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[en] PROTEOMIC ASPECTS AND TRACE-ELEMENT ACCUMULATION IN FISH BILE: POTENTIAL BIOLOGICAL MARKER FOR ENVIRONMENTAL EXPOSURE? / [pt] ASPECTOS PROTEÔMICOS E DETERMINAÇÃO DE ELEMENTOS-TRAÇO EM BÍLIS DE PEIXE: POTENCIAL MARCADOR BIOLÓGICO DE EXPOSIÇÃO AMBIENTAL?RACHEL ANN HAUSER DAVIS 01 June 2012 (has links)
[pt] A bílis de peixe é um fluido biológico com grande potencial como biomarcador de exposição ambiental a metais e misturas complexas. Este trabalho verificou o potencial desta matriz em situações de contaminação ambiental por metais e analisou aspectos proteômicos. Duas espécies de peixe (Mugil liza – Tainhas, e Tilapia rendalli – Tilápias) foram coletadas de diferentes locais. Indivíduos de tilápia foram expostos a concentrações sub-letais de Cu em laboratório. A bílis foi analisada com relação ao seu conteúdo de elementos-traço (Cd, Cu, Pb, Zn, Ni, Mn), e testes estatísticos demonstram que esta matriz pode ser utilizada para biomonitoramento de exposição de metais ao invés do fígado, sendo mais vantajosa que esta última por ser uma matriz menos complexa, o que facilita o seu pré-tratamento, além de não haver necessidade de sacrificar o animal. Diferentes protocolos de clean-up foram testados com objetivo de analisar a bílis por eletroforese 1D e 2D e zimografias para a detecção de enzimas. O protocolo final de clean-up inclui sonicação, centrifugação, deslipidificação e dessalinização. A presença de metalotioneínas foi analisada nesta matriz, através de eletroforeses 1D, 2D, espectrofotometria e SEC-HPLC-ICP-MS, e foi verificada que existe realmente expressão destas proteínas na bílis, inclusive com o mesmo comportamento reportado no fígado, de expressão aumentada em situações de contaminação ambiental. Também foi verificada a existência de metaloproteases de matriz na bílis através de zimografias gelatinolíticas, que também apresentaram potencial biomarcador de contaminação ambiental, pois algumas bandas gelatinolíticas estiveram presentes apenas em um local contaminado quando comparado a um lugar controle. Este trabalho verificou a existência de dois biomarcadores protéicos em bílis de peixe, um específico à contaminação por metais (metalotioneínas) e um não-específico (metaloproteases de matriz), além da possibilidade de uso da bílis de peixe como biomarcador à exposição a metais pela determinação das concentrações de elementos-traço nesta matriz. / [en] Fish bile is a biological fluid with great potential as a biomarker of environmental exposure to metals and complex mixtures. This study verified the potential of this matrix in environmental contamination situations by metals and analysed proteomic aspects. Two fish species (Mugil liza – mullets, and Tilapia rendalli – tilapias) were sampled from different locations. Tilapia individuals were exposed to sub-lethal Cu concentrations in the laboratory. Bile was analyzed regarding trace-element concentrations (Cd, Cu, Pb, Zn, Ni, Mn), and statistical techniques indicate that this matrix can be used in biomonitoring for metal exposure instead of liver, being more advantageous than the latter, since this is a less complex matrix, leading to easier pre-treatment, besides the possibility of not having to sacrifice the animal. Different clean-up protocols were tested with the aim of analysing bile by 1D and 2D electrophoresis and zymographies for protein and enzyme detection. The final clean-up protocol consists of sonication, centrifugation, delipidation and desalting. The presence of metallothioneins was analyzed in this matrix by 1D and 2D electrophoresis, spectrophotometry and SEC-HPLC-ICP-MS, and it was verified that there really is metallothionenin expression in bile, showing similar behaviour to that reported in liver, of increased expression in environmental contamination situations. We also verified the existence of matrix metalloproteases in bile by gelatinolinic zymographies, and these also showed potential as biomarkers for environmental contamination, since some gelatinolinic bands were present only in a contaminated site in comparison to a reference location. This study verified the existence of two protein biomarkers in fish bile, one specific to metal contamination (metallothioneins) and one non-specific (matrix metalloproteinases), besides the possibility of utilizing fish bile as a biomarker to metal exposure by the determination of trace-elements in this matrix.
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[en] EVALUATION OF THE OIL CONTAMINATION OF THE ESTUARINE ENVIRONMENT OF THE GUANABARA BAY (RJ) BY THE FLUOROMETRIC DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS (PAHS) IN THE MUGIL LIZA FISH BILE / [pt] AVALIAÇÃO DA CONTAMINAÇÃO DE ÓLEO NO AMBIENTE ESTUARINO DA BAÍA DE GUANABARA (RJ) PELA DETERMINAÇÃO FLUORIMÉTRICA DE HIDROCARBONETOS POLICÍCLICOS AROMÁTICOS (HPAS) NA BÍLIS DE PEIXES MUGIL LIZAROBERTA LYRIO SANTOS NEVES 17 July 2006 (has links)
[pt] Os Hidrocarbonetos Policíclicos Aromáticos (HPAs) são
poluentes ubíquos
na natureza sendo os derrames de óleo uma das principais
fontes destes compostos
para os ambientes aquáticos costeiros. Este trabalho
avalia a possibilidade de uso
dos metabólitos de HPAs na bílis do peixe Mugil liza
(tainha) como biomarcadores
no monitoramento ambiental de ecossistemas aquáticos.
Para
esta avaliação
realizou-se um monitoramento sazonal na Baía de
Guanabara,
RJ, área cronicamente
contaminada por óleo, e em Itaipu, Niterói, RJ, como
área
controle. Nos locais
de coleta foram medidos parâmetros físico-químicos tais
como, pH, oxigênio
dissolvido, entre outros e selecionados os peixes
variando
entre 35 e 51 cm para
maior homogeneidade das amostras. Em laboratório foram
feitas medidas morfométricas
dos indivíduos e retirado o seu líquido biliar. O método
analítico foi otimizado
e seus parâmetros de desempenho analítico foram
determinados. As amostras
de líquido biliar foram diluídas em etanol 48% (1:2000
v/v) e analisadas por
fluorescência nos comprimentos de onda de excitação e
emissão, respectivamente,
332 nm e 383 nm. A média das concentrações de HPAs
totais
na bílis dos peixes
coletados na Baía de Guanabara foi significativamente
diferente da área controle,
Itaipu. Na Baía de Guanabara, valores de 7,0 ± 3,4
(n=19)
e 10,4 ± 6,4 (n=12) mg
de equivalentes (eqv.) de pireno L-1 foram obtidos,
respectivamente, no inverno e
no verão. Em Itaipu, a concentração de HPAs foi de 1,8 ±
0,7 (n=11) mg de eqv.
de pireno L-1. Estes resultados indicam que o método é
capaz de diferenciar áreas
recentemente contaminadas por óleo de áreas não
contaminadas, sendo o peixe
Mugil liza um possível biomonitor para esta área. Este
método analítico apresenta
vantagens em relação a outros métodos, tais como tempo e
custo de análise reduzidos,
podendo ser usado em levantamento de dados preliminares
nos programas
de monitoramento ambiental. / [en] Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in
the
environment. The main source of contamination is
antropogenic, and oil spills are
one of the main PAHs sources for the aquatic environment.
This work evaluates
the usage of PAH metabolites in fish bile (Mugil liza) as
biomarkers in the aquatic
environment. For this evaluation two distinct areas were
monitores: Guanabara
Bay, RJ, known for its chronic oil contamination, and
Itaipu, Niterói, RJ, the
control area. Physico-chemical measurements: pH, dissolved
oxygen, conductivity
and transparency were made in situ and fish varying from
35 to 50 cm were
selected for sampling homogeneity and sexual maturity. In
the laboratory,
morphometric measures were taken and the fish bile was
extracted. After
optimizing the analytical method the samples were analysed
by diluting each bile
sample in ethanol 48% (1:2000 v/v) and fluorimetric
measurements were made in
excitation/emission wavelengths of 332 nm/383 nm. The mean
total PAHs
concentrations in the bile samples collected in the
Guanabara Bay were
significantly different from the control area, Itaipu. In
the Guanabara Bay the
means were 7,0 ± 3,4 (n=19) and 10,4 ± 6,4 (n=12) mg L-1
pyrene equivalents, in
winter and summer, respectively. In Itaipu, the mean HPA
concentration was 1,8
± 0,7 (n=11) mg L-1 pyrene equivalents. These results
indicate that this method
can differentiate contaminated areas from non contaminated
ones, making the fish
Mugil liza one possible biomonitor in the Guanabara Bay,
RJ. Additionally, this
analytical method has advantages compared to other methods
because it is less
time consuming and is inexpensive and therefore could be
used as a preliminary
monitoring tool.
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Entwicklung eines indirekten ELISA zum serologischen Nachweis von Opisthorchis felineus (Rivolta, 1884) und Metorchis bilis (Braun, 1790) (Trematoda: Opisthorchiidae) beim Fuchs : mit einem Beitrag zu den Leberenzymaktivitäten bei experimenteller Opisthorchiidose /Dell, Kerstin. January 2001 (has links)
Berlin, Freie Universität, Thesis (doctoral), 2001.
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[en] METHOD VALIDATION FOR THE DETERMINATION OF 1-HYDROXYPYRENE IN FISH BILE / [pt] VALIDAÇÃO DE MÉTODOS PARA DETERMINAÇÃO DE 1- HIDROXIPIRENO EM BÍLIS DE PEIXESDANIELLE LOPES DE MENDONCA 25 August 2008 (has links)
[pt] O petróleo é uma mistura complexa de diversos compostos,
sendo os hidrocarbonetos seus principais constituintes,
chegando a atingir 98% da composição total. Entre os
hidrocarbonetos, os hidrocarbonetos policíclicos
aromáticos (HPA) destacam-se por serem, em geral,
resistentes à biodegradação e persistentes no ambiente,
sendo potencialmente carcinogênicos ao homem e aos
organismos. A presença destes compostos nos
ecossistemas, de origem natural ou proveniente de fontes
antropogênicas, e seus efeitos podem ser monitorados
utilizando organismos do próprio ambiente.
Nos ecossistemas aquáticos, a determinação de metabólitos de
HPA na bílis de peixes que habitam o ambiente pode ser usada
como indicadora de exposição ambiental recente. Assim, o
desenvolvimento e a validação de métodos usualmente
empregados na determinação de metabólitos de HPA em bílis de
peixes têm contribuição e aplicação na elaboração de
diagnósticos ambientais sobre a contaminação por petróleo e
derivados nos sistemas aquáticos. Este trabalho otimizou,
validou e comparou dois métodos para a determinação de 1-
hidroxipireno em bílis de peixes. Um método usando a
cromatografia a liquido de alta eficiência com detector de
fluorescência (HPLC-F) e o outro usando a espectroscopia de
fluorescência convencional. Os métodos foram validados com
base nos parâmetros linearidade, sensibilidade, limite de
detecção, limite de quantificação, repetitividade,
reprodutibilidade e incluindo os cálculos das
incertezas associadas. Nos dois casos, as fontes de
contribuição mais significativas para a incerteza dos
métodos foram as soluções-padrão. A incerteza padrão
combinada associada ao método utilizando a cromatografia a
líquido foi menor que a associada ao método utilizando
fluorescência molecular. Entretanto, o método em
fluorescência, apesar de ser semi-quantitativo e de
apresentar incerteza maior, tem tempo de análise e custos de
operação e manutenção menores, sendo indicado para análises
preliminares. / [en] Petroleum is a complex mixture of several compounds. Among
its principal components there are hydrocarbons which
account to up to 98% of the total composition. Among the
hydrocarbons, there are polycyclic aromatic hydrocarbons
(PAH), which are more resistant to biodegradation, quite
persistent in the environment and potentially carcinogenic
to humans and marine organisms. The presence of these
compounds on ecosystems, from natural origin or from
anthropogenic sources, and their efluorescênciaects, can be
monitored using living organisms of the environment. In
aquatic ecosystems, the determination of PAH metabolites, in
fish bile from the environment can be used
as an indicator of recent environmental exposure. Thus, the
development and validation of methods usually employed in
the determination of PAH metabolites in the bile of fish
have assistance in the preparation and implementation of
diagnoses on the environmental contamination by petroleum
and derivatives in aquatic systems. In this work two methods
for the determination of 1- hydroxypyrene in fish bile were
improved, validated and compared. A method using the high
liquid chromatography with fluorescence detector (HPLC-F)
and the other using fixed fluorescence spectroscopy. The
methods were validated based on parameters linearity,
sensitivity, limit of detection, limit of quantification,
repeatability, reproducibility. In addition, calculations of
the uncertainties associated to the 1-hydroxypyrene
measurement were made. In both cases, the
sources of most significant contribution to the methods
uncertainty were the standard solutions. The combined
standard uncertainty associated with the method using the
liquid chromatography was lower than that associated with
the method using molecular fluorescence. The fluorimetric
method can be used as a semi-quantitative tool and presented
greater uncertainty. However, this method has lower analysis
time and costs of operation and maintenance, being quite
suitable for preliminary analysis.
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Molecular studies of the response of Helicobacter hepaticus to bile, and the effect of Helicobacter bilis on human hepatoma cellsOkoli, Arinze Stanley, Medical Sciences, Faculty of Medicine, UNSW January 2009 (has links)
Enterohepatic Helicobacter species (EHS) are emerging infectious disease agents. Infection of the enterohepatobiliary tract of several mammals by this group of bacteria results in various pathological disorders. The availability of the Helicobacter hepaticus sequenced and annotated genome, allowed molecular characterisation of the responses of H. hepaticus to host factors such as bile. The adaptation/responses of the bacterium to bovine, porcine and human bile were investigated using proteomics and transcriptomics. Ninety-one different proteins were identified in the responses of H. hepaticus response to the three types of bile. These proteins participate in several key cellular processes including DNA replication; protein transcription, translation and folding; oxidative stress response; motility; virulence; and metabolism. In particular, the bacteria deployed several strategies such as inhibition of the TCA cycle and the electron transport chain as well as iron sequestration to ensure control of the levels of hydroxyl radicals. The results of this study revealed also the modulation by bile of the expression of H. hepaticus genes involved in response to oxidative stress and virulence. The responses of human HEp-2 and Huh7-derived cell-lines to H. hepaticus and Helicobacter bilis, respectively, were investigated employing proteomics and transcriptomics. One-hundred and twenty different proteins were differentially expressed in the responses of the human cells to the presence of Helicobacter spp. in the cell cultures. These proteins are involved in regulation of cell proliferation and structure; metabolism; protein transcription, translation and modification; stress response; and tumour induction. For example, in co-cultures of Huh7-derived cells and H. bilis, the activation of several mitochondrial and endoplasmic reticulum stress-related proteins and the dysregulation of several apoptosis effectors were suggested as mechanisms that could result in the death of the liver cells. Importantly, the differential expression of several tumour-related proteins by the Huh7 cells supported a possible role for Helicobacter spp. in liver cancer.
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Molecular studies of the response of Helicobacter hepaticus to bile, and the effect of Helicobacter bilis on human hepatoma cellsOkoli, Arinze Stanley, Medical Sciences, Faculty of Medicine, UNSW January 2009 (has links)
Enterohepatic Helicobacter species (EHS) are emerging infectious disease agents. Infection of the enterohepatobiliary tract of several mammals by this group of bacteria results in various pathological disorders. The availability of the Helicobacter hepaticus sequenced and annotated genome, allowed molecular characterisation of the responses of H. hepaticus to host factors such as bile. The adaptation/responses of the bacterium to bovine, porcine and human bile were investigated using proteomics and transcriptomics. Ninety-one different proteins were identified in the responses of H. hepaticus response to the three types of bile. These proteins participate in several key cellular processes including DNA replication; protein transcription, translation and folding; oxidative stress response; motility; virulence; and metabolism. In particular, the bacteria deployed several strategies such as inhibition of the TCA cycle and the electron transport chain as well as iron sequestration to ensure control of the levels of hydroxyl radicals. The results of this study revealed also the modulation by bile of the expression of H. hepaticus genes involved in response to oxidative stress and virulence. The responses of human HEp-2 and Huh7-derived cell-lines to H. hepaticus and Helicobacter bilis, respectively, were investigated employing proteomics and transcriptomics. One-hundred and twenty different proteins were differentially expressed in the responses of the human cells to the presence of Helicobacter spp. in the cell cultures. These proteins are involved in regulation of cell proliferation and structure; metabolism; protein transcription, translation and modification; stress response; and tumour induction. For example, in co-cultures of Huh7-derived cells and H. bilis, the activation of several mitochondrial and endoplasmic reticulum stress-related proteins and the dysregulation of several apoptosis effectors were suggested as mechanisms that could result in the death of the liver cells. Importantly, the differential expression of several tumour-related proteins by the Huh7 cells supported a possible role for Helicobacter spp. in liver cancer.
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[fr] LE MARQUAGE DES PEPTIDES AVEC DES MÉTAUX ET DÉTECTION PAR MS ET L OPTIMISATION DES PROCÉDURES DE L EXTRACTION DE MÉTALLOPROTÉINES DANS LES ÉCHANTILLONS BIOLOGIQUES À DES FINS DE PROTÉOMIQUE / [pt] MARCAÇÃO DE PEPTÍDEOS COM METAIS USANDO DETECÇÃO POR MS E OTIMIZAÇÃO DE PROCEDIMENTOS DE EXTRAÇÃO DE METALOPROTEÍNAS EM AMOSTRAS BIOLÓGICAS COM PROPÓSITOS PROTEÔMICOS / [en] PEPTIDE-LABELING WITH METALS USING MS DETECTION AND OPTIMIZATION OF METALLOPROTEIN EXTRACTION PROCEDURES IN BIOLOGICAL SAMPLES WITH PROTEOMIC PURPOSES19 November 2021 (has links)
[pt] Método de identificação e quantificação de peptídeos, através da otimização de estratégias para a marcação de peptídeos com metais e subsequente separação por nano-HPLC-UV, MALDI MS. Primeiramente, peptídeos foram marcados com 3 diferentes metais lantanídeos usando um reagente funcional NHS-DOTA. Os resultados demonstraram que a reação de derivatização usando o reagente quelante DOTA foi eficiente para peptídeos simples e misturas dos mesmos, verificada através do MALDI MS a partir da relação m/z. Em paralelo, análises ambientais foram realizadas pela otimização de um procedimento de extração de metalotioneína em bílis de peixe, uma vez que esta matriz tem sido reportada como um biomarcador ambiental de contaminação por metal. Diferentes procedimentos e agentes de redução foram aplicadas para a extração de metalotioneína em bílis e fígado de peixe (Oreochromis niloticus. Análises espectrofotométricas foram realizadas a fim de quantificar os extratos de MT, e gel SDS-PAGE foi usado para avaliação qualitativa dos diferentes procedimentos usados. Cada procedimento foi avaliado estatisticamente. Metodologia de superfície de resposta foi aplicada para amostras de bílis, a fim de avaliar a resposta desta matriz. Em um contexto ambiental, concentrações de MT biliar foi mais baixa que MT do fígado, no entanto, a primeira mostrou-se mais adequada para um monitoramento ambiental. / [en] This work developed a new method for the identification and quantification of peptides, by optimizing some of the available strategies suitable for labeling peptides with lanthanide metals with subsequent separation by nano-HPLC with UV detection, matrix-assisted laser desorption ionization-mass spectrometry (MALDI MS). First, peptides were labeled with the three different lanthanide metals using a functional DOTA-based reagent. The results demonstrate that the derivatization reaction using the chelating reagent DOTA-NHS-ester was effective for single peptides and peptide mixtures, verified from the m/z relation obtained by MALDI MS. In parallel, environmental analyses were conducted, by performing the standardization of metalloprotein purification in fish bile, since this matrix has been reported as a biomarker for environmental metal contamination. Different procedures and reducing agents were applied to purify MT isolated from fish (Oreochromis niloticus) bile and liver. Spectrophotometrical analyses were used to quantify the resulting MT samples, and SDS-PAGE gels were used to qualitatively assess the different procedure results. A response surface methodology was applied for bile samples. In an environmental context, biliary MT was lower than liver MT, and, bile MT seems to be more adequate in environmental monitoring scopes. / [fr] Ce travail a développé une nouvelle méthode pour l identification et la quantification des peptides, par l optimisation de certaines stratégies disponibles appropriées pour le marquage des peptides avec des métaux lanthanide, une séparation par nano-HPLC et détection UV, et suivi par MALDI MS. Tout d abord, les peptides ont été marqués avec les trois métaux lanthanides différents et un réactif fonctionnel - DOTA. Les résultats montrent que la réaction de transformation en dérivé à l aide du réactif chélateur DOTA-NHS-ester a été efficace pour des peptides individuels et des mélanges de peptides, vérifiées à partir de la relation m/z obtenue par MALDI MS. En parallèle, nous avons effectué l optimisation pour la purification de métalloprotéine dans la bile de poisson, qui est signalée entant que biomarqueurs de contamination métallique de l environnement. Des procédures différentes et les agents réduisant ont été apliqués pour purifier les MT isolées de la bile et du foie des poissons (Oreochromis niloticus). Des analyses spectrophotométriques ont été utilisées pour quantifier les échantillons de MT, et le gel SDS-PAGE a été utilisé pour évaluer qualitativement les différents résultats de la procédure. Chaque procédure a en suíte été évaluée statistiquement, une méhtode des surfaces de réponse a été appliquée. Les MT de la bile semblent être plus adéquate pour la surveillance de l environnement en ce qui concerne l exposition récente à des xénobiotiques qui peuvent influer sur l expression protéomique et metalloproteomique de cette matrice biologique.
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