Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 5 uppsatser.

Orientadores: Nelci Fenalti Hoehr, Roberto Rittner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T17:11:44Z (GMT). No. of bitstreams: 1 Oliveira_AndreNazariode_M.pdf: 1693019 bytes, checksum: c0e1f56be54157760e94e4822c8d9f5f (MD5) Previous issue date: 2009 / Resumo: Este projeto tem a finalidade de analisar as características toxicológicas de novos compostos quinazolínicos, que foram sintetizados recentemente no Instituto de Química-UNICAMP. Uma série de derivados de 4-fenilamino quinazolinas foi sintetizada como potentes inibidores da proteína quinase e sua citotoxicidade foi demonstrada através de técnicas de Inibição de Crescimento de 50% da população celular, onde procuramos estabelecer alguns efeitos biológicos desses novos compostos quinazolínicos, através de cultura de células PC12 e de cultura de células tumorais humanas, recomendadas pelo NCI (The U.S. National Cancer Institute) para ensaios anti-proliferiativos para estudos do câncer. A utilização da metodologia de "docking molecular" nos levou ao descobrimento do sítio de ação dos compostos quinazolínicos em células tumorais de mama (MCF07). Estudos com camundongos foram realizados, utilizando a metodologia recomendada pela OECD (Organisation for Economic Co-operation and Development), para determinação in vivo da toxicidade aguda pelos compostos quinazolínicos, descrevendo os efeitos causados pelas diferentes concentrações dos compostos quinazolínicos em estudo aplicadas aos animais. Através da Espectrometria de Ressonância Magnética Nuclear de Hidrogênio (¹H) pôde-se identificar o metabólito final das catecolaminas, o ácido homovanílico em urina, escolhido por ser um composto que aparece em grandes concentrações na urina e por ser de excelente precisão diagnóstica em algumas doenças neurológicas. / Abstract: This project aims to analyze the toxicological characteristics of new quinazoline compounds, which were summarized recently in the Institute of Chemistry-UNICAMP. The compound 4-phenylamino quinazoline was synthesized as a potent inhibitor of protein kinase and its cytotoxicity was demonstrated by an inhibitory activity on growth of human tumor cell lines. Through techniques of 50% Growth Inhibition of cell lines we provide some biological effects of these new quinazoline compounds through PC12 cell culture and culture of human tumor cells, as recommended by the NCI (The U.S. National Cancer Institute) for testing anti-proliferative for studies of cancer. The use of the methodology of "molecular docking" has led to the discovery the binding site of action of quianzoline compounds in breast tumor cells (MCF07). Studies with mice were performed, using the methodology recommended by the OECD (Organisation for Economic Co-operation and Development), to determine in vivo toxicity of the quinazoline compounds, describing the effects caused by different concentrations of quinazoline compounds under study applied to animals. Through the Nuclear Magnetic Resonance Spectrometry of Hydrogen (¹H) was able to identify the final metabolite of catecholamines, the acid in urine homovanílico, chosen to be a compound that appears in high concentrations in urine and to be an excellent diagnostic marker in some neurological diseases. / Mestrado / Ciencias Biomedicas / Mestre em Ciências Médicas

Lead (Pb²⁺) is an environmental toxin that is known to cause lasting cognitive deficits following early life exposure. Previously, our laboratory demonstrated increased sensitivity to the psychostimulant effects of cocaine in animals with elevated blood Pb²⁺ levels (BLL). This effect was abolished following introduction of dopamine (DA) receptor antagonists, indicating that the dopaminergic (DAergic) system may be a target of Pb²⁺’s toxic effects. However, the biological mechanisms through which Pb²⁺ increased sensitization to cocaine’s psychostimulant effects have not been fully elucidated. There is some disagreement regarding the magnitude and direction of Pb²⁺’s effects on the DAergic system. Furthermore, many studies to date have measured the effects of Pb²⁺ in only one sex (usually male), one exposure, and one or two time-points, making it difficult to determine any potential sex-, age-, and exposure-dependent effects. In the present study, we used a well-validated animal model and Pb²⁺ exposure paradigm that uses chronic dietary exposure to 180ppm and 1500ppm Pb²⁺ acetate (PbAC) in the diet. These levels of Pb²+ in the diet resulted in low and moderate levels of BLLs that on average approximated 4.5 and 22.0µg/dl in young adult rats. These levels of Pb²⁺ exposure are relevant to contemporary levels of BLL in intoxicated children in many cities in the United States and in many parts of the world where Pb²⁺ exposure continues to be a major public health concern. It should be noted that at the low level of Pb²⁺ exposure, the resulting BLL of 4.5µg/dl is just below the current CDC level of action. Using this well-defined rat model of chronic Pb²⁺ exposure, in Aim 1, we measured DA concentration and turnover in the dorsal striatum (STR) of juvenile (PN14), adolescent (PN28), and young adult (PN50) male and female rats. Tyrosine hydroxylase (TH) protein, the rate-limiting step in the synthesis of DA, and phosphorylation of TH at serine 40 (pser40TH) were assessed as an indirect measure of TH activity. Thus, we measured the ratio of pser40TH to total TH protein. We also measured vesicular monoamine transporter-type 2 (VMAT2) levels in the STR, nucleus accumbens (NAC), and olfactory tubercle (OT) since this protein is critical for the sequestration of DA in presynaptic vesicles and has been used as a biomarker for DA terminal integrity. In Aim 2, we examine the effect of chronic Pb²⁺ exposure on D1 and D2 dopamine receptor (D1R and D2R) in the OT, NAC, and STR. Analysis of D1R and D2R is important since the downstream effects of DA are dependent on the DA receptor subtype it activates. In Aim 1, we observed significant increases in DA and its metabolites homovanillic acid (HVA) and 3,4-Dihydroxyphenylacetic acid (DOPAC) in the STR of adolescent and young adult male rats with BLL as low as 4.5µg/dl in the absence of phosphorylation at the serine 40 residue of TH or altered VMAT2 levels. In Aim 2, a significant increase in D2R was detected in the juvenile male rat STR. We also observed increases in D1R expression in adolescent male rats in the NAC, OT, STR, and in the OT of adolescent female rats. Together, these results demonstrate that chronic Pb²⁺ exposure alters DA receptor levels in a manner characteristic of a hyperactive DAergic state. The observations presented in this work suggest that a hyperactive DAergic system underlies the heightened sensitization to cocaine we previously observed in Pb²⁺-exposed animals. This work builds upon the current understanding of how Pb²⁺ modulates the DAergic system and provides some elucidation of the mechanisms underlying increased drug sensitization our laboratory has previously observed in rats exposed to Pb²⁺.

The objective of the present work is the study of the electrochemical behaviour of two diagnostic tumor markers of great importance, vanillylmandelic acid (VMA) and homovanillic acid (HVA), on carbon-based electrodes by using cyclic and differential pulse voltammetry. A comparison was made among non-modified glassy carbon electrode (GCE) and GCE modified by multi-walled carbon nanotubes, Nafion (Nafion/GCE) and poly(neutral red) (PNR/GCE), and further boron doped diamond (BDD) electrode which was activated by anodic polarization (Eakt = +2,4 V, t = 30 s) or by polishing on alumina slurry. Significant differences in the voltammetric responses of VMA and HVA were found, not only among utilized electrode materials which also influenced the way of controlling the oxidation process, but also they depend on the pH value of aqueous media in which these acids occurred. An acidic environment is the most suitable for their determination. Calibration dependences were measured in 0.1 mol∙l−1 phosphate buffer pH 3.0 which was chosen as an optimal supporting electrolyte for differential pulse voltammetric determination. Achieved detection limits were 0.6, 0.9, 0.8 and 1.2 μmol∙l−1 for HVA and 0.4, 1.5, 2.4 and 1.1 μmol∙l−1 for VMA at BDD electrode, non-modified GCE, Nafion/GCE and PNR/GCE, respectively. Limits...

In this master s thesis, electrochemical determination of homovanillic acid (HVA) andˈ vanillylmandelic acid (VMA) was performed on carbon paste electrode. The purpose of this thesis was finding out optimal conditions for determination of these analytes by ndifferential pulse voltammetry (DPV) and high pressure liquid chromatography with a carbon paste electrode as an electrochemical detector (HPLC-ED). The electrochemical behavior of the compounds depends mainly on pH of the solution; the best results are obtained in acidic medium. The limits of detection for DPV method were for HVA 0,38 μmol·dm-3 and for VMA 0,25 μmol·dm-3 . The limits of detection for HPLC-ED were for HVA 0,7 μmol·dm-3 and for VMA 0,2 μmol·dm-3 . Developed method was successfully used for the determination of homovanillic and vanillylmandelic acid in urine samples. With HPLC - ED the limits of detection 0,8 μmol·dm-3 (HVA) and 0,5 μmol·dm-3 (VMA) were achieved. Key words: Homovanillic acid, vanillylmandelic acid, carbon paste electrode, DPV, HPLC

Presented dissertation thesis is focused on the development of electrochemical methods for the determination of three important tumour biomarkers, namely homovanillic acid (HVA), vanillylmandelic acid (VMA), and 5-hydroxyindole-3-acetic acid (5-HIAA). First part of the study is focused on electrochemical behaviour of these analytes in batch arrangement using differential pulse voltammetry (DPV) at screen-printed carbon electrodes (SPCEs). It has been proved that presented method is sufficiently sensitive for monitoring above mentioned analytes. Moreover, it can be used for determination of HVA and VMA in mixture. Obtained limits of detection (LODs) were 0.24 µmol·L-1 for HVA, 0.06 µmol·L-1 for VMA, and 0.12 µmol·L-1 for 5-HIAA. The requirements to speed up the analysis and at the same time to reduce its price initialized our study of the determination of tested biomarkers in flow systems. Firstly, flow injection analysis with amperometric detection was investigated for the determination of all three biomarkers at the same SPCE, and then an analogous determination of structural more similar pair, HVA and VMA, was performed at a boron doped diamond electrode (BDDE). Obtained LODs of optimized methods were as follows: at SPCE 0.07 µmol·L-1 for HVA, 0.05 µmol·L-1 for VMA, and 0.03 µmol·L-1 for 5-HIAA,...