Dissertations / Theses on the topic 'OTX proteins'

The microbiological safety of avian eggs is a major concern for the poultry industry and for consumers due to the potential for severe impacts on public health. Innate immune defense is formed by proteins with antimicrobial and immune-modulatory activities and ensures the protection of the chick embryo against pathogens. The objective of this project was to identify the chicken eggshell membrane (ESM) proteins that play a role in these innate immune defense mechanisms. We hypothesized that ESM Ovocalyxin-36 (OCX-36) is a pattern recognition protein, and characterized purified ESM OCX-36. OCX-36 has antimicrobial activity against S. aureus and binds E. coli lipopolysaccharide (LPS) and S. aureus lipoteichoic acid (LTA). We additionally investigated the OCX-36 nonsynonymous single nucleotide polymorphisms (SNPs) at cDNA position 211. The corresponding isoforms (proline-71 or serine-71) were purified from eggs collected from genotyped homozygous hens. A significant difference between Pro-71 and Ser-71 OCX-36s for S. aureus LTA binding activity was observed. From these experiments, we confirmed the hypothesis that OCX-36 is a pattern recognition molecule. We also found that OCX-36 has anti-endotoxin properties and is a macrophage immunostimulator to produce NO and TNF-α. Digested OCX-36 down-regulated the expression of genes involved in LPS signaling and inflammatory responses. Moreover, OCX-36-derived peptides inhibited the production of LPS-induced pro-inflammatory mediators associated with endotoxemia in vivo. Quantitative proteomics analysis of ESMs was performed to evaluate changes in ESM protein abundance during chick embryonic development. Bioinformatics analysis revealed enrichment of proteins associated with antimicrobial and immune protection, vascularization, calcium mobilization and lipid transport, which are vital for chick embryonic development. In unfertilized eggs, protease inhibitors and antimicrobial proteins were enriched. In summary, the ESMs are enriched in proteins with antimicrobial, antioxidant and immune-modulatory properties, which aid in the development of the chick embryo and protect the embryo and unfertilized egg against pathogen invasion.

Bacteria are one of the largest causes of human disease, with millions of deaths every year attributed to bacterial infections, and they have become more difficult to tackle with the widespread emergence of antibiotic resistance. In this thesis, I describe my studies that pursued two approaches: one focus was on using antimicrobial histones as an alternative to treatment for antibiotic resistant bacteria; in another approach the recombinant version of an eggshell cuticle protein was expressed and purified for testing against food-safety pathogens. One major pathogen that is contributing to this challenge of antibiotic resistance is Staphylococcus aureus. The methicillin-resistant strain of S. aureus leads to increased hospital stays and increased mortality in patients. The impact of such pathogens is worsened when bacteria form surface-attached aggregates known as biofilms. Development of new approaches to eradicate antibiotic- resistant biofilms will benefit human health. This study looked at an alternative method to eradicate bacteria compared to traditional antibiotics. Histones with antimicrobial activity were extracted from chicken blood and tested against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus biofilm (MSSA and MRSA). The histone mixture completely eradicated both strains in biofilm form at relatively low concentrations. In addition, the histone mixture also displayed fast kill kinetics against planktonic forms of the two strains. Finally, the interaction of the histone mixture with the bacterial membrane in MRSA biofilms was observed by scanning electron microscopy (SEM). Bacteria treated with the histone mixture showed clear morphological changes, including pore formation and cell collapse. Therefore, the histone mixture purified from chicken red blood cells could prove to be a good alternative to traditional antibiotics for protection against antibiotic-resistant strains of bacteria in their planktonic and biofilm forms. Reduction of food-borne illness is another important aspect in the promotion of human health. A significant contributor to food-borne illness is contaminated table eggs. The unfertilized egg can be contaminated by a variety of pathogens including Salmonella spp. and Bacillus spp. The egg is protected by the eggshell which is traversed by respiratory pores that are normally covered by a cuticle plug to restrict pathogen entry. This cuticle consists of several proteins including ovocaxlyin-32 (OCX-32). OCX-32 has a large number of naturally occurring haplotypes due non-synonymous single nucleotide polymorphisms (SNPs). In this study, the goal was to express five of the most common haplotypes of OCX-32 in Escherichia coli and purify the recombinant protein for assay of its antimicrobial activity. Five constructs that contain the cDNA of common OCX-32 haplotypes (A, B, C, D, and O) with a histidine tag at the C-terminus were generated. The constructs were subcloned into pGEX4T-1 vector which encodes Glutathione-S-transferase (GST) upstream of the multiple cloning site. My study developed methods to optimize the expression conditions, and to increase the solubility of the recombinant protein. Various expression strains of E. coli and solubility buffers were tested. In addition, the construct was subcloned into a plasmid containing the small ubiquitin-like modifier (SUMO) fusion tag; the solubility of the new SUMO-OCX-32 haplotype A recombinant fusion protein was evaluated. The best results were obtained by slow dialysis refolding of denatured SUMO-OCX-32 fusion protein. This recombinant protein showed almost complete solubility with minimal precipitation and was tested against the egg-related pathogen, Bacillus cereus. Unfortunately, the SUMO-OCX-32 recombinant protein did not inhibit growth of B. cereus. In my studies reported in this thesis, two very different approaches were taken. A histone mixture was isolated from an abundant starting material, which proved to be highly effective and promising in the eradication of S. aureus biofilms at relatively low concentrations. Alternatively, expression of a soluble recombinant protein for functional activity assay was very challenging and required the optimization of a number of methods to prepare soluble protein for testing. One of the methods tested proved effective in obtaining large amounts of soluble protein. However, further developmental work will be essential to determine if this approach is a viable strategy in acquiring functional protein.

Le plexus choroïde est une structure fortement vascularisée. Situé dans les ventricules cérébraux, il est responsable de la production du liquide céphalorachidien (LCR) contenant un large panel de molécules essentielles aux diverses fonctions cérébrales. Ma thèse a porté sur la découverte de deux protéines exprimées par le plexus choroïde qui modulent la neurogenèse adulte chez la souris : homéoprotéine OTX2 et la protéine précurseur de l'amyloide (PPA). OTX2 est fortement exprimé dans le plexus choroïde et est sécrété dans le LCR. Nous avons découvert qu'il peut réguler la neurogenèse chez la souris adulte en transférant depuis les cellules du plexus choroïde dans les astrocytes de soutien de la zone sous-ventriculaire (ZSV) et de la chaine migratoire rostral (CMR). Nous avons mis en évidence un mécanisme potentiel par lequel OTX2 modifie l'expression des protéines de la matrice extracellulaire spécifiques des astrocytes, ce qui ralentit la migration des neuroblastes dans la CMR et diminue le nombre de nouveaux neurones dans le bulbe olfactif. De plus, grâce à une analyse transcriptomique du plexus choroïde à partir de modèles de souris knockout conditionnels et constitutifs pour Otx2 chez l'adulte, nous avons montré qu'OTX2 régule l'expression des gènes impliqués dans de nombreuses fonctions homéostatiques. Associé à une approche d’élimination virale d'OTX2, nous avons confirmé son implication dans la réponse immunitaire, la réponse au stress oxydatif, l’épissage et homéostasie des métaux. Ces résultats démontrent qu'OTX2 est un régulateur majeur des fonctions essentielles du plexus choroïde pour le maintien de homéostasie cérébrale. Enfin, la PPA est également fortement exprimée dans le plexus choroïde et son domaine soluble est libéré dans le LCR. Lorsque nous avons réduit les concentrations de PPA dans le plexus choroïde chez la souris adulte, nous avons observé une diminution de la prolifération dans les deux niches neurogéniques : la ZSV et le gyrus denté de l'hippocampe (GD). L'augmentation des concentrations de PPA a entraîné une prolifération accrue dans ces deux niches. Pour mesurer l'impact sur les fonctions cérébrales de la PPA extracellulaire provenant du plexus choroïde, nous avons exprimé spécifiquement dans le plexus choroïde de souris sauvage adultes, la PPA humaine porteuse de mutations SwInd de la maladie d'Alzheimer familiale. Après 6 mois, la prolifération dans la ZSV et le GD a été considérablement réduite. Après 12 mois, des souris ont montré des déficits d'apprentissage inverse dans des expériences de comportement en champ ouvert et ont montré une plasticité altérée dans la potentialisation à long terme (PLT) mesurée par stimulation à haute fréquence de tranches d'hippocampe
The choroid plexus is a richly vascularized structure located in brain ventricles and is responsible for the production of cerebrospinal fluid (CSF) containing a large panel of molecules essential for various brain functions. My thesis focused on the discovery of two proteins expressed by the choroid plexus that modulate adult neurogenesis in mice: the OTX2 homeoprotein transcription factor and the amyloid precursor protein (APP). OTX2 is highly expressed in the choroid plexus and is secreted into the CSF. We found that it can regulate neurogenesis in the adult mouse by transferring into supporting astrocytes of the subventricular zone (SVZ) and rostral migratory stream (RMS). We revealed a potential mechanism whereby OTX2 alters the expression of astrocyte-specific extracellular matrix proteins, resulting in slowed neuroblast migration in RMS and decreased newborn neurons in the olfactory bulb. Moreover, by transcriptomic analysis of the choroid plexus from conditional and constitutive Otx2 knockdown adult mouse models, we shown that OTX2 regulates the expression of genes implicated in numerous homeostatic functions. Coupled with a viral Otx2 knockdown approach, we confirmed its involvement in immune response, oxidative stress response, splicing and metal homeostasis. These findings provide evidence that OTX2 is a major regulator of essential choroid plexus functions for the maintenance of brain homeostasis. Finally, APP is also highly expressed in the choroid plexus and its soluble domain is released into the CSF. When we reduced APP levels in the adult mouse choroid plexus, we observed decreased proliferation in both neurogenic niches: SVZ and hippocampus dentate gyrus (DG). Increasing APP levels resulted in increased proliferation in both niches. To gage the impact on brain function of extracellular APP coming from choroid plexus, we expressed, specifically in choroid plexus of adult wild type mice, the human APP bearing SwInd mutations of familial Alzheimer disease. After 6 months, proliferation in SVZ and DG were significantly reduced. After 12 months, mice showed reversal learning deficits in open field behavior experiments and showed impaired plasticity in long term potentiation (LTP) measured by high frequency stimulation of hippocampus slices

Octa-heme cytochrome c tetrathionate reductase (OTR) from Shewanella oneidensis MR-1 is a periplasmic protein and shows several extraordinary structural features around its active-site heme. OTR has been found able to catalyse the in vitro reduction of tetrathionate, nitrite, hydroxylamine and hydrogen peroxide. However the physiological function of this novel protein remains unknown. The subject of this thesis is the in vitro catalytic mechanism and the in vivo function of OTR. As OTR displays great similarity with bacterial penta-heme cytochrome c nitrite reductase (NrfA) in several aspects, it has been proposed that OTR might be physiologically involved in the metabolism of nitrite or other nitrogenous compounds. However kinetics assays and phenotypes studies carried out in this project suggest this is not the case. In vitro kinetic assays of the reduction of nitrite and hydroxylamine catalysed by OTR showed no significant difference in enzyme activities among the wild-type OTR and its mutant forms which have one active site residue replaced by alanine, namely OTR K153A, C64A, N61A and D150A. And the nitrite reductase activity of OTR (kcat/Km = 1.0×105 M-1•s-1) are much lower than that of NrfA (kcat/Km = ~108 M-1•s-1). These results indicate that OTR is not specifically adapted to reduce nitrite and it cannot compete for nitrite against NrfA in vivo. No phenotype difference was identified between the wild-type and the Δotr strain of Shewanella oneidensis MR-1 when nitrite or nitrate served as the sole electron acceptor. OTR appears not to be involved in the respiration or detoxification of nitrite, which is consistent with previous transcriptional and phenotype reports that involve OTR or its homologues. The in vitro tetrathionate reduction activity of OTR was unable to be reproduced in this project for unknown reasons. Although transcriptomic data from the literature suggest that OTR may be related to the metabolism of sulphur-containing compounds, kinetic and phenotype studies reveal that OTR does not directly participate in the respiration of thiosulfate, sulfite, tetrathionate, polysulfide or elemental sulphur. Cysteine 64 is a highly-conserved amino acid residue of OTR close to the active site and its side-chain sulphur atom is covalently bonded by either an oxygen or a sulphur atom as observed in the crystal structure. Such a modification is potentially important to the function of OTR. ESI mass spectroscopy results show that in native OTR the modified form is around 48 Da heavier than the unmodified form, and the MALDITOF peptide mass spectra show that the modified form could be converted into the unmodified form by reducing agent DTT. These results suggest that the modification could be a cysteine persulfide attaching an extra oxygen atom in the form of water or hydroxide anion.

Pour comprendre comment les gènes du développement exercent de multiples fonctions temporelles, nous prenons comme modèle le facteur de transcription Otx2. Celui-ci est impliqué dans la gastrulation, le développement de l'œil, du système olfactif, de la glande pinéale, du thalamus et de la région cranio-faciale. Dans la rétine adulte, deux tissus distincts expriment Otx2 : l'épithélium pigmenté (RPE) et la rétine neurale, contenant les photorécepteurs. L'ablation globale du gène Otx2 entraîne la dégénérescence exclusive des photorécepteurs alors qu'elle modifie l'expression de gènes surtout dans le RPE. Ces faits suggèrent un mécanisme non autonome, confirmé par des expériences de gain et perte de fonction restreintes au RPE. Pour approcher les fonctions de la protéine Otx2 dans la rétine neurale et le RPE, une étude à grande échelle de ses cibles génomiques a été menée. Les profils distincts d'occupation du génome du RPE et de la rétine neurale suggèrent des fonctions différentes d'Otx2. Dans la rétine neurale, ce profil est très proche de celui du facteur paralogue Crx, indiquant une redondance fonctionnelle entre Otx2 et Crx. Nous avons émis l'hypothèse qu'une combinatoire de partenaires protéiques différents permet de moduler l'action d'Otx2 en sélectionnant des cibles génomiques distinctes. Pour identifier cette combinatoire in vivo et la corréler aux fonctions exercées par Otx2, nous avons créé une lignée de souris exprimant une protéine de fusion Otx2-TAP-tag à un niveau physiologique. Cet outil permettra la purification des complexes protéiques Otx2 in vivo et leur identification par analyse protéomique.

Thesis (M.Sc. (Horticulture)) --University of Limpopo, 2009
Field experiments were conducted at the University of Limpopo experimental farm, Syferkuil during 2005/06 and 2006/07 production seasons. This was initiated to examine the effect of leaf removal on cowpea biomass, protein content and grain yield under sole and binary cultures. Treatments consisted of cowpea varieties (Pan 311 and Red caloona), cropping systems (sole and intercropping) and cowpea-leaf pruning regimes (pruning and un-pruned). Sweet corn was planted, as a component crop in the intercropped plots while sole sweet corn plot was included as a treatment. All treatment combinations were laid out as Randomize complete block design (RCBD) with four replicates. Supplementary irrigation was carried out during the plant growth period. Fully expanded leaves were harvested once on all cowpea plants in the two middle rows from designated plots at seven weeks after planting for each year. Growth and yield data were collected from component crops during the course of the trial while the protein content of harvested leaves and immature pods as well as the different cowpea plant parts at harvest were determined. Results of the study revealed that leaves of cowpea variety, Pan 311 harvested prior to the reproductive stage had significantly higher protein content than those of Red caloona. Protein content of immature Pan 311 pods had higher (18.8 to 25.1%) than Red caloona (17.9 to 20.7%) during both planting seasons. The percent protein content of cowpea stem obtained at harvest for Pan 311 varied between 9.3 and 9.4%, and between 9.9 and 12.3% for Red caloona during both planting seasons. Grain yield obtained for Pan 311 and Red caloona were 1703.7 kg ha-1 and 1479.8 kg ha-1, respectively during 2005/06 and 1290.7 kg ha-1 and 511.7 kg ha-1 respectively during 2006/07 planting seasons. Sweet corn intercropped with Red caloona during both planting seasons had higher average grain yield than when intercropped with Pan 311. Although intercropping decreased the partial land equivalent ratio (LER) value of individual component crops, the combined LER values of between 1.1 and 2.3 under intercrop for the different treatment combinations implies that the practice is advantageous. The results of post harvest soil analyses revealed that topsoil has the pH value of 7.11-7.29 indicating neutral soil while subsoil pH value of 6.27-6.91 indicated slightly acidic to neutral soil during both planting seasons. Based on the findings of this study, cowpea variety Pan 311 can be recommended as a better vegetable crop than Red caloona since it has higher leaf and immature pod protein content. It also had higher grain yield than Red caloona when intercropped with sweet corn. Sweet corn had high grain yield when intercropped with Red caloona than when intercropped with Pan 311. Keywords: Cropping systems, protein content, grain yields, leaf pruning and cowpea.

La carcinose péritonéale est une atteinte néoplasique métastatique de la séreuse péritonéale caractérisée par la diffusion de multiples nodules tumoraux. Son pronostic est sombre, marqué par une chimiorésistance. Les traitements intrapéritonéaux, développés pour délivrer des drogues de chimiothérapie anti-cancéreuse directement au contact de ces nodules, ont permis d’améliorer en partie les résultats oncologiques de cette pathologie. Le principe est de mettre à profit la barrière péritonéo-plasmatique pour administrer des posologies plus élevées de drogues, directement au contact des nodules, et ainsi majorer leur cytotoxicité. En stratégie curative, la chimiothérapie intrapéritonéale est associée à une chirurgie de cytoréduction (CRS) complète et son efficacité est majorée par l’adjonction d’une hyperthermie (ChimioHyperthermie IntraPéritonéale - CHIP). Si ce traitement combiné a transformé le pronostic de patients sélectionnés, les résultats restent insatisfaisants. Par exemple les patients atteints de carcinose d’origine colorectale présentent un taux de survie globale à 5 ans de 40% lorsqu’ils sont éligibles à la CRS-CHIP et une médiane de survie de l’ordre de 16 mois quand le traitement se cantonne à de la chimiothérapie systémique.Une meilleure compréhension des mécanismes cellulaires impliqués dans cette chimiorésistance est donc nécessaire pour déterminer de nouvelles cibles thérapeutiques. Les protéines de choc thermique jouent un rôle fondamental dans l’homéostasie protéique intracellulaire en agissant comme protéines chaperonnes et en régulant l’architecture du cytosquelette. L’Hsp27 (ou HspB1) en particulier est impliquée dans la réponse à différents stress cellulaires comme le choc thermique, le stress oxydatif et l’exposition aux drogues de chimiothérapie. Via des mécanismes finement régulés, Hsp27 exerce une protection garantissant la survie cellulaire, en adaptant ses niveaux d’expression, d’oligomérisation et de phosphorylation. Le taux d’Hsp27 est dès lors augmenté dans la plupart des cancers et apparaît comme marqueur fort de mauvais pronostic. Cela en fait un acteur clé de la chimiorésistance et une cible thérapeutique potentielle.Parmi les thérapeutiques ciblées basées sur l’ARN, les oligonucléotides antisens (ASO) sont des molécules issues du génie génétique capables de bloquer spécifiquement la traduction d’un ARN messager cible en protéine. L’apatorsen, un ASO anti-Hsp27 de deuxième génération, a été développé pour bloquer la synthèse d’Hsp27 au sein de la cellule cancéreuse et ainsi rétablir la chimiosensibilité. Après avoir mis en place un modèle de carcinose péritonéale colorectale traitée par CRS et CHIP chez le rat, nous avons étudié in vitro et in vivo, l’effet de l’adjonction de l’apatorsen au traitement standard de cette maladie. Nos résultats ne montrent pas de gain significatif de survie et donnent lieu à une discussion sur cette stratégie de traitement
Peritoneal carcinomatosis is a neoplasic metastatic process of the peritoneal serous lining characterized by the spread of multiple tumoral nodules. The prognosis of such attempt is very poor, characterized by a global chemoresistance. Intraperitoneal treatments were developed to improve drug’s cytoxicity by delivering them directly on nodules. The principle is to take advantage of the peritoneal-plasma barrier that allows to deliver higher drug’s concentration directly onto nodules and so to improve cytotoxicity. In curative intent strategy intraperitoneal chemotherapy is combined to a complete surgical cytoreduction (CRS) and to hyperthermia to enhance efficiency (Hyperthermic Intraperitoneal Chemotherapy - HIPEC). Thanks to this strategy overall survival improved in selected patients but still be flawed. For example, patients with colorectale peritoneal carcinomatosis present a 40% five-year overall survival, whereas those not eligible to that aggressive treatment present a 16 months median survival. So a better understanding of cellular molecular mechanisms responsible for this chemoresistance that will allow identifying new therapeutic targets is needed. Heat shock proteins play a fundamental role in intracellular protein homeostasis by acting as chaperone and regulating cytoskeleton architecture. In particular, Hsp27 acts as a regulator of the cellular response to various stress, such as thermic choc, oxidative stress, exposition to antineoplasic drugs. Through finely regulated process, Hsp27 exerts a cytoprotective role to guaranty cell survival, by adapting its level of expression, oligomerization and phosphorylation. As so Hsp27 is a key actor of chemoresistance and a designated therapeutic target.Antisens oligonucleotides are a new class of molecular targeted treatment able to specifically block the traduction into protein of a messenger RNA. Apatorsen, a second generation anti-Hsp27 ASO, has been developed to decrease Hsp27 levels in neoplastic cells and so restore chemosensitivity.After establishing a colorectal peritoneal carcinomatosis rat model with CRS and HIPEC, we studied in vitro and in vivo the effect of the apatorsen adjunction to this standard treatment. Our results did not show a significant survival improvement and give rise to a discussion upon this treatment strategy

These studies were aimed at investigating the potential application of nanoparticles engineered from oil-in-water microemulsion precursors for enhancing immune responses to HIV-1 Tat and Gag p24 proteins. Both of the HIV-1 proteins have been reported to be critical in the virus life cycle and are being evaluated in clinical trials as vaccine candidates. Anionic nanoparticles were prepared using emulsifying wax as the oil phase and Brij 78 and sodium dodecyl sulfate as the surfactants. The resulting nanoparticles were coated with Tat and were demonstrated to produce superior immune responses after administration to BALB/c mice compared to Tat adjuvanted with Alum. Similarly, cationic nanoparticles were prepared using emulsifying wax and Brij 78 and cetyl trimethyl ammonium bromide as the surfactants. The cationic nanoparticles were investigated for delivery of immunostimulatory adjuvants, namely three Toll-like receptor ligands, for obtaining synergistic enhancements in immune responses to a model antigen, Ovalbumin (OVA). In vitro and in vivo studies were carried out to elucidate possible mechanisms by which nanoparticles may result in enhancements in immune responses. In vitro studies were carried out to evaluate the uptake of nanoparticles into dendritic cells and to assess the release of pro-inflammatory cytokines from dendritic cells in the presence of nanoparicles. In vivo studies were carried out using a MHC class I restricted transgenic mouse model to investigate the potential for nanoparticles coated with OVA to enhance presentation of the protein to CD8+ T cells compared to OVA alone. Finally, the preparation of nanoparticles with a low amount of surface chelated nickel for high affinity binding to histidine-tagged (his-tag) proteins was investigated. It was hypothesized that this strengthened interaction of his-tag protein to the nickel chelated nanoparticles (Ni-NPs) would result in a greater uptake of antigen in vivo; therefore, enhanced immune responses compared to protein bound to anionic nanoparticles. In vivo evaluation of his-tag HIV-1 Gag p24 bound to Ni-NPs resulted in enhanced immune responses compared to protein either adjuvanted with Alum or coated on the surface of nanoparticles.

Introdução: A incidência de baixa estatura devido a deficiência do hormônio do crescimento (DGH) ocorre em 1:4.000-10.000 nascidos vivos. Diversos fatores de transcrição são necessários para a diferenciação dos cinco tipos de células produtoras de 6 hormônios hipofisários. Mutações nos fatores de transcrição HESX1, GLI2, LHX3, LHX4, SOX2, SOX3, PROP1 e POU1F1 foram descritas em pacientes com deficiência hormonal hipofisária isolada ou múltipla associada ou não a outras malformações. Mutações no gene OTX2, um fator de transcrição responsável pela formação da vesicula ocular e pela hipófise, podem causar malformações oculares tais como anoftalmia e microftalmia, isoladamente ou em associação com DGH isolado (DGHI) ou deficiência hipofisária hormonal múltipla (DHHM). Recentemente, dois pacientes não relacionados com DHHM e neuroipófise ectópica, sem anormalidades oculares foram descritos com mutações em heterozigose no OTX2 sugerindo um papel deste gene na etiologia do hipopituitarismo sem outras características sindrômicas. Objetivo: O objetivo desse trabalho foi o de analisar o gene OTX2 em pacientes com DGHI ou DHHM e correlacionar os achados moleculares com o fenótipo. Pacientes: Foram estudados 125 pacientes com DHHM (6 filhos de pais consangüíneos e 33 com parentes com baixa estatura) e 33 com DGHI (7 filhos de pais consangüíneos e 8 com parentes com baixa estatura). Materiais e métodos: Amostras de DNA dos pacientes foram submetidas à reação de polimerização em cadeia utilizando-se primers intrônicos desenhados para amplificar os 3 exons e as regiões flanqueadoras do gene OTX2. Os produtos de PCR foram purificados e sequenciados pelo método de Sanger. Resultados: Uma nova variante alélica c.689A>T, p.H230L em heterozigose no exon 5 foi encontrado em um único paciente com deficiência de GH, TSH, LH/FSH e ACTH associada a neuroipófise ectópica, sem malformação ocular. A histidina na posição 230 é altamente conservada em todas as espécies de vertebrados, e a análise in silico prediz um efeito prejudicial à estrutura da proteína. A análise da variante na família revelou 8 parentes não afetados como portadores heterozigotos, sugerindo uma doença autossômica dominante com padrão de penetrância incompleta. Esta variante não foi encontrada em 400 alelos de 200 controles brasileiros, porém foi descrito como polimorfismo no banco de dados de SNP em uma população européia americana, com incidência de 1 alelo T, entre 8600 alelos, sendo assim considerado raro. Encontramos também outras quatro variantes alélicas na casuística (c.98-70C> A; c.420G> C, p.P148P; c.435C> T, p.S145S; C * 10G> A), não conservadas entre as espécies. Duas delas levando a troca silenciosa de amino ácidos, sem efeito deletério no sítio exonic splice enhancer. Conclusão A nossa coorte de 158 pacientes é a maior população rastreada para mutações no OTX2 e a detecção de uma variante suspeita em heterozigose em um único paciente portador de hipopituitarismo e neuroipófise ectópica sugere que mutações no OTX2 são uma causa rara de DHHM ou DGHI sem malformação ocular na população estudada. O achado molecular da variante c.689A>T, p.H230L em heterozigose, com padrão de penetrância incompleta é consistente com a observação de que as características fenotípicas de camundongos heterozigotos com perda de função do Otx2 são fortemente influenciados pela background genético, não podendo dessa forma, descartar que outros moduladores genéticos possam ser responsáveis pela penetrância incompleta nessa família. Essa hipótese deverá ser investigada pela análise do exoma do paciente e seus familiares. O fato de a variante estar localizada numa região altamente conservada entre as espécies, sugere que a mesma seja causadora do fenótipo em questão, porém serão necessários os estudos funcionais de transfecção transitória para determinar se se trata de perda de função ou efeito negativo dominante de genes alvos expressos no ectoderme oral ou neural
Introduction: The incidence of short stature due to growth hormone deficiency (GHD) occurs in 1:4.000-10.000 live births. Several transcription factors are required for differentiation of five types of cells producing 6 pituitary hormones. Mutations in the transcription factors HESX1, GLI2, LHX3, LHX4, SOX2, SOX3, PROP1 and POU1F1 have been described in patients with isolated pituitary hormone deficiency or multiple associated or not with other malformations. Mutations in OTX2, a transcription factor responsible for the formation of the eye vesicle and the pituitary gland, can cause ocular malformations such as anophthalmia and microphthalmia, alone or in association with isolated GHD (IGHD) or combined pituitary hormone deficiency (CPHD). Recently, two unrelated patients with CPHD and ectopic neurohypophysis without ocular abnormalities were described with heterozygous mutations in OTX2 suggesting a role of this gene in the etiology of hypopituitarism without other syndromic features. Objective: The aim of this study was to analyze the OTX2 gene in patients with GHD or CPHD and correlate the molecular findings with the phenotype. Patients: We studied 125 patients with CPHD (6 children of consanguineous parents and 33 relatives with short stature) and 33 with IGHD (7 children of consanguineous parents and 8 relatives with short stature). Materials and methods: DNA samples from the patients were subjected to polymerase chain reaction using intronic primers designed to amplify the 3 exons and flanking regions of the gene OTX2. The PCR products were purified and sequenced by the Sanger method. Results: A new heterozygous allelic variant c.689A> T, p.H230L in exon 5 was found in one patient with deficiencies of GH, TSH, LH / FSH and ACTH associated with ectopic neurohypophysis without ocular malformation. The histidine at position 230 is strongly conserved in all vertebrate species, and in silico analysis predicts a detrimental effect on protein structure. The analysis of the variant in the family revealed eight unaffected relatives as heterozygous carriers, suggesting an autosomal dominant pattern with incomplete penetrance. This variant was not found in 400 alleles of 200 Brazilian controls, but it was described as a polymorphism in the SNP database in a European American population, with an incidence of 1 T allele among 8600 alleles, and therefore considered rare. We also found four other allelic variants in the samples (c.98-70C> A; c.420G> C, p.P148P; c.435C> T, p.S145S; C * 10G> A), not conserved between species. Two of them leading to a silent amino acid exchange, without deleterious effect in the exonic splice enhancer site. Conclusion Our cohort of 158 patients is the largest population screened for mutations in OTX2 and the detection of a suspected heterozygous variant in one patient with hypopituitarism and ectopic neurohypophysis suggests that OTX2 mutations are a rare cause of CPHD/IGHD without ocular malformation in the studied population. The molecular finding of a heterozygous variant c.689A> T, p.H230L, with incomplete penetrance pattern is consistent with the observation that the phenotypic characteristics of mice with heterozygous loss of function of Otx2 are strongly influenced by genetic background, suggesting that other genetic modulators may be responsible for the incomplete penetrance in this family. This hypothesis should be investigated by analysis of exoma in the patient and their family. The fact that the variant is located in a region highly conserved among species suggests that it is causing the phenotype in question, but it will require the functional studies with transient transfections to determine whether it is the loss of function or effect of dominant negative target genes expressed in neural or oral ectoderm

Les micelles inverses d'aerosol ot sont des muroemulsions eau/huile dont la taille est controlee par la quantite d'eau solubilisee. La solubilisation des petites molecules ainsi que des petites proteines entrainent des perturbations dans la structure et la reactivite. Un modele de localisation des sondes en micelle est propose a partir des mesures de structure, par diffusion des rayons x, et de reactivite par radiolyse pulse. De plus, une synthese in situ des semiconducteurs en micelle inverse est exposee

Le récepteur hypophysaire de la GnRH (RGnRH) joue un rôle crucial dans le contrôle de la fonctionde reproduction. Dans le promoteur distal du Rgnrh, j’ai caractérisé un élément de réponsebifonctionnel répondant aux protéines LIM à homéodomaine ISL1/LHX3 et à GATA2. D’autre part,deux motifs TAAT situés dans la région plus proximale confèrent à ce gène la capacité de répondreaux facteurs Paired-like PROP1 et OTX2. Tous ces facteurs, exprimés précocement au cours del’ontogenèse hypophysaire, pourraient participer à l’émergence de l’expression du Rgnrh. Hors del’hypophyse, j’ai découvert que le Rgnrh est exprimé au cours du développement postnatal dansl’hippocampe de rat, où il module la plasticité synaptique. Par ailleurs, j’ai identifié deux nouveauxsites d’expression, la rétine et la glande pinéale. Ces résultats mettent en lumière l’importancefonctionnelle de ce récepteur et de son ligand et les rôles multiples qu’il ont acquis au cours del’évolution des Vertébrés
In the pituitary, the GnRH receptor (GnRHR) plays a crucial role in the neuroendocrine control ofreproductive function. Within the distal region of the Gnrhr promoter, I have characterized abifunctional response element modulated by the LIM homeodomain proteins ISL1/LHX3 and byGATA2. Besides, in the proximal region of the promoter, two TAAT motifs conferred response toPaired-like factors PROP1 and OTX2. All these factors are expressed during pituitary ontogenesis andcould participate in the onset and regulation of Gnrhr expression. Outside of the pituitary, I havediscovered that the Gnrhr was expressed during postnatal development in the rat hippocampus, whereit modulated synaptic plasticity. Furthermore, I have identified two novel sites of Gnrhr expression, theretina and the pineal gland. Altogether, these data highlight the functional importance of this receptorand its ligand as well as the multiple roles they have acquired during vertebrate evolution

Nous avons applique des méthodes immunocytochimiques à l'analyse des domaines membranaires dans deux systèmes différents : la membrane plasmique des cellules du cristallin de bovidé et la membrane des thylakoïdes. Dans le premier système, nous montrons que la mp26, protéine majoritaire des jonctions communicantes des fibres, est aussi présente dans les domaines non fonctionnels de la membrane plasmique des fibres, et absente de la membrane plasmique des cellules épithéliales du cristallin. La membrane des thylakoïdes, quant à elle, comporte deux domaines de compositions bien distinctes: les régions accolées renferment l'essentiel du photosystème II et de son antenne, alors que le photosystème I, l'atpase et, à moindre degré, la ferrédoxine NADP-réductase sont ségréges dans les régions non accolées de la membrane. Le cytochrome b6/f est présent dans les deux domaines. Par ailleurs, nous avons étudié l'accessibilité aux anticorps sur les faces interne et externe de la membrane de divers polypeptides de la membrane des thylakoïdes.

Dissertação de mestrado em Biotechnology
Wine is a widely consumed product that is often associated with contaminations of toxic metabolites called mycotoxins. The most important mycotoxin associated to wine is ochratoxin A (OTA), and its detection usually involves the clean-up of samples with immunoaffinity columns (IAC) and quantification by HPLC coupled with fluorescence detection (FL). However, the several drawbacks associated with the use of the IAC led to the search for alternative clean-up methods. Thus, in this work, new platforms for OTA clean-up from wine were developed, based on proteins whose affinity towards OTA makes them suitable candidates to mimic the binding properties of the antibodies used in the IAC method. In a first approach, a protein with high affinity towards OTA, was used to develop a new solid phase extraction (SPE) method for the extraction of the mycotoxin from wine and subsequent quantification by HPLC-FL. The capture of OTA by the columns constructed with agarose-immobilized OTA binding protein was optimized to allow the full recovery of OTA in wine, and the method was further validated by the evaluation of various parameters such as recovery rates, selectivity and limits of detection (LOD) and quantification (LOQ). The developed method was selective enough for a reliable determination of OTA in wine, presenting recovery rates superior to 98% and LOD and LOQ of 0.02 and 0.05 μg L-1, respectively. Furthermore, the performance of the developed method revealed no significant differences in relation to the IAC method in concentrations up to 2 μg L-1 of OTA. In comparison with other conventional SPE methods reported in the literature, the developed method has proved to be suitable to be employed in the determination of OTA in wine. In a second approach, the domain where lies the primary binding site of OTA in the OTA-binding protein used in the first approach was evaluated as OTA ligand for developing OTA extraction platforms based on this domain. For that, the domain was recombinantly produced, fused to a 6xHis tag, with and without the thioredoxin (TrxA) solubility partner, in two E. coli strains, BL21 (DE3) and Origami 2 (DE3). Soluble proteins, with and without TrxA, were produced by both strains, but the Origami strain provided higher yields of production (18.7 and 23.4 mg per litre of culture, respectively). In addition, differences observed in the affinity of the proteins for the nickel resin in the purification suggested that the structures acquired by the recombinant proteins produced in each strain were different. Furthermore, the fact that fusion proteins were less prone to degradation suggested that TrxA contributed for their stability. Studies of fluorescence spectroscopy revealed that only the recombinant proteins produced by the Origami strain were capable of interacting with OTA, thus indicating that these proteins were functional. The ability of the proteins produced from this strain, with and without TrxA, immobilized in nickel via the 6xHis tag, to capture the mycotoxin in buffer solutions was evaluated. SPE columns constructed with the nickel-immobilized recombinant proteins did not show the ability to effectively capture OTA. On the other hand, incubation assays performed in eppendorfs allowed decreasing OTA in solution up to 54 and 63%, respectively for immobilized proteins, with and without TrxA. These results open perspectives for the development of OTA extraction platforms based on the recombinant domain of this OTA-binding protein with matrixes less expensive than the agarose used in the first approach by means of specific purification tags.
O vinho é um produto largamente consumido que está frequentemente associado a contaminações com metabolitos tóxicos denominados de micotoxinas. A micotoxina mais importante associada ao vinho é a ocratoxina A (OTA), e a sua deteção envolve normalmente um passo de concentração das amostras com colunas de imunoafinidade (IAC) e deteção por HPLC acoplada com deteção por fluorescência (FL). Contudo, as diversas desvantagens associadas ao uso das IAC levaram à procura de métodos de concentração alternativos. Assim, neste trabalho, foram desenvolvidas novas plataformas de concentração de OTA do vinho, baseadas no uso de proteínas cuja afinidade para a OTA as torna candidatos adequados para mimetizar as propriedades de ligação dos anticorpos usados no método IAC. Numa primeira abordagem, uma proteína com alta afinidade para a OTA foi usada para desenvolver um novo método de extração em fase solida (SPE) para extrair esta micotoxina do vinho e subsequente quantificação por HPLC-FL. A captura de OTA por colunas construídas com a proteína em estudo imobilizada em agarose foi otimizada de forma a permitir uma recuperação total da micotoxina presente no vinho, e o método foi posteriormente validado através da avaliação de parâmetros como taxas de recuperação, seletividade e limites de deteção (LOD) e quantificação (LOQ). O método desenvolvido foi suficientemente seletivo para permitir uma quantificação confiável de OTA no vinho, apresentando taxas de recuperação superiores a 98% e um LOD e LOQ de 0.02 e 0.05 μg L-1, respetivamente. Em adição, o desempenho do método desenvolvido não apresentou diferenças significativas em relação ao método das IAC em concentrações até 2 μg L-1 de OTA. Em comparação com outros métodos convencionais de SPE reportados na literatura, o método desenvolvido revelou ser adequado para aplicação na determinação de OTA no vinho. Numa segunda abordagem, o domínio da proteína usada na primeira abordagem que contém o principal local de ligação da micotoxina, foi avaliado como ligando da OTA para o desenvolvimento de plataformas de extração de OTA baseadas nesse domínio. Para isso, este foi produzido de forma recombinante, em fusão com um 6xHis tag, com e sem tiorredoxina (TrxA) como parceiro de solubilidade, em duas estirpes de E. coli, BL21 (DE3) e Origami 2 (DE3). O domínio proteico solúvel, com e sem TrxA, foi produzido por ambas as estirpes, mas a estirpe Origami obteve maiores rendimentos de produção (18.7 e 23.4 mg por litro de cultura, respetivamente). Em adição, as diferenças observadas na afinidade das proteínas para a resina de níquel na purificação sugeriram que as estruturas adquiridas pelas proteínas produzidas em cada estirpe eram diferentes. Além disso, o facto de que que as proteínas de fusão se mostraram menos suscetíveis a degradação sugere que a TrxA contribuiu para a sua estabilidade. Estudos de espectroscopia de fluorescência revelaram que apenas as proteínas recombinantes produzidas pela estirpe Origami foram capazes de interagir com a OTA, indicando assim que estas se encontravam funcionais. A capacidade das proteínas produzidas por esta estirpe, com e sem TrxA, imobilizadas em níquel a partir do 6xHis tag, para capturarem a micotoxina em soluções tampão foi avaliada. Colunas SPE construídas com estas proteínas recombinantes imobilizadas em níquel não mostraram a capacidade de capturar efetivamente a OTA. Por outro lado, os ensaios realizados em “eppendorfs” permitiram reduções de OTA em solução até 54 e 63%, respetivamente para as proteínas imobilizadas com e sem TrxA. Estes resultados abrem perspetivas para o desenvolvimento de plataformas de extração de OTA baseadas neste domínio da proteína estudada com matrizes menos caras que a agarose usada na primeira abordagem por meio de tags de purificação específicos.

碩士
長庚大學
生藥科學研究所
90
It has been demonstrated that oxidized low-density lipoprotein (ox-LDL) is a risk factor in atherosclerosis by stimulating vascular smooth muscle cells (VSMCs) proliferation. Ox-LDL stimulates multiple signaling pathways including activation of PI3K/Akt and p42/p44 MAPK, which play a key role of regulation of cell proliferation and survival. However, the mechanism of PI3K/Akt and p42/p44 MAPK activation triggered by ox-LDL-induced is not well understood in VSMCs. In this study, we investigated the effects of ox-LDL on cell proliferation by exploring the relationship between PI3K/Akt and p42/p44 MAPK activation. Ox-LDL induced time- and concentration-dependent manner, as determined by Western blotting analysis using a specific phospho-Akt. Pretreatment of these cells with pertussis toxin, cholera toxin, and foskolin for 24h attenuated the ox-LDL-induced responses. Phosphorylaiton of Akt stimulated by ox-LDL and EGF was attenuated by the inhibitors of PI3K (LY294002 and wortmannin), tyrosine kinase (genistein), Src family, (PP1), PKC (staurosporine), Ca2+ chelator (BAPTA/AM) plus EGTA. Pretreatment of these cells with epidermal growth factor receptor (EGFR) inhibitor AG1478 and platelet-derived growth factor receptor (PDGFR) inhibitor AG1296 attenuated the ox-LDL-induced Akt phosphorylation. This indicated that ox-LDL-induced Akt activation was mediated through transactivation of EGFR and PDGFR in rat VSMCs. We further determined whether activation of PI3K/Akt is necessary for activation of MAPK. Pretreatment of these cells with wortmannin inhibited ox-LDL stimulated p42/p44 MAPK activation and [3H]-thymidine incorporation. Furthermore, treatment of cells with MEK1/2 inhibitor (U0126), did not affect Akt phosphorylation in response to ox-LDL. In addition, overexpression of dominant negative mutants of the regulatory subunit p85 of PI3K and Akt inhibited MEK1/2 and p42/44 MAPK activation induced by ox-LDL. Therefore, we suggested PI3K/Akt is an upstream component of MEK1/2 and p42/44 MAPK induced by ox-LDL in rat VSMCs. Taken together, these results suggested that the mitogenic effect of ox-LDL is mediated through PTX-sensitive G protein-coupled receptor that involved transactivation of EGFR and PDGFR and PI3K/Akt/MAPK pathway in rat VSMCs.

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.