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Behavioural, neurochemical, inflammatory and mitichondrial markers following social isolation rearing in rats before and after selected deug intervention / Marisa MöllerMöller, Marisa January 2012 (has links)
Purpose:
Schizophrenia is a progressive degenerative illness that has been causally linked to mitochondrial dysfunction, oxidative stress and a pro-inflammatory state. Social isolation rearing (SIR) in rats models the neurodevelopmental aspects of schizophrenia. The antioxidant and glutamate modulator, N-acetyl cysteine (NAC), has demonstrated therapeutic potential in schizophrenia as adjunctive treatment, although this has not been tested in the SIR model. The purpose of this study was to assess whether SIR induces changes in mitochondrial function (adenosine triphosphate (ATP)), pro- vs. anti-inflammatory cytokine balance, tryptophan metabolism, a disturbance in cortico-striatal monoamines and related metabolites, and associated alterations in behaviors akin to schizophrenia, viz. social interaction, object recognition memory and prepulse inhibition (PPI). Moreover, I evaluated whether these bio-behavioral alterations could be reversed with sub-chronic clozapine, or NAC, and whether NAC may bolster the response to clozapine treatment.
Methods: The objectives of the study were pursued through separately conducted studies. Male Sprague-Dawley (SD) rats (10 rats/group) were used in this study (Ethics number: NWU-0035-08-S5). Rats were randomly allocated to either social rearing or SIR for 8 weeks receiving either no treatment, vehicle, NAC (150 mg/kg/day), clozapine (5 mg/kg/day) or a combination of clozapine + NAC (CLZ + NAC) during the last 11 or 14 days of social rearing or SIR. After the 8 weeks, rats were tested for social interactive behaviors, object recognition memory and prepulse inhibition (PPI). Peripheral tryptophan metabolites (determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)) and pro- and anti-inflammatory cytokines (IL-4, IL-6, TNF-α, IFN-γ) (enzyme-linked immunosorbent assay (ELISA)) were determined. Cortico-striatal ATP (bioluminescence assay) and monoamines (high performance liquid chromatography (HPLC)) were also determined.
Results:
SIR-induced significant deficits in social interactive behaviours, object recognition memory and PPI, associated with increased peripheral kynurenine, quinolinic acid (QA), and pro-inflammatory cytokines, as well as a decrease in kynurenic acid (KYNA), neuroprotective ratio and anti-inflammatory cytokines. I also observed an increase in striatal, but reduced frontal cortical ATP, dopamine, serotonin as well as their metabolites and noradrenaline’s metabolite, with noradrenaline increased in both brain regions in SIR rats. A separate dose-response study of NAC (50, 150, 250 mg/kg/day) found 150 mg/kg to be the most appropriate dose for the NAC and CLZ + NAC studies. Clozapine, NAC as well as CLZ + NAC reversed all these changes, with NAC being less effective than CLZ alone. CLZ + NAC was found to be more effective than clozapine alone in reversing certain bio-behavioral alterations induced by SIR. In addition NAC alone dose dependently reversed most of the SIR induced alterations.
Conclusion:
SIR induces behavioral alterations, a pro-inflammatory state, mitochondrial dysfunction and cortico-striatal monoamine alterations, closely resembling evidence in schizophrenia. Importantly, all these bio-behavioral alterations were reversed with clozapine, NAC and CLZ + NAC treatment. However, CLZ + NAC was more effective than clozapine alone in reversing some bio-behavioral alterations, supporting the therapeutic application of NAC as adjunctive treatment in schizophrenia. In addition, NAC dose dependently reversed SIR-induced cortico-striatal serotonin, noradrenaline and metabolites, emphasizing NAC’s potential use in other anxiety and stress- related disorders. / Thesis (PhD (Pharmacology))--North-West University, Potchefstroom Campus, 2013
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Behavioural, neurochemical, inflammatory and mitichondrial markers following social isolation rearing in rats before and after selected deug intervention / Marisa MöllerMöller, Marisa January 2012 (has links)
Purpose:
Schizophrenia is a progressive degenerative illness that has been causally linked to mitochondrial dysfunction, oxidative stress and a pro-inflammatory state. Social isolation rearing (SIR) in rats models the neurodevelopmental aspects of schizophrenia. The antioxidant and glutamate modulator, N-acetyl cysteine (NAC), has demonstrated therapeutic potential in schizophrenia as adjunctive treatment, although this has not been tested in the SIR model. The purpose of this study was to assess whether SIR induces changes in mitochondrial function (adenosine triphosphate (ATP)), pro- vs. anti-inflammatory cytokine balance, tryptophan metabolism, a disturbance in cortico-striatal monoamines and related metabolites, and associated alterations in behaviors akin to schizophrenia, viz. social interaction, object recognition memory and prepulse inhibition (PPI). Moreover, I evaluated whether these bio-behavioral alterations could be reversed with sub-chronic clozapine, or NAC, and whether NAC may bolster the response to clozapine treatment.
Methods: The objectives of the study were pursued through separately conducted studies. Male Sprague-Dawley (SD) rats (10 rats/group) were used in this study (Ethics number: NWU-0035-08-S5). Rats were randomly allocated to either social rearing or SIR for 8 weeks receiving either no treatment, vehicle, NAC (150 mg/kg/day), clozapine (5 mg/kg/day) or a combination of clozapine + NAC (CLZ + NAC) during the last 11 or 14 days of social rearing or SIR. After the 8 weeks, rats were tested for social interactive behaviors, object recognition memory and prepulse inhibition (PPI). Peripheral tryptophan metabolites (determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS)) and pro- and anti-inflammatory cytokines (IL-4, IL-6, TNF-α, IFN-γ) (enzyme-linked immunosorbent assay (ELISA)) were determined. Cortico-striatal ATP (bioluminescence assay) and monoamines (high performance liquid chromatography (HPLC)) were also determined.
Results:
SIR-induced significant deficits in social interactive behaviours, object recognition memory and PPI, associated with increased peripheral kynurenine, quinolinic acid (QA), and pro-inflammatory cytokines, as well as a decrease in kynurenic acid (KYNA), neuroprotective ratio and anti-inflammatory cytokines. I also observed an increase in striatal, but reduced frontal cortical ATP, dopamine, serotonin as well as their metabolites and noradrenaline’s metabolite, with noradrenaline increased in both brain regions in SIR rats. A separate dose-response study of NAC (50, 150, 250 mg/kg/day) found 150 mg/kg to be the most appropriate dose for the NAC and CLZ + NAC studies. Clozapine, NAC as well as CLZ + NAC reversed all these changes, with NAC being less effective than CLZ alone. CLZ + NAC was found to be more effective than clozapine alone in reversing certain bio-behavioral alterations induced by SIR. In addition NAC alone dose dependently reversed most of the SIR induced alterations.
Conclusion:
SIR induces behavioral alterations, a pro-inflammatory state, mitochondrial dysfunction and cortico-striatal monoamine alterations, closely resembling evidence in schizophrenia. Importantly, all these bio-behavioral alterations were reversed with clozapine, NAC and CLZ + NAC treatment. However, CLZ + NAC was more effective than clozapine alone in reversing some bio-behavioral alterations, supporting the therapeutic application of NAC as adjunctive treatment in schizophrenia. In addition, NAC dose dependently reversed SIR-induced cortico-striatal serotonin, noradrenaline and metabolites, emphasizing NAC’s potential use in other anxiety and stress- related disorders. / Thesis (PhD (Pharmacology))--North-West University, Potchefstroom Campus, 2013
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Analyse des agents de chimiothérapie par extraction sur phase solide automatisée couplée à la chromatographie liquide et la spectrométrie de masse en tandem (SPE-LC-ESI-MS/MS)Rabii, Farida 12 1900 (has links)
Les dernières décennies ont été marquées par une augmentation du nombre des cas de cancers, ce qui a subséquemment conduit à une augmentation dans la consommation des agents de chimiothérapie. La toxicité et le caractère cancérogène de ces molécules justifient l’intérêt crucial porté à leur égard. Quelques études ont fait l’objet de détection et de quantification des agents de chimiothérapie dans des matrices environnementales.
Dans ce projet, une méthode utilisant la chromatographie liquide couplée à la spectrométrie de masse en tandem (LC-MS/MS) précédée d’une extraction sur phase solide (SPE) automatisée ou en ligne a été développée pour la détection et la quantification d’un groupe de six agents de chimiothérapie. Parmi ceux-ci figurent les plus utilisés au Québec (gemcitabine, méthotrexate, cyclophosphamide, ifosfamide, irinotécan, épirubicine) et présentant des propriétés physico-chimiques et des structures chimiques différentes. La méthode développée a été validée dans une matrice réelle représentant l’affluent d’une station d’épuration dans la région de Montréal. Deux des six composés cytotoxiques étudiés en l’occurrence (cyclophosphamide et méthotrexate) ont été détectés dans huit échantillons sur les neuf qui ont été recensés, essentiellement au niveau de l’affluent et l’effluent de quelques stations d’épuration de la région de Montréal. Les résultats des analyses effectuées sur les échantillons réels ont montré qu’il n’y avait pas de différence significative dans la concentration entre l’affluent et l’effluent, et donc que les systèmes d’épuration semblent inefficaces pour la dégradation de ces molécules. / The last few decades have been marked by an increase in the number of cancer cases, which subsequently led to an increase in the consumption of chemotherapeutic agents. The toxicity and the carcinogenicity of these molecules justify the increased interest. Few studies have been conducted to detect and quantify chemotherapeutic agents in environmental matrices.
In this project, a method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) preceded by an online solid-phase extraction (SPE) has been developed for the detection and quantification of a group of six chemotherapeutic agents, which are among the most commonly used in Quebec (gemcitabine, methotrexate, cyclophosphamide, ifosfamide, irinotecan, epirubicin) and having different physico-chemical properties and different chemical structures. The developed method was validated in a real water matrix representing the influent of a sewage treatment plant in the Montreal area. Two of the six studied cytotoxic agents (cyclophosphamide and methotrexate) were detected in eight samples of the nine taken mainly at the influent and effluent of some treatment plants in the Montreal area. The results of the analysis of real samples showed that there was no significant difference in concentration between the influent and effluent. This also demonstrates the inadequacy of the current wastewater treatment approaches to remove those compounds.
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Mass Spectrometric Applications for Diagnosing Metabolic and Endocrine DiseasesKushnir, Mark M. January 2008 (has links)
Disease-specific compounds (biomarkers) are analyzed in clinical laboratories to assist with diagnosing diseases. This thesis describes development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS) based tests for diagnosing a diverse group of endocrine and metabolic diseases. The analytical methods used on-line and off-line sample extraction and analytical derivatization as means of enhancing the analytical sensitivity, specificity and clinical utility. All developed methods were extensively validated and reference intervals for the biomarker concentrations were established in blood samples of healthy adults and children. Advantages of the LC-MS/MS as an analytical technique include possibility of simultaneous measurement of multiple analytes and ability of confirming their identity. In this thesis we proposed and evaluated approaches for the assessment of the specificity of analysis in the methods that use tandem mass spectrometry detection. To enhance throughput of the LC-MS/MS tests for the biomarkers that have endogenous or exogenous isomers an approach was developed for quantitation of isomers from unresolved chromatographic peaks. Using methods developed in this thesis we performed a study of the steroidogenesis in ovarian follicles of healthy women and women with polycystic ovary syndrome (PCOS). Obtained data on the steroid concentrations and associations between the steroid metabolites in the pathway would be helpful for better understanding of the ovarian pathophysiology. Potential biomarkers of PCOS were identified in the thesis; further studies will be necessary to confirm their clinical utility.
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Developmental Aspects of Drug Transport Across the Blood-Brain BarrierBengtsson, Jörgen January 2009 (has links)
The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice. A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport. In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.
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Correlation of fecal ergovaline, lolitrem B, and their metabolites in steers fed endophyte infected perennial ryegrass strawMurty, Lia D. 21 November 2012 (has links)
Perennial ryegrass (PRG, Lolium perenne) is a hardy cool-season grass that is infected with the endophytic fungus Neotyphodium lolii, which enables the plant to be insect repellant and drought resistant, lowering the use of insecticides and fertilizers. However, this fungus produces the compound lolitrem B (LB, m/z 686.4) which causes the tremorgenic neurotoxicity syndrome 'ryegrass staggers' in livestock consuming forage which contains <2000 ppb LB. Ergovaline (EV, m/z 534) is a vasoconstrictor normally associated with tall fescue (Festuca arudinacea), but has also been found in endophyte-infected PRG. Past research has shown a strong linear correlation between levels of LB and EV in PRG. The purpose of this study was to examine the linear relationship between EV and LB in feces and determine common metabolites. To accomplish this, four groups of steers (n=6/group) consumed endophyte- infected PRG over 70 days consumed the following averages of LB and EV: group I 2254ppb LB/633 ppb EV; group II 1554ppb LB/ 373ppb EV, group III 1011ppb LB/259ppb EV, and group IV 246ppb LB/<100ppb EV. Group I in week 4 was inadvertently given a washout period at which time the steers consumed the amount of LB and EV given to group IV (control). Both feed and feces samples were extracted using difference solid phase extraction methods and quantified by
HPLC-fluorescence for LB and EV. Concentrations of EV and LB obtained through HPLC-fluorescence in both PRG and feces showed a linear relationship. Additional screening for metabolites was conducted LC-MS/MS and showed possible oxidation and reduction metabolites for both toxins. / Graduation date: 2013
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Analytical Method Development of Fluorinated Silanes using Mass SpectrometryEklundh Odler, Tea January 2018 (has links)
The aim of this study was to develop an analytical method for fluorinated silanes. Furthermore, as a secondary aim, to explore if there would be possible to detect 1H,1H,2H,2H-perfluorooctyl triethoxysilane (6:2 PTrEtSi) and 1H,1H,2H,2H-perfluorodecyl triethoxysilane (8:2 PTrEtSi) in two different matrices, sludge and cosmetic extract. The method development included experiments using LC-MS, LC-MS/MS, UPC2, GC-MS and APGC-MS/MS and was carried out using standards containing 6:2 PTrEtSi and 8:2 PTrEtSi. The analytical method that worked best for the compounds was GC-MS/MS and an analytical method using APGC-MS/MS was developed for fluorinated silanes. The IDL for 6:2 PTrEtSi was 0.0012 μg/mL and 1.32 μg/mL for 8:2 PTrEtSi. This makes the developed method suitable for high contaminated samples, such as extracts from cosmetic products. It was concluded that a method using LC as the analytical instrument would not work for the two target compounds since they were too reactive with the mobile phase. However, LC could be a good choice for siloxanes, compounds that are formed from hydrolysis and condensation of fluorinated silanes. The samples analyzed in this study were three sludge extracts and one extract from a cosmetic product. 6:2 PTrEtSi was expected to be detected in the cosmetic sample since the compound was stated on the table of contents of the cosmetic product. No detection of 6:2 TrEtSi or 8:2 TrEtSi could be made in either of the samples. The reason for this was suspected to be transformation or degradation of the compounds into other compounds. Therefore, a full scan of the cosmetic sample using LC-MS/MS was included in the experiment as an addition to verify the suspicions that compounds such as siloxanes could have been formed. An interesting peak was discovered with m/z 947 which could be a disiloxane of 6:2 PTrEtSi.
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Biotransformação de corantes dispersos do tipo azo pela ação de enzimas redutoras e oxidação fotoeletrocatalítica após pré-concentração por MIP / Biotransformation of disperse azo dyes by the action of reducing enzymes and photoelectrocatalytic oxidation after preconcentration by MIPFranco, Jefferson Honorio [UNESP] 21 November 2016 (has links)
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Previous issue date: 2016-11-21 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Corantes sintéticos do tipo azo têm sido um assunto de grande preocupação ambiental devido ao potencial genotóxico e mutagênico dos produtos de biotransformação. Deste modo, nos últimos anos a consequência da ingestão destes corantes presentes na agua potável servida à população é discutida por diversos autores. Este estudo avalia a ação de microssomas de fígado de rato, enzimas redutoras produzidas pela bactéria Escherichia coli (E. coli) e nitroredutase imobilizada na biotransformação de três corantes dispersos que possuem grupos azo, Disperse Red 73 (DR 73), Disperse Red 78 (DR 78) e Disperse Red 167 (DR 167). A técnica de Espectrofotometria de absorção molecular na região do Uv- visível, Cromatografia Líquida de Alta Eficiência com detector de arranjo de diodos (CLAE-DAD) e Cromatografia líquida acoplada à espectrometria de massas (LC-MS/MS) foram técnicas usadas para identificar os principais produtos gerados após os processos de degradação dos corantes. Polímeros de impressão molecular magnéticos (MMIPs) foram investigados usando reações de polimerização por precipitação para pré-concentração do corante DR 73, juntamente com a degradação por fotoeletrocatálise e subsequente análise dos produtos por LC-MS/MS. Os estudos in vitro do metabolismo de biotransformação dos corantes têxteis com microssoma de fígado de rato mostraram que as reações ocorreram preferencialmente no grupo azo e nitro dos corantes, indicando a redução destes grupos pelas enzimas do citocromo P-450. Foram obtidos dois produtos de degradação para cada corante após reação com a bactéria E. coli; o corante DR 73 originou os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 4-nitroanilina, os produtos 3-((4-aminofenil)(etil)amino)propanitrila e 2-cloro-4-nitroanilina foram obtidos após reação com o corante DR78 e o DR 167 originou dimetil 3,3`-((3-acetamido-4aminofenil)azanediyl)dipropanoato e 2-cloro-4-nitroanilina, indicando a clivagem do grupo azo, possivelmente, pela enzima azoredutase, produzida pela bacteria. A enzima nitroredutase, imobilizada em partículas magnéticas modificadas com tosil, mostrou que a redução dos corantes ocorreu preferencialmente no grupo nitro, enquanto que a enzima livre no meio reacional resultou em mais de um produto de biotransformação para cada corante, atuando em mais de um sítio da molécula, comprovando a eficácia da imobilização enzimática para estudos de biotransformação e formação de produtos majoritários. A mutagenicidade dos corantes foi avaliado pelo ensaio de Salmonella/microssoma realizado nas estirpes TA 98 e TA 100, com e sem S9. De acordo com este ensaio, DR 73 foi o mais mutagênico. O MMIP para o corante DR 73 apresentou excelentes valores de religação (16 mg g−1 e 6 mg g−1, para MMIP e MNIP, respectivamente) indicando que o polímero molecularmente impresso formou cavidades específicas para retenção do corante. Através dos resultados obtidos por LCMS/MS, observou-se 100% de degradação do corante em apenas 60 min de tratamento via fotoeletrocatálise para soluções mais diluidas do mesmo, comprovando a eficiência da técnica na degradação de poluentes. Sendo assim, estes resultados sugerem que o MMIP mostrou uma excelente especificidade e seletividade para o corante DR 73 e uma técnica promissora na captação de corantes mutagênicos de águas superficiais, com grande potencial de aplicação e exploração na pré-concentração antes do tratamento. Além disso, a redução destes corantes por sistemas biológicos representa uma grande preocupação ambiental devido ao aumento da genotoxicidade para os seres vivos, em especial a seres humanos, produzindo compostos nocivos, tais como aminas condenadas pela Agência Internacional de Pesquisa sobre o Câncer. / Synthetic azo dyes have been a matter of great concern due to the genotoxic and mutagenic potential of the products originating from azo dye biotransformation. Thus, in recent years the result of the intake of these dyes present in drinking water supplied to a population is discussed by several authors. This work evaluates the action of rat liver microsomes, reducing enzymes produced by the Escherichia coli (E. coli) and nitroreductase immobilized on biotransformation of three disperse dyes bearing azo groups, namely Disperse Red 73 (DR 73), Disperse Red 78 (DR 78), and Disperse Red 167 (DR 167). UV-Vis spectrophotometry, high-performance liquid chromatography with diode array detector (HPLC-DAD), and liquid chromatography coupled to mass spectrometry (LC-MS/MS) were techniques used to identify the main products generated after the process degradation of dyes. Magnetic molecularly imprinted polymers (MMIPs) were investigated using precipitation polymerization reactions for preconcentration of the dye DR 73, together with the photoelectrocatalysis degradation and subsequent analysis of the products by LC-MS/MS. In vitro studies of biotransformation metabolism of textile dyes with rat liver microsome showed that the reactions occur preferentially in the group of azo and nitro dyes, indicating the reduction of these groups by enzymes of the cytochrome P-450. There were obtained two degradation products for each dye after reaction with E. coli; the dye DR 73 gave the product 3 - ((4-aminophenyl) (ethyl) amino) propanitrila and 4-nitroaniline, the product 3 - ((4-aminophenyl) (ethyl) amino) propanitrila and 2-chloro-4-nitroaniline were obtained after reaction with the dye DR78 and DR 167 gave 3,3`-dimethyl-((3-acetamido-4-aminophenyl) azanediyl) dipropanoato and 2chloro-4-nitroaniline; indicating cleavage of the azo group, possibly by azoredutase enzyme produced by bacteria. The nitroreductase enzyme immobilized on modified magnetic particles Tosyl showed that the reduction of dyes occurred preferentially in the nitro group, while the free enzyme in the reaction medium resulted in more than a product of biotransformation for each dye, acting in more than one site of the molecule, proving the efficacy of enzyme immobilization for biotransformation studies and formation of major products. The mutagenicity of the dyes was evaluated by the Salmonella/microsome assay performed on strains TA 98 and TA 100, with and without S9. According to this assay, DR 73 was the most mutagenic. The MMIP to the dye DR 73 showed excellent rebinding values (16 mg g−1 and 6 mg g−1, for MMIP and MNIP, respectively) indicating that the molecularly imprinted polymer formed cavities for specific dye retention. Through the results obtained by LC-MS/MS, it was observed 100% dye degradation in 60 min treatment for more dilute solutions thereof, proving the efficiency of technique in pollutant degradation. Thus, these results suggest that MMIP showed excellent specificity and selectivity for the dye DR 73 and a promising technique in capturing mutagenic dyes of surface water, with great potential for application and operation in the pre-concentration before treatment. Moreover, the reduction of these dyes by biological systems is a major environmental concern due to increased of genotoxicity for living beings, especially humans, producing harmful compounds, such as condemned amines by the International Agency for Research on Cancer.
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Desenvolvimento, validação e aplicação de metodos para analise multrirresidual de agrotoxicos em suco de laranja e tangerina utilizando CLAE-DAD, CL-EM-EM E CLUE-DAD / Development, validation and application of methods for multirresidual pesticide determination of orange juice tangerine juice by HPLC-DAD, LC-MS-MS and UPLC-DADChiaradia, Mariza Campagnolli 13 August 2018 (has links)
Orientador: Isabel Cristina Sales Fontes Jardim / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-13T14:12:56Z (GMT). No. of bitstreams: 1
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Previous issue date: 2009 / Resumo: O Brasil é o maior produtor mundial de citrus e o principal exportador de suco. Dentre as espécies cultivadas no Brasil, há a predominância de laranjas Pera e tangerinas Murcote. Porém, os agricultores brasileiros tem enfrentado a incidência de várias pragas agrícolas na cultura de citrus, de forma que, para manter a produção e a qualidade, tem sido necessário aplicar agrotóxicos de forma constante e cada vez maior. Por outro lado, devido ao risco resultante da exposição dos consumidores aos resíduos de agrotóxicos presentes nos alimentos, agencias governamentais reguladoras tem estabelecido limites máximos de resíduos (LMR) para todos os agrotóxicos. Neste contexto, três métodos para a determinação de resíduos dos principais agrotóxicos (aldicarbe, difenoconazol, diflubenzurom, diurom, imidacloprido e tiofanato metílico) aplicados no cultivo de laranjas Pera e tangerinas Murcote foram desenvolvidos e validados. Vários métodos de preparo de amostras foram avaliados e o método QuEChERS foi o que se mostrou mais eficiente, rápido e simples. Foram utilizadas diferentes técnicas cromatográficas para a validação do método: cromatografia líquida de alta eficiência com detecção por arranjo de diodos (CLAE-DAD), cromatografia líquida acoplada a espectrometria de massas em série (CL-EM-EM) e cromatografia líquida de ultra eficiência com detecção por arranjo de diodos (CLUE-DAD). Os parâmetros analíticos mostraram que o método proposto extrai de maneira satisfatória os agrotóxicos das matrizes, permitindo sua detecção por todas as técnicas analíticas utilizadas. Entretanto, devido a complexidade da matriz estudada foi necessário fazer a calibração na matriz. As recuperações obtidas foram de 79 a 124% com CLAE-DAD, de 83 a 119% com CL-EM-EM e de 78 a 113% com CLUEDAD, com coeficientes de variação menores que 15% para todas as tecnicas cromatograficas. Os limites de quantificacao obtidos mostraram que os métodos podem ser utilizados para a detecção dos agrotóxicos em concentrações abaixo dos LMR estabelecidos pela legislação brasileira e dos EUA. Com a CLUE-DAD obtiveram-se os menores tempos de análise, 7 min, e a CL-EM-EM foi a técnica cromatografica mais seletiva e com a melhor detectabilidade (4 mg L) utilizada neste trabalho. O método foi aplicado a amostras coletadas no comercio da cidade de Campinas-SP e o agrotóxico encontrado com maior frequência foi o imidacloprido, mas em concentrações abaixo dos LMR / Abstract: Brazil is the world's largest producer of oranges and the largest exporter of juice. Among the species grown in Brazil, there is a predominance of Pera oranges and Murcote tangerines. Brazilian agriculturist have faced several diseases during orange and tangerine cultivation that demand constant and even expensive applications of pesticides to mantain production and quality. On the other hand, because of potential health risk to the consumers, resulting from acute and/or chronic dietary exposure, government regulatory agencies have established maximum residue limits (MLR) for all the pesticides. Thus, this work developed and validated three methods to determine the main pesticides applied to Pera orange and Murcote tangerine cultivation in Brazil: aldicarb, difenoconazole, diflubenzuron, diuron, imidacloprid and thiophanate-methyl. Several sample preparation methods were evaluated and the QuEChERS method was the more suitable, rapid and simplest extraction method for the matrices studied. The methods were validated using different chromatographic techniques: high performance liquid chromatography ¿ diode array detection (HPLC-DAD), liquid chromatography ¿ tandem mass spectrometry (LC-MSMS) and ultra-performance liquid chromatography ¿ diode array detection (UPLC-DAD). The analytical parameters demonstrate that the proposed methodologies satisfactorily extract the pesticides from the matrices, allowing detection using all the quantification techniques employed. Because of the complexity of the matrices studied, it was necessary to employ matrix-matched calibration. When using HPLC-DAD recoveries of 79 to 124% were obtained, with LC-MS-MS the recoveries were 83 to 119% and when using UPLC they were 78 to 113%, with coefficients of variation below to 15% for all chromatographic techniques. These limits of quantification show that the methods developed can be used to detect these pesticides at concentrations below the MRL established by Brazilian and USA legislation. UPLC-DAD had a more rapid analysis time, 7 min, and LC-MS-MS had the best selectivity and detectivity (4 mg L). The methods were applied to samples collected in markets of Campinas city, and the pesticide most frequently found was imidacloprid, with concentrations well below the listed MRL / Doutorado / Quimica Analitica / Doutor em Ciências
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ATIVIDADE FITOESTROGÊNICA DE Morus nigra L., MORACEAE, EM RATAS OVARIECTOMIZADAS / ACTIVITY OF FITOESTROGÊNICA, MORUS NIGRA L. MORACEAE, IN RATS VARIECTOMIZEDSilva, Selma do Nascimento 02 October 2012 (has links)
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Previous issue date: 2012-10-02 / The hypoestrogenism in climacteric is associated with vasomotor symptoms, cardiovascular disease, osteoporosis and urogenital changes. At this stage of life of women, hormone replacement HRT can alleviate some consequences of decreased estrogen caused by ovarian failure. However, estrogen therapy may cause adverse effects such as breast tenderness, uterine bleeding and increase the relative risk for cancers of the breast and endometrium. Morus nigra L. (mulberry) is a plant species most used in Brazil for the treatment of menopausal symptoms. Thus, this study aims to assess the likely effects phytoestrogenics the hydroalcoholic extract (HE) from the leaves of M. nigra in ovariectomized female rats. Therefore, the dried leaves were pulverized and soaked in 70% ethanol in the proportion 1:3 (v/v) to obtain the HE (yield=21.90%). The HE was subjected to evaluation of the antioxidant activity by capturing free radical 2,2-diphenyl-1-picryl-hidrazil, analyzed by liquid chromatography coupled to mass spectrum (LC-MS/MS) for identification of compound and then partitioned with hexane, chloroform, ethyl acetate and methanol/water. The safety of extract was determined by the test of acute toxicity in mice at doses of 0.1 to 10.0 g/kg orally (p.o.). To evaluate the estrogenic activity of the extract from M. nigra leaves, the rats were divided into two control groups: sham-operated (SHAM) and ovariectomized (OVX), which received 0.1 mL/100 g saline, and two test groups: ovariectomized and treated with a solution estroprogestative (OVX-EP-50g/kg) and ovariectomized and treated with HE M. nigra 500mg/kg (OVX-HE500), n = 8-10, daily, p.o., for 14 weeks. Throughout the treatment period were analyzed the frequency stage of the estrous cycle, food intake and body weight. At the end of treatment were evaluated biochemical parameters and hormone, histomorphometry of the uterus, vagina and breast. Furthermore, the influence of M. nigra on the proliferation of breast tumor cell line MCF-7 was determined by MTT method. HE showed high antioxidant activity when compared to standard quercetin. The analysis by LC-MS/MS EH compared with literature data allowed the identification of flavonoids (kaempferol and quercetin) and quinic acid derivatives (caffeoylquinic acid and isomers dicaffeoylquinic acid). In the analysis of the estrous cycle, the group OVX-HE500 showed an increase in proestrous and estrous phases at 15.25% and 26.6%, respectively, when compared to OVX group. Ovariectomy caused an increase in body mass, which was prevented by treatment with HE and EP solution. The weight of abdominal adipose tissue was also significantly lower in groups OVX-HE and OVX-EP compared to the OVX group. Ovariectomy also induced atrophy of uterine tissue (OVX group) compared to SHAM group, indicating the efficiency of the surgical procedure, and the administration of EP significantly increased uterine weight compared with OVX group. Average uterine weight of the OVX-HE group was also higher than the OVX group, but smaller than the OVX-EP group. In the histological analysis, it was observed that the characteristics of the squamous epithelium of the vagina of OVX-EP group (57.79 ± 1.49m), relative to thickness, were similar to that of SHAM group (50 66 ± 1.60m). After 14 weeks of administration of HE was a partial reversal of vaginal atrophy (37.34 ± 1.77m), when compared to the OVX group (12.92 ± 0.53m), showing maturation of this tissue with the treatment, however, the HE did not alter breast tissue, unlike the stimulus EP-induced.
Regarding biochemistry was observed that the treatments (HE and EP) reduced concentrations of triglycerides in 27.5% and 23.8% respectively, when compared to OVX. In in vitro tests, the data indicate that the HE M. nigra acts as a weak phytoestrogen and protects against cell proliferation of human breast carcinoma (MCF-7). In acute toxicity study, the treatment of mice with HE did not produce behavioral changes or deaths. Together, the data demonstrate that the HE M. nigra L. has beneficial effects in models of induced menopause in rats, decreased uterine and vaginal atrophy, without changing the mammary structure, improving triglyceride levels and shows up secure and potent oxidant activity. These effects may be related to their flavonoid constituents, and thus the plant species may be useful in controlling symptoms of menopause as an alternative to Hormone Replacement Therapy. / O hipoestrogenismo no climatério associa-se com sintomas vasomotores, doenças cardiovasculares, osteoporose e alterações urogenitais. Nesta fase da vida da mulher, a reposição hormonal pode amenizar algumas consequências da diminuição estrogênica ocasionada pela falência ovariana. Porém, a terapia estrogênica pode ocasionar efeitos adversos como mastalgia, sangramentos uterinos, além de aumentar o risco relativo para neoplasias de mama e endométrio. Morus nigra L. (amora) é uma das espécies vegetais mais utilizadas no Brasil para o tratamento dos sintomas do climatério. Assim, o presente estudo objetiva avaliar os prováveis efeitos fitoestrogênicos do extrato hidroalcoólico (EH) das folhas de M. nigra em ratas Wistar ovariectomizadas. Para tanto, as folhas secas foram pulverizadas e maceradas em etanol a 70% na proporção 1:3 (v/v), para obtenção do EH (rendimento=21,90%). O EH foi submetido à avaliação da atividade antioxidante pela captura do radical livre 2,2-difenil-1-picril-hidrazila, analisado por cromatografia líquida acoplada ao espectro de massa (LC-MS/MS) para identificação de composto e, em seguida, particionado com hexano, clorofórmio, acetato de etila e metanol/água. A segurança do extrato foi determinada pelo teste de toxidade aguda em camundongos, nas doses de 0,1 10,0g/kg, por via oral (v.o.). Para avaliar a atividade estrogênica do extrato das folhas de M. nigra, as ratas foram divididas em dois grupos controle: falso-operados (SHAM) e ratas ovariectomizadas (OVX), que receberam 0,1mL/100g de solução salina; e dois grupos teste: ovariectomizadas e tratadas com solução estroprogestativa (OVX-EP-50g/Kg) e ovariectomizadas e tratadas com EH de M. nigra 500mg/kg (OVX-EH500), n=8-10, diariamente, por v.o., durante 14 semanas. Durante todo o período de tratamento foram analisadas a frequência das fases do ciclo estral, a ingestão de alimentos e o peso corporal. Ao final do tratamento foram avaliados os parâmetros bioquímicos e hormonais, histomorfometria do útero, vagina e mama. Além disso, a influência de M. nigra sobre a proliferação de células tumorais de mama da linhagem MCF-7 foi determinada pelo método MTT. O EH apresentou alta atividade antioxidante quando comparada ao padrão quercetina. Na análise do EH por LC-MS/MS em comparação com dados da literatura permitiu a identificação de flavonoides (caempferol e quercetina) e derivados do ácido quínico (ácido cafeoilquínico e isômeros de ácido dicafeoilquínico). Na análise do ciclo estral, o grupo OVX-EH500 apresentou um aumento nas fases estro e proestro em 15,25% e 26,6%, respectivamente, quando comparado ao grupo OVX. A ovariectomia promoveu um aumento no peso corporal, que foi inibido pelo tratamento com o EH e solução EP. O peso do tecido adiposo abdominal também foi significativamente menor nos grupos OVX-EP e OVX-EH, quando comparados ao grupo OVX. A ovariectomia também induziu atrofia do tecido uterino (Grupo OVX) em comparação ao grupo SHAM, indicando a eficiência do procedimento cirúrgico; e a administração de EP aumentou significativamente o peso do útero em comparação com grupo OVX. A média do peso uterino do grupo OVX-EH também foi maior do que o grupo OVX, porém menor que o grupo OVX-EP. Quanto à análise histológica, observou-se que as características do epitélio escamoso da vagina do grupo OVX-EP (57,79 ± 1,49m), em relação à espessura, se assemelharam à das ratas do grupo SHAM (50,66 ± 1,60m). Após 14 semanas de administração de EH houve uma reversão parcial da atrofia vaginal (37,34 ± 1,77m), quando comparado ao grupo OVX (12,92 ± 0,53 m), mostrando maturação deste tecido com o tratamento; entretanto, o EH não alterou o tecido mamário, diferente do estímulo induzido pelo EP. Em relação
à bioquímica foi observado que os tratamentos (EH e EP) reduziram as concentrações de triglicérides em 27,5% e 23,8% respectivamente, quando comparado ao grupo OVX. Nos testes in vitro, os dados indicam que o EH de M. nigra atua como um fraco fitoestrógeno e protege contra a proliferação de células de carcinoma de mama humano (MCF-7). No estudo toxicológico agudo, o tratamento de camundongos com o EH não produziu alterações comportamentais nem mortes. Em conjunto, os dados demonstram que o EH de M.nigra apresenta efeitos benéficos em modelos de hipoestrogenismo induzida em ratas, diminuindo a atrofia uterina e vaginal, sem alterar a estrutura mamária, melhorando os níveis de triglicérides, tendo potencial antioxidante, além de mostrar-se seguro. Esses efeitos podem estar relacionados com seus constituintes flavonoídicos, e dessa forma, a espécie vegetal pode ser útil no controle de sintomas da menopausa como uma alternativa para Terapia de Reposição Hormonal.
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