) is singly the most important tyrosine as the immediate electron donor to the oxidised ZnCe6+, and (iii) Y25 and Y58 are both redox-active in this system, but appear to be interchangebale. A high-resolution (~1.5 Ã…) crystal structure of the three tyrosine mutants was obtained and these structures showed there to be no mutation-induced effects on the overall 3-D structure of the protein. Minimal effects observed in the Y45F mutant are reported. The molecular design of a "second generation" of the BFR series is also presented where the symmetry of the BFR homodimer is broken to generate a BFR heterodimer to contain genetically distinct electron donor and acceptor subunits. The BFR heterodimer was made by introducing a small (20 aa) peptide linker between the two subunits. The ultimate goal will be to demonstrate light-induced directional electron flow in the BFR heterodimer.

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 15, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Kent S. Gates. Vita. Includes bibliographical references.

Photosystem II is unique! It remains the only enzyme that can oxidize water using light as energy input. Water oxidation in photosystem II is catalyzed by the CaMn4 cluster. The electrons extracted from the CaMn4 cluster are transferred to P680+ via the tyrosine residue D1-Tyr161 (YZ). Favorable oxidation of YZ is coupled to a proton transfer along a hydrogen bond to the nearby D1-His190 residue, resulting in the neutral radical YZ•. By illuminating photosystem II at cryogenic temperatures, YZ• can be trapped in a stable state. Magnetic interaction between this radical and the CaMn4 cluster gives rise to a split electron paramagnetic resonance (EPR) signal with characteristics that depend on the oxidation state (S state) of the cluster. The mechanism by which the split EPR signals are formed is different depending on the S state. In the S0 and S1 states, split signal induction proceeds via a P680+-centered mechanism, whereas in the S2 and S3 states, our results show that split induction stems from a Mn-centered mechanism. This S state-dependent pattern of split EPR signal induction can be correlated to the charge of the CaMn4 cluster in the S state in question and has prompted us to propose a general model for the induction mechanism across the different S states. At the heart of this model is the stability or otherwise of the YZ•–(D1-His190)+ pair during cryogenic illumination. The model is closely related to the sequence of electron and proton transfers from the cluster during the S cycle. Furthermore, the important hydrogen bond between YZ and D1-His190 has been investigated by following the split EPR signal formation in the different S states as a function of pH. All split EPR signals investigated decrease in intensity with a pKa of ~4-5. This pKa can be correlated to a titration event that disrupts the essential hydrogen bond, possibly by a direct protonation of D1-His190.  This has important consequences for the function of the CaMn4 cluster as this critical YZ–D1-His190 hydrogen bond steers a multitude of reactions at the cluster.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2010.
Committee Chair: Bridgette Barry; Committee Member: Ingeborg Schmidt-Krey; Committee Member: Jake Soper; Committee Member: Nils Kroger; Committee Member: Wendy Kelly. Part of the SMARTech Electronic Thesis and Dissertation Collection.

The kinase interaction motif (KIM) family of protein-tyrosine phosphatases (PTPs) includes hematopoietic protein-tyrosine phosphatase (HePTP), striatal-enriched protein-tyrosine phosphatase (STEP), and protein-tyrosine phosphatase receptor type R (PTPRR). KIM-PTPs bind and dephosphorylate mitogen-activated protein kinases (MAPKs) and thereby critically modulate cell proliferation and differentiation. PTP activity can readily be diminished by reactive oxygen species (ROS), e.g. H2O2, which oxidize the catalytically indispensable active-site cysteine. This initial oxidation generates an unstable sulfenic acid intermediate that is quickly converted into either a sulfinic/sulfonic acid (catalytically dead and irreversible inactivation) or a stable sulfenamide or disulfide bond intermediate (reversible inactivation). Critically, our understanding of ROS-mediated PTP oxidation is not yet sufficient to predict the molecular responses of PTPs to oxidative stress. However, identifying distinct responses will enable novel routes for PTP-selective drug design, important for managing diseases such as cancer and Alzheimer's disease. Therefore, we performed a detailed biochemical and molecular study of all KIM-PTP family members to determine their H2O2 oxidation profiles and identify their reversible inactivation mechanism(s). We show that despite having nearly identical 3D structures and sequences, each KIM-PTP family member has a unique oxidation profile. Furthermore, we also show that whereas STEP and PTPRR stabilize their reversibly oxidized state by forming an intramolecular disulfide bond, HePTP uses an unexpected mechanism, namely, formation of a reversible intermolecular disulfide bond. In summary, despite being closely related, KIM-PTPs significantly differ in oxidation profiles. These findings highlight that oxidation protection is critical when analyzing PTPs, for example, in drug screening.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.
Committee Chair: Bridgette Barry; Committee Member: El-Sayed, Mostafa; Committee Member: Fahrni, Christoph; Committee Member: Kröger, Nils; Committee Member: McCarty, Nael. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Title from PDF of title page (University of Missouri--Columbia, viewed on March 2, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dr. Kent S. Gates, Dissertation Supervisor. Vita. Includes bibliographical references.

Proton coupled electron transfer reactions often involve tyrosine residues, because when oxidized, the phenolic side chain deprotonates. Tyrosine Z (YZ) is responsible for extracting electrons in a stepwise fashion from the oxygen evolving-complex in order to build enough potential to oxidize water. This process requires that each step YZ must deprotonate and reprotonate in order to maintain the high midpoint potential that is necessary to oxidize the oxygen-evolving complex, which makes YZ highly involved in proton coupled electron transfer reactions. In this thesis YZ has been studied within oxygen-evolving photosystem II utilizing electron paramagnetic resonance spectroscopy to monitor the tyrosyl radical that is formed upon light excitation. Kinetic analysis of YZ has shed light on the factors that are important for PSII to carry out water oxidation at the oxygen-evolving complex. Most notably the strong hydrogen-bonding network and the midpoint potential of YZ have been shown to be integral aspects of the water splitting reactions of PSII. By studying YZ within oxygen-evolving PSII, conclusions are readily applied to the native system.

Orientador: Hiroshi Aoyama
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-06T06:33:37Z (GMT). No. of bitstreams: 1 Sousa_RobertaReginaRuelade_M.pdf: 3257089 bytes, checksum: 29f24a432f8c0bfd7dd71af48cc7608a (MD5) Previous issue date: 2005
Resumo: As proteínas tirosina fosfatases (PTP) (EC 3.1.3.48) são enzimas regulatórias chaves que participam na transdução de sinal e são essenciais na regulação do crescimento, diferenciação, ciclos celulares, na transcrição gênica, resposta imune e outros processos. Esta classe de enzimas, que contém cisteína no sítio ativo, pode ser inativada por agentes oxidantes. Neste trabalho, estudamos os efeitos de peróxido de hidrogênio e t-butil hidroperóxido, compostos que induzem estresse oxidativo, na atividade de uma PTP purificada de membranas de linfócitos humanos, indicativamente a CD45. A PTP foi purificada de membranas de linfócitos humanos através de cromatografias de troca iônica (DEAE Sepharose) e exclusão molecular (Sephacryl S-200). A purificação enzimática foi acompanhada por SDS-PAGE e eletroforese bidimensional. A atividade enzimática foi determinada através de incubação a 37°C por 30 min em pH 5,0 em presença de 5 mM de p-nitrofenil fosfato (pNPP) como substrato. A enzima obtida da cromatografia de exclusão molecular apresentou uma massa molecular relativa de aproximadamente 200 kDa, reconheceu mais eficientemente tirosina fosfato (cerca de 3,2 vezes) como substrato quando comparado ao pNPP, e foi inibida por inibidores específicos de PTP, tais como vanadato (40%), pervanadato (100%), p-cloromercuribenzoato (20%) and Cu2+ (95%). Ácido okadáico, um inibidor específico de serina e treonina proteína fosfatases, não afetou a atividade da PTP de membranas de linfócitos. Estes resultados de caracterização sugerem fortemente que a PTP purificada de membranas de linfócitos humanos é a CD45. Peróxido de hidrogênio e t-butil hidroperóxido inibiram a atividade dessa proteína com valores de IC50 (concentração do composto que produz 50% de inibição enzimática) de 50 µM e 16 mM, respectivamente. Glutationa reduzida (GSH) protegeu parcialmente a enzima contra estes oxidantes, porém proteções totais foram obtidas quando se adicionava 250 mM de desferoxamina ao meio de ensaio. Nossos resultados sugerem que essa proteína pode ser regulada por alteração do estado de oxidação dos grupos funcionais do sítio ativo e que esta regulação poderia fornecer um mecanismo de controle celular através de espécies reativas de oxigênio
Abstract: Protein phosphatases, that dephosphorylate proteins in residues of threonine, serine and tyrosine, are a class of enzymes that can be stressed by compounds present in oxidant or reductant environments. In particular, the protein tyrosine phosphatases (PTP) (EC 3.1.3.48) are key regulatory enzymes that participate in signal transduction and are essential for regulating cellular growth, differentiation, cell cycle, gene transcription, immune response and other processes. This class of enzymes, which contain cysteine in the active site, can be inactivated by oxidant reagents. In this work we have studied the effect of hydrogen peroxide and t-butyl hydroperoxide, compounds that induce oxidative stress, on a purified PTP (CD45) from membrane human lymphocytes. PTP was purified from human lymphocyte membranes through ion exchange (DEAE Sepharose) and molecular exclusion (Sephacryl S-200) chromatographies. The enzyme purification was followed by SDS-PAGE and 2D electrophoresis. The enzyme activity was determined by incubation at 37oC for 30 minutes at pH 5.0 in presence of 5 mM p-nitrophenylphosphate (pNPP) as substrate. The enzyme obtained from molecular exclusion chromatography had a relative molecular mass of approximately 200 kDa, recognized more efficiently tyrosine phosphate (about 3.2-fold) as substrate when compared with p-NPP, and was inhibited by specific PTP inhibitors, such as, vanadate (40%), pervanadate (100%), p-chloromercuribenzoate (20%) and Cu2+ (95%). Okadaic acid, a specific serine and threonine protein phosphatases inhibitor, did not significantly affect the lymphocyte membrane PTP activity. These characterization results strongly suggest that the membrane PTP purified from human lymphocytes was the CD45. Hydrogen peroxide and t-butyl hydroperoxide inhibited the CD45 activities with IC50 (concentration of compound that produces 50% enzyme inhibition) values of 50 µM and 16 mM, respectively. Reduced glutathione (GSH) partially protected the enzyme against these oxidations, but full protections were observed when 250 mM deferoxamine were added to the assay medium. Our results suggest that CD45 can be regulated by altering the oxidation state of active site functional groups, and that this regulation could provide a mechanism of cell control by reactive oxygen species
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

Tyrosyl radicals can facilitate proton-coupled electron transfer (PCET) reactions that are linked to catalysis in many biological systems. One such protein system is ribonucleotide reductase (RNR). This enzyme is responsible for the conversion of ribonucleotides to deoxyribonucleotides. The beta2 subunit of class Ia RNRs contains a diiron cluster and a stable tyrosyl radical (Y122*). Reduction of ribonucleotides is dependent on reversible, long-distance PCET reactions involving Y122* located 35 Å from the active site. Protein conformational dynamics are postulated to precede diiron cluster assembly and PCET reactions in RNR. Using UV resonance Raman spectroscopy, we identified structural changes to histidine, tyrosine, and tryptophan residues with metal cluster assembly in beta2. With a reaction-induced infrared spectroscopic technique, local amide bond structural changes, which are associated with the reduction of Y122*, were observed. Moreover, infrared spectroscopy of tyrosine-containing pentapeptide model compounds supported the hypothesis that local amide bonds are perturbed with tyrosyl radical formation. These findings demonstrate the importance of the amino acid primary sequence and amide bonds on tyrosyl radical redox changes. We also investigated the function of a unique tyrosine-histidine cross-link, which is found in the active site of cytochrome c oxidase (CcO). Spectrophotometric titrations of model compounds that mimic the cross-link were consistent with a proton transfer role in CcO. Infrared spectroscopic data support the formation of tyrosyl radicals in these model compounds. Collectively, the effect of the local structure and the corresponding protein dynamics involved in tyrosyl radical-mediated PCET reactions are illustrated in this work.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Oct. 5, 2009). "... in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

Yes
Phenol and its derivatives are hazardous, teratogenic and mutagenic, and have gained significant attention in recent years due to their high toxicity even at low concentrations. Phenolic compounds appear in petroleum refinery wastewater from several sources, such as the neutralized spent caustic waste streams, the tank water drain, the desalter effluent and the production unit. Therefore, effective treatments of such wastewaters are crucial. Conventional techniques used to treat these wastewaters pose several drawbacks, such as incomplete or low efficient removal of phenols. Recently, biocatalysts have attracted much attention for the sustainable and effective removal of toxic chemicals like phenols from wastewaters. The advantages of biocatalytic processes over the conventional treatment methods are their ability to operate over a wide range of operating conditions, low consumption of oxidants, simpler process control, and no delays or shock loading effects associated with the start-up/shutdown of the plant. Among different biocatalysts, oxidoreductases (i.e., tyrosinase, laccase and horseradish peroxidase) are known as green catalysts with massive potentialities to sustainably tackle phenolic contaminants of high concerns. Such enzymes mainly catalyze the o-hydroxylation of a broad spectrum of environmentally related contaminants into their corresponding o-diphenols. This review covers the latest advancement regarding the exploitation of these enzymes for sustainable oxidation of phenolic compounds in wastewater, and suggests a way forward.

Introduction: Chronic myeloid leukaemia is a blood cancer which progresses from a chronic phase to an acute blast crisis if untreated. Disease progression and treatment resistance may be precipitated by the mutator action of BCR/ABL protein tyrosine kinase (PTK), but only few protein phosphosites involved in the DNA damage response have been investigated with respect to BCR/ABL action. Aim: The aim of this PhD project was to demonstrate that BCR/ABL PTK expression can affect the response to genotoxic stress signalling at the protein phosphorylation level. Methodology: Etoposide-induced DNA damage response has been studied in control and BCR/ABL PTK-expressing Ba/F3 cells using apoptosis and γH2AX assays. Quantitative phosphoproteomics was performed with iTRAQ peptide labelling to discover putative modulated phosphorylation sites. Absolute quantification (AQUA ) performed with selected reaction monitoring was used to validate discovery phosphoproteomics. The effect of genotoxic stress on the THO complex protein Thoc5/Fmip was studied using western blots. Results: The expression of BCR/ABL PTK induced γH2AX phosphorylation after etoposide exposure. This was associated with the modulation of H2AX tyrosine 142 phosphorylation, MDC1 (serines 595 and 1053) and Hemogen serine 380 phosphorylation among proteins regulated by both BCR/ABL PTK and etoposide. We identified that leukaemogenic PTKs mediate Thoc5/Fmip phosphorylation on tyrosine 225 via Src proto-oncogene and oxidative stress, while ATM and MEK1/2 may control its phosphorylation. Human CD34+ CD38- leukaemic stem cells showed pronounced level of THOC5/FMIP tyrosine phosphorylation. Expression of phosphomutant Thoc5/Fmip Y225F might reduce apoptosis mediated by etoposide and H2O2. Conclusion: BCR/ABL PTK can sustain, create, block and change the intensity of protein phosphorylation related to genotoxic stress. Modulation of H2AX, MDC1, Hemogen and Thoc5/Fmip post-translational modifications by BCR/ABL PTK might promote unfaithful DNA repair, genomic instability, anti-apoptotic signalling or abnormal cell differentiation, resulting in leukaemia progression.

Chronic myeloid leukemia (CML) is characterized by clonal expansion of hematopoietic progenitor cells, result from the translocation (9:22). The oncogene BCR-ABL, in the Ph chromosome, is transcribed and translated into a fusion protein BCR / ABL. The ABL tyrosine kinase (TK) in the fusion protein is constitutively activated and is needed for the initial leukemogenic event of CML and its activity induces production of reactive oxygen species (ROS). Of particular relevance to CML is the fact that an increase of ROS can have consequences, facilitating genomic instability may contribute to disease progression. The aim of this study was to determine the oxidative status in patients with CML, in attendance at a university hospital (HUWC). This is a cross-sectional study consisted of 30 adult patients of both sexes with clinical and laboratory diagnosis of CML on treatment with inhibitors (TK) 1st and 2nd generation. The concentrations of malondialdehyde (MDA) and nitrite (NO2-) were performed by spectrophotometric method. The activities of enzymes glutathione peroxidase (GSH-Px) and catalase (CAT) were determined in hemolysate by Glutathione Peroxidase Cellular Activity Kit  Assay (Sigma-Aldrich) and spectrophotometry, respectively. Total glutathione, reduced glutathione (reduced GSH), glutathione (GSSG) were determined by Total Glutathione Activity Kit  (Assay Designs, Inc) and calculated the ratio GSH / GSSG. For statistical analysis of nonparametric data was used  and ANOVA test for multiple comparisons Tukey. It was considered the minimum level of significance of 5%. The average concentrations of NO2- and MDA were increased in CML patients compared to control, regardless of disease activity. The antioxidant profile was characterized by decreased CAT and GSH-Px increased also independent of disease activity. The reduced GSH is presented decreased, the GSSH, increased and GSH / GSSG decreased. It was observed that patients using protease inhibitors of TK 2nd generation of oxidative stress parameters were significantly elevated compared to controls. In the analysis of patients on imatinib were not detected significant changes in oxidative status. We conclude that patients with CML are under oxidative stress and impaired antioxidant activity.
A Leucemia mielÃide crÃnica (LMC) à caracterizada pela expansÃo clonal de cÃlulas progenitoras hematopoÃticas, resultante da translocaÃÃo (9:22). O oncogene de fusÃo BCR-ABL, no cromossomo Ph, à transcrito e traduzido numa proteÃna de fusÃo BCR/ABL. A tirosina quinase (TK) ABL na proteÃna de fusÃo à constitutivamente ativada sendo necessÃria para os eventos leucemogÃnicos iniciais da LMC e sua atividade induz a produÃÃo de espÃcies reativas de oxigÃnio (EROs). De particular relevÃncia para LMC à o fato de que um aumento de EROs pode ter consequÃncias, facilitando a instabilidade genÃmica podendo contribuir para a progressÃo da doenÃa. O objetivo do estudo foi determinar o perfil oxidativo em pacientes com LMC, em acompanhamento ambulatorial no Hospital UniversitÃrio Walter CantÃdio (HUWC). Trata-se de um estudo transversal constituÃdo de 30 pacientes adultos, com diagnostico clÃnico e laboratorial de LMC, em tratamento com inibidores de (TK) de 1 e 2 geraÃÃo. As concentraÃÃes de malonaldeÃdo (MDA) e de nitrito (NO2-) foram realizadas por mÃtodo espectofotomÃtrico. As atividades das enzimas Glutationa peroxidase (GSH-Px) e catalase (CAT) foram determinadas no hemolisado, por kit Glutathione Peroxidase Cellular Activity Assay (Sigma-Aldrich) e por espectofotometria, respectivamente. Glutationa total, glutationa reduzida (GSH reduzida), glutationa oxidada (GSSG) foram determinadas por kit Total Glutathione Activity (Assay Designs, Inc) e calculada a relaÃÃo GSH/GSSG. Para a anÃlise estatÃstica de dados nÃo paramÃtricos foi utilizado o ANOVA e o teste de mÃltiplas comparaÃÃes de Tukey. Foi considerado o nÃvel mÃnimo de significÃncia de 5%. As concentraÃÃes mÃdia de MDA e de NO2- foram aumentadas nos pacientes com LMC em relaÃÃo ao controle, independente da atividade da doenÃa. O perfil antioxidante foi caracterizado pela diminuiÃÃo da CAT e aumento da GSH-Px tambÃm independente da atividade da doenÃa. A GSH reduzida se apresentou diminuÃda, a GSSH, aumentada e a relaÃÃo GSH/GSSG diminuÃda. Os pacientes em uso de inibidores de TK de 2 geraÃÃo apresentaram parÃmetros do estresse oxidativo significativamente elevados em relaÃÃo ao grupo controle. Conclui-se que os pacientes com LMC estÃo sob estresse oxidativo e com atividade antioxidante comprometida.

o,o???-Dityrosine (dityrosine), an oxidation product of tyrosine produced by reaction between tyrosyl radicals, is becoming established as a biomarker of free radical oxidative protein damage in vivo. Attempts to measure dityrosine concentrations in various physiological and pathological systems have produced varied and often contradictory results. Dityrosine concentrations in urine, plasma, cerebrospinal fluid (CSF) and brain tissue varying over three orders of magnitude have been reported, together with inconsistent claims of significant dityrosine elevation in several ageing-related pathologies. Some of these findings have contributed to the implication of free radical activity in the pathology of several neurodegenerative disorders, vascular and ocular abnormalities and in phagocyte response to infection. The aim of this study was to test the hypothesis that dityrosine levels are elevated in ageing and ageing-related disease. The study also aims to determine the utility of dityrosine measurement as an index of oxidative damage, and elucidate possible explanations for the inconsistent levels reported. An assay for the quantification of dityrosine was developed using capillary HPLC with electrospray tandem quadrupole mass spectrometry (HPLC-MS/MS). The assay was highly specific for dityrosine and has the highest absolute sensitivity for dityrosine of any method reported to date, with a detection limit of 3 femtomoles of dityrosine on-column. Urine samples from volunteers of different age and from hospital patients with various pathologies were analysed. Plasma protein hydrolysates from control, Alzheimer???s and stroke subjects were analysed, together with hydrolysates of post mortem brain tissue from Alzheimer???s and control subjects. Urinary dityrosine level is elevated in states of acute infection and inflammation, but does not correlate with age or chronic disease. Protein dityrosine in four sections of Alzheimer???s brain was not significantly different from control sections. Dityrosine was present in human plasma and tissue proteins at approximately 5-35 residues per million tyrosine residues, and in normal urine at 5-25 micromol/mol creatinine or 20-200 nM. Most of the discrepancies in the literature relate to inadequate specificity of the analytical method. Interpretation of published data with critical appraisal of measurement technology specificity is essential in developing an accurate understanding of the role of free radicals in ageing and disease.

La mitochondrie est une organelle très importante vu son implication dans plusieurs processus cellulaires. Elle produit notamment la majeure partie de l'énergie qui est consommée par la cellule, grâce aux processus d'oxydation phosphorylante (OXPHOS). La phosphorylation des enzymes impliquées dans les OXPHOS apparait comme une voie de régulation importante de la production énergétique. L'objectif de ce thèse était donc de comprendre comment les phosphorylations, et plus particulièrement, les phosphorylations sur tyrosine induites par la Src kinase influencent les OXPHOS. Il a donc été démontré qu'il existe, à l'intérieur des mitochondries, des voies de régulation de ces processus de phosphorylation induits par la Src kinase. Ces processus pouvant induire la phosphorylation de plusieurs enzymes mitochondriales, notamment plusieurs sous-unités des complexes du système des électrons et ainsi, grandement influencer les OXPHOS. Il a aussi été démontré que la Src kinase semble aussi présente dans les mitochondries de cellules cancéreuses, induisant la phosphorylation d'une sous-unité de la NADH-oxidoréductase et une augmentation du métabolisme énergétique mitochondrial. Cette régulation des OXPHOS dans les cellules cancéreuses par la Src kinase pourrait participer à l'établissement du phénotype hautement prolifératif de ces cellules
Mitochondria are implicated in several key cellular processes. They are producing most part of the energy that is consumed by the cell via oxidative phosphorylation processes (OXPHOS). Phosphorylation of different components implicated in OXPHOS are known to constitute an important regulation pathway of energetic production. The objective of this thesis was to understand how tyrosine phosphorylation induced by the Src kinase could influence OXPHOS. First, it was shown that Src kinase mediated phosphorylation can be regulated directly in mitochondria, inducing phosphorylation of several mitochondrial proteins and different effects on OXPHOS. I also demonstrated that Src kinase is also present in mitochondria of cancer cells where it can lead to phosphorylation of NADH-oxidoreductase. This phosphorylation site is associated with increase of OXPHOS which could be implicated in the establishment of global phenotype of cancer cells

A deficiência da semi-aldeído succínico desidrogenase (SSADH) e a tirosinemia tipo II caracterizam-se pela presença de elevadas concentrações teciduais e plasmáticas de ácido γ-hidroxibutírico (GHB) e tirosina, respectivamente. Tanto os pacientes afetados pela deficiência da SSADH quanto aqueles com tirosinemia tipo II apresentam sinais e sintomas neurológicos. Embora os mecanismos responsáveis pela disfunção neurológica sejam pouco conhecidos, sabe-se que podem estar relacionados ao acúmulo de GHB e tirosina e seus possíveis efeitos tóxicos sobre o sistema nervoso central (SNC). Considerando ainda que o estresse oxidativo parece estar envolvido em diversas doenças ou alterações patológicas que afetam o SNC, os efeitos do GHB e da L-tirosina sobre vários parâmetros de estresse oxidativo foram investigados em homogeneizados de córtex cerebral de ratos jovens. Os efeitos in vivo do GHB, ou de seu precursor 1,4-butanodiol, foram semelhantes. Ambos mostraram comprometimento das defesas antioxidantes não-enzimáticas e indução da lipoperoxidação. Os efeitos in vitro e in vivo da L-tirosina, por sua vez, foram diferentes: in vitro, a L-tirosina provocou dano oxidativo ao DNA, diminuiu as defesas antioxidantes não-enzimáticas e enzimáticas e alterou o estado redox (razão SH/SS), enquanto que a administração aguda de L-tirosina causou dano oxidativo a lipídios e proteínas, modificou a razão SH/SS, diminuiu as defesas antioxidantes não-enzimáticas e estimulou a atividade da glicose-6-fosfato desidrogenase. Esses resultados sugerem que o GHB e a L-tirosina são capazes de promover o estresse oxidativo em córtex cerebral de ratos jovens. Desta forma, caso esses mesmos efeitos ocorrerem em seres humanos, é provável que o estresse oxidativo seja um dos mecanismos responsáveis pela neuropatologia característica da deficiência da SSADH e tirosinemia tipo II.
Succinic semialdehyde dehydrogenase (SSADH) deficiency and tyrosinemia type II are characterized by predominant tissue and blood accumulation of γ-hydroxybutyric acid (GHB) and tyrosine, respectively. Patients with SSADH deficiency and tyrosinemia type II present neurological signs and symptoms. Although mechanisms of brain damage remain unclear, they are probably related to the accumulation of GHB or tyrosine leading to possible noxious effects on central nervous system (CNS) development in those patients. Considering that the damaging consequences of oxidative stress have been implicated in a variety of disorders of CNS, the effect of GHB and L-tyrosine were investigated on some oxidative stress parameters in cerebral cortex homogenates of young rats. The in vitro and in vivo effects of GHB, or its precursor 1,4-butanediol (1,4-BD), were similar. It was observed that GHB or 1,4-BD impairs non-enzymatic antioxidant defenses and induces lipid peroxidation. On the other hand, the in vitro and in vivo effects of L-tyrosine were different. Oxidative damage to DNA was promoted while non-enzymatic and enzymatic antioxidant defenses, and thiol-disulfide redox state (SH/SS ratio) were markedly diminished by L-tyrosine in vitro. In contrast, the acute administration of L-tyrosine causes lipid peroxidation and protein oxidation, decreases non-enzymatic antioxidant defenses, alters SH/SS ratio and stimulates glucose-6-phosphate dehydrogenase activity. Taken together, it may be presumed that GHB and L-tyrosine elicit oxidative stress in cerebral cortex of young rats. If these effects also occur in the brain of patients affected by SSADH deficiency or tyrosinemia type II, it is possible that oxidative stress may contribute, at least in part, to the neurological dysfunction characteristic of these diseases.

Os compostos fenólicos, quando em altas concentrações na água, são poluentes de alta periculosidade, difíceis de serem eliminados até mesmo por métodos convencionais, como por exemplo, a biorremediação microbiológica dos lodos ativados e os métodos físico-químicos. Sabendo da presença dos fenóis em muitos efluentes industriais se faz necessário o estudo de novos processos para o tratamento desses efluentes que possuam aplicações eficazes e, sobretudo, ecologicamente corretas. A presente pesquisa visou a degradação de poluentes fenólicos em soluções aquosas através da oxidação enzimática com tirosinase imobilizada em quitosana. A contribuição tecnológica relaciona-se com a possibilidade de aplicação do estudo em escala industrial em processos de tratamento de efluentes industriais que contenham poluentes fenólicos. Fenóis como o 4-clorofenol, 4-cresol, 4-nitrofenol e o próprio fenol, foram utilizados em ensaios de oxidação enzimática em vaso agitado, em regime dinâmico, em processo de batelada, temperatura de 45°C e três faixas de concentração enzimática (40, 60 e 80 U/ml). Além disso, foram testadas três formas de quitosana utilizadas na imobilização enzimática e aplicadas nos processos de oxidação de fenol: esferas, flocos e micro partículas de quitosana. A enzima tirosinase foi eficiente na degradação de fenol, 4-cresol e 4-clorofenol, diminuindo consideravelmente a concentração destes poluentes nas soluções aquosas. Porém, a tirosinase não oxidou significativamente o 4-nitrofenol. Verificou-se que o efeito de alguns substituintes do anel aromático do fenol e a posição desse substituinte no anel aromático, exerce influência direta na atividade da enzima durante as reações oxidativas envolvendo os compostos fenólicos. Embora o presente estudo tenha apresentado bons resultados na remoção de alguns compostos fenólicos em soluções aquosas por meio da oxidação de tais poluentes pela enzima tirosinase imobilizada em quitosana, o complexo enzima-suporte não apresentou a mesma eficiência nos ensaios subseqüentes, nos quais foi estudada a reutilização da enzima imobilizada.
The phenolic compounds when in high concentrations in water are extremely dangerous pollutants and difficult to be eliminated even by conventional methods such as the microbiological bioremediation with activated sludge and physico-chemical methods. Phenols are present in many industrial effluents and is necessary to develop new and effective processes for the treatment of that effluents and, if possible, environmentally friendly. This research aimed the degradation of phenolic pollutants in aqueous solutions by enzymatic oxidation with tyrosinase immobilized in chitosan. Phenols such as 4-chlorophenol, 4-cresol, 4-nitrophenol and phenol, had been used in assays of enzymatic oxidation in an agitated reactor, in dynamic regime, in a batch process, temperature of 45°C and three values of enzymatic concentration ( 40, 60 and 80 U/ml). In addition, three forms of chitosan had been used in enzymatic immobilization and applied in the processes of oxidation of phenol: pellets, flakes and small particles of chitosan. Tyrosinase was efficient in the degradation of phenol, 4-cresol and 4-chlorophenol, reducing significantly the concentration of these pollutants in aqueous solutions. However, tyrosinase did not oxidized 4-nitrophenol. It was verified that the effect of some substitutes and their position in the aromatic ring has a direct influence on the activity of the enzyme during the oxidative reactions involving phenolic compounds. Although this study has shown good results in the removal of some phenolic compounds in aqueous solutions through the oxidation of such pollutants by the tyrosinase immobilized on chitosan, the enzyme-support did not present the same efficiency in subsequent assays, in which we studied the reuse of the immobilized enzyme.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder which affects over a million people in the United States. This disease is marked by the selective loss of dopaminergic neurons in the substantia nigra, leading to a decrease in the important neurotransmitter dopamine (DA), which is essential for the initiation and execution of coordinated movement. Currently, the pathogenesis behind PD is unknown, but there is evidence that both exogenous causes, such as pesticides and metals, as well as endogenous causes, such as reactive oxygen species or reactive metabolism intermediates, may play a role in the onset and progression of the disease. DA is catabolized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further metabolized by aldehyde dehydrogenase and aldehyde reductase to the acid and alcohol products, respectively. Studies have demonstrated the reactivity of DOPAL with peptides and proteins, leading to covalent modification which may be detrimental to protein action. Furthermore, studies have shown that DOPAL is toxic, leading to a decrease in cell viability. Due to this, it was of interest to further study DOPAL and how it may play a role in the onset and progression of PD. It was of particular interest to determine protein targets of DOPAL modification. Until recently, no protein targets were identified and the cellular consequence of elevated DOPAL had not been fully studied. It has been previously shown that the important enzyme, tyrosine hydroxylase (TH) is inhibited by other catechols, including DA. This enzyme catalyzes the rate-limiting step in DA synthesis, oxidizing tyrosine to L-DOPA which is further metabolized to DA. Therefore, it was of interest to determine the effect of DOPAL on TH activity. It was hypothesized that DOPAL modifies and inhibits TH, leading to a decrease in the production of L-DOPA and DA. This work employed the use of a dopaminergic cell model (PC6-3 cells), to positively identify TH as a protein target of DOPAL modification. It also used both cell lysate as well as PC6-3 cell studies to investigate the effect of DOPAL modification on TH activity. Mass spectrometry was also utilized to determine sites of protein modification on TH. Results show that TH is potently inhibited by DOPAL modification, leading to a significant decrease in both L-DOPA and DA. Furthermore, DOPAL inhibition appears to be slowly-irreversible, with enzyme activity showing a time- and concentration dependent in recovery after preincubation with DOPAL. A novel cloning and purification procedure was used to clone human recombinant TH, which was used in mass spectrometry studies in which five sites of DOPAL modification were discovered. Furthermore, a real-time assay for TH activity was developed using a plate reader to spectrophotometrically observe the formation of L-DOPA over time. These data demonstrate the toxicity and potent enzyme inhibition by DOPAL and implicate DOPAL as a neurotoxin relevant in the pathogenesis of PD.

L’hypertension artérielle pulmonaire (HTAP) correspond à un groupe de maladies qui se caractérise par une obstruction vasculaire suite à une vasoconstriction excessive, une prolifération cellulaire et la création de thromboses in situ, conduisant à une augmentation progressive des résistances vasculaires pulmonaires puis au décès. De nombreuses avancées dans la prise en charge de l’HTAP ont été réalisées ces dernières années avec la mise à disposition de médicaments principalement vasodilatateurs. Cependant, aucun de ces médicaments n’est curatif de la maladie témoignant de la nécessité à obtenir de nouvelles thérapeutiques. Des molécules axant leur effet sur la lutte contre la prolifération cellulaire liée à l’activation des récepteurs à tyrosine-kinases (RTK) ou le stress oxydant (SO) paraissent aujourd’hui comme de potentielles innovations thérapeutiques dans l’HTAP. Cependant, à l’heure actuelle, les données sur le SO dans l’HTAP sont trop peu détaillées pour cibler correctement cette voie physiopathologique. De même, les inhibiteurs de tyrosine-kinases ont montré un bénéfice dans la prise en charge de l’HTAP mais associé à des effets indésirables graves tels qu’une toxicité cardiaque. Dans ce travail, nous avons approfondi le mécanisme d’action du SO dans la physiopathologie de l’HTAP et complété l’identification des RTK dans le remodelage vasculaire pulmonaire afin de permettre la mise au point de thérapeutiques efficaces avec un rapport bénéfice risque favorable pour le patient
Pulmonary arterial hypertension (PAH) corresponds to a group of diseases characterized by a vascular obstruction due to vasoconstriction, cellular proliferation and in situ thrombosis, leading to a progressive increase in pulmonary vascular resistances. New knowledge in the PAH management were performed in the last few years, specifically for vasodilators. However, none of those treatments cure the disease and new drugs are still needed. Molecules targeting cellular proliferation induced by tyrosine kinases receptors (TKR) activation or oxidative stress (OS) seem to be potential therapeutic innovations. However, knowledge on OS in PAH is not enough accomplished in PAH to target accurately this pathophysiologic pathway. Similarly, tyrosine kinase inhibitors have shown efficacy in PAH management but associated with severe adverse events as cardiac toxicity. In this study, mechanism of action of OS in pathophysiology of PAH was detailed and identification of TKR involved in vascular remodeling was completed in order to find efficient therapeutics with a favorable risk benefit ratio for PAH patient

Nowdays there is a plethora of evidence for H2O2-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase / xanthine oxidase (XDH / XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H2O2 and O2 ¿- , yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H2O2 in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H2O2 in the mM range. H2O2-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H2O2. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H2O2, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H2O2 contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to ¿/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H2O2-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45¿ leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H2O2-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

Nowdays there is a plethora of evidence for H₂O₂-mediated oxidative stress in the epidermis as well as in the system in patients with vitiligo (for review see (Schallreuter, Bahadoran et al. 2008). Xanthine dehydrogenase/xanthine oxidase (XDH/XO) catalyses the oxidative hydroxylation of hypoxanthine to xanthine followed by xanthine to uric acid, the last two steps in purine degradation pathway. Under oxidative conditions, XDH is converted to XO. The reactions catalysed by this enzyme generate H₂O₂ and O₂̇⁻, yielding in the presence of ROS accumulation, allantoin from uric acid. Therefore XO has been considered a major biologic source of oxygen-derived free radicals in many organs. The presence of XO in the human epidermis has not been shown so far. In this study several techniques were utilised to nail the presence and activity of XO in epidermal melanocytes and keratinocytes. The enzyme is regulated by H₂O₂ in a concentration dependent manner, where concentrations of 10-6M upregulate activity. Importantly, the results showed that the activity of XO is little affected by H₂O₂ in the mM range. H₂O₂-mediated oxidation of tryptophan and methionine residues in the sequence of XO yields only subtle alterations in the enzyme active site. These findings are in agreement with enzyme kinetics in the presence of 10-3M H₂O₂. Since uric acid is the end product of XO activity and this can be oxidised to allantoin by H₂O₂, we wanted to know whether allantoin is formed in the epidermis of patients with vitiligo. In order to address this issue, we utilised HPLC/mass spectrometry analysis. Analysis of epidermal cell extracts from suction blister tissue identified the presence of allantoin in patients with acute vitiligo, while this product was absent in healthy controls. In conclusion, our results provide evidence for functioning epidermal XO in the human epidermis which 4 can be a major source for the production of H₂O₂ contributing to oxidative stress in vitiligo. In addition, this thesis also demonstrates for the first time the presence of XO in melanosomes, and we showed that both 7BH4 and 7-biopterin inhibit XO activity in a concentration dependent manner. Moreover, XO has the potential to bind to 6/7BH4 and 6/7-biopterin from the pterin/tyrosinase inhibitor complex. This discovery adds another receptor independent mechanism for regulation of tyrosinase within the melanocyte similar to α/ß-MSH as shown earlier (Moore, Wood et al. 1999; Spencer, Chavan et al. 2005). Since the entire epidermis of patients with vitiligo is under H₂O₂-mediated oxidative stress, oxidative DNA damage would be highly expected. This thesis shows for the first time that epidermal 8-oxoG levels as well as plasma level of this oxidised DNA base are significantly increased in patients compared to healthy controls. We have shown that epidermal cells from patients with vitiligo respond to oxidative DNA damage via the overexpression of p21 and Gadd45α leading to a functioning increased short-patch base-excision repair (BER), while increased apoptosis can be ruled out due to lower caspase 3 and cytochrome c response compared to healthy controls. Our results show that patients develop effective DNA repair machinery via hOgg1, APE1 and DNA polymeraseß. Taking into consideration that these patients do not have an increased prevalence for solar-induced skin cancers, our data suggest that BER is a major player in the hierarchy to combat H₂O₂-mediated oxidative stress preventing ROS-induced tumourigenesis in the epidermis of these patients.

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The medicinal Alternanthera (Amaranthaceae) species, such as A. philoxeroides, present a great variety of bioactive compounds, among which are the betacyanins, nitrogen pigments that belong to the betalains class. These compounds are widely used as additives for food and drugs, due to their antioxidant action and lack of toxicity, which have already been proven. Techniques have been developed in order to improve productivity and performance of this pigment, one of those is the use of in vitro elicitors or in vivo stressing agents. Both have an important role in the transduction process of signals that regulate the defense genes in plants, acting as stimulators of production or degradation of several primary and secondary metabolites. This work aimed to assess, in A. philoxeroides plants, the growth and production characteristics of betacyanin in in vitro plants; the levels of photosynthetic pigments, betacyanins, lipid peroxidation and antioxidant enzymes activity in in vivo plants under salt stress, and also to quantify the level of betacyanin and the 5GT-DBs gene expression in the biosynthetic route of this compound in in vitro plants submitted to elicitation by NaCl and by tyrosine. For this, three trials were conducted. In the first one, A. philoxeroides explants were inoculated in MS medium with increasing NaCl concentrations (0, 50, 100, 150, 200 and 250 mM) for 35 days. In the second one, plants from in vitro cultures were acclimatized in greenhouses and irrigated with a sodium chloride solution (0, 200 and 400 mM) for 30 days. The third trial had two essays, one composed of in vitro A. philoxeroides plants in a liquid MS medium in vermiculite substrate for 35 days. After this period, a NaCl solution (400 mM) was added to the medium and the shoots were collected after 0, 12, 24, 36 and 48 hours of exposure. In the second one, nodal segments were inoculated in MS medium with and without tyrosine (0 e 75 µM), and its aerial parts were collected after 35 days. In the growth analysis, reduction of the averages was observed for the following variables: height, number of shoots, number of sprouts and root number and length; for the plants that have grown in the sodium chloride medium. The highest concentrations of betacyanins were found in the stalks of plants from MS medium, with 50 mM of NaCl. After 30 days of in vivo cultivation, the levels of chlorophyll a, chlorophyll b and carotenoids decreased as the salt concentration increased, while the reason of chlorophyll a/b in plants submitted to a higher salt concentration presented a difference in comparison to the control. Higher levels of betacyanin were observed on stalks, when compared to the leaves, in the highest salt concentrations. On the leaves, there was a significant increase of lipid peroxidation and superoxide dismutase activity. On the roots, there was an increase of enzymes catalase and ascorbate peroxidase. Regarding the analysis of differential expression (qRT-PCR), it was possible to observe that from 12 to 24 hours of salt stress, the 5-GT gene expression firstly increased, then there was a decrease in 36 hours and a new increase in 48 hours. The 5-GT gene also showed increased expression as a response to tyrosine. It was possible to conclude that A. philoxeroides elicited in vitro with sodium chloride present a decrease of the assessed morphological parameters, but in low concentrations betacyanin synthesis is stimulated. Salt stress causes greater degradation in the photosynthetic pigments, increment of betacyanin synthesis in stalks and damage to the cell membranes of the leaves. The increase of antioxidant enzymes activity stimulated the protective system against oxidative stress on in vivo A. philoxeroides plants. It is suggested that in this species, the enzyme bethanidine 5-Oglucosyltransferase reaches its highest expression in 48 hours of exposure to salt elicitation and also when grown in a medium containing tyrosine.
As espécies medicinais do gênero Alternanthera (Amaranthaceae) como A. philoxeroides apresentam uma variedade de compostos bioativos, entre eles as betacianinas, que são pigmentos nitrogenados pertencentes à classe das betalaínas. Esses compostos são amplamente utilizados como aditivos de produtos alimentícios e medicamentos devido à sua comprovada ação antioxidante e ausência de toxicidade. Técnicas têm sido desenvolvidas para aprimorar a produtividade e o rendimento deste pigmento, sendo uma delas o uso de elicitores in vitro ou agentes estressantes in vivo. Ambos apresentam um importante papel no processo de transdução de sinais que regulam os genes de defesa nas plantas, agindo como estimuladores para a produção e ou degradação de diversos metabólitos, tanto primários quanto secundários. Assim, o objetivo do presente trabalho foi avaliar em plantas de A. philoxeroides, as características de crescimento e produção de betacianina em plantas cultivadas in vitro; os teores dos pigmentos fotossintéticos, betacianinas, peroxidação lipídica e atividade de enzimas antioxidantes em plantas cultivadas in vivo, sob estresse salino, além de, quantificar o teor de betacianina e a expressão do gene 5GT-DBs envolvido na rota biossintética, deste composto, em plantas in vitro submetidas à elicitação por NaCl e pelo aminoácido tirosina. Para isso, foram conduzidos três experimentos. No primeiro, explantes de A. philoxeroides foram inoculados em meio MS, com concentrações crescentes de NaCl (0, 50, 100, 150, 200 e 250 mM), durante 35 dias. No segundo, plantas provenientes da cultura in vitro foram aclimatizadas em casa de vegetação e irrigadas com solução de cloreto de sódio (0, 200 e 400 mM), por 30 dias. O terceiro experimento contou com dois ensaios, sendo o primeiro composto de plantas de A. philoxeroides cultivadas in vitro, em meio MS líquido, no substrato vermiculita, durante 35 dias. Após esse período, foi adicionada ao meio, solução de NaCl (400 mM) e coletada a parte aérea das plantas após 0, 12, 24, 36 e 48 horas de exposição ao sal, Já o segundo, segmentos nodais foram inoculados em meio MS, na presença e ausência de tirosina (0 e 75 µM), tendo sua parte aérea coletada após 35 dias de cultivo. Nas análises de crescimento observou-se redução das médias para as variáveis altura, número de gemas, número de brotos e no número e comprimento de raiz, nas plantas crescidas nos meios contendo cloreto de sódio. As maiores concentrações de betacianinas foram encontradas nos caules de plantas cultivadas em meio MS com 50 mM de NaCl. Após 30 dias de cultivo in vivo os teores de clorofilas a, clorofila b, e carotenóides decresceram à medida que aumentou a concentração de sal, enquanto a razão clorofila a/b das plantas submetidas à maior concentração de sal apresentou

Doutoramento em Bioquímica
Os polissacarídeos são os componentes maioritários dos grãos de café verde e torrado e da bebida de café. Os mais abundantes são as galactomananas, seguindo-se as arabinogalactanas. Durante o processo de torra, as galactomananas e arabinogalactanas sofrem modificações estruturais, as quais estão longe de estar completamente elucidadas devido à sua diversidade e à complexidade estrutural dos compostos formados. Durante o processo de torra, as galactomananas e arabinogalactanas reagem com proteínas, ácidos clorogénicos e sacarose, originando compostos castanhos de alto peso molecular contendo nitrogénio, designados de melanoidinas. As melanoidinas do café apresentam diversas atividades biológicas e efeitos benéficos para a saúde. No entanto, a sua estrutura exata e os mecanismos envolvidos na sua formação permanecem desconhecidos, bem como a relação estrutura-atividade biológica. A utilização de sistemas modelo e a análise por espectrometria de massa permitem obter uma visão global e, simultaneamente, detalhada das modificações estruturais nos polissacarídeos do café promovidas pela torra, contribuindo para a elucidação das estruturas e mecanismos de formação das melanoidinas. Com base nesta tese, oligossacarídeos estruturalmente relacionados com a cadeia principal das galactomananas, (β1→4)-Dmanotriose (Man3), e as cadeias laterais das arabinogalactanas, (α1→5)-Larabinotriose (Ara3), isoladamente ou em misturas com ácido 5-Ocafeoilquínico (5-CQA), o ácido clorogénico mais abundante nos grãos de café verde, e péptidos compostos por tirosina e leucina, usados como modelos das proteínas, foram sujeitos a tratamento térmico a seco, mimetizando o processo de torra. A oxidação induzida por radicais hidroxilo (HO•) foi também estudada, uma vez que estes radicais parecem estar envolvidos na modificação dos polissacarídeos durante a torra. A identificação das modificações estruturais induzidas por tratamento térmico e oxidativo dos compostos modelo foi feita por estratégias analíticas baseadas principalmente em espectrometria de massa, mas também em cromatografia líquida. A cromatografia de gás foi usada na análise de açúcares neutros e ligações glicosídicas. Para validar as conclusões obtidas com os compostos modelo, foram também analisadas amostras de polissacarídeos do café obtidas a partir de resíduo de café e café instantâneo. Os resultados obtidos a partir dos oligossacarídeos modelo quando submetidos a tratamento térmico (seco), assim como à oxidação induzida por HO• (em solução), indicam a ocorrência de despolimerização, o que está de acordo com estudos anteriores que reportam a despolimerização das galactomananas e arabinogalactanas do café durante a torra. Foram ainda identificados outros compostos resultantes da quebra do anel de açúcares formados durante o tratamento térmico e oxidativo da Ara3. Por outro lado, o tratamento térmico a seco dos oligossacarídeos modelo (individualmente ou quando misturados) promoveu a formação de oligossacarídeos com um maior grau de polimerização, e também polissacarídeos com novos tipos de ligações glicosídicas, evidenciando a ocorrência de polimerização através reações de transglicosilação não enzimática induzidas por tratamento térmico a seco. As reações de transglicosilação induzidas por tratamento térmico a seco podem ocorrer entre resíduos de açúcares provenientes da mesma origem, mas também de origens diferentes com formação de estruturas híbridas, contendo arabinose e manose como observado nos casos dos compostos modelo usados. Os resultados obtidos a partir de amostras do resíduo de café e de café instantâneo sugerem a presença de polissacarídeos híbridos nestas amostras de café processado, corroborando a ocorrência de transglicosilação durante o processo de torra. Além disso, o estudo de misturas contendo diferentes proporções de cada oligossacarídeo modelo, mimetizando regiões do grão de café com composição distinta em polissacarídeos, sujeitos a diferentes períodos de tratamento térmico, permitiu inferir que diferentes estruturas híbridas e não híbridas podem ser formadas a partir das arabinogalactanas e galactomananas, dependendo da sua distribuição nas paredes celulares do grão e das condições de torra. Estes resultados podem explicar a heterogeneidade de estruturas de melanoidinas formadas durante a torra do café. Os resultados obtidos a partir de misturas modelo contendo um oligossacarídeo (Ara3 ou Man3) e 5-CQA sujeitas a tratamento térmico a seco, assim como de amostras provenientes do resíduo de café, mostraram a formação de compostos híbridos compostos por moléculas de CQA ligadas covalentemente a um número variável de resíduos de açúcar. Além disso, os resultados obtidos a partir da mistura contendo Man3 e 5-CQA mostraram que o CQA atua como catalisador das reações de transglicosilação. Por outro lado, nas misturas modelo contendo um péptido, mesmo contendo também 5-CQA e sujeitas ao mesmo tratamento, observou-se uma diminuição na extensão das reações transglicosilação. Este resultado pode explicar a baixa extensão das reações de transglicosilação não enzimáticas durante a torra nas regiões do grão de café mais ricas em proteínas, apesar dos polissacarídeos serem os componentes maioritários dos grãos de café. A diminuição das reações de transglicosilação na presença de péptidos/proteínas pode dever-se ao facto de os resíduos de açúcares redutores reagirem preferencialmente com os grupos amina de péptidos/proteínas por reação de Maillard, diminuindo o número de resíduos de açúcares redutores disponíveis para as reações de transglicosilação. Além dos compostos já descritos, uma diversidade de outros compostos foram formados a partir dos sistemas modelo, nomeadamente derivados de desidratação formados durante o tratamento térmico a seco. Em conclusão, a tipificação das modificações estruturais promovidas pela torra nos polissacarídeos do café abre o caminho para a compreensão dos mecanismos de formação das melanoidinas e da relação estrutura-atividade destes compostos.
Polysaccharides are the major components of green and roasted coffee beans, and coffee brew. The most abundant ones are galactomannans, followed by arabinogalactans. During the roasting process, galactomannans and arabinogalactans undergo structural modifications that are far to be completely elucidated due to their diversity and complexity of the compounds formed. During the roasting process, galactomannans and arabinogalactans react with proteins, chlorogenic acids, and sucrose, originating high molecular weight brown compounds containing nitrogen, known as melanoidins. Several biological activities and beneficial health effects have been attributed to coffee melanoidins. However, their exact structures and the mechanisms involved in their formation remain unknown, as well as the structure-biological activity relationship. The use of model systems and mass spectrometry analysis allow to obtain an overall view and, simultaneously, detailed, of the structural modifications in coffee polysaccharides promoted by roasting, contributing to the elucidation of the structures and formation mechanisms of melanoidins. Based on this thesis, oligosaccharides structurally related to the backbone of galactomannans, (β1→4)-D-mannotriose, and the side chains of arabinogalactans, (α1→5)-Larabinotriose, alone or in mixtures with 5-O-caffeoylquinic acid, the most abundant chlorogenic acid in green coffee beans, and dipeptides composed by tyrosine and leucine, used as models of proteins, were submitted to dry thermal treatments, mimicking the coffee roasting process. The oxidation induced by hydroxyl radicals (HO•) was also studied, since these radicals seem to be involved in the modification of the polysaccharides during roasting. The identification of the structural modifications induced by thermal and oxidative treatment of the model compounds was performed mostly by mass spectrometry-based analytical strategies, but also using liquid chromatography. Gas chromatography was used in the analysis of neutral sugars and glycosidic linkages. To validate the conclusions achieved with the model compounds, coffee polysaccharide samples obtained from spent coffee grounds and instant coffee were also analysed. The results obtained from the model oligosaccharides when submitted to thermal treatment (dry) or oxidation induced by HO• (in solution) indicate the occurrence of depolymerization, which is in line with previous studies reporting the depolymerization of coffee galactomannans and arabinogalactans during roasting. Compounds resulting from sugar ring cleavage were also formed during thermal treatment and oxidative treatment of Ara3. On the other hand, the dry thermal treatment of the model oligosaccharides (alone or when mixed) promoted the formation of oligosaccharides with a higher degree of polymerization, and also polysaccharides with new type of glycosidic linkages, evidencing the occurrence of polymerization via non-enzymatic transglycosylation reactions induced by dry thermal treatment. The transglycosylation reactions induced by dry thermal treatment can occur between sugar residues from the same origin, but also of different origins, with formation of hybrid structures, containing arabinose and mannose in the case of the model compounds used. The results obtained from spent coffee grounds and instant coffee samples suggest the presence of hybrid polysaccharides in these processed coffee samples, corroborating the occurrence of transglycosylation during the roasting process. Furthermore, the study of mixtures containing different proportions of each model oligosaccharide, mimicking coffee bean regions with distinct polysaccharide composition, subjected to different periods of thermal treatment, allowed to infer that different hybrid and non-hybrid structures may be formed from arabinogalactans and galactomannans, depending on their distribution in the bean cell walls and on roasting conditions. These results may explain the heterogeneity of melanoidins structures formed during coffee roasting. The results obtained from model mixtures containing an oligosaccharide (Ara3 or Man3) and 5-CQA and subjected to dry thermal treatment, as well as samples derived from spent coffee grounds, showed the formation of hybrid compounds composed by CQA molecules covalently linked to a variable number of sugar residues. Moreover, the results obtained from the mixture containing Man3 and 5-CQA showed that CQA acts as catalyst of transglycosylation reactions. On the other hand, in the model mixtures containing a peptide, even if containing 5-CQA and subjected to the same treatment, it was observed a decrease in the extent of transglycosylation reactions. This outcome can explain the low extent of non-enzymatic transglycosylation reactions during roasting in coffee bean regions enriched in proteins, although polysaccharides are the major components of the coffee beans. The decrease of transglycosylation reactions in the presence of peptides/proteins can be related with the preferential reactivity of reducing residues with the amino groups of peptides/proteins by Maillard reaction, decreasing the number of reducing residues available to be directly involved in the transglycosylation reactions. In addition to the compounds already described, a diversity of other compounds were formed from model systems, namely dehydrated derivatives formed during dry thermal treatment. In conclusion, the identification of the structural modifications in coffee polysaccharides promoted by roasting pave the way to the understanding of the mechanisms of formation of melanoidins and structure-activity relationship of these compounds.

Tyrosyl phosphorylation plays an important role in many fundamental cellular processes, including cell growth, differentiation and proliferation. The levels of phosphotyrosine (pY) are regulated by the opposing actions of protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs). A limitation to understanding the roles of PTPs in physiological and pathological cell signaling has been the absence of global proteomic approaches that enable the systematic and comprehensive analysis of PTP expression, regulation and function. This dissertation describes the development and application of novel proteomic methodologies that permit the global analysis of PTP expression (qPTPome), regulation (by oxidation and nitrosylation; q-oxPTPome) and substrates/binding proteins. These methods provide a workflow to begin assessing PTP function at a systems level, rather than its current targeted format. Application of these techniques will provide invaluable information to begin bridging the gap in our understanding of PTP and PTK function in normal and malignant cell signaling.

碩士
弘光科技大學
化妝品科技研究所
97
Scopoletin (6-methoxy-7-hydroxycoumarin) is a pharmacologically active coumarin compound that has been isolated from several traditional Chinese herbs, such as Lycium chinense Mill and Angelicae dahuricae Radix. It also has been widely existed in the plant, such as Rutaceae Dictamnus、Rutaceae Murrayapaniculata. Several studies have shown that scopoletin show hepatoprotective activity, antihypertension, anti-bacteriae, down fever and relieve pain. In addition, It was shown that scopoletin show the anti-hemoglutination effect on the domestic rabbit. The aim of this research is to evaluate whether scopoletin whether has antioxidation and white efficacy. Previous study reported that scopoletin has antioxidation activity. However, there is no report about interaction between scopoletin and other antioxidants such as vitamin C or vitamin E. Therefore, we aim to study possible synergistic antioxidation effects between scopoletin and vitamin C or vitamin E. Antioxidation activity was assayed by determining the DPPH radical scavenging activity , total phenol content, ABTS+ radical cation decoloriation and reducing power. The experimental results showed that DPPH radical capture ability is higher along with scopoletin concentration. The capture ability of scopoletin is also more obvious when combined with vitamin C or vitamin E. When scopoletin was combined with vitamin C, the total phenol content also increased in a dose-dependent pattern, whereas similar result was not found in the case of vitamin E. Additionally, scopoletin and vitamin C also showed remarkable synergistc effects on reducing power and the elimination of ABTS+. Afterwards, the effects of scopoletin on mushroom tyrosinase activity, intracellular tyrosinase activity and melanin content of murine B16F10 melanoma cells were evaluated. Kojic acid was used as a positive standard in the above experiments. The results showed that high concentration of scopoletin(2 mM~10 mM)could suppress mushrom tyrosinase activity in a dose-dependent pattern. However, when scopoletin combined with kojic acid, there is no obvious synergistic effect on the inhibition of tyrosinase activity. Interestingly, intracellular tyrosinase activity of B16F10 cells was suppressed by low concentration of scopoletin(25 μM, 50 μM, 100μM). Besides, the melanin content in B16F10 cells was also decreased by scopoletin in a dose-dependent pattern. Afterwards, we also evaluated the effects of scopoletin on protein content of melanin related proteins- Tyrosinae、Tyrosinase-related protein-1(TRP-1) and Tyrosinase-related protein-2(TRP-2) by western blot. The intracellular tyrosinase、TRP-2 was also decreased by scopoletin in a dose-dependent pattern. The further work will elucidate the whitening mechanism of scopoltin, and we will also evaluate the possibility of scopoletin applied in the antioxidation and whitening cosmetic formulations.

碩士
中華科技大學
健康科技研究所
102
Abstract Recently, natural skin care products and cosmetics are popular. Therefore, biotechnologycombined with Chinese herbal medicine has the large potential. In the past, many researchers screened many new ingredients to inhibit tyrosinase activity and increase antioxidant capacity from Chinese herbal plantsIn this experiment, Walnuts, Moutan, and Asparticwere selected and they were extracted by distilled water, 95% ethanol, 50% ethanol, 100% ethyl acetate and 50% ethyl acetate.These extracted liquid was then fermented by Bifidobacterium bifidumat different times.Some chemical properties including anti-tyrosinase activity, reducing power and scavenging activity of DPPH radical were measured.Additionally, cytotoxicity was evaluated by the survival rate of CCD-966SK cells. HPLC chromatogram reveled that the different compounds were existed in the liquid fermented or not by B. bifidum. The 100% anti-tyrosinase activity was found in the operating condition (Aspartic extracted by distilled water and fermented for 12 hours).DPPH scavenging was achieved 89.4% when Moutanextracted by 95% ethanol and then fermented for 8 hours. In conclusion, Moutanand Asparticextracted by solvents and then fermented by B. bifidum have potential as whitening agent and antioxidants. Keywords: whitening, anti-oxidation, Aspartic, Moutan, lactic acid bacteria, tyrosinase

The work presented in this thesis describes the occurrence and properties of two multicopper oxidases derived from lichens. Despite numerous data on laccases and tyrosinases in fungi and flowering plants, this is the first report of the occurrence of these enzymes in lichenized ascomycetes. Extracellular laccase and tyrosinase activity was measured in 50 species of lichens from different taxonomic groupings and contrasting habitats. Out of 27 species tested from suborder Peltigerineae, all displayed laccase and tyrosinase activity that correlated to each other, while activity was absent in species tested from other lichen groups. Identification of the enzymes as laccases and tyrosinases was confirmed by the ability of lichen thalli or leachates to readily metabolize substrates such as 2,2’-azino(bis-3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine and o-tolidine in case of laccase and L-dihydroxyphenylalanine (L-DOPA), Ltyrosine and epinephrine in case of tyrosinase in the absence of hydrogen peroxide. The activities of both enzymes were highly sensitive to cyanide and azide, and tyrosinase activity was sensitive to hexylresorcinol. Laccase activity had typical pH and temperature optima and an absorption spectrum with a peak at 614 nm. Tyrosinases could be activated by sodium dodecyl sulphate (SDS) and had typical tyrosinase molecular masses of approx. 60 kDa. The diversity of laccase isoforms in 20 lichen species from suborder Peltigerineae was investigated. The molecular masses of the active forms of most laccases varied between 135 and 190 kDa, although some lichens within the family Peltigeraceae had laccases with higher masses, typically varying from 200 to over 350 kDa. Most species contained one oligomeric laccase isoform. Desiccation and wounding stimulated laccase activity, while only wounding stimulated tyrosinase activity. The ability of laccases to decolorize dye is a classic attribute of laccases, and one with biotechnological potential. The ability of eight lichen species to decolourize different types of dyes was therefore tested. Interestingly, results showed that not only species belonging to suborder Peltigerineae but also species from other lichen group effectively decolourised dyes after 48 h suggesting that other oxidases appear to have ability to decolorize. Hopefully, our work could contribute to the better knowledge and application of lichen multicopper oxidases.
Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2012.

碩士
大葉大學
生物產業科技學系
96
The three caffeic acid derivatives contents, anti-oxidative and anti-tyrosinase activities of extracts with 100% pure water (WEs), and 10%, 40%, 70% ethanol (0.1EtEs, 0.4EtEs, 0.7EtEs, respectively), and 10%, 40% and 70% glycerol (0.1GlEs, 0.4 GlEs, 0.7GlEs, respectively) of the powders (PCfSPs) of purple coneflower seeds produced in Taiwan were investigated in this study. The maximum caffeic acid derivatives contents (1.13% dried weight) was obtained from the freeze-dried 0.7EtEs of PCfSPs, while those (90.6 g/mL 70% glycerol) was obtained from the 0.7GlEs of PCfSPs for glycerol extraction. Among the WEs and EtEs of PCfSPs, only the freeze-dried 0.7EtEs had the best DPPH-scavenging effect (IC50 activity: 97g 0.7EtEs/mL), while the 0.7GlEs had the strongest effect (IC50: 1.89% 0.7GlEs) for glycerol extraction. In general, the freeze-dried WEs and EtEs had better anti-tyrosinase activities than those of the hot-dried extracts. The anti-tyrosinase activities increased as increasing the extraction concentrations of ethanol. Among the WEs and EtEs of PCfSPs, only the freeze-dried 0.7EtEs had the best anti-tyrosinase activity (IC50: 625g 0.7EtEs/mL), while the 0.7GlEs had the strongest activity (IC50: 2.32% 0.7GlEs).

碩士
大葉大學
生物產業科技學系
95
The resveratrol contents, anti-oxidative ( DPPH-free-radical-scavenging effect ) and anti-tyrosinase activities of Polygonum cuspidatum ( one of Chinese traditional medicine ) extracts with 100% pure water ( Wes ) and 10% and 70% ethanol solutions ( 0.1 EtEs and 0.7 EtEs, respectively ) were investigated in this study. The maximum extract content ( 30% extraction yield ) and resveratrol ( 1.57% dried weight ) was obtained from the freeze-dried 0.7 EtEs of P. cuspidatum. Among the P. cuspidatum extracts, only the freeze-dried 0.7 EtEs had strong DPPH-scavenging effects or anti-oxidative activities, while the activities of the freeze-dried 0.7 EtEs first gradually decreased to a certain value as increasing their addition concentrations or resveratrol contents, and then increased as increasing the concentrations or contents. For the other P. cuspidatum extracts, the anti-oxidative activities first increased and then decreased to some certain values as increasing their addition concentrations or resveratrol contents. Except that the tyrosinase relative activities of the hot-dried WEs and 0.1 EtEs were kept at 80%~90%, the freeze-dried 0.7 EtEs also had stronger anti-tyrosinase activities than the others. However, the tyrosinase relative activities started decreasing from original 70% or the anti-tyrosinase activities became much significant and effective when the addition concentrations or resveratrol contents of the freeze-dried 0.7 EtEs were over 1000 g/mL or 15.7 g, respectively. In this study, only the freeze-dried 0.7 EtEs had the higher extract and resveratrol contents and stronger anti-oxidative anti-tyrosinase activities than the others.

碩士
國立臺灣大學
生化科學研究所
93
Reactive oxygen species (ROS) have emerged as intracellular signaling molecules in multiple cellular processes such as proliferation, apoptosis, and senescence. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, particularly protein tyrosine phosphatases (PTPs). Recent studies suggested that the abnormally high levels of cellular ROS production are concurrent with the development of human diseases such as cancers. We examined the role of ROS in the control of signal transduction in Ras- and Src-transformed NIH-3T3 cells which constitutively produce more ROS and possess more active tyrosine phosphorylation signals than parental NIH-3T3 cell. Treatment of transformed cells with diphenyleneiodonium (DPI), a NADPH oxidase (Nox) inhibitor, led to the suppression of cellular ROS production. Under this condition, we found that the activity of endogenous PTPs was elevated, whereas the tyrosine phosphorylation level of cellular proteins was decreased. Such an effect was more pronounced in Ras-transformed cells, compared with that in Src-transformed cells, suggesting that Ras-mediated signal transduction is essentially ROS-dependent. This hypothesis was further investigated. In Ras-transformed cells treated with DPI, a 120 kDa protein, which showed a significant decrease of tyrosine phosphorylation, was subsequently identified as focal adhesion kinase (FAK). Upon the inhibition of cellular ROS production, FAK was dephosphorylated at tyrosine 397 (Tyr397), an integrin-induced auto-phosphorylation site whose phosphorylation activates FAK and enhances cell migration. Treatment of DPI also led to the decrease of phosphorylation levels of Tyr576, Tyr577, Tyr861 and Tyr925 of FAK, concurrent with the decreased kinase activity of Src that recognizes and phosphorylates those Tyr residues in FAK. We also observed that the overexpression of dominant negative Rac1N17, which has been shown to block Nox activity, resulted in the dephosphorylation of FAK and Src. Furthermore, we showed that Ras-transformed cells lost their membrane protrusions in a time-dependent manner in response to DPI treatment, concomitant with the reactivation of cellular PTPs that were originally undergoing reversible oxidation. Our data thus suggested that the Ras-mediated migration signal is regulated by the ROS-dependent activation of FAK and Src through constitutive oxidation and inactivation of endogenous PTPs.

碩士
國立清華大學
生物科技研究所
95
Previously, our group found that geldanamycin (GA) with sublethal dose provokes the ER stress in rat brain tumour 9L (9L RBT) cells, which followed by the generation of reactive oxygen species (ROS). However, the effect of GA on the unfolded protein response (UPR) signaling pathway remains unclear. Recently, activation of JAK/STAT pathway has been observed in response to generation of intracellular ROS and exogenous hydrogen peroxide (H2O2). Here, we investigated the effect of AG490, the specific inhibitor of Janus kinase-2 (JAK2), on the expression of grp78 coding for ER stress protein and the mechanistic relationship of GA signaling to ER stress. The mRNA and protein level of grp78 and grp94 were examined by real-time quantitative RT-PCR, Western blotting analysis and metabolic labeling experiment in 9L RBT cells treated with GA or AG490. In this study, we firstly discovered that AG490 only could specifically transactivate the ER-resident molecular chaperones GRP78 and GRP94 in 9L RBT cells. In addition, calcium chelator BAPTA-AM, mitochondrial uniporter inhibitor ruthenium red (RR), antioxidant N-acetylcysteine (NAC), and the inhibitor of mitochondrial PT pore, cyclosporin A (CyA), abolished the grp78 and grp94 induction by AG490. Therefore, it suggests that intracellular calcium disturbances and oxidative stress are involved in AG490-induced ER stress. Furthermore, serine/threonine kinase inhibitor H7, PKA inhibitor KT5720, and additional PKC isozyme-selective inhibitors including Gö6983 and Gö6976 could diminished the AG490-induced upregulation of grp78 and grp94 genes. However, the similar suppression effect of Gö6983 and Gö6976 was not observed on the GA-mediated induction of grp78 and grp94 mRNA. Thus, we suggest that the signaling pathway of AG490-induced ER stress response might different from GA. In conclusion, AG490, as an ER stress inducer, might evoke intracellular calcium disturbances and generation of ROS, which lead to activation of cPKC and PKA for upregulation of grp78 and grp94 genes in 9L RBT cells.

博士
國立臺灣大學
生化科學研究所
106
Reactive oxygen species (ROS) has been shown to play a critical role in the development and progression of cardiac pathophysiology. Protein is one of the prime targets of cellular ROS generated in cardiomyocytes. Among the 20 common amino acid, cysteine (Cys) residue, which has a remarkably low pKa characteristic, is highly susceptible to oxidation. Identification of the redox role of thiol modifications has gained significant importance because Cys residue plays an important role in protein function under pathophysiological conditions. Here, our study focuses on the redox-dependent regulation of Cys-sulfonation (SO3H), which belongs irreversible oxidation, on protein tyrosine phosphatases (PTPs). Because of its high nucleophilic property, catalytic Cys residue within PTPs is the direct targets of cellular ROS. To investigate the effect of ROS-induced Cys-sulfonation on PTPs and the mechanism of proteolysis in protein quality control machinery. Using the antibody-based method that could detect protein Cys-sulfonation, we found that endogenous PTPs already oxidized irreversibly and undergoing proteasome-mediated degradation in H9c2 cells. Protein tyrosine phosphatase 1B (PTP1B) is one of PTPs oxidation well-studied, so we chose PTP1B as our experiment model. Upon H2O2 stimuli, through the in vivo and in vitro immunoprecipitation assays, we found that PTP1B catalytic Cys215 residue was sulfonated and it facilitated PTP1B for ubiquitination and proteolysis. Inhibition of PTP1B oxidation by site-directed mutagenesis of PTP1B at active-site Cys215 abrogates the effects of protein ubiquitination and degradation on PTP1B. Through the high-throughput Yeast proteome chips, we identified CDC53, which has homology to human Cul1 E3 ligase, could strongly interact with the chemically synthesized peptide containing Cys-sulfonation. Subsequent investigation with the dominant-negative construct of Cul1 E3 ligase suggested that PTP1B serve as the substrate for Cul1 via Cys-sulfonation recognition. Impairment of Cul1-mediated degradation of Cys-sulfonated PTPs potentiated cardiotoxicity. The findings of this study provide important insight into how biologically significant Cys oxidation directs myocardial protein turnover through ubiquitin/proteasome-mediated degradation.

碩士
嘉南藥理科技大學
生物科技系暨研究所
91
Microwave aqueous extracts (MAE) from twelve Chinese herbs and fifteen commercial pure compounds were evaluated for their (1) melanin biosynthesis anti-oxidative activities by tyrosinase inhibition, (2) nitric oxide (NO) scavenging activities by SNP, (3) radical scavenging effects by the DPPH and ( 4) peroxynitrite scavenging activities by the SNP / H2O2 / luminal chemiluminescence’s methods. The results of our investigation show that glycyrrhizae radix for the preliminary assay optimal tyrosinase inhibition (90. 73%) and NO scavenging effects (82.39%) of microwave Chinese herbs extracts can be concluded that the duration of microwave radiation is 6 min, the microwave energy is 720w. Our studies also show three Chinese herbs by MAE ( Ramulus mori, Glycyrrhizae radix and Cortex mori) exhibited high anti- tyrosinase (SC50=0.3,0.9 and 3.11mg/ml, respectively) and NO scavenging effect (SC50=0.238, 0.249 and 0.236mg/ml, respectively). On the other hands, Acanthopanax senticosus (SC50=0.755, 0.713mg/ml, respectively) and Ginkgo biloba (SC50=0.583, 0.756mg/ml, respectively) scavenged DPPH free radical and peroxynitrite production, most effectively. Commercial pure compounds (such as licorice-derived, flavonoids, antioxidants and tyrosinase inhibitors compounds) were referenced index for their anti-tyrosinase, free radical scavenging and antioxidant properties. The results indicated that under 100μM concentration, Kojic acid against tyrosinase (63.14%), Hydroquinone scavenged NO (40.17%), Catechin scavenged DPPH free radical (97.99%), and Quercetin decreased peroxynitrite production (52.25%) present more effect for bioactivities. Collectively, these data suggest that Chinese herbs by MAE maybe extract some effect such as anti-oxidative and anti-tyrosinase. Consequently, some detail bioactive mechanisms and MAE in order to isolate and identify photochemical compounds, involved in some biological activities will be studied.

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologias, Universidade do Algarve, 2014
A saúde Humana é tema de grande importância, merecendo assim uma enorme atenção por parte da comunidade científica. As condições ambientais às quais o Homem se encontra sujeito nos dias de hoje, como por exemplo os agravados níveis de poluição e intensos níveis de radiação UV, são por si só potenciais causadores de inúmeros distúrbios e condições perigosas que podem facilmente evoluir para doenças, muitas delas irreversíveis e até mesmo incuráveis. Em associação aos hábitos de vida pouco saudáveis de uma enorme percentagem da população como o tabaco, má nutrição, vida sedentária e o intenso nível stress a que estamos sujeitos diariamente, todos juntos contribuem de uma forma assustadora para o desenvolvimento e progressão de doenças como cancros, Alzheimer, Parkinson e uma série de outras doenças. Tal facto, gera um stress oxidativo no organismo, que como consequência trará uma série de distúrbios ou até mesmo doenças graves. Visando aumentar a esperança média de vida, fontes naturais de compostos bioativos têm sido intensamente estudadas, de forma a prevenir, retardar e até mesmo curar determinadas doenças. Inúmeros compostos bioactivos foram já isolados de frutas, vegetais, plantas e algas, essencialmente oriundos de organismos pertencentes ao Reino das plantas. Parte destes metabolitos naturais fazem ou fizeram parte de ensaios clínicos, os quais inclusive obtiveram para alguns casos obtiveram resultados bastante positivos levando á produção farmacológica. O Oceano é um dos ecossistemas mais ricos do planeta Terra, albergando uma vasta diversidade de organismos permitindo-nos um espetáculo de cor, texturas, tamanhos e diferentes complexidades. Diariamente são produzidos inúmeros metabolitos, muitos deles por descobrir, os quais apresentam as mais variadas bioatividades e aplicações medicinais. Estudos têm vindo a provar que os compostos naturais produzidos nos Oceanos são de facto uma potencial fonte para o desenvolvimento da indústria Farmacológica. Devido á sua interessante e exuberante fisiologia e adaptações ao meio ambiente os invertebrados marinhos são talvez uma das mais promissoras fontes de compostos bioativos. Muito poucos estudos foram ainda realizados nesta área e apesar de ainda existir muito por descodificar e descobrir, estudos prévios apontam para uma forte possibilidade na utilização de invertebrados marinhos como fonte de compostos bioativos. Contudo á que ter em atenção que o estudo dos organismos como fonte de compostos bioativos é ainda bastante recente, tendo sido iniciado à sensivelmente 60 anos, pelo que ainda necessita de muitas reformulações no que toca á extração de compostos. Atualmente aproximadamente 1500 metabolitos de origem marinha já foram descritos, dos quais alguns deram inclusive origem a medicamentos como: Ziconitide (Prialt™), Yondelis™ e Halaven™, bastante referenciados para a prevenção e tratamento do cancro. Sendo que os referidos medicamentos são derivados da descoberta de compostos bioativos em esponjas. Assim sendo e considerando o potencial que os invertebrados marinhos representam para a indústria farmacológica, neste trabalho foi estuda uma espécie de lesma do mar (B.leachii), a qual é considerada invasora no Mar Mediterrâneo. Com o intuito de avaliar o potencial da mesma como fonte de metabolitos naturais foram realizados diferentes ensaios, todos eles complementares, permitindo a quantificação da atividade antioxidante (Inibição do DPPH, atividade quelante do ferro e cobre, atividade redutora do ferro e a inibição do oxido nítrico), neuroprotectora, avaliando o efeito dos extratos de B.leachii na inibição enzimática (acetilcolinesterase, butirilcolinesterase e tirosinase) e ainda o seu efeito in vitro para culturas celulares, avaliando a capacidade anti-inflamatória dos extratos (avaliando a viabilidade celular após ser induzida uma resposta inflamatória pelo LPS) e o seu potencial na proteção das células contra o efeito de H2O2. Os resultados deste estudo mostram que a B. leachii apresenta uma atividade antioxidante bastante relevante. Estes resultados são bastante promissores, pois os compostos antioxidantes têm a capacidade de prevenir o stress oxidativo, que quando descontrolado despoleta a iniciação e desenvolvimento de doenças. Os extratos mostraram ainda uma elevada afinidade para inibir a tirosinase, mostrando-se assim bastante promissores para indústria da cosmética (inibindo a produção de melanina) ou até mesmo para a indústria farmacológica. Uma vez que a tirosinase está implícita na progressão de doenças neurológicas como Parkinson, devido à libertação de compostos tóxicos associados á sua atividade, extratos de B. leachii podem portanto representar uma esperança na possibilidade do desenvolvimento de tratamento da doença. Ainda referente à atividade neuroprotectora, o extrato de acetona mostrou uma enorme habilidade na inibição do óxido nítrico, apresentando assim uma potente atividade anti-inflamatória, a qual é essencial para a prevenção de doenças neurológicas. De acordo com os dados obtidos para o seu perfil de ácidos gordos, a espécie é de facto possuidora de uma considerável percentagem de ácidos polinsaturados, especialmente ómega-3, destacando-se o EPA, o que vai de encontro á sua capacidade anti-inflamatória. De uma forma geral a espécie de lesma estudada mostrou ser uma potencial fonte de metabolitos naturais os quais apresentaram as mais diversas atividades biológicas sendo possivelmente capazes de beneficiar a saúde Humana, dada a possibilidade de atuação perante determinadas doenças. Além disso este estudo demonstrou que a lesma do mar estudada apresenta um teor proteico relevante. Pelo que uma possível alimentação em recursos obtidos destes organismos pode beneficiar em muito o sistema imunitário do Homem. Os dados obtidos nesta tese de certa forma acabam por ir ao encontro da teoria que tem vindo a ser desenvolvida pela comunidade científica: Que os compostos presentes na maioria dos invertebrados marinhos na verdade são metabolitos secundários (oriundos da dieta). Finalmente o facto de se falar de uma espécie invasora, e uma vez que a espécie tem todo este potencial, os dados justificam que no âmbito do controle da sua densidade e abundância para os locais onde é invasora, em vez de simplesmente remover o excesso de biomassa, que seria depois desperdiçado, que seja feito o reencaminhamento devido da biomassa para locais onde possa ser devidamente estuda e aproveitada. Este estudo potencializa a abertura de novas portas e oportunidades para o desenvolvimento da saúde humana. Sendo um dos primeiros estudos realizados, não existem estudos comparáveis pelo que todos os resultados são válidos para descartar ou conduzir a novas oportunidades e teorias.
Nowadays diseases are evolving and progressing so quickly, every single day thousands and thousands of people die due to several untreatable conditions. The environmental that we live on is full of dangerous agents such as pollution and radioactivity, which contribute for the emerging of new diseases. Considering the risks that those conditions represent for humans health, the research community is doing a huge effort to prevent and hopefully shut down several diseases. Scientists have been using natural sources as a way to extract natural bioactive compounds, with a range of different bioactivities, which can potently be used by the pharmaceutical industry for the new drug development. Oceans are one of the more diverse ecosystems in the world. Aquatic systems are stuffed with a huge diversity of organisms where a magnificent world of colors, shapes, textures and interesting metabolites can be seen. Marine animals had evolved in a complete different environmental and for that they contain a considerable amount of bioactive compounds due to an accurate chemical defensive system, diet and other adaptations to the extreme conditions of oceans. By its interesting physiology, marine invertebrates namely sponges, tunicates, sea cucumbers and sea hare, are considered as a potential resource of bioactive compounds. In the recent years around 1500 marine natural products have been identified, 45 were tested during preclinical and clinical trials. A few of them have been approved for clinical use: this is the case of Ziconitide (Prialt™) used for the chronic pain; ecteinascidin 743 (Yondelis™) used for the treatment of ovarian cancer soft tissue sarcoma and eribulin mesylate (Halaven™) for the treatment of recurrent breast cancer. Another example is bryostatin, which was originally studied for anticancer activity and led to the development of analogues with a high potential to alleviate the symptoms related with Alzheimer’ disease. Considering the background and the urgency of the identification of new bioactive compounds, the main goal of this thesis project is to evaluate the biological activities of extracts from Bursatella leachii, which is an invasive species of sea hare in the Mediterranean. For the following extracts it was evaluated the antioxidant and neuroprotective activity using different and complementary assays. As for the antioxidant activity, it was evaluated through the RSA of DPPH, nitric oxide inhibition, metal chelating activity and iron reducing/antioxidant power. For the neuroprotective activity it was quantified the ability to inhibition toome specific enzymes and the anti-inflammatory power of the extracts. This work proved that the B. leachii could be considered as a potent source of bioactive compounds with a positive effect in health. As for the results the extracts exhibit a potent antioxidant activity, a high inhibitory power of tyrosinase and a very high affinity to in vitro inhibit the NO production, in other words a potent anti-inflammatory activity. In a near Future it is probably that B.leachii could be used by the food, cosmetic or pharmaceutical industry, as a source of bioactive molecules in favor of human’s health and quality. Also the study gives propose to the excess of B. leachii biomass found in Mediterranean Sea.