Dissertations / Theses: 'Urine humaine' – Grafiati (2024)

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Pour nourrir les humains, l’agriculture actuelle est fortement tributaire de l’utilisation de fertilisants issus de ressources fossiles. Or la majorité des nutriments de l’alimentation est ensuite excrétée dans les urines. Celles-ci sont usuellement mélangées aux eaux usées dont la gestion ne permet qu’un faible recyclage de ces nutriments et entraîne de nombreux impacts environnementaux. L’objectif de cette thèse est de caractériser les filières envisageables de valorisation de l’urine humaine en agriculture au niveau agronomique et de leurs impacts environnementaux. Une revue de la littérature des différents traitements de l’urine et des urinofertilisants obtenus montre que : (i) l'efficacité agronomique de la plupart des urinofertilisants est haute et nécessite d’être davantage étudiée ; (ii) la majorité des pathogènes peuvent être facilement inactivés, les résidus de pharmaceutiques sont plus difficilement dégradés ; (iii) la consommation d'énergie et de réactifs des traitements peut être élevée. L’efficacité fertilisante d’une dizaine d’urinofertilisants a ensuite été mesurée au champ et en serre. Elle est élevée pour la majorité et proche de celle des engrais minéraux (équivalence engrais de 52% à 120%). Elle est liée à une forte teneur en azote minéral dans la majorité des urinofertilisants. La volatilisation ammoniacale peut potentiellement être importante (e.g. 34% de l‘azote en conditions propices), le pH élevé et la teneur en azote ammoniacal, selon les urinofertilisants, étant des facteurs de risque importants. Enfin, une évaluation par analyse du cycle de vie des impacts environnementaux associés à la production de céréales a été réalisée selon le mode de fertilisation : biologique et conventionnelle versus trois urinofertilisants. Les impacts sont plus faibles pour la majorité des indicateurs en comparaison aux pratiques actuelles, en grande partie grâce aux impacts évités de l’épuration des eaux usées et de la production d’engrais minéraux. La volatilisation ammoniacale et la consommation d’énergie des traitements sont les deux éléments les plus sensibles du bilan environnemental. Ces résultats montrent que le déploiement de filières de valorisation de l’urine humaine peut contribuer à une transition vers une gestion systémique et soutenable des nutriments
To feed humans, agriculture mostly relies on the use of fertilizers derived from fossil resources. Yet, most nutrients from food are excreted in urines and mixed in wastewaters. Wastewater treatment allows only a weak recycling of the nutrients and has many environmental impacts. The objective of this thesis is to characterize the possible management options for the use of human urine in agriculture considering their fertilizing efficiency and their environmental impacts. A literature review of the various urine treatments and urine-based fertilizers shows that: (i) the fertilizing efficiency of most urine-based fertilizers is high but needs to be further studied; (ii) most pathogens in urine can be easily inactivated but pharmaceutical residues are more difficult to degrade; (iii) the energy and chemical consumption of treatments can be high. The fertilizing efficiency of ten urine-based fertilizers has been measured under greenhouse and field conditions. It is high for most of the urine-based fertilizers and close to that of mineral fertilizers (equivalent ranging from 52% to 120%). It is linked to high mineral nitrogen content in the majority of urine-based fertilizers. Ammonia volatilization after field application can potentially be high (e.g. 34% of total nitrogen in favorable conditions). High pH and ammoniacal nitrogen content according to the urine-based fertilizers are important risk factors. Finally, a life cycle assessment of the environmental impacts for cereal production was carried out considering three urine-based fertilizers and two agricultural systems (conventional and organic). The impacts are lower for the majority of the indicators compared to the current practices. It is mainly due to the avoided impacts due to wastewater treatment and mineral fertilizers synthesis. Ammonia volatilization and the energy consumption of the treatments appear as the main environmental hotspots. These results show that the implementation of human urine management options can contribute to a transition towards a more sustainable and systemic management of nutrients

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La barrière entre l’aspect bénéfique du sélénium (Se) et son aspect toxique est étroite. Afin de mieux contrôler les apports de cet élément dans l’alimentation, de nombreux travaux s’intéressent à la compréhension de son métabolisme. Cette thèse présente le développement et l’optimisation de méthodes d’analyse des espèces séléniées dissoutes et volatiles présentes dans l’urine de sujets non supplémentés. Le couplage entre la chromatographie liquide et la spectrométrie de masse atomique précédé d’un prétraitement de l’échantillon par extraction sur phase solide a permis non seulement de confirmer la présence de métabolites séléniés précédemment détectés dans l’urine de sujets supplémentés mais également, de mettre en évidence des composés inconnus. Un de ces composés a été identifié par spectrométrie de masse moléculaire. Les couplages entre la chromatographie gazeuse et les spectrométries de masse atomique et moléculaire, précédés d’une micro-extraction sur phase solide ont été utilisés pour l’analyse des formes volatiles de Se. Ces méthodes ont permis de confirmer la présence de composés séléniés et mixtes Se/S dans les urines de sujets non supplémentés et, de définir des conditions de stockage permettant de préserver la spéciation originelle dans l’échantillon
The concentration range between beneficial and toxic effects for selenium (Se) is very narrow. In order to monitor selenium intake and to improve knowledge of Se metabolism, studies on selenium species ingested and excreted are performed. This report presents the development and the optimization of speciation analysis of dissolved and volatile Se species in urine of non supplemented subjects by coupling liquid chromatography or gas chromatography to inductively coupled plasma mass spectrometry. In the case of dissolved species, solid phase extraction of the sample as sample pretreatment allowed us to confirm the presence of known selenium compounds and to highlight unknown selenium species. One of these new species was further identified by molecular mass spectrometry. The analysis of volatile selenium compounds was performed by gas chromatography coupled to ICP-MS after sample extraction by SPME. This method led to the identification of some selenium and mixed selenium/sulphur species in urine from non supplemented subjects and allowed us to define suitable storage conditions to maintain original speciation

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La barrière entre l'aspect bénéfique du sélénium (Se) et son aspect toxique est étroite. Afin de mieux contrôler les apports de cet élément dans l'alimentation, de nombreux travaux s'intéressent à la compréhension de son métabolisme. Cette thèse présente le développement et l'optimisation de méthodes d'analyse des espèces séléniées dissoutes et volatiles présentes dans l'urine de sujets non supplémentés. Le couplage entre la chromatographie liquide et la spectrométrie de masse atomique précédé d'un prétraitement de l'échantillon par extraction sur phase solide a permis non seulement de confirmer la présence de métabolites séléniés précédemment détectés dans l'urine de sujets supplémentés mais également, de mettre en évidence des composés inconnus. Un de ces composés a été identifié par spectrométrie de masse moléculaire. Les couplages entre la chromatographie gazeuse et les spectrométries de masse atomique et moléculaire, précédés d'une micro-extraction sur phase solide ont été utilisés pour l'analyse des formes volatiles de Se. Ces méthodes ont permis de confirmer la présence de composés séléniés et mixtes Se/S dans les urines de sujets non supplémentés et, de définir des conditions de stockage permettant de préserver la spéciation originelle dans l'échantillon.

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L’alimentation et l’excrétion constituent deux besoins physiologiques fondamentaux de tout être humain. En analysant leur matérialisation depuis l’échelle cellulaire jusqu’à celle des grands cycles biogéochimiques planétaires, nous proposons de considérer que l’alimentation et l’excrétion humaines participent d’un système dont les modalités de réalisations dans les différentes sociétés humaines permettent de caractériser des régimes socio-écologiques. Nous avons plus particulièrement analysé les systèmes alimentation/excrétion des territoires urbains au regard de leur soutenabilité et proposons une méthodologie de caractérisation fondée principalement sur l’analyse du flux de la substance qui nous paraît la plus pertinente, à savoir l’azote, et sur les modalités de gestion des urines humaines qui représentent près des trois quarts de ce flux. Nous montrons que les systèmes alimentation/excrétion des différentes communautés humaines présentent une très grande variété selon les lieux et époques considérés et proposons de les distinguer entre autres en fonction de leur circularité, c’est-à-dire par le taux de retour sur des sols agricoles des excrétats. En prenant l’agglomération parisienne comme cas d’étude, nous montrons que son système alimentation/excrétion a été de plus en plus circulaire au cours du XIXe siècle, culminant au tout début du XXe siècle aux alentours de 50 % de circularité, avant de se linéariser progressivement au cours du XXe siècle. En ce début de XXIe siècle, nous caractérisons le système alimentation/excrétion de l’agglomération parisienne comme non soutenable car linéaire à plus de 95 %, intensif, inefficace et polluant aux échelles locales et globales. Ces caractéristiques sont généralisées au sein du monde occidental et interpellent sur la possibilité d’une transition socio-écologique vers des systèmes alimentation/excrétion soutenables. Or, depuis les années quatre-vingt-dix, une prise de conscience relative à l’urine a réémergé, principalement en Suède puis dans l’Europe germanique. Elle s’est traduite par de nombreuses réalisations et recherches autour de la séparation à la source des urines. Nous montrons que ce dispositif est actuellement le seul, dans le monde occidental, à avoir permis de nouveau la mise en œuvre de systèmes alimentation/excrétion circulaires. Pouvant être déclinée sous de multiples formes en fonction des contextes, la séparation à la source des urines bénéficie, malgré le verrouillage socio-technique de l’agglomération parisienne autour du tout-à-l’égout, d’un contexte favorable à son développement. Nous avons élaboré un scénario prospectif explorant ainsi la possibilité que l’agglomération parisienne dépasse, en quelques décennies, l’extremum de circularité qu’elle avait connu à la Belle Époque et que les acteurs de ce territoire réalisent, en cohérence avec une transition socio-écologique des autres systèmes énergétiques, hydrauliques et de transport, un régime socio-écologique soutenable de leur système alimentation/excrétion. Cette thèse fait partie du programme de recherche et action OCAPI (www.leesu.fr/OCAPI)
Nutrition and excretion are fundamental physiological needs for all human beings. Analysis of their materiality, from the cellular scale up to the great planetary-scale biogeochemical cycles, shows that nutrition and excretion form a system. The focus of our study is the sustainability of the nutrition/excretion systems of urban areas, which we have sought to assess by analysing substance flows. The most relevant of these substances seems to be nitrogen, so by assessing urban nitrogen flows we can characterise the different possible socioecological regimes and their sustainability. We identify a wide diversity of nutrition/excretion systems depending on the places and eras considered. We propose to distinguish them in terms of their circularity, in other words by the rate at which nitrogen from excreta returns to agricultural land. Using the Paris urban area as our case study, we show that its nutrition/excretion system became increasingly circular in the 19th century, reaching maximum circularity right at the start of the 20th century, before becoming steadily more linear in the course of the 20th century. In these early years of the 21st century, the nutrition/excretion system of the Paris urban area is essentially linear, and still generates significant pollution at both local and global scales. Its environmental footprint is exacerbated by a diet that is very protein rich, mostly animal in origin, and by the non-consumption of a significant proportion of the food produced. All these factors make it unsustainable. These characteristics are found throughout the Western world and raise questions about the possibility of a socioecological transition to sustainable systems of nutrition and excretion. Since the 1990s, initially in Sweden, followed by Nordic and German-speaking Europe, awareness has been growing of the role of urine. Urine is responsible for three-quarters of urban nitrogenous excretions and is a safe substance: following a period of storage, it can be used as agricultural fertiliser. This new awareness has been followed by extensive experimentation and research on urine source separation. We show that this is currently the only method in the Western world to have accomplished a return to circular systems of nutrition/excretion. Urine source separation can be done in multiple ways, depending on circ*mstances, and conditions in France are favourable to its development, despite the sociotechnical lock-in to mixed sewage management systems. In a forward-looking scenario, we therefore explore the possibility that the Paris urban area could return to, and within a few decades even surpass, the heights of circularity that it attained during the Belle Époque. In that case, alongside a socioecological transition in the other systems – water, energy, transport – the people of this territory could establish a sustainable regime for their system of nutrition/excretion. This thesis is part of the OCAPI research and action programme (www.leesu.fr/OCAPI)

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Les expositions professionnelles agricoles, et notamment l’exposition aux pesticides, ont été associées à divers effets négatifs, sur la fertilité, le déroulement de la grossesse et le développement de l’enfant. Peu d’études se sont intéressées à des activités agricoles spécifiques excepté le travail sous serre et la floriculture et aucune n’a été à ce jour conduite en France. (1) L’interrogation de plus de 800 femmes, incluses dans la cohorte AGRIculture & CANcer (AGRICAN) et ayant eu une grossesse depuis l’inclusion (2005), grâce à 2 questionnaires rétrospectifs a montré un allongement du délai nécessaire à concevoir en lien avec l’emploi sur une exploitation agricole, le travail de nuit ou l’exposition à des vibrations. Des augmentations de risque d’avortements spontanés et de malformations ont également été observées mais restent à confirmer. (2) Le développement d’une méthode d’analyse multi-résidus a permis de mesurer 25 pesticides différents parmi 116 recherchés dans les urines de femmes travaillant sur des exploitations de poly-culture élevage. Les herbicides étaient les plus fréquemment détectés, notamment en lien avec la présence de maïs sur l’exploitation ou de tâches réalisées au contact des animaux d’élevage. Le glyphosate ou son métabolite AMPA étaient retrouvés dans 85% des échantillons.Un projet de recherche poursuivra les travaux engagés en s’intéressant au développement cognitif des enfants nés depuis 2005
Agricultural exposures, including pesticide exposure, have been associated with several negative effects on fertility, pregnancy and child development. Few studies focused on specific agricultural activity excepted floriculture and working in greenhouse and none was conducted in France. (1) More than 800 women, enroled in the AGRIculture & CANcer (AGRICAN) cohort and who reported a pregnancy since enrolment (2005) agreed to fill in 2 questionnaires. An increase of time to pregnancy was observed for women who worked on a farm, for those exposed to night work and to vibrations. Increased risks of spontaneous abortions or abnormalities were also observed in relation to agricultural work but these results need to be confirmed. (2) Multi-residue analytical method was developed and applied to women of childbearing age, working in crop-livestock farms. Twenty-five pesticides or metabolites were detected among 116 measured in urine samples. Herbicides were the most frequently detected, especially when women worked on corn-crop farms or were involved in breeding tasks. Glyphosate or its metabolite AMPA were detected in 85% of urine samples.Future project will allow us to investigate cognitive development of children born since 2005

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L’objectif de ce travail de thèse est de développer une base de données spectrale pour faciliter l’annotation et l’interprétation biologique des jeux de données d’analyse métabolomique obtenus en utilisant la chromatographie liquide couplée à la spectrométrie de masse. Deux approches ont été utilisées : l’identification par comparaison aux spectres de masse de composés de références et l’identification directement à partir des données biologiques. Pour la première approche une chiomiothèque de métabolite a été constituée et analysée. L’identification à partir de données biologiques a été réalisée sur une cohorte de volontaires de 227 individus travaillant au CEA. 244 métabolites ont ainsi été identifiés dans les urines humaines, donc 78 jamais été décrits comme faisant parti du métabolome urinaire. 139 métabolites ont également était caractérisés sur la base de leur masse précise mais sans identification formelle. Ces 383 métabolites représentent environ 1000 ions dans chacun des modes d’ionisation. Les variations physiologiques au sein de la cohorte, en fonction de l’âge, du poids et du genre, de ces différents métabolites ont été étudiées afin de construire une base de données relationnelle. Enfin, le métabolome urinaire pouvant être affecté par les conditions de prélèvement des échantillons d’urines, nous avons réalisé des études de stabilité dans les conditions de prélèvement des métabolites précédemment caractérisés. Ces études nous ont permis de proposer des recommandations en termes de conditions de prélèvement et de stockage à court terme des urines et de mesurer l’impact de la contamination bactérienne sur les concentrations de différents métabolites urinaires

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Le cancer du rein est un problème majeur de santé publique. Un diagnostic précoce améliore les chances de survie. Le diagnostic repose essentiellement sur les examens d'imagerie comme l'échographie, la tomodensitométrie et l'IRM. Ces examens sont parfois associés à la biopsie et sont couteux et parfois invasifs. De plus, l'imagerie n'est pas capable de faire la distinction entre les tumeurs bénignes et les tumeurs malignes et entre les sous-types histologiques de carcinome à cellules claires qui est le plus fréquent. Il n'existe pas dans le cancer rénal de marqueur comme la PSA dans le cancer de la prostate ou la Foetoprotéine et l'HCG dans le cancer du testicule. Le but de cette étude est concentré sur la recherche des marqueurs mARNs ou miARNs dans les liquides biologiques (sérum, plasma, urine) pour le cancer du rein à cellules claires. Nous avons montré que les petit* ARNs dans le sérum et les urines et l'intégrité des ARNs dans les urines étaient des outils diagnostiques dans le cancer du rein à cellules claires. Si ces petit* ARNs circulants sont validés, on peut éventuellement imaginer l'intérêt pratique en clinique comme la détection des petit* ARNs circulants dans l'urine pour prédire le cancer, la classification de la tumeur pour aider le clinicien, la prédiction d'une récidive ou d'une progression soit après néphrectomie soit au cours d'un traitement médical

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
A large part of nutrients such as nitrogen, phosphorus and potassium, which are essential in agriculture, is found in human urine being their amounts, in most cases, significant, more suitable and sustainable than those in commercial fertilizers. Due to the demand for sustainable alternatives in the context of ecological sanitation, reuse of human urine can be faced as a potentially feasible alternative in the context of decentralized wastewater treatment systems. The experimental research which based this work was divided into two stages, in the first the effect of storage, within open and closed recipients, as a treatment of pure urine and yellow water, being used two containers for pure and two for a 7% dilution of urine. In order to verify the effect of storage, monitoring and evaluation were performed by means of physical, chemical and microbiological indicators. Storage of urine proved to be a cost-effective cleaning method of pure and diluted urine and. Containers with lid were more efficient in physical-chemical and biological stabilization process controlling loss of ammonia by volatilization, and contamination by the influence of the external environment. Concentrations of nutrients were maintained in high levels and fecal contamination tended to elimination in a period of 20 days, at room temperature. In the second step, the aim was the recovery of phosphorus by precipitation, carried out in a jar-test apparatus with MgO addition, at several concentrations, to pure and 7%-diluted urine to 7%. Two ranges of concentrations were used - phase A (0; 0.05; 0.15; 0.30 and 0.45 g of MgO/L) and phase B (0; 0.45; 0.60; 0.75 and 0.90 g MgO/L) - under stirring at 120 rpm, for a period of 2 hours. Precipitation and hence the phosphorus recovery was directly related to the concentration of MgO in both phases.
Grande parte dos nutrientes que são essenciais na agricultura, como nitrogênio, fósforo e potássio, é encontrada na urina humana e suas quantidades significativas são, na maioria das vezes, mais apropriadas e sustentáveis do que as encontradas nos fertilizantes químicos comerciais. Devido à demanda por alternativas sustentáveis, no contexto do esgotamento sanitário ecológico, o reuso da urina humana pode ser citado como descentralizados de tratamento de águas residuárias. A pesquisa que fundamentou este uma alternativa viável e de grande potencial na escala de sistemas trabalho foi dividida em duas etapas, tendo a primeira o objetivo de analisar o efeito de modos diversos (aberto e fechado) de armazenamento, como forma de tratamento da urina pura e de águas amarelas. Para tal foram utilizados quatro recipientes de urina pura e diluída a 7%, em dois recipientes com tampa e dois sem tampa. Para verificar o efeito do armazenamento foi realizada a caracterização, por meio de indicadores físicos, químicos e microbiológicos. O armazenamento da urina mostrou-se um método de higienização de baixo custo e bastante eficiente. Quanto às formas de armazenamento (aberta e fechada), a utilização de urina pura em recipientes com tampa mostrou-se mais eficiente no processo de estabilização físico-química e biológica, pois não ocorreu perda de amônia por volatilização, nem contaminações por influência do ambiente externo, a concentração de nutrientes foi considerada satisfatória e os níveis de coliformes termotolerantes tenderam à nulidade em um período de 20 dias, em temperatura ambiente (25 a 26ºC). Na segunda etapa, o objetivo foi a recuperação de fósforo por precipitação levada a efeito em aparelho jar-test, no qual foram adicionadas diferentes concentrações de MgO para cada 1,0 L de urina pura e de diluída a 7%. Duas faixas de concentrações foram utilizadas - fase A (0; 0,05; 0,15; 0,30 e 0,45 g de MgO/L) e fase B (0; 0,45; 0,60; 0,75 e 0,90 g de MgO/L) -, sob agitação a 120 rpm, por um período de 2 horas.A precipitação e, consequentemente a recuperação de fósforo, foi diretamente relacionada às concentrações de MgO, nas duas fases.

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Analyses of amino levels in human urine were studied in order to evaluate their potential use in tests which could be used for the early detection of cancer. Three suitable reagents for derivatisation were studied and it was found that of the three reagents considered for use in this study, {dansyl chloride, dabsyl chloride and OPA}, dansyl chloride proved the most suitable as it was the most sensitive, enabled accurate reproducible separation of a greater number of amino acids than the other reagents and was the easiest of the three with which to work. A control group of 27 healthy individuals, were provided with five sample bottles in order for them to provide a number of separate samples taken throughout the day. The data that were obtained from these sample were used to establish the amino acid levels in the control group of healthy individuals. The results also demonstrate that the daily variation in amino acid levels in a healthy individual was not responsible for the difference between individuals. In the Discussion and Summary it can be seen that the pattern of concentrations of individual amino acids obtained is different for each form of cancer. Each pattern adds further evidence for the presence of a particular form of tumour. The final conclusion is that the lower average range of the amino acid alanine in cancer patients is statistically significant, it is therefore possible to use the depressed average level of alanine along with raised average level of isoleucine in data obtained from a number of samples taken from any individual patient over a period of 2-3 days in comparison with the average levels in the control group of healthy volunteers to assess a patient for the presence of any form of cancerous tumour within that individual.

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As aflatoxinas são substâncias naturais que apresentam efeitos tóxicos aos humanos e são reconhecidamente carcinogênicas. Estas substâncias podem estar presentes na dieta humana ou, em casos específicos, no ar respirado. Desta maneira, a exposição humana às aflatoxinas é objeto de muita preocupação. Uma das maneiras mais eficazes de avaliar a exposição humana as aflatoxinas é através da mensuração da presença de biomarcadores da exposição a estas substâncias em fluidos biológicos. Dentre as possibilidades de biomarcadores de exposição às aflatoxinas tem-se que aflatoxina M1 (AFM1), presente na urina e leite humano, é considerada um biomarcador válido. Assim sendo, o objetivo deste trabalho de pesquisa foi avaliar a presença de AFM1 em amostras de urina provenientes de indivíduos residentes na região urbana e rural da cidade de Piracicaba-SP, assim como, de leite de gestantes de Piracicaba e cidades da região. Nos indivíduos doadores de amostras de urina foi levantado também o padrão de ingestão de alimentos com alto risco de conter aflatoxinas, através da aplicação de inquéritos de freqüência alimentar e recordatórios 24 horas. A análise de AFM1 em urina e leite foi realizada por cromatografia liquida de alta eficiência (CLAE) com detecção por fluorescência. A extração e purificação do extrato foram realizadas com auxílio de colunas de imunoafinidade. No total 69 amostras de urina e 18 de leite foram analisadas. Entre as amostras de urina detectou-se a presença de AFM1 em 54 (78%) das amostras, com concentrações variando de 1,8 até 39,9 pg/mL. Não foi observada diferença estatística entre as concentrações médias detectadas entre urinas de indivíduos da zona urbana e rural, bem como no nível de consumo de produtos de risco. Apesar das concentrações de AFM1 detectadas serem inferiores as concentrações médias reportadas em outros países a freqüência de amostras positivas foi bastante elevada mostrando que as populações estudadas estão sendo expostas às aflatoxinas. Assim, melhores avaliações dos níveis de exposição necessitam ser realizados considerando que a amostragem utilizada foi pontual, pode existir variação de contaminação sazonal com aflatoxinas na dieta e a contaminação é heterogênea dentro no alimento. Não foi observada uma correlação entre o nível do consumo de produtos de risco e as concentrações detectadas em amostras de urina. Apenas uma amostra de leite apresentou contaminação detectada; entretanto, o nível de contaminação estava entre o limite de detecção (LD) e o limite de quantificação (LQ).
Aflatoxins are natural substances that present toxic and carcinogenic effects to humans. These substances may be present in human diet or, in specific cases, in the breathing air. Thus, the human exposition to aflatoxins is object of concern. One of the most effective ways to evaluate human exposition to aflatoxins is to measure the presence of biomarkers in biological fluids. Among the possibilities of aflatoxin presence biomarkers, the aflatoxin M1 (AFM1), present in human urine and milk, is considered a valid biomarker. The objective of this work was to evaluate the presence of AFM1 in urine samples from individuals who live in urban and rural areas in the county of Piracicaba, state of São Paulo, Brazil, and in milk of pregnant women from Piracicaba and neighbor cities. Urine-donor individuals were researched in relation to the ingestion of food with high risk of containing aflatoxins through the application of a food frequency questionnaire and 24-hour recall. The analysis of AFM1 in urine and milk was performed through high-performance liquid chromatography (HPLC) with fluorescence detection. The extract purification and extraction were performed with the aid of immunoaffinity columns. Overall, 69 urine and 18 human breast milk samples were analyzed. Among urine samples, the presence of AFM1 was detected in 54 (78%), with concentrations ranging from 1.8 to 39.9 pg/mL. No statistical difference was observed between average concentrations detected in the urine of individuals from urban and rural areas, as well as the consumption of aflatoxin risky food. Although the AFM1 concentrations detected are lower than those reported for other countries, the frequency of positive samples was quite high, showing that the populations studied are exposed to aflatoxins. Thus, further evaluations on the exposition levels should be performed, and considering that the sampling used in this work was punctual, there may be seasonal contamination variations in diet and the contamination level is heterogeneous within a food. No correlation between the consumption of risky food and concentrations detected in urine samples was observed. Only one milk sample presented detected contamination; however, the contamination level was between the limit of detection (LOD) and the limit of quantification (LOQ).

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No Brasil, ainda não há a realização de pesquisas envolvendo a determinação de elementos químicos em fluidos biológicos e a elaboração de valores de referência para a sua população infantil. O biomonitoramento de elementos químicos apresenta essencial importância na avaliação da saúde humana, no entanto, na análise dos dados dos estudos brasileiros de biomonitoramento, os resultados obtidos são geralmente comparados com valores estipulados para outros países, o que pode gerar uma estimativa equivocada do risco. Sendo assim, o objetivo do presente estudo foi descrever as concentrações médias urinárias de elementos químicos essenciais e não essenciais em crianças brasileiras em fase escolar (6-14 anos), propondo valores de referência para Cd, Co, Li, Mo, Pt e Sb. Para o desenvolvimento do estudo, foram utilizadas amostras de urina obtidas pela \"Pesquisa Nacional para Avaliação do Impacto da Iodação do Sal\" (PNAISAL), sendo tomada uma amostragem de 6.965, escolhidas aleatoriamente, abrangendo 19 unidades da federação e comtemplando as 5 regiões brasileiras. As determinações dos elementos químicos foram realizadas por método de ajuste de matriz, com simples diluição de urina e análise direta por espectrometria de massas com plasma acoplado indutivamente (ICP-MS). Foi realizada dosagem de creatinina nas amostras para ajuste de matriz e correção de possíveis efeitos de diluição. As concentrações médias obtidas para os elementos Cd, Co, Li, Mo, Pt e Sb foram 0,267, 0,769, 7,949, 66,839, 0,022 e 2,389 ?g/g de creatinina, respectivamente. Os dados obtidos foram comparados com resultados de estudos de biomonitoramento para população adulta brasileira e de outros países, evidenciando a necessidade da estipulação de valores próprios para a população infantil brasileira.
There is no research involving the determination of chemical elements in biological fluids and the development of reference values in Brazil for its child population. Human biomonitoring of chemical elements has great importance in human health assessment, however, in analysis of Brazilian biomonitoring studies, the results are usually compared with values established for other countries, which can lead to an erroneous estimate of the risk. Thus, the aim of this study was to determine the concentration of essential and nonessential elements in Brazilian children (6-14 years), proposing reference values for Cd, Co, Li, Mo, Pt and Sb. To develop the study, urine samples obtained by the \"Pesquisa Nacional para Avaliação do Impacto da Iodação do Sal\" (PNAISAL) were used, taking a sample of 6,965 randomly chosen, covering all Brazilian regions. Samples were directly analyzed by inductively coupled plasma mass spectrometry ICP-MS against matrix-matching calibration. Creatinine measurement was done to correct possible effects of sample dilution. The mean concentrations obtained for Cd, Co, Li, Mo, Sb and Pt elements were 0.267, 0.769, 7.949, 66.839, 0.022 and 2.389 ?g/g of creatinine, respectively. The data were compared with results from biomonitoring studies for Brazilian adult and foreign populations, highlighting the need for stipulation of reference values for Brazilian child population.

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A fumonisina B1 (FB1) e uma micotoxina produzida pelo metabolismo secundário de espécies de Fusarium, principalmente F. verticillioides e F. proliferatum, os quais contaminam diversos alimentos antes e apos o processamento, sobretudo o milho e derivados, gerando graves problemas para a Saúde Pública e a qualidade dos alimentos. O objetivo deste trabalho foi avaliar a exposição humana a FB1 presente nos alimentos através da estimativa de ingestão da toxina na dieta e da análise de diferentes biomarcadores presentes em amostras de sangue, urina e cabelo. Além disso, foram investigados os efeitos da toxina através da avaliação de ácido fólico presentes em alimentos e em soro, e os níveis de uréia e creatinina presentes em soro. O estudo foi realizado em dois municípios dos Estados de São Paulo e Santa Catarina, cujos respectivos voluntários foram categorizados como de baixo consumo de derivados de milho (Grupo A, voluntários de Pirassununga/SP) e de alto consumo de derivados de milho (Grupo B, voluntários de Erval Velho/SC). As amostras de alimentos do Grupo A (Pirassununga/SP) foram fornecidas pelos voluntários (n=100) nos meses de Junho/2011, Setembro/2011, Dezembro/2011 e Marco/2012. Os voluntários do Grupo B (Erval Velho/SC) (n=20) forneceram amostras de alimentos no mês de Abril/2012. Em cada grupo, uma lista com 20 alimentos a base de milho foi entregue aos voluntários, para fornecimento de amostras daqueles disponíveis em suas respectivas residências em cada mês de amostragem, totalizando 122 amostras de derivados de milho no Grupo A e 17 amostras no Grupo B coletadas durante o estudo. Adicionalmente, aplicou-se um Questionário de Frequência Alimentar (QFA) e um Inquérito Recordatório de 24 horas (QIR - 24 h) no momento das coletas de amostras. Em cada mês de amostragem de alimentos, foram coletadas amostras de sangue, urina (somente Grupo A) e cabelo dos voluntários, sendo as amostras armazenadas a -20ºC (urina e cabelo) ou -80ºC (sangue) até o momento das análises. As amostras de alimentos foram submetidas a análise de FB1, sendo que as de farinha de milho foram também analisadas quanto ao teor de ácido fólico. Ambas as análises foram feitas através de cromatografia líquida de alta eficiência (CLAE). Em soro, foram avaliadas a relação esfinganina/esfingosina (Sa/So), resíduos de FB1, ácido fólico, uréia e creatinina. Em urina, foram analisados os níveis de FB1, creatinina para correção do volume urinário e a relação Sa/So. Em cabelo, foram analisados os resíduos de FB1 através de CLAE acoplada a espectrometria de massas. Todos os métodos de análise foram submetidos a procedimento de otimização e validação intra--laboratorial. A incidência de FB1 nos alimentos foi, em média, 72% (n=122) nas amostras do Grupo A (Pirassununga/SP) e 35% (n=17) no Grupo B (Erval Velho/SC). Os maiores níveis foram encontrados em amostras de pipoca provenientes do Grupo B, com uma amostra excedendo o limite de tolerância estabelecido no Brasil (2,500 µg kg-1). A ingestão diária provável média (IDPM) de FB1 no Grupo A foi de 63,3 ng kg-1 peso corpóreo (p.c.) dia-1, que corresponde a 3,1% da ingestão provisória máxima tolerável (IPMT) recomendada para fumonisinas (2.000 ng kg-1 p.c. dia-1). A IDPM do Grupo B apresentou uma média de 190,1 ng kg-1 p.c. dia-1 o que corresponde a 9,5% da IDMT. As concentrações de ácido fólico nas amostras de farinha de milho variaram de < 0,3 µg kg-1 (limite de quantificação do método) a 1.705 µg kg-1, com média de 713 ± 435 µg kg-1. Somente uma amostra apresentou nível de ácido fólico acima do valor mínimo estabelecido pela ANVISA. Em urina, a incidência de FB1 foi de 33,4% (n=251), com níveis médios de 3,19 ± 3,15 ng mg-1 de creatinina. Não houve correlação (P>0,05) entre as concentrações de FB1 na urina e nos alimentos. Os níveis de esfinganina foram mais elevados em mulheres, com 25,0% (n=116) de amostras positivas, em comparação à urina de homens, 10,4% (n=96). A relação Sa/So apresentou em média 0,91, 0,77 e 0,89 para urina de mulheres, homens e em combinação, respectivamente. Em soro, os níveis de esfingosina foram em média 2,48 ng mL-1 para o Grupo A e 5,01 ng mL-1 para o Grupo B. A relação Sa/So variou de 0,06 a 3,19 com média de 0,79 para o Grupo A e 0,78 para o Grupo B. Embora tenha havido correlação positiva (r=0,574, P<0,05) entre a relação Sa/So no soro e os dados de consumo de milho e derivados obtidos no QIR-24 h, não foram observadas correlações (P>0,05) entre a ingestão de FB1 e a relação Sa/So na urina ou soro. A concentração de ácido fólico no soro variou de 6,7 a 24,0 ng mL-1 (média de 13,4 ± 5,4 ng mL-1), com ambos os grupos (A e B) apresentando resultados dentro dos valores de referências. Não foram observados níveis detectáveis de FB1 nas amostras de soro. No entanto, FB1 foi detectada em 4 amostras de cabelo humano (7,2%) dos Grupos A e B, cuja concentração média foi de 21,3 ± 12,1 ng g-1. Em síntese, os resultados obtidos nas análises de biomarcadores de FB1 no presente trabalho estão de acordo com os valores de IDPM encontrados, indicando que a exposição a FB1 nas populações estudadas não representa um risco a saúde.
Fumonisin B1 (FB1) is a mycotoxin produced by the secondary metabolism of Fusarium species, mainly F. verticillioides and F. proliferatum, which contaminates foods before and after processing and causes serious problems to public health and food quality. The aim of this study was to evaluate the human exposure to FB1 in food by means of estimated intake of toxin in the diet, and analysis of different biomarkers in serum, urine and hair. In addition, folic acid in food and blood as well urea and creatinin in serum were investigated to evaluate the toxin effects. The study was conducted in two cities of Sao Paulo and Santa Catarina States, where the respective volunteers were categorized as low-consumers of corn products (Group A, volunteers from Pirassununga/SP) and high-consumers of corn products (Group B, volunteers from Erval Velho/SC). Food samples from Group A (Pirassununga/SP) were provided by volunteers (n=100) in June/2011, September/2011, December/2011 and March/2012. The volunteers from Group B (Erval Velho/SC) (n=20) provided food samples in April/2012. In each group, a list of 20 corn products was given to volunteers, to allow them to check and collect the food items available in their homes at each sampling time. The total number of samples of corn products provided by the volunteers were 122 and 17 in Group A and Group B, respectively. Addicionally, a Food Frequency Questionnaire (FFQ) and a 24-Hours Dietary Recall Questionnaire (24h-DRQ) were applied by the time of sample collections. In each month of food samples collection, samples of blood, urine (only Group A) and hair from the volunteers were collected and storage at -20ºC (urine and hair) or -80ºC (blood) until analysis. Food samples were submitted to determination of FB1, and corn meal samples were also evaluated for folic acid levels. Both analysis were performed by high performance liquid chromatography (HPLC). In serum, analyses included sphinganine/sphingosine ratio (Sa/So), FB1 residue, folic acid, urea and creatinine. In urine, the levels of FB1, creatinine to correct urinary volume and Sa/So ratio were evaluated. In hair, FB1 residues were analysed by HPLC coupled to mass spectrometry. All the analytical methods were submitted to optimization and intra-laboratorial validation procedures. The mean incidences of FB1 in corn products were 72% (n=122) in samples of Group A (Pirassununga/SP), and 35% (n=17) of Group B (Erval Velho/SC). The higher levels were found in popcorn from Group B, with one sample exceeding the tolerance limit established in Brazil (2,500 µg kg-1). The mean probable daily intake (PDIM) of FB1 in Group A was 63.3 ng kg-1 body weigh (b.w.) day-1, which corresponds to 3.1% of provisional maximum tolerable intake (PMTDI) recommended for fumonisins (2,000 ng kg-1 b.w. day-1). PDIM of Group B was 190.1 ng kg-1 b.w. day-1, which represents 9.5% of PMTDI. Folic acid levels in corn meal ranged from &LT; 0,3 µg kg-1 (quantification limit) to 1.705 µg kg-1, with a mean of 713 ± 435 µg kg-1. Only one sample had levels of folic acid above the minimum established by ANVISA. In urine, the incidence of FB1 was 33,4% (n=251), at mean levels of 3,19 ± 3,15 ng mg-1 of creatinine. There wasn\'t correlation (P>0.05) between concentrations of FB1 in urine and foods. Sphinganine levels were higher in woman, with 25.0% (n=116) of positive samples in comparison to urine of men, 10.4% (n=96). The mean Sa/So ratios were 0.91, 0.77 and 0.89 for urine of women, men and in combination, respectively. In serum, sphingosine presented a mean of 2.48 ng mL-1 to Group A and 5.01 ng mL-1 to Group B. Sa/So ratio ranged from 0.06 to 3.19 with a mean of 0.79 to Group A and 0.78 to Group B. Although a positive correlation (r=0.574, P<0.05) was found between Sa/So ratio in serum and corn consumption data obtained by 24h-DRQ, no correlation was observed (P>0,05) with FB1 intake and Sa/So ratio in urine or serum. Folic acid concentration in serum ranged from 6.7 to 24.0 ng mL-1 (mean of 13.4 ± 5.4 ng mL-1), with both groups (A and B) presenting levels within the reference valuies. There were no detectable levels of FB1 in serum samples. However, FB1 was detected in 4 human hair samples (7.2%) of Groups A and B, at a mean concentration was 21.3 ± 12.1 ng g-1. In summary, the results obtained in the analyses of FB1 biomarkers in the present study are in agreement with the PDIM values found, hence indicating that FB1 exposure in the populations studied do not represent a health concern.

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A critical evaluation of the published data for chromium levels in serum and urine shows major discrepancies, indicating that further work to establish normal values for these parameters is necessary. Methods have been developed for the determination of total protein-bound, and alpha-2-globulin-bound, chromium in serum, and chromium in urine. The sample pretreatment for serum is based on concurrent protein precipitation and dehydration using propan-2-ol for the total protein-bound chromium, and 0.5M hydrochloric acid in propan-2-ol for the alpha-2-globulin-bound metal. The precipitates are washed in propan-2-ol, then in toluene. Urine aliquots equivalent to 20 umol creatinine are dried at 75° C. Acetic acid (plus 7.5% v.v. sulphuric acid) and 1,1,1,5,5,5-hexafluoropenta-2,4-dione are added to the serum precipitates and urine residues. The chromium in the specimens is converted to the beta-diketonate at 75° C, and the complex extracted with petroleum spirit. The excess diketone is removed by washing with phosphate buffer. The chromium is back-extracted with ammonia in EDTA solution and, after an evaporation step, dissolved in ammonium acetate solution. Atomic absorption spectrometry with electrothermal atomisation is used to measure the chromium, and because of the matrix simplification achieved, background correction is not necessary. The mean results on serum from normal subjects were 0.11 ug/L for total protein-bound chromium, and 0.07 ug/L for alpha-2-globulin-bound chromium. The detection limit was 0.03 ug Cr/L for both serum parameters. The mean normal value for urinary chromium was 0.44 ug/ 10 mmol creatinine, with a detection limit of 0.05 ug Cr/10 mmol creatinine. The analytical relative standard deviations for the three parameters at the above levels were: 7%, 9% and 13%respectively. The serum chromium parameters did not show a significant response to a glucose challenge. Precautions against sample contamination were taken, and techniques for reagent purification, and equipment cleaning to a high standard were developed.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
Cocaine is a stimulant that features a strong ability to cause dependence. Often adulterants are added to this drug in order to mimic its action or minimize its adverse effects. When there are other pharmacologically active components in the drug composition, severe problems can occur to users’ health, such as intoxication symptoms. Thus, the aim of this study was to develop a method for the determination of the main adulterants of cocaine (caffeine, levamisole, lidocaine, phenacetin, diltiazem, and hydroxyzine) in human urine. The high-performance liquid chromatography with a photodiode array detector and the dispersive liquid-liquid microextraction based on solidification of floating organic drop were used as analysis technique and as sample preparation technique, respectively. The reversed-phase chromatographic separation was obtained with a C18 column (250 x 4.6 mm; 5 μm; 80 Å) in gradient elution mode using acetonitrile-trifluoroacetic acid 0.026% (v/v) at 1 mL min-1 as mobile phase, at 25°C, and detection at 235 nm. The analysis time was 25 min. Under optimum conditions, human urine samples were alkalized with 0.5 mol.L-1 sodium phosphate buffer (pH 10) and added sodium chloride (20% m/v). Acetonitrile (150 μL) and 1-dodecanol (30 μL) were used as dispersive and extraction solvent, respectively. The method presented linear range of 312.5 – 3125 ng.mL−1 for caffeine and levamisole and 187.5 – 1875 ng.mL−1 for lidocaine, phenacetin, diltiazem, and hydroxyzine, with limit of quantification of 187.5 ng.mL-1 to lidocaine, phenacetin, diltiazem, and hydroxyzine and 312.5 ng.mL-1 for caffeine and levamisole. Recovery mean values were between 6.0 and 42.6%. The method showed good precision and accuracy, with within- and between-run relative standard deviation and relative error less than 15%. The samples were stable after freeze-thaw cycle and short-term room temperature stability tests. Additionally, this method was applied in samples of urine of five cocaine users and at least one adulterant was identified in all samples. It is expected that this method will contribute to the precision in the diagnosis of cocaine adulterants’ intoxication and to the proper planning of therapeutic measures.
A cocaína é uma droga estimulante que apresenta capacidade de causar dependência. Frequentemente são adicionados a esta droga adulterantes com o intuito de mimetizar sua ação ou minimizar seus efeitos adversos. Quando há nessa droga outros componentes farmacologicamente ativos, agravos à saúde dos usuários podem ocorrer, como quadros de intoxicação. Assim, o objetivo deste trabalho foi desenvolver um método de determinação dos principais adulterantes da cocaína (cafeína, levamisol, lidocaína, fenacetina, diltiazem e hidroxizina) em urina humana. A cromatografia líquida de alta eficiência com detector de arranjo de fotodiodos foi utilizada como técnica de análise e a microextração líquido-líquido dispersiva com solidificação da gota orgânica flutuante, como técnica de preparo das amostras. A separação cromatográfica dos analitos em fase reversa foi obtida em uma coluna C18 (250 x 4,6 mm; 5 μm; 80 Å) em modo gradiente e usando acetonitrila-ácido trifluoroacético 0,026% (v/v) a 1 mL.min-1 como fase móvel (25°C e detecção a 235 nm). O tempo de análise foi de 25 min. Para o preparo da amostra, a urina foi alcalinizada com tampão fosfato de sódio 0,5 mol.L-1 (pH 10) e adicionada de cloreto de sódio (20% m/v). Acetonitrila (150 μL) e 1-dodecanol (30 μL) foram utilizados como solvente dispersor e extrator, respectivamente. O método apresentou intervalos lineares de concentração de 312,5 – 3125 ng.mL−1 para cafeína e levamisol e de 187,5 – 1875 ng.mL−1 para lidocaína, fenacetina, diltiazem e hidroxizina, com limite de quantificação de 187,5 ng.mL-1 para lidocaína, fenacetina, diltiazem e hidroxizina e 312,5 ng.mL-1 para cafeína e levamisol. Os valores médios de recuperação variaram de 6,0 a 42,6%. O método mostrou boa precisão e exatidão intra e intercorrida com coeficientes de variação e erros relativos menores que 15%. As amostras apresentaram-se estáveis após ciclos de congelamento-descongelamento e após serem mantidas por 24h em temperatura ambiente. Ainda, o método foi aplicado em cinco amostras de urina de usuários de cocaína e pelo menos um adulterante foi identificado em todas as amostras. Espera-se que este método possa contribuir para a precisão no diagnóstico das intoxicações por adulterantes da cocaína e para o adequado planejamento das medidas terapêuticas.

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Um grupo especial de indivíduos transplantados renais mantém a função do enxerto estável após a total retirada da terapia imunossupressora, alcançando um estado imunológico chamado de Tolerância operacional. Ainda não há marcadores moleculares e celulares que discriminem a tolerância no transplante humano, e os seus mecanismos estão sendo investigados. O perfil de peptídeos presentes na urina pode trazer informações importantes sobre o estado fisiopatológico renal. Nós investigamos se a tolerância operacional apresenta um perfil diferencial de peptídeos urinários, potencialmente, relevante para diagnóstico deste estado imunológico, assim como para a compreensão de seus mecanismos. Realizamos análises qualitativas do extrato de peptídeos da urina de indivíduos dos diferentes grupos do estudo: saudáveis (SA, n=6), tolerantes operacionais (TO, n=5), rejeição crônica (RC, n=8) e estável sob terapia imunossupressora convencional (EST, n=5), utilizando a abordagem proteômica de Shotgun. Identificamos um total de 15283 diferentes peptídeos em todos os grupos, correspondentes a 646 proteínas distintas, distribuídas nos diferentes grupos: TO = 189, RC = 296, EST = 205 e no grupo SA 219 proteínas. Observamos proteínas exclusivas dos diferentes grupos: TO teve 87 proteínas exclusivas, RC 168, EST 106 e SA 108 proteínas. Apesar das proteínas exclusivas não terem sido compartilhadas por todos os indivíduos do mesmo grupo, a totalidade dos indivíduos de cada grupo apresentou várias dessas proteínas (cada indivíduo apresentou em média 15% das proteínas exclusivas de seu grupo). Das 646 proteínas identificadas, apenas 2,3% foram classificadas pelo Gene ontology como relacionadas ao sistema imune e os compartimentos celulares mais frequentes foram: núcleo 36% e citoplasma 23%. Destacamos algumas proteínas relacionadas à resposta imune, exclusivas de alguns grupos, como no grupo TO, a C-C motif chemokine 24 (CCL24) e Endothelin-1 e no grupo RC, a beta 2 microglobulina. Essas moléculas podem ter relevância nos mecanismos imunológicos desses estados clínicos. A abordagem proteômica aplicada neste trabalho permitiu a identificação de um perfil diferencial de peptídeos na urina de cada grupo diferente de estudo. Os peptídeos urinários diferenciais e suas respectivas proteínas podem ter relevância funcional ou como biomarcadores - em relação ao estado fisiológico e às diferentes evoluções clínicas no transplante renal
A special group of renal transplant recipients maintain stable graft function after the complete withdrawal of immunosuppression, achieving a state called operational tolerance. To date, there are no cellular or molecular biomarkers to discriminate human transplantation tolerance and the underlying mechanisms are being investigated. The profile of urinary peptides may provide important information about different renal physiopathological statuses. We investigated whether operational tolerance displays a differential urinary peptide profile, potentially relevant as biomarkers or for the understanding of mechanisms involved in tolerance. We performed qualitative analysis of peptide urinary extracts in individuals from different study groups: healthy (HI, n=6), operational tolerance (OT, n=5), chronic rejection (CR, n=8) e stable under conventional immunosuppression (Sta, n=5), using Shotgun proteomics. Altogether, we identified 15283 different peptides, corresponding to 646 distinct proteins, distributed in all groups: OT = 189, CR = 296, Sta = 205, and 219 proteins in HI. Several proteins were exclusively detected in specific groups: OT showed 87 exclusive proteins, CR 168, Sta 106 and HI 108 proteins. Although the exclusive proteins were not shared by all individuals from that specific group, all individuals from each group presented several of the group-exclusive proteins (each individual presented an average of 15% of the proteins exclusive to his group). Of the 646 proteins identified, only 2.3% were classified in Gene Ontology as related to the immune system and the most frequent cellular compartments were: 36% from nucleus and 23% cytoplasmic. Of notice, some proteins related to the immune response were also group-exclusive, such as, in OT, a C-C motif chemokine 24 (CCL24) and Endothelin-1, and beta 2 microglobulin in CR. These proteins may display relevant roles in the mechanisms involved in these clinical statuses. In conclusion, the proteomic approach used in this study allowed the identification of a differential urinary peptide profile in each different study groups. The differential urinary peptides and their corresponding proteins may display a relevant role functional or as biomarkers - in the state of homeostasis and in different clinical outcomes in renal transplantation

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The field of metabonomics is beginning to grow rapidly due to the ability to analyse biofluids, providing a 'snapshot' of biological processes that have happened (cf: proteomic/transcriptomic studies, which predict what may happen), making it possible to profile responses over time. The work described in this thesis was motivated by the aim of profiling clinical urine samples obtained from fracture patients, with a view to identifying potential biomarkers related to failed fracture healing. This led to the need to develop and evaluate metabonomic approaches, specifically a orthogonal separation approach complementary to the commonly-used reversed phase (RP) separation methods, namely hydrophilic interaction liquid chromatography (HILI C). Urine samples from healthy volunteers were collected and used to develop an LCMS 'metabonomic toolbox'. This development evaluated various aspects of a· metabonomic study that are commonly poorly reported within the literature: 'study design, sample collection storage and handling considerations, data extraction, normalisation and scaling methods, and multivariate data analysis tools. From the literature, the commonly-used method of normalising to creatinine was ' found to be unsuitable due to perturbations in the urinary excretion of creatinine due to factors such as illness. Methods used to evaluate system ~tability were also developed and added to the 'toolbox'. HILIC was successfully used as a separation technique orthogonal to RP, producing comparable results but using different metabolites; this highlights the fact that much potential information is P?ssibly being lost when only RP-LC-MS methods are used for analysis. The need to use both modes of ionisation polarity were also addressed for an increased coverage in biofluid metabolite profiles. , The knowledge gained in the development of the 'metabonomic toolbox' was used for the analysis of clinical urine samples. Despite the lack of properly time-setted samples and none of the recruited patients suffering delayed fracture healing, potential metabolites related to fracture healing were found. However, the samples were very different to previously-analysed samples from healthy volunteers; they , showed very large amounts of protein, which had a large range of molecular weights. These were' identified- proteomically. Finally, ESI-Q-o-ToF MS/MS, MALDI-ToFlToF MS/MS and racemic amino acid analysis were used for the structural determination of a pseudomonad biosurfactant, which was identified, unexpectedly, as the cyclic Iipopeptide white line inducing principle, WLIP.

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Includes abstract.
Includes bibliographical references.
Two thirds of new TB cases in sub-Saharan Africa are HIV coinfected. HIV-TB co-infection increases the incidence of extra-pulmonary, sputum smear-negative and sputum-scarce TB. In these vulnerable patientgroups with high mortality rates, sputum-based diagnostic tools are unhelpful. Urine-based diagnostics offer an attractive, easily available alternative for rapid diagnosis. We evaluated the point-of-care urine LAM strip test (Determine TB LAM Ag test, Alere) and urine-based Xpert MTB/RIF for TB diagnosis in two patient cohorts with high HIV prevalence. A spot urine sample was collected from two cohorts of persons with suspected TB. The first cohort consisted of ambulatory primary care clinic patients suspected of having TB (group 1) whilst the second comprised hospitalised patients with suspected HIV co-infection (group 2). The urine LAM ELISA, LAM strip test and Xpert MTB/RIF were performed according to the manufacturer’s instructions. In addition, the effects of using an alternative ‘rulein’ cut-point for the urine LAM strip test and a pelleted (2-10ml) urine sample for Xpert MTB/RIF testing on diagnostic accuracy and inter-reader reliability was assessed. The diagnostic reference standard was M. tuberculosis culture positivity.

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In cities today, vast amounts of nutrients are being wasted. Improvement in nutrient management within agriculture can contribute to a more sustainable society. Reusing nutrients in agriculture could aid in creating a more circular system, where organic fertilizers can be used instead of chemical fertilizers. Urine is a liquid which has a high nutrient content. According to the Swedish environmental protection agency, human urine can replace mineral fertilizers, by using methods such as source separation, where urine is divided from faeces. This is a cheap, effective and sustainable fertilizer management system that can be easily achieved. In this study, urine fertilizers were compared with ecological and conventional fertilizers (NPK and cow manure). The study examined the effect of different urine fertilizers compared with organic and inorganic ones on plant growth, nutrient content, pH value and microbial growth. The plant growth experiment was carried out in the greenhouse facilities in Alnarp, Sweden. The results from the experiment show that cow manure has a better outcome when it comes to plant growth, but Aurin, one of the urine fertilizers, had the highest uptake of nitrate. Non-diluted urine had a stable result in all analyses. According to this study human urine is a fertilizer which can be used in crop cultivation systems, and can deliver good agricultural results.

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The objectives of this study were: (1) to confirm that transfer factor (TF) (also known as dialyzable leukocyte extract) can be extracted from human urine; (2) to develop a more efficient method of extraction, and; (3) to determine the molecular weight of the TF in urine. The study used urine from donors with known skin test reactions to purified protein derivative of Seiberts (PPD-S) and coccidoidin immitis (cocci). For the first study, a forty-eight hour catch of urine and bovine leukocytes were divided into aliquots and processed using dual acetone precipitation followed by dialysis through a 6,000 molecular weight cut off membrane under negative pressure for 4 to 5 days to extract the TF-like substance. Results confirmed that TF activity could be derived from human urine, and that this procedure could be used with blood to extract TF. In the second study, tangential flow filtration combined with stirred cell final filtration and dual acetone precipitation required only 10 hours to produce TF activity nearly identical to the first study. The stirred cell and acetone procedure could also be used to extract TF from leukocyte extracts. Concentrated urine aliquots placed in equilibrium dialysis membranes demonstrated that the most active fraction was the 2,000 dialysate which was consequently subjected to Fast Atom Bombardment (FAB) ionization using a four sector mass spectrometer. This produced two groups of activity: one between 1270-1510, and the other between 1880-2142 daltons. In conclusion, TF can be extracted from human urine. Tangential flow filtration followed by stirred cell filtration can be used to extract the active substance, and the molecular weight of the active substance in urine TF is between 1279-1510 or 1880-2142 daltons.

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Introdução: O Papilomavirus Humano ( HPV) é um vírus de DNA que inclui 118 genótipos, sendo o tipo 16 responsável por 80% dos casos de câncer cervical nas mulheres. Os homens são um importante reservatório de HPV e os principais responsáveis pela transmissão às suas parceiras. Objetivo: Detectar HPV DNA e determinar a prevalência de HPV-16, 18, 6 e 11 em amostras de urina de homens infectados pelo HIV-1. Material e Métodos: Homens adultos infectados pelo HIV proveniente de clínica de Urologia/doenças sexualmente transmissíveis e clínica de HIV foram convidados a participar da pesquisa. O estudo foi conduzido entre março de 2006 e abril de 2008. Cerca de 20 ml de urine foi coletada em uma sala específica. As amostras foram submetidas a um PCR Real Time utilizando-se Sybr Green® com primers degenerados PGMY09/11 para detecção de HPV DNA. As amostras positivas, então, foram submetidas a uma PCR convencional utilizando-se primers específicos para cada tipo de HPV. Resultados: Um total de 223 homens infectados pelo HIV-1 foi testado, sendo que 81% deles utilizavam HAART. Sessenta e nove (30.9%) homens apresentaram positividade para HPV DNA na urina pelo método de PCR Real Time. Vinte e dois (31.9%) deles foram positivos para o HPV-16, pelo método da PCR convencional. Dezoito (26.5%) homens apresentaram o DNA do HPV-11; quatro (5.8%) apresentaram o HPV-6 e cinco (7.2%) apresentaram o HPV-18. Vinte (29%) apresentaram DNA de algum tipo de HPV que não aqueles que foram propostos nos objetivos do trabalho. Os homens HIV/HPV mostraram atividade sexual precoce menor que os não infectados por HPV (33.4% vs 40.3%, p=0.22), porém alto número de parceiros sexuais no último ano (40.6% vs 36.4%, p=0.50) do que os homens HPV negativos. A média de carga viral de HIV foi de 10663 cópias/mL e 14104 cópias/mL (p=0.0002), para HPV positivo e HPV negativo, respectivamente. A contagem média de células TCD4+ foi de 441 cels/ml e 517 cels/ml (p=0.30), respectivamente. Conclusões: Foi detectada alta prevalência (30.9%) de HPV na urina de homens assintomáticos, infectados pelo HIV-1. Um terço deles foi infectado pelo HPV-16. O alto número de parceiros sexuais foi um fator importante para a aquisição de HPV nesta população. Nós descrevemos o primeiro estudo utilizando PCR Real Time para detectar HPV em amostras de urina no Brasil, sugerindo a urina como um espécime confiável para a triagem de HPV em larga escala na população
Background: Human Papillomaviruses (HPV) is a DNA virus that includes 118 genotypes and the type 16 is the responsible for 80% of the cervical cancer in women. Men are an important reservoir of HPV and major responsible for the transmission for their partners. Aim: To detect HPV DNA and to determine the HPV-16, 18, 6 and 11 prevalence in the urine samples of HIV-1-infected men. Methods: Adult men HIV-infected subjects in the Urology/Sexually Transmitted Disease (STD) Clinic and HIV Out clinic were invited to participate. The collect was conducted between March 2006 and april 2008. About 20 ml of urine was collected in a specific room, previously cleaned within the last 3 hours. The samples were performed to a real time PCR, using Sybr Green® with PGMY09/11 degenerate primers to detect HPV DNA. The positive samples were also submitted to conventional PCR using type-specific primers for each HPV type. Results: A total of 223 HIV-infected men were tested, 81% of whom were on HAART. Sixty-nine (30.9%) men were positive for HPV DNA in their urine samples by Real Time PCR. Twenty-two (31.9%) of them were positive for HPV-16 by conventional PCR. Eighteen (26.1%) men showed the HPV-11 DNA; four (5.8%) showed HPV-6 DNA and five (7.2%) showed HPV-18 DNA. Twenty (29%) presented DNA of some type of HPV than those which have been proposed in the goals of the work. The HIV/HPV co-infected men showed similar early sexual activity (33.4% vs 40.3%, p=0.22), but higher number of sexual partners in the last year (40.6% vs 36.4%, p=0.50) than HPV negative men. The mean HIV RNA plasma viral load was 10663 copies/mL and 14104 copies/mL (p=0.0002), for HPV positive and HPV negative, respectively. The mean T CD4+ cells count was 441 cells/mL and 517 cells/mL (p=0.30), respectively. Conclusions: High (30.9%) prevalence of HPV in urine of asymptomatic HIV-1-infected men was detected. One third of them were infected by HPV-16 type. Higher numbers of sexual partners was an important factor for HPV acquisition in this population. We described the first study using real time PCR to detect HPV in urine samples in Brazil and suggest that urine as a reliable specimen for HPV screening in large sample size population

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The production of building materials is a significant contributor to anthropogenic greenhouse gas emissions with conventional kiln brick production being one of the most energy intensive processes. In addition, phosphorus is a resource that is required by all living organisms and is a key ingredient in many fertilisers. The demand for building materials and global natural phosphate rock (phosphorous) are increasing and decreasing respectively as urbanization increases. Naturally occurring phosphorous is expected to experience a peak in the near future after which it will be completely depleted. Urine has been identified as a potential source of phosphorous for fertiliser production as well as urea for microbial induced calcium carbonate precipitation (MICP) applications. MICP is a natural process that has the ability to produce bio-building material. Urine accounts for a small percentage of the total volume of domestic wastewater but contains a large percentage of the nutrients wastewater treatment plants (WWTP) seek to remove before they adversely affect receiving water bodies. The unprecedented rate of climate change and the associated pressures, coupled with the increased awareness around the depletion of natural resources, presents a significant challenge for which innovative and sustainable solutions are required. The reason for engaging in this project was to investigate if the urea present in human urine could be used in the natural MICP for the production of bio-bricks while at the same time recovering phosphorus from urine. Firstly, a thorough review of literature was conducted to assess current innovations pertaining to the dissertation topic. The process of bio-brick production by MICP requires a urea rich solution which could be recovered from urine. However, the urea present in urine naturally degrades and this process needs to be delayed if urine is to be used as a urea source for MICP. This was achieved by “stabilising” the urine with calcium hydroxide. Sporosarcina pasteurii (S. pasteurii) was the bacteria strain used to help drive the MICP process. The bacteria degraded the urea present in the urine to form carbonate ions which then combined with the calcium ions present in the urine solution to produce calcium carbonate. This calcium carbonate was then used as a bio-cement to glue loose sand particles together in the shape of a brick. The cementation media was made by adding calcium chloride and nutrient broth to the stabilised urine, and lowering its pH to 11.2. The purpose of adding calcium chloride was to improve the efficiency of the process since the stabilised urine did not have enough calcium ions. Ordinary sand mixed with Greywacke aggregate and inoculated with S. pasteurii bacteria was used as the media for the MICP process. Bio-brick moulds were filled with the sand mixture and sealed. The cementation media was pumped through the bio-brick mould to fill its’ pore volume. The media was retained in the moulds for a defined retention time ranging from 1-8 hours. At the end of every retention time, new cementation media was pumped through the bio-brick to fill it’s pore volume again. iv To establish an optimal starting influent calcium concentration the influent calcium concentration changed between experiments. Additionally, in subsequent experiments, the calcium concentration was raised in a stepwise manner during an experiment to establish the maximum amount the influent calcium concentration could be raised to before the microbial community experienced adverse effects. Additionally, experiments explored the effects a range of retention times had on the bio-brick system in order to establish an optimal retention time. Another experiment was set up to investigate the relationship between the number of treatments and the resultant compressive strength. The findings from the above-mentioned experiments further guided subsequent experiments which singled out and tested certain factors thought to be affecting the bio-brick system. The factors tested include after treatment washing, ionic strength, pH and calcium concentration of the influent cementation media. Possible alternative nutrient medias (ANMs) were also investigated for a cheaper alternative to the laboratory grade growth media used to grow the bacteria. Lastly, an integrated system that produced both fertilisers and bio-bricks was developed. Its basic economics of raw material inputs and outputs were used to assess the financial implications of the proposed system, and the social and policy barriers likely to affect the implementation of an integrated urine treatment system were examined. Urine treated with calcium hydroxide offers a urea-rich solution that can be used for MICP processes. This resulted in the worlds’ first bio-brick “grown” from human urine. The starting influent calcium concentration reached a maximum of 0.09 M before adverse effects to the microbial community were experienced. Furthermore, in terms of a stepwise increase during the treatment cycle, the influent calcium concentration could be raised to 0.12 M without any adverse bacteria effects. The minimum retention time the bio-brick system could withstand was 2 hours which allowed the treatment cycle to be completed in a shorter time. The highest compressive strength obtained was equal to 2.7 MPa. To produce this strength about 31.2 L of stabilised urine was used. The relationship between the number of treatments and the compressive strength showed that an increase in the number of treatments increased the compressive strength. Both the pH and ionic strength of the urine were identified to have an inhibiting effect on the ureolytic activity and MICP process. Additionally, using an influent cementation media with an optimal pH for urea hydrolysis, improved the bacteria’s ability to operate at higher ionic strengths. However, when the stabilised urine was stored, urea hydrolysis occurred earlier likely because of external contamination by naturally occurring bacteria in the lab. LML (Lactose mother liquor) was identified as alternative growth media for S. pasteurii growth which could reduce raw material costs considerably. The bio-brick production process was found to be more cost-effective if it was incorporated into the integrated urine treatment process system. The integrated system included fertiliser production by recovering calcium phosphate fertilisers and ammonium sulphate fertilisers before and after the bio-brick production respectively. Producing 1000 bio-bricks a day would require 23% of Cape Towns’ population daily urine production and would incur a profit of ZAR 7330 per day between the raw material cost and the revenue from sales. For implementation in a South African context, certain policy barriers need to be overcome. Potential paths for implementation are reclassifying the urine for its use in an industrial process and obtaining an operating permit or seeking an exemption for a permit through the ECA (Environment Conservation Act). Research suggests that products from the integrated system are likely to be socially v accepted and that a combined appeal to people's environmental sensitivities and targeted marketing messages would enhance people’s acceptance. Finally, recommendations for further paths to take to build on the research established in this dissertation were made. It is recommended that additional characteristics of the bio-bricks should be tested, recycled material should be used as media for bio-bricks, the bacteria strain should be modified and methods for reducing the ionic strength of urine should be investigated. Additionally, it is recommended that consumers’ willingness to use urine-based products should be further studied, the legislative options for implementing bio-brick and fertiliser production should be investigated and a more detailed and expansive economic analysis should be performed.

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Millions of people are exposed to arsenic in the United States and worldwide. Commonly found arsenic species in human urine are AsIII (arsenite), AsV (arsenate), MMA (monomethyl arsenic acid), DMA (dimethylarsinic acid) and AB (arsenobetaine). Evidence has shown that these species vary in toxicity, and since each of these metabolites can be detected through analysis, they have the potential to be used as biomarkers for human exposure. For human exposure assessments in areas that have naturally occurring arsenic contaminated sources, or those who live or work near contaminated environmental sites where arsenic has been used, it is important to fully understand what species of arsenic residents are being exposed to in order to grasp the risk of arsenic exposure specifically and in its entirety.

Since it is difficult to determine direct human exposures, a swine model was used as a surrogate. Swine urine was collected from two different swine studies where animals were given non-toxic doses of arsenic contaminated soil and another group receiving a soluble reference dose using sodium arsenate for comparison. The urine samples from these studies were used to modify an arsenic speciation method using high-performance liquid chromatography and inductively coupled plasma mass spectrometry (LCICPMS). It is evident that when comparing the percent of arsenic species found in swine urine samples with what is found in humans a correlation can be made. There was a range of 64–74% DMA in swine samples for all test soils where a range of 60–75% DMA has been reported in human urine samples. This further illustrates the importance of arsenic speciation in swine urine since it does appear that it could correlate to human exposure. If proper measurement systems are utilized to quantify As species of health concern, dosed swine can be used to assess and predict human toxicological effects of arsenic exposure.

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Human urine is a valuable resource which has good potential to be used as a fertilizer all over the world. In the developing countries sanitation and food security are both issues that need urgent attention. A urine separation toilet can be constructed with minimal investment in the Nicaraguan context, and the usage of the urine as a fertilizer can help establish higher yields and is a good alternative to chemical fertilizers. This field experiment is trying this in practice in the context of rural Nicaragua, to determine the effect of urine on two plants on. For this study, the common bean (Phaseolus vulgaris) and the Chaya (Cnidoscolus aconitifolius) was selected and the results confirm that urine has potential as a fertilizer in the Nicaragua context. The common bean yield was twice as large after urine fertilization and the Chaya reacted positively to urine fertilization. For urine separation purposes, two different separators were constructed on the site to showcase the benefits with separating the urine from the faeces, creating lower latrine volume and better sanitation in the outhouse. The risks associated with human urine are low if the urine is separated securely to avoid crosscontamination from faeces. If a safety-barrier system is adopted, the overall risks with using urine as a fertilizer are negligible. The spreading potential of urine separation and fertilization in rural Nicaragua is high, but more experiments and demonstrations are needed to reach adopters of the technology.
La orina humana es un recurso valioso que tiene un buen potencial para ser utilizado como fertilizante en el mundo entero. En los países en vías de desarrollo, el saneamiento y la seguridad alimentaria son dos temas que necesitan atención urgente. Un inodoro de separación de orina puede ser construido con una inversión mínima en el contexto Nicaragüense, y el uso de la orina como fertilizante puede ayudar a establecer mayores rendimientos y es una buena alternativa a los fertilizantes químicos. Este experimento de campo está probando esto en la práctica en el contexto de Nicaragua rural, para determinar la diferencia en crecimiento entre dos cultivos con y sin fertilización de orina. Para este estudio se seleccionó el frijol común (Phaseolus vulgaris) y la Chaya (Cnidoscolus aconitifolius) El rendimiento de frijol fue dos veces mayor después de la fertilización de la orina y el Chaya reaccionó positivamente a la fertilización de la orina. Para fines de separación de orina, se construyeron dos separadores diferentes en el sitio para mostrar los beneficios con la separación de la orina de las heces, creando un menor volumen de letrina y un mejor saneamiento. Los riesgos asociados con la orina humana son bajos si la orina se separa con seguridad para evitar la contaminación cruzada de las heces. Si se adopta un sistema de barrera de seguridad, los riesgos generales con el uso de orina como fertilizante son insignificantes. El potencial de propagación de la separación de orina y la fertilización en Nicaragua rural es alto, pero se necesitan más experimentos y demostraciones para llegar a los usuarios de la tecnología.

2017-06-02

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Les cellules souches humaines induites à la pluripotence (hiPSCs) ont été conçues pour la première fois en 2007 par l’équipe du Docteur Yamanaka, au Japon. Ce sont des cellules somatiques reprogrammées par un virus permettant, par exemple, la différenciation neuronale à des fins d'étude de maladies neuro-développementales telle que la Schizophrénie. Le prélèvement des cellules somatiques se fait aujourd'hui majoritairement par des méthodes assez invasives, notamment les biopsies de peau ou prélèvements sanguins. Ceci peut représenter un frein à leur utilisation notamment chez les enfants et surtout les enfants malades. La différenciation neuronale privilégiée est la voie dopaminergique (DA) car c'est ce type cellulaire qui est principalement atteint chez les schizophrènes. C'est pourquoi on priorise pour ce projet l'utilisation de cellules contenues dans l'urine, qui seront reprogrammées via un virus non-intégratif, le virus de Sendaï (SeV). La différenciation neuronale nous permettra d'obtenir des neurones DA fonctionnels, caractérisés par électrophysiologie. Les expériences ont montré une très grande efficacité de reprogrammation cellulaire au niveau des cellules d'urine, ainsi qu'un grand potentiel de différenciation neuronale, malgré quelques différences observées entre les lignées saines et schizophréniques. Grâce à ce projet, la réalisation d'un modèle cellulaire pour la Schizophrénie a pu être établie. Les différences notées entre les lignées pendant la différenciation ouvrent une nouvelle voie pour approfondir l'étude de la maladie au niveau cellulaire et moléculaire.
Human Induced Pluripotent Stem Cells (hiPSCs) were conceived for the first time in 2007 in Japan, by Doctor Yamanaka’s team. These are somatic cells reprogrammed thanks to a retrovirus allowing, for example, neuronal differentiation for the purpose of neurodevelopmental disorders studies such as Schizophrenia. Today, the removal of somatic cells is mainly made by enough invasive methods, including skin and blood biopsies. This can represent a brake in their use predominantly children, mainly sick children. The preferred neuronal differentiation is the dopaminergic (DA) way because it's the mostly cell type affected in schizophrenics. That's why we prioritize the use of urine cells for this project, reprogrammed via a non integrative virus, the Sendai virus (SeV). The neuronal differentiation enables us to get functional DA neurons characterized by electrophysiology. Experimentations show a huge efficiency of urine cells reprogramming as well as a great potential of neuronal differentiation despite some distinctions between the two lines. Thanks to this project, the achievement of a cellular model for Schizophrenia could be established. The differences noticed between the two lines during the differentiation open up a new way to make cellular and molecular studies of this disease deeper.

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O reciclo de nutrientes entre as áreas urbanas e as áreas cultiváveis é uma etapa crítica em direção ao desenvolvimento ecológico sustentável. A maior parte dos nutrientes que são essenciais na agricultura (N, P, K) é encontrada na urina humana e, na maioria dos casos, sua quantidade total é mais apropriada do que as encontradas nos fertilizantes artificiais. Este trabalho teve por objetivo a caracterização quali-quantitativa, avaliar as formas de estocagem (aberta, fechada e aerada) e a evaporação como técnica de redução de volume e concentração de nutrientes. Para isso foi realizada uma etapa de caracterização, com urina de homens, mulheres, idosos e crianças. Os resultados da caracterização, analisando volume e concentração de nutrientes, mostram que a produção per capita de urina fica em torno de 1,23L/dia para homens, mulheres e idosos e em 0,7L/dia para crianças, e que esta apresenta 7,5g/L de nitrogênio, 0,5g/L de fósforo e 1,6g/L de potássio. Quanto às formas de estocagem, a utilização em reservatórios fechados foi a forma mais eficiente no processo de estabilização físico-química e biológica, pois não ocorreu perda de amônia por volatilização, e nem possíveis contaminações por influência do ambiente externo, a concentração dos nutrientes nessa forma também foi mais satisfatória. Os níveis de coliformes termotolerantes tenderam a praticamente nulo em um período de 15 dias e em temperatura ambiente. O processo de evaporação consistiu na utilização da energia solar como única fonte de calor, e assim, houvesse a diminuição no volume e aumento na concentração dos nutrientes. A evaporação da urina humana foi realizada, utilizando urina fresca e estocada. A urina fresca foi coletada com o apoio do grupo de pesquisa da UFES e a estocada de um reservatório de 200L localizado na ETE - UFES. Foram utilizados dois recipientes para cada tipo de urina, sendo que em um deles foi adicionado ácido sulfúrico concentrado a fim de minimizar a perda de amônia. A taxa de evaporação foi de 2,3 a 2,8L/m².d. Quanto aos nutrientes (N, P, K, Ca e Mg), o resíduo formado ao final apresenta concentrações significativas em quantidades que chegam a 91% das concentrações desses nutrientes nos fertilizantes artificiais. Após a evaporação a média foi de 21 Kg de material residual para 500 Kg de urina líquida. Conclui-se que utilizar a evaporação para esses fins é bastante interessante do ponto de vista econômico e técnico.

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Urolithiasis occurs in 20% of males and 5--10% of females, and 75% of kidney stones contain calcium oxalate (CO) mineral. To analyze mineral-binding proteins and to make gender comparisons, using the model of Doyle et al. (Clin Chem, 37: 1589--1594, 1991), CO crystals were generated in whole and centrifuged urine samples and then washed with water or sodium hydroxide. Crystals and mineral-binding proteins were analyzed by SDS-PAGE, Western blotting and electron microscopy (SEM). Regardless of urine or crystal treatment, osteopontin and UPTF1 proteins were consistently present in the samples, whereas THP and albumin were partially removed. SEM showed larger crystals precipitated from female than from male urine. Western blotting demonstrated more albumin bound to crystals from females. In other experiments, CO crystals were grown in the presence of poly-L-aspartic acid (PA) and albumin. SEM demonstrated that these proteins affected CO crystallization. Competitive protein-binding assays and fluorescence activated cell sorter analysis after binding of PA and albumin to hydroxyapatite indicated that PA binds hydroxyapatite with a stronger affinity than albumin.

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Dissertation submitted to Faculdade de Ciências e Tecnologia - Universidade Nova de Lisboa in fulfilment of the requirements for the degree of Doctor of Philosophy (Biochemistry - Biotechnology)
The use of anabolic androgenic steroids (AAS) and other banned substances to enhance athletic performance has important health and social implications. The AAS are a major group included in the prohibited list of the world anti-doping agency (WADA) as well as of major sports authorities. This class of drugs, along with other anabolic agents, represent 64,9 % of all adverse analytical findings reported by WADA accredited laboratories, as stated in the WADA statistic report for 2009. The AAS are a class of hormones that include the natural male sex hormone, testosterone, and its many synthetic derivatives. They exert multiple actions affecting both the physiology of the human body and the individual behaviour. Under intensive training, the AAS induce the synthesis of proteins in muscle and bone causing an accelerated growth of these organs. Furthermore, during acute endurance workout, as well as during competition, androgen’s action seems to be critical to enhance the performance capacity, since they affect the production of red blood cells and increase neural conduction. In addition, after intense exercises, androgens are thought to prevent muscle catabolism and exhaustion and to speed up the recovery process.In general, the normal proceeding for AAS determination includes chromatographicseparation coupled to mass spectrometry detectors. The use of GC-MS methodologies is the most employed strategy for AAS control. However, over the last years, with the development of suitable LC-MS and LC-MS/MS systems, some AAS presenting poor chromatographic properties for GC-MS analysis, even after derivatisation, are being analysed by LC-MS(/MS) procedures.The aim of the research programme presented in this thesis was, primarily, the development of a new screening method based on mass spectrometry (MS) using the soft ionisation technique matrix-assisted laser desorption/ionisation (MALDI). The major goals to be achieved were the development of an accurate, sensitive and robust methodology able to improve the screening of AAS for doping control in both analysis time and sample throughput. Additionally, the developed method should be capable to overcome the GC-MS limitation related to thermo-labile and polar AAS, so that the initial screening method could be extended to all AAS included in the prohibited list.In parallel with the development of a screening procedure based on MALDI-MS(/MS)techniques, and applying the deep expertise of the research group on reaction enhancement by delivery energy based techniques, the improvement of the global sample preparation for the analysis of AAS by anti-doping control laboratories was also included in the research programme.
Fundação para a Ciência e a Tecnologia”, for financial support through the grant SFRH/BD/31652/2006

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Apples are a major dietary source of polyphenols in the Western diet and contain procyanidins, hydroxycinnamic acids, flavanols, dihydrochalcones and flavonols. Despite their abundance and familiarity very little research into their metabolism has been performed; research is required to elucidate the metabolic products of these polyphenols and characterise their absorption and excretion pathways. A human intervention study was designed specifically to investigate the absorption, metabolism, excretion and biokinetcs of apple polyphenols. Male volunteers (n = 9) consumed a supermarket apple juice substituted with water as the control phase, and the same apple juice substituted with a high polyphenol cider apple extract as the test phase. Blood samples were taken over 0-24 h and urine samples were collected at 0-4 h, 4-8 h and 8-24 h. A rapid, validated and novel single LC/ES/IMS/MS method was developed and validated for the analysis of a wide range of polyphenols and their metabolites in these urine and plasma samples (after sample preparation). Apple polyphenolrelated metabolites were identified using LC/MS/MS and MS2; nine urinary metabolites and seven plasma metabolites were identified, mostly for the first time after apple consumption. Data on the excretion, bioavailability and biokinetics of these metabolites, including products of the colonic micro flora, were obtained. In urine, the major apple-related polyphenolic metabolites identified were dihydroxyphenyl valerolactone sulfate and 5- (3', 4'- dihydroxyphenyl) -y- valerolactone glucuronide; both colonic bacterial metabolites which appear at their maximum concentrations 4-8 h post apple ingestion. Minor metabolites included (-) epicatechin sulfate and glucuronide conjugates. In plasma, 3, 4-dihydroxyphenylacetic acid, 5- (3',4'-dihydroxyphenyl) -y- valerolactone glucuronide and dihydroxyphenyl valero lactone sulfate predominate; Tmax values of 5-6 h were observed. Minor plasma metabolites included phloretin (Cmax291 ± 175 nM) and p-coumaric acid (Cmax 634 ± 225 nM). In conclusion, the project has identified apple-related polyphenol metabolites in human urine and plasma; many for the first time after apple consumption. Important biokinetic parameters have also been reported for these metabolites.

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Seven billion people are urinating every day, excreting 28 million tonnes of nitrogen (N) a year (Vinneräs, 2002). This mass of N excreted is equal to 26% of the annual global N fertilizer demand, a value of $21.3 trillion USD (based on $350 USD per metric tonne of urea) (Alibaba, 2012, FAO, 2011). The use of human excreta as N fertilizer would allow the recovery of nutrients, resulting in savings for farmers, and counter the threat to people's health by reducing contact with untreated human waste. This practice, termed ecological sanitation (EcoSan), has a long history of application in traditional agricultural contexts, but has a limited scientific knowledge base. This thesis builds upon the scientific knowledge base by: (1) analyzing the chemical changes in soil from long-term use (nine years) of human urine as a fertilizer compared to mineral fertilizer; (2) optimizing the human urine application rate for spinach in Himachal Pradesh, India; and (3) performing a theoretical quantitative microbial risk assessment (QMRA) for the use of human urine on spinach in northern rural India. A sensitivity experiment, comparing synthetic human urine, mineral fertilizer and in combination and with the increasing application rates, observed that spinach was able to withstand significantly higher EC soil levels than were commonly reported. Brown mustard biomass production rates did decrease with the increased application rates of the three fertilizer treatments. Tissue samples from field trials in India had no significant difference between N concentrations for the three fertilizer treatments (human urine, mineral fertilizer and combination of the two) and were all significantly higher than the control (no fertilizer). The dry biomass production of spinach from the human urine treatments were significantly higher than the control and were not significantly different to mineral fertilizer treatments, confirming that human urine can substitute mineral fertilizer. The optimal human urine application rate in Himachal Pradesh, India, was 59.3 m3 ha-1 of human urine, resulting in 360 kg N, 570 kg P2O5 and 720 kg K2O per ha per season which is higher than the current guidelines. Of the three fertilizer treatment, the optimal fertilizer was the combination treatment (human urine with additional phosphate (P) and potassium (K)) at 360 kg N ha-1 as it had significantly higher biomass productions to that of the human urine and the mineral fertilizer treatments. The novel finding was the use of sodium (Na) by the spinach plants as a supplement for K deficiencies in the human urine fertilizer treatments. The tissue samples from the human urine treatments had significantly higher Na concentrations than the other treatments, with no signs of toxicity. This finding illustrates that other crops with the ability to compensate for limited K will perform well with EcoSan systems by using the Na contained in urine. The results of this thesis illustrated that the 28 million tonnes of excreted human N is an effective substitute to expensive mineral fertilizer. Though, based on the QMRA, areas prone to high occurrence of diarrheal disease should use human urine for crops that will be processed, such as rice, or for cash-crops and not for edible crops grown close to the ground, such as spinach.
Sept milliards de personnes urinent chaque jour, excrétant 28 millions de tonnes d'azote (N) par an (Vinnerås 2002). Cette masse d'azote excrétée est égale à 26% de la demande annuelle d'engrais azoté global, d'une valeur de $ 21,3 trillions USD (sur la base de 350 $ USD par tonne métrique d'urée) (Alibaba 2012, FAO, 2011). L'utilisation des excréments humains comme engrais permettrait la récupération des nutriments, ce qui permettrait aux agriculteurs de faire des économies, et contrer la menace pour la santé des personnes en réduisant le contact avec les déchets humains non traités. Cette pratique, appelée assainissem*nt écologique (EcoSan), a une longue histoire d'utilisation dans des contextes agricoles traditionnelles, mais dispose d'une base de connaissances scientifiques limitée. Cette thèse s'appuie sur la base de connaissances scientifiques par: (1) l'analyse des changements chimiques dans le sol à partir de l'utilisation à long terme de l'urine humaine comme engrais par rapport aux engrais minéraux, (2) optimiser le taux d'application de l'urine humaine pour les épinards dans l'Himachal Pradesh, Inde, et (3) d'effectuer une évaluation quantitative des risques microbiologiques théorique (QMRA) pour l'utilisation de l'urine humaine sur les épinards dans le nord de l'Inde rurale. Une expérience de sensibilité, en comparant l'urine humaine synthétique, d'engrais minéraux et la combinaison avec l'augmentation des taux d'application, ont montré que les épinards étaient capable de résister à des niveaux de CE dans le sol nettement plus élevés que ce qui a fréquemment été rapporté. Les taux de production de biomasse de moutarde brune ont diminué avec l'augmentation du taux d'application des trois traitements de fertilisation. Des échantillons de tissus provenant d'essais sur le terrain en Inde n'avaient pas de différence significative entre les concentrations d'azote pour les trois traitements de fertilisation (urine humaine, engrais minéraux et la combinaison des deux) et étaient significativement plus élevés que le contrôle (sans engrais). La production de biomasse sèche des épinards dans les traitements de l'urine humaine a été significativement plus élevée que le contrôle et n'était pas significativement différente aux traitements d'engrais minéraux, confirmant que l'urine humaine peut remplacer les engrais minéraux. Le taux d'application de l'urine humaine optimale dans l'Himachal Pradesh, en Inde, était de 59,3 m3 par hectare d'urine humaine, résultant en 360 kg d'azote, 570 kg de P2O5 et 720 kg K2O par hectare et par saison, ce qui est plus élevé que les lignes directrices actuelles. Parmi les trois traitement d'engrais, l'engrais optimal était le traitement combiné (urine humaine avec du phosphate supplémentaire (P) et le potassium (K)) à 360 kg N ha-1 puisque les taux de production de la biomasse étaient sensiblement plus élevés pour celle de l'urine humaine et de l'engrais minéraux. La nouvelle découverte a été l'utilisation de sodium (Na) par les plants d'épinards comme un supplément pour les lacunes en K dans les traitements de fertilisation d'urine humaine. Les échantillons de tissus provenant des traitements d'urine humaine avaient des concentrations de Na significativement plus élevés que les autres traitements, sans aucun signe de toxicité. Ce résultat montre que d'autres cultures qui ont la capacité à compenser la quantité limité en K se développeront bien avec les systèmes ecosan en utilisant le Na contenu dans l'urine. Les résultats de cette thèse ont illustré que les 28 millions de tonnes de N humain excrété est un substitut efficace à l'engrais minéral cher. Bien que, sur la base du QMRA, les zones sujettes à la fréquence élevée des maladies diarrhéiques devraient utiliser l'urine humaine pour les cultures qui seront transformées, tels que le riz, ou de cultures de rente, et non pour des cultures comestibles poussant au ras du sol, comme les épinards.

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Background: Arsenic (As) is a naturally-occurring element with known toxicant effects. The primary exposure pathway is through ingestion, but the overall contribution of food versus water and the impact of specific dietary nutrients on urinary As excretion is not well understood. Methods: Secondary analyses of laboratory results from food, water and urine samples, questionnaire and anthropometric data, and dietary records were performed on four study populations: the National Health Exposure Assessment Survey (NHEXAS)-Arizona, Arizona Border Survey (ABS), the Arizona sub-group of the Binational Arsenic Exposure Survey (BAsES), and the 2003-2004 National Health and Nutrition Examination Survey (NHANES). Dietary As intake was measured in duplicate food samples and/or modeled from dietary records for each population using the U.S. Total Diet Study (TDS) arsenic residue database and a published market basket survey. Urinary total As, As⁵, As³, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were analyzed, and sum of species As was calculated as the sum of As⁵, As³, MMA and DMA. Regression analyses modeled the relation between urinary As biomarkers (total, sum of species, MMA:sum of species, and DMA:MMA) and dietary As, adjusted for drinking and cooking water As intake, current smoking, sex, age, ethnicity, body mass index, and nutrient intake. Results: Modeled dietary As based on TDS mean As residue data greatly underestimated exposure as compared with measured As in duplicate diet samples and estimates based on other residue data. Dietary As was a significant predictor of urinary total As in all four populations, of sum of species As in both BAsES and NHANES, and of %MMA and DMA:MMA in NHANES. Dietary protein intake was associated with decreased sum of species As in both BAsES and NHANES, but dietary folate was not. Conclusions: Dietary As contributes a markedly greater proportion of total ingested As and is a better predictor of urinary As than water As intake in the U.S. Among subjects who did not consume seafood, total As exposure from food and water exceeded the provisional tolerable daily intake of 2.1 µg/kg body weight/day in 3-15% of these study populations. Increased protein intake may mitigate the effects of As.

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Plastic debris is defined on the basis of their dimensions in microplastics (between 5 mm and 1 µm) and nanoplastics (< 1µm). Micro and nano-plastic (NPs) pollution is growing exponentially in the last decades, and there are increasing evidences of the ubiquitous distribution of plastic contaminants in the environment. potential deleterious effects of MPs on human beings. This is the first study aimed to investigate the presence of MPs in human kidney. Moreover, we analyzed the effect of microparticles of PE on HK2 kidney tubular cells. In vivo we investigated the presence of MPs in urine and kidney tissues and we characterized the microparticles found through Micro-Raman Spectroscopy. In vitro we analyzed the effect of PE-MPs, BPA, and co-treatment BPA-MPs on HK2 kidney tubular cells. We performed different cells culture conditions and we analyzed various biomarkers of inflammation, oxidative stress, and mitochondrial activity.To the best of our knowledge, this is the first study that demonstrated the presence of MPs and their compounds in human urine and kidney tissue. In particular, this is the first study that advance evidence of MPs clearance in humans. Moreover, we added new evidences of their potential nephrotoxic effect in vitro on HK2 tubular cells using PE-microspheres and BPA.

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A urina humana contém a maior parte dos nutrientes essenciais à agricultura. Porém, a urina é, também, via de excreção de fármacos inalterados e seus metabólitos e disruptores endócrinos. O presente trabalho teve como objetivo avaliar o perfil cromatográfico do diclofenaco de sódio, prednisolona, progesterona e sulfametoxazol na urina humana durante o processo de tratamento de estocagem da urina humana com e sem acidificação, avaliar a influência da temperatura e variação de pH durante o processo de tratamento, quantificar por cromatografia líquida de alta eficiência acoplada a um detector de arranjo de fotodiodos (CLAE-DAD) os compostos estudados na urina humana acidificada durante processo tratamento de estocagem e determinar o método de detecção. O método foi aplicado para as amostras de urina humana acidificada e não acidificada submetidas ao processo de estocagem durante ciclos de 30 dias sob diferentes temperaturas. Foi observado que a variação de temperatura empregada não alterou o perfil cromatográfico das amostras analisadas. A urina que não foi submetida ao processo de acidificação demonstrou alteração no seu perfil cromatográfico, provavelmente devido ao processo de hidrólise da uréia, não sendo, portanto, possível a quantificação dos fármacos e disruptores endócrinos na mesma. O método para CLAE em fase reversa desenvolvido nesse estudo é sensível, seletivo e reprodutível para determinação dos 4 fármacos presentes na urina humana durante o período de estocagem. A fase móvel mais adequada para a eluição dos fármacos e disruptores endócrinos estudados (SULFA, PRED, DICLO E PRO) na urina humana, com menor dispersão do analito, foi a fase móvel 4 (FM4). Nessa fase móvel a eluição foi realizada por gradiente, fluxo de 1,0 mL.min-¹ e concentração de acetonitrila (ACN) variando entre 10 e 15%, permitindo que essa fase móvel apresentasse a força cromatográfica e a seletividade adequada para a separação dos fármacos e disruptores estudados.O método CLAE-DAD utilizado apresentou satisfatória linearidade (r > 0,99 para todos os compostos analisados) e precisão (CV < 5% para todos os compostos). Os limites de detecção (LD) e quantificação (LQ) apresentaram valores menores que aqueles utilizados no processo de estocagem, portanto adequado para as análises realizadas. Desse modo a quantificação dos compostos estudados foi realizada apenas na urina humana acidificada. Conclui-se que as condições de tratamento utilizadas no presente trabalho, ou seja, acidificação da urina e temperatura de estocagem, não foram suficientes para reduzir a concentração dos compostos estudados. A concentração adicionada inicialmente manteve-se até o final do experimento, não havendo diminuição da mesma.

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Bacteriuria, or the presence of bacteria in urine, is associated with both asymptomatic, as well as symptomatic urinary tract infection (UTI) and underpins much of the dynamic of microbial colonization of the urinary tract. The prevalence of bacteriuria in dissimilar patient groups such as healthy adults, institutionalized elderly, pregnant women, and immune-compromised patients varies widely. In addition, assessing the importance of ‘significant bacteriuria’ in infected individuals represents a diagnostic challenge, partly due to various causal microbes, and requires careful consideration of the distinct etiologies of bacteriuria in different populations and circ*mstances. Recent molecular discoveries have revealed how some bacterial traits can enable organisms to grow in human urine, which, as a fitness adaptation, is likely to influence the progression of bacteriuria in some individuals. This study was designed as a comprehensive analysis of asymptomatic bacteriuria (ABU) causal organisms in dissimilar populations, and an in-depth microbiological analysis of the mechanisms used by one such causal organism, Streptococcus agalactiae. This organism causes UTI including ABU; however, growth of S. agalactiae in human urine has not been reported. In the first part of this study, we evaluate the prevalence and etiology of bacteruria, and discuss recent advances in the molecular detection of bacteriuria from a diagnostic viewpoint.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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The aim of this dissertation is to analyse the use of narratives informed by the discourse of human security in the context of the Colombian conflict during the government of President Alvaro Uribe Velez (2002-2010). Its main contribution is to map the transformation of these narratives from the site of their formulation in the international institutions to the site of their appropriation into domestic settings; and then consider their role in the formation of the actors' strategies and the construction of the subjectivities of the individuals affected by the conflict dynamics. The research proceeds to this analysis through an investigation of the policies for the internally displaced and those relating to the rights of the victims informed by the framework of transitional justice. It shows that, with a combination of narratives of empowerment and reconciliation, they fulfill complementary roles in the construction of the subjectivities of individuals affected by the conflict in Colombia. The dissertation also concludes that the flexibility of the human security discourse allowed the Uribe government to reinforce its position.

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This thesis analyses individual and societal relations to human excrement by looking at historical and contemporary examples of symbolics and management systems of human sh*t and piss. It furthermore connects urban culture to a particular type of perception of the meaning of human waste. End-of-pipe, large scale sewerage solutions for densely populated areas and cities are analysed for their historical origins and contemporary ramifications, and contrasted with examples of classical, mediaeval, early modern and contemporary times in different regions of Europe and India. The cases were presented in a non-chronological order to avoid simple narratives of progress. The focus is on questions of agricultural recycling of excrement and the relevance of human waste for public health issues. Analytical tools during the cross-temporal and cross-cultural case comparison are the categorisations of human excrement as e.g. waste, threat or resource, the technique of dualism-deterritorialisation and occasionally the Entanglement approach. Main results are that the large-scale introduction of sewerage systems in European cities around the world coincides with urbanisation and industrialisation, that pre-industrial dense settlement faced essentially the same excrement management challenges as modern cities do and that the stability of certain management systems has been severely influenced by factors such as power structures, paradigms of purity and piety as well as economic developments. The future relevance of this topic is seen in the predicted rise of urban regions worldwide, but especially in the developing world, a development which is expected to complicate human excrement management issues considerably.

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Objective New biomarkers for acute kidney injury are needed and urinary Cystatin C is one alternative. The objective was to validate a urinary Cystatin C method on Mindray BS-380 comparing urine samples from the Uppsala Longitudinal Study of Adult Men (ULSAM-77) and urine samples from a reference group for Cystatin C. A visual control for a relationship between Cystatin C and C-reactive protein (CRP) and interleukin 6 (IL-6) respectively was made. Methods Precision, linearity, recovery, interference, and stability of the urine cystatin C method were investigated. Comparisons were made between ULSAM-77 samples and a reference group samples consisting of ordinary people. Results The highest total imprecision was 10.24 % for the sample with the lowest concentration. The second lowest concentration had 4.21 % total variation coefficient. The linearity equation was y = 0.99x – 0.01 with an R2-value of 0.99. The recovery for all concentrations was always 91 % or more. No interference from hemoglobin at a concentration of 10 g/L was found. The samples were stable at +5°C for seven days. The median for the samples from ULSAM-77 was 0.09 mg/L and the median for the reference samples was 0.06 mg/L. There was no obvious relationship between Cystatin C and CRP/IL-6 from ULSAM-77. Conclusion Reliable data of urinary Cystatin C can be analyzed on a Mindray BS-380. The level of urinary Cystatin C was higher for people age 77 than for those with a median age of 49. There was no correlation between the concentration of Cystatin C in urine and the levels of CRP and IL-6.

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Orientador: Leonardo Pezza
Banca: Jose Anchieta Gomes Neto
Banca: Aristeu Gomes Tininis
Resumo: A proposta apresentada por este trabalho foi estudar e desenvolver um método analítico para a determinação do herbicida paraquat em urina humana, de modo que este seja rápido, sensível, econômico e com uma menor geração de resíduos em relação aos métodos disponíveis, atualmente empregados para esta finalidade, atendendo assim os princípios e requisitos da Química Verde (Green Chemistry) e da Química Analítica Verde (Green Analytical Chemistry). Outro aspecto também abordado, é o emprego da técnica de análise por imagem digital utilizando software analítico através da geração de imagens digitais por meio das fotografias obtidas dos spot-tests, tornando a metodologia portátil e consequentemente mais acessível. O método desenvolvido consiste na reação de redução do dicátion paraquat (PQ2+) para a formação de um cátion radicalar (PQ*+) de coloração azul, utilizando uma solução de glicose (Glc) em meio alcalino (NaOH) como reagente redutor. As concentrações dos reagentes foram otimizadas para obter a maior resposta analítica possível, com o auxílio de ferramentas quimiométricas. O produto colorido possui máxima absorbância em torno de 600 nm, e, devido a coloração azul intenso desenvolvida na sua redução, tornou-se possível sua detecção e quantificação por análise das imagens digitais dos spot-tests da reação no canal de cor vermelho (canal Red do sistema RGB), utilizando-se o software livre "ImageJ - Image Processing and Analysis in Java". Através da análise da intensidade efetiv... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: This work was proposed study and develop an analytical method for determination and quantification of paraquat herbicide in human urine samples, so that this is rapid, sensible, economic, and lesser waste generating that the related methods in literature, currently employed for this purpose, following with Green Chemistry (G.C.) and Green Analytical Chemistry (GAC) principles. Another approached aspect is employ of Digital Image Analysis (DIA) technique using analytical software through the generating digital images photographed of the spot-tests, making the methodology portable and consequently most accessible. The method developed is based in reduction reaction between paraquat dicátion (PQ2+) for this formation of the radicalar cation (PQ*+) of blue coloring, using a glucose (Glc) solution in alkaline mean (NaOH) as reducing reagent, the reagent concentrations was being optimized for the highest analytical response possible, with the help of the chemometric tools. The colored product has max absorbance at 600 nm, and, because intense blue coloring developed in the reduction, was possible to the detection and quantification from analysis of the digital images of the spot-tests of reaction in red color channel (Red channel of the model RGB), employing the free software ImageJ - Image Processing and Analysis in Java. Through of the analysis of effective intensity (Ar) of the coloring, measured in the red color channel, was plotted an analytical curve that present linearity in... (Complete abstract click electronic access below)
Mestre

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Abstract:

The body of this dissertation is focused on understanding the pathomechanisms and paving the way for new treatment paradigms in human metabolic disease, particularly phenylketonuria (PKU), maple syrup urine disease (MSUD), and succinic semialdehyde dehydrogenase (SSADH) deficiency.

Phenylketonuria and MSUD are heritable aminoacidopathies displaying aberrant cerebral transport of large neutral aminoacids. This work presents evidence that non-physiological amino acids (NPAAs) have pharmacodynamic efficacy in selective exclusion of phenylalanine from the brain of phenylketonuric mice. Data is presented for feeding and intraperitoneal injection studies of various NPAA's including methyl-aminoisobutyric acid (MAIB), and some selected MAIB-related alkanoic acid analogues. My data indicates that MAIB is the most selective phenylalanine transport inhibitor identified thus far. Regional brain amino acid studies in intermediate MSUD mice fed low (6%) and high (19%) protein chow suggest that despite varying improvements in the pathophysiological branched-chain amino acids (leucine, isoleucine and valine) in serum, glutamine, aspartate, glutamate, gamma-aminobutyric acid (GABA), asparagine, citrulline, and serine levels remained unchanged in the brain, demonstrating that dietary correction of MSUD monitored in blood does not accurately reveal corrections in brain biochemistry, providing important insights for human patients. Moreover, I have documented similar findings in PKU mice.

The final chapters of this work contain a review of the treatment prospects for SSADH disorder (a defect in GABA metabolism), and our collaborative work with the University of California focused on hyperphysiological GABA's on mTOR-driven selective autophagy. SSADH-deficient mouse studies utilizing electron microscopy to quantify mitochondria in liver and brain tissues suggest mitophagic inhibition may play a causal role in the findings of oxidative stress in patients and mice. The impact of these findings are discussed from a pharmacological viewpoint including the scope of treatment of hyperGABAergic disorders.

Lastly, I have included my literature characterization of hepatocyte transplantation (HTx) for inborn errors of metabolism which suggests that we can attempt therapeutic HTx in a murine model of a new disease, transaldolase deficiency, with a goal of gaining almost complete hepatic repopulation with gene-replete (wild-type) cells. My final article is a preclude to future postdoctoral work in the area of liver repopulation and novel therapeutic approaches.