The objective of the research was to investigate proton-coupled electron transfer (PCET) reactions involving antioxidants to gain insight into the detailed mechanisms of glutathione (GSH), Trolox, and α-tocopherol (α-TOH). PCET reactions are complex redox reactions that transfer electrons and protons sequentially or in concert. These reactions are ubiquitous in natural or artificial processes that produce electrochemical energy that is extractable as electricity or as chemical fuels of high energy content. Examples of processes based on PCET are photosynthesis, respiration, nitrogen fixation, carbon dioxide reduction, redox fuel cells, and artificial photosynthesis. Antioxidants were selected as a PCET model to understand the coupling between proton transfer (PT) and electron transfer (ET) in order to elucidate structure-reactivity relationships under different experimental conditions. PCET reactions were studied with a set of electrochemical techniques to propose a preliminary mechanism that could be validated with digital simulations matching the electrochemical response. In some cases, other analytical techniques were used to aid in the system characterization. This thesis presents the results and discussion of the effects of oxidant-base pairs on the mediated oxidation of GSH, the -2e-/-H+ process of Trolox in aqueous and nonaqueous solvents with various pH values, and the particle collision electrolysis of α-tocopherol in oil-in-water emulsion droplets on an ultramicroelectrode. Ultimately our goal was to determine the kinetic and thermodynamic factors that control PCET reactions so that they can be applied in designing artificial systems for the production of energy using more abundant reagents with lower cost and better yields.

Arachidonic acid (ARA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids required for proper embryonic development, specifically neurodevelopment. However, little is known regarding their conversion to other metabolites during embryogenesis. The oxidation of ARA gives rise to the biologically active eicosanoids and the oxidation of DHA gives rise to the biologically active docosanoids. The oxidation of ARA and DHA occurs through enzymatic processes, via lipoxygenase (LOX), or non-enzymatic processes, via radical-mediated lipid peroxidation. We hypothesize that oxidation of ARA and DHA via LOX is required for proper embryonic development. Additionally, we hypothesize that α-tocopherol, a potent lipid soluble antioxidant, mediates the conversion of ARA and DHA to their respective oxidized metabolites. Using zebrafish as a model of vertebrate embryogenesis, we found that the selective knockdown of either 12-LOX or 5-LOX decreased the production of docosanoids, altered fatty acid homeostasis, and increased the incidence of malformations and mortality in embryos by 24 hours post fertilization. α-Tocopherol deficiency also increased the incidence of malformations and mortality during embryogenesis, and in its absence, increased oxidized metabolites of ARA and DHA and decreased fatty acids concentrations. Therefore, oxidized metabolites of ARA and DHA perform crucial functions during embryonic development, but the production of oxidized fatty acids must be balanced with antioxidant bioavailability for proper embryogenesis. / Graduation date: 2012

For more than 80 years vitamin E has been in the focus of scientific research. Most of the progress concerning non-antioxidant functions, nevertheless, has only arisen from publications during the last decade.<br> Most recently, the metabolic pathway of vitamin E has been almost completely elucidated. Vitamin E is metabolized by truncation of its side chain. The initial step of an omega-hydroxylation is carried out by cytochromes P450 (CYPs). This was evidenced by the inhibition of the metabolism of alpha-tocopherol by ketoconozole, an inhibitor of CYP3A expression, whereas rifampicin, an inducer of CYP3A expression increased the metabolism of alpha-tocopherol. Although the degradation pathway is identical for all tocopherols and tocotrienols, there is a marked difference in the amount of the release of metabolites from the individual vitamin E forms in cell culture as well as in experimental animals and in humans. Recent findings not only proposed an CYP3A4-mediated degradation of vitamin E but also suggested an induction of the metabolizing enzymes by vitamin E itself.<br> In order to investigate how vitamin E is able to influence the expression of metabolizing enzymes like CYP3A4, a pregnane X receptor (PXR)-based reporter gene assay was chosen. PXR is a nuclear receptor which regulates the transcription of genes, e.g., CYP3A4, by binding to specific DNA response elements. And indeed, as shown here, vitamin E is able to influence the expression of CYP3A via PXR in an in vitro reporter gene assay. Tocotrienols showed the highest activity followed by delta- and alpha-tocopherol. An up-regulation of Cyp3a11 mRNA, the murine homolog of the human CYP3A4, could also be confirmed in an animal experiment. The PXR-mediated change in gene expression displayed the first evidence of a direct transcriptional activity of vitamin E. PXR regulates the expression of genes involved in xenobiotic detoxification, including oxidation, conjugation, and transport. CYP3A, e.g., is involved in the oxidative metabolism of numerous currently used drugs. This opens a discussion of possible side effects of vitamin E, but the extent to which supranutritional doses of vitamin E modulate these pathways in humans has yet to be determined. <br><br> Additionally, as there is arising evidence that vitamin E's essentiality is more likely to be based on gene regulation than on antioxidant functions, it appeared necessary to further investigate the ability of vitamin E to influence gene expression. Mice were divided in three groups with diets (i) deficient in alpha-tocopherol, (ii) adequate in alpha-tocopherol supply and (iii) with a supranutritional dosage of alpha-tocopherol. After three months, half of each group was supplemented via a gastric tube with a supranutritional dosage of gamma-tocotrienol per day for 7 days. Livers were analyzed for vitamin E content and liver RNA was prepared for hybridization using cDNA array and oligonucleotide array technology. A significant change in gene expression was observed by alpha-tocopherol but not by gamma-tocotrienol and only using the oligonucleotide array but not using the cDNA array. The latter effect is most probably due to the limited number of genes represented on a cDNA array, the lacking gamma-tocotrienol effect is obviously caused by a rapid degradation, which might prevent bioefficacy of gamma-tocotrienol.<br> Alpha-tocopherol changed the expression of various genes. The most striking observation was an up-regulation of genes, which code for proteins involved in synaptic transmitter release and calcium signal transduction. Synapsin, synaptotagmin, synaptophysin, synaptobrevin, RAB3A, complexin 1, Snap25, ionotropic glutamate receptors (alpha 2 and zeta 1) were shown to be up-regulated in the supranutritional group compared to the deficient group. The up-regulation of synaptic genes shown in this work are not only supported by the strong concentration of genes which all are involved in the process of vesicular transport of neurotransmitters, but were also confirmed by a recent publication. However, a confirmation by real time PCR in neuronal tissue like brain is now required to explain the effect of vitamin E on neurological functionality. The change in expression of genes coding for synaptic proteins by vitamin E is of principal interest thus far, since the only human disease directly originating from an inadequate vitamin E status is ataxia with isolated vitamin E deficiency. Therefore, with the results of this work, an explanation for the observed neurological symptoms associated with vitamin E deficiency can be presented for the first time. / Chemisch handelt es sich bei Vitamin E um acht lipophile Derivate des 6 Chromanols mit einer Seitenkette. Nach dem Sättigungsgrad der Seitenkette lassen sich die Derivate in die Tocopherole (gesättigte Seitenkette) und die Tocotrienole (ungesättigte Seitenkette mit drei Doppelbindungen) einteilen. Entsprechend der Methylierung des Chromanrings lassen sie sich in alpha-, beta-, gamma- und delta-Tocopherol, bzw. Tocotrienol unterscheiden. Davon besitzt alpha-Tocopherol, das gleichzeitig die im Plasma dominierende Form darstellt, die höchste biologische Aktivität. Aufnahme wie auch der Transport von Vitamin E im Körper sind vergleichsweise gut erforscht. Die Kenntnisse zu Metabolismus und Elimination waren jedoch bis vor kurzem sehr lückenhaft. Lange Zeit waren nur Vitamin E-Metabolite mit geöffnetem Chromanring, die sogenannten Simon-Metabolite Tocopheronsäure und Tocopheronolacton bekannt. Diese Metabolite können nur aus oxidativ gespaltenem Vitamin E entstehen und galten daher auch als Beweis für die antioxidative Wirkung von Vitamin E. Mit verbesserter Analytik wurde vor einigen Jahren gezeigt, dass die Simon-Metabolite größtenteils Isolierungsartefakte sind. Stattdessen wurden Metabolite mit intaktem Chromanring identifiziert. Tocopherole wie auch Tocotrienole werden im Körper durch eine Verkürzung der Seitenkette abgebaut. Die Endprodukte sind in jedem Fall CEHCs (Carboxyethyl Hydroxychromane). Die Seitenkettenverkürzung startet mit einer omega-Hydroxylierung gefolgt von 5 Schritten &#61472;beta-Oxidation. Die omega Hydroxylierung der Seitenkette durch Cytochrom P450 (CYP) Enzyme wurde indirekt bestätigt. CYP3A4 gilt dabei als eines der wahrscheinlichsten Enzyme im Abbau von Vitamin E, die Beteiligung weiterer CYPs wird jedoch gleichfalls angenommen. Auffällig ist, dass nicht alle Vitamin E-Formen in gleichem Ausmaß abgebaut werden. Die Ausscheidung von CEHCs aus alpha-Tocopherol ist, verglichen zu andern Vitamin E-Formen, in kultivierten Zellen wie auch in vivo sehr gering. Die Art der Seitenkettenverkürzung von Vitamin E spricht für einen Abbau über das Fremdstoff-metabolisierende System, welches auch eine Vielzahl von Medikamenten verstoffwechselt.<br> Im ersten Teil der vorliegenden Arbeit konnte mittels Reportergenassay in HepG2 Zellen gezeigt werden, dass Vitamin E einen nukleären Rezeptor, den Pregnan X Rezeptor (PXR), zu aktivieren und die Expression von PXR-regulierten Genen zu beeinflussen vermag. PXR reguliert eine Reihe von Genen für Fremdstoff-metabolisierende Enzyme wie z.B. Cytochrom P450 3A4 durch Bindung an sein responsives Element im Promotor der Zielgene. Die untersuchten Vitamin E-Formen unterschieden sich deutlich hinsichtlich ihrer PXR-Aktivierung. Die Tocotrienole zeigten die höchste PXR-Aktivierung - vergleichbar mit Rifampicin, einem bekannt guten PXR-Aktivator - gefolgt von delta / alpha- und gamma-Tocopherol. Im Tierversuch an Mäusen konnte die erhöhte Expression von Cyp3a11, dem Homolog des humanen CYP3A4 in Abhängigkeit von der alpha-Tocopherol-Zufuhr bestätigt werden. Somit konnte erstmals gezeigt werden, dass Vitamin E die Expression von Genen direkt beeinflussen kann. Darüber hinaus unterstreicht diese Beobachtung die Möglichkeit einer Wechselwirkung von pharmakologischen Dosen Vitamin E mit dem Abbau von Medikamenten. Eine genregulatorische Funktion von Vitamin E ist auf den ersten Blick überraschend. Denn wenngleich Vitamin E vor über 80 Jahren als Fertilitätsfaktor bei Ratten entdeckt wurde, steht die erst später beschriebene antioxidative Eigenschaft von Vitamin E bis heute im Fokus der meisten Publikationen. Die molekularen Mechanismen der Essentialität von Vitamin E wurden dagegen wenig untersucht. Erst in den letzten Jahren finden Funktionen von Vitamin E Interesse, die über seine antioxidative Wirkung hinausgehen. Dabei konnte gezeigt werden, dass Vitamin E in vitro die Expression von Genen wie dem Scavenger Rezeptor CD36, dem Connective Tissue Growth Factor oder dem Peroxisomen-Proliferator aktivierten Rezeptor gamma beeinflussen kann.<br> Um weitere Zielgene von Vitamin E in vivo identifizieren zu können, wurden im zweiten Teil der vorliegenden Arbeit Mäuse in drei Fütterungsgruppen mit einer a) defizientem b) adäquatem sowie c) mit einer supranutritiven alpha Tocopherol-Versorgung über 3 Monate gefüttert. Zusätzlich erhielt die Hälfte der Tiere aus jeder Gruppe während der letzten Lebenswoche eine supranutritive Dosis gamma-Tocotrienol pro Tag. Aus den Lebern der Tiere wurde die RNA präpariert und die differentielle Genexpression mittels a) cDNA und b) Oligonukleotide enthaltenden GenChips analysiert.<br> Eine signifikante Änderung in der Genexpression zwischen den verschiedenen Fütterungsgruppen fand sich jedoch nur in den Analysen der Oligonukleotid GenChips. Dies kann auf die begrenzte Anzahl von Genen zurückzuführen sein, die auf den cDNA GenChips repräsentiert waren. Auch ein signifikanter Effekt von gamma-Tocotrienol auf die Genexpression konnte nicht beobachtet werden. Wahrscheinlich ist die hohe Ausscheidung von gamma-CEHC, dem Abbauprodukt von gamma-Tocotrienol, die im Urin der Tiere gemessen wurde und die damit womöglich verringerte Bioverfügbarkeit von gamma-Tocotrienol dafür verantwortlich.<br> Mit Hilfe der Oligonukleotid GenChips konnte jedoch ein signifikanter Effekt von alpha-Tocopherol auf die Expression einer Vielzahl von Genen beobachtet werden. Herausstechend war dabei die erhöhte Expression von für den vesikulären Transport essentiellen Genen, die für den synaptischen Signaltransfer benötigt werden. So wurden z.B. Synapsin, Synaptotagmin, Synaptophysin, Synaptobrevin, RAB3A, Complexin 1, Snap25, die ionotrophen Glutamat Rezeptoren alpha 2 und zeta 1 in Abhängigkeit von der alpha Tocopherol-Versorgung über die Diät erhöht exprimiert. Die Beobachtung, dass Vitamin E bei neurologischen Prozessen eine Rolle zu spielen scheint ist jedoch nicht neu. Bei Patienten mit einem Mangel an funktionellem alpha-Tocopherol-Transfer-Protein (alpha-TTP) kann es zu stark verringerten Plasmakonzentrationen an Vitamin E kommen, da alpha-TTP eine zentrale Rolle in der Aufnahme und Verteilung von Vitamin E im Körper einnimmt. An diesen Patienten können charakteristische Vitamin E-Mangelzustände beobachtet, die durch eine Reihe von neurologischen Störungen wie Ataxien, Hyporeflexie sowie eine verringerte propriozeptive und vibratorische Sensitivität gekennzeichnet sind. Mit den vorliegenden Ergebnissen kann nun erstmals eine mechanistische Erklärung für diese Symptome diskutiert werden. Eine Bestätigung der vorliegenden Ergebnisse via RT-PCR und Western Blot, z.B. in neuronalem Gewebe wie dem Gehirn, sowie anschließende funktionellen Untersuchungen ist daher dringend geboten.

Vitamin E wird immer noch als das wichtigste lipophile Antioxidanz in biologischen Membranen betrachtet. In den letzten Jahren hat sich jedoch der Schwerpunkt der Vitamin E-Forschung hin zu den nicht-antioxidativen Funktionen verlagert. Besonderes Interesse gilt dabei dem α-Tocopherol, der häufigsten Vitamin E-Form im Gewebe von Säugetieren, und seiner Rolle bei der Regulation der Genexpression. Das Ziel dieser Dissertation war die Untersuchung der genregulatorischen Funktionen von α-Tocoperol und die Identifizierung α-Tocopherol-sensitiver Gene in vivo. Zu diesem Zweck wurden Mäuse mit verschiedenen Mengen α-Tocopherol gefüttert. Die Analyse der hepatischen Genexpression mit Hilfe von DNA-Microarrays identifizierte 387 α-Tocopherol-sensitive Gene. Funktionelle Clusteranalysen der differentiell exprimierten Gene zeigten einen Einfluss von α-Tocooherol auf zelluläre Transportprozesse. Besonders solche Gene, die an vesikulären Transportvorgängen beteiligt sind, wurden größtenteils durch α-Tocopherol hochreguliert. Für Syntaxin 1C, Vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and Syntaxin binding protein 1 konnte eine erhöhte Expression mittels real time PCR bestätigt werden. Ein funktioneller Einfluss von α-Tocopherol auf vesikuläre Transportprozesse konnte mit Hilfe des in vitro β-Hexosaminidase Assays in der sekretorischen Mastzelllinie RBL-2H3 gezeigt werden. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer konzentrationsabhängigen Erhöhung der PMA/Ionomycin-stimulierten Sekretion der β-Hexosaminidase. Eine erhöhte Expression ausgewählter Gene, die an der Degranulation beteiligt sind, konnte nicht beobachtet werden. Damit schien ein direkter genregulatorischer Effekt von α-Tocopherol eher unwahrscheinlich. Da eine erhöhte Sekretion auch mit β-Tocopherol aber nicht mit Trolox, einem hydrophilen Vitamin E-Analogon, gefunden wurde, wurde vermutet, dass α-Tocopherol die Degranulation möglicherweise durch seine membranständige Lokalisation beeinflussen könnte. Die Inkubation der Zellen mit α-Tocopherol resultierte in einer veränderten Verteilung des Gangliosids GM1, einem Lipid raft Marker. Es wird angenommen, dass diese Membranmikrodomänen als Plattformen für Signaltransduktionsvorgänge fungieren. Ein möglicher Einfluss von Vitamin E auf die Rekrutierung/Translokation von Signalproteinen in Membranmikrodomänen könnte die beobachteten Effekte erklären. Eine Rolle von α-Tocopherol im vesikulären Transport könnte nicht nur seine eigene Absorption und seinen Transport beeinflussen, sondern auch eine Erklärung für die bei schwerer Vitamin E-Defizienz auftretenden neuronalen Dysfunktionen bieten. Im zweiten Teil der Arbeit wurde die α-Tocopheroltransferprotein (Ttpa) Knockout-Maus als genetisches Modell für Vitamin E-Defizienz verwendet, um den Effekt von Ttpa auf die Genexpression und die Gewebeverteilung von α-Tocopherol zu analysieren. Ttpa ist ein cytosolisches Protein, das für die selektive Retention von α-Tocopherol in der Leber verantwortlich ist. Die Ttpa-Defizienz resultierte in sehr geringen α-Tocopherol-Konzentrationen im Plasma und den extrahepatischen Geweben. Die Analyse der α-Tocopherol-Gehalte im Gehirn wies auf eine Rolle von Ttpa bei der α-Tocopherol-Aufnahme ins Gehirn hin. / Vitamin E is still considered the most important lipid-soluble antioxidant within biological membranes. However, in the last years the non-antioxidant functions of vitamin E have become the focus of vitamin E research. From the eight members of the vitamin E family, specific emphasis is given to α-tocopherol, the most abundant vitamin E form in mammalian tissues, and its role in the regulation of gene expression. The aim of this thesis was the analysis of the gene regulatory functions of α-tocopherol and the identification of α-tocopherol sensitive genes in vivo. For this purpose mice were fed diets differing in α-tocopherol content. The analysis of hepatic gene expression using DNA microarrays identified 387 α-tocopherol-sensitive genes. Functional cluster analyses of these differentially expressed genes demonstrated an influence of α-tocopherol on cellular transport processes. Especially the expression of genes involved in vesicular trafficking was largely upregulated by α-tocopherol. Upregulation of syntaxin 1C, vesicle-associated membrane protein 1, N-ethylmaleimide-sensitive factor and syntaxin binding protein 1 was verified by real time PCR. A role of α-tocopherol in exocytosis was shown by the in vitro β-hexosaminidase release assay in the secretory mast cell line RBL-2H3. Incubation with α-tocopherol resulted in a concentration dependent increase of PMA/ionomycin-stimulated secretion of β-hexosaminidase. Induction of selected genes involved in degranulation was not observed at any time point. Thus, a direct gene-regulatory effect of α-tocopherol seemed rather unlikely. Since increased secretion was also observed with ß-tocopherol but not with trolox, a water-soluble analog of vitamin E, it was hypothesized that α-tocopherol might affect degranulation through its localization at the plasma membrane. Incubation of cells with α-tocopherol changed the distribution of the gangliosid GM1, a Lipid raft marker. These membrane microdomains are assumed to function as signaling platforms. An possible influence of vitamin E on the recruitment/translocation of signaling proteins into membrane microdomains could explain the observed effects. A role of α-tocopherol in the vesicular transport might not only affect its own absorption and transport but also explain the neural dysfunctions observed in severe α-tocopherol deficiency. In the second part of this dissertation the α-tocopherol transfer protein (Ttpa) knockout-mouse as a model of genetic vitamin E deficiency was used to analyze the effect of Ttpa gene expression and tissue distribution of α-tocopherol. Ttpa is a cytosolic protein, which is responsible for the selective retention of α-tocopherol in the liver. Its deficiency resulted in very low α-tocopherol concentrations in plasma and extrahepatic tissues. Analysis of α-tocopherol contents in brain indicated a role for Ttpa in the uptake of α-tocopherol into the brain.

Diet and oral supplements comprise two distinct sources of antioxidants known to prevent oxidative stress. Beneficial effects from antioxidants have been seen for patients at risk for type 2 diabetes. The aim of this thesis was to evaluate the positive effects of antioxidants against oxidative stress and DNA damage in type 2 diabetes subjects. We also used antioxidants as tools to determine the mechanisms behind genotoxicity induced by mutagenic pro-oxidative agents in mouse lymphoma cells. Several techniques were used to measure oxidative stress and DNA damage, but the main technique used was alkaline comet assay. The results showed that the fruit and vegetable intake was inversely related to oxidative stress in type 2 diabetes subjects. However, oral supplementary intake of 20 antioxidants did not decrease oxidative stress biomarkers. In studies on mouse lymphoma cells, using the alkaline comet assay, DNA damage was induced by catechol and o-phenylenediamine (OPD), while 4-nitro-o-phenylenediamine (4-NOPD) induced only oxidative damage, showing different mechanisms of action behind the mutagenicity of the compounds. Also, oxidative stress was induced by catechol and 4-NOPD, whereas imbalances in the nucleotide pool were seen after exposure to OPD or 4-NOPD. Addition of antioxidants together with these pro-oxidants showed that β-carotene was able to reduce DNA damage at low concentrations of catechol, but increased DNA damage at high concentration. In comparison, addition of α-tocopherol slightly decreased catechol-induced DNA damage at all concentrations of catechol. However, no effect of α-tocopherol was seen on OPD-or 4-NOPD-induced DNA damage. In conclusion, antioxidants from fruits and vegetables, but not from oral supplements, reduced oxidative stress in type 2 diabetes patients, suggesting fruits and vegetables being a healthier source for antioxidant-intake, as compared to oral supplements. Different mechanisms of action for mutagenic pro-oxidants were shown in mouse lymphoma cells, introducing the nucleotide pool as an interesting target for oxidative stress. Reduction of catechol-induced DNA damage by β-carotene or α-tocopherol was shown, with a pro-oxidative action of β-carotene at high concentration of catechol, Interestingly, α-tocopherol was not able to decrease OPD- or 4-NOPD-induced DNA damage, supporting different mechanisms of action behind the genotoxicity from the three pro-oxidants.

Made available in DSpace on 2015-07-22T20:49:58Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-30. Added 1 bitstream(s) on 2015-08-11T03:25:27Z : No. of bitstreams: 1 Publico-00264.pdf: 1668171 bytes, checksum: dbdf365617164c11a9a209e69d18f997 (MD5) / O ƒÑ-tocoferol tem sido o foco das pesquisas dentre os demais componentes da vitamina E por ser a forma predominantemente encontrada nos tecidos de mamiferos, e por possuir uma extensa gama de atividades biologicas. O ƒÑ-tocoferol pode atuar como regulador de enzimas especificas e de fatores de transcricao de forma a influenciar estruturas celulares como membranas e dominios lipidicos, desencadeando respostas celulares que muitas vezes se mostram independentes de sua funcao antioxidante. No sistema hematopoetico, seus efeitos foram favoraveis em casos de anemias hemoliticas, aumentando a resistencia dos eritrocitos a lise. Tambem foi observado que a suplementacao previa com ƒÑ-tocoferol a irradiacao resulta em aumento da sobrevida de camundongos por induzir aumento no numero de unidades formadoras de colonias (CFUs). Entretanto, os mecanismos biologicos ativados pelo ƒÑ-tocoferol nas celulas hematopoeticas ainda nao foram descritos. Assim, os bjetivos deste trabalho foram verificar os efeitos do ƒÑ-tocoferol na hematopoese murina e os mecanismos intracelulares relacionados a estes efeitos. Para tal, camundongos foram tratados intraperitonealmente com doses de 40 mg/kg/dia de ƒÑ-tocoferol durante 2 semanasem dias intercalados, sendo sacrificados 24 horas apos a ultima dose; condicoes estas que nao causaram toxicidade as celulas da medula ossea. Amostras histologicas dos femures dos animais que receberam o tratamento com ƒÑ-tocoferol apresentaram hiperplasia medular. O tratamento com ƒÑ-tocoferol induziu aumento na porcentagem das celulas progenitoras hematopoeticas (Lin-Sca-1+c-Kit+ e Lin-Sca-1-c-Kit+), assim como o aumento do estado proliferativo destas populacoes (com mais celulas primitivas na fase S/G2/M do ciclo celular), com o consequente aumento da capacidade de formar CFUs de granulocitos e macrofagos. Dentre as distintas populacoes de celulas maduras da medula ossea, houve um favorecimento da linhagem granulocitica/monocitica (Mac-1+Gr-1+) em detrimento das linhagens eritrociticas (Ter119+) e linfociticas (B220+ e CD3+). Como forma de avaliar o mecanismo de acao do ƒÑ-tocoferol, investigou-se tambem a ativacao das proteinas relacionadas com a sinalizacao das celulas hematopoeticas. Assim, observou-se que as populacoes primitivas medulares apresentaram uma menor ativacao da cinase regulada por sinais extracelulares 1/2 (ERK1/2), da proteina cinase C (PKC), do ¡§ativador de transcricao e transdutor de sinal ¡V 5¡§ (STAT-5), mas nao da proteina cinase B/Akt. Tambem foi verificado que a diminuicao do estado fosforilado da ERK1/2 ocorreu desde os primeiros dias de tratamento. Interessante destacar quer o ƒÑ-tocoferol potencializou o efeito da interleucina-3 (IL-3) sobre a ativacao da ativacao da ERK1/2 nas celulas primitivas hematopoeticas. O inibidor da MEK (PD98059) foi capaz de restabelecer as porcentagens normais das linhagens eritrocitica e granulocitica/monocitica, assim como os niveis normais da fosfo- ERK1/2, alem da resposta da ERK1/2 ao estimulo com IL-3. Entretanto, o PD98059 nao restabeleceu as porcentagens normais das celulas primitivas hematopoeticas, nem da linhagem linfocitica. A quantificacao das especies reativas de oxigenio nas diferentes populacoes da medula ossea mostrou que, nas condicoes de tratamento estabelecidas, o ƒÑ-tocoferol nao exerceu funcao proou antioxidante, pois nao houve alteracao significativa dos niveis de especies reativas de oxigenio entre os grupos controle e tratado com ƒÑ-tocoferol. Desta forma, foi mostrada uma nova propriedade do ƒÑ-tocoferol independente de sua acao redox: a inducao de hiperplasia na medula ossea pelo aumento dos progenitores hematopoeticos e favorecimento da diferenciacao destes em granulocitos e macrofagos, pela potencializacao da resposta da ERK1/2 ao estimulo com IL-3. / TEDE / BV UNIFESP: Teses e dissertações

Made available in DSpace on 2014-12-17T14:03:42Z (GMT). No. of bitstreams: 1 HeleniAC_DISSERT.pdf: 2512013 bytes, checksum: e69e375e25ebb38b43385a02d5ecc6c7 (MD5) Previous issue date: 2013-06-10 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The Vitamin E consists of eight chemically homologous forms, designated alpha, beta, gamma and delta tocopherols and tocotrienols. Biologically, the alpha-tocopherol (&#945;-TOH) is the most important. Commercially, are found two types of &#945;-TOH a natural (RRR-alpha-tocopherol) and another synthetic (all-rac-alpha-tocopherol). Both forms are absorbed in the intestine, the liver is a preference in favor of forms 2R, due to transfer protein &#945;-TOH. It has higher affinity to these stereoisomers. Newborns are considered high risk for vitamin E deficiency, mainly premature, these have breast milk as a food source for maintenance of serum &#945;-TOH. Clinical signs such as thrombocytosis, hemolytic anemia, retrolental fibroplasia, intraventricular hemorrhage, bronchopulmonary dysplasia and spinocerebellar degeneration can be found in case of a low intake of &#945;-TOH. Thus, maternal supplementation on postpartum with &#945;-TOH can be an efficient way to increase levels of vitamin E in breast milk and thus the consequently increase the supply of micronutrient for the newborn. However, most studies with vitamin E supplementation have been conducted in animals and little is known about the effect of maternal supplementation in humans, as well as on its efficiency to increase levels of &#945;-TOH in human milk, depending on the shape natural or synthetic. The study included 109 women, divided into three groups: control without supplementation (GC) (n=36), supplemented with natural capsule (GNAT) (n=40) and the synthetic capsule (GSINT) (n=33). Blood samples were collected for determination of maternal nutritional status, and colostrums at initial contact and after 24 hours post-supplementation. Analyses were performed by High Performance Liquid Chromatography. Values of &#945;-TOH in serum below 499.6mg/dL were considered deficient. We used the Kruskal-Wallis test and Tukey test to confirm the increase of alpha-tocopherol in milk and efficiency of administered capsules. Daily consumption of &#945;-TOH was based on daily intake of 500 mL of colostrum by the newborn and compared with the nutritional requirement for children from 0 to 6 months of age, 4 mg / day. The mothers had mean concentration of serum &#945;-TOH in 1016 ? 52, 1236 ? 51 and 1083 ? 61 mg / dL, in CG, GNAT and GSINT respectively. There were no women with deficiiency. The GC did not change the concentrations of &#945;-TOH in colostrum. While women supplemented with natural and synthetic forms increased concentrations of &#945;-TOH colostrum in 57.6% and 39%, respectively. By comparing supplemented groups, it was observed a significant difference (p=0.04), the natural capsule more efficient than the synthetic, approximately 49.6%. Individually, 21.1% of the women provided below 4mg/day of &#945;-TOH, after supplementation for this index declined4.1%. Thus, maternal supplementation postpartum raised the levels of alpha-tocopherol in colostrum, and increased efficiency was observed with the natural form / A vitamina E consiste em oito formas quimicamente hom?logas, denominadas, alfa, beta, gama e delta, tocofer?is e tocotrien?is. Biologicamente, o alfa-tocoferol (&#945;-TOH) ? o mais importante. Comercialmente s?o encontradas duas formas de &#945;-TOH, uma natural (RRR-alfa-tocoferol) e outra sint?tica (all-rac-alfa-tocoferol). Ambas as formas s?o absorvidas no intestino, entretanto, no f?gado ocorre uma prefer?ncia em favor das formas 2R, devido ? prote?na de transfer?ncia de &#945;-TOH, apresentar maior afinidade a estes estereois?meros. Os neonatos s?o considerados grupo de risco para defici?ncia de vitamina E, principalmente os pr?-maturos, estes t?m o leite materno como a fonte alimentar para manuten??o dos n?veis s?ricos de &#945;-TOH. Sinais cl?nicos como trombocitose, anemia hemol?tica, fibroplasia retrolental, hemorragia intraventricular, displasia bronco pulmonar e degenera??o espinocerebelar podem ser encontrados caso ocorra uma baixa ingest?o de &#945;-TOH. Sendo assim, a suplementa??o materna nos p?s-parto com &#945;-TOH pode ser uma forma eficiente de aumentar os n?veis de vitamina E no leite materno e, consequentemente aumento no fornecimento do micronutriente para o rec?m-nascido. Entretanto, a maioria dos estudos com suplementa??o de vitamina E foram realizados em animais e s?o escassos os conhecimentos de sua suplementa??o em humanos, bem como, sobre sua efici?ncia para aumentar os n?veis de &#945;-TOH no leite humano, em fun??o da forma natural ou sint?tica. Participaram do estudo 109 mulheres, distribu?das em tr?s grupos: controle sem suplementa??o (GC) (n=36), suplementadas com a c?psula natural (GNAT) (n=40) e com a c?psula sint?tica (GSINT) (n=33). Foram coletadas amostras de sangue para determina??o do estado nutricional materno, e de colostro no contato inicial e ap?s 24 horas p?s-suplementa??o. As an?lises foram realizadas por Cromatografia L?quida de Alta Efici?ncia. Valores de &#945;-TOH no soro inferiores a 499,6 &#956;g/dL foram considerados como deficientes. Foram utilizados o teste de Kruskal Wallis e teste de Tukey para confirmar o aumento de alfa-tocoferol no leite e a efici?ncia das c?psulas administradas. O consumo di?rio de &#945;-TOH foi baseado na ingest?o di?ria de 500 mL de colostro pelo rec?m-nascido e comparada com o requerimento nutricional para crian?as de 0 ? 6 meses de idade, 4 mg/dia. As parturientes apresentaram concentra??o m?dia de &#945;-TOH no soro de 1016 ? 52, 1236 ? 51 e 1083 ? 61 &#956;g/dL, nos grupos GC, GNat e GSINT, respectivamente. N?o foram encontradas mulheres com defici?ncia. O GC n?o apresentou varia??o nas concentra??es de &#945;-TOH no colostro. Enquanto mullheres suplementadas com as formas natural e sint?tica aumentaram as concentra??es de &#945;-TOH no colostro em 57,6% e 39%, respectivamente. Ao comparar os grupos suplementados foi observado uma diferen?a significativa (p=0,04), sendo a c?psula natural mais eficiente que a sint?tica em aproximadamente 49,6%. Individualmente 21,1% das mulheres forneceram valores inferiores as 4mg/dia de &#945;-TOH, ap?s a suplementa??o este ?ndice declinou para 4,1%. Sendo assim, a suplementa??o materna no p?s-parto elevou os n?veis de alfa-tocoferol no colostro e maior efici?ncia foi observada com a forma natural

Submitted by (edna.saturno@ufrpe.br) on 2017-05-17T13:15:12Z No. of bitstreams: 1 Marcos Jose Batista dos Santos.pdf: 1103645 bytes, checksum: 5cac226fa34979ad4d0f47844cb516c6 (MD5) / Made available in DSpace on 2017-05-17T13:15:12Z (GMT). No. of bitstreams: 1 Marcos Jose Batista dos Santos.pdf: 1103645 bytes, checksum: 5cac226fa34979ad4d0f47844cb516c6 (MD5) Previous issue date: 2013-02-25 / This thesis was proposed in order to: verify the performance and egg quality of japanese quails supplemented with selenium (Se) and vitamin E, investigate the performance, egg quality and modeling the deposition of selenium (Se) and vitamin E in eggs of Japanese quails supplemented with these nutrients, evaluate by meta-analysis, supplementation (Se) chelated and non-chelated, evaluating the deposition in eggs, organs and performance of laying hens supplemented with these nutrients. At first objective 200 quails were used with 45 days of life, the experimental diets were supplemented with selenomethionine and vitamin E acetate, dl-α-tocopherol in the proportion of 0.2 ppm Se, 20 mg/kg vitamin E, 0.2 ppm up to 20 mg/kg vitamin E and basal diet (RR). In Objective 2, 600 quails were housed at 45 days of life in cages, four functions were adjusted for the deposition of Se in eggs, the treatments were: (RR) RR + 0.1 ppm Se + 20 mg/kg vitamin E (Se1) RR + 0.2 ppm Se + 20 mg/kg vitamin E (SE2), RR + 0.3 ppm Se 20 mg/kg vitamin E (SE3), RR + 0.4 ppm Se 20 mg/kg vitamin E (SE4). For objective 3 Data were composed of eight articles published between 2004 and 2010. If the levels of chelated and non-chelated included in the analysis were in the range of 0.15 to 0.9 ppm of this mineral in the diet Meta-analysis was performed by three sequential analyzes: correlation, graphical and variance - covariance. The conclusions reached were: Se supplementation and vitamin E is effective in increasing the concentration of these nutrients in the yolk and albumen of eggs of Japanese quail, but not influence the deposition of Se in the egg shell. Se supplementation and vitamin E did not influence the performance of quails, but influence the variables of quality. The models that best describes the deposition of Se in eggs and the shell is sigmoidal logistic and with 0.29 and 0.33 ppm for maximum deposition If the eggs respectively. The best model for deposition of Vitamin E is the requirement with sigmoidal 0.36 ppm Se for maximum deposition of vitamin E in eggs. The association of Se and vitamin E did not affect the production variables, but the variables of the quality of the eggs was influencing . The supplementation of the Se chelated and non-chelated influence the deposition in hens eggs, the laying rate and egg weight, however, does not influence the deposition of this nutrient in organs. The chelated form of Se is more effective deposition of eggs and has a greater rate of laying. / Objetivou-se com esta pesquisa verificar o desempenho e a qualidade dos ovos de codornas japonesas suplementadas com selênio e vitamina E; averiguar o desempenho, qualidade dos ovos e modelar a deposição de selênio e vitamina E nos ovos de codornas japonesas suplementadas com esses nutrientes; avaliar por metaanálise, a suplementação de selênio quelatado e não-quelatado, avaliando o desempenho de poedeiras comerciais, a qualidades interna e externa dos ovos e a deposição desses nutrientes nos ovos e órgãos. No primeiro experimento utilizou-se 200 codornas com 45 dias de idade distribuídas de acordo com um delineamento inteiramente casualizado, as rações experimentais foram suplementadas com Sequelatado e vitamina E acetato de dl-α-tocoferol na proporção de 0,2 ppm Se, 20 mg/kg vitamina E, 0,2 ppm Se e 20 mg/kg de vitamina E e Ração Referência (RR). No objetivo dois foram alojadas 250 codornas com 45 dias de vida em gaiolas, foram ajustados quatro funções matemáticas a resposta de deposição de Se nos ovos, os tratamentos foram: (RR); RR + 0,1 ppm de Se mais 20 mg/kg de DL-α- tocoferil (Se1), RR + 0,2 ppm de Se + 20 mg/kg de DL-α- tocoferil (Se2), RR + 0,3 ppm de Se mais 20 mg/kg de DL-α- tocoferil (Se3), RR + 0,4 ppm de Se mais 20 mg/kg de DL-α- tocoferil (Se4). No objetivo três os dados foram compostos por oito artigos publicados entre 2004 e 2010. Os níveis de selênio quelatado e não-quelatado incluídos na análise estavam no intervalo de 0,15 á 0,9 ppm desse mineral na dieta. A meta-análise foi realizada por três análises sequenciais: correlação, gráfica e variância - covariância. As conclusões obtidas foram: A suplementação de Se e vitamina E é eficaz para aumentar a concentração desses nutrientes na gema e albúmen dos ovos de codornas japonesas, mas não influenciam na deposição de Se na casca dos ovos. A suplementação de Se e Vitamina E não influenciam no desempenho das codornas, mas influenciam as variáveis de qualidade dos mesmos. Os modelos que melhor descrevem a deposição de Se nos ovos e na casca é o sigmoidal e o logístico com 0,29 e 0,33 ppm de Se para máxima deposição nos ovos respectivamente. O melhor modelo para deposição de vitamina E é o sigmoidal com exigência de 0,36 ppm de Se para deposição máxima de vitamina E nos ovos. A associação de Se e vitamina E não influenciam as variáveis de produção, mas influenciam as variáveis de qualidade dos ovos. A suplementação de selênio quelatado e não quelatado influenciam a deposição nos ovos, a taxa de postura, peso e massa dos ovos, porém não influenciam a deposição desse nutriente nos órgãos.

Made available in DSpace on 2014-07-29T15:07:45Z (GMT). No. of bitstreams: 1 Dissertacao Catia Ciencia Animal 2011.pdf: 762064 bytes, checksum: bf88768cc40c5c8bee991ff416b2a29d (MD5) Previous issue date: 2011-08-03 / The function of antioxidants is control the free radicals to improve the quality of fresh and cryopreserved semen. The objective this work was to study the effect of antioxidants catalase, &#945;-tocopherol and sodium pyruvate added in bull semen in order to control the reactive oxygen species and improve sperm quality after cryopreservation. Were used six adult Nelore bulls previously selected from other animals by evaluation of body condition and andrological examination. Semen was collected and immediately evaluated for motility and vigor at the laboratory. After, the semen were diluted with Tris-egg yolk-glycerol medium with the antioxidants molecules in different concentrations. The work was divided into four experimental groups: Group 1 (control without addition of antioxidants), Group 2 (with catalase antioxidant in three concentrations 20 IU, 80 IU and 200 IU), Group 3 (with &#945;-tocopherol antioxidant in the concentrations of 50 mM, 100 mM and 150 mM) and Group 4 (with sodium pyruvate antioxidant in the concentrations of 1.5 mM, 3.5 mM and 5 mM). After semen thawing the computerized analysis (CASA) of motility, plasm membrane, and acrosome integrity, mitochondrial activity and measurement of thiobarbituric acid reactive substances (TBARS). It was observed that the antioxidants had no significant effect on most parameters measured, but the lower antioxidants concentrations were more efficient than the control group for the total motility, progressive motility, average path velocity, and plasm membrane integrity parameters. In the TBARS evaluation was observed lower lipid peroxidation when higher antioxidants concentrations were used. Thus more research is needed to determine the functionality of sperm, as well as indentify the optimal concentration of the antioxidants that benefits all sperm parameters after thawing. / Os antioxidantes têm como função neutralizar os efeitos dos radicais livres proporcionando uma melhor qualidade do sêmen fresco e criopreservado. O objetivo deste trabalho foi estudar o efeito da adição dos antioxidantes catalase, &#945;-tocoferol e piruvato de sódio no sêmen bovino visando um controle das espécies reativas de oxigênio para melhorar a qualidade espermática pós-criopreservação. Foram utilizados seis touros adultos da raça Nelore selecionados previamente entre outros animais por meio da avaliação de condição corporal e exame andrológico. O sêmen foi coletado e imediatamente avaliado quanto a motilidade e vigor no laboratório. Em seguida, o sêmen foi diluído com o meio Tris-gema-glicerol com as moléculas antioxidantes nas diferentes concentrações. O trabalho foi estruturado em quatro grupos experimentais: Grupo1 (controle sem adição de antioxidantes); grupo 2 (com antioxidante catalase em três concentrações 20 UI, 80 UI e 200 UI); grupo 3 (com antioxidante &#945;-tocoferol nas concentrações de 50 &#956;M, 100 &#956;M e 150 &#956;M) e o grupo 4 (com antioxidante piruvato de sódio nas concentrações de 1.5 &#956;M, 3.5 &#956;M e 5 &#956;M). Após a descongelação foram realizadas as análises computadorizada (CASA) da motilidade, da integridade de membrana plasmática, de integridade acrossomal, da atividade mitocondrial e mensuração de substâncias reativas ao ácido tiobarbitúrico (TBARS). Observou-se que os antioxidantes não tiveram efeito significativo na maioria dos parâmetros avaliados, porém nas menores concentrações tiveram melhor resultado comparando-se com o grupo controle para os parâmetros de motilidade total, motilidade progressiva, velocidade de trajeto e integridade de membrana. Em relação a avaliação de TBARS observou-se que as maiores concentrações dos antioxidantes têm menor peroxidação lipídica. Desta forma, tornam-se necessárias mais pesquisas em relação a avaliação funcional dos espermatozóides, bem como a identificação da concentração ideal que beneficie todos os parâmetros espermáticos pós-descongelação.

Submitted by Franciele Moreira (francielemoreyra@gmail.com) on 2017-11-29T15:47:24Z No. of bitstreams: 2 Tese - Itallo Conrado Sousa de Araújo - 2017.pdf: 13043895 bytes, checksum: c3f16d3550fa6f7cec4898c3e9256066 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-11-30T10:22:35Z (GMT) No. of bitstreams: 2 Tese - Itallo Conrado Sousa de Araújo - 2017.pdf: 13043895 bytes, checksum: c3f16d3550fa6f7cec4898c3e9256066 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-11-30T10:22:35Z (GMT). No. of bitstreams: 2 Tese - Itallo Conrado Sousa de Araújo - 2017.pdf: 13043895 bytes, checksum: c3f16d3550fa6f7cec4898c3e9256066 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-11-09 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Two experiments were conducted to avaluate the effect of in ovo supplementation of antioxidants to broiler chickens on neonatal oxidative status, incubation results, chick quality and broiler performance. In the first experiment, five levels of vitamin E (0.0, 25.0, 35.0, 45.0 and 55.0 mg) were diluted in 0.5 mL of sunflower oil and in the second experiment five levels were used of cantaxanthin (0.000, 0.035, 0.045, 0.055 and 0.065 mg) obtained from a commercial product (canthaxanthin 10%), diluted with 0.5 mL of distilled water. In both experiments were used 780 eggs, distributed in three incubators (block), 260 eggs in each. Vitamin E supplementation improved egg hatchability, lower birth rate of chicks and better physical quality of chicks. There was also a positive response in the small intestine weight and villus height of the duodenum of the chicks, which provided improvement in feed conversion for all the periods studied during the performance. The results of protein concentration in the liver and striated muscle were higher for the chicks that received vitamin E. It was concluded that vitamin E supplementation in ovo improves the oxidative state of the chick and this improves the incubation results, quality of the chick and performance in the initial phase. On the other hand, supplementation with the commercial product of canthaxanthin showed worsening for the hatching and birth window variables, with a consequent increase in the number of neonatal chicks with physical quality below 71 points. Supplementation of canthaxanthin did not influence the weight or length of neonatal chicks. Furthermore, it was possible to verify a higher amount of total proteins in the liver of the chicks supplemented with the commercial product of canthaxanthin, it was also possible to verify improvement in the catalase activity present in the chicks liver. It can be concluded that the commercial product is not indicated for inoculation in ovo because it contains compounds that hinder chicks hatching. However, the improvement in oxidative status was evident, and further studies could be indicated with the use of pure canthaxanthin in ovo for broiler chickens. / Foram conduzidos dois experimentos com o objetivo de verificar o efeito da suplementação de agentes antioxidantes in ovo para frangos de corte sobre o estado oxidativo do neonato, os resultados de incubação, qualidade do pinto e desempenho inicial do frango. No primeiro experimento foram utilizados cinco níveis de vitamina E (0,0; 25,0; 35,0; 45,0 e 55,0 mg) diluídos em 0,5 mL de óleo de girassol e no segundo experimento foram utilizados cinco níveis de cantaxantina (0,000; 0,035; 0,045; 0,055 e 0,065 mg), obtida de um produto comercial (cantaxantina 10%), diluídos dem 0,5 mL de água destilada. Em ambos os experimentos foram utilizados 780 ovos, distribuídos em três incubadoras (bloco), sendo 260 ovos em cada uma. A suplementação de vitamina E promoveu melhora da eclosão dos ovos, menor janela de nascimento dos pintos e melhor qualidade física dos pintos. Também houve resposta positiva no peso do intestino delgado e altura de vilos do duodeno dos pintos o que proporcionou melhoria na conversão alimentar para todos os períodos estudados durante o desempenho. Os resultados de concentração de proteína no fígado e músculo estriado esquelético do peito foram superiores para os pintos que receberam a vitamina E. Conclui-se que a suplementação de vitamina E in ovo melhora o estado oxidativo do pinto e isso propicia melhora dos resultados de incubação, qualidade do pinto e desempenho na fase inicial. Já a suplementação com o produto comercial com cantaxantina demonstrou piora para as variáveis de eclosão e janela de nascimento, com consequente aumento do número de pintos neonatos com qualidade física abaixo de 71 pontos. A suplementação da cantaxantina não influenciou o peso ou o comprimento dos pintos neonatos. Ainda, foi possível verificar maior quantidade de proteínas totais no fígado dos pintos suplementados com o produto comercial de cantaxantina e também foi possível verificar melhora na atividade de catalase presente no fígado dos pintos. Pode-se concluir que o produto comercial não é indicado para a inoculação in ovo por conter compostos que prejudicaram a eclosão dos pintos. Entretanto, ficou evidente a melhoria no estado oxidativo, podendo assim ser indicado mais estudos com o uso da cantaxantina pura in ovo para frangos de corte. Palavras-chave:,Antioxidantes, Ovos férteis, Proteção oxidativa, Suplementação exógena