Investigations towards the synthesis of isotope labelled analogues of tocopherols and tocotrienols /

Vitamin E is considered as the most effective lipophilic chain breaking antioxidant. a-Tocopherol and its analogues have been studied thoroughly with regards to its biokinetics and bioavailabily. Deuterated tocopherols have been synthesized and utilized in such studies. Tocotrienols are arousing more and more interest because of their high efficiency as antioxidants. However, to date, there is no effective synthetic method reported for deuterated tocotrienols. This thesis is focused on the investigation of the synthetic methods of deuterated tocotrienols and their analogues: 5-trideuteromethyl-a-tocotrienol, 5- trideuteromethyl-p-tocotrienol, tocotrienol acetate, silyl tocotrienol ether, etc. Several synthetic procedures for the preparation of poly-deuterated tocopherols are known. Mainly the deuterium is introduced by use of labelled formaldehyde and deuterated hydrogen chloride under Lewis acid catalysis. However, these methods are not effective in tocotrienols due to exchange of protons for deuterium at other sites under the acidic conditions. We developed several different approaches to generate polydeuterated tocotrienols by using both morpholinomethylation followed by reduction with NaCNBDs as deuterated reducing reagents and transmetalation strategy. The 5-trideuteromethyl-a-tocotrienol was finally obtained in a satisfactory yield of 60%. In addition, this thesis also discussed the study of structural comparison and the chemical property difference of tocopherols and tocotrienols, which provides hints to explain the reactivity difference of them towards oxidation at the C3-C4 positions.Furthermore, the methodology of halogenation and dehydrohalogenation of tocotrienol was explored to prepare a hexaene tocotrienol derivative as a florescent reporter of tocopherol.

NMR studies of the exchange reactions of CH3CN.BX3 with excess CH3CN /|nJoseph Fogelman. -- 260 St. Catharines, Ont. : [s. n.],

Exchange reactions between molecular complexes and excess acid or base are well known and have been extensively surveyed in the literature(l). Since the exchange mechanism will, in some way involve the breaking of the labile donor-acceptor bond, it follows that a discussion of the factors relating to bonding in molecular complexes will be relevant. In general, a strong Lewis base and a strong Lewis acid form a stable adduct provided that certain stereochemical requirements are met. A strong Lewis base has the following characteristics (1),(2) (i) high electron density at the donor site. (ii) a non-bonded electron pair which has a low ionization potential (iii) electron donating substituents at the donor atom site. (iv) facile approach of the site of the Lewis base to the acceptor site as dictated by the steric hindrance of the substituents. Examples of typical Lewis bases are ethers, nitriles, ketones, alcohols, amines and phosphines. For a strong Lewis acid, the following properties are important:( i) low electron density at the acceptor site. (ii) electron withdrawing substituents. (iii) substituents which do not interfere with the close approach of the Lewis base. (iv) availability of a vacant orbital capable of accepting the lone electron pair of the donor atom. Examples of Lewis acids are the group III and IV halides such (M=B, AI, Ga, In) and MX4 - (M=Si, Ge, Sn, Pb). The relative bond strengths of molecular complexes have been investigated by:- (i) (ii) (iii) (iv) (v] (vi) dipole moment measurements (3). shifts of the carbonyl peaks in the IIIR. (4) ,(5), (6) .. NMR chemical shift data (4),(7),(8),(9). D.V. and visible spectrophotometric shifts (10),(11). equilibrium constant data (12), (13). heats of dissociation and heats of reactions (l~), (16), (17), (18), (19). Many experiments have bben carried out on boron trihalides in order to determine their relative acid strengths. Using pyridine, nitrobenzene, acetonitrile and trimethylamine as reference Lewis bases, it was found that the acid strength varied in order:RBx3 > BC1 3 >BF 3 • For the acetonitrile-boron trihalide and trimethylamine boron trihalide complexes in nitrobenzene, an-NMR study (7) showed that the shift to lower field was. greatest for the BB~3 adduct ~n~ smallest for the BF 3 which is in agreement with the acid strengths. If electronegativities of the substituents were the only important effect, and since c~ Br ,one would expect the electron density at the boron nucleus to vary as BF3acetate adducts and the shift in the carbonyl I.R. stretch, Drago (22) et al have proposed that the angular di~tortion of the X-B-X bonds from sp2 (12 ) to sp3 (10 hybridization is proportional to the amount of charge transferred, i.e. to the nature of the base, and they have rejected the earlier concept of reorganization energy in explaining the formation of the adduct bond (19).

The determination of sinigrin in Brassica, and investigations into the use of allyl-isothiocyanate as a nematicide

The goal of this thesis was to study factors related to the development of Brassica juncea as a sustainable nematicide. Brassica juncea is characterized by the glycoside (glucosinolate) sinigrin. Various methods were developed for the determination of sinigrin in Brassica juncea tissue extracts. Sinigrin concentrations in plant tissues at various stages of growth were monitored. Sinigrin enzymatically breaks down into allylisothiocyanate (AITC). AITC is unstable in aqueous solution and degradation was studied in water and in soil. Finally, the toxicity of AITC against the root-lesion nematode (Pratylenchus penetrans) was determined. A method was developed to extract sinigrin from whole Brassica j uncea tissues. The optimal time of extraction wi th boiling phosphate buffer (0.7mM, pH=6.38) and methanol/water (70:30 v/v) solutions were both 25 minutes. Methanol/water extracted 13% greater amount of sinigrin than phosphate buffer solution. Degradation of sinigrin in boiling phosphate buffer solution (0.13%/minute) was similar to the loss of sinigrin during the extraction procedure. The loss of sinigrin from boiling methanol/water was estimated to be O.Ol%/minute. Brassica juncea extract clean up was accomplished by an ion-pair solid phase extraction (SPE) method. The recovery of sinigrin was 92.6% and coextractive impurities were not detected in the cleaned up extract. Several high performance liquid chromatography (HPLC) methods were developed for the determination of sinigrin. All the developed methods employed an isocratic mobile phase system wi th a low concentration of phosphate buffer solution, ammonium acetate solution or an ion-pair reagent solution. A step gradient system was also developed. The method involved preconditioning the analytical column with phosphate buffer solution and then switching the mobile phase to 100% water after sample injection.Sinigrin and benzyl-glucosinolate were both studied by HPLC particle beam negative chemical ionization mass spectrometry (HPLCPB- NCI-MS). Comparison of the mass spectra revealed the presence of fragments arising from the ~hioglucose moiety and glucosinolate side-chain. Variation in the slnlgrin concentration within Brassica juncea plants was studied (Domo and Cutlass cuItivars). The sinigrin concentration in the top three leaves was studied during growth of each cultivar. For Cutlass, the minimum (200~100~g/g) and maximum (1300~200~g/g) concentrations were observed at the third and seventh week after planting, respectively. For Domo, the minimum (190~70~g/g) and maximum (1100~400~g/g) concentrations were observed at the fourth and eighth week after planting, respectively. The highest sinigrin concentration was observed in flower tissues 2050±90~g/g and 2300±100~g/g for Cutlass and Domo cultivars, respectively. Physical properties of AITC were studied. The solubility of AITC in water was determined to be approximately 1290~g/ml at 24°C. An HPLC method was developed for the separation of degradation compounds from aqueous AITC sample solutions. Some of the degradation compounds identified have not been reported in the literature: allyl-thiourea, allyl-thiocyanate and diallyl-sulfide. In water, AITC degradation to' diallyl-thiourea was favored at basic pH (9.07) and degradation to diallyl-sulfide was favored at acidic pH (4 . 97). It wap necessary to amend the aqueous AITC sample solution with acetonitrile ?efore injection into the HPLC system. The acetonitrile amendment considerably improved AITC recovery and the reproducibility of the results. The half-life of aqueous AITC degradation at room temperature did not follow first-order kinetics. Beginning with a 1084~g/ml solution, the half-life was 633 hours. Wi th an ini tial AITC concentration of 335~g/ml the half-life was 865 hours. At 35°C the half-life AITC was 76+4 hours essentially independent of the iiisolution pH over the range of pH=4.97 to 9.07 (1000~g/ml). AITC degradation was also studied in soil at 35°C; after 24 hours approximately 75% of the initial AITC addition was unrecoverable by water extraction. The ECso of aqueous AITC against the root-lesion nematode (Pratylenchus penetrans) was determined to be approximately 20~g/ml at one hour exposure of the nematode to the test solution. The toxicological study was also performed with a myrosinase treated Brassica juncea extract. Myrosinase treatment of the Brassica juncea extract gave nearly quantitative conversion of sinigrin into AITC. The myrosinase treated extract was of the same efficacy as an aqueous AITC solution of equivalent concentration. The work of this thesis was focused upon understanding parameters relevant to the development of Brassica juncea as a sustainable nematicide. The broad range of experiments were undertaken in support of a research priority at Agriculture and Agri-Food Canada.

Total synthesis of (S)-4-hydroxy-[a]-Lapachone

(S)-4-Hydroxy-a-lapachone has been prepared for the first time. The commercially available compound 2-acetyl-1-naphthol was used as the starting material. The synthesis involved methylation, followed by Baeyer-Villiger oxidation, and hydrolysis of the acetate to give 1-methoxy-2-naphthol. After protecting of the hydroxyl group, t-BuLi was used to form 3-(3',3'-dimethyl-acryloyl)-1- meth oxy-2- (meth oxymethoxy)-naphthalen e. eycl izationand oxidation then gave 4-keto-a-lapachone. Finally enzymic biotransformation by Mortierella isabellina ATCC 42613 was used to yield the target compound. The enantiomeric excess of the product was determined to be ~98% by using 1H NMR chiral shift analysis. The overall yield is 80/0. The biological activity of (S)-4-hydroxy-alapachone and its acetate are under investigation.

Investigations on the production of long chain fatty acids in Choanephora cucurbitarum (Berk.

The fatty acid composition of the total cellular lipids of Choanephora cucurbitarum incubated for 96 hrs on either glucose-ammonium sulfate or malt-weast extract media was determined. The major fatty acids were palmitic, palmitoleic, stearic and linoleic acids. The saturated fatty acid possessing the longest acyl chain was stearate (C 18:0). The presence of glutamic acid (2.0 x 10-1% or 1.36 x la-2M) in either of the above growth media resulted in increase in percent of 1f-linolenic acid, decrease in percent of linoleic ~iCid and appearance of a new series of fatty acid> C ~8 e.g. C ",,,,'V' C2k:O, C26,O. The addition of glutamic acid had no effect on the lipid yield but slightly decreased the degree of unsaturation. Compounds which duplicated the effect of glutamic acid were acetate, malate, citrate, succinate, 0( -ketoglutarate, prOline, -y -aminobutyric acid and glucose (3%) but not aspartic acid or alanine. ~o correlation was found between glutamic acid pool concentration and the presence in the growth medium of those compounds which stimulate long chain fatty acid production. Four hours of incubation with 27 JJ 1-1 glutamate supported the production of long chain fatty acids. This stimulation is inhibited if 272 .u M isophthalic acid is added with 27 AJ M glutamate. But, long chain fatty acids were detected when 27 JJ M eX -ketoglutarate is also present in the incubation mixture. Five hours of incubation with 100 ,Mg/ml of cycloheximide resulted in over 9CY/o inhibition of cytoplasmic :protein synthesise Glutamate (27 .uM) enhanced the synthesis of long chain fatty acids under these conditions. These findings are discussed in an attempt to provide a plausible explanation COmmon to compounds that support the production of long chain fatty acids.

Honey proteins and their interaction with polyphenols

This project aimed to determine the protein prof i les and concent rat ion in honeys, ef fect of storage condi t ions on the protein content and the interact ion between proteins and polyphenols. Thi r teen honeys f rom di f ferent botanical or igins were analyzed for thei r protein prof i les using SDS-PAGE, protein concent rat ion and phenol ic content , using the Pierce Protein Assay and Fol in-Ciocal teau methods, respectively. Protein-polyphenol interact ions were analyzed by a combinat ion of the ext ract ion of honeys wi th solvents of di f ferent polar i t ies fol lowed by LCjMS analysis of the obtained f ract ions. Results demonst rated a di f ferent protein content in the tested honeys, wi th buckwheat honey possessing the highest protein concent rat ion. We have shown that the reduct ion of proteins dur ing honey storage was caused, partially, by the protein complexat ion wi th phenolics. The LCjMS analysis of the peak elut ing at retent ion t ime of 10 to 14 min demonst rated that these phenolics included f lavonoids such as Pinobanksin, Pinobanksin acetate, Apigenin, Kaemferol and Myricetin and also cinnamic acid.

Icewine fermentation by Saccharomyces cervisiae : fundamental stress responses and comparative fermentation dynamics

Please consult the paper edition of this thesis to read. It is available on the 5th Floor of the Library at Call Number: Z 9999.5 B63 P54 2007

Functional genomics of BAHD acyltransferases in different varieties of the wine grape, Vilis vinifera

The characteristic "foxy" aroma of Vilis labrusca Concord grapes is due in large part to methyl anthranilate, a volatile ester formed by the enzyme anthraniloyl- CoA:methanol anthraniloyltransferase (VIAMAT) of the superfamily of BARD acyltransferases. The publication of the genome ofthe closely related wine grape Vilis vinifera, which does not accumulate methyl anthranilate, permitted the searching for any putative VlAU4T-like genes, with the result of 5 highly homologous candidates being found, with one candidate sharing 95% identity to VlAU4T. Probing the gene expression of 18 different cultivars of V. vinifora ripe berries by RT -PCR showed that many varieties do indeed express VlAU4T-like genes. Subsequent cloning of the full-length open reading frame of one of these genes from eDNA prepared from the cultivar Sauvignon Blanc permitted preliminary biochemical characterization of the enzyme after heterologous expression in E. coli. It was determined that this alcohol acyltransferase (named VvsbAATl) catalyzes the formation of cis-3-hexenyl acetate, a "green-leaf' volatile. Although the cloned gene from Sauvignon Blanc had 95% identity at the amino acid level to VIAMAT, it displayed an altered substrate specificity and expression pattern. These results highlight the difficulty in predicting substrate specificity and function of enzymes through the basis of sequence homology, which is a common finding in the study of BARD acyltransferases. Also, the determination of function of VvsbAATl and other BARD acyltransferases in V. vinifera could be used as a genetic marker for certain aroma characteristics in grape breeding programs.

Hyperosmotic Stress and the Impact on Metabolite Formation and Redox Balance in Saccharomyces cerevisiae and Saccharomyces bayanus strains

Wine produced using an appassimento-type process represents a new and exciting innovation for the Ontario wine industry. This process involves drying grapes that have already been picked from the vine, which increases the sugar content due to dehydration and induces a variety of changes both within and on the surface of the grapes. Increasing sugar contents in musts subject wine yeast to conditions of high osmolarity during alcoholic fermentations. Under these conditions, yeast growth can be inhibited, target alcohol levels may not be attained and metabolic by-products of the hyperosmotic stress response, including glycerol and acetic acid, may impact wine composition. The further metabolism of acetic acid to acetylCoA by yeast facilitates the synthesis of ethyl acetate, a volatile compound that can also impact wine quality if present in sufficiently high concentrations. The first objective of this project was to understand the effect of yeast strain and sugar concentration on fermentation kinetics and metabolite formation, notably acetic acid and ethyl acetate, during fermentation in appassimento-type must. Our working hypotheses were that (1) the natural isolate Saccharomyces bayanus would produce less acetic acid and ethyl acetate compared to Saccharomyces cerevisiae strain EC-1118 fermenting the high and low sugar juices; (2) the wine produced using the appassimento process would contain higher levels of acetic acid and lower levels of ethyl acetate compared to table wine; (3) and the strains would be similar in the kinetic behavior of their fermentation performances in the high sugar must. This study determined that the S. bayanus strain produced significantly less acetic acid and ethyl acetate in the appassimento wine and table wine fermentations. Differences in acetic acid and ethyl acetate production were also observed within strains fermenting the two sugar conditions. Acetic acid production was higher in table wine fermented by S. bayanus as no acetic acid was produced in appassimento-style wine, and 1.4-times higher in appassimento wine fermented by EC-1118 over that found in table wine. Ethyl acetate production was 27.6-times higher in table wine fermented by S. bayanus, and 5.2-times higher by EC-1118, compared to that in appassimento wine. Sugar utilization and ethanol production were comparable between strains as no significant differences were determined. The second objective of this project was to bring a method in-house for measuring the concentration of pyridine nucleotides, NAD+, NADP+, NADH and NADPH, in yeast cytosolic extract. Development of this method is of applicative interest for our lab group as it will enable the redox balance of the NAD+/ NADH and NADP+/ NADPH systems to be assessed during high sugar fermentations to determine their respective roles as metabolic triggers for acetic acid production. Two methods were evaluated in this study including a UV-endpoint method using a set of enzymatic assay protocols outlined in Bergmeyer (1974) and a colorimetric enzyme cycling method developed by Sigma-Aldrich® using commercial kits. The former was determined to be limited by its low sensitivity following application to yeast extract and subsequent coenzyme analyses, while the latter was shown to exhibit greater sensitivity. The results obtained from the kits indicated high linearity, accuracy and precision of the analytical method for measuring NADH and NADPH, and that it was sensitive enough to measure the low coenzyme concentrations present in yeast extract samples. NADtotal and NADPtotal concentrations were determined to be above the lower limit of quantification and within the range of the respective calibration curves, making this method suitable for our research purposes.

The positive impact of red palm oil in school meals on vitamin A status: study in Burkina Faso

Affiliation: Augustin Zeba & Hélène Delisle : Département de nutrition, Faculté de médecine, Université de Montréal

Les transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 contribuent au maintien de l’homéostasie du cholestérol intratesticulaire

Le testicule assure la production des spermatozoïdes et la sécrétion de la testostérone. Chaque fonction est assumée par un compartiment cellulaire distinct: l’épithélium séminifère et le tissu interstitiel. Le cholestérol, présent dans les deux compartiments, est un composé indispensable aux membranes cellulaires et un précurseur essentiel de la testostérone. Dans le compartiment interstitiel, environ 40 % du cholestérol utilisé pour la production hormonale est importé du sang à partir des lipoprotéines HDL et/ou LDL. Dans l’épithélium séminifère, la cellule de Sertoli assure le contrôle et le maintien de la spermatogenèse. Elle a la capacité de synthétiser du cholestérol à partir de l’acétate in vitro, néanmoins, il n’y a pas d’évidence qu’elle le fait in vivo. De plus il existe, au niveau des tubules séminifères, une barrière hémato-testiculaire qui empêche le libre passage de plusieurs composés sanguins, y compris le cholestérol. Nous avons testé l’hypothèse qu’il existe des moyens d’importation du cholestérol sanguin, mais aussi l’exportation du cholestérol intra-tissulaire, qui contourneraient cette barrière et qui contribueraient au maintien du taux intratubulaire du cholestérol compatible avec le bon déroulement de la spermatogenèse. Nous avons comparé les taux de variation de l’expression de l’ARNm et de la protéine des transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 aux taux de variation du cholestérol libre et estérifié au cours de la spermatogenèse chez les souris normales durant le développement postnatal. Afin de mieux apprécier le niveau d’implication de chacun de ces récepteurs, nous avons examiné comment la suppression du gène d’une enzyme comme la lypase hormono-sensible (HSL) ou de celui d’un transporteur de cholestérol comme SR-BI, CD36 ou NPC1 était compensée et comment cette suppression affectait le taux de cholestérol libre et estérifié dans chacun des deux compartiments cellulaires du testicule. Nous avons dans un premier temps mis au point une nouvelle technique d’isolation des testicules en fraction enrichie en tissu interstitiel (ITf) et en tubules séminifères (STf) qui a l’avantage de mieux préserver l’intégrité des formes phosphorylées et glycosylées des protéines comparée aux techniques préexistantes. Les résultats de nos analyses ont montré que l’expression de SR-BI et CD36 étaient maximales dans les ITf au moment où les souris ont complété leur maturité sexuelle et où le niveau de synthèse de la testostérone était maximal. Dans les tubules séminifères, l’expression maximale de SR-BI et le taux le plus élevé de cholestérol estérifié étaient mesurés de façon concomitante à 35 jours après la naissance, au moment où la première vague de l’activité spermatogénétique était complétée. L’expression de l’ABCA1 était maximale au moment où le taux de cholestérol était élevé et minimale au moment où le taux de cholestérol était le plus bas, alors que le niveau d’expression de CD36 était maximal chez l’adulte au moment où le taux de spermiation était le plus élevé. L’expression de SR-BII variait peu dans les deux compartiments cellulaires durant le développement. La suppression génétique de la HSL et de NPC1, qui cause une infertilité chez les souris mâles, était accompagnée d’une accumulation de cholestérol libre et estérifié dans les tubules séminifères. Par contre, la suppression génétique de SR-BI et CD36, qui ne causent pas d’infertilité chez les souris mâles était sans impact significatif sur le taux de cholestérol intratubulaire. Nous avons montré que l’invalidation génétique d’un transporteur sélectif ou d’une enzyme du métabolisme du cholestérol était accompagnée d’un ensemble de mécanismes de compensation visant à maintenir le taux de cholestérol libre aux niveaux semblables à ceux mesurés dans les fractions tissulaires de souris normales. Ensemble, nos résultats ont montré que l’expression des transporteurs sélectifs de cholestérol SR-BI, SR-BII, CD36 et ABCA1 variait en fonction de la spermatogenèse et du taux intratesticulaire du cholestérol suggérant leur contribution au maintien de l’homéostasie du cholestérol intratesticulaire.

Huile de palme rouge au Burkina Faso: Qualité et consommation par les femmes de la zone de production et impact sur leur statut en vitamine A

Dans le cadre de la recherche d’appui à la phase III du projet huile de palme rouge (HPR) au Burkina Faso, une étude de base a porté sur 150 femmes de la zone de production échantillonnées par la méthode aléatoire géographique. Leur statut en vitamine A (VA) a été évalué par HPLC et leurs apports alimentaires par un questionnaire de fréquence de consommation. Les connaissances, les perceptions, les habitudes d’utilisation et de consommation de l’HPR ont été explorées par un questionnaire pré-testé administré au domicile des participantes. Une étude comparative sur la qualité nutritionnelle, physico-chimique, microbiologique et sensorielle de 13 échantillons d’HPR de différentes provenances a été également réalisée. La prévalence de faibles rétinolémies était de 10,7% et les apports en VA provenaient à 90% des aliments d’origine végétale. Seules 5,9% des femmes productrices présentaient une faible rétinolémie, comparativement à 20,8% des femmes non-productrices d’HPR. Les échantillons d’HPR présentaient un profil satisfaisant mais quelques-uns étaient limites au plan microbiologique. En outre, aucun échantillon ne se distinguait nettement selon tous les paramètres de qualité étudiés. Cette étude démontre que les aliments d’origine végétale riches en caroténoïdes provitaminiques A, dont l’HPR qui en est la meilleure source, peuvent permettre d’avoir un statut adéquat en VA. Les risques de contamination de l’HPR au stade de la vente au détail impliquent une sensibilisation et une formation aux pratiques exemplaires de manipulation. Mots clés : Huile de palme rouge, vitamine A, diversification alimentaire, qualité, Burkina Faso.

Adhesion and transcellular migration of neutrophils and B lymphocytes on fibroblasts

During tissue inflammation, infiltrated leukocytes may have physical contacts with fibroblasts. We observed that neutrophils and B lymphocytes adhered in a larger proportion than T cells on cultured fibroblasts. Microscopy showed that adhesion was also characterized by leukocyte engulfment by the fibroblasts. In migration assays, only neutrophils and B lymphocytes were selectively able to migrate through a fibroblast barrier. Adhesion and migration were increased by stimulation with tumor necrosis factor-alpha (TNF-alpha) and phorbol-12-myristate-13-acetate (PMA). Antibodies against ICAM-1/beta2 integrin blocked the interaction of neutrophils to fibroblasts. For B lymphocytes the couple VCAM-1/alpha4 integrin was also involved in this interaction. Human skin fibroblasts presented similar adhesion characteristics as rat cardiac fibroblasts. By measuring the distance between the border of migration holes and cadherin-positive adherens junctions, more than 65% of the holes correspond to the transcellular route over the paracellular route. Furthermore, vimentin staining revealed that the migration holes were highly nested by intermediate filaments in accordance with the transcellular route. Our results demonstrated that engulfment of neutrophils and B lymphocytes by fibroblasts resulted in selective passage by a transcellular route.

Rôle du dimère Gbetagamma dans l’organisation des systèmes de signalisation cellulaire

Selon le modèle classique, le signal reçu par les récepteurs couplés aux protéines G (RCPG) se propage suite à des interactions transitoires et aléatoires entre les RCPGs, les protéines G et leurs effecteurs. Par les techniques de transfert d’énergie de résonance de bioluminescence (BRET), de complémentation bimoléculaire de protéines fluorescentes (BiFC) et de co-immunoprécipitation, nous avons observé que les récepteurs, les protéines G et les effecteurs forment un complexe stable, avant et après l’activation des récepteurs. L’interaction entre l’effecteur Kir3 et le dimère Gbetagamma se produit initialement au réticulum endoplasmique et est sensible à un agoniste liposoluble des récepteurs beta2-adrénergiques. Bien que peu de spécificité pour les nombreux isoformes des sous-unités Gbetagamma ait été observée pour l’activation du canal Kir3, les interactions précoces au RE sont plus sensibles aux différentes combinaisons de Gbetagamma présentes. En plus de son rôle dans la régulation des effecteurs, le dimère Gbetagamma peut interagir avec de nombreuses protéines possédant des localisations cellulaires autres que la membrane plasmique. Nous avons identifié une nouvelle classe de protéines interagissant avec la sous-unité Gbeta, autant en système de surexpression que dans des extraits de cerveaux de rats, soit les protéines FosB et cFos, qui forment le complexe de transcription AP-1, suite à leur dimérisation avec les protéines de la famille des Jun. La coexpression du dimère Gbetagamma réduit l’activité transcriptionnelle du complexe AP-1 induit par le phorbol 12-,myristate 13-acetate (PMA), sans toutefois interférer avec la formation du complexe Fos/Jun ou son interaction avec l’ADN. Toutefois, le dimère Gbetagamma colocalise au noyau avec le complexe AP-1 et recrute les protéines histones déacétylases (HDAC) afin d’inhiber l’activité transcriptionnelle du complexe AP-1.

Rôle de la synthèse de l'acide rétinoïque dans le contrôle de la prolifération et de la différenciation des cellules épithéliales mammaires

L’acide rétinoïque (AR) est le ligand des récepteurs nucléaires RAR et RXR qui agissent comme facteurs de transcription ligand-inductibles et médient ses effets biologiques. Il est connu que l’AR a des propriétés prodifférenciatrices et antiprolifératives, notamment sur les cellules de l’épithélium mammaire. Une perte de sensibilité de l’AR a toutefois été mise en évidence dans plusieurs lignées cellulaires mammaires cancéreuses, ce qui pourrait faciliter la croissance des tumeurs. Or jusqu’ici cette perte de sensibilité avait été attribuée à des défauts de la voie de signalisation de l’AR, causée par la perte de l’expression des récepteurs à l’AR dans la tumeur, bien que plusieurs lignées de cellules cancéreuses y soient tout de même très sensibles. Peu d’études se sont intéressées au rôle de la voie de synthèse de l’AR dans la transformation des cellules mammaires. En effet, l’AR est synthétisé à partir de la vitamine A, ou rétinol, son précurseur sanguin provenant de la diète. Les cellules de l’épithélium mammaire normales ont la capacité de synthétiser l’AR à partir du rétinol. Nos rapportons pour la première fois que l’épithélium mammaire est probablement le siège de la synthèse et de la signalisation de l’AR. Cela est dû, au moins en partie, à l’expression d’une enzyme de synthèse de l’AR, RALDH3, dans l’épithélium mammaire normal. Dans cette étude, nous démontrons que les cellules cancéreuses de type luminal, qui ont sensibles à l’AR (et qui expriment le récepteur des estrogènes ER, catégorie qui regroupe 75 % des tumeurs diagnostiquées) n’ont au contraire pas la capacité de sythétiser l’AR, probablement en raison d’une faible expression de RALDH3 dans les tumeurs, sous l’effet des estrogènes. Cela pourrait représenter un nouveau mécanisme favorisant la croissance des tumeurs luminales dont les cellules proliférent en présence du rétinol sanguin. RALDH1, une autre enzyme de la voie de synthèse de l’AR, et qui partage 70 % d’identité de séquence avec RALDH3, est un marqueur de tumeurs plus agressives et de la formation de métastase. Nous montrons au contraire, que RALDH3 est un marqueur d’une moindre probabilité de développer des métastases chez les patientes atteintes d’une tumeur luminale. Cela suggère des rôles different pour ces deux enzymes dans la glande mammaire. Nos résultats indiquent que RALDH1 et 3 ont des propriétés enzymatiques très différentes, ce qui est en accord avec cette dernière hypothèse. Nos données suggèrent aussi que RALDH1 et 3 pourraient être des marqueurs de populations distinctes de cellules dans la glande mammaire normale. Nous proposons d’exploiter les diffèrences entre RALDH1 et 3 afin de mettre au point des méthodes de séparation des différentes population de cellules de l’épithélium mammaire ce qui pourrait aider à comprendre le rôle de la synthèse d’AR dans ce tissu.