Thesis advisor: Daniel A. Kirschner / In the present study, the ability of small compounds to inhibit the fibrillogenesis of beta-amyloid 12-28 was explored. Beta-amyloid 12-28 is a synthetic fragment of Alzheimer's beta-amyloid, which contains the core hydrophobic residues thought to be significant for fiber formation. Using x-ray diffraction, preliminary screening of over sixteen compounds was performed. Cotinine, PTI-00703®, and tetracycline were chosen because of their ease of solubility, the effect on the coherent domain size of the beta-crystallite subunit in the presence of chosen small molecules as shown by x-ray diffraction, as well as their presence in previously published literature. This conformational-driven inhibition of fibrillogenesis was explored in the current research using circular dichroism spectroscopy and x-ray diffraction. Circular dichroism spectroscopy revealed the nascent beta-sheet structure of beta-amyloid12-28 when first dissolved and only cotinine, out of all three inhibitors, was able to shift the equilibrium away from the fibrillogenic beta-sheet structure toward a random coil secondary structure after 36 hours of incubation. X-ray diffraction in this study demonstrated no change in hydrogen bond spacing at ~4.7Å and intersheet spacing at ~10-12Å both alone and in the presence of all small molecules. With increasing concentration of inhibitor, however, the widths of these reflections increased, indicating a decrease in the coherent domain size. / Thesis (MS) — Boston College, 2010. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Second-hand smoke exposure (SHSe) causes significant morbidity and mortality in children. A large proportion of children with smoking parents do not live in smoke-free homes, however, to date, little is known about the prevalence of partial smoking restrictions and their efficacy in reducing children’s SHSe. Given the lack of convincing evidence on how to achieve further reductions in children’s SHSe in the home, the identification of the modifiable factors associated with childhood SHSe is imperative to reduce the burden of disease resulting from childhood SHSe. Analysis of the Omnibus Survey (OS) revealed that the prevalence of smoke-free homes in England did not increase significantly between 2006 and 2008. Only 30% of smokers reported a smoke-free home in 2008. However, during the same time period, the proportion of smokers (who did not have a smoke-free home) reporting that they did not smoke when in the same room as a child increased significantly from 62.5% to 74.8%. Using the Health Survey for England, biologically validated self-reported measures of child SHSe revealed that in 2008 and 2009 approximately 50% of children living with a smoking parent were not exposed to SHSe in the home (0.30ng/ml, 95% confidence interval 0.27-0.32ng/ml). Of the 50% of children who remained exposed inside the home, 29% had a parent that smoked in one room only in the home. These children had significantly lower cotinine concentrations (1.13ng/ml, 95% CI 1.05-1.22) than the 21% of children with smoking parents who smoked in 2 or more rooms in the home (2.36ng/ml, 95% CI 2.08-2.68ng/ml). Although smoking in one room equates to lower risk it does not equate to no risk and so interventions are required to change indoor smoking to outdoor smoking. The OS data found that good knowledge of SHS-related illnesses was predictive of both full and partial smoking restrictions in the home. Increases in the proportion of respondents with good knowledge occurred during 2003-2006, a period when frequent anti-SHS mass media campaigns were aired. A case-study evaluation of a brief mass media campaign in the North West and North East of England, which aimed to move smoking parents to smoke outside, was found to have no statistically significant effect on home smoking behaviour in the short term, however knowledge that SHS caused both heart attack and Sudden Infant Death Syndrome increased in this region following the campaign whilst simultaneous decreases were found in the rest of England. Following the identification of those children most exposed to SHS, and the modifiable factors associated with this exposure, this thesis suggests that a comprehensive multi-level approach to tobacco control policy, which includes emotive media campaigns which include information on SHS-related illnesses, will contribute to the continued reduction of childhood SHSe.

No description available.

No description available.

The median prevalence of smoking among women of childbearing age in the United States is 22.4%. Of women who identify themselves as smokers in the three months prior to conception, 55% quit during pregnancy; however, 40% of those who quit relapse and return to smoking within six months after delivery. Smoking has been identified as an important means of stress management among smokers in general, and though limited to the perinatal period, pregnancy-specific stress adds to a woman’s typical day-to-day stress burden. Little data exists as to the effect of SHS exposure on smoking status during pregnancy and the impact of SHS exposure on the maternal perception of stress is unknown. Due to limited evidence, a critical need exists to examine the relationships of perceived maternal stress, SHS exposure, and perinatal smoking status in order to better understand perinatal smoking behaviors. The purposes of this dissertation were to: 1) evaluate the literature examining the relationship between the variables of maternal stress, SHS exposure, and perinatal smoking status; 2) determine the reliability and validity of the Everyday Stressors Index (ESI) use in pregnant women; and 3) to investigate the impacts of maternal perception of everyday stress, and SHS exposure on perinatal smoking status. Evidence obtained from the critical review of the literature supported an association between psychosocial stress and smoking during pregnancy or postpartum. Little information regarding the role of SHS exposure on perinatal smoking status was discovered. Psychometric testing of the ESI demonstrated strong internal consistency reliability, and factor analysis yielded three factors capturing three important domains of everyday stress. SHS exposure emerged as the most significant predictor of smoking status. Persistent smokers/relapsers had the highest ESI scores, followed by quitters, and then nonsmokers. While ESI means decreased in all smoking status groups from the first to the third trimester, the magnitude of decrease was not predictive. A significant interaction effect of SHS exposure in the home and decrease in ESI score occurred in the quit group only with quitters 1.14 times more likely to experience a decrease in ESI score compared to smokers/relapsers.

Upregulation of nicotinic acetylcholine receptors (nAChRs) is a well-documented response to chronic nicotine exposure. Nicotinic acetylcholine receptors are pentameric ligand-gated ion channels consisting of alpha (α2-10) and beta (β2-4) subunits. Nicotine, an agonist of nAChRs, alters trafficking and assembly of some subtypes of nAChRs, leading to an increase in expression of high sensitivity receptors on the plasma membrane. These physiological changes in nAChRs are believed to contribute to nicotine addiction, although the mechanism of these processes has not been resolved. Recently, many studies have converged on the idea that nicotine induces upregulation by an intracellular mechanism. In this dissertation, expression levels of nAChRs were quantified upon exposure to nicotine and its primary metabolite, cotinine. A pH sensitive variant of GFP, super ecliptic pHluorin (SEP), was integrated with a nAChR subunit to study expression and trafficking of nAChRs by differentiating intracellular and plasma membrane inserted receptors. In this work, cotinine is shown to increase the number of α4β2 nAChRs within a cell. Cotinine also affects trafficking of α4β2, evident by a redistribution of intracellular receptors and an increase in single vesicle insertion events on the plasma membrane. This work shows both nicotine and cotinine alter the overall assembly of α4β2 to favor the high sensitivity (α4)2(β2)3 version. Since cotinine and nicotine induce similar physiological changes in nAChRs, the metabolite potentially plays a role in the mechanism of nicotine addiction. Although an intracellular mechanism for upregulation has been supported, a shift in assembly to the high sensitivity (α4)2(β2)3 version exclusively in the endoplasmic reticulum has not previously been detected. In order to study organelle specific changes in stoichiometry, a novel method was developed to isolate single nAChRs in nanovesicles derived from native cell membranes. Separation of nanovesicles originating from the endoplasmic reticulum and plasma membrane, encompassing isolated nAChRs, allows precise changes in stoichiometry to be monitored in subcellular regions. In this work, single molecule bleaching steps of green fluorescent protein (GFP) encoded in each alpha subunit of the pentamer are detected. The number of bleaching steps, or transitions to a nonfluorescent state upon continuous excitation, corresponds to the number of GFP-labeled alpha subunits present. Therefore, the stoichiometry can be deduced by detection of two bleaching steps, as in (α4)2(β2)3, or three bleaching steps, seen in (α4)3(β2)2. Using this method on isolated nAChRs, a shift to assembly of high sensitivity (α4)2(β2)3 receptors is detected definitively within the endoplasmic reticulum. In addition, an increase in (α4)2(β2)3 receptors located on the plasma membrane is shown when nicotine is present. This work provides convincing evidence that nicotine acts intracellularly, within the endoplasmic reticulum, to alter stoichiometry of nAChRs.

Introduction: Asthma is a common debilitating disease of the airways that afflicts an estimated 300 million worldwide, causing reduction in physical activity, lost school/work days, and even death. There are many known and suspected risk factors of asthma; however, there is much controversy over prior and current cigarette smoking. Approximately 25% of the United States population currently smokes, with a quarter of these being asthma patients. Another 22 to 43 percent of asthmatics are ex-smokers. Objectives: (1) To estimate the prevalence for lifetime asthma in the adult US population; (2) to determine prevalence odds ratios (POR) of lifetime asthma based on questionnaire (smoking status, tobacco consumption) after adjustment of potential confounding variables; (3) to determine POR of lifetime asthma based on laboratory values (serum cotinine); and (4) to assess the validity of self-reported measures (smoking status and tobacco consumption) using serum cotinine as the gold standard.Methods: The National Health and Nutrition Examination Survey (NHANES) 1999-2002 is a proportional cross-sectional sample that uses weights to be representative. Crude odds ratios were obtained through univariate analysis; multiple logistic regression analysis was used to obtain adjusted odds ratios of asthma. Interactions for age, gender, and race/ethnicity were explored. Validity measures included sensitivity and specificity tests for self-reported smoking and non-parametric correlation of tobacco consumption with serum cotinine levels.Results: The overall prevalence of lifetime asthma among n=10,252 adults was 11.56% (95%CI 10.45-12.66). Analyses were stratified by race/ethnicity due to significant interaction. After adjusting for age, gender, body mass index, and family history of asthma, ex-smoking non-Hispanic Whites, non-Hispanic Blacks, and other races had odds ratios of 1.57 (95%CI 1.26-1.97), 1.52 (95%CI 1.01-2.27), and 1.97 (95%CI 1.01-3.83), respectively, relative to never smokers within their respective race/ethnic groups. Sample persons with a family history of asthma and increasing body mass index were significant predictors for lifetime asthma among all race/ethnic groups. Based on laboratory values, non-Hispanic White respondents with serum cotinine levels of 0.011 to Discussion: Self-reported smoking and tobacco consumption are valid measures of tobacco use. The present study found no significant relationship between current smoking and lifetime asthma. Despite the limited findings, asthmatic smokers make up a distinct, difficult-to-treat subgroup for which future treatment research should address.

The human biomonitoring is today an important tool for monitoring of people exposition to environmental pollutants. Mercury and cadmium are long-term monitored biomarkers in the Czech population. A developmental trend of mercury concentration level in urine and hair of children population as well as in urine and blood of adult population is investigated. These biomarkers were monitored in the years 1996 - 2011. Data originate from the NIPH (National Institute of Public Health) databases and from the international project COPHES/DEMOCOPHES. Adult blood donors at age of 18 to 64 were the first monitored group, the second one were children at age of 6 to 12, which were selected based on an agreement with elementary schools or with the paediatricians' cooperation. Available data were statistically processed; trends are presented in graphs as a dependence of geometric means on the time (the year of the biological samples collection). Results show an unstable development of biomarkers levels in different matrixes. A moderate decreasing trend of cadmium level in urine of adults was observed. GM values for adults decreased from 0.43 μg/g creatinine in 2009 to 0.24 μg/g creatinine in 2009. For year 2011, data are available only for women (GM = 0.21 μg/g creatinine). The downward trend was also found in the...

Human biomonitoring represents today an important tool for the monitoring of the human beings exposition to the environmental pollutants. COPHES and DEMOICOPHES projects are address to harmonized approach to human biomonitoring within Europe. This process enables receiving comparable data on the international level. Four biomarkers (mercury, cadmium, kotinin and phthalate metabolites) were selected for this pilot study; all of them are extensively monitored at present. 27 European states at all participated in these projects and procedures were tested in 16 countries of the European Union and the Switzerland. Two localities different in the population density - Prague and Liberec were selected for the Czech Republic. Children of age 6 - 11 and their mothers were selected as the sensitive population groups. Samples of biological material (hair and urine) were taken from the representative individuals. 120 pairs of mother - child were involved in the study. Information about participant's alimentation and life style, inhabitation, education, and smoking habits was gained in questioners. Obtained information was joined with results of analyses of biological material and was evaluated statistically. It was found from results that recorded levels of monitored biomarkers do not exceed substantial healthy limits...

O tabagismo durante a gravidez é o principal fator de risco previsível associado com complicações tanto no parto quanto na gestação. Estão relacionados, por exemplo, partos prematuros, baixo peso ao nascer, doenças no trato respiratório do feto e, com a morte infantil. A exposição à fumaça do tabaco pode ser avaliada através da presença de nicotina e cotinina nos fluidos biológicos, incluindo plasma, soro, urina, saliva, cabelo e unha. A saliva é uma matriz alternativa que apresenta como vantagens a facilidade de obtenção, não é uma amostra invasiva, a coleta pode ser assistida, é de baixo custo, o risco de contração de infecções durante a coleta e manuseio da amostra é mínimo e pode ser utilizada em situações que é difícil a obtenção de outro tipo de amostra. Como a prevalência de gestantes fumantes de cigarro é relativamente alta, é importante uma confirmação por um laboratório de análise para classificar de forma correta e confiável se a gestante é fumante ou não. O objetivo desta pesquisa foi desenvolver e validar um método para análise de nicotina e cotinina em amostras de saliva de gestantes. Foi utilizada a extração líquido-líquido para o preparo das amostras de saliva e foi realizada a cromatografia em fase gasosa com detector termiônico específico para a separação, identificação e quantificação dos analitos nicotina, cotinina e do padrão interno ketamina. O limite de detecção foi de 10 ng/mL para nicotina e de 6 ng/mL para cotinina. Os limites inferiores de quantificação foram de 40 ng/mL e 20 ng/mL para a nicotina e cotinina, respectivamente. O método foi considerado linear na faixa de concentração de 40 a 2000 ng/mL (R=0,996) para a nicotina e de 20 a 2000 ng/mL (R=0,999) para a cotinina. Os valores de recuperação dos analitos variaram de 66,1% a 92,2%. A precisão intradia apresentou uma variação de 6,06% a 11,50% e a precisão interdia variou de 6,89% a 10,57%. A exatidão intradia variou de -11,29% a 19,81%, e os valores da exatidão interdia variaram de -11,78% a 13,08%. Ambos os analitos apresentaram estabilidade nas amostras biológicas e em solução. Na primeira avaliação (período gestacional de 1 a 25 semanas), o método foi aplicado em 259 amostras de saliva de gestantes fumantes e não fumantes. E na segunda (período gestacional de 36 a 40 semanas e até 113 dias após o parto), foi aplicado em 45 amostras de gestantes fumantes. Foi observada uma diferença significativa ao comparar o autorrelato das gestantes fumantes e não fumantes, na primeira avaliação, com a concentração de nicotina e cotinina nas amostras de saliva (p<0,001). As concentrações de cotinina em amostras de saliva correlacionaram positivamente com o número de cigarros consumidos diariamente pelas gestantes fumantes, na primeira avaliação (p<0,001). Não foram obtidas diferenças estaticamente significativas para as variáveis: número de cigarros diários (p=0,255), concentração de nicotina (p=0,949) e cotinina (p=0,665). O ponto de corte da nicotina foi maior que zero, com sensibilidade de 36,8% e especificidade de 97,7%. Para a cotinina, o ponto de corte foi maior que 17,2 ng/mL, com sensibilidade de 94,3% e especificidade de 98,8%. O método analítico desenvolvido e validado atendeu aos critérios exigidos pela resolução 27/2012 e à 899/2003 da Agência Nacional de Vigilância Sanitária. / Smoking during pregnancy is the main factor associated with complications in both and gestation. It is related, for example, premature births, low birth weight, diseases in the respiratory tract of the fetus and, with child death. Exposure to tobacco smoke can be assessed by the presence of nicotine and cotinine in biological fluids including plasma, serum, urine, saliva, hair and nails. The saliva is an alternative matrix that presents as advantages the ease of obtaining, is not an invasive sample, the collection can be assisted, it is of low cost, the risk of contraction of infections during the collection and handling of the sample is minimal and can be used in situations where it is difficult to obtain another type of sample. As the prevalence of pregnant cigarette smokers is relatively high, confirmation by an analysis laboratory is important to correctly and reliably classify whether the pregnant woman is a smoker or not. The objective of this research was to develop and validate a method for the analysis of nicotine and cotinine in saliva samples from pregnant women. Liquid-liquid extraction was used for the preparation of saliva samples and gas chromatography with specific thermionic detector was performed for the separation, identification and quantification of the analytes nicotine, cotinine and the internal standard ketamine. The limit of detection was 10 ng/mL for nicotine and 6 ng/mL for cotinine. The lower limits of quantification were 40 ng/mL and 20 ng/mL for nicotine and cotinine, respectively. The method was considered linear in the concentration range of 40 to 2000 ng/mL (R=0.996) for nicotine and 20-2000 ng/mL (R=0.999) for cotinine. The recovery values of the analytes range from 66.1% to 92.2%. The intraday precision ranged from 6.06% to 11.50% and the interday precision ranged from 6.89% to 10.57%. The intraday accuracy ranged from -11.29% to 19.81%, and the interday accuracy ranged from -11.78% to 13.08%. Both analytes presented stability in the biological samples and in solution. At the first evaluation (gestational period of 1 to 25 weeks), the method was applied to 259 saliva samples from pregnant smokers and nonsmokers. And in the second (gestational period of 36 to 40 weeks and up to 113 days postpartum), it was applied in 45 samples of pregnant smokers. A significant difference was observed when comparing the self-report of pregnant smokers and nonsmokers in the first evaluation with the concentration of nicotine and cotinine in saliva samples (p<0.001). The concentrations of cotinine in saliva samples correlated positively with the number of cigarettes consumed daily by pregnant smokers in the first evaluation (p<0.001). No statistically significant differences were found for the variables: number of daily cigarettes (p=0.255), nicotine concentration (p=0.949) and cotinine (p=0.665). The cutoff point of nicotine was greater than zero, with sensitivity of 36.8% and specificity of 97.7%. For cotinine, the cutoff point was greater than 17.2 ng/mL, with sensitivity of 94.3% and specificity of 98.8%. The analytical method developed and validated met the criteria required by resolution 27/2012 and 899/2003 of the National Sanitary Surveillance Agency.