Análise do impacto da deleção do gene p94 (ac134) de autographa californica multiple nucleopolyhedovirus

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós Graduação em Biologia Molecular, 2015.

Polymorphismes dans des gènes de métabolisme des corticostéroïdes : rôle dans la réponse thérapeutique

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Covalent Protein Adduction by Drugs of Abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Verification of the mechanism of glyphosate resistance in Italian ryegrass biotypes.

The intense use of glyphosate for weed control led to the emergence of several cases of resistance to this herbicide. Weeds can survive the application of herbicides due to several factors, which may or may not be related to the herbicide site of action. The objectives of this study were to quantify the accumulation of shikimate in ryegrass biotypes in response to glyphosate application; investigate possible mutations on the EPSPs gene in susceptible and resistant biotypes; and evaluate the response of ryegrass biotypes to the application of glyphosate after treatment with a metabolism inhibitor of cyt P450 monooxygenase.

Controle de Helicoverpa armigera (Lepidoptera: Noctuidae) em soja com inseticidas químicos e biológicos.

RESUMO: O objetivo deste trabalho foi avaliar o desempenho de inseticidas autorizados emergencialmente para o controle de Helicoverpa armigera (Lepidoptera: Noctuidae) em soja. Sete inseticidas foram pulverizados em campo e, após 24 horas, folhas do ponteiro foram coletadas e oferecidas para lagartas de 2o instar em laboratório. Lagartas do 4o instar receberam a última folha trifoliolada que se encontrava completamente expandida no momento da pulverização. Outro grupo foi exposto a folhas coletadas a partir de 72 horas da pulverização. Em campo, seis inseticidas foram pulverizados e, em seguida, as plantas foram infestadas com lagartas de 2o e 3o instar. No primeiro estudo, flubendiamida, clorantraniliprole, clorfenapir, indoxacarbe e metoxifenozida causaram 100% de mortalidade do 4o instar aos oito dias após o início da exposição, enquanto baculovírus e Bacillus thuringiensis (Bt) propiciaram mortalidade de 60-75%, que evoluiu para 88?90% ao final da fase de pupa. Para o 2o instar, apenas flubendiamida e clorantraniliprole proporcionaram mortalidade de 100%. Flubendiamida, clorantraniliprole e clorfenapir apresentaram o menor tempo letal para o 4o instar, e flubendiamida e clorantraniliprole, para o 2o instar. Após 72 horas da pulverização, o desempenho dos inseticidas foi insatisfatório. Em campo, houve eficiência satisfatória de flubendiamida, espinosade, baculovírus e Bt sobre lagartas de 2o e 3o instar. ABSTRACT:The objective of this work was to evaluate the performance of insecticides authorized on an emergency basis to control of Helicoverpa armigera (Lepidoptera: Noctuidae) in soybean. Seven insecticides were sprayed on the field and, 24 hours after that, soybean pointer leaves were collected and offered to 2nd instar larvae in the laboratory. Fourth instar larvae received the last trifoliate leaf that was fully expanded at the time of spraying. Another larvae group was exposed to leaves collected from 72 hours onwards after spraying. In the field, six insecticides were sprayed, and then the plants were infested with 2nd and 3rd instar larvae. In the first study, flubendiamide, chlorantraniliprole, chlorfenapyr, indoxacarb, and methoxyfenozide caused 100% mortality of the 4th instar, eight days after the beginning of exposure, while baculovirus and Bacillus thuringiensis (Bt) caused 60?75% mortality, which reached 88?90% at the end of the pupal stage. For 2nd instar larvae, only flubendiamide and chlorantraniliprole caused 100% mortality. Flubendiamide, chlorantraniliprole, and chlorfenapyr showed the lowest lethal time for the 4th instar, and flubendiamide and chlorantraniliprole for the 2nd instar. Seventy-two hours after spraying, the performance of insecticides was not satisfactory. In the field, there was satisfactory efficiency of flubendiamide, spinosad, baculovirus, and Bt on 2nd and 3rd instar larvae.

Development of effect-based bio-chemiluminescent cell bioassays for nutraceutical and biomedical applications

The final goal of the bioassay developed during the first two years of my Ph.D. was its application for the screening of antioxidant activity of nutraceuticals and for monitoring the intracellular H2O2 production in peripheral blood mononuclear cells (PBMCs) from hypercholesterolemic subjects before and after two months treatment with Evolocumab, a new generation LDL-cholesterol lowering drug. Moreover, a recombinant bioluminescent protein was developed during the last year using the Baculovirus expression system in insect cells. In particular, the protein combines the extracellular domain (ECD) of the Notch high affinity mutated form of one of the selective Notch ligands defined as Jagged 1 (Jag1) with a red emitting firefly luciferase since a pivotal role of “aberrant” Notch signaling activation in colorectal cancer (CRC) was reported. The probe was validated and characterized in terms of analytical performance and through imaging experiments, in order to understand if Jagged1-FLuc binding correlates with a Notch signaling overexpression and activation in CRC progression.