Immunoassays for 2-methylisoborneol (MIB) and monensin in water were developed, devised and tested to see if the sensitivity could be established and improved. MIB and monensin are hydrophobic haptens with molecular weights of 168 and 671 Da, respectively. Rabbits were immunized with (-) camphor-BSA and (-) borneol-BSA for the production of polyclonal antibodies (pAbs) to MIB. Monoclonal antibodies (mAbs) were produced in Mus musculus using (-) camphor-BSA as immunogen. (+) Bornylamine-thyroglobulin (TG) and MIB-TG were synthesized and used as plate coatings. For the monensin immunoassay, monensin was conjugated to BSA and OVA for immunogen and plate coating, respectively. Several physical parameters that affect the sensitivity of immunoassays including pre-incubation of antibody and antigen, incubation time and temperature, detergent, organic solvents, and ionic strength were evaluated. Improvement of immunoassay sensitivity was also performed by reducing the concentrations of coating antigen and antibodies and using alternative reporter systems such as chemiluminescence (CICL-ELISA), tyramide signal amplification (TSA) and biotin-streptavidin. Different assay formats, i.e., competitive indirect and competitive direct were also compared. Usability of both pAb-based immunoassays for MIB and monensin was evaluated in fortified water samples. A polyclonal-based (pAb) ELISA for MIB had a detection limit of 4.8 ng mL-1 and an IC50 of 105 ng mL-1. Rabbits immunized with (-) camphor-BSA showed a higher immune response than rabbits immunized with (-) borneol-BSA. One clone (i.e., 4F11) of fourteen characterized clones was used to create the monoclonal antibody (mAb)-based ELISA, which had an IC50 of 100.2 ng mL-1 and an LOD of 1.9 ng mL-1. The pAb- and mAb-based CI-ELISA were not specific to MIB alone and cross reacted with camphor and camphor-like compounds. Meanwhile, a pAb-based ELISA for monensin produced a detection limit of 0.1 ng mL-1 and had an IC50 of 1.056-1.090 ng mL-1 with high specificity to monensin. Other reporter systems did not improve the sensitivity of the immunoassays significantly. MIB and monensin polyclonal-based assays showed good correlation to analytical instrumental methods (i.e., GC-MS and LC-MS) in fortified water samples. With a detection limit of ca. 5 ng mL-1 and 0.1 ng mL-1 for MIB and monensin, respectively, both polyclonal-based assays can be used for detection of these analytes in water from different sources and employed as screening tools to complement GC/HPLC-MS instrument methods.

The phytotoxicities of monensin, the 10:2 saturated fluorotelomer carboxylic acid (10:2 FTCA), and atrazine to freshwater macrophytes were investigated in three microcosm-based assessments. Both responses of plants grown as individuals in “cone-tainers” and those grown in mixed and monocultures were examined to permit comparison of the toxicological sensitivities of macrophytes under each planting design, and investigation of whether interactions between neighbours may modify plant response to an environmental contaminant. Exposures of monensin and the 10:2 FTCA at environmentally relevant concentrations were found to produce few significant effects in the higher aquatic plants across all growth conditions, thus direct comparisons of effective concentrations were not conducted. Significant differences between relative growth rates (RGR) of plant grown in assemblages versus individually indirectly indicate that over longer exposure durations toxicity may be underestimated using the individual “cone-tainer” method. RGRs and sensitivities of plants to atrazine were found to be in the same range across planting methods, demonstrating that responses of aquatic plants in the individual-test system reflected those observed in model populations and two-species communities. A lack of observed relations between plants in the mixed and monoculture tests, however, meant that the potential for modification of toxicity through plant interactions was not investigated.

The phytotoxicities of monensin, the 10:2 saturated fluorotelomer carboxylic acid (10:2 FTCA), and atrazine to freshwater macrophytes were investigated in three microcosm-based assessments. Both responses of plants grown as individuals in “cone-tainers” and those grown in mixed and monocultures were examined to permit comparison of the toxicological sensitivities of macrophytes under each planting design, and investigation of whether interactions between neighbours may modify plant response to an environmental contaminant. Exposures of monensin and the 10:2 FTCA at environmentally relevant concentrations were found to produce few significant effects in the higher aquatic plants across all growth conditions, thus direct comparisons of effective concentrations were not conducted. Significant differences between relative growth rates (RGR) of plant grown in assemblages versus individually indirectly indicate that over longer exposure durations toxicity may be underestimated using the individual “cone-tainer” method. RGRs and sensitivities of plants to atrazine were found to be in the same range across planting methods, demonstrating that responses of aquatic plants in the individual-test system reflected those observed in model populations and two-species communities. A lack of observed relations between plants in the mixed and monoculture tests, however, meant that the potential for modification of toxicity through plant interactions was not investigated.

Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-16Bitstream added on 2014-06-13T18:31:27Z : No. of bitstreams: 1 badin_f_me_jabo.pdf: 588683 bytes, checksum: 95acfa363bf6d9a1c4fd8c48c39fdd26 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As indústrias sucroenergéticas têm como preocupação o controle de contaminantes da fermentação, responsáveis por afetar a viabilidade da levedura, provocando diversos transtornos no processo, comprometendo a eficiência fermentativa e o rendimento industrial. Dentre as alternativas para o controle das contaminações, destacam-se o uso de antimicrobianos sintéticos. Sua utilização continua pode favorecer o desenvolvimento de cepas resistentes, contribuindo para o incremento do custo de produção, além da possibilidade de incorporação de resíduos no produto final. Objetivou-se avaliar o efeito do biocida convencional (monensina sódica) e biocidas naturais preparados à base de própolis (Extrato Hidroalcoólico de Própolis - EHP e Extrato Oleoso de Própolis- EOP) sobre a fisiologia das leveduras, o controle dos contaminantes do processo fermentativo e composição do destilado. O delineamento experimental utilizado foi o Inteiramente Casualizado com parcelas subdivididas, com 4 repetições. Os Tratamentos Principais foram: Testemunha, EOP, EHP e monensina sódica (Kamoran WP). Os Tratamentos Secundários constituíram-se nos 10 ciclos fermentativos. Avaliaram-se as características químico-tecnológicas do caldo, mosto e vinho, parâmetros microbiológicos das leveduras e composição do destilado obtido. Os resultados obtidos demonstraram que os biocidas avaliados apresentaram efeito similar, sendo efetivos no controle dos contaminantes da fermentação, não afetando negativamente suas características fisiológicas. Não afetaram a composição a composição do destilado final obtido / The control of fermentation contaminants is one of the sugar mills concerns. The fermentation contaminants are responsible to affect the yeast viability, generating several overturns to the process, compromising the fermentative efficiency as well the industrial yield. Among the alternatives to control contamination, the use of synthetic antimicrobials can be highlighted. Its progressed use may favor the development of resistant strains, contributing in production cost improving, besides the possibility of residues incorporation into the final product. This work aimed evaluate the effect of conventional biocides (sodic monensin) and natural ones based on propolis (Propolis Hydroalcoholic Extract – EHP and Propolis Oily Extract – EOP) under the yeasts physiology, the fermentative process contaminants control, and the distilled composition. The experimental design used was the split-plot with four replications. The main treatments were: Control, EOH, EHP, and sodic monensin (Kamoran WP). The secondary treatments were the 10 fermentative cycles. The evaluated characteristics were: juice, must, and wine chemical-technical characteristics, yeasts microbiologic parameters, and the distillated obtained composition. The results obtained showed that the evaluated biocides presented similar effect, being effectives to control the fermentation contaminants, not affecting negatively its physiologic characteristics. They did not affect the composition of the distilled obtained

Made available in DSpace on 2014-06-11T19:27:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-01-11Bitstream added on 2014-06-13T18:31:47Z : No. of bitstreams: 1 millen_dd_me_botfmvz.pdf: 639274 bytes, checksum: cd47f616a6044fb18da77875d3325af1 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Universidade Estadual Paulista (UNESP) / Esse estudo, conduzido no confinamento experimental da UNESP – Botucatu, foi realizado para testar anticorpos contra as bactérias ruminais S. bovis e F. necrophorum e várias cepas de bactérias proteolíticas sobre o desempenho e incidências de paraqueratose ruminal (IPR) e abscesso hepático em bovinos jovens com diferentes graus de sangue Zebu. O delineamento experimental foi inteiramente casualizado em arranjo fatorial 3X2 em parcelas subdivididas com três repetições (4 animais/baia), aonde três grupos genéticos (24 Tri-Cross (TC) - ½ Brangus, ¼ Angus, ¼ Nelore; 24 Canchim (CC) - ⅝ Charolais, ⅜ Nelore e 24 Nelores (NE)) e dois aditivos alimentares (anticorpos policlonais – 10 ml/cabeça/dia (AP) e monensina sódica – 30 mg/kg de dieta (MN)) foram as variáveis estudadas em três períodos de coleta de dados, correspondentes as três dietas com níveis crescentes de concentrado (73, 82 e 85%). Não houve interação (P>0,05) entre os grupos genéticos e os aditivos alimentares estudados. Animais suplementados com AP apresentaram similares (P>0,05) ganho de peso diário (GPD), ingestão de matéria seca em quilos (IMSKG), conversão alimentar (CA) e custo para ganhar um quilo de peso vivo que aqueles suplementados com MN. A ingestão de matéria seca em porcentagem do peso vivo (IMSPV) foi maior (P<0,05) para animais suplementados com AP que aqueles suplementados com MN. As IMSPV e IMSKG foram maiores na dieta de 73% de concentrado (P<0,05) para animais suplementados com AP e não diferiram entre os aditivos alimentares para as outras duas dietas (P>0,05). Conforme as dietas propostas, GPD e IMSPV diminuíram (P<0,05) e CA e IMSKG aumentaram, linearmente, durante o estudo (P<0,05). Animais suplementados com AP apresentarem menor (P=0,09) IPR que àqueles suplementados com MN. TC e CC apresentaram maiores (P<0,05) IMS e GDP e melhor (P<0,05) CA que NE. Rumens de NE mostraram, significativamente (P<0,05), maiores IPR que TC e CC. / This study, conducted at the São Paulo State University feedlot, Botucatu Campus, Brazil, was designed to test antibodies against S. bovis, F. necrophorum, and several strains of proteolytic bacteria (RMT) on performance and parakeratosis and liver abscesses incidences of Bos indicus-based types (BIBT). The experiment was designed as a split plot 3X2 factorial scheme, replicated thrice (4 bulls/pen), in which 24 8-mo-old bulls (297 kg) of each of three BIBT: 3-way cross (½ Brangus, ¼ Angus, ¼ Nellore; TC), Canchim (⅝ Charolais, ⅜ Nellore; CC), or Nellore (NE) were fed one of two diets containing either monensin (MO) at 30 mg/kg of diet or RMT at 10 mL/head/d and evaluated in increasing levels of concentrate (73, 82 and 85%). There were no interactions (P>0.05) between breed type and feed additives. Bulls fed RMT had similar (P>0.05) average daily gain (ADG), dry matter intake in kilos (DMIKG), cost to gain one kilo of body weight and feed conversion (FC) as those fed MO. When analyzed as percentage of BW, bulls fed RMT consumed (P<0.05) more feed than those fed MO. DMIKG and DMIBW were higher in 73% concentrate but did not differ in the others two diets evaluated. According to diets offered, ADG and DMIBW were reduced (P<0.05) and DMIKG and FC (P<0.05) were increased as the level of concentrate in the diet got higher. Bulls fed RMT to have lesser (P=0.09) rumen parakeratosis scores than those fed MO. TC and CC had greater (P<0.05) DMI and ADG, and better (P<0.05) FC than NE. Rumens from NE bulls had greater (P<0.05) parakeratosis scores than CC and TC. Just one liver abscess was found in this study what led to conclude as no abscesses incidence. Feeding RMT enhanced intake of Bos indicus-based bulls fed 73% concentrate diet while maintaining rumen health, and permitting performance similar to that of bulls fed monensin. RMT could be an eventual substitute if monensin was really prohibited.

Receptor Tyrosine Kinase (RTK) inhibitors, such as erlotinib/tarceva, have been introduced in the past decade as a promising therapeutic option in Head and Neck Squamous Cell Carcinoma (HNSCC), however, they lack significant efficacy as single agents. As a result, RTK inhibitors require a combination based therapeutic approach with other treatment modalities. To uncover such a combination of agents, we performed a high throughput Prestwick library screen that included 1200 compounds approved by the FDA on HNSCC cell lines and found that monensin, a coccidial antibiotic, synergistically enhanced the cytotoxicity of erlotinib. RT-PCR revealed that monensin induced the expression of Activation of Transcription Factor (ATF) 3 and its downstream target C/EBP homologous protein (CHOP) which are key regulators of apoptosis. Furthermore, RNA-Seq analysis suggests that monensin augments erlotinib cytotoxicity by disturbing lipid and sterol biosynthesis. Therefore, identifying the mechanism of action exerted by monensin may open alternative avenues of cancer treatment.

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