Estudo do promotor do gene da cadeia alfa 2 do colageno tipo I em portadores de fibromatoge gengival hereditaria

Orientador: Sergio Roberto Peres LineDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: Há grande variação interindividual de fibroblastos gengivais em relação à síntese de proteínas da matriz. Esta variabilidade está bem documentada em casos de reação fibrótica gengival induzida por drogas, mas o mesmo não acontece em relação à Fibromatose Gengival Hereditária (FGH), condição em que há aument9 da secreção de colágeno normal na matriz extracelular. Condições particulares individuais como polimorfismos genéticos ou mutações podem influir na resposta de fibroblastos a estímulos do ambiente. Assim, procuramos identificar polimorfismos na região do promotor da cadeia alfa 2 do colágeno tipo 1 (COLIA2) em um grupo de pacientes normais (n=12) e outro de portadores de FGH (n=13). Além disso, também testamos a hipótese de alteração no padrão de metilação destaseqüência estar relacionada ao aumento da expressão do colágeno do tipo I em portadores de FGR. Primeiramente foi feita a amplificação por PCR da região do promotor da cadeia alfa 2 do colágeno entre -340 e +2 pares de base(bp), porção do promotor que possui mais seqüências reconhecidas como determinantes na interação com fatores de transcrição. A ste passo se seguiu a análise de heteroduplexes para a identificação de polimorfismos. Esta análise foi feita do seguinte modo: aquecimento das amostras a 98°C por 5 minutos, resfi-iamento a O°C e manutenção a 20°C por uma hora. A detecção de heteroduplexes foi feita em gel de poliacrilamida a 6%/ Tampão TBE, corado pelo método da prata. A análise de metilação foi feita através do uso de enzimas de restrição (HAL In e HP A n, que não clivam as suas seqüências de reconhecimento de DNA quando estas seqüências estiverem metiladas). Previamente à reação de PCR, o DNA foi digerido com as enzimas citadas. A metilação permitiria a amplificação da seqüência de DNA que estivesse metilada entre as regiões dos primers utilizados. Após a amplificação, as amostras foram submetidas a eletroforese em gel de poliacrilamida a 6%/ Tampão TBE, corado pelo método da prata.Não houve diferença na migração dos fi-agmentos' de DNA após a reação de heteroduplexes, nem foi detectada alteração no padrão de metilação entre os dois grupos de indivíduos. Estes achados indicam que mutações ou alteração no padrão de metilação da região do promotor do COLIA2 compreendida entre -340 e +2 bp provavelmente não estejam relacionadas ao crescimento gengival de portadores de FGRAbstract: There is a great interindividual variation in gingival fibroblasts capacity for synthesizing extracellular matrix proteins. This variability is well-documented in patients exhibiting drug-induced gingival overgrowth, but it is still poorly understood in patients with Hereditary Gingival Fibromatosis (HGF). There is an increase in the amount of normal collagen secreted into the extracellular matrix of the gingiva of patients with HGF. IndividuaIs may carry genetic polymorphisms or mutations, which could determine the partem of fibroblasts response to environmental stimuli.hus, we tried to identify genetic polymorphisms in the promoter of the collagen type I alfa 2 chain (COLIA2) in a group of normal subjects (n=12) and another group of patients with HGF (n=13). Besides, we also tested the hypothesis that undermethylation of this sequence could be related to the overexpression of type I collagen in these patients. lnicially we amplified the COLIA2 promoter region from -340 to +2 bp by PCR, since this is the promoter region were most cis-acting-factors have been identified until now. Thereafter, heteroduplex analysis was performed by heating the samples 5 rninutes to 98°C, chilling at O°C, and maintaining them one hour at 20°C. The samples were run in a 6% polyacrilarnidel TBE gel, which was stained with silver for heteroduplexes detection. Methylation analysis was carried out using restriction enzymes which do not cut DNA when their recognition sites are methylated (HAL 111 e HPA 11). DNA was digested by these enzymes prior to PCR. Successful amplification of a selected fragment of the promoter where restriction sites of these two enzymes are found would mean that the sequence was methylated. The samples were run in a 6% polyacrilarnidel TBE gel, which was stained with silver. There was no difference in the rnigration of DNA fragments after the heteroduplex reaction, neither methylation partem alteration was detected between the two groups analysed. This may indicate that mutations or methylation pattern alteration in the promoter region of the COLIA2 reaching from -340 to +2 bp are probably not involved in the gingival overgrowth affecting patients with HGFMestradoBiologia e Patologia Buco-DentalMestre em Ciência

Sex determination of human remains from peptides in tooth enamel

The assignment of biological sex to archaeological human skeletons is a fundamental requirement for the reconstruction of the human past. It is conventionally and routinely performed on adults using metric analysis and morphological traits arising from postpubertal sexual dimorphism. A maximum accuracy of ∼95% is possible if both the cranium and os coxae are present and intact, but this is seldom achievable for all skeletons. Furthermore, for infants and juveniles, there are no reliable morphological methods for sex determination without resorting to DNA analysis, which requires good DNA survival and is time-consuming. Consequently, sex determination of juvenile remains is rarely undertaken, and a dependable and expedient method that can correctly assign biological sex to human remains of any age is highly desirable. Here we present a method for sex determination of human remains by means of a minimally destructive surface acid etching of tooth enamel and subsequent identification of sex chromosome-linked isoforms of amelogenin, an enamel-forming protein, by nanoflow liquid chromatography mass spectrometry. Tooth enamel is the hardest tissue in the human body and survives burial exceptionally well, even when the rest of the skeleton or DNA in the organic fraction has decayed. Our method can reliably determine the biological sex of humans of any age using a body tissue that is difficult to cross-contaminate and is most likely to survive. The application of this method will make sex determination of adults and, for the first time, juveniles a reliable and routine activity in future bioarcheological and medico-legal science contexts

Biomarkers of exposure to lead

This article aims at describing some of the most important biomarkers of exposure to lead, showing its advantages and limitations, as well as how they relate to present, past or cumulative exposure to lead. This basic knowledge is essential for the comprehension of the effects of lead, the difficulties of the diagnosis of excessive exposure, and the problem of exposure to low levels of lead. The biomarkers of exposure discussed in depth in this article are blood, bone, and teeth.O objetivo deste artigo é descrever alguns dos principais biomarcadores de exposição a chumbo, mostrando suas vantagens e limitações, assim como sua relação com exposição presente, passada ou cumulativa. Essas noções básicas são fundamentais para a compreensão dos efeitos do chumbo, da dificuldade do diagnóstico e do problema da exposição a baixas doses. Os biomarcadores de exposição a chumbo mais discutidos neste artigo são sangue, osso e dentes

The use of a DNA stabilizer in human dental tissues stored under different temperature conditions and time intervals

Objective: The present study evaluated the use of a reagent to stabilize the DNA extracted from human dental tissues stored under different temperature conditions and time intervals. Material and Methods: A total of 161 teeth were divided into two distinct groups: intact teeth and isolated dental pulp tissue. The samples were stored with or without the product at different time intervals and temperature. After storage, DNA extraction and genomic DNA quantification were performed using real-time PCR; the fragments of the 32 samples that represented each possible condition were analyzed to find the four pre-selected markers in STR analysis. Results: The results of the quantification showed values ranging from 0.01 to 10,246.88 ng/μL of DNA. The statistical difference in the quantity of DNA was observed when the factors related to the time and temperature of storage were analyzed. In relation to the use of the specific reagent, its use was relevant in the group of intact teeth when they were at room temperature for 30 and 180 days. The analysis of the fragments in the 32 selected samples was possible irrespective of the amount of DNA, confirming that the STR analysis using an automated method yields good results. Conclusions: The use of a specific reagent showed a significant difference in stabilizing DNA in samples of intact human teeth stored at room temperature for 30 and 180 days, while the results showed no justification for using the product under the other conditions tested

Contrasting effects of aliskiren versus losartan on hypertensive vascular remodeling

Background: Hyperactivation of the renin-angiotensin system contributes to hypertension-induced upregulation of vascular matrix metalloproteinases (MMPs) and remodeling, especially in the two kidney, one clip (2K1C) hypertension model. We hypothesized that the AT(1)R antagonist losartan or the renin inhibitor aliskiren, given at doses allowing similar antihypertensive effects, could prevent in vivo vascular MMPs upregulation and remodeling, and collagen/elastin deposition found in 2K1C hypertension by preventing the activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and transforming growth factor-beta(1) (TGF-beta(1)). We also hypothesized that aliskiren could enhance the effects of losartan.Methods: 2K1C rats were treated with aliskiren (50 mg.kg(-1).day(-1)), or losartan (10 mg.kg(-1).day(-1)), or both by gavage during 4 weeks.Results: Aliskiren, losartan, or both drugs exerted similar antihypertensive effects when compared with 2K1C rats treated with water. Aliskiren reduced plasma renin activity in both sham and 2K-1C rats. Losartan alone or combined with aliskiren, but not aliskiren alone, abolished 2K1C-induced aortic hypertrophy and hyperplasia, and prevented the increases in aortic collagen/elastin content, MMP-2 levels, gelatinolytic activity, and expression of phospho-ERK 1/2 and TGF-beta(1). No significant differences were found in the aortic expression of the (pro) renin receptor.Conclusions: These findings show that although losartan and aliskiren exerted similar antihypertensive effects, only losartan prevented the activation of vascular profibrotic mechanisms and MMP upregulation associated with vascular remodeling in 2K1C hypertension. Our findings also suggest that aliskiren does not enhance the protective effects exerted by losartan. (C) 2012 Elsevier Ireland Ltd. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ São Paulo, Fac Med Ribeirao Preto, Dept Pharmacol, BR-14049900 Ribeirao Preto, SP, BrazilUniv São Paulo, Fac Med Ribeirao Preto, Dept Biochem & Immunol, BR-14049900 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, BrazilUniv São Paulo, Dent Sch Ribeirao Preto, Dept Morphol Estomatol & Physiol, BR-14049900 Ribeirao Preto, SP, BrazilUniversidade Federal de São Paulo, Dept Med, Div Nephrol, São Paulo, BrazilWeb of Scienc

Chemical and physical factors influencing lead and copper contamination in drinking water: approach for a case study in analytical chemistry

CHEMICAL AND PHYSICAL FACTORS INFLUENCING LEAD AND COPPER CONTAMINATION IN DRINKING WATER: APPROACH FOR A CASE STUDY IN ANALYTICAL CHEMISTRY. Lead and copper concentrations in drinking water increase considerably on going from municipality reservoirs to the households sampled in Ribeirao Preto (SP-Brazil). Flushing of only 3 liters of water reduced metal concentrations by more than 50%. Relatively small changes in water pH rapidly affected corrosion processes in lead pipes, while water hardness appeared to have a long-term effect. This approach aims to encourage University teachers to use its content as a case study in disciplines of Instrumental Analytical Chemistry and consequently increase knowledge about drinking water contamination in locations where no public monitoring of trace metals is in place

Systemically alendronate was incorporated into dental tissues but did not cause morphological or mechanical changes in rats teeth

This study evaluated the effect of the systemic use of sodium alendronate in rats in vivo. Forty-five Wistar rats aged 36 to 42 days and weighing 200 to 230 g were randomly assigned to a control group (n = 20), which received distilled water, and an experimental group (n = 25), which received 2 weekly doses of 1 mg/kg of chemically pure sodium alendronate. The animals were killed after 60 days of treatment. The tibias were removed for analysis of bone mineral density by dual-energy X-ray absorptiometry (DXA). Then, the maxillary incisors were extracted for analysis of the mineralized dental tissues using fluorescence spectroscopy (FS), scanning electron microscopy (SEM), bright field microscopy (BFM), and cross-sectional microhardness (CSMH) testing. DXA and CSMH data were subjected to statistical analysis by Kruskal-Wallis test (5% significance level). The experimental group presented higher bone mineral density than the control group by DXA. FS analysis revealed presence of alendronate in the mineralized dental tissues of the specimens of the experimental group. Significant morphological differences were not found by SEM and BFM. Enamel and dentin (100 and 300 mu m from the dentinoenamel junction) CSMH data did not show significant difference between the control and experimental groups. Based on the obtained results, we conclude that while alendronate increased the bone mineral density and was incorporated into the mineralized dental tissues it did not cause significant alterations in the morphology and microhardness of rat incisor enamel and dentin. Microsc. Res. Tech. 75:12651271, 2012. (C) 2012 Wiley Periodicals, Inc.Brazilian Ministry of Healths Federal Agency for Support and Evaluation of Graduate Education (CAPES

A Critical Review of Biomarkers Used for Monitoring Human Exposure to Lead: Advantages, Limitations, and Future Needs

Lead concentration in whole blood (BPb) is the primary biomarker used to monitor exposure to this metallic element. The U.S. Centers for Disease Control and Prevention and the World Health Organization define a BPb of 10 μg/dL (0.48 μmol/L) as the threshold of concern in young children. However, recent studies have reported the possibility of adverse health effects, including intellectual impairment in young children, at BPb levels < 10 μg/dL, suggesting that there is no safe level of exposure. It appears impossible to differentiate between low-level chronic Pb exposure and a high-level short Pb exposure based on a single BPb measurement; therefore, serial BPb measurements offer a better estimation of possible health outcomes. The difficulty in assessing the exact nature of Pb exposure is dependent not so much on problems with current analytical methodologies, but rather on the complex toxicokinetics of Pb within various body compartments (i.e., cycling of Pb between bone, blood, and soft tissues). If we are to differentiate more effectively between Pb stored in the body for years and Pb from recent exposure, information on other biomarkers of exposure may be needed. None of the current biomarkers of internal Pb dose have yet been accepted by the scientific community as a reliable substitute for a BPb measurement. This review focuses on the limitations of biomarkers of Pb exposure and the need to improve the accuracy of their measurement. We present here only the traditional analytical protocols in current use, and we attempt to assess the influence of confounding variables on BPb levels. Finally, we discuss the interpretation of BPb data with respect to both external and endogenous Pb exposure, past or recent exposure, as well as the significance of Pb determinations in human specimens including hair, nails, saliva, bone, blood (plasma, whole blood), urine, feces, and exfoliated teeth

Efeito do fluor e de alguns metais na atividade de proteinases da matriz esmalte in vitro

Orientador: Sergio Roberto Peres LineTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O esmalte dental é um tecido epitelial altamente mineralizado, que recobre os dentes dos vertebrados. Em contraste com os tecidos mineralizados conjuntivos, os cristais no esmalte não são depositados sobre uma matriz rica em colágeno, mas crescem concomitantemente à remoção da maioria das proteínas da matriz do esmalte, as quais são gradualmente degradadas durante a mineralização. Existem múltiplas evidências para um papel importante das proteases presentes na matriz do esmalte nesta degradação. A inibição da atividade de proteases presentes no esmalte poderia comprometer a perfeita mineralização do esmalte. Neste trabalho testou-se in vítro o efeito de flúor e de alguns metais na atividade de proteases presentes na matriz do esmalte. Três ensaios foram utilizados: um ensaio de ativídade enzimática usando um substrato colori métrico (azocaseína), um ensaio de degradação de proteínas estruturais do esmalte e zimografia. Os resultados mostraram que o flúor (625 uM to 10 mM) não exibe efeito inibitório sobre a atividade das proteases, o que é um dado relevante para a discussão dos possíveis mecanismos patogênicos da fluorose dentária. Os resultados da inibição com alguns metais revelaram que zinco, cádmio e chumbo têm um efeito inibitório em concentrações de até 110 uM. Estes últimos resultados são interessantes à medida em que se sabe que estes metais são incorporados ao esmalte durante o desenvolvimento em concentrações que refletem a exposição durante a formação dos tecidos dentais, e, ainda, porque existe uma associação ainda não explicada entre aumento de cárie e exposição a metais pesados, particularmente cádmio e chumboAbstract: Dental enamel is a highly mineralized epithelial tissue found on mammalian teeth. In contrast to the connective mineralized tissues, enamel crystallites are not deposited onto a collagenous matrix, but grow concomitantly with the removal of most of the enamel matrix proteins, which are gradually degraded during mineralization. There are many evidences indicating that enamel resident proteinases play an important role in the degradation of proteins. Inhibition of enamel matrix proteinases could impair normal enamel mineralization In this work, we investigated the effect of fluoride and some metais on the activity of enamel matrix proteinases in vitro using a colorimetric assay (azocasein), an enamel protein degradation assay and zymography. The results showe that fluoride (625 uM to 10 mM) does not have an inhibitory effect on proteinase activity. This is a relevant finding conceming possible pathogenic mechanisms underlying dental fluorosis. The results of the inhibition by some metais revealed that zinc, cadmium and lead cause inhibition at concentrations as lowas 110 uM. These results are interesting due to the fact that dental tissues are known to harbor these metais in concentrations related to the exposure at the time of dental tis sues formation, and, furthermore, because there is an unexplained link between increased caries prevalence and exposure to heavy metais, particularly cadmium and leadDoutoradoBiologia CelularDoutor em Biologia Celular e Estrutura