Antioxidant activity in vivo and in vitro of Halimeda incrassata aqueous extracts

The aim of the present paper was to provide the evidences for the antioxidant activity in Halimeda incrassata (Ellis) Lamouroux aqueous extracts obtained after simple water extraction of the fresh algae at room temperature (23°C). Previously in the literature, only antioxidant activity associated to carotenoids fractions of seaweeds has been reported. From different species of seaweeds, Halimeda incrassata aqueous extract exhibited the highest antioxidant activity on the inhibition of TBARS formed during the spontaneous lipid peroxidation of rat brain homogenates with an IC50 of 0.340mg.mL-1. Halimeda incrassata aqueous extract (0.5mg.mL-1), was also capable of decreasing the in vitro generation of hydrogen peroxide by two distinct metabolic pathways involving glutamic and malonic acids. Also, Halimeda incrassata (at doses of 50, 100 and 200mg.Kg-1) showed a neuroprotective effect in vivo on the gerbil model of bilateral carotid occlusion because of decreasing the locomotor and exploratory activity induced by ischemia. In summary, Halimeda incrassata aqueous extracts exhibit antioxidant properties in different in vitro as well as in vivo models which could be explained by the presence of several hydrosoluble compounds. Further studies on this way are necessary to elucidate the precise structure of these compounds. Low toxicity of most seaweeds to humans, but particularly of Halimeda genus may favor its use as functional food.

Chemical sanitizers to control biofilms formed by two Pseudomonas species on stainless steel surface

The biofilm formation of Pseudomonas aeruginosa and Pseudomonas fluorescens on AISI 304 stainless steel in the presence of reconstituted skim milk under different temperatures was conducted, and the potential of three chemical sanitizers in removing the mono-species biofilms formed was compared. Pseudomonas aeruginosa cultivated in skim milk at 28 °C presented better growth rate (10.4 log CFU.mL-1) when compared with 3.7 and 4.2 log CFU.mL-1 for P. aeruginosa and P. fluorescens cultivated at 7 °C, respectively. Pseudomonas aeruginosa formed biofilm when cultivated at 28 °C. However, only the adhesion of P. aeruginosa and P. fluorescens was observed when incubated at 7 °C. The sodium dichloroisocyanurate was the most efficient sanitizer in the reduction of the adhered P. aeruginosa cells at 7 and 28 °C and those on the biofilm, respectively. The hydrogen peroxide was more effective in the reduction of adhered cells of P. fluorescens at 7 °C.

Investigation of glutathione peroxidase activity in chicken meat under different experimental conditions

Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and results, this study aimed to investigate the influence of different analytical conditions on GSH-Px activity in chicken thighs from broilers that were fed different diets with different sources and concentrations of selenium. GSH-Px activity was evaluated six hours after slaughter and 120 days after frozen storage at -18 ºC. The different analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction medium, types of substrate (H2O2 (0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and different buffer concentrations (buffer 1, potassium phosphate 50 mM pH 7.0 + EDTA 1 mM + mercaptoethanol 1 mM, and buffer 2, tris-HCl 50 mM pH 7.6 + EDTA 1 mM + mercapthanol 5 mM). The results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at 22 ºC without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme and inhibited its activity. The enzyme presented higher affinity to hydrogen peroxide when compared to terc-butil peroxide, and the addition of a buffer containing mercaptoethanol did not increase GSH-Px enzymatic activity. The activity of GSH-Px was not influenced by the source and concentration of selenium in the diet either. The obtained results allowed the determination of the best temperature of contact between the enzyme and substrate (22 ºC), the optimum concentration, and the type of substrate and buffer to be used. This information is extremely useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

Correlation, by multivariate statistical analysis, between the scavenging capacity against reactive oxygen species and the bioactive compounds from frozen fruit pulps

The contents of total phenolic compounds (TPC), total flavonoids (TF), and ascorbic acid (AA) of 18 frozen fruit pulps and their scavenging capacities against peroxyl radical (ROO•), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) were determined. Principal Component Analysis (PCA) showed that TPC (total phenolic compounds) and AA (ascorbic acid) presented positive correlation with the scavenging capacity against ROO•, and TF (total flavonoids) showed positive correlation with the scavenging capacity against •OH and ROO• However, the scavenging capacity against H2O2 presented low correlation with TF (total flavonoids), TPC (total phenolic compounds), and AA (ascorbic acid). The Hierarchical Cluster Analysis (HCA) allowed the classification of the fruit pulps into three groups: one group was formed by the açai pulp with high TF, total flavonoids, content (134.02 mg CE/100 g pulp) and the highest scavenging capacity against ROO•, •OH and H2O2; the second group was formed by the acerola pulp with high TPC, total phenolic compounds, (658.40 mg GAE/100 g pulp) and AA , ascorbic acid, (506.27 mg/100 g pulp) contents; and the third group was formed by pineapple, cacao, caja, cashew-apple, coconut, cupuaçu, guava, orange, lemon, mango, passion fruit, watermelon, pitanga, tamarind, tangerine, and umbu pulps, which could not be separated considering only the contents of bioactive compounds and the scavenging properties.

Effect of modified atmosphere applied to minimally processed radicchio (Cichorium intybus L.) submitted to different sanitizing treatments

Stability of minimally processed radicchio (Cichorium intybus L.) was evaluated under modified atmosphere (2% O2, 5% CO2, and 93% N2) on 3, 5, 7 and 10 days of storage at 5°C. The samples were hygienized in sodium hypochlorite or hydrogen peroxide solutions to identify the most effective sanitizing solution to remove microorganisms. Microbiological analysis was conducted to identify the presence of coliforms at 35°C and 45°C, mesophilic microorganisms, and yeast and mold. Physicochemical analyses of mass loss, pH, soluble solids, and total acidity were conducted. The color measurements were performed using a Portable Colorimeter model CR-400. The antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic methods. The sensory evaluation was carried out using a hedonic scale to test overall acceptance of the samples during storage. The sodium hypochlorite (150 mg.L-1) solution provided greater safety to the final product. The values of pH ranged from 6.17 to 6.25, total acidity from 0.405 to 0.435%, soluble solids from 0.5 to 0.6 °Brix, mass loss from 1.7 to 7.2%, and chlorophyll from 1.068 to 0.854 mg/100g. The antioxidant activity of radicchio did not show significant changes during the first 3 days of storage. The overall acceptance of the sample stored in the sealed package without modified atmosphere was 70%, while the fresh sample was obtained 77% of approval. Although the samples packaged under modified atmosphere had a higher acceptance score, the samples in sealed packages had satisfactory results during the nine days of storage. The use of modified atmosphere, combined with cooling and good manufacturing practices, was sufficient to prolong the life of minimally processed radicchio, Folha Larga cultivar, for up to ten days of storage.

The kinetics and induced decomposition on the thermal decomposition of hydroperoxides /

This project is focussed on the thermsLl decomposition of t-butyl hydroperoxide and sec-butyl hydroperoxide at 120°C to 160°C in three alcohol solvents. These are methanol, ethajiol and isopropyl alcohol. The aim of the project was to examine the process of induced decomposition. Thermal decomposition of t-hutyl hydroperoxide and sec-butyl hydroperoxide indicate that these reactions have first-order kinetics with activation energies on the order of 20 to 28 K cal/mole, Styrene was used as a free radical trap to inhibit the induced decomposition. The results permitted calculation of how much induced decomposition occurred in its absence. The experimental resvilts indicate that the induced decomposition is important for t-butyl hydroperoxide in alcohol solvents, as shown by both the reaction rate suid product studies. But sec-butyl hydroperoxide results show that the concerted mechanism for the interaction of two sec-butylperoxy radicals occurs in addition to the induced decomposition. Di-sodium E.D,T.A. was added to reduce possible effects of trace transition metal ion .impurities. The result of this experiment were not as expected. The rate of hydroperoxide decomposition was about the same but was zero-order in hydroperoxide concentration.

Sample preparation and heavy metal determination by atomic spectrometry /

Microwave digestions of mercury in Standards Reference Material (SRM) coal samples with nitric acid and hydrogen peroxide in quartz vessels were compared with Teflon® vessel digestion by using flow injection cold vapor atomic absorption spectrometry. Teflon® vessels gave poor reproducibiUty and tended to deliver high values, while the digestion results from quartz vessel show good agreement with certificate values and better standard deviations. Trace level elements (Ag, Ba, Cd, Cr, Co, Cu, Fe, Mg, Mn, Mo, Pb, Sn, Ti, V and Zn) in used oil and residual oil samples were determined by inductively coupled plasma-optical emission spectrometry. Different microwave digestion programs were developed for each sample and most of the results are in good agreement with certified values. The disagreement with values for Ag was due to the precipitation of Ag in sample; while Sn, V and Zn values had good recoveries from the spike test, which suggests that these certified values might need to be reconsidered. Gold, silver, copper, cadmium, cobalt, nickel and zinc were determined by continuous hydride generation inductively coupled plasma-optical emission spectrometry. The performance of two sample introduction systems: MSIS™ and gas-liquid separator were compared. Under the respective optimum conditions, MSIS^"^ showed better sensitivity and lower detection limits for Ag, Cd, Cu, Co and similar values for Au, Ni and Zn to those for the gas-liquid separator.

A study of thermal decompositions of an allylic hydroperoxide /|nby Thomas A. McCarrick. -- 260 St. Catharines [Ont.] : Dept. of Chemistry, Brock University,

The thermal decomposition of 2,3-di~ethy l - J-hydr operox y- 1 - butene , p r epared f rol") singl e t oxygen, has been studied i n three solvents over the tempe r a ture r ange from 1500e to l o00e and t!1e i 111 t ial ~oncentrfttl nn r Ange from O. 01 M to 0.2 M. Analys i s of the kine tic data ind ica te s i nduced homolysis as the n ost probRble mode of d e composition, g iving rise to a 3/2 f S order dependence upon hy d.roperoxide concent :r8.tl on . Experimental activation e nergies for the decomposition were f ound to be between 29.5 kcsl./raole and 30.0 k cal./mole .• \,iith log A factors between 11 . 3 and 12.3. Product studies were conducted in R variety of solvents a s well as in the pr esence of a variety of free r adical initiators . Investigation of the kinetic ch a in length indicated a chain length of about fifty. A degenerat i ve chain branching mechanism 1s proposed which predicts the multi t ude of products which Rre observed e xperimentally as well as giving activation energies and log A factors si~il a r to those found experimentally .

Factors affecting DNA uptake by mammalian cells

The ability to introduce DNA and express custom DNA sequences in bacteria opened the door for improvements in a large number of fields including agriculture, pharmacology, medicine, nutrition, etc. The ability to introduce foreign DNA sequences into mammalian cells in an efficient manner would have a large impact on therapeutic applications especially gene therapy. The methods in use today suffer from low efficiencies and sometimes toxicity. In this work a number of factors were evaluated for their effect onONA uptake efficiency. The factors studied included exposure to sublethal concentration of hydrogen peroxide which have been show to lead to destabilisation ofthe lysosomes. These exposures have proven to be very toxic to cells when combined with either the calcium phosphate or the lipofectAMINE® transfection methods. Another factor evaluated was exposure to Electro-Magnetic Fields (EMF). This was fuelled by the fact that EMF have been shown to mediate a number of effects on cell structure and/or physiology. EMF exposure by itself was not sufficient to induce the cells to pick up the DNA, therefore its effect on calcium phosphate and lipofectAMINE® was tested. Although some positive results were obtained, the variability of these results exceeded by far any observed enhancements which discouraged any further work on EMF. Also tested was the possible effect the presence of the cytomegalovirus (CMV) sequence might have on DNA uptake (based on previous results in this lab). It was found that the presence ofCMV in the DNA sequence does not enhance uptake or slow down degradation of the internalised DNA. The final factor tested was the effect of basic amino acids on transfection efficiency. It was found that arginine can enhance DNA uptake by about 170% v/ith calcium phosphate and about 200% with LipofectAMINE®. A model was proposed to explain the effect of arginine as well as the lack of effect from other amino acids.

Investigations into the determinations of hydride-forming elements

Analytical methods for the determination of trace amounts of germanium, tin and arsenic were established using hydride generation coupled with direct current plasma atomic emission spectrometry. A continuous gas flowing batch system for the hydride generation was investigated and was applied to the determination of germanium(Ge), tin(Sn), antimony(Sb) and lead(Pb) (Preliminary results suggest that it is also applicable to arsenic)As) ). With this system, the reproducibility of signals was improved and the determination was speeded up, compared with the conventional batch type hydride generation system. Each determination was complete within one minute. Interferences from a number of transition metal ions, especially from Pd(II), Pt(IV), Ni(II), Cu(II), Co(II), and Fe(II, III), have proven to be very serious under normal conditions, in the determination of germanium, tin, and arsenic. These interference effects were eliminated or significantly reduced in the presence of L-cystine or L-cysteine. Thus, a 10-1000 fold excess of Ni(II), Cu(II), Co(II), Fe(II), Pt(IV), Pd(II), etc. can be tolerated without interference, In the presence of L-cystine or L-cysteine, compared with absence of interference reducing agent. The methods for the determination of Ge, Sn, and As were examined by the analyses of standard reference materials. Interference effects from the sample matrix, for example, in transition metal-rich samples, copper, iron and steel, were eliminated by L-cystine (for As and Sn) and by LI cysteine (for Ge). The analysis of a number of standard reference materials gave excellent results of As and Sn contents in agreement with the certified values, showing there was no systematic interference. The detection limits for both germanium and tin were 20 pg ml- I . Preliminary studies were carried out for the determination of antimony and lead. Antimony was found to react with NaBH4, remaInIng from the previous determinations, giving an analytical signal. A reversed injection manner, i.e., injection of the NaBH4 solution prior to the analyte solution was used to avoid uncertainty caused by residual NaBH4 present and to ensure that an excess of NaB H4 was available. A solution of 0.4% L-cysteine was found to reduce the interference from selected transition metal ions, Co(II), Cu(II), Ni(II) and Pt(IV). Hydrochloric acid - hydrogen peroxide, nitric acid - ammonium persulphate, and potassium dichromate malic acid reaction systems for lead hydride generation were compared. The potassium dichromate - malic acid reaction medium proved to be the best with respect to reproducibility and minimal interference. Cu(II), Ni(II), and Fe(II) caused strong interference In lead determinations, which was not reduced by L-cysteine or Lcystine. Sodium citrate, ascorbic acid, dithizone, thiosemicarbazide and penicillamine reduced interferences to some extent. Further interference reduction studies were carried out uSIng a number of amino acids, glycine, alanine, valine, leucine and histidine, as possible interference reducing agents in the determination of germanium. From glycine, alanine, valine to leucine, the interference reduction effect in germanium determinations decreased. Histidine II was found to be very promising In the reduction of interference. In fact, histidine proved more efficient than L-cystine and L-cysteine In the reduction of interference from Ni(II) in the determination of germanium. Signal enhancement by easily ionized elements (EIEs), usually regarded as an interference effect in analysis by DCP-AES, was studied and successfully applied to advantage in improving the sensitivity and detection limit in the determination of As, Ge, Sn, Sb, and Pb. The effect of alkali and alkaline-earth elements on the determination of the above five hydride forming elements was studied. With the appropriate EIE, a signal enhancement of 40-115% was achieved. Linear calibration and good reproducibility were also obtained in the presence of EIEs. III

A Survey of hMLH1 mRNA Splicing Profiles in Human Cell Lines: Comparing Primary Cultured Fibroblasts Before and After Oxidative Stress and Transiently versus Stably Demethylated Colon Cancer Derived Cell Lines

Most human genes undergo alternative splicing and loss of splicing fidelity is associated with disease. Epigenetic silencing of hMLH 1 via promoter cytosine methylation is causally linked to a subset of sporadic non-polyposis colon cancer and is reversible by 5-aza-2' -deoxycytidine treatment. Here I investigated changes in hMLHI mRNA splicing profiles in normal fibroblasts and colon cancer-derived human cell lines. I established the types and frequencies of hMLHI mRNA transcripts generated under baseline conditions, after hydrogen peroxide induced oxidative stress, and in acutely 5-aza-2' -deoxycytidine-treated and stably derepressed cancer cell lines. I found that hMLHI is extensively spliced under all conditions including baseline (50% splice variants), the splice variant distribution changes in response to oxidative stress, and certain splice variants are sensitive to 5- aza-2' -deoxycytidine treatment: Splice variant diversity and frequency of exon 17 skipping correlates with the level of hMLHI promoter methylation suggesting a link between promoter methylation and mRNA splicing.

Investigation into the relationships between molecular chaperones, mitochondrial antioxidant enzymes, and endothermic vertebrate longevity

The maximum lifespan (MLSP) of endothermic vertebrates can range from as little as a year to over two centuries, yet the underlying phenotype of aging is very similar amongst this group of organisms. One organelle that may be important in the phenotype of aging is the mitochondrion. When damaged, this organelle is thought to contribute to many of the neurodegenerative diseases of aging. For this thesis, mitochondria from brain tissues of 7 mammalian and 2 avian species were isolated to assess whether the antioxidant glutathione system and major molecular chaperone, HSP60, is correlated to species MLSP. Furthermore, HSP60, and the major endoplasmic reticulum chaperone, GRP78, were measured under basal conditions, and following the introduction of an oxidative stress (hydrogen peroxide) in cultured mammalian myoblasts from 10 different species. My results indicate that the enzymes involved in the glutathione defense system are not correlated to species MLSP in brain mitochondria; however HSP60 levels are indeed higher in the longer-lived species. HSP60 levels are also higher at the basal level in cultured mammalian myoblasts and after 1 hour of hydrogen peroxide exposure. GRP78 induction is not correlated to species MLSP at the basal level or following hydrogen peroxide exposure. Therefore, these results suggest that HSP60 is a correlate of longevity in endothermic vertebrate species, but neither the glutathione antioxidant defense system, nor GRP78, correlates to species longevity.

Activités antidiabétiques et neuroprotectrices du jus de bleuet biostransformé

Le bleuet démontre un potentiel thérapeutique dans le traitement du cancer et des maladies cardiovasculaires et neurodégénératives. Ces effets bénéfiques sont attribuables aux composés phénoliques abondants dans le bleuet, tels que les anthocyanines et les flavonoïdes. La biotransformation du jus de bleuet avec les bactéries Serratia vaccinii augmente sa teneur en composés phénoliques et son activité anti-oxydante, et modifie ses activités physiologiques. L’objectif de la présente étude est d’évaluer l’activité neuroprotectrice et le potentiel antidiabétique du jus de bleuet biostranformé (BJ). Le BJ est étudié dans différents tests dont : 1) La protection des neurones (N2a) contre le stress oxydatif (SO) induit par le peroxyde d’hydrogène; 2) La stimulation de la prise de glucose par les cellules musculaires (C2C12) et adipeuses (3T3-L1); 3) L’activité anti-hyperglycémique chez les souris obèses diabétiques KKAy. En effet, tandis que le jus de bleuet normal n’a aucun effet, le BJ augmente l’activité des enzymes anti-oxydantes, comme la catalase et la SOD (Superoxide Dimutase) et protège les neurones contre les changements de la signalisation des MAPKs et contre la toxicité induite par le peroxyde d’hydrogène. Le BJ augmente aussi la prise de glucose de 48% dans les cellules C2C12 et de 142% dans les cellules 3T3-L1. Cette augmentation n’est pas expliquée par une augmentation du calcium cytosolique mais plutôt par une stimulation de la phosphorylation de l’AMPK. De plus, le BJ inhibe l’adipogenèse chez les 3T3-L1. Le BJ diminue également l’hyperglycémie chez les souris obèses diabétiques KKAy et protège les jeunes souris pré-diabétiques contre le développement de l’obésité et du diabète. L’activité anti-hyperglycémique du BJ pourrait impliquer les adipokines puisque le BJ augmente le niveau d’adiponectine chez les souris diabétiques. Le BJ représente ainsi une approche prometteuse pour le traitement du diabète et les maladies neurodégénératives et une source de nouveaux agents thérapeutiques contre ces maladies.

Deletion of the RNR4 gene causes hyperresistance to the carcinogen 4-NQO in the yeast model

La stabilité génomique, qui est essentielle à la vie, est possible grâce à la réplication et la réparation de l’ADN. Une des enzymes responsables de la réplication et de la réparation de l’ADN est la ribonucleotide reductase (RNR), qui est retrouvée chez la levure et chez l’humain. Cette enzyme catalyse la formation de déoxyribonucléotides et maintien le pool de dNTP requis pour la réparation et la réplication de l’ADN. L’enzyme RNR est un tétramère α2β2 constitué d’une grande (R1, α2) et d’une petite (R2, β2) sous-unité. Chez S. cerevisiae, les gènes RNR1 et RNR3 encodent la sous-unité α2 (R1). L’activité catalytique de RNR dépend d’une interaction avec le fer et de la formation d’un complexe entre R1 et R2. L’expression de toutes les sous-unités est inductible par les dommages causés à l’ADN. Dans cette étude, nous démontrons que des cellules qui n’expriment pas une des sous-unités, Rnr4, du complexe RNR sont sensibles à divers agents endommageant l’ADN, tels que le méthyl méthane sulfonate, la bléomycine, le péroxyde d’hydrogène et les rayons ultraviolets (UVC 254 nm). Au contraire, le mutant est résistant au 4-nitroquinoline-1- oxide (4-NQO), un composé qui engendre des lésions encombrantes. Par conséquent, le mutant rnr4Δ démontre une réduction marquée en mutations induites par le 4-NQO comparativement à la souche parentale. Nous voulions identifier la voie de réparation de l’ADN qui conférait cette résistance au 4-NQO ainsi que les protéines impliquées. Les voies BER, NER et MMR n’ont pas aboli la résistance au 4-NQO de la souche rnr4Δ. La protéine recombinante Rad51 ne joue pas un rôle critique dans la réparation de l’ADN et dans la résistance au 4-NQO. La délétion du gène REV3, qui encode une polymérase de contournement, impliquée dans la réparation post-réplication, a partiellement aboli la résistance au 4-NQO dans rnr4Δ. Ces résultats suggèrent que la polymérase Rev3 et possiblement d’autres polymérases translésion (Rev1, Rev7, Rad30) pourraient être impliquées dans la réparation de lésions encombrantes dans l’ADN dans des conditions de carence en dNTP. La réparation de l’ADN, un mécanisme complexe chez la levure, implique une vaste gamme de protéines, dont certaines encore inconnues. Nos résultats indiquent qu’il y aurait plus qu’une protéine impliquée dans la résistance au 4-NQO. Des investigations plus approfondies seront nécessaires afin de comprendre la recombinaison et la réparation post-réplication.

Modulation de la voie de signalisation de Gαq par l’hyperglycémie : mécanisme moléculaire

Les complications vasculaires telles que l’augmentation de la contractilité et la prolifération cellulaire sont les complications les plus communes observées dans le diabète et l’hyperglycémie chronique est un facteur important dans ces processus. La voie de signalisation de Gαq joue un rôle important dans la régulation du tonus vasculaire et l’altération de celle-ci peut contribuer aux complications vasculaires observées dans les cas de diabète et d’hyperglycémie. Il a été observé que les taux et l’activité des protéines kinase C (PKC) et du diacylglycérol (DAG) sont augmentés dans ces conditions. Cependant, aucune étude n’a démontré l’implication de Gαq/11 et des PLCβ, molécules de signalisation en amont de PKC/DAG. Plusieurs études révèlent que l’augmentation des taux et de l’activité des PKC et du DAG induite par l’hyperglycémie dans des cellules du muscle lisse vasculaire (CMLV) est attribuée à l’augmentation du stress oxydatif. De plus, les niveaux de certains peptides vasoactifs, tels que l’angiotensine II et l’endothéline-1, augmentés dans les conditions de diabète/d’hyperglycémie, peuvent contribuer à l’augmentation du stress oxydatif observée. Le travail présenté dans cette thèse avait pour but d’examiner les effets de l’hyperglycémie sur les niveaux d’expression protéique de Gαq/11 et de ses molécules associées, ainsi que d’étudier le mécanisme moléculaire par lequel l’hyperglycémie module la voie de signalisation de Gαq dans les CMLV. Dans la première étude, nous avons examiné si l’hyperglycémie pouvait moduler l’expression des protéines Gαq, Gα11, PLCβ1 et PLCβ2. Le prétraitement des CMLV A10 avec 26 mM de glucose durant 72 heures augmente l’expression des protéines Gαq, Gα11, PLCβ-1 et PLCβ-2 en comparaison avec les CMLV témoins. Le traitement avec des antagonistes aux récepteurs AT1 de l’Ang II, et ETA/ETB de l’ET-1, atténue la hausse de Gαq, de Gα11, de PLCβ1 et de PLCβ2 induite par l’hyperglycémie. De plus, la formation d’IP3 stimulée par l’ET-1 était plus élevée dans les CMLV exposée à 26 mM de glucose. Le traitement des CMLV A10 avec l’Ang II et l’ET-1 augmente également les niveaux d’expression des protéines Gα q/11 et PLCβ. Cette augmentation de l’expression est restaurée au niveau des CMLV témoins par les antagonistes des récepteurs AT1, ETA et ETB. Ces résultats suggèrent que l’augmentation de l’expression des protéines Gαq/11 et PLCβ dans les CMLV induite par l’hyperglycémie est attribuée à l’activation des récepteurs AT1, ETA et ETB. Dans la seconde étude, nous avons examiné l’implication du stress oxydatif dans l’augmentation des niveaux d’expression des protéines Gαq/11 et PLCβ et de leur signalisation induite par l’hyperglycémie. Nous avons également déterminé le mécanisme responsable de l’augmentation du stress oxydatif induite par l’hyperglycémie. L’augmentation de l’expression des protéines Gαq/11 et PLCβ des CMLV A10 exposées à 26 mM de glucose est revenue au niveau basal après un traitement avec l’antioxydant diphenyleneiodonium (DPI), et la catalase, un chélateur du peroxyde d’hydrogène, mais pas par le 111Mn-tetralis(benzoic acid porphyrin) (MnTBAP) ni par l’acide urique, des chélateurs du peroxynitrite. De plus, l’augmentation de la formation d’IP3 stimulée par l’ET-1 dans les CMLV exposées à 26 mM de glucose est revenue au niveau basal après un traitement avec le DPI et la catalase. Ces résultats suggèrent que l’augmentation du stress oxydatif induite par l’hyperglycémie contribue à l’augmentation de l’expression des protéines Gαq/11 et les molécules associées à la voie de signalisation de Gq. De plus, l’augmentation de la production d’anion superoxyde (O2-), de l’activité de la NADPH oxydase et de l’expression des protéines p22(phox) et p47(phox) induite par l’hyperglycémie est revenue à un niveau basal après un traitement avec les antagonistes des récepteurs AT1, ETA et ETB. Ces résultats suggèrent que l’hyperglycémie augmente les niveaux endogènes de l’Ang II et de l’ET-1, ce qui augmente le stress oxydatif par la formation d’O2- et de H2O2 et peut contribuer à l’augmentation des niveaux de Gq/11α et de leurs molécules de signalisation. Puisqu’il a été observé que l’hyperglycémie transactive les récepteurs aux facteurs de croissance tels que le récepteur au facteur de croissance épidermique (EGF-R) et le récepteur au facteur de croissance dérivé des plaquettes (PDGF-R), nous avons entrepris d’examiner, dans la troisième étude, l’implication d’EGF-R et de PDGF-R dans l’augmentation des niveaux de Gαq/11, de PLCβ et de leur signalisation induite par l’hyperglycémie. L’augmentation des niveaux d’expression des protéines Gαq, Gα11, PLCβ-1 et PLCβ-2 induite par l’hyperglycémie est revenue au niveau basal après un traitement avec les inhibiteurs d’EGF-R (AG1478) et de PDGF-R (AG1295) et par l’inhibiteur de c-Src, PP2. L’augmentation de la phosphorylation d’EGF-R et de PDGF-R induite par l’hyperglycémie a été abolie par AG1478, AG1295 et PP2. De plus, l’augmentation des niveaux de Gαq/11, et de PLCβ induite par l’hyperglycémie est atténuée par l’inhibiteur des MAPK, le PD98059, et par l’inhibiteur d’AKT, le wortmannin. L’augmentation de la phosphorylation d’ERK et d’AKT était également atténuée par AG1478 et AG1295. Ces résultats suggèrent que la transactivation des récepteurs aux facteurs de croissance induite par c-Src peut contribuer à l’augmentation des niveaux de Gα q/11/PLC et de leur signalisation par la voie des MAPK/PI3K. En conclusion, les études présentées dans cette thèse indiquent que l’hyperglycémie augmente les niveaux de Gαq/11 et de PLCβ. Nous avons émis des évidences qui démontrent que l’augmentation endogène de l’Ang II et de l’ET-1 par l’hyperglycémie peut contribuer à l’augmentation de la production d’O2- et de H2O2 résultant ainsi en une augmentation du stress oxydatif qui pourrait être responsable de l’augmentation de Gαq/11/PLC et de leur signalisation dans les conditions d’hyperglycémie. Finalement, nous avons démontré que la transactivation des récepteurs aux facteurs de croissance induite par l’hyperglycémie peut être responsable de l’augmentation de Gαq/11/PLC et les molécules associées à la voie de signalisation de Gq dans les cas de diabète et d’hyperglycémie.