This study examines D.P.S Monyaise’s short story titled Omphile Umphi Modise which were scrutinized by Mogapi (1992), Pretorius (1990), Manyaka (1992), Shole (1997) and Malimabe (1998). Their investigation did not only focus on the content of the short story, but also concentrated on the structure and style of the author. When scrutinizing Omphile Umphi Modise, the focus mainly falls on content and characterization of the characters. This research is based on an adopted narratological model, which is used by Groenewald (1993) and Mojalefa (1994).. When investigating the content, emphasis is put on the topic as a theme and part of the plot, which is imperative in the analysis of Omphile Umphi Modise. Two terms which are to define and describe are used to explain and unravel ideas that were used by the writer to simplify the problems a reader would encounter in this investigation. When analyzing the content as theory, the following four elements are noticed: characters, events, time and place. Another important aspect on the level of content is the division of the characters into two subgroups, namely quarrelsome and kindhearted.. In Omphile Umphi Modise, the kindhearted is Motlalepule, while the quarrelsome is rraModise who blatantly refuses to see Motlalepule’s good side. These two characters are described by the following: intention, patronage abilities, success and resistance. The investigation explains that the theme, which is ‘keeping a secret can lead to one’s doom’, is the one that connects the events and the plot, with the intention of highlighting the message to attract the reader. Design is one of the most important aspects of the plot. It is herewithin divided into two sections which highlight the characterization of the protagonist and antagonist as well as the revelation of the aspect of tragedy in Omphile Umphi Modise. The characters are divided into two sections, namely main and minor characters. The main characters are the protagonist, the antagonist and the tritagonist. The antagonist in Omphile Umphi Modise is rraModise/Mosimane yo o tedu tsebeng/ tedu tsa katse who is portrayed to be evil because he mandated Thandi, a minor character, to put drugs in the tea Motlalepule was about to drink, and sadly ended up drinking it herself. After she was drugged, mosimane yo tedu tsebeng/tedu tsa katse raped and impregnated her. The writer uses techniques to explain the personality traits of the characters. He employs a distinguishing technique to highlight personality traits of Motlalepule which are beauty, misfortune, suffering, and poverty. These are explained by the following techniques: focus, differentiation, conflict, use of the possessive, use of the adjective form, point of view, use of diminutive form, exaggeration, use of the pronoun, use of idioms, proverbs and, similes and omission. Differentiation is employed to highlight leadership as a personality trait of the antagonist, mosimane yo o tedu tsebeng/ tedu tsa katse/rraModise is which is explained by two techniques: indicative mood and point of view. AFRIKAANS : In hierdie ondersoek word D.P.S Monyaise se kortverhaal getiteld Omphile Umphi Modise ontleed. Dié kortverhaal is krities ontleed deur Mogapi (1992), Pretorius (1990), Manyaka (1992), Shole (1997) en Malimabe (1998). Hulle ondersoeke het nie slegs die inhoud van die kortverhaal betrek nie, maar het ook op die struktuur van die kortverhaal en die styl van die skrywer gefokus. Met die ontleding van Omphile Umphi Modise, word daar gekonsentreer op twee aspekte, naamlik inhoud en karaktarisering van die karakters. Hierdie navorsing word gebaseer op die aangenome narratologiese model wat gebruik word deur Mojalefa (1994) en Groenewald (1993).. Wanneer die inhoud ondersoek word, word daar klem gelê op die onderwerp as ‘n tema en as deel van die plot, wat van belang is in die analise van Omphile Umphi Modise. Idees wat deur die skrywer gebruik is om die probleme van die leser ten opsigte van die verstaan van die ondersoek te vereenvoudig, word twee terme naamlik en defineer en te interpreteer verduidelik en geanaliseer. Wanneer die inhoud as teorie ontleed word, word die volgende vier elemente betrek: karakters, gebeure, tyd en plek. ‘n Ander belangrike aspek is die vlak van inhoud – die karakters word in twee subgroepe verdeel, naamlik. antagonis en protagonis. In Omphile Umphi Modise, is Motlalepule die protagonis, terwyl rraModise die antagonis is wat blatant weier om Motlalepule se goeie kant raak te sien. Hierdie twee karakters word beskryf deur intensie, vermoë, sukses en teenstand. Die ondersoek verduidelik dat die tema ‘om ‘n geheim te hou wat tot ‘n mens se ondergang kan lei’, die een is wat gebeure en plot koppel, met die doel om die boodskap uit te lig om sodoende die leser te interesseer. Ontwerp is een van die belangrikste aspekte van die plot. Dit word onderverdeel in twee afdelings wat die karakterisering van die protagonis en antagonis, sowel as die tragedieaspek van Omphile Umphi Modise beklemtoon. Die karakters word in twee groepe verdeel, naamlik. hoofkarakters en newekarakters. Die hoofkarakters is die protagonis, die antagonis en die tritagonis. Die antagonis in Omphile Umphi Modise is rraModise/Mosimane wa tedu tsebeng/ tedu tsa katse wat uitgebeeld word as boos, omdat hy Thandi, ‘n newekarakter, oorreed het om ‘n dwelmmiddel in Motlalepule se tee te gooi, en dit toe self gedrink het. Nadat sy bedwelm is, het mosimane yo tedu tsebeng/tedu tsa katse haar verkrag en swanger gemaak. Die skrywer gebruik verskillende tegnieke om die persoonlikheidskenmerke van die karakters uit te beeld. Hy gebruik ‘n spesifieke tegniek om die persoonlikheidskenmerke van Motlalepule uit te lig, naamlik skoonheid, ongeluk, swaarkry en armoede. Hierdie kenmerke word uitgelig deur die volgende tegnieke: fokus, differensiasie, konflik, die possessiewe vorm die adjektiewe vorm, perspektief, die diminutiewe vorm,, oordrywing, die gebruik van die voornaamwoord, die gebruik van idiome, spreekwoorde en vergelykings en elisie. Differensiasie is gebruik om leierskap as ‘n persoonlikheidskenmerk van die antagonis, mosimane yo o tedu tsebeng/ tedu tsa katse/rraModise uit te lig en wat verder verduidelik word deur twee tegnieke naamlik indikatiewe modus en perspektief. / Dissertation (MA)--University of Pretoria, 2011. / African Languages / unrestricted

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterised by a reduction of dopamine concentration in the striatum due to degeneration of dopaminergic neurons in the substantia nigra. Currently, first line treatment of PD includes the use of dopamine precursors, dopamine agonists and inhibitors of enzymatic degradation of dopamine, in an effort to restore dopamine levels and/or its effects. However, all these therapeutic strategies are only symptomatic and unfortunately do not slow, stop or reverse the progression of PD. From the discovery of adenosine A2A receptor-dopamine D2 receptor heteromers and the antagonistic interaction between these receptors, the basis of a new therapeutic approach towards the treatment of PD emerged. Adenosine A2A receptor antagonists have been shown to decrease the motor symptoms associated with PD, and are also potentially neuroprotective. The possibility thus exists that the administration of an adenosine A2A antagonist may prevent further neurodegeneration. Furthermore, the antagonism of adenosine A1 receptors has the potential of treating cognitive deficits such as those associated with Alzheimer's disease and PD. Therefore, dual antagonism of adenosine A1 and A2A receptors would be of great benefit since this would potentially treat both the motor as well as the cognitive impairment associated with PD. The affinities (Ki-values between 0.6 mM and 38 mM) of a series of 1,4-dihydropyridine derivatives were previously illustrated for the adenosine A1, A2A and A3 receptor subtypes by Van Rhee and co-workers (1996). These results prompted this pilot study, which aimed to investigate the potential of the structurally related 3,4-dihydropyrimidin-2(1H)-ones (dihydropyrimidones) and 2-amino-1,4-dihydropyrimidines as adenosine A1 and A2A antagonists. In this pilot study, a series of 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines were synthesised and evaluated as adenosine A1 and A2A antagonists. Since several adenosine A2A antagonists also exhibit MAO inhibitory activity, the MAO-inhibitory activity of selected derivatives was also assessed. A modified Biginelli one pot synthesis was used for the preparation of both series of compounds under solvent free conditions. A mixture of a β- diketone, aldehyde and urea/guanidine hydrochloride was heated for an appropriate time to afford the desired compounds in good yields. MAO-B inhibition studies comprised of a fluorometric assay where kynuramine was used as substrate. A radioligand binding protocol described in literature was employed to investigate the binding of the compounds to the adenosine A2A and A1 receptors. The displacement of N-[3H]ethyladenosin-5’-uronamide ([3H]NECA) from rat striatal membranes and 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) from rat whole brain membranes, was used in the determination of A2A and A1 affinity, respectively. The results showed that both 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines had weak adenosine A2A affinity, with the p-fluorophenyl substituted dihydropyrimidone derivative (1h) in series 1, exhibiting the highest affinity for the adenosine A2A receptor (28.7 μM), followed by the p-chlorophenyl dihydropyrimidine derivative (2c) in series 2 (38.59 μM). Both series showed more promising adenosine A1 receptor affinity in the low micromolar range. The p-bromophenyl substituted derivatives in both series showed the best affinity for the adenosine A1 receptor with Ki-values of 7.39 μM (1b) and 7.9 μM (2b). The pmethoxyphenyl dihydropyrimidone (1d) and p-methylpneyl dihydropyrimidine (2e) derivatives also exhibited reasonable affinity for the adenosine A1 receptor with Ki-values of 8.53 μM and 9.67 μM, respectively. Neither the 3,4-dihydropyrimidones nor the 2-amino-1,4- dihydropyrimidines showed MAO-B inhibitory activity. Comparison of the adenosine A2A affinity of the most potent derivative (1h, Ki = 28.7 μM) from this study with that of the previously synthesised dihydropyridine derivatives (Van Rhee et al., 1996, most potent compound had a Ki = 2.74 mM) reveals that an approximate 100- fold increase in binding affinity for A2A receptors occurred. However, KW6002, a known A2A antagonist, that has already reached clinical trials, has a Ki-value of 7.49 nM. The same trend was observed for adenosine A1 affinity, where the most potent compound (1b) of this study exhibited a Ki-value of 7.39 μM compared to 2.75 mM determined for the most potent dihydropyridine derivatives (Van Rhee et al., 1996). N6-cyclopentyladenosine (CPA), a known adenosine A1 agonist that was used as a reference compound, however had a Kivalue of 10.4 nM. The increase in both adenosine A1 and A2A affinity can most likely be ascribed to the increase in nitrogens in the heterocyclic ring (from a dihydropyridine to a dihydropyrimidine) since similar results were obtained by Gillespie and co-workers in 2009 for a series of pyridine and pyrimidine derivatives. In their case it was found that increasing the number of nitrogens in the heterocyclic ring (from one to two nitrogen atoms for the pyridine and pyrimidine derivatives respectively) increased affinity for the adenosine A2A and adenosine A1 receptor subtypes, while three nitrogen atoms in the ring (triazine derivatives) were associated with decreased affinity. It thus appears that two nitrogen atoms in the ring (pyrimidine) are required for optimum adenosine A1 and A2A receptor affinity. The poor adenosine A2A affinity exhibited by the compounds of this study can probably be attributed to the absence of an aromatic heterocyclic ring. The amino acid, Phe-168 plays a very important role in the binding site of the A2A receptor, where it forms aromatic - - stacking interactions with the heterocyclic aromatic ring systems of known agonists and antagonists. Since the dihydropyrimidine ring in both series of this pilot study was not aromatic, the formation of aromatic - -stacking interactions with Phe-168 is unlikely. In conclusion, the 3,4-dihydropyrimidone and 2-amino-1,4-dihydropyrimidine scaffolds can be used as a lead for the design of novel adenosine A1 and A2A antagonists, although further structural modifications are required before a clinically viable candidate will be available as potential treatment of PD. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Parkinson’s disease (PD) is a neurodegenerative disorder that is characterised by a reduction of dopamine concentration in the striatum due to degeneration of dopaminergic neurons in the substantia nigra. Currently, first line treatment of PD includes the use of dopamine precursors, dopamine agonists and inhibitors of enzymatic degradation of dopamine, in an effort to restore dopamine levels and/or its effects. However, all these therapeutic strategies are only symptomatic and unfortunately do not slow, stop or reverse the progression of PD. From the discovery of adenosine A2A receptor-dopamine D2 receptor heteromers and the antagonistic interaction between these receptors, the basis of a new therapeutic approach towards the treatment of PD emerged. Adenosine A2A receptor antagonists have been shown to decrease the motor symptoms associated with PD, and are also potentially neuroprotective. The possibility thus exists that the administration of an adenosine A2A antagonist may prevent further neurodegeneration. Furthermore, the antagonism of adenosine A1 receptors has the potential of treating cognitive deficits such as those associated with Alzheimer's disease and PD. Therefore, dual antagonism of adenosine A1 and A2A receptors would be of great benefit since this would potentially treat both the motor as well as the cognitive impairment associated with PD. The affinities (Ki-values between 0.6 mM and 38 mM) of a series of 1,4-dihydropyridine derivatives were previously illustrated for the adenosine A1, A2A and A3 receptor subtypes by Van Rhee and co-workers (1996). These results prompted this pilot study, which aimed to investigate the potential of the structurally related 3,4-dihydropyrimidin-2(1H)-ones (dihydropyrimidones) and 2-amino-1,4-dihydropyrimidines as adenosine A1 and A2A antagonists. In this pilot study, a series of 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines were synthesised and evaluated as adenosine A1 and A2A antagonists. Since several adenosine A2A antagonists also exhibit MAO inhibitory activity, the MAO-inhibitory activity of selected derivatives was also assessed. A modified Biginelli one pot synthesis was used for the preparation of both series of compounds under solvent free conditions. A mixture of a β- diketone, aldehyde and urea/guanidine hydrochloride was heated for an appropriate time to afford the desired compounds in good yields. MAO-B inhibition studies comprised of a fluorometric assay where kynuramine was used as substrate. A radioligand binding protocol described in literature was employed to investigate the binding of the compounds to the adenosine A2A and A1 receptors. The displacement of N-[3H]ethyladenosin-5’-uronamide ([3H]NECA) from rat striatal membranes and 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) from rat whole brain membranes, was used in the determination of A2A and A1 affinity, respectively. The results showed that both 3,4-dihydropyrimidones and 2-amino-1,4-dihydropyrimidines had weak adenosine A2A affinity, with the p-fluorophenyl substituted dihydropyrimidone derivative (1h) in series 1, exhibiting the highest affinity for the adenosine A2A receptor (28.7 μM), followed by the p-chlorophenyl dihydropyrimidine derivative (2c) in series 2 (38.59 μM). Both series showed more promising adenosine A1 receptor affinity in the low micromolar range. The p-bromophenyl substituted derivatives in both series showed the best affinity for the adenosine A1 receptor with Ki-values of 7.39 μM (1b) and 7.9 μM (2b). The pmethoxyphenyl dihydropyrimidone (1d) and p-methylpneyl dihydropyrimidine (2e) derivatives also exhibited reasonable affinity for the adenosine A1 receptor with Ki-values of 8.53 μM and 9.67 μM, respectively. Neither the 3,4-dihydropyrimidones nor the 2-amino-1,4- dihydropyrimidines showed MAO-B inhibitory activity. Comparison of the adenosine A2A affinity of the most potent derivative (1h, Ki = 28.7 μM) from this study with that of the previously synthesised dihydropyridine derivatives (Van Rhee et al., 1996, most potent compound had a Ki = 2.74 mM) reveals that an approximate 100- fold increase in binding affinity for A2A receptors occurred. However, KW6002, a known A2A antagonist, that has already reached clinical trials, has a Ki-value of 7.49 nM. The same trend was observed for adenosine A1 affinity, where the most potent compound (1b) of this study exhibited a Ki-value of 7.39 μM compared to 2.75 mM determined for the most potent dihydropyridine derivatives (Van Rhee et al., 1996). N6-cyclopentyladenosine (CPA), a known adenosine A1 agonist that was used as a reference compound, however had a Kivalue of 10.4 nM. The increase in both adenosine A1 and A2A affinity can most likely be ascribed to the increase in nitrogens in the heterocyclic ring (from a dihydropyridine to a dihydropyrimidine) since similar results were obtained by Gillespie and co-workers in 2009 for a series of pyridine and pyrimidine derivatives. In their case it was found that increasing the number of nitrogens in the heterocyclic ring (from one to two nitrogen atoms for the pyridine and pyrimidine derivatives respectively) increased affinity for the adenosine A2A and adenosine A1 receptor subtypes, while three nitrogen atoms in the ring (triazine derivatives) were associated with decreased affinity. It thus appears that two nitrogen atoms in the ring (pyrimidine) are required for optimum adenosine A1 and A2A receptor affinity. The poor adenosine A2A affinity exhibited by the compounds of this study can probably be attributed to the absence of an aromatic heterocyclic ring. The amino acid, Phe-168 plays a very important role in the binding site of the A2A receptor, where it forms aromatic - - stacking interactions with the heterocyclic aromatic ring systems of known agonists and antagonists. Since the dihydropyrimidine ring in both series of this pilot study was not aromatic, the formation of aromatic - -stacking interactions with Phe-168 is unlikely. In conclusion, the 3,4-dihydropyrimidone and 2-amino-1,4-dihydropyrimidine scaffolds can be used as a lead for the design of novel adenosine A1 and A2A antagonists, although further structural modifications are required before a clinically viable candidate will be available as potential treatment of PD. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Many irreversible antagonists are known to bind irreversibly to pharmacological receptors. However, few studies suggest that these irreversible antagonists may also display irreversible non-specific antagonism by binding irreversibly to non-syntopic binding sites on the receptor macromolecule, whereby they modulate the signal transduction of these receptors or reduce the agonist binding affmity. The aim of this study was to investigate whether the classical irreversible antagonists phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific antagonism at various G protein-coupled receptor (GPCR) types. In addition, the subcellular mechanism whereby benextramine displays irreversible non-specific antagonism was investigated. Three cell lines were employed to investigate the antagonism by these irreversible antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were pre-treated at the appropriate concentrations and incubation times with an appropriate irreversible antagonist, with or without an appropriate reversible competitive antagonist at a sufficient concentration to protect the specific receptors. This was followed by washing procedures with drug-free media to rinse any unbound or reversibly bound drugs from the cells. When appropriate, cell membranes were prepared. Receptor function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation, or the binding of [35S]-GTPyS to membraness. Receptor concentrations were determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated. Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10 uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent. When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively. 4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism was observed when the incubation time was increased (100 uM, 60 minutes). In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible non-specific antagonism at typical experimental conditions. These findings confirm concerns in literature and supports the possibility that more irreversible antagonists could display irreversible non-specific antagonism, and that could influence the interpretation of data obtained with such drugs. In addition, benextramine may prove to be a useful experimental drug in studying GPCR signalling. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

Many irreversible antagonists are known to bind irreversibly to pharmacological receptors. However, few studies suggest that these irreversible antagonists may also display irreversible non-specific antagonism by binding irreversibly to non-syntopic binding sites on the receptor macromolecule, whereby they modulate the signal transduction of these receptors or reduce the agonist binding affmity. The aim of this study was to investigate whether the classical irreversible antagonists phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible nonspecific antagonism at various G protein-coupled receptor (GPCR) types. In addition, the subcellular mechanism whereby benextramine displays irreversible non-specific antagonism was investigated. Three cell lines were employed to investigate the antagonism by these irreversible antagonists: Chinese hamster ovary (CHO-K1) cells transfected to express the porcine a2A-adrenoceptor (a2A-AR) at higher (a2A-H) or lower (a2A-L) numbers, human neuroblastoma (SH-SY5Y) cells that endogenously express muscarinic acetylcholine receptors (mACh-Rs), and SH-SY5Y cells transfected (5HT2A-SH-SY5Y)o express the human 5HT2A-serotonirne ceptor (5HTZA-R).C ells of the appropriate cell line were pre-treated at the appropriate concentrations and incubation times with an appropriate irreversible antagonist, with or without an appropriate reversible competitive antagonist at a sufficient concentration to protect the specific receptors. This was followed by washing procedures with drug-free media to rinse any unbound or reversibly bound drugs from the cells. When appropriate, cell membranes were prepared. Receptor function was evaluated by measuring whole-cell [3H]-cAMP or [3H]-IPx acumulation, or the binding of [35S]-GTPyS to membraness. Receptor concentrations were determined from radioligand-binding assays. In addition, the constitutive [35S]-GTPyS binding to Go protein before and after pre-treatment with benextramine was investigated. Results suggest that phenoxybenzamine (100 uM, 20 minutes) and benextramine (10 uM, 20 minutes) display irreversible non-specific antagonism at a2A-ARs when measuring Gi-mediated effects in a2A-L cells, but the affinity for a2A-ARs in a2A-H cells was not changed. In addition, it was found that the observed irreversible nonspecific antagonism by benextramine appears to be time- and concentration-dependent. When the mechanism of irreversible antagonism by benextramine was further investigated, benextramine reduced the binding of [35S]-GTPyS to a2A-H membranes with protected a2A-ARs, but did not modulate the constitutive binding of [35S]-GTPyS to Go. In addition, benextramine displays irreversible non-specific antagonism by inhibiting the G,-mediated effects of a2A-ARs in a2A-H cells and the Gq-mediated effects of mACh-Rs or 5HT2A-Rs in SH-SY5Y or 5HT2A-SH-SY5Y cells respectively. 4-DAMP mustard (100 uM, 20 minutes) did not display irreversible non-specific antagonism at mACh-Rs in SH-SY5Y cells, but irreversible non-specific antagonism was observed when the incubation time was increased (100 uM, 60 minutes). In conclusion it was found that phenoxybenzamine, benextramine and 4-DAMP mustard display irreversible non-specific antagonism at typical experimental conditions. These findings confirm concerns in literature and supports the possibility that more irreversible antagonists could display irreversible non-specific antagonism, and that could influence the interpretation of data obtained with such drugs. In addition, benextramine may prove to be a useful experimental drug in studying GPCR signalling. / Thesis (Ph.D. (Pharmacology))--North-West University, Potchefstroom Campus, 2004.

L’accumulation de triglycérides (TG) dans les hépatocytes est caractéristique de la stéatose hépatique non-alcoolique (SHNA). Cette dernière se produit dans diverses conditions dont le facteur commun est le métabolisme anormal des lipides. Le processus conduisant à l'accumulation des lipides dans le foie n’a pas encore été totalement élucidé. Toutefois, des lipides s'accumulent dans le foie lorsque les mécanismes qui favorisent leur exportation (oxydation et sécrétion) sont insuffisants par rapport aux mécanismes qui favorisent leur importation ou leur biosynthèse. De nos jours il est admis que la carence en œstrogènes est associée au développement de la stéatose hépatique. Bien que les résultats des études récentes révèlent l'implication des hormones ovariennes dans l'accumulation de lipides dans le foie, les mécanismes qui sous-tendent ce phénomène doivent encore être étudiés. En conséquence, les trois études présentées dans cette thèse ont été menées sur des rates ovariectomizées (Ovx), comme modèle animal de femmes post-ménopausées, pour étudier les effets du retrait des œstrogènes sur le métabolisme des lipides dans le foie, en considérant l'entraînement physique comme étant un élément positif pouvant contrecarrer ces effets. Il a été démontré que l'entraînement physique peut réduire l'accumulation de graisses dans le foie chez les rates Ovx. Dans la première étude, nous avons montré que chez les rates Ovx nourries à la diète riche en lipides (HF), les contenus de TG hépatiques étaient élevées (P < 0.01) comparativement aux rates Sham, 5 semaines après la chirurgie. Le changement de la diète HF par la diète standard (SD) chez les rates Sham a diminué l’accumulation de lipides dans le foie. Toutefois, chez les rates Ovx, 8 semaines après le changement de la HF par la SD le niveau de TG dans le foie était maintenu aussi élevé que chez les rates nourries continuellement avec la diète HF. Lorsque les TG hépatiques mesurés à la 13e semaine ont été comparés aux valeurs correspondant au retrait initial de la diète HF effectué à la 5e semaine, les niveaux de TG hépatiques chez les animaux Ovx ont été maintenus, indépendamment du changement du régime alimentaire; tandis que chez les rats Sham le passage à la SD a réduit (P < 0.05) les TG dans le foie. Les mêmes comparaisons avec la concentration des TG plasmatiques ont révélé une relation inverse. Ces résultats suggèrent que la résorption des lipides au foie est contrée par l'absence des œstrogènes. Dans cette continuité, nous avons utilisé une approche physiologique dans notre seconde étude pour investiguer la façon dont la carence en œstrogènes entraîne l’accumulation de graisses dans le foie, en nous focalisant sur la voie de l'exportation des lipides du foie. Les résultats de cette étude ont révélé que le retrait des œstrogènes a entraîné une augmentation (P < 0.01) de l’accumulation de lipides dans le foie en concomitance avec la baisse (P < 0.01) de production de VLDL-TG et une réduction l'ARNm et de la teneur en protéines microsomales de transfert des triglycérides (MTP). Tous ces effets ont été corrigés par la supplémentation en œstrogènes chez les rates Ovx. En outre, l'entraînement physique chez les rates Ovx a entraîné une réduction (P < 0.01) de l’accumulation de lipides dans le foie ainsi qu’une diminution (P < 0.01) de production de VLDL-TG accompagnée de celle de l'expression des gènes MTP et DGAT-2 (diacylglycérol acyltransférase-2). Des études récentes suggèrent que le peptide natriurétique auriculaire (ANP) devrait être au centre des intérêts des recherches sur les métabolismes énergétiques et lipidiques. Le ANP est relâché dans le plasma par les cellules cardiaques lorsque stimulée par l’oxytocine et exerce ses fonctions en se liant à son récepteur, le guanylyl cyclase-A (GC-A). En conséquence, dans la troisième étude, nous avons étudié les effets du blocage du système ocytocine-peptide natriurétique auriculaire (OT-ANP) en utilisant un antagoniste de l’ocytocine (OTA), sur l'expression des gènes guanylyl cyclase-A et certains marqueurs de l’inflammation dans le foie de rates Ovx. Nous avons observé une diminution (P < 0.05) de l’ARNm de la GC-A chez les rates Ovx et Sham sédentaires traitées avec l’OTA, tandis qu’une augmentation (P < 0.05) de l'expression de l’ARNm de la protéine C-réactive (CRP) hépatique a été notée chez ces animaux. L’exercice physique n'a apporté aucun changement sur l'expression hépatique de ces gènes que ce soit chez les rates Ovx ou Sham traitées avec l’OTA. En résumé, pour expliquer l’observation selon laquelle l’accumulation et la résorption de lipides dans le foie dépendent des mécanismes associés à des niveaux d’œstrogènes, nos résultats suggèrent que la diminution de production de VLDL-TG induite par une déficience en œstrogènes, pourrait être un des mecanismes responsables de l’accumulation de lipides dans le foie. L’exercice physique quant à lui diminue l'infiltration de lipides dans le foie ainsi que la production de VLDL-TG indépendamment des niveaux d'œstrogènes. En outre, l'expression des récepteurs de l’ANP a diminué par l'OTA chez les rates Ovx et Sham suggérant une action indirecte de l’ocytocine (OT) au niveau du foie indépendamment de la présence ou non des estrogènes. L’axe ocytocine-peptide natriurétique auriculaire, dans des conditions physiologiques normales, protègerait le foie contre l'inflammation à travers la modulation de l’expression de la GC-A. / Excessive accumulation of triglycerides (TGs) in hepatocytes is the characteristic of non-alcoholic hepatic steatosis (NAHS). NAHS occurs in various conditions in which abnormal fat metabolism is a common factor. The primary processes leading to lipid accumulation in the liver are not well understood. However, lipid in the form of TG accumulates within liver cells when mechanisms that promote their removal (by oxidation or secretion) cannot keep pace with mechanisms that promote lipid import or biosynthesis. Today, it is well accepted that estrogen deficiency is associated with the development of a state of hepatic steatosis. Although recent findings indicated the implication of ovarian hormones in liver lipid accumulation, mechanisms underlying this phenomenon need to be further investigated. Therefore, the three studies presented in this thesis have been conducted in ovariectomized (Ovx) rats, as animal model of post-menopausal women, to investigate the effects of estrogen withdrawal on liver fat metabolism and considering the effects of exercise training as a positive counteractive factor. It has been shown that exercise training can reduce liver fat accumulation in Ovx rats. In the first study, we showed that in high fat (HF) fed animals, liver TG content was higher (P < 0.01) in Ovx compared to Sham rats as soon as 5-week after the surgery. Switching from the HF to a standard (SD) diet resulted in a decrease in liver fat accumulation in Sham animals. However, 8 weeks after the diet switch, liver fat accumulation was as high in Ovx rats as those maintained on the HF diet. When liver TG content measured at week 13 was compared to initial pre-switching values (week 5), liver TG levels in Ovx animals were maintained at the same level independently of the diet switch, while in Sham rats switching to a SD diet reduced liver TG accumulation (P < 0.05). The same comparisons with plasma TG levels revealed an opposite relationship. These results may be taken as evidence that indeed liver fat resorption is hampered in the absence of estrogens. To go one step further, we used a physiological approach in our second study to investigate how estrogen deficiency affects liver fat accumulation putting an emphasis on the pathway of lipid exportation from the liver. Results of this study showed that estrogen withdrawal resulted in higher (P < 0.01) liver fat accumulation concomitantly with lower (P < 0.01) very low density lipoprotein-triglyceride (VLDL-TG) production and lower mRNA and protein content of hepatic microsomal triglyceride transfer protein (MTP). All of these effects in Ovx rats were corrected with estrogen supplementation. Moreover, exercise training in Ovx rats reduced (P < 0.01) liver fat accumulation and further reduced (P < 0.01) hepatic VLDL-TG production along with gene expression of MTP and diacylglycerol acyltransferase-2 (DGAT-2). A recent growing body of literature suggests that atrial natriuretic peptide (ANP) hormone should be the interest of new investigations in the field of energy and lipid metabolism. ANP is released from the heart into plasma by oxytocin (OT) stimulation and exerts its biological action by binding to its receptor, guanylyl cyclase-A (GC-A: ANP receptor). Therefore, in the third study, we investigated the effects of blocking the oxytocin-atrial natriuretic peptide (OT-ANP) system, using an OT antagonist (OTA), on the gene expression of hepatic guanylyl cyclase-A and some inflammatory markers in the liver of Ovx rats. Hepatic GC-A mRNAs were decreased (P < 0.05) in Ovx and Sham OTA-treated rats in the sedentary state, contrary to hepatic C-reactive protein (CRP) mRNA expression that increased in these animals (P < 0.05). Exercise training had no effect on hepatic expression of these genes in both Sham and Ovx rats receiving OTA. Overall, our results point to the interpretation that hepatic fat accumulation and resorption are dependent on mechanisms associated with a normal estrogenic status; indicating that a decrease in VLDL-TG production might be a contributing factor responsible for the hepatic fat accumulation induced by estrogen deficiency. Exercise training lowers liver fat accretion and VLDL-TG production independently of the estrogen levels. Moreover, hepatic expression of ANP receptors is decreased by OTA in both Sham and Ovx rats suggesting an indirect action of the OT system on the liver independently of the estrogenic status of the animal. Oxytocin-atrial natriuretic peptide axis may contribute to the protection of hepatic tissue under normal physiological conditions such as reducing inflammatory markers within the hepatocytes by exerting its role through guanylyl cyclase-A expression.

L’accumulation de triglycérides (TG) dans les hépatocytes est caractéristique de la stéatose hépatique non-alcoolique (SHNA). Cette dernière se produit dans diverses conditions dont le facteur commun est le métabolisme anormal des lipides. Le processus conduisant à l'accumulation des lipides dans le foie n’a pas encore été totalement élucidé. Toutefois, des lipides s'accumulent dans le foie lorsque les mécanismes qui favorisent leur exportation (oxydation et sécrétion) sont insuffisants par rapport aux mécanismes qui favorisent leur importation ou leur biosynthèse. De nos jours il est admis que la carence en œstrogènes est associée au développement de la stéatose hépatique. Bien que les résultats des études récentes révèlent l'implication des hormones ovariennes dans l'accumulation de lipides dans le foie, les mécanismes qui sous-tendent ce phénomène doivent encore être étudiés. En conséquence, les trois études présentées dans cette thèse ont été menées sur des rates ovariectomizées (Ovx), comme modèle animal de femmes post-ménopausées, pour étudier les effets du retrait des œstrogènes sur le métabolisme des lipides dans le foie, en considérant l'entraînement physique comme étant un élément positif pouvant contrecarrer ces effets. Il a été démontré que l'entraînement physique peut réduire l'accumulation de graisses dans le foie chez les rates Ovx. Dans la première étude, nous avons montré que chez les rates Ovx nourries à la diète riche en lipides (HF), les contenus de TG hépatiques étaient élevées (P < 0.01) comparativement aux rates Sham, 5 semaines après la chirurgie. Le changement de la diète HF par la diète standard (SD) chez les rates Sham a diminué l’accumulation de lipides dans le foie. Toutefois, chez les rates Ovx, 8 semaines après le changement de la HF par la SD le niveau de TG dans le foie était maintenu aussi élevé que chez les rates nourries continuellement avec la diète HF. Lorsque les TG hépatiques mesurés à la 13e semaine ont été comparés aux valeurs correspondant au retrait initial de la diète HF effectué à la 5e semaine, les niveaux de TG hépatiques chez les animaux Ovx ont été maintenus, indépendamment du changement du régime alimentaire; tandis que chez les rats Sham le passage à la SD a réduit (P < 0.05) les TG dans le foie. Les mêmes comparaisons avec la concentration des TG plasmatiques ont révélé une relation inverse. Ces résultats suggèrent que la résorption des lipides au foie est contrée par l'absence des œstrogènes. Dans cette continuité, nous avons utilisé une approche physiologique dans notre seconde étude pour investiguer la façon dont la carence en œstrogènes entraîne l’accumulation de graisses dans le foie, en nous focalisant sur la voie de l'exportation des lipides du foie. Les résultats de cette étude ont révélé que le retrait des œstrogènes a entraîné une augmentation (P < 0.01) de l’accumulation de lipides dans le foie en concomitance avec la baisse (P < 0.01) de production de VLDL-TG et une réduction l'ARNm et de la teneur en protéines microsomales de transfert des triglycérides (MTP). Tous ces effets ont été corrigés par la supplémentation en œstrogènes chez les rates Ovx. En outre, l'entraînement physique chez les rates Ovx a entraîné une réduction (P < 0.01) de l’accumulation de lipides dans le foie ainsi qu’une diminution (P < 0.01) de production de VLDL-TG accompagnée de celle de l'expression des gènes MTP et DGAT-2 (diacylglycérol acyltransférase-2). Des études récentes suggèrent que le peptide natriurétique auriculaire (ANP) devrait être au centre des intérêts des recherches sur les métabolismes énergétiques et lipidiques. Le ANP est relâché dans le plasma par les cellules cardiaques lorsque stimulée par l’oxytocine et exerce ses fonctions en se liant à son récepteur, le guanylyl cyclase-A (GC-A). En conséquence, dans la troisième étude, nous avons étudié les effets du blocage du système ocytocine-peptide natriurétique auriculaire (OT-ANP) en utilisant un antagoniste de l’ocytocine (OTA), sur l'expression des gènes guanylyl cyclase-A et certains marqueurs de l’inflammation dans le foie de rates Ovx. Nous avons observé une diminution (P < 0.05) de l’ARNm de la GC-A chez les rates Ovx et Sham sédentaires traitées avec l’OTA, tandis qu’une augmentation (P < 0.05) de l'expression de l’ARNm de la protéine C-réactive (CRP) hépatique a été notée chez ces animaux. L’exercice physique n'a apporté aucun changement sur l'expression hépatique de ces gènes que ce soit chez les rates Ovx ou Sham traitées avec l’OTA. En résumé, pour expliquer l’observation selon laquelle l’accumulation et la résorption de lipides dans le foie dépendent des mécanismes associés à des niveaux d’œstrogènes, nos résultats suggèrent que la diminution de production de VLDL-TG induite par une déficience en œstrogènes, pourrait être un des mecanismes responsables de l’accumulation de lipides dans le foie. L’exercice physique quant à lui diminue l'infiltration de lipides dans le foie ainsi que la production de VLDL-TG indépendamment des niveaux d'œstrogènes. En outre, l'expression des récepteurs de l’ANP a diminué par l'OTA chez les rates Ovx et Sham suggérant une action indirecte de l’ocytocine (OT) au niveau du foie indépendamment de la présence ou non des estrogènes. L’axe ocytocine-peptide natriurétique auriculaire, dans des conditions physiologiques normales, protègerait le foie contre l'inflammation à travers la modulation de l’expression de la GC-A. / Excessive accumulation of triglycerides (TGs) in hepatocytes is the characteristic of non-alcoholic hepatic steatosis (NAHS). NAHS occurs in various conditions in which abnormal fat metabolism is a common factor. The primary processes leading to lipid accumulation in the liver are not well understood. However, lipid in the form of TG accumulates within liver cells when mechanisms that promote their removal (by oxidation or secretion) cannot keep pace with mechanisms that promote lipid import or biosynthesis. Today, it is well accepted that estrogen deficiency is associated with the development of a state of hepatic steatosis. Although recent findings indicated the implication of ovarian hormones in liver lipid accumulation, mechanisms underlying this phenomenon need to be further investigated. Therefore, the three studies presented in this thesis have been conducted in ovariectomized (Ovx) rats, as animal model of post-menopausal women, to investigate the effects of estrogen withdrawal on liver fat metabolism and considering the effects of exercise training as a positive counteractive factor. It has been shown that exercise training can reduce liver fat accumulation in Ovx rats. In the first study, we showed that in high fat (HF) fed animals, liver TG content was higher (P < 0.01) in Ovx compared to Sham rats as soon as 5-week after the surgery. Switching from the HF to a standard (SD) diet resulted in a decrease in liver fat accumulation in Sham animals. However, 8 weeks after the diet switch, liver fat accumulation was as high in Ovx rats as those maintained on the HF diet. When liver TG content measured at week 13 was compared to initial pre-switching values (week 5), liver TG levels in Ovx animals were maintained at the same level independently of the diet switch, while in Sham rats switching to a SD diet reduced liver TG accumulation (P < 0.05). The same comparisons with plasma TG levels revealed an opposite relationship. These results may be taken as evidence that indeed liver fat resorption is hampered in the absence of estrogens. To go one step further, we used a physiological approach in our second study to investigate how estrogen deficiency affects liver fat accumulation putting an emphasis on the pathway of lipid exportation from the liver. Results of this study showed that estrogen withdrawal resulted in higher (P < 0.01) liver fat accumulation concomitantly with lower (P < 0.01) very low density lipoprotein-triglyceride (VLDL-TG) production and lower mRNA and protein content of hepatic microsomal triglyceride transfer protein (MTP). All of these effects in Ovx rats were corrected with estrogen supplementation. Moreover, exercise training in Ovx rats reduced (P < 0.01) liver fat accumulation and further reduced (P < 0.01) hepatic VLDL-TG production along with gene expression of MTP and diacylglycerol acyltransferase-2 (DGAT-2). A recent growing body of literature suggests that atrial natriuretic peptide (ANP) hormone should be the interest of new investigations in the field of energy and lipid metabolism. ANP is released from the heart into plasma by oxytocin (OT) stimulation and exerts its biological action by binding to its receptor, guanylyl cyclase-A (GC-A: ANP receptor). Therefore, in the third study, we investigated the effects of blocking the oxytocin-atrial natriuretic peptide (OT-ANP) system, using an OT antagonist (OTA), on the gene expression of hepatic guanylyl cyclase-A and some inflammatory markers in the liver of Ovx rats. Hepatic GC-A mRNAs were decreased (P < 0.05) in Ovx and Sham OTA-treated rats in the sedentary state, contrary to hepatic C-reactive protein (CRP) mRNA expression that increased in these animals (P < 0.05). Exercise training had no effect on hepatic expression of these genes in both Sham and Ovx rats receiving OTA. Overall, our results point to the interpretation that hepatic fat accumulation and resorption are dependent on mechanisms associated with a normal estrogenic status; indicating that a decrease in VLDL-TG production might be a contributing factor responsible for the hepatic fat accumulation induced by estrogen deficiency. Exercise training lowers liver fat accretion and VLDL-TG production independently of the estrogen levels. Moreover, hepatic expression of ANP receptors is decreased by OTA in both Sham and Ovx rats suggesting an indirect action of the OT system on the liver independently of the estrogenic status of the animal. Oxytocin-atrial natriuretic peptide axis may contribute to the protection of hepatic tissue under normal physiological conditions such as reducing inflammatory markers within the hepatocytes by exerting its role through guanylyl cyclase-A expression.