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An investigation of the factors influencing the inhibitory and toxic effects of ethanol and higher alcohols, byproducts of alcoholic fermentation, on yeast, is presented. The relative potency of alcohols was found to correlate strongly with the carbon chain-length or molecular size and the lipid solubility of the respective alcohols. Higher alcohols act synergistically with each other and with ethanol in causing cell death of suspensions of non-growing Saccharomyces cerevisiae. The presence of higher alcohols in fermented broth, even at low concentrations, and other by-products of alcoholic fermentation, could explain the higher potency of ethanol produced during fermentation compared to added ethanol. The kinetics of uptake of labelled ethanol supplied at different concentrations gave no evidence of enzymic involvement in the ethanol uptake process. The rate of release of labelled ethanol by cells fed labelled glucose paralled the rate of p14sC-C0b2s release. This does not support the view that ethanol accumulates within the cells to higher concentrations than occur in the medium. Supplementation of a basal synthetic medium with various nutrients did not confer additional survival capacity on yeast against the adverse effects of alcohol. Osmotic pressure did not influence alcohol toxicity below 10% (w/v) sorbitol equivalent of osmotic pressure. Alcohol toxicity is not influenced by hydrogen ion concentration (pH) over a range of pH 5.3 to 3.5.

Raman spectroscopy, together with multivariate statistical analyses, has proven to be a near real-time analytical technique capable of phenotyping cells, tissues and organs. This dissertation will show exclusively the application of the Raman spectroscopy phenotypic profiling method to; (i) microbial toxicity, (ii) ex-vivo organ perfusion, and (iii) subcellular location targeting. Real-time analytical methods for monitoring living biological systems will enable study of the physiological changes associated with growth, genetic manipulations, and adverse environmental conditions. Most existing analytical methods (NMR exempt), though highly accurate, must be performed off-line and most require destruction of the studied sample. These attributes make these methodologies less desirable to the study of physiological changes of cells, tissues, and organs. In this work, Raman spectroscopy has been identified and shown to be a good candidate for real-time analysis mainly because it can be performed: (i) in near real-time, (ii) non-destructively and with minimal sample preparation, (iii) through a glass barrier (i.e., can be performed in situ), and (iv) with minimal spectral interference from water. Here, Raman spectroscopy was used in combination with multivariate statistics to analyze the differing toxic effects of 4-C chain alcohols on E. coli. Good correlations were established between Raman spectra and off-line analytical techniques used to measure: (i) saturated, unsaturated, and cyclopropane fatty acids; (ii) amino acid composition of total protein; and (iii) cell membrane fluidity. Also, Raman 'fingerprint' analysis was used to discriminate among different phenotypic responses of cells. In addition, this methodology was applied to analyze perfusates of organs maintained by the VasoWave® organ perfusion system. Raman fingerprints can be used to assess organ health, and it is believed this data can be used to inform decisions such as whether or not to transplant an organ. Finally, molecular biology techniques were used to design and produce specific protein targets harboring a silver binding domain fusion, which upon release migrate to specific subcellular locations. By employing the related technique of surface-enhanced Raman scattering (SERS), which produces a highly amplified Raman signal in the presence of metallic nanoparticle substrates (e.g., silver nanoparticles), different regions of the E. coli cell structure were studied. The target regions studied by the technique included: (i) outer cell membrane, (ii) periplasm, and the (iii) cytoplasm. / Ph. D.

O álcool é uma das substâncias psicoativas mais consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, tem representado grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de ingerir bebidas alcoólicas durante a gestação ser bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. O consumo excessivo de álcool durante a gravidez tem sido associado com a síndrome fetal pelo álcool (FAS), caracterizada por crianças com dificuldades comportamentais e de aprendizado. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado álcool durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal ao etanol. Dentre os marcadores utilizados estão os ésteres etílicos de ácidos graxos (FAEE), que podem ser detectados em amostras de mecônio de recém-nascidos. No presente trabalho, um método analítico foi desenvolvido visando a detecção de oito FAEEs em amostras de mecônio e aplicada em amostras coletadas de recém-nascidos cujas mães admitiram ou não o uso de etanol durante a gestação. A microextração em fase sólida por Headspace (HS-SPME), uma técnica de preparação de amostras relativamente recente, foi utilizada para análise. Os FAEEs foram identificados e quantificados por cromatografia gasosa/espectrometria de massas (GC/MS), operado no modo de ionização química. Os correspondentes ésteres etílicos deuterados foram sintetizados e utilizados como padrão interno. Os limites de quantificação (LOQ) obtidos foram abaixo de 150 ng/g e limites de detecção (LOD) foram abaixo de 100ng/g para todos os analitos. O método mostrou boa linearidade na concentração estudada (LOQ-2000ng/g), com coeficiente (r2) melhor que 0.98. Os valores de precisão apresentaram coeficientes de variação menores que 15% para todos os FAEEs estudados. Quando o método foi aplicado em amostras de mecônio, foi possível detectar níveis de alguns FAEEs de recém-nascidos não suspeitos a exposição fetal ao etanol. / Alcohol is the main psychoactive drug consumed worldwide and its increasing use by young women has been a great problem point out by specialists in the subject. Although the adverse effects associated to the habit of drinking alcoholic beverages during gestation being very much documented, it is known that a considerable number of pregnant women have difficulties to abandon the habit. Excessive alcohol use during pregnancy has been associated with Fetal Alcohol Syndrome (FAS) characterized by children with cognitive and behavioral disorders. However, because of denial, embarrassment and fear, maternal reports of gestational use of alcohol are often inaccurate. Consequently, a series of biomarkers have been studied to diagnose fetal exposure to alcohol. Recently, fatty acid ethyl esters (FAEE) have been studied as biomarkers found in meconium of neonates exposed in utero. In the present work, an analytical method was developed aiming the detection of eight FAEEs in meconium samples and applied to real specimens collected from newborns whose mothers admitted or not the use of alcohol during pregnancy. Headspace solid-phase microextraction (HS-SPME), a relatively recent sample preparation technique, was used for analysis. FAEEs were identified and quantified by gas chromatography/mass spectrometry (GC/MS), operated in chemical ionization mode. The corresponding deuterated ethyl esters were synthesized and used as internal standards. The lower limits of quantification (LOQ) obtained were below 150ng/g and limits of detection (LOD) were bellow 100ng/g for all analytes. The method showed good linearity in the range of concentration studied (LOQ-2000ng/g), with coefficient of linearity better than 0.98. The precision assay, given by the relative standard deviation (RSD) of the method was lower than 15% for all FAEEs studied. When the method was applied to real samples, it was possible to detect trace levels of some FAEEs from non-suspected ethanol exposed newborns.

O álcool é uma das substâncias psicoativas mais consumidas mundialmente e seu uso por mulheres em idade reprodutiva, em particular, tem representado grande preocupação por parte de especialistas e da sociedade em geral. Apesar dos efeitos adversos associados ao ato de ingerir bebidas alcoólicas durante a gestação ser bastante documentados e conhecidos, sabe-se que uma parcela de mulheres grávidas tem dificuldades em abandonar o hábito. O consumo excessivo de álcool durante a gravidez tem sido associado com a síndrome fetal pelo álcool (FAS), caracterizada por crianças com dificuldades comportamentais e de aprendizado. Entretanto, devido ao sentimento de culpa e medo de ações punitivas, mulheres raramente admitem terem utilizado álcool durante a gestação. Como resultado, uma série de marcadores biológicos tem sido estudada para se diagnosticar a exposição fetal ao etanol. Dentre os marcadores utilizados estão os ésteres etílicos de ácidos graxos (FAEE), que podem ser detectados em amostras de mecônio de recém-nascidos. No presente trabalho, um método analítico foi desenvolvido visando a detecção de oito FAEEs em amostras de mecônio e aplicada em amostras coletadas de recém-nascidos cujas mães admitiram ou não o uso de etanol durante a gestação. A microextração em fase sólida por Headspace (HS-SPME), uma técnica de preparação de amostras relativamente recente, foi utilizada para análise. Os FAEEs foram identificados e quantificados por cromatografia gasosa/espectrometria de massas (GC/MS), operado no modo de ionização química. Os correspondentes ésteres etílicos deuterados foram sintetizados e utilizados como padrão interno. Os limites de quantificação (LOQ) obtidos foram abaixo de 150 ng/g e limites de detecção (LOD) foram abaixo de 100ng/g para todos os analitos. O método mostrou boa linearidade na concentração estudada (LOQ-2000ng/g), com coeficiente (r2) melhor que 0.98. Os valores de precisão apresentaram coeficientes de variação menores que 15% para todos os FAEEs estudados. Quando o método foi aplicado em amostras de mecônio, foi possível detectar níveis de alguns FAEEs de recém-nascidos não suspeitos a exposição fetal ao etanol. / Alcohol is the main psychoactive drug consumed worldwide and its increasing use by young women has been a great problem point out by specialists in the subject. Although the adverse effects associated to the habit of drinking alcoholic beverages during gestation being very much documented, it is known that a considerable number of pregnant women have difficulties to abandon the habit. Excessive alcohol use during pregnancy has been associated with Fetal Alcohol Syndrome (FAS) characterized by children with cognitive and behavioral disorders. However, because of denial, embarrassment and fear, maternal reports of gestational use of alcohol are often inaccurate. Consequently, a series of biomarkers have been studied to diagnose fetal exposure to alcohol. Recently, fatty acid ethyl esters (FAEE) have been studied as biomarkers found in meconium of neonates exposed in utero. In the present work, an analytical method was developed aiming the detection of eight FAEEs in meconium samples and applied to real specimens collected from newborns whose mothers admitted or not the use of alcohol during pregnancy. Headspace solid-phase microextraction (HS-SPME), a relatively recent sample preparation technique, was used for analysis. FAEEs were identified and quantified by gas chromatography/mass spectrometry (GC/MS), operated in chemical ionization mode. The corresponding deuterated ethyl esters were synthesized and used as internal standards. The lower limits of quantification (LOQ) obtained were below 150ng/g and limits of detection (LOD) were bellow 100ng/g for all analytes. The method showed good linearity in the range of concentration studied (LOQ-2000ng/g), with coefficient of linearity better than 0.98. The precision assay, given by the relative standard deviation (RSD) of the method was lower than 15% for all FAEEs studied. When the method was applied to real samples, it was possible to detect trace levels of some FAEEs from non-suspected ethanol exposed newborns.