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Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-17T11:51:08Z No. of bitstreams: 2 Dissertação - Alessandro Arruda Alves - 2017.pdf: 2612408 bytes, checksum: c21fd834dc73ac66ba55f32ba689f6f3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-04-17T11:51:37Z (GMT) No. of bitstreams: 2 Dissertação - Alessandro Arruda Alves - 2017.pdf: 2612408 bytes, checksum: c21fd834dc73ac66ba55f32ba689f6f3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-04-17T11:51:37Z (GMT). No. of bitstreams: 2 Dissertação - Alessandro Arruda Alves - 2017.pdf: 2612408 bytes, checksum: c21fd834dc73ac66ba55f32ba689f6f3 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-10 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / In Brazilian agriculture, the State of Goiás is one of the largest producers of corn, beans, cotton, sorghum, sugarcane and soybeans, with 75% of its exports composed of products related to agriculture and minerals. Thus, a large part of the economy of the State of Goiás is focused on rural activities, justifying the intense use of pesticides in the region. These pesticides will be metabolized in the liver in two main phases, the first one of bioactivation converting the original substances into more polar metabolites, through oxidation reduction reactions, hydrogenation and dehalogenation, most of the reactions are made by the cytochrome P450 (CYP) superfamily And the second phase in which conjugation reactions occur, in which metabolites are converted into non-toxic products. Agrochemicals are also known to cause various human health damage, such as cancer, cholinergic syndrome, Parkinson's disease and Alzheimer's as well as cytogenetic changes such as DNA damage and chromosome translocations. Thus, polymorphisms in enzymes acting in the path of detoxification and DNA repair may be important biomarkers of diseases in individuals exposed to pesticides. The present study sequenced 3 regions of the following genes: CYP2E1 (promoter region), CYP1A1 (exon 7) and OGG1 (intron region) of 260 individuals from Goiânia and Aparecida de Goiânia, GO. 20 ml of blood was collected, DNA was extracted using commercial kits and PCR was performed. Subsequently, the amplicons were sequenced by the automated Sanger method and compared to the 1000 Genome project. The population of this study was composed of 159 men and 101 women, mean age of 38 years. A total of 5 SNP's were found in the Goias population, being rs2031922 (change of C> T and frequency of the T allele of 0.05) of the CYP2E1 gene, rs1048943 (change of A> G and frequency of the G allele of 0.1 ) And rs1799814 (change of C> A and frequency of allele A of 0.07) of the CYP1A1 gene and rs1052133 (change of C> G and frequency of allele G of 0.2) and rs293795 (change of A> G and frequency Of the 0.18 T allele) of the OGG1 gene. All loci did not deviate from the Hardy-Weinberg equilibrium, nor was a statistically significant difference between age and genotype composition. The rs1048943 and rs1799814 loci of the CYP1A1 gene showed a moderate disequilibrium linkage and the rs1052133 and rs293795 loci of the OGG1 gene showed a strong disequilibrium linkage. The haplotype inference revealed a frequency of the haplotypes of loci rs1048943 and rs1799814 of A / C = 0.8, G / A = 0.12, A / A = 0.07 of the CYP1A1 gene. The haplotypes of the loci rs293795 and rs1052133 had A / G = 0.07, A / C = 0.8, G / C = 0.13. Due to the important participation in detoxification or DNA repair and relationship with pesticides, SNPs rs2031920 of the CYP2E1 gene, rs1048943 of CYP1A1 and rs1052133 of the OGG1 gene are potential biomarkers of susceptibility to disease development in individuals who have contact with these xenobiotics / Na agricultura brasileira o Estado de Goiás é um dos maiores produtores de milho, feijão, algodão, sorgo, cana-de-açúcar e soja, tendo 75% de suas exportações compostas de produtos ligados às áreas de agricultura, agropecuária e minérios. Dessa forma, grande parte da economia do Estado de Goiás é voltada para atividades rurais, justificando o intenso uso de agrotóxicos na região. Esses agrotóxicos serão metabolizados no fígado em 2 fases principais, a primeira de bioativação convertendo as substâncias originais em metabolitos mais polares, por meio de reações de oxidação redução, hidrogenação e desalogenação, grande parte das reações são feitas pela superfamília de enzimas citocromo P450 (CYP) e a segunda fase na qual ocorrem reações de conjugação, na qual metabolitos são convertidos em produtos atóxicos. Os agrotóxicos também são conhecidos por causarem diversos danos à saúde humana, tais como, câncer, síndrome colinérgica, mal de Parkinson e Alzheimer assim como alterações citogenéticas, tais como dano no DNA e translocações cromossômicas. Assim, polimorfismos nas enzimas atuantes em vias de detoxificação e reparo de DNA podem ser importantes biomarcadores de doenças em indivíduos com exposição a agrotóxicos. O presente estudo sequenciou 3 regiões dos seguintes genes: CYP2E1 (região promotora), CYP1A1 (éxon 7) e OGG1 (região de íntron) de 260 indivíduos de Goiânia e Aparecida de Goiânia, GO. Foram coletados 20 ml de sangue, o DNA foi extraído com o uso de kits comercias e foi realizada a PCR. Posteriormente, os amplicons foram sequenciados pelo método de Sanger automatizado e comparados com a população do projeto 1000 genomas. A população deste estudo foi composta por 159 homens e 101 mulheres, médias de idade de 38 anos. Foram encontrados na população goiana um total de 5 SNP’s, sendo eles rs2031922 (mudança de C>T e frequência do alelo T de 0,05) do gene CYP2E1, rs1048943 (mudança de A>G e frequência do alelo G de 0,1) e rs1799814 (mudança de C>A e frequência do alelo A de 0,07) do gene CYP1A1 e rs1052133 (mudança de C>G e frequência do alelo G de 0,2) e rs293795 (mudança de A>G e frequência do alelo T de 0,18) do gene OGG1. Todos os locos não desviaram do equilíbrio de Hardy-Weinberg, também não houve diferença estatisticamente significativa entre a idade e a composição genotípica. Os locos rs1048943 e rs1799814 do gene CYP1A1 apresentaram um desequilíbrio de ligação moderado e os locos rs1052133 e rs293795 do gene OGG1 apresentaram um desequilíbrio de ligação forte. A inferência haplotípica revelou uma frequência dos haplótipos dos locos rs1048943 e rs1799814 de A/C=0,8, G/A= 0,12, A/A=0,07 do gene CYP1A1. Os haplótipos dos locos rs293795 e rs1052133 apresentaram frequência de A/G=0,07, A/C=0,8, G/C=0,13. Devido a importante participação em vias de detoxificação ou reparo de DNA e relação com agrotóxicos, os SNP’s rs2031920 do gene CYP2E1, rs1048943 do CYP1A1 e o rs1052133 do gene OGG1 são potenciais biomarcadores de susceptibilidade ao desenvolvimento de doenças em indivíduos que possuem contato com estes xenobióticos.

Atualmente, há um grande incentivo governamental para a produção de biodiesel a partir de óleo de mamona. O aumento na fabricação desse óleo irá aumentar a produção de torta de mamona, que tem grande potencial de emprego na tecnologia de material biodegradável e utilização na alimentação animal, se destoxificada. Os objetivos desta tese foram a caracterização química da torta de mamona, a extração de suas proteínas para desenvolvimento de material biodegradável, e a caracterização do resíduo sólido do processo de extração, visando seu uso na alimentação animal. A extração das proteínas da torta de mamona foi feita por solubilização em meio alcalino. Inicialmente, diversos parâmetros (velocidade de agitação, concentração da torta na solução extratora e temperatura de extração) foram testados no intuito de aumentar o rendimento de extração das proteínas, utilizando-se NaOH (pH = 9). Em seguida, diversos experimentos foram realizados para se avaliar os efeitos do pH (8-12) e/ou do tipo de agente alcalino (NaOH, KOH e Ca(OH)2) na extração das proteínas da torta de mamona, sempre à temperatura de 50°C, velocidade de agitação de 400rpm, e concentração da torta na solução extratora de 20%. Os extratos proteicos obtidos foram liofilizados (EPL), e os resíduos foram desidratados em estufa (40°C/24 horas). Análises para determinação da composição bromatológica, minerais, amido, compostos fenólicos totais, ácidos graxos, aminoácidos, fibra dietética, da microestrutura, atividade alergênica, eletroforese dimensional e identificação de ricina foram feitas na matéria prima, nas proteínas extraídas e nos resíduos. A composição de aminoácidos foi analisada no extrato proteico liofilizado (EPL) e nos resíduos somente nas amostras preparadas com NaOH. A composição bromatológica das matérias primas (semente, torta e farelo) mostrou elevados teores de óleo (43,6%) e fibras (29,5%) para as sementes; bem como altos teores de proteína (36-40%) e fibras (29-30%) para a torta e o farelo de mamona. A semente de mamona apresentou valores de minerais considerados razoáveis para oleaginosas, e a torta de mamona apresentou alta proporção de K; Ca e P, importantes para alimentação animal. Verificou-se alto teor de ácido ricinoléico (79-90%) nas matérias primas avaliadas. A torta de mamona apresentou altos teores de ácido glutâmico (15%), arginina (11%) e triptofano (9%), sendo que os teores dos outros aminoácidos variaram entre 2 e 7%. Os resultados da caracterização química dos EPL e dos resíduos foram dependentes principalmente do pH de extração das proteínas. Observou-se aumento da concentração de proteína (64-68%) e de cinzas (13-19%), nos EPL, em função do aumento do pH (10 - 12), independente do tipo de agente alcalinizante. Os resíduos obtidos da extração dos EPL apresentaram teores de proteína, cinzas e fibras variando entre 20 e 34%, 12 e 17% e 39 e 42%, respectivamente. Em relação ao perfil de minerais dos EPL, verificou-se aumento nas concentrações de Na e Mn com o aumento do pH para ambos agentes alcalinizantes. O perfil de minerais dos resíduos mostrou aumento significativo nos teores de Na e K com aumento do pH de extração, ajustado com NaOH e KOH, respectivamente. O teor de ácido ricinoléico foi menor no resíduo, e maior no EPL, obtidos em pH 12 ajustado com NaOH. A composição de aminoácidos dos resíduos sofreu efeito do pH apenas para o pH 12. Os aminoácidos presentes em maior concentração nos EPL e nos resíduos foram ácido glutâmico, triptofano e arginina. Os EPL e os resíduos obtidos da extração das proteínas apresentaram menor poder alergênico avaliado pelo teste de desgranulação de mastócitos obtidos do lavado peritoneal de ratos. O perfil eletroforético dos EPL e dos resíduos mostrou maior proporção de proteínas com massa molecular entre 29-36kDa, seguidos por proteínas de massa molecular ao redor de 20kDa e menor proporção de proteínas de massa molecular entre 45-66kDa. Não foi observada a presença da ricina nos resíduos obtidos em pH 12. Os resultados de todos os experimentos de extração das proteínas da torta de mamona permitiram escolher os melhores parâmetros de extração: temperatura = 50°C, velocidade de agitação = 400rpm, concentração da torta na solução extratora = 20%, pH = 12 e agente alcalinizante = NaOH. Em conclusão, o processo de extração de proteínas por solubilização alcalina permitiu a produção de concentrado proteico com características interessantes para a produção de material biodegradável (agricultura e biofilmes), e de resíduos ainda com alto teor de proteínas e de fibras, isento de ricina, podendo, portanto ser utilizado na alimentação animal. / Presently, there is large government incentive for biodiesel production from castor seed oil. The increase of this oil fabrication will raise the production of castor seed pulp, which has great potential for utilization for biodegradable material technology and for animal feed, if detoxified. The objectives of this research were the chemical characterization of the castor seed pulp, the extraction of the proteins for biodegradable material developing, and the characterization of the solid residue of the extraction process, aiming its use for animal feed. The protein extraction from the castor seed pulp was made through solubilization in alkaline medium. Initially, several parameters (stirring speed, pulp concentration in the extraction solution and extraction temperature) were tested aiming the optimization of the protein extraction yield, using NaOH (pH 9). Subsequently, several experiments were realized to evaluate the effects of pH (8-12) and/or of the type of alkaline agent (NaOH, KOH and CaOH) on the castor seed pulp protein extraction, always at 50 °C, 400rpm stirring speed, and 20 % pulp concentration in the extraction solution. The obtained protein extracts were lyophilized (EPL), and the residues were dehydrated in stove (40 °C/24 h). The raw material, the extracted proteins and the residues were analyzed for bromatologic composition, minerals, starch, total phenolic compounds, fatty acids, amino acids, dietetic fiber, microstructure, allergenic activity, dimensional electrophoresis and ricin identification. The amino acids composition was analyzed on the lyophilized extract (EPL) and in the residues of the samples prepared with NaOH. The bromatologic composition of the raw materials (seed, pulp and meal) showed high content of oil (43.6 %) and fiber (29.5 %) for the seeds; as well as high amounts of protein (36-40 %) and fiber (29-30 %) for the pulp and the castor seed meal. The castor seed had average mineral values for oil seeds, and the castor seed pulp showed high percentage of K, Ca and P, valuable for animal feed. The evaluated raw materials had high percentage of ricinoleic acid (79-90 %). The castor seed pulp showed high percentage of glutamic acid (15 %), arginine (11 %) and triptophane (9 %). The percentage of the other amino acids varied between 2 and 7 %. The results of the chemical characterization of the EPL and of the residues were mainly dependent on the pH during protein extraction. It was observed an increase of the protein concentration (64-68 %) and of the ash (13-19 %), within the EPL, due to the pH raise (10 - 12), independently of the type of alkaline agent. The residues of the EPL extraction showed protein, ash and fiber content varying between 20 and 34%, 12 and 17% e 39 and 42%, respectively. Concerning the mineral profile of the EPL, an increase of Na and of Mn was observed with the increase of pH for both alkaline agents. The mineral profile of the residues showed a significant increase of the Na and the K content with the increase of the pH of the extraction solution, adjusted with NaOH and KOH, respectively. The ricinoleic acid content was lower in the residue, and higher in the EPL, obtained at pH 12 using NaOH. The amino acid composition of the residues was affected by the pH only at pH 12. The higher content of amino acid in the EPL and in the residues was of glutamic acid, triptophane and arginine. The EPL and the residues of the protein extraction showed low allergenic capacity evaluated by the mastocyte degranulation test obtained from the peritoneal wash of rats. The electrophoresis profile of the EPL and of the residues showed higher content of proteins with molecular weight varying between 29-36 kDa, followed by proteins with molecular weight around 20 kDa and lower proportion of proteins with molecular weight between 45 and 66 kDa. Ricin was not observed in the residues obtain at pH 12. The results of all the experiments of protein extraction of the castor seed pulp allowed to select the best extraction parameters: temperature = 50 °C, stirring speed = 400rpm, castor seed pulp concentration in the extraction solution = 20 %, pH = 12 and alkaline agent = NaOH. In conclusion, through the alkaline protein extraction process it was possible to achieve a protein concentrate with interesting properties for biodegradable material production (agriculture and biofilms), and of ricinless residues which still have high content of proteins and fiber that can be used for animal feed.

O presente trabalho teve como principal objetivo avaliar o potencial de linhagens termotolerantes da espécie Kluyveromyces marxianus para produção de xilitol a partir do hidrolisado hemicelulósico de palha de arroz. Foi também objetivo deste trabalho estabelecer condições de tratamento alcalino diluído (desacetilação) previamente ao processo de hidrólise ácida da palha de arroz com vistas à obtenção de um hidrolisado com menor toxicidade. Numa primeira etapa do trabalho, foram avaliadas 4 cepas de K.marxianus (Y-6860, Y-6373, Y- 265 e Y-8287) quanto a produção de xilitol em meio semidefinido variando a temperatura de 30 a 40 °C. Com base nos resultados obtidos, foi selecionada a linhagem Y-6373, que apresentou os maiores valores de conversão de xilose em xilitol (YP/S = 0,70 g/g) e produtividade volumétrica (QP = 0,60 g/L.h) na temperatura de 40 °C. Esta linhagem foi então utilizada nos ensaios fermentativos a partir dos hidrolisados obtidos. Para obtenção do hidrolisado hemicelulósico, a palha de arroz foi primeiramente submetida a um tratamento alcalino, em escala de frascos Erlenmeyer, onde se avaliou o efeito da temperatura (50 - 70 °C) e da carga de NaOH (20 - 80 mg/g de biomassa) sobre a remoção de grupos acetil. De acordo com o modelo obtido, a máxima remoção de acetil (97,2 ± 3,4%) pôde ser alcançada empregando 70 °C; 80 mg de NaOH /g de palha por 45 min. Nestas condições do processo, o modelo previu remoções de lignina e de cinzas de 41,3 ± 4,3 e 62,3 ± 5,3%, respectivamente, sem perda significativa das frações açucaradas. Em maior escala (reator de 50L), o tratamento alcalino mostrou resultados de remoção de acetil próximos aos previstos pelo modelo. Com a palha desacetilada, foram então realizados ensaios de hidrólise ácida variando a concentração de H2SO4 (0,5 - 1,5% m/v) e tempo de residência (30 - 90 min) visando estabelecer as condições que maximizem a eficiência de hidrólise da hemicelulose (EHH). Nas condições otimizadas, (H2SO4 1,0% m/v e 85 minutos) foi alcançada uma EHH de 75% em frascos e de 76% em reator. Os resultados referentes à fermentabilidade do hidrolisado empregando a K. marxinuas Y-6373 demonstraram que independentemente da suplementação nutricional, a conversão de xilose em xilitol foi elevada ~ 0,87 g/g e superior a obtida em meio semidefinido contendo apenas xilose como fonte de carbono, porém os valores de produtividade volumétrica foram cerca de 6,5 vezes inferiores ~ 0,13 g/L.h, devido a baixa eficiência de utilização da xilose (XC) ~ 20%, após 72 h de cultivo. A fermentabilidade dos hidrolisados foi melhorada após o procedimento de destoxificação e os resultados variaram com o método empregado. Os melhores resultados foram obtidos em hidrolisado tratado com CaO (YP/S= 0,74 g/g; QP = 0,32 g/L.h e XC =92%). Neste trabalho, foi também demonstrado o efeito repressivo da glicose sobre a assimilação e conversão da xilose em xilitol por K. marxianus, uma vez que em meio semidefinido simulando as concentrações de xilose e glicose presentes no hidrolisado, os valores de XC e YP/S foram reduzidos em torno de 40%. De uma forma geral, conclui-se que a linhagem de K. marxianus selecionada neste estudo, apresenta grande potencial para a produção de xilitol em elevadas temperaturas, porém são ainda necessários estudos para otimizar as condições de produção de xilitol a partir de hidrolisados hemicelulósicos. / In this work the potential of thermotolerant strains of Kluyveromyces marxianus to produce xylitol from rice straw hemicellulosic hydrolyzed was evaluated. In addition, were also established conditions of diluted alkaline treatment (deacetylation) previously to the process of rice straw acid hydrolysis, aiming to decrease the toxicity of the hydrolysate . Initially, four yeast strains of K.marxianus (Y-6860, Y-6373, Y-2265 e Y-8287) were evaluated regarding to the xylitol production in a semi-defined medium, varying the temperature from 30 to 40 ºC. From these results, the strain Y-6373 was selected, due to high values of xylose conversion into xylitol (YP/S = 0.70 g/g) and volumetric productivity (QP = 0.60 g/L.h) at 40ºC. This strain was thus used in the fermentative tests employing the hemicellulosic hydrolysate obtained. The hemicellulosic hydrolysate of rice straw was prepared in two steps; firstly the rice straw was submitted to the alkaline treatment, which was carried out in Erlenmeyer flasks scale. In these experiments, the effects of temperature from 50 to 70 °C and NaOH load from 20 to 80 mg/g biomass on the removal of acetyl groups were evaluated. According to the model obtained, the maximum acetyl removal (97.2 ±3.4%) could be achieved by employing 70 ºC; 80 mg of NaOH/g of straw during 45 minutes. Under these conditions, the model predicted lignin and ashes removals of 41.3±4.3 and 62.3±5.3%, respectively, without significant loss of sugars. On a large scale (reactor of 50L), the alkaline treatment showed outcomes of acetyl removal near the ones predicted by the model. After, the deacetylating straw was submitted to acid hydrolysis process, varying the H2SO4 concentration from 0.5 to 1.5% w/v, and the residence time from 30 to 90 minutes, aiming to stablish the conditions that maximize the efficiency of hemicellulose hydrolysis (EHH). Under optimized conditions, (H2SO4 1.0% w/v and 85 minutes) 75% EHH was achieved in Erlenmeyers flasks and 76% in reactor. As regarding the hydrolysate fermentability employing K. marxinuas Y-6373, the results showed that, regardless of the nutritional supplementation, the conversion of xylose into xylitol was high ~ 0.87 g/g and superior in relation to the one obtained at a semidefined medium containing only xylose as a carbon source. Nevertheless, the values ofvolumetric productivity were approximately 6.5 times inferior ~ 0.13 g/L.h, due to the lowefficiency of xylose consumption (XC) ~ 20%, after 72h of cultivation. The hydrolysate fermentability was improved after detoxification and the results varied with the method employed. The best results were obtained on hydrolysate treated with CaO (YP/S= 0.74 g/g; QP = 0.32 g/L.h and XC = 92%). The glucose effect was further examined in semi-defined medium. The results showed that xylose assimilation and xylitol production by K. marxianus was repressed by glucose, since in a semi-defined media simulating the concentrations of xylose/glucose found in the hydrolyzed, the values of XC e YP/S were reduced in approximately 40%. Based on the results, was concluded that the strain of K. marxianus selected on this study, presents a great potential to produce xylitol at elevated temperatures. However, studies are still necessary to optimize the conditions of this bioconversion from the hemicellulosic hydrolysate.

Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.

Este estudo teve como objetivo avaliar a interação entre substâncias húmicas e xenobióticos através de estudos ecotoxicológicos. O principal foco foi avaliar a resposta entre diferentes níveis tróficos em organismos aquáticos (fitoplanctônicos, zooplanctônicos, peixes e macroinvertebrados bentônicos) e terrestres (vegetais superiores, insetos e anelídeos) avaliando os efeitos diretos e indiretos das SH e de sua mistura com xenobióticos sobre os organismos. O reconhecido efeito das SH aumentarem ou reduzirem o efeito tóxico de algumas substâncias foi estudado. Os principais pontos para discussão são: a) SH podem reduzir ou estimular o crescimento algal (P. subcapitata); b) A presença de SH podem proteger os organismos contra efeitos tóxicos de metais, no entanto, a presença de Cd/Cu afeta negativamente o crescimento de C. xanthus. d) Em uma análise integrada de processos de remediação solo/água, a presença de SH afetou negativamente ou positivamente os efeitos tóxicos da atrazina em alguns organismos. A significância das SH como tecnologia é discutida. / This study focuses the interaction of humic substances and xenonbiotics, throw ecotixicological studies. The main point was quantify and qualify the ecotoxicological responses of several throphic levels of freshwater (algae, zooplankton, fishes and benthic organisms) and soil organisms (higth plants, insects and annelids) analyzing the direct and indirect effects of humic substances (HS) and their mixture on organisms. The recognized ability of HS on improve or reduce the toxic effect of same substances has been studied. The main points of discussion are: a) humic can be both reduce or stimulate the algal growth (P. subcapitata); b) The presence of HS can be protect aquatic organisms to negative effects of metals. However, the presence of mixture of Cd/Cu affect negatively the growth of C. xanthus; d) In a integrated analysis of remediation process in soil/water microcosm, the presence of HS displayed negative or positive effects on atrazine toxicity for some organisms. The value of humic technology was discussed.

Le but du projet était d'acquérir des connaissances plus approfondies sur les mécanismes impliqués dans l'absorption et l'accumulation des éléments traces métalliques (ETM) par les plantes et dans la résistance à leurs effets toxiques. Arabidopsis halleri, peut vivre sur des sols fortement pollués par des ETM et a la capacité d'accumuler des concentrations extraordinairement élevées de ces métaux dans les tissus aériens. A. halleri est utilisée comme espèce modèle pour étudier les mécanismes biologiques et moléculaires impliqués dans la tolérance et l'accumulation de fortes concentrations de métaux dans le sol. Cette espèce est caractérisée par une tolérance élevée à de fortes concentrations de Cd et Zn. Elle est également capable d'accumuler de très grandes quantités de Zn dans les pousses, appelées hyperaccumulateurs de Zn, et également de Cd dans certaines populations, appelées hyperaccumulateurs de Cd. Arabidopsis arenosa, une espèce étroitement liée à A. halleri, montre également une tolérance élevée aux deux métaux mais est signalée comme non-hyperaccumulatrice. Dans le sud de la Pologne, les deux espèces peuvent être observées sur les mêmes sites pollués. Les expériences ont été conçues pour étudier la variabilité inter et intra spécifique entre les populations métallisées (M) et non métallisées (NM) d'A. halleri et A. arenosa afin d'identifier les paramètres physiologiques et les gènes responsables du comportement contrasté de leur adaptation à la contamination métallique. L'accumulation des ETM, l'activité de l'appareil photosynthétique et le contenu pigmentaire ont été analysés dans les populations M et NM d'A. halleri et A. arenosa, poussant dans leur habitat naturel. Des expériences hydroponiques ont également été réalisées afin de déterminer les différences physiologiques entre les populations étudiées dans des conditions contrôlées. De plus, le niveau d'expression des gènes impliqués dans l'absorption des métaux, le transport radial, la translocation et la détoxification a également été étudié. Mes résultats montrent que la population M de A. arenosa est aussi tolérante aux métaux lourds que la population M de A. halleri. Il a été démontré que la population M de A. arenosa hyperaccumule le Cd et le Zn. De plus, les deux espèces diffèrent dans la réponse de l'appareil photosynthétique lorsqu'elles sont exposées à des concentrations élevées de Cd et de Zn, ce qui suggère des mécanismes différents impliqués dans l'homéostasie des métaux. Le niveau d'expression des gènes a montré des différences non seulement entre les espèces mais aussi entre les populations. Le type de traitement (Cd ou Zn) a également eu un effet différent sur toutes les populations en ce qui concerne certains gènes. Les résultats acquis dans ce projet comblent les lacunes dans les caractéristiques physiologiques des espèces d'A. halleri à la fois sur le terrain et dans des conditions hydroponiques contrôlées. La caractérisation physiologique approfondie de la population M de A. arenosa a identifié cette population comme un nouvel hyperaccumulateur de Cd et Zn. Ces résultats peuvent servir de base à de futures études sur le phénomène d'hyperaccumulation. De plus, l'observation de l'expression contrastée des gènes impliqués dans l'absorption, la translocation et la détoxification des métaux entre A. halleri et A. arenosa suggère que A. arenosa pourrait être un bon nouveau modèle pour étudier l'homéostasie et la tolérance aux métaux chez les plantes. / Doctorat en Sciences agronomiques et ingénierie biologique / info:eu-repo/semantics/nonPublished

Objective: This study was completed as a follow up to research regarding buprenorphine Medication Assisted Therapy (MAT) in Johnson City Tennessee for-profit clinics. We wished to determine the practice patterns over the last three years. Methods: Johnson City for-profit Medical Assisted Therapy clinics were called with a telephone survey. When the clinic representative answered the phone, they were asked questions regarding patient costs for therapy, insurance coverage, counseling offered on site, and opportunities for tapering while 20 weeks pregnant. Results: At all the MAT clinics contacted, the representative informed us that tapering in pregnancy could be considered contrary to current national guidelines. 43% of the clinics are now accepting insurance as compared to 0% in the 2016 study. The average weekly cost per visit remained consistent. Conclusion: The concept of tapering buprenorphine during pregnancy appears to have become a standard of care for this community and it is offered at all of the clinics that were contacted, some even require it, even though national organizations such as American College of Obstetricians and Gynecologists and American Society of Addiction Medicine; do not recommend this approach. Patients who have insurance including government funded insurance, are now able to obtain buprenorphine with no out of pocket expense at numerous clinics. The high cost for the uninsured patient continues to create an environment conducive to buprenorphine diversion.

The worldwide rise of diabetes and obesity has spurred research investigating the molecular mechanisms that mediate the deleterious effects associated with these diseases. Individuals with diabetes and/or obesity are at increased risk from a variety of health consequences, including heart attack, stroke and peripheral vascular disease; all of these complications have oxygen deprivation as the central component of their pathology. The nematode Caenorhabditis elegans has been established as a model system for understanding the genetic and molecular regulation of oxygen deprivation response, and in recent years methods have been developed to study the effects of excess glucose and altered lipid homeostasis. Using C. elegans, I investigated transcriptomic profiles of wild-type and hyl-2(tm2031) ( a ceramide biosynthesis mutant) animals fed a standard or a glucose supplemented diet. I then completed a pilot RNAi screen of differentially regulated genes and found that genes involved in the endobiotic detoxification pathway (ugt-63 and cyp-25A1) modulate anoxia response. I then used a lipidomic approach to determine whether glucose feeding or mutations in the ceramide biosynthesis pathway or the insulin-like signaling pathway impact lipid profiles. I found that gluocose alters the lipid profile of daf-2(e1370) (an insulin-like receptor mutant) animals. These studies indicate that a transcriptomic approach can be used to discover novel pathways involved in oxygen deprivation response and further validate C. elegans as a model for understanding diabetes and obesity.

Sphingolipids are a class of lipid molecules that function both as structural membrane components and as bioactive signaling molecules. Sphingolipids can be produced de novo or salvaged and recycled. Despite the established roles of sphingolipids such as sphingosine 1-phosphate and ceramides in regulating signaling involved in pro- and anti-tumorigenic cellular processes, the role of the de novo sphingolipid biosynthesis pathway in cancer is unclear. The main objective of this thesis study was to determine whether there is an essential role for this pathway in cancer and whether its disruption can be a cancer-specific metabolic vulnerability. Here, we find that de novo sphingolipid synthesis through the rate-limiting enzyme serine palmitoyltransferase (SPT) is not required in cancer cells due to their salvage capacity. However, upregulation of SPT in cancer cells creates a requirement to detoxify its product, 3-ketodihydrosphingosine (3KDS), via the downstream enzyme 3-ketodihydrosphingosine reductase (KDSR). We demonstrate that KDSR is essential in cancer cells both in vitro and in vivo to restrain the levels of its substrate 3KDS, the accumulation of which can disrupt ER structure and function, resulting in proteotoxic stress and cell death. Our findings also reveal that KDSR is essential specifically in cancer cells and not normal cells and that upregulation of SPT in cancer may act as a biomarker for sensitivity to targeting KDSR. Altogether, this thesis study provides new insights into the role of KDSR in the de novo sphingolipid biosynthesis pathway in both cancer and ER homeostasis and demonstrates the potential to exploit this for therapeutic purposes in a cancer-specific manner.