Spelling suggestions: "subject:"oxidases."" "subject:"axidases.""
81 |
Investigações sobre os mecanismos de resistência em larvas e adultos de Aedes aegypti, Linnaeus, 1762Medeiros, Priscila Fernandes Viana January 2011 (has links)
Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-09-21T12:34:57Z
No. of bitstreams: 1
priscila_f_v_medeiros_ioc_bp_0058_2011.pdf: 6129115 bytes, checksum: a9a22a0f84ef8206e0afcbc77535046a (MD5) / Made available in DSpace on 2012-09-21T12:34:57Z (GMT). No. of bitstreams: 1
priscila_f_v_medeiros_ioc_bp_0058_2011.pdf: 6129115 bytes, checksum: a9a22a0f84ef8206e0afcbc77535046a (MD5)
Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O uso de inseticidas neurotóxicos contra o mosquito Aedes aegypti ainda é um
componente importante nos programas de controle de dengue. No Brasil,
organofosforados (OP) e piretróides (PI) são usados para o controle de larvas e
adultos de A. aegypti, respectivamente, desde 1967 e 2000. O uso frequente
destes produtos selecionou populações resistentes do vetor, e motivou o
Ministério da Saúde (MS) a coordenar, desde 1999, uma rede nacional de
monitoramento da resistência de A. aegypti (MoReNAa), da qual nosso
laboratório participa desde o início. A resistência é principalmente derivada de
fatores metabólicos (enzimas que detoxificam o inseticida) ou de mutações nos
sítios-alvo dos inseticidas, no Sistema Nervoso Central. Bioensaios com larvas
revelaram a resistência de várias populações ao OP temephos, larvicida
empregado há mais de 40 anos no país. A Rede MoReNAa conta também com
bioensaios para adultos, com testes moleculares para avaliação de alteração
no alvo de PI, o canal de sódio regulado por voltagem (AaNav) e com ensaios
bioquímicos para quantificação, em mosquitos adultos, da atividade enzimática
de Glutationa S-Transferases (GST), Esterases e Oxidases de Função Mista
(MFO) (relacionadas à resistência metabólica), e de Acetilcolinesterase (Ace)
(alvo de OP e carbamatos - CB). Além de serem usadas classes distintas de
inseticidas contra larvas e adultos, as aplicações são feitas de maneira
diferenciada: em 4-6 ciclos anuais sobre as larvas, e apenas em situações de
emergência sobre os mosquitos adultos - procedimento que tem o potencial de
elicitar mecanismos (e intensidades) de resistência diferentes. Além disso,
bioensaios para quantificação da resistência a OP são feitos com larvas,
enquanto os ensaios bioquímicos estavam disponíveis apenas para mosquitos
adultos. Estes foram os principais motivos para adaptar, no âmbito desta
dissertação, ensaios bioquímicos para o estágio larvar do vetor. Em relação ao
ensaio da Ace, que conta com duas reações, uma na presença e a outra na
ausência do CB propoxur verificamos, por meio de curvas do tipo dose-
resposta, diferenças entre larvas e adultos. Além disto identificamos, em
algumas populações do vetor resistentes a OP, alterações na atividade total
desta enzima. Uma vez que algumas destas enzimas participam também de
processos endógenos, que ocorrem naturalmente nos insetos, quantificamos
sua atividade ao longo do desenvolvimento da cepa referência de
suscetibilidade, Rockefeller, e de duas populações de campo. Nestes ensaios
foram observadas quatro grandes “categorias” de perfis de atividade
enzimática: 1) maiores atividades no estágio adulto (AChE); 2) maiores
atividades no estágio larval (Esterases “α-EST” e “β-EST”); 3) atividades que
aumentam no decorrer de cada estágio avaliado (MFO) e 4) atividades que tendem
a aumentar no estágio larvar e a diminuir nos primeiros dias de vida adulta
(DVA) (Esterase “ρNPA” e GST). Posteriormente, ensaios bioquímicos com
larvas e adultos de populações de campo revelaram alterações de Ace e
Esterases preferencialmente no estágio larvar, alterações de GST mais
restritas ao estágio adulto, e alteração de MFO nos dois estágios do vetor.
Estes ensaios possibilitam conhecer com detalhe os mecanismos de
resistência em diferentes populações do vetor e podem contribuir com a
definição de estratégias racionais para o controle de A. aegypti. / The use of neurotoxic insecticides against the mosquito Aedes aegypti is still an
important component in dengue control programs. In Brazil, organophosphates
(OP) and pyrethroids (PI) are used for the control of A. aegypti larvae and
adults since, respectively, 1967 and 2000. The frequent use of these products
has selected resistant vector populations, and prompted the Ministry of Health
(MS) to start the coordination, in 1999, of an Aedes aegypti insecticide
resistance monitoring network (MoReNAa); our laboratory participates in the
network since its beginning. Resistance is mainly derived from metabolic factors
(enzymes detoxifying the insecticides) or from mutations at the target sites of
insecticides (in the Central Nervous System). Bioassays with larvae disclosed
resistance of various populations to the OP temephos, larvicide employed for
over 40 years in the country. MoReNAa network also performs bioassays with
adults, molecular tests to assess substitution at the PI target site, the voltage
regulated sodium channel (AaNAv), and biochemical assays that quantify, in
adult mosquitoes, the activity of Glutathione S-Transferases (GST), Esterases
and Mixed Function Oxidases (MFO) (related to metabolic resistance), and of
Acetylcholinesterase (Ace) (target of OP and carbamates - CB). Besides using
different insecticide classes against larvae and adults, the treatment are
performed differently: in 4-6 times per year for larvae and only in emergency
situations in the case of adult mosquitoes – a procedure that has the potential to
elicit different mechanisms (and intensities) of resistance. Furthermore,
bioassays for quantification of resistance to OP are made with larvae, while
biochemical assays were available only for adult mosquitoes. These were the
main reasons to adapt, in the context of this dissertation, biochemical assays for
the larval stage of the vector. Considering the Ace test, consisting of two
reactions, in the presence or in the absence of the CB propoxur, we identified -
through the use of inhibition curves - differences between larvae and adults. We
also detected, in some OP resistant vector populations, changes in the total
activity of this enzyme. Since some of the enzymes of metabolic resistance are
also involved in endogenous processes, that occur naturally in insects, we
quantified their activity during the development of the reference strain of
susceptibility, Rockefeller, and of two field populations. In these experiments we
observed four major "categories " of enzyme activity profiles: 1) higher activity in
the adult stage (AChE); 2) higher activity in the larval stage (“α-EST” and “β-
EST” Esterases); 3) activities that increase during each stage evaluated (MFO)
and 4) activities tending to increase in the end of the larval stage and to
decrease in the first days of adult life (DVA) (Esterase “ρNPA” and GST).
Subsequently, biochemical assays with larvae and adults of field populations
revealed main changes in Ace and Esterases in the larval stage, GST changes
preferably in the adult stage, and MFO alterations on both vector stages. These
assays enable the detailed knowledge of resistance mechanisms of different
vector populations and can contribute to define rational strategies for A. aegypti
control.
|
82 |
Caractérisation électrochimique et spectroscopique de protéines membranaires immobilisées sur des nanomatériaux / Electrochemical and spectroscopic characterization of membrane proteins immobilized on nanomaterialsMeyer, Thomas 19 February 2015 (has links)
Le domaine de la bioénergétique concerne l’étude des échanges et des transformations de l’énergie au sein des organismes vivants. Cette thèse propose une étude électrochimique et spectroscopique de protéines issues de la chaine respiratoire, les oxydases terminales, afin de comprendre l’influence de différentes propriétés de ces enzymes (potentiels des cofacteurs, dépendance pH…) sur leur mécanisme réactionnel. La première partie de ce travail décrit le développement d’une méthode d’immobilisation permettant de conserver l’intégrité et l’activité de ces enzymes. Cette technique a d’abord été utilisée pour étudier l’inhibition de la cytochrome aa3 oxydase de P. denitrificans et a permis de mettre en avant l’importance du transfert de protons sur la réaction de réduction de l’oxygène. Une deuxième étude propose de comparer deux isoformes de la cytochrome cbb3 oxydase dont aucune différence n’a été observée à ce jour. La spectroscopie IRTF couplée à l’électrochimie montre l’implication de résidus acides différents au cours de la réaction d’oxydoréduction suggérant des différences mécanistiques. La dernière partie propose une étude comparative d’oxydases terminales de différents types et met en perspective l’influence des potentiels relatifs des hèmes sur la réaction de réduction de l’oxygène. / The field of bioenergetics concerns the study of exchange and transformation of energy in living organisms. This manuscript proposes an electrochemical and spectroscopic study of the fourth complex of the respiratory chain, the terminal oxidases. The aim of this study was to understand the influence of some properties of these enzymes (potential of the cofactors, pH dependency…) on the catalytic mechanism. The first part describes an immobilization procedure which retains the protein activity and structure. This procedure has been applied for the study the inhibition of the proton pathways of cytochrome aa3 oxidase from P. denitrificans and shows the importance of proton transfer on the oxygen reduction. In a second study, two isoforms of cytochrome cbb3 oxidase were compared. No differences were observed between them until now. Our electrochemically induced FTIR spectroscopy study suggests the implication of different acidic residues during the redox reaction implying differences in the mechanism of these enzymes. The last part deals with the comparison of terminal oxidases of different types and shows the influence of the relative order of the midpoint potentials of the hemes on the oxygen reduction.
|
Page generated in 0.0261 seconds