Orientador: Luís Fernando Barbisan / Resumo: O ferro heme presente na carne vermelha está associado ao aumento da incidência do câncer colorretal (CCR). O heme pode catalisar a formação de compostos nitrosos e a peroxidação lipídica no lúmen intestinal. No entanto, os efeitos pró-carcinogênicos do heme podem ser inibidos por alguns compostos como os sais de cálcio, clorofila entre outros. Sabe-se que o indol-3-carbinol (I3C), presente nas plantas da família das Brassicas e os simbióticos são compostos promissores na prevenção do câncer de cólon, atuando em via de proliferação, apoptose e modulação da microbiota intestinal. Dessa forma, o objetivo desse estudo foi o de avaliar os efeitos da ingestão de simbiótico (prebiótico inulina associado ao probiótico Bifidobacterium lactis bb-12) e de I3C, isolados ou em associação sobre o processo de carcinogênese de cólon induzido pela 1,2-dimetilhidrazina (DMH) em ratos Wistar alimentados ou não com dieta suplementada com heme. Os animais foram alocados em 9 grupos, os grupos 1 a 8 (n=12) receberam quatro doses de DMH (40 mg/Kg) nas duas semanas iniciais do experimento. Os grupos 1 e 9 (n=12 e 5) receberam ração basal até o final do experimento e os grupos 2 a 8 receberam ração basal suplementada com heme, heme+I3C, heme+simbiótico, heme+I3C+simbiótico, I3C, simbiótico e I3C+simbiótico, respectivamente. A eutanásia ocorreu ao final da 25ª semana. Neste momento foi realizada a coleta do cólon com os respectivos tumores e amostras de fezes do ceco. Em seguida, procedeu-se a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Colorectal cancer (CRC) is the third most common type of cancer worldwide. Hemin iron, which is found in red meat, catalyzes the formation of carcinogenic N-nitroso compounds and lipid peroxidation end-products in the colon lumen. The procarcinogenic effect of hemin is known to be inhibited by molecules, such as calcium, chlorophyll and others. However, the preventive effect of indole 3-carbinol and synbiotics on colon carcinogenesis remains uninvestigated. The aim of this study was to assess the modifying effects of a synbiotic (inulin+ Bifidobacterium lactis) and/or I3C against dimethylhidrazine (DMH)-induced colon carcinogenesis in hemin-fed male Wistar rats. Nine groups of animals were evaluated. Groups 1–8 received a total of four s.c. DMH injections (40 mg/kg b.w.) over 2 weeks, whereas group 9 was given EDTA solution (vehicle). Two weeks after DMH-initiation, G1 and G9 were fed a basal diet while groups G2, G3, G4, G5, G6, G7 and G8 received a basal diet containing hemin, hemin+I3C, hemin+synbiotic, hemin+I3C+synbiotic, I3C, synbiotic and I3C+synbiotic, respectively, during 23 weeks. At 25 week, all animals were killed and their colons were removed. Cecal contents were collected to determine fecal water cytotoxicity and genotoxicity (DNA damage) in Caco-2 cells. Colon tumors were measured and samples were collected and stored at -800C. The colons were fixed flat in 10% buffered formalin for 24 h and stained with 1.0% methylene blue for classical ACF analysis and qua... (Complete abstract click electronic access below) / Doutor

In this work electrochemical studies are broached as well the development of an analytical methodology to determinate ketoconazole in pharmaceuticals formulations with the help of electrode of carbon paste was modified with hemin. The tests were carried out in solution tampão Britton-Robinson (BR) in pH 2, with an electrode compounded by 50% of carbon past and 50% of hemin, for having the best analytical answer to the compound in question. Based on the electrochemical parameters that were taken of the technique cyclic voltammetry, differential pulse and of the square wave, it was possible diagnostic some information that was connected to the reaction mechanism such as: the reversibility of the process, the existence or not of adsorption process and the number of electrons that is involved in the electrochemical reactions. The resulted said that the reaction of ketoconazole reduction is irreversible, with one definite peaks at 1,44V device phenomenon of reduction do CTZ. The analytical methodology that was developed is fast, sensible and precise, to routine analysis on pills of CTZ. The analytic curve was linear in the concentration area between 1,8 and 5,5.10 6 mol/L, with detection limit 1,7.10 7 mol/L and quantification limit in 5,6.10 7 mol/L. / Neste trabalho são abordados os estudos eletroquímicos bem como o desenvolvimento de metodologia analítica para a determinação de cetoconazol em formulações farmacêuticas com auxílio de eletrodo de pasta de carbono modificado com hemina. Os experimentos foram realizados em solução tampão Britton-Robinson (BR) no pH 2, com uma composição do eletrodo de 50% de pasta de carbono e 50% de hemina, pois apresenta a melhor resposta analítica para o referido composto. Com base nos parâmetros eletroquímicos extraídos das técnicas de voltametria cíclica, de pulso diferencial e de onda quadrada, foi possível diagnosticar algumas informações relacionadas com o mecanismo da reação como: a reversibilidade do processo, a presença ou não de processos de adsorção e também o número de elétrons envolvidos nas reações eletroquímicas. Os resultados indicaram que a reação de redução do cetoconazol é irreversível com um pico a 1,44V devido ao fenômeno de redução de CTZ. A metodologia analítica desenvolvida é rápida, sensível e precisa para a análise de rotina de comprimidos de CTZ. A curva analítica foi linear na região de concentração entre 1,8 a 5,5.10 6 mol/L, com limite de detecção de 1,7.10 7mol/L e com limite de quantificação de 5,6.10 7 mol/L.

Acute kidney injury (AKI) is a major public health concern, which contributes to serious hospital complications, chronic kidney disease (CKD) and even death. Renal ischemia- reperfusion injury (IRI) remains a leading cause of AKI.IRI combines major cell stress, significant burst of free radicals, and strong inflammatory responses leading to extensive cell injury, necrosis, and late interstitial fibrosis. Moreover, IRI- induced AKI releases pro-inflammatory cytokines (e.g. IL-1β, TNF-α, IL-6) that induce a systemic inflammatory response, resulting in pro-inflammatory cells recruitment and remote organ damage. AKI is associated with poor outcomes, particularly when extrarenal complications or distant organ injuries occur.The stress-responsive enzyme, heme oxygenase-1 (HO-1) mediates protection against renal IRI and may be preventively induced using hemin prior to renal insult. This HO-1 induction pathway called hemin preconditioning is largely known in the literature to be effective.We first confirmed that hemin-induced HO-1 improved renal outcomes after IRI (i.e. fewer renal damage, renal inflammation and oxidative stress). We then demonstrated that this protective pathway mitigated AKI-induced ALI, a major extrarenal complication after renal IRI, through modulation of systemic and lung inflammation.Afterwards, we focused on the specific contribution of myeloid HO-1 to renal IRI, which remains poorly characterized. We therefore investigated the contribution of myeloid HO-1 to renal IRI using mice with myeloid-restricted deletion of HO-1 (HO-1M-KO). We observed that myeloid HO-1 appeared to be a critical regulator of the earliest phases of IRI (i.e. higher plasma creatinine, tubular damage, and renal inflammation/oxidative stress in HO-1M-KO mice).As a link between the severity of renal injury and the risk maladaptive repair leading to CKD has been established, we thereby decided to focus on tubular repair and fibrosis deposition upon IRI. We identified that myeloid HO-1 prevented maladaptive repair and subsequent CKD through modulation of cell-cycle and autophagy regulatory proteins.We then showed that hemin-mediated protection requires specific expression of HO-1 within myeloid cells. We therefore identified CD11b+ F4/80lo macrophages as the main protective myeloid source of HO-1 upon renal IRI. Interestingly, we observed this myeloid cell sub- population in the kidney and spleen, suggesting that protective effects might be provided by both tissue-resident and infiltrating/circulating HO-1+ myeloid cells.Based on its promising cytoprotective effects when giving preemptively, we investigated the use of hemin-induced myeloid HO-1 as a strategy to mitigate established AKI. However, due to its chemical structure and oxidative properties, hemin worsened IRI-induced AKI. We thereby identified that hemin had a dual effect on renal IRI, protective or deleterious, depending on the timing of its administration.Altogether, this work suggests that myeloid HO-1 plays a critical role in the modulation of IRI- induced AKI by improving short- and long-term functional outcomes after renal IRI. We conclude that hemin-induced myeloid HO-1 pathway might be an efficient preventive strategy in many renal IRI situations with predictable AKI such as renal transplantation or partial nephrectomy. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished

Rhizobium leguminosarum is a Gram negative, motile, nitrogen-fixing soil bacterium. Due to the scarcity of iron in the soil bacteria have developed a wide range of iron scavenging systems. The two types of iron scavenging systems used are indirect and direct. In-silico analysis of the genome identified a unique direct iron scavenging system the Hmu operon. This system has been identified in other closely related rhizobium species and is believed to be involved in utilizing heme compounds as a sole source of iron. We have attempted to characterize the role of the Hmu operon in iron utilization by monitoring the growth of R. leguminosarum ATCC 14479 in hemin supplemented media. Growth curves show that it is capable of using hemin as a sole source of iron. The outer membrane profiles were analyzed for the presence of hemin binding proteins.

Formés à partir de brins d’ADN ou d’ARN riches en guanines, les quadruplexes résultent de l’empilement de tétrades de guanines constituées chacune par l’auto-assemblage dans un même plan de quatre guanines, stabilisées entre elles par un réseau de liaisons hydrogènes. En s’inspirant de cet édifice naturel, il est présenté au long de ce manuscrit de thèse la synthèse et l’étude de molécules de type TASQ (pour template-assembled synthetic G-quartet) hydrosolubles capables de former de manière intramoléculaire une tétrade de guanines synthétique : les DOTASQ, le PorphySQ et le PNADOTASQ. La première application développée pour ces composés est le ciblage des quadruplexes d’ADN et d’ARN, présents dans des régions clefs du génome (télomères, promoteurs d’oncogènes) et du transcriptome (5’-UTR et TERRA), et dont la stabilisation par un ligand pourrait ouvrir de nouvelles perspectives en terme de thérapie antitumorale ciblée. Les résultats in vitro sont présentés et permettent de démontrer que les TASQ hydrosolubles développés sont des composés offrant une bonne sélectivité pour les quadruplexes mais surtout une excellente sélectivité grâce à un mode d’action bioinspiré basé sur une reconnaissance biomimétique. La seconde application mise au point est l’utilisation des TASQ comme catalyseurs pour des réactions de peroxydation : leur architecture même leur permet de mimer l’activité catalytique de l’ADN (ou DNAzyme) ainsi que celle de protéines (enzyme) comme la horseradish peroxidase. Ce processus est dépendant de la formation intramoléculaire de la tétrade de guanines synthétique et ouvre de nombreuses perspectives en terme d’utilisation en biologie ainsi qu’en nanotechnologie. / Natural G-quartets, a cyclic and coplanar array of four guanine residues held together via Hoogsteen H-bond network, have recently received much attention due to their involvement in G-quadruplex-DNA, an alternative higher-order DNA structure strongly suspected to play important roles in key cellular events (chromosomal stability, regulation of gene expression). Besides this, synthetic G-quartets, which artificially mimic native G-quartets, have also been widely studied for their involvement in nanotechnological applications (i.e. nanowires, artificial ion channels, etc.). In contrast, intramolecular synthetic G-quartets, also named template-assembled synthetic G-quartet (TASQ), have been more sparingly investigated, despite a technological potential just as interesting.In this way, we designed and synthesized three series of innovative hydrosoluble TASQ: DOTASQ (for DOTA-Templated Synthetic G-Quartet), PorphySQ (containing a porphyrin template) and the most effective PNADOTASQ where PNA-guanine arms replace native DOTASQ alkyl-guanine arms. We report herein the results of both DNA and RNA interactions (notably their selective recognition of quadruplex-DNA according to a bioinspired process) and peroxidase-like hemin-mediated catalytic activities (either in an autonomous fashion as precatalysts for TASQzyme reactions, or in conjunction with quadruplex-DNA as enhancing agents for DNAzyme processes). These results provide a solid scientific basis for TASQ to be used as multitasking tools for bionanotechnological applications.

This research had as main objective to develop an electroanalytical procedure for the determination of amodiaquine in human milk, using a carbon paste electrode modified with hemin, hemoglobin functional group responsible for transporting oxygen. The amodiaquine (a derivative 4-aminoquinoquinolínico) is a drug used to treat malaria with anti-inflammatory and antipyretic properties. The characterization and preparation of carbon paste electrode modified with hemin was investigated. The electrochemical behavior of modified electrode was explored using differential pulse voltammetry. The best voltammetric response was obtained for the composition of carbon paste 10% hemin solution in Britton-Robson buffer pH 7.0. To study the electrochemical behavior of amodiaquine was used the technique of cyclic voltammetry, which presented in a neutral medium oxidation process at 0.054 V vs. Ag / AgCl at a rate of 20 mV s-1. The voltammetric techniques used to develop the methodology where the differential pulse voltammetry and square wave voltammetry. In this study the optimized conditions were: pH 7, scan rate = 40 mV s-1, amplitude = 200 mV and frequency = 150 Hz, where the parameters indicated the square wave voltammetry as the best technique for determining the voltammetric amodiaquine has a minor detection limit. The limits of detection and quantification were obtained in the determination of 8.7 x 10-7 mol L-1 and 2.9 x 10-6 mol L-1, respectively. After optimization of all relevant parameters, the analytical method developed was successfully applied in samples of breast milk. The concentration of amodiaquine obtained was 1.7 x 10-5 mol L-1. The method was efficient and reproducible in the determination of amodiaquine present in breast milk, there is no need any pretreatment of the sample. / Este trabalho teve como objetivo desenvolver uma metodologia eletroanalítica para a determinação de amodiaquina em leite materno, utilizando um biossensor a base de hemina, uma porfirina presente na hemoglobina e responável pelo transporte de oxigênio. A amodiaquina, por sua vez, é um derivado 4-aminoquinoquinolínico amplamente utilizada no tratamento da malária, com propriedades antiinflamatória e antipirética. Sua transferência para o leite materno é moderada, porém mesmo em baixas dosagens pode ser considerada prejudicial ao lactante. A caracterização e a preparo do biossensor foi a primeira etapa deste trabalho. O comportamento eletroquímico do biossensor foi explorado pelo emprego da técnica voltametria de pulso diferencial. As melhores respostas voltamétricas foram obtidas com a composição da pasta de carbono contendo 10% de hemina em solução-tampão Britton-Robson de pH 7,0. Para o estudo do comportamento eletródico da amodiaquina foi utilizada a técnica de voltametria cíclica, que apresentou em meio neutro um processo de oxidação em 0,054 V vs. Ag/AgCl na velocidade de 20 mV s-1 em pH 7,0. As técnicas voltamétricas utilizadas para o desenvolvimento da metodologia foram a voltametria de pulso diferencial e a voltametria de onda quadrada. Nesse estudo as condições otimizadas encontradas foram: pH = 7; velocidade de varredura = 40 mV s-1; amplitude = 200 mV e freqüência = 150 Hz, onde os parâmetros indicaram a voltametria de onda quadrada como a melhor técnica para a determinação voltamétrica da amodiaquina por apresentar melhor desempenho. Os limites de detecção e quantificação obtidos na determinação da amodiaquina por voltametria de onda quadrada foram de 8,7 x 10-7 mol L-1 e 2,9 x 10-6 mol L-1, respectivamente. Após a otimização de todos os parâmetros relevantes, o método analítico desenvolvido foi aplicado com sucesso em amostras de leite materno. A concentração de amodiaquina analisada foi de 1,7 x 10 -5 mol L-1. O método desenvolvido mostrou-se eficiente e reprodutível na determinação de amodiaquina presente no leite materno, não sendo necessário qualquer tratamento prévio da amostra.

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