Disposable immunosensor using a simple method for oriented antibody immobilization for label-free real-time detection of an oxidative stress biomarker implicated in cancer diseases

This work proposes a novel approach for a suitable orientation of antibodies (Ab) on an immunosensing platform, applied here to the determination of 8-hydroxy-2′-deoxyguanosine (8OHdG), a biomarker of oxidative stress that has been associated to chronic diseases, such as cancer. The anti-8OHdG was bound to an amine modified gold support through its Fc region after activation of its carboxylic functions. Non-oriented approaches of Ab binding to the platform were tested in parallel, in order to show that the presented methodology favored Ab/Ag affinity and immunodetection of the antigen. The immunosensor design was evaluated by quartz-crystal microbalance with dissipation, atomic force microscopy, electrochemical impedance spectroscopy (EIS) and square-wave voltammetry. EIS was also a suitable technique to follow the analytical behavior of the device against 8OHdG. The affinity binding between 8OHdG and the antibody immobilized in the gold modified platform increased the charge transfer resistance across the electrochemical set-up. The observed behavior was linear from 0.02 to 7.0 ng/mL of 8OHdG concentrations. The interference from glucose, urea and creatinine was found negligible. An attempt of application to synthetic samples was also successfully conducted. Overall, the presented approach enabled the production of suitably oriented Abs over a gold platform by means of a much simpler process than other oriented-Ab binding approaches described in the literature, as far as we know, and was successful in terms of analytical features and sample application.

Myoglobin-biomimetic electroactive materials made by surface molecular imprinting on silica beads and their use as ionophores in polymeric membranes for potentiometric transduction

Myoglobin (Mb) is among the cardiac biomarkers playing a major role in urgent diagnosis of cardiovascular diseases. Its monitoring in point-of-care is therefore fundamental. Pursuing this goal, a novel biomimetic ionophore for the potentiometric transduction of Mb is presented. It was synthesized by surface molecular imprinting (SMI) with the purpose of developing highly efficient sensor layers for near-stereochemical recognition of Mb. The template (Mb) was imprinted on a silane surface that was covalently attached to silica beads by means of self-assembled monolayers. First the silica was modified with an external layer of aldehyde groups. Then, Mb was attached by reaction with its amine groups (on the external surface) and subsequent formation of imine bonds. The vacant places surrounding Mb were filled by polymerization of the silane monomers 3-aminopropyltrimethoxysilane (APTMS) and propyltrimethoxysilane (PTMS). Finally, the template was removed by imine cleavage after treatment with oxalic acid. The results materials were finely dispersed in plasticized PVC selective membranes and used as ionophores in potentiometric transduction. The best analytical features were found in HEPES buffer of pH 4. Under this condition, the limits of detection were of 1.3 × 10−6 mol/L for a linear response after 8.0 × 10−7 mol/L with an anionic slope of −65.9 mV/decade. The imprinting effect was tested by preparing non-imprinted (NI) particles and employing these materials as ionophores. The resulting membranes showed no ability to detect Mb. Good selectivity was observed towards creatinine, sacarose, fructose, galactose, sodium glutamate, and alanine. The analytical application was conducted successfully and showed accurate and precise results.

Novel LTCC-potentiometric microfluidic device for biparametric analysis of organic compounds carrying plastic antibodies as ionophores: Application to sulfamethoxazole and trimethoprim

Monitoring organic environmental contaminants is of crucial importance to ensure public health. This requires simple, portable and robust devices to carry out on-site analysis. For this purpose, a low-temperature co-fired ceramics (LTCC) microfluidic potentiometric device (LTCC/μPOT) was developed for the first time for an organic compound: sulfamethoxazole (SMX). Sensory materials relied on newly designed plastic antibodies. Sol–gel, self-assembling monolayer and molecular-imprinting techniques were merged for this purpose. Silica beads were amine-modified and linked to SMX via glutaraldehyde modification. Condensation polymerization was conducted around SMX to fill the vacant spaces. SMX was removed after, leaving behind imprinted sites of complementary shape. The obtained particles were used as ionophores in plasticized PVC membranes. The most suitable membrane composition was selected in steady-state assays. Its suitability to flow analysis was verified in flow-injection studies with regular tubular electrodes. The LTCC/μPOT device integrated a bidimensional mixer, an embedded reference electrode based on Ag/AgCl and an Ag-based contact screen-printed under a micromachined cavity of 600 μm depth. The sensing membranes were deposited over this contact and acted as indicating electrodes. Under optimum conditions, the SMX sensor displayed slopes of about −58.7 mV/decade in a range from 12.7 to 250 μg/mL, providing a detection limit of 3.85 μg/mL and a sampling throughput of 36 samples/h with a reagent consumption of 3.3 mL per sample. The system was adjusted later to multiple analyte detection by including a second potentiometric cell on the LTCC/μPOT device. No additional reference electrode was required. This concept was applied to Trimethoprim (TMP), always administered concomitantly with sulphonamide drugs, and tested in fish-farming waters. The biparametric microanalyzer displayed Nernstian behaviour, with average slopes −54.7 (SMX) and +57.8 (TMP) mV/decade. To demonstrate the microanalyzer capabilities for real applications, it was successfully applied to single and simultaneous determination of SMX and TMP in aquaculture waters.

1,1-enediamines and β-substituted enamines in heterocyclic synthesis

The work in this thesis mainly deals with l,l-enediamines and ~ -substituted enamines (push-pull olefines) and their reactions, leading to the formation of a number of heterocycles. Various ~-substituted enamines were prepared by a 'one pot synthesis' in which a l,l-enediamine presumably acts as an intermediate. These enamines, various substituted crotonamides and propenamides, were made by using two different orthoesters, various secondary and primary amines and cyanoacetamide. Their structures, mechanism of formation and geometry are discussed. A synthetic route to various unsymmetrically substituted pyridines was examined. Two substituted pyridinones were obtained by using two different ~-substituted enamines and cyanoacetamide. In one case a dihydropyridine was isolated. This dihydropyridine, on heating in acidic conditions, gave a pyridinone, which confirmed this dihydropyridine as an intermediate in this pyridine synthesis. A new synthetic method was used to make highly substituted pyridinones, which involved the reaction of l,l-enediamines with the ~-substituted enamines. A one pot synthesis and an interrupted one pot synthesis were used to make these pyridinones. Two different orthoesters and three different secondary amines were used. Serendipitous formation of a pyrimidinone was observed when pyrrolidine was used as the secondary amine and triethyl orthopropionate was used as the orthoester. In all cases cyanoacetamide was used as the carbon acid. This pyridine synthesis was designed with aI, l-enediamine as the Michael donor and the ~ -substituted enamines as Michael acceptors. Substituted ureas were obtained in two cases, which was a surprise. Some pyrimidines were made by reacting two substituted enamines with two different amidines. When benzamidine was used, the expected pyrimidines were obtained. But, when 2-benzyl-2-thiopseudourea (which is also an amidine) was used, of the two expected pyrimidines, only one was obtained. In the other case, an additional substitution reaction took place in which the S-benzyl group was lost. An approach to quinazolone and benzothiadiazine synthesis is discussed. Two compounds were made from 1, I-dimorpholinoethene

An aryne route to aporphine alkaloids and related topics / |nIjaz Ahmad Chanda. -- 260 St. Catharines, Ont. : [s. n.],

This research was directed towards the investigation and development of an aryne route to the syntheses of aporphi ne and dibenzopyrrocolinium (dibenzoindolizinium) alkaloids and to the stability of the latter under the conditions used for aryne formation. The work c an be divided into three main sections . i) - Synthesis of Glaucine 6-Bromo-3,4-dimethoxyphenylacetic acid, prepared by the action of bromine i n acetic acid on3,4-dimethoxyphenylacetic a cid, was converted into its acid chloride by t he action of thionyl chloride. This on treatment with 3,4- dimethoxyphenylethylamine pr ovided N-(3, 4-dimethoxyphenylethyl)- 2-(2-bromo-4,S-dimethoxyphenyl)-acetamide which on dehydration with phosphoryl chloride (Bischler Napieralski reaction) in dry benzene afforded l -(2-bromo-4,S-dimethoxybenzyl)- 3,4-dihydro-6,7-dimethoxyisoquinoline, isolated as hydrochl oride. A new method o f destroying the excess of phosphoryl chloride was developed which proved to be quite useful. Methylation of the dihydroisoquinoline'with methyl iodide in methanol , and subsequent reduction with sodium borohydride provided (±)-6-bromolaudanosine. Act ion of potassamide or sodamide in anhydrous liquid ammonia on (±)-6-bromolaudanosine yielded the corresponding amino derivative along with other products. Diazotization and ring closure of (±)-6-aminolaudanosine then a f forded (±)-glaucine which was isolated as methiodide. ii) - Intramolecular Capture of Aryne During Glaucine Synthesis, and Subsequent Reactions . This section deals with the by-products formed under the conditions of the aryne stage of t he glaucine synthesis. The crude product, obtained in the reaction of potassamide or sodamide in liquid ammonia on (±)-6-bromolaudanosine, was s eparated by chromatography, Three products were separated and identified. a ) - 5,6-Dimethoxy-2-( 3,4-dimethoxy-6-ethylphenyl)-lmethylindole. Two mechanisms are proposed for the formation of this interesting product. This compound also was prepared by the action of potassamide in l,iquid ammonia on 5,6 ,l2,l2atetrahydro- 2,3,9,lO-tetramethoxy-7-methyldibenz[b,g]indolizinium i odide . b) - 5,6-Dimethoxy-2-(3,4-dimethoxy-6-vinylphenyl)-lmethylindoline. Its formation represented a new method of Hofmann degradation . Further confirmation of structure was done by performing the normal Hofmann reaction on 5, 6,12,12a-tetrahydro -2/3,9,lO-tetramethoxy ~7-methyldibe nz[ b,g]indolizinium iodide. The indoline prepared i n this way was identical in all respects with that prepared above . c) - 1- (2-amino-4,5-dimethoxybenzyl ) -l,2,3,4-tetrahydro-2- methyl-6,7-dimethoxyisoquinoline, was converted t o glaucine as stated in section 1 . iii) - Attempt:,ed Sxnthesis of Liriodenine Piperonal was converted into 3,4-methylenedioxyinitrostyrene which on reduction with lithium aluminium hydride provided 3,4-methylenedioxyphenylethylamine. The method of extraction after the reduction was improved t o some extent. The amine on condensation with m-chlorophenylacetyl chloride, prepared by the action of oxalyl chloride on 3,4-methylenedioxyphenylacetic acid, provided N-[ ~ -(3,4-methylenedioxyphenyl)- e thyl)-3-chlorophenylacetamide. This on dehydration with phosphoryl chloride in dry benzene followed by air oxidation afforded l-(3-chlorobenzoyl)-6,7-methylenedioxyi soquinoline. This compound on r eaction with potassamide in liquid ammonia afforded a crude product from which. one product was separated by chromatography i n a pure condition . This yellow compound analysed as,c17Hl ON2021 and was t he main product i n the reaction ; a t entative structure is proposed. A second compound, not obtained in pure condition, was submitted to Pschorr reaction in the hope of obtaining liriodenine, but without success.

Chelated fluoroboron cations : synthesis and multinuclear nmr studies

The preparation of chelated difluoroboron cations (DD)BF2+, where DD is a saturated polydentate tertiary-amine or polydentate aromatic ligand, has been systematically studied by using multinuclear solution and solid state nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Three new methods of synthesis of (DD)BF2+ cations are reported, and compared with the previous method of reacting a chelating donor with Et20.BF3. The methods most effective for aromatic donors such as 1,1O-phenanthroline are ineffective for saturated polydentate tertiary-amines like N,N,N' ,Nil ,Nil-pentamethyldiethylenetriamine. Polydentate tertiary-amine donors that form 5-membered rings upon bidentate chelation were found to chelate effectively when the BF2 source contained two leaving groups (a heavy halide and a Lewis base such as pyridine =pyr or isoxazole =ISOX), i.e., pyr.BF2X (X = CI or Br), ISOX.BF2X and (pyr)2BF2+. Those that would form 6membered rings upon chelation do not chelate by any of the four methods. Polydentate aromatic ligands chelate effectively when the BF2 source contained a weak Lewis base, e.g., ISOX.BF3, ISOX.BF2X and Et20.BF3. Bidentate chelation by polydentate tertiaryamine and aromatic donors leads to nmr parameters that are significantly different then their (D)2BF2+ relatives (D =monod~ntate t-amines or pyridines). The chelated haloboron cations (DD)BFCI+, and (DD)BFBr+ were generated from D.BFX2 adducts for all ligands that form BF2+ cations above. In addition, the (DD)BCI2+ and (DD)BBr2+ cations were formed from D.BX3 adducts by the chelating aromatic ligands, except for the aromatic ligand 1,8-bis(dimethylamino)naphthalene, which formed only the (DD)BF2+ cation, apparently due to its extreme steric hindrance. Chelation by a donor is a two-step reaction. For polydentate tertiary-amine ligands, the two rates appear to be very dependent on the two possible leaving groups on the central boron atom. The order of increasing ease of displacement for the donors was: pyr < Cl < Br < ISOX. The rate of chelation by polydentate aromatic ligands appears to be dependent on the displacement of the first ligand from the boron. The order of increasing ease of displacement for the donors was: pyr < CI < ISOX ~ Br < Et20.

Studies of some cocondensations of nickel atoms with unsaturated organic ligands and with mixed ligand systems

The cocondensation of nickel with a number of unsaturated ligands was studied, as was the cocondensation with a number of mixed ligand systems. Enamines were found not to react with nickel while acrylonitrile was polymerized. In the mixed ligand syst.ems different products were obtained than when the ligands were cocondensed individually. Cocondensations of benzyl halide/allyl halide mixtures gave unstable products that were not observed when the halides were cocondensed individually. The effect of Kao-Wool insulation on nickel/benzyl halide cocondensations was found to be significant. Kao-Wool caused the bulk of the benzyl halide to be polymeri zed to a number of poly-benzylic species. An alkali metal reactor was designed for the evaporation of sodium and potassium atoms into cold solutions of metal halide and an or ganic substrate. This apparatus was used to synthesize Ni(P¢3 )3' but proved unsuccessful for synthesizing a nickel-enamine compound.

The preparation and reactions of some 1, 2-dipolar species /|nI. D. Brindle. -- 260 St. Catharines, Ont. : [s. n.],

This research was directed mainly towards the investigation of the reactions of allylic amineimides. The work can be divided into two main sections. Section 1 of the thesis deals mainly with thermolysis studies of amineimides. Sections 1a and 1b represent a comprehensive survey of amineimide literature up to 1971. N-A1ly1-N,N-dirnethylarnine-benzirnide was prepared and rearranged at 1400 to l-allyl-1-benzoyl-2,2-dimethylhydrazine. A tentative mechanism involving an initial migration to the carbonyl oxygen was disproved by incorporating the amineimide system into a five-membered ring. N,N~Dimethyl-N-propargylamine-benzimidedid not rearrange on heating; but the hydrobromide, on heating, disproportionated to give 1-benzoyl~2,2,2-trimethylhydraziniumbromide and I-benzoyl-2,2~ dimethylhydrazine. l-Ally'l--l, I-dimethyl-2-benzoy-lhydrazinium bromide and 1~benzoy-1-2,2, 2-trimethy-lhydrazinium iodide both disproportionated to give l~benzoyl-2,2-dimethylhydrazine. Section 1 concludes with a discussion of the mechanisms of ally'lic migrations in amineimides proposed by J. E. Baldwin. Section 2 deals with the formation of five-membered heterocyclic compounds from amineimides by bromination. 1,1-Dimethyl-2benzoyl- 4-bromopyrazolidinium bromide was formed from N-allyl-N,Ndime thy-lamtne-benzimide , 1,1-dimethyl-2-benzoyl-4-bromopyrazol-3enium bromide from N,N~dimethyl-N-propargylamine~benzimidevia the unusual acetylenic "bromonium" ion. Hydrogenolysis of both heterocyclic compounds gave the same product. The preparation was extended by forming 2,2-dimethyl-4-bromoisoxazolinium bromide from N-allylN, N-dimethylamine-N-oxide. Sections 3 and 4 cover a number of unsuccessful attempts to synthesise other amineimides and l,2-dipolar species.

Synthesis and utilization of guanidine catalysts for applications directed towards the preparation of butenolide and modified amino acid derivatives

Development of guanidine catalysts is explored through direct iminium chloride and amine coupling, alongside a 2-chloro-l,3-dimethyl-IH-imidazol-:-3-ium chloride (DMC) induced thiourea cyclization. Synthesized achiral catalyst N-(5Hdibenzo[ d,t][1,3]diazepin-6(7H)-ylidene)-3,5-bis(trifluoromethyl) aniline proved unsuccessful towards O-acyl migrations, however successfully catalyzed the vinylogous aldol reaction between dicbloro furanone and benzaldehyde. Incorporating chirality into the guanidine catalyst utilizing a (R)-phenylalaninol auxiliary, generating (R)-2-((5Hdibenzo[ d,t] [1,3 ]diazepin-6(7H)-ylidene ) amino )-3 -phenylpropan-l-ol, demonstrated enantioselectivity for a variety of adducts. Highest enantiomeric excess (ee) was afforded between dibromofuranone and p-chlorobenzaldehyde, affording the syn conformation in 96% ee and the anti in 54% ee, with an overall yield of30%. Attempts to increase asymmetric induction were focused on incorporation of axial chirality to the (R)phenylalaninol catalyst using binaphthyl diamine. Incorporation of (S)-binaphthyl exhibited destructive selectivity, whereas incorporation of (R)-binaphthyl demonstrated no effects on enantioselectivity. Current studies are being directed towards identifying the catalytic properties of asymmetric induction with further studies are being aimed towards increasing enantioselectivity by increasing backbone steric bulk.

Molecular Magnetism: The Design, Synthesis, and Characterization of New Building Blocks for Molecule-based Magnetic Materials

Two classes of building blocks have been prepared and characterized and their coordination chemistry explored working towards the preparation of new molecule-based magnetic materials. In the first project, the amine functionality of 3,3'-diamino-2,2'- bipyridine was exploited for the preparation of a new family of ligands (H2L 1)-(H2L 4). The molecular structures of three ligands have been fully characterized by X-ray crystallography. [molecular structure diagram will not copy here, but is available in full pdf.] The coordination chemistry of these ligands with divalent first row transition metal ions was investigated. For ligand (H2L1), the molecular structures of four coordination complexes with stoichiometries [Zn2(Ll)(OAc)(MeO)]2 (I), [Cu2(L1)(OAc)2 (II), [Li(L1)]3 (III), and [Ni(L1)]3 (IV) were determined by X-ray crystallography. For ligand (H2L2), a Cu(II) complex of stoichiometry [Cu3(L2)(OAc)3MeO] (V) was determined by X-ray crystallography. The magnetic properties of complexes (II), (III), and (V) have been fully elucidated. In project two, synthetic strategies for the preparation of porphyrin molecules bearing triol substituents is presented. Following this approach, three new porphyrin derivatives have been prepared and characterized [Zn(HPTPP-CH2C(CH20H)3)] (VI), [P(TPP)(OCH2C(CH2)H)3)2]+CL- (VII), and [P(OEP)(C6H5)(OCH2C(CH2OH)3)]+Cl- (VIII). Attempts to exchange the labile methoxide bridges of a tetraironIIl single molecule magnet of stoichiometry [Fe4(OMe)6(dpm)6] (Hdpm = dipivaloylmethane) with the triol appended porphyrins will be discussed. [molecular structure diagram will not copy here, but is available in full pdf.]

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10−5 and 10−4 M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10−5 M OA increased synaptically driven contractions by ∼1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

The action of octopamine on muscles of Drosophila melanogaster larvae

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10(-5) and 10(-4) M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10(-5) M OA increased synaptically driven contractions by ≈ 1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

Cell-selective modulation of the neuromuscular system in Drosophila

The capacity for all living cells to sense and interact with their environment is a necessity for life. In highly evolved, eukaryotic species, like humans, signalling mechanisms are necessary to regulate the function and survival of all cells in the organism. Synchronizing systemic signalling systems at the cellular, organ and whole-organism level is a formidable task, and for most species requires a large number of signalling molecules and their receptors. One of the major types of signalling molecules used throughout the animal kingdom are modulatory substances (e.x. hormones and peptides). Modulators can act as chemical transmitters, facilitating communication at chemical synapses. There are hundreds of circulating modulators within the mammalian system, but the reason for so many remains a mystery. Recent work with the fruit fly, Drosophila melanogaster demonstrated the capacity for peptides to modulate synaptic transmission in a neuron-specific manner, suggesting that peptides are not simply redundant, but rather may have highly specific roles. Thus, the diversity of peptides may reflect cell-specific functions. The main objective of my doctoral thesis was to examine the extent to which neuromodulator substances and their receptors modulate synaptic transmission at a cell-specific level using D. melanogaster. Using three different modulatory substances, i) octopamine - a biogenic amine released from motor neuron terminals, ii) DPKQDFMRFa - a neuropeptide secreted into circulation, and iii) Proctolin - a pentapeptide released both from motor neuron terminals and into circulation, I was able to investigate not only the capacity of these various substances to work in a cell-selective manner, but also examine the different mechanisms of action and how modulatory substances work in concert to execute systemic functionality . The results support the idea that modulatory substances act in a circuit-selective manner in the central nervous system and in the periphery in order to coordinate and synchronize physiologically and behaviourally relevant outputs. The findings contribute as to why the nervous system encodes so many modulatory substances.

Évaluation du risque cancérigène associé à la contamination de l’eau potable de puits municipaux par les nitrates/nitrites dans certaines régions rurales du Québec

La spécialisation des techniques agricoles que nous connaissons ces dernières décennies, particulièrement dans les régions rurales, est à l’origine de l’abus de fertilisants. Ces derniers sont actuellement reconnus comme étant les causes principales de la contamination de l’eau souterraine par les nitrates. Suite à leur ingestion via l’eau potable, les nitrates sont transformés en nitrites par la flore buccale. Une fois dans l’estomac les nitrites réagissent avec certaines amines provenant de l’alimentation pour générer des nitrosamines cancérogènes. L’objectif de notre étude était d’estimer quantitativement l’excès de risque de cancer (ER) pour les populations de sept régions rurales du Québec qui consomme l’eau potable provenant de réseaux municipaux alimentés en eau souterraine. Le territoire à l’étude était caractérisé par une agriculture intensive d’élevage. Les médianes (et 95e centiles) régionales des concentrations de nitrates mesurées dans les réseaux de ces régions étaient de : 0,18 (2,74); 0,48 (10,35); 0,15 (1,28); 0,32 (11); 0,05 (0,76); 0,10 (4,69); 0,09 (2,13) mg N-NO3-/l. Nous avons envisagé un scénario de transformation complète des nitrites et de certaines amines (diméthylamine, diéthylamine, n-butylamine, méthyléthylamine) en nitrosamines spécifiques : N-diméthylnitrosamine (NDMA), N-diéthylnitrosamine (NDEA), N-n-dibutylnitrosamine (NDBA) et N-méthyléthylnitrosamine (NMEA). Pour estimer la concentration de nitrites formés dans l’estomac, nous avons considéré une consommation définie d’eau potable, le volume de l’estomac et un taux de transformation des nitrates en nitrites. Supposant les quantités de nitrites et de chaque amine constantes pendant 1h, nous avons considéré la constante de nitrosation spécifique à chaque amine pour évaluer la dose d’exposition journalière à chaque nitrosamine équivalente formée. Par la suite, la combinaison de cette dose à un estimateur de potentiel cancérogène qhumain spécifique à chaque nitrosamine, nous a permis d’évaluer l’ER associé à chacune d’elles. Globalement l’analyse a démontré que les ER les plus élevés, estimés pour le NDBA, étaient de l’ordre de 10-6, ne contribuant pas de façon significative à une augmentation du risque de cancer pour ces populations.

Evaluation du systeme nerveux autonome dans l'hypertension arterielle essentielle

L’analyse spectrale de la fréquence cardiaque, de la pression artérielle systolique, de la pression artérielle diastolique ainsi que de la respiration par la transformée de Fourier rapide, est considérée comme une technique non invasive pour la détermination de l’activité du système nerveux autonome (SNA). Dans une population de sujets normaux volontaires, nous avons obtenu à l’état basal, des oscillations de basses fréquences (0,05-0,15Hz) reliées au système nerveux sympathique autonome et des oscillations de hautes fréquences (0,2Hz) représentant sur les intervalles entre chaque ondes R de l’électrocardiogramme (RR), l’arythmie sinusale respiratoire correspondant à une activité vagale. Nous avons comparé les tests de stimulation du système nerveux sympathique autonome déclenché par le passage de la position de repos (en décubitus dorsal), à la position orthostatique volontaire et le passage de la position de repos à la position orthostatique avec la table basculante à 60o. Nous avons également comparé un groupe normotendu à un groupe hypertendu qui a été soumis au passage du repos à l’orthostation volontaire et pour lesquels nous avons évalué la sensibilité du baroréflexe et la réponse sympathique par la mesure des catécholamines circulantes. Dans un groupe de sujets ayant une hypertension artérielle essentielle, nous avons évalué l’effet de la thérapie hypotensive, par le Trandolapril qui est un Inhibiteur de l’enzyme de conversion (IEC) de l`angiotensine. Dans ce groupe hypertendu, nous avons procédé, en plus de la stimulation sympathique par l’orthostation volontaire, à un exercice isométrique de trois minutes à 30 % de la force maximale. Nous avons également complété notre évaluation par la mesure de la densité de récepteurs ß2 adrénergiques sur lymphocytes et par la mesure des indices de contractilité à l’aide de l’échocardiographie en M mode. Les résultats ont montré, dans les groupes normaux volontaires, dans les deux types de stimulation du système nerveux sympathique par la position orthostatique, une augmentation significative des catécholamines plasmatiques avec une augmentation de la fréquence cardiaque et des basses fréquences de RR, confirmant ainsi que l’on est en état de stimulation sympathique. On observe en même temps une diminution significative des hautes fréquences de RR, suggérant un retrait vagal lors de cette stimulation. On a observé au test de la table basculante six cas d’hypotension orthostatique. On a comparé la position orthostatique volontaire entre le groupe de sujets normaux et le groupe de sujets hypertendus. L’analyse spectrale croisée de RR et de la pression artérielle systolique a permis d’évaluer dans l’hypertension artérielle (HTA), essentielle une sensibilité du baroréflexe atténuée, accompagnée d’une réactivité vagale réduite en présence d’une activité et d’une réactivité sympathique augmentées suggérant une altération sympathovagale dans l’HTA. Dans le groupe de sujets hypertendus traités (Trandolapril 2mg/jour), nous avons identifié un groupe de répondeurs au traitement par le Trandolapril et un groupe de non répondeurs à ce type de thérapie anti-hypertensive. Le groupe répondeur avait un profil hyper-adrénergique avec une hyper-réactivité sympathique, une fréquence cardiaque et des pressions artérielles diastolique et systolique plus élevées au repos. Dans le groupe total traité au Trandolapril, la densité des récepteurs ß2 adrénergiques a doublé, après thérapie, alors que la réactivité des basses fréquences obtenues à l’analyse spectrale a augmenté. Nous avons montré dans notre étude qu’un IECA a pu inhiber le mécanisme facilitateur de l’angII sur les terminaisons nerveuses sympathiques et a permis ainsi de réduire l’hyperactivité sympathique et le mécanisme de « down regulation » des récepteurs ß2 adrénergiques rendant ainsi l’expression de l’influence du SNA post synaptique plus efficace. Dans l’ensemble de nos protocoles cliniques, par l’utilisation de l’analyse spectrale des signaux RR, de la pression artérielle systolique,de la pression artérielle diastolique et de la respiration, nous avons montré que cette technique non invasive permet de décrire et de mieux comprendre les mécanismes physiologiques, physiopathologiques et pharmacologiques reliés au système nerveux autonome et à l’hypertension artérielle essentielle.