In this work we developed an electrochemical sensor modified with multi-walled carbon nanotubes and 5-nitroindole (5-NI) for the simultaneous determination of ascorbic (AA) and uric (UA) acids in urine and serum samples and a biosensor based on dsDNA for determination of 3-nitro-L-tyrosine ethyl ester (3NO2TEE), as a biomarker of biological peroxynitration. The polymer of 5-NI was electrogenerated in situ on the carbon nanotubes deposited on glassy carbon electrode (GCE). Thereafter, the aromatic nitro group, present in the molecule, was reduced, generating a hydroxylamine/nitroso redox couple, then used for detection and quantification of the analytes in the biological samples. The electrochemical behavior of the modified electrodes were studied using cyclic voltammetry, differential pulse voltammetry and chronoamperometry, which were used for the detection of the analytes and to obtain the kinetic parameters and the analytical characterization of the platforms. The chronoamperometric studies were performed in order to obtain more information about the redox processes between AA and the sensor, since it proved to be an electrocatalytic process. Thus, by means of graphs and Cottrell equations, it was possible to obtain values for the diffusion coefficient (DAA) and the catalytic constant (kcat) for the AA electrocatalytic oxidation. The values for DAA and kcat, determined for AA were 4.2 x10-6 cm-2 s-1 and 1.1 x 106 M-1 s-1, respectively. The platform for the detection of AA e AU showed good sensitivity and stability in urine and serum samples, with LOD of 1.9 mol L-1 for AA and 2.1 mol L-1 for UA. The analytical performance obtained for the nanostructured platform GCE/MWCNT/poly-5-NID justifies its use as a sensor for the simultaneous determination of AA and UA. Studies of 3NO2TEE in protic media (pH 4.5 acetate buffer and pH 7.4 and 10.0, in phosphate buffer) and in aprotic media (DMF/TBAPF6, 0.1 mol L-1), using Pt and glassy carbon electrodes, were necessary before the construction of the CT-DNA biosensor. The spectrolectrochemistry of 3NO2TEE, held in aprotic media was useful for rationalizing the electrochemical behavior of 3NO2TEE and intermediates generated during the reduction. For the biosensor construction, the amount of dsDNA, the conditioning time for reduction of the analyte, the pH value, on GCE, were optimized. The developed biosensor was used to determine the concentration of 3NO2TEE and showed a linear range from 60 to 320 nmol L-1 and LOD and LOQ of 58 nmol L-1 and 200 nmol L-1, respectively. These results indicate that the prepared sensor and biosensor were effective for the detection of substances with biological interest. (P.S.: some expressions out of formatting) / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Neste trabalho foram desenvolvidos um sensor eletroquímico modificado com nanotubos de carbono de paredes múltiplas (MWCNT) e 5-nitroindol (5-NI) para determinação simultânea dos ácidos ascórbico (AA) e úrico (AU), em amostras de urina e soro, e um biossensor à base de dsDNA para determinação do éster etílico da 3-nitro-L-tirosina (3NO2TEE), como biomarcador de peroxinitração biológica. O trímero do 5-nitroindol foi eletrogerado in situ sobre os nanotubos de carbono depositados em eletrodo de carbono vítreo (ECV). Após esse processo, o grupo nitro aromático, presente na molécula foi reduzido gerando o par redox hidroxilamina/nitroso, utilizado para detecção e quantificação dos analitos presentes nas amostras biológicas. Os comportamentos eletroquímicos dos eletrodos modificados foram estudados, empregando as técnicas de voltametria cíclica, voltametria de pulso diferencial e cronoamperometria, as quais foram utilizadas para a detecção dos analitos e para obtenção dos parâmetros cinéticos e caracterização analítica da plataforma. Os estudos cronoamperométricos foram realizados com o objetivo de obter maiores informações dos processos redox entre AA e o sensor, uma vez que este demonstrou ser um processo eletrocatalítico. Assim, por meio de gráficos e equações de Cottrell, foi possível obter os valores para o coeficiente de difusão (DAA) e a constante catalítica (kcat) da reação para o AA. Os valores do DAA e de kcat, determinados para AA, foram de 4,2 x10-6 cm-2 s-1 e 1,1 x 106 M-1 s-1, respectivamente. O método desenvolvido de detecção de AA e AU apresentou boa sensibilidade e estabilidade em amostras de urina e soro, com limite de detecção (LD) de 1,9 mol L-1 para AA e 2,1 mol L-1 para AU. A partir do desempenho analítico obtido da plataforma nanoestruturada ECV/MWCNT/poli-5-NID, justifica-se a sua utilização deste como sensor para a determinação simultânea de AA e AU. Antes da construção do biossensor de DNA, para nitroaromáticos de importância biológica, foi necessária a realização dos estudos da 3NO2TEE, em meios prótico: tampão acetato pH 4,5 e tampão fosfato pH 7,4 e 10,0 e, em meio aprótico (DMF/ TBAPF6, 0,1 mol L-1), utilizando eletrodo de Pt e carbono vítreo, como eletrodos de trabalho. A espectroeletroquímica de 3NO2TEE, realizada em meio aprótico, foi útil para racionalizar o comportamento eletroquímico de 3NO2TEE e de seus intermediários gerados durante a redução. Para a construção do biossensor, sobre o eletrodo de carbono vítreo, foram otimizados: concentração de dsDNA, tempo de condicionamento para a redução do nitrocomposto, valor de pH. O biossensor ECV/dsDNA apresentou faixa linear de 60 - 320 nmol L-1 e LD e LQ (limite de quantificação) encontrados foram 58 nmol L-1 e 200 nmol L-1. Estes resultados indicaram que o sensor e o biossensor foram produzidos e aplicados de forma eficaz para detecção de substâncias de interesse biológico. (obs.: algumas expressões fora da formatação)

A doença renal crônica é uma doença mediada pelo sistema imune e caracterizada por fibrose. Camundongos deficientes em TLR2, TLR4, MyD88 e NLRP3 se mostraram protegidos frente ao dano renal e à deposição de colágeno após serem submetidos à obstrução unilateral do ureter (UUO). Além disso, os camundongos protegidos exibiram menor produção de citocinas relacionadas com um perfil imune Th2 e apresentaram menor acúmulo de macrófagos do subtipo M2. Inicialmente, creditamos aos macrófagos M2 o papel de macrófagos formadores de fibrose uma vez que tal subpopulação é encontrada em maior número aos sete dias após a UUO em animais WT, porém, vimos que os personagens centrais no desenvolvimento da fibrose são macrófagos M1, encontrados no início da lesão renal. Também vimos que o ácido úrico é a molécula capaz de induzir a troca de fenótipo de M1 para M2 ao longo da UUO, além de ser capaz de ativar a via do inflamassoma. O ácido úrico solúvel é liberado em um contexto de hipóxia e ativa o complexo do inflamassoma NLRP3 por mecanismos diferentes, mas complementares. / Chronic kidney disease is an immune mediated disease characterized by fibrosis development. The damaged tissue releases molecules such as soluble uric acid resulting from the degradation of extracellular matrix or dead cells, which activate TLR and NLR, leading to the translocation of MyD88 in many cell types. This modulation of the immune system interferes with the activation of different subtypes of macrophages and activity of CD4+ T cells, with the Th1/Th2 paradigm as a possible effector mechanism of fibrosis. TLR2, TLR4, MyD88, and NLRP3 deficient mice are protected against renal damage and collagen deposition after being submitted to unilateral ureteral obstruction (UUO), when compared to wild type animals. Moreover, protected mice exhibited less production of Th2 related cytokines and reduced accumulation of M2 macrophages. Initially, we hypothesized M2 macrophages are responsible for fibrosis formation since this subset is found in greater numbers seven days after UUO in WT mice, however, we observed the central characters on the development of fibrosis are M1 macrophages found in the onset of renal injury. These data were confirmed by the injection of Stat6 KO M1 macrophages into Rag deficient mice previously depleted of macrophages and subjected to UUO, in which we observed higher proteinuria and increased collagen deposition. We also observed that uric acid is able to induce the exchange of phenotype from M1 to M2 along the UUO, besides being able to activate the inflammasome pathway. The soluble uric acid is released in the context of hypoxia and activates the NLRP3 inflammasome complex by different, but complementary mechanisms. Therefore, the renal damage releases soluble uric acid, which signals via innate immune receptors, and the damage brings as a consequence the deposition of proteins in the renal interstitium, culminating in fibrosis.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-04-27T11:55:53Z No. of bitstreams: 1 analuisasilveiravieiratollendal.pdf: 1031624 bytes, checksum: 2a4a0647b9d22be1da3f719765d6d470 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-04-27T12:09:21Z (GMT) No. of bitstreams: 1 analuisasilveiravieiratollendal.pdf: 1031624 bytes, checksum: 2a4a0647b9d22be1da3f719765d6d470 (MD5) / Made available in DSpace on 2018-04-27T12:09:21Z (GMT). No. of bitstreams: 1 analuisasilveiravieiratollendal.pdf: 1031624 bytes, checksum: 2a4a0647b9d22be1da3f719765d6d470 (MD5) Previous issue date: 2018-01-19 / Introdução: A doença renal crônica (DRC) se tornou uma preocupante questão de saúde pública em todo o mundo devido às suas crescentes incidência e prevalência e ao impacto em morbimortalidade por ela desencadeado. O tratamento da DRC se baseia na intervenção em seus fatores de risco. Entretanto, os fatores atualmente conhecidos e sua abordagem não têm sido suficientes para conter a doença. Por esse motivo, torna-se imprescindível a busca por outros fatores associados à sua patogênese. Nesse sentido, a hiperuricemia tem sido apontada, nas últimas décadas, como uma condição associada à DRC, porém sem que ainda tenha sido estabelecida uma associação causal entre ambas. Objetivos: 1. Avaliar as evidências sobre o impacto da hiperuricemia na incidência e progressão da DRC, através de revisão sistemática da literatura; 2. Avaliar o impacto dos níveis séricos de ácido úrico (AU) sobre o declínio da taxa de filtração glomerular (TFG) em uma população de pacientes com DRC. Métodos: Primeiramente, realizou-se revisão sistemática da literatura com busca por artigos publicados no período entre Janeiro de 2005 e Dezembro de 2016, utilizando-se a combinação de palavraschave “chronic renal insufficiency AND hyperuricemia AND uric acid” nos bancos de dados Lilacs e Pubmed. Os resumos dos artigos foram avaliados por dois pesquisadores, de acordo com os critérios de inclusão e exclusão estabelecidos. Na segunda fase do estudo, 788 pacientes incidentes no ambulatório de DRC do Centro Hiperdia Minas/Juiz de Fora tiveram seus registros eletrônicos analisados e o impacto dos níveis de AU na progressão da DRC foi avaliado. Resultados: Relativamente à revisão sistemática, foram encontrados 150 estudos envolvendo seres humanos, dos quais 22 foram elegíveis, 13 estudos avaliaram incidência e 11 avaliaram progressão da DRC (aumento de creatinina, variação da taxa de filtração glomerular, início de terapia renal substitutiva); dois avaliaram ambos os desfechos. Todos os treze artigos que avaliaram associação entre hiperuricemia e incidência de DRC mostraram associação positiva entre ambas. Uma metanálise avaliou impacto da hiperuricemia em 190.718 indivíduos e encontrou relação causal independente para incidência de DRC. Em relação à progressão da DRC, os estudos longitudinais apresentaram resultados conflitantes e três estudos randomizados controlados foram identificados, comparando um grupo tratado com alopurinol e um grupo controle, todos com melhora dos desfechos renais no grupo tratado. Os resultados da análise do banco de dados do Centro HIPERDIA mostraram que pacientes admitidos com hiperuricemia, ou seja, AU maior do que 6,8mg/dL, apresentaram risco quase duas vezes maior (IRR=1,91 95% IC: 1,21-3,00, p=0,005) de progressão rápida da DRC (TFG>5mL/min/ano). Além disso, para cada 1 mg/dL de aumento nos níveis basais de AU houve risco anual 48% maior de progressão rápida (IRR=1,48 95% IC:1,16-1,88, p=0,001). Conclusão: A revisão sistemática sugeriu que hiperuricemia se associa de forma independente com incidência de DRC, porém seu papel na progressão da doença ainda é controverso. Entre os pacientes com DRC do Centro Hiperdia Minas/Juiz de Fora, os níveis séricos aumentados de AU associaramse a maior risco de progressão rápida da doença renal crônica. / Introduction: Chronic kidney disease (CKD) has become a worrisome public health problem worldwide due to its increasing incidence and prevalence as well as its impact on morbidity and mortality. Treatment of CKD is based on risk factor intervention. However, currently known factors and their approach are insufficient to stop the disease. For this reason, it is imperative to search for other factors associated with its pathogenesis. Hyperuricemia has been identified as a condition associated with CKD, but causal association between them has not yet been proved. Objectives: 1. To evaluate the impact of hyperuricemia on the incidence and progression of CKD through a systematic review of the literature; 2. To evaluate the impact of serum uric acid levels on the decline of the glomerular filtration rate (GFR) in a population of chronic renal patients. Methods: Initially a systematic review of the literature was carried out between January 2005 and December 2016. The combination of keywords "chronic renal insufficiency AND hyperuricemia AND uric acid" was used to search in the Lilacs and Pubmed databases. The articles’ abstracts were evaluated by two researchers according to established inclusion and exclusion criteria. Secondly, the electronic records of 788 patients of the CKD outpatient clinic of the Hiperdia Minas/Juiz de Fora Center were analyzed and the impact of uric acid levels on the progression of CKD was evaluated. Results: A total of 150 studies involving humans were found. Twenty two were eligible; 13 studies evaluated incidence and 11 evaluated progression of CKD (increase in creatinine, variation of glomerular filtration rate, initiation of renal replacement therapy); two of the articles evaluated both outcomes. All thirteen articles that assessed the association between hyperuricemia and incidence of CKD showed a positive association between both. A further meta-analysis of 190,718 individuals evaluated the impact of hyperuricemia on the incidence of CKD and found an independent causal relationship. Regarding the progression of CKD, longitudinal studies presented conflicting results; three randomized controlled trials compared a group treated with allopurinol and a control group, all with improvement of the renal outcomes within the treated group. The Hiperdia Center database analysis results showed that patients admitted with hyperuricemia, that is, uric acid higher than 6.8mg/dL, presented almost twice the risk (IRR = 1.91 95% CI: 1, 21-3.00, p = 0.005) of rapid progression of CKD (TFG> 5mL/min/year). In addition, for each 1 mg/dL increase in the uric acid levels baseline, there was an additional 48% annual risk of progression (IRR = 1.48 95% CI: 1.16-1.88, p = 0.001). Conclusion: The systematic review suggested that hyperuricemia is independently related to the incidence of CKD, however, its role in disease progression is still controversial. Among patients with CKD, increased serum uric acid levels were associated with an increased progression of chronic kidney disease.

Uric acid excretion disorders are the most common cause of primary dysuricemia. The kidneys eliminate two-thirds of uric acid production and the other third is eliminated in the gastrointestinal tract. Renal reabsorption and secretion occur through the polarised epithelial cells in the proximal tubules. Uric acid transporters are expressed on these cell membranes. Reabsorption deficiency leads to hypouricemia and elevated fraction excretion associated with urolithiasis, nephrolithiasis or acute renal injury. Decreased uric acid secretion in the kidneys and small intestine leads to hyperuricemia, which develops into gout in 10% of individuals. Genome wide association studies detected a strong effect of SLC22A12 (URAT1), SLC2A9 (GLUT9) reabsorbing transporters and ABCG2 (ABCG2) secreting transporter on uric acid serum concentration variability. This thesis aimed to map out urate transporter allelic variants in a cohort of primary dysuricemia patients and identification of the variants causing defective uric acid excretion. Six non-synonymous variants were described in SLC22A12 (URAT1) and SLC2A9 (GLUT9) genes in hypouricemic individuals, which had not been identified previously in any population studies. Significant decreases in uric acid transport have been demonstrated experimentally in vitro,...

Uric acid excretion disorders are the most common cause of primary dysuricemia. The kidneys eliminate two-thirds of uric acid production and the other third is eliminated in the gastrointestinal tract. Renal reabsorption and secretion occur through the polarised epithelial cells in the proximal tubules. Uric acid transporters are expressed on these cell membranes. Reabsorption deficiency leads to hypouricemia and elevated fraction excretion associated with urolithiasis, nephrolithiasis or acute renal injury. Decreased uric acid secretion in the kidneys and small intestine leads to hyperuricemia, which develops into gout in 10% of individuals. Genome wide association studies detected a strong effect of SLC22A12 (URAT1), SLC2A9 (GLUT9) reabsorbing transporters and ABCG2 (ABCG2) secreting transporter on uric acid serum concentration variability. This thesis aimed to map out urate transporter allelic variants in a cohort of primary dysuricemia patients and identification of the variants causing defective uric acid excretion. Six non-synonymous variants were described in SLC22A12 (URAT1) and SLC2A9 (GLUT9) genes in hypouricemic individuals, which had not been identified previously in any population studies. Significant decreases in uric acid transport have been demonstrated experimentally in vitro,...

Katecholamine (Dopamin, Adrenalin, Noradrenalin) und Serotonin sind wichtige Monoamin-Neurotransmitter im menschlichen zentralen Nervensystem, deren quantitative Bestimmung von großem medizinischen Interesse ist, weil damit Aussagen zum Verlauf von Nervenkrankheiten und zur Tumorgefährdung des sympathoadrenalen bzw. neuroendokrinen Systems möglich sind. Ascorbinsäure und Harnsäure finden sich in vielen Körperflüssigkeiten. Ihre Bestimmung ist klinisch ebenfalls bedeutend, da deren Konzentration als Indikatoren bekannter Krankheitsbilder dienen. Etablierte Standardmethoden, wie die Hochleistungsflüssigkeitschromatographie (HPLC) und immunologische Nachweisverfahren (ELISA) werden im klinischen Bereich zur Bestimmung der Neurotransmitter genutzt. Diese sind kostenintensiv und zeitaufwändig und daher für die Anwendung in den Arztpraxen, vor allem in Entwicklungsländern nicht geeignet. Elektrochemische Verfahren, insbesondere voltammetrische Messmethode haben den Vorteil, solche Bestimmungen in einfacher Weise zu ermöglichen. In der Literatur finden sich Angaben zu eingesetzten Elektroden auf Kohlenstoffbasis mit hoher Sensitivität für die Katecholamine. Allerdings wurden diese Elektroden meist einzeln hergestellt. Der kommerzielle Durchbruch ist deshalb bisher, hauptsächlich infolge der mangelnden Reproduzierbarkeit der Elektrodeneigenschaften und der Verfügbarkeit einfacher elektronischer Geräte ausgeblieben. Es war daher Ziel dieser Arbeit, durch industrienahe Herstellungsverfahren Graphitelektroden mit reproduzierbaren Eigenschaften zu entwickeln und diese auf ihre Eignung für den quantitativen Nachweis bioaktiver Stoffe zu erproben. Dazu waren Verfahrensschritte zu optimieren, die es erlauben, diese siebgedruckten Graphitelektroden reproduzierbar und kostengünstig zu fertigen und sie auf verschiedene Weise, z.B. durch halbleitende Polymere und nanoskalige Metalle zu modifizieren. Neben den Neurotransmittern enthalten Körperflüssigkeiten unter anderem Ascorbinsäure und Harnsäure in hohen Konzentrationen. Daher waren zunächst Modellanalyten unter Verwendung dieser Stoffe herzustellen. Die voltammetrischen Methoden, wie die zyklische Voltammetrie (CV), die Differentielle Puls-Voltammetrie (DPV) und die Square-Wave-Voltammetrie (SWV) sollten auf ihre Eignung zum Nachweis der bioaktiven Substanzen erprobt werden. Schließlich waren die Elektroden in realen Analyten zu testen. Insgesamt konnte in der vorliegenden Arbeit gezeigt werden, dass ausgewählte Neurotransmitter, Ascorbinsäure und Harnsäure sich mit differentiellen voltammetrischen Verfahren an industrienah hergestellten modifizierten Dickschichtelektroden bestimmen lassen. Es ist erstmalig gelungen, eine modifizierte Dickschichtelektrode zu entwickeln, mit der es möglich ist, Katecholamine unabhängig von Ascorbinsäure (3 mM) und Harnsäure (2 mM) quantitativ nachzuweisen. Damit eröffnen sich neue Wege für den Einsatz von elektrochemischen Sensoren für die einfache Bestimmung der Neurotransmitter vor Ort. Die beschriebenen modifizierten Dickschichtelektroden sind ohne Verlust an elektrochemischer Aktivität an der Luft oder im Grundelektrolyten monatelang lagerfähig. Die Elektroden lassen sich im Gegensatz zu den in der Literatur beschriebenen Elektroden mit Einzelfertigung kostengünstig in großer Stückzahl mit hoher Reproduzierbarkeit herstellen.:Inhaltsverzeichnis I Abkürzungen V 1 Einleitung und Zielsetzung der Arbeit 1 2 Theoretischer Teil 5 2.1 Elektrochemische Verfahren in der Analytik 5 Klassifizierung elektroanalytischer Methoden 5 2.1.1 Voltammetrie 5 Cyclovoltammetrie (CV) 6 Differential-Puls-Voltammetrie (DPV) 9 Square-Wave-Voltammetrie (SWV) 10 2.1.2 Chronocoulometrie (ChrC) 11 2.1.3 Impedanzmessung (EIS) 12 2.1.4 Elektrochemische Quarzmikrowaage (EQCM) 14 2.2 Poly-3,4-Ethylendioxythiophen, ein leitfähiges Polymer 19 2.2.1 Leitfähige Polymere 19 2.2.2 Das Poly-3,4-ethylendioxythiophen 20 Elektrochemische Synthese und Dotierung 20 2.3 Bioaktive Stoffe 24 2.3.1 Katecholamine 24 Dopamin 25 Noradrenalin und Adrenalin 25 Abnorme Konzentration der Katecholamine 25 2.3.2 Serotonin 26 2.3.3 Interaktion von Katecholaminen und Serotonin 26 2.3.4 Ascorbinsäure und Harnsäure 27 2.3.5 Elektrochemisches Verhalten der bioaktiven Stoffe 28 Katecholamine 28 Serotonin 30 Ascorbinsäure 30 Harnsäure 30 3 Experimenteller Teil 32 3.1 Chemikalien 32 3.2 Lösungen 33 3.2.1 Ausgangslösungen 33 Grundelektrolyte 33 Lösungen der bioaktiven Stoffe 33 3.2.2 Lösungen für Elektrodenmodifizierungen 33 EDOT-haltige Lösungen 33 Neurotransmitter-Lösungen 34 HAuCl4-Lösungen 34 Goldkolloide 34 Eisenhexacyanoferrat(II)-Goldsäurehaltige Lösung 35 3.3 Elektrochemische Messmethoden 35 3.3.1 Voltammetrie, Chronocoulometrie und Impedanz 35 3.3.2 Elektrochemische Quarzmikrowaage 38 3.4 Elektroden und Präparation der Elektroden 39 3.4.1 Untersuchte Elektroden, deren Aktivierung und Konditionierung 39 3.4.3 Modifizierungen der Elektroden 41 Poly-3,4-Ethylendioxythiophen (PEDOT) 41 Goldnanopartikel 41 Komposite aus Goldnanopartikeln und Preußisch Blau (Au/PB) 42 Polymerfilme aus Monoamin-Neurotransmittern 42 3.5 Präparation der UP für Untersuchungen in realen Medien 43 3.6 Spektroskopische Methoden 43 4 Ergebnisse und Diskussion 45 4.1 Unmodifizierte Elektrodenoberflächen 45 4.1.1 Einfluss der Aktivierung der Elektrodenoberflächen auf das Messverhalten 45 4.1.2 Bestimmung bioaktiver Stoffe an unmodifizierten Elektroden 48 Ermittlung des Peakpotenzials 48 Messeffekte an Gold- und Graphitelektroden in Neurotransmitter-Lösungen hoher Konzentrationen 50 Bestimmung bioaktiver Stoffe im Gemisch 52 4.2 Au- und Au/PB-modifizierte Elektroden 54 4.2.1 Abscheidung 54 4.2.2 Untersuchungen bioaktiver Stoffe an Au-modifizierten Elektroden 56 4.3 PEDOT-modifizierte Elektroden 58 4.3.1 Abscheidungen der PEDOT-Schichten 58 CV-Abscheidungen der PEDOT-Schichten 59 ChrC-Abscheidungen der PEDOT-Schichten 62 4.3.2 Voruntersuchungen an PEDOT-modifizierten Elektroden 66 Ermittlung des optimalen Potenzialbereiches für voltammetrische Messungen an PEDOT-modifizierten Elektroden 66 Ermittlung der optimale PEDOT-Schichten für die Bestimmung bioaktiver Stoffe 68 Peakpotenziale bioaktiver Stoffe 71 Einfluss des pH-Wertes des Elektrolyten und der Scangeschwindigkeit auf voltammetrische Messsignale bioaktiver Stoffe 72 Einfluss der Messmethoden auf die Messsignale bioaktiver Stoffe an PEDOT-modifizierten Elektroden 74 4.3.3 Bestimmung bioaktiver Stoffe an PEDOT-modifizierten Elektroden 78 Bestimmung der Neurotransmitter (Dopamin, Adrenalin, Noradrenalin und Serotonin) 78 Bestimmung von Ascorbinsäure und Harnsäure 81 Bestimmung der Neurotransmitter mit Zusatz von Ascorbinsäure und Harnsäure 82 Stabiltität der PEDOT-modifizierten Elektroden 83 Vergleich der Ergebnisse an PEDOT-Elektroden mit Literaturangaben 84 4.3.4 Spektroskopische Untersuchungen der PEDOT-Oberflächen 85 4.3.5 Zusammenfassung der Ergebnisse an PEDOT-Elektroden 87 4.4 Au-PEDOT-modifizierte Elektroden 88 4.4.1 Abscheidungen der Goldnanopartikel auf PEDOT-Oberflächen 88 Abscheidung der Goldnanopartikel durch Adsorption aus Goldkolloiden 88 Abscheidung der Goldnanopartikel auf PEDOT-modifizierten Elektroden mittels Cyclovoltammetrie 92 4.4.2 Bestimmung bioaktiver Stoffe an Au-PEDOT-Elektroden 94 Peakpotenziale bioaktiver Stoffe an Au-PEDOT-Elektroden 94 Bestimmung von Neurotransmittern in 0,1 M Phosphatpufferlösungen 96 Bestimmung von Neurotransmittern mit Zusatz von Ascorbinsäure und Harnsäure 98 Bestimmung von Ascorbinsäure und Harnsäure 99 Stabilität der Sensitivitäten und Reproduzierbarkeit der Elektrodenherstellung 102 Vergleich der Ergebnisse an Au-PEDOT-Elektroden mit Literaturangaben 102 4.4.3 Zusammenfassung der Ergebnisse an Au-PEDOT-Elektroden 104 4.5 Polymonoamin-modifizierte Elektroden bzw. PEDOT-Elektroden 105 4.5.1 Abscheidungen der Polymerschichten aus Monoaminen an Graphitelektroden 106 4.5.2 Abscheidungen der Polymerschichten aus Monoaminen an PEDOT-Elektroden 106 CV-Abscheidung 106 SWV-Abscheidung 108 4.5.3 Bestimmung bioaktiver Stoffe an Polyserotonin-modifizierte PEDOT-Elektroden 111 Peakpotenziale bioaktiver Stoffe 111 Bestimmung der Neurotransmitter 112 Bestimmung von Ascorbinsäure und Harnsäure 114 Bestimmung der Neurotransmitter mit Zusatz von AS und HS 114 Bestimmung von Harnsäure in Gegenwart von Dopamin 116 4.5.4 Möglicher Einsatz der 5-HT-PEDOT-Elektroden als pH-Elektroden 117 4.5.5 Zusammenfassung der Ergebnisse an Polyserotonin-PEDOT-Elektroden 118 4.6 Bestimmung bioaktiver Stoffe in UM 119 4.6.1 Bestimmung von Harnsäure 119 Bestimmung von Harnsäure im Modellanalyten 119 Bestimmung von Harnsäure in präparierten UP 119 4.6.2 Bestimmung von Dopamin 120 DA-Bestimmung im Modellanalyten 120 Bestimmung von Dopamin in präparierten UP 121 5 Zusammenfassung und Ausblick 123 Zusammenfassung 123 Ausblick 126 Tabellenverzeichnis 127 Abbildungsverzeichnis 130 Anhang 138 Literaturverzeichnis 152 VERSICHERUNG 157

Uric acid is a main metabolite of purine degradation in humans and in higher primates. Its increased plasmatic level is called hyperuricemia and may be the cause of gout and many other similar diseases. Uricemia is controlled by many transporters, which are located in proximal tubule of human kidney. When some transporter have abnormal function, the physiological plasmatic level of uric acid may be impaired. In genome wide association study (GWAS) it was discovered that some hyperuricemia or gout patients have ABCG2 protein damaged. This protein carries out uric acid from epithelial cell to the urine. The goal of this diploma thesis is the determination of transport capacity of ABCG2 allelic variants found via GWAS (Institute of Rheumatology of 1st medical faculty UK in Prague) in vitro with Xenopus laevis oocyte expression system. Uric acid secretion was compared with wild type variant. Keywords: Uric acid, GWAS study, Xenopus laevis, membrane transport protein, ABCG2

Background: Periodontal disease has been associated with gestational complications and both conditions have a high prevalence in rural populations from developing regions. A cross-sectional study was carried out to explore the relationship between periodontal inflamed surface area (PISA), blood pressure (BP), and, serum uric acid levels (UA) in a group of rural North Chinese pregnant women in the third trimester of pregnancy. Methods: Three hundred and thirty-five rural women aged 20–34 years, with normal body mass index (BMI) were examined in a cross-sectional study during their third trimester of gestation. Exclusion criteria were history of pregnancy complications,multiple pregnancy, smoking habits, diabetes, hypertension or any known infectious disease. Socio-demographic variables, including age and socioeconomic status (SES), systolic blood pressure (SBP) and diastolic blood pressure (DBP) readings, serum UA levels, and PISA values were recorded. A structural equation model was implemented with two constructed latent variables including “Dem” (comprising of age and SES category to represent unobserved demographic variables) and, “BP” (comprising of SBP and DBP to account for measurement error and lack of multiple BP readings). The model accounted for co-variance of BP and UA, and implemented simultaneous regressions for BP and UA as outcomes, upon Dem and PISA values as exogenous variables. Results: The median PISA score was 1,081.7 (IQR = 835.01), reflecting high levels of periodontal inflammation in the sample. SEM showed a significant association of PISA with BP (estimate = 0.011, 95%CI = 0.009–0.012 p < 0.001) and UA (estimate = 0.001, 95% CI = 0.001–0.001, p < 0.001). Conclusion: Higher PISA values were significantly associated with higher blood pressure and uric acid levels among rural pregnant women in a cross-sectional sample from a center in North China after accounting for a latent demographic construct derived from age and SES.

A oxidação do ácido úrico (7,9-diidro-1H-purina-2,6,8(3H)-triona) pela mieloperoxidase (MPO) gera o radical de urato que, em presença do radical ânion superóxido combinam para formar o hidroperóxido de urato (HOOU). Considerando os altos níveis de MPO, ácido úrico e superóxido na placa de ateroma espera-se que o ácido úrico seja oxidado a HOOU neste microambiente inflamatório. O HOOU é um oxidante mais forte que o peróxido de hidrogênio e pode oxidar grupos tiólicos de proteínas como a proteína dissulfeto isomerase (PDI). Como consequência à oxidação da PDI, ocorre uma modulação positiva sobre a NADPH oxidase (Nox) com aumento da produção de superóxido por neutrófilos. Sendo assim, a formação do HOOU no leito vascular poderia elucidar os mecanismos moleculares pelos quais o urato colabora para a progressão da aterosclerose em pacientes com hiperuricemia. Para investigar a interação do HOOU com a PDI, padronizou-se a síntese química do HOOU através de um sistema de fotooxidação do tipo I, utilizando urato, riboflavina (fotossensibilizador) e luz UVA. Inicialmente padronizou-se o tipo de irradiação e tempo de reação com o melhor rendimento para a síntese do HOOU. O HOOU formado e seu produto de redução o álcool 5-hidroxiisourato foram separados, identificados e caracterizados através de cromatografia liquida acoplada à espectrometria de massa (LC/MS). Após a purificação, determinou-se o coeficiente de absortividade molar em 308 nm (&#949;308 = 6537 &#177; 377 M-1.cm-1) e o tempo de meia-vida, aproximadamente 41 minutos à 22&#176;C do HOOU. O HOOU foi capaz de reagir seletivamente com o aminoácido metionina e com o tripeptídeo glutationa. Além disso, o HOOU não forma adutos estáveis com a glutationa, sendo que toda glutationa consumida foi transformada em glutationa dissulfeto. Quando incubado com a PDI (10 &#181;M), cerca de 70 e 100% do total de HOOU (3 &#181;M) foi consumido após 30 e 120 segundos, respectivamente, enquanto que a PDI (23 &#181;M) teve seus grupos tiólicos oxidados após a incubação com 140 &#181;M HOOU por 30 min a 22&#176;C. O HOOU oxidou os resíduos de cisteína dos dois sítios catalíticos da PDI com uma constante de velocidade da reação de 1,5 &#177; 0,04 x 103 M-1.s-1, demonstrando uma interação favorável com essa proteína no meio biológico e um possível papel modulatório do HOOU sobre a via PDI-Nox. Interessantemente, o ácido úrico aumentou a produção de superóxido e o consumo de oxigênio de células HL-60 diferenciadas em neutrófilos (dHL-60) e ativadas com acetato de miristato de forbol (PMA). Essa regulação poderia ser mediada através da formação do HOOU durante o \"burst\" oxidativo dos neutrófilos que oxidaria a enzima PDI induzindo um consequente aumento da atividade da Nox. / The oxidation of the uric acid (7,9-dihidro-1H-purine-2,6,8(3H)-trione) by myeloperoxidase (MPO) generates the urate radical. In inflammatory conditions the superoxide reacts with urate radical to form the urate hydroperoxide (HOOU). Taking into account the high amount of MPO, urate and superoxide in the atheroma plaque, it is likely that HOOU is being formed in this inflammatory environment. The HOOU is a strong oxidizing agent and can react with thiol groups from proteins, like the protein disulfide isomerase (PDI). As a consequence of its oxidation, PDI positively modulates NADPH oxidase (Nox) and increases superoxide production by neutrophils. Therefore, the formation of HOOU in the vascular sheet would contribute to tissue damage and would explain the positive correlation between hyperuricemia and the risk for cardiovascular disease. To investigate the interaction of HOOU with PDI, we performed the chemical synthesis of the compound by the Type I photooxidation, using UVA irradiation and riboflavin as a photosensitizer. Initially, we standardized the irradiation light and the time of the reaction that produced the highest income. The HOOU and its reduced product 5-hydroxiisourate were separate, identified and characterized by liquid chromatography coupled to mass spectrometry (LC/MS). We also determine the molar extinction coefficient of HOOU at 308 nm (&#949;308 = 6537 &#177; 377 M-1.cm-1). The half-life of the compound was 41 min at 22&#176;C. The HOOU selectively oxidized methionine and glutathione. The reaction of HOOU with glutathione did not form any stable adducts. Thus, all consumed glutathione generated glutathione disulfide. When incubated with PDI (10 &#181;M), 70 and 100% of the total amount of HOOU (3 &#181;M) was consumed within 30 and 120 seconds, respectively. Besides, the PDI (23 &#181;M) had its thiol groups oxidized after incubation with 140 &#181;M HOOU for 30 min at 22&#176;C. The HOOU oxidized the cysteine residues from the catalytical sites of PDI with a second order rate constant of 1.5 &#177; 0.04 x 103 M-1.s-1. This result suggests a favorable interaction of HOOU with this protein in the biological system, as well as a possible modulatory role of HOOU on the PDI-Nox pathway. Interestingly, urate increased superoxide production and oxygen consumption by neutrophil-like cells (differentiated HL-60 cells). This effect could be mediated by the formation of HOOU during the neutrophil oxidative burst, followed by the oxidation of PDI, a positive regulation of Nox and an increase in superoxide production.

A nefropatia diabética (ND) é uma das complicações microvasculares do diabetes e consiste no dano ao parênquima renal por consequência de uma série de fatores hemodinâmicos e moleculares. A ocorrência de ND e de outras complicações mesmo em indivíduos sob adequado controle glicêmico tem sido associada a um fenômeno conhecido como memória metabólica. Neste trabalho foram investigadas vias bioquímicas e moleculares persistentemente alteradas no rim de animais diabéticos tratados após um período inicial de hiperglicemia, com o propósito de entender os mecanismos envolvidos na memória metabólica. Para tanto, ratos com diabetes induzida por estreptozotocina foram mantidos hiperglicêmicos durante 4 semanas (período curto) ou 12 semanas (período longo) e posteriormente tratados com insulina isoladamente ou combinada com metformina (100mg/kg/dia) durante as 4 (período curto) ou 12 (período longo) semanas seguintes. Todos os animais tratados tiveram os seus níveis glicêmicos e função renal normalizados. Os tratamentos também foram capazes de normalizar os níveis elevados de malonaldeído no rim, bem como a excreção aumentada dos adutos de DNA 8-oxo-2\'-desoxiguanosina (8-oxodG) e N2-carboxietil-2\'- desoxiguanosina (CEdG) na urina observados nos animais diabéticos. Níveis aumentados de 8-oxodG foram detectados em DNA mitocondrial (mtDNA), mas não em DNA nuclear, de animais diabéticos apenas no período curto de estudo e também foram normalizados após o controle glicêmico. Nós identificamos uma via gradualmente alterada durante o curso do diabetes que permanece persistentemente alterada após o controle glicêmico tardio. Essa via compreende um declínio precoce do clearance de ácido úrico e expressão da pAMPK, seguida pelo acúmulo de fumarato, expressão aumentada de TGF-&#946;, expressão reduzida de PGC-1&#945; e redução da metilação e hidroximetilação do mtDNA. A redução persistente do clearance de ácido úrico em animais diabéticos tratados pode sustentar as alterações bioquímicas renais prolongadas observadas após o controle glicêmico, e essa regulação é provavelmente mediada pela redução sustentada da expressão de pAMPK e pela indução de inflamação. Este trabalho propõe a primeira consideração do possível papel da hiperuricemia e das alterações bioquímicas subjacentes como parte da memória metabólica na nefropatia diabética. / Diabetic nephropathy is one of the diabetes microvascular complications, and it consists on the damage to the renal parenchyma due to several hemodynamic and molecular factors. The occurrence of diabetic nephropathy and other complications even in those individuals under tight glycemic control has been associated to a phenomenon known as metabolic memory. Here we investigated biochemical and molecular pathways persistently altered in the kidney of diabetic animals treated after a previous period of hyperglycemia, aiming to understand underlying mechanisms in metabolic memory. Streptozotocin-induced diabetic rats were maintained hyperglycemic during 4 (short period) or 12 weeks (long period), and then they were treated with insulin alone or combined with metformin (100 mg/kg/day) for the following 4 or 12 weeks, respectively. All the treated animals had them glycemic levels and renal function normalized. The treatments were also able to control enhanced kidney malondialdehyde levels, as well as the increased urine excretion of the DNA adducts 8-oxo-2\'- deoxyguanosine (8-oxodG) and N2-carboxyethyl-2\'-deoxyguanosine seen in diabetic animals. Increased levels of 8-oxodG were detected in mitochondrial DNA, but not in nuclear DNA of diabetic animals in the short period, and were also recovered after glycemic control. We have identified a kidney pathway that is gradually altered during the course of diabetes and remains persistently changed after late glycemic control. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-&#946; expression, reduced PGC-1&#945; expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This work proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy.