Recherche des partenaires d'ATAD3 et étude de l'effet de l'invalidation sur la différenciation adipocytaire

ATAD3 is a vital inner membrane mitochondrial ATPase with unknown function but widely expressed in animals and plants. The main project was to study the role of ATAD3 in a cellular model of differentiation, the one used being adipocyte-like 3T3-L1 cells induced by insuline. Induction of these cells provokes the start of the adipogenesis program which precedes lipid droplets synthesis (lipogenesis). We observed that along differentiation, a synthesis and a remodelling of mitochondria occur. ATAD3, which is expressed in pre-adipocytes, is increased before lipid droplets accumulation and before mitochondrial synthesis and remodelling and is maintained all along differntiation. We have generated cell lines with constitutive invalidation of ATAD3 (siRNA) and studied the differentiation of these cells. We have shown that ATAD3 invalidation inhibits lipogenesis and mitochondrial remodelling without any inhibition of insulin transduction signal neither inhibition of mitochondrial protein synthesis. The phenotype was rescued by ATAD3 transfection and complemented by over-expression of Drp1. These results show that ATAD3 is a necessary and limiting factor in adypocyte differentiation and lipogenesis. Three parallel projects completed this thesis work: The first was to contribute to purification of recombinant human ATAD3 produced in yeast. These experiments didn't allow us to reveal the ATPase activity of ATAD3 but allowed us to obtain a significant quantity of purified protein that will be useful to generate antibodies and also for others applications. The second was to study ATAD3 isoforms expression profiles in human and rodent samples. These experiments allowed the description of several isoforms of ATAD3 with tissue-specific expression. A third project was the identification of ATAD3 protein partners in order to try to understand its function. Using labelled ATAD3 protein probes (in vitro transcription/traduction), we performed interaction tests by Far Western. These experiments led to the identification of seveLe projet principal a consisté à étudier le rôle d'ATAD3 dans un modèle de différenciation cellulaire en culture, les cellules adipocytaires 3T3-L1 induites par l'insuline. L'induction de ces cellules provoque la mise en route du programme d'adipogenèse qui précède la synthèse de gouttelettes de triglycérides (lipogenèse). Nous avons observé qu'au cours de la différenciation, une synthèse et un remodelage du réseau mitochondrial ont lieu. ATAD3, exprimée dans les pré-adipocytes, s'accumule avant la synthèse et le remodelage du réseau mitochondrial et est maintenue élevée tout au long de la différenciation. Nous avons réalisé des lignées invalidées constitutivement pour ATAD3 (siRNA) et étudié la différenciation de ces cellules. Nous avons montré que l'invalidation d'ATAD3 inhibe la lipogenèse et le remodelage du réseau mitochondrial sans affecter la signalisation par l'insuline ni la synthèse des protéines mitochondriales. Le phénotype a été récupéré par transfection d'ATAD3 et une complémentation par Drp1 a été mise en évidence. Ces résultats montrent qu'ATAD3 est nécessaire et limitant dans la différenciation adipocytaire. Trois projets annexes ont complétés ce travail de thèse : Le premier a consisté à contribuer à la purification d'ATAD3 humaine, produite chez la levure. Ces expériences n'ont pas permis de révéler l'activité ATPasique d'ATAD3 mais ont permis d'obtenir une quantité significative de protéine recombinante qui pourra servir à la production d'anticorps ainsi qu'à d'autres applications. Le second a consisté à étudier les isoformes d'ATAD3 chez l'homme et les rongeurs. Ces expériences ont permis de révéler l'existence de plusieurs isoformes tissulaires. Un troisième projet a consisté à identifier des partenaires protéiques d'ATAD3 afin de tenter de comprendre la fonction de cette protéine. A l'aide de sondes protéiques d'ATAD3 radiomarquées (transcription/traduction in vitro), nous avons réalisé des tests d'interaction de type Far Western. Ces expériences ont aboutis à quelques candidats potentiels interagissant avec les parties N- ou C-terminale d'ATAD3

Recherche des partenaires d'ATAD3 et étude de l'effet de l'invalidation sur la différenciation adipocytaire

ATAD3 is a vital inner membrane mitochondrial ATPase with unknown function but widely expressed in animals and plants. The main project was to study the role of ATAD3 in a cellular model of differentiation, the one used being adipocyte-like 3T3-L1 cells induced by insuline. Induction of these cells provokes the start of the adipogenesis program which precedes lipid droplets synthesis (lipogenesis). We observed that along differentiation, a synthesis and a remodelling of mitochondria occur. ATAD3, which is expressed in pre-adipocytes, is increased before lipid droplets accumulation and before mitochondrial synthesis and remodelling and is maintained all along differntiation. We have generated cell lines with constitutive invalidation of ATAD3 (siRNA) and studied the differentiation of these cells. We have shown that ATAD3 invalidation inhibits lipogenesis and mitochondrial remodelling without any inhibition of insulin transduction signal neither inhibition of mitochondrial protein synthesis. The phenotype was rescued by ATAD3 transfection and complemented by over-expression of Drp1. These results show that ATAD3 is a necessary and limiting factor in adypocyte differentiation and lipogenesis. Three parallel projects completed this thesis work: The first was to contribute to purification of recombinant human ATAD3 produced in yeast. These experiments didn't allow us to reveal the ATPase activity of ATAD3 but allowed us to obtain a significant quantity of purified protein that will be useful to generate antibodies and also for others applications. The second was to study ATAD3 isoforms expression profiles in human and rodent samples. These experiments allowed the description of several isoforms of ATAD3 with tissue-specific expression. A third project was the identification of ATAD3 protein partners in order to try to understand its function. Using labelled ATAD3 protein probes (in vitro transcription/traduction), we performed interaction tests by Far Western. These experiments led to the identification of seveLe projet principal a consisté à étudier le rôle d'ATAD3 dans un modèle de différenciation cellulaire en culture, les cellules adipocytaires 3T3-L1 induites par l'insuline. L'induction de ces cellules provoque la mise en route du programme d'adipogenèse qui précède la synthèse de gouttelettes de triglycérides (lipogenèse). Nous avons observé qu'au cours de la différenciation, une synthèse et un remodelage du réseau mitochondrial ont lieu. ATAD3, exprimée dans les pré-adipocytes, s'accumule avant la synthèse et le remodelage du réseau mitochondrial et est maintenue élevée tout au long de la différenciation. Nous avons réalisé des lignées invalidées constitutivement pour ATAD3 (siRNA) et étudié la différenciation de ces cellules. Nous avons montré que l'invalidation d'ATAD3 inhibe la lipogenèse et le remodelage du réseau mitochondrial sans affecter la signalisation par l'insuline ni la synthèse des protéines mitochondriales. Le phénotype a été récupéré par transfection d'ATAD3 et une complémentation par Drp1 a été mise en évidence. Ces résultats montrent qu'ATAD3 est nécessaire et limitant dans la différenciation adipocytaire. Trois projets annexes ont complétés ce travail de thèse : Le premier a consisté à contribuer à la purification d'ATAD3 humaine, produite chez la levure. Ces expériences n'ont pas permis de révéler l'activité ATPasique d'ATAD3 mais ont permis d'obtenir une quantité significative de protéine recombinante qui pourra servir à la production d'anticorps ainsi qu'à d'autres applications. Le second a consisté à étudier les isoformes d'ATAD3 chez l'homme et les rongeurs. Ces expériences ont permis de révéler l'existence de plusieurs isoformes tissulaires. Un troisième projet a consisté à identifier des partenaires protéiques d'ATAD3 afin de tenter de comprendre la fonction de cette protéine. A l'aide de sondes protéiques d'ATAD3 radiomarquées (transcription/traduction in vitro), nous avons réalisé des tests d'interaction de type Far Western. Ces expériences ont aboutis à quelques candidats potentiels interagissant avec les parties N- ou C-terminale d'ATAD3

ATAD3, une ATPase membranaire mitochondriale vitale impliquée dans la progression tumorale

ATAD3 (ATPase family AAA domain-containing protein 3) est une ATPase mitochondriale membranaire dont la fonction n’est pas connue à ce jour mais qui est indispensable au développement embryonnaire. Le gène codant pour ATAD3 existe chez les organismes pluricellulaires à tissus spécialisés et a été conservé sous la forme d’un gène unique jusqu’aux vertébrés. Chez les primates et les humains, deux autres gènes sont apparus (appelés ATAD3B et ATAD3C par rapport à ATAD3A, le gène ancestral). L’invalidation d’ATAD3 dans différentes lignées cellulaires non transformées est associée à une modification drastique de la structure du réseau mitochondrial, à une inhibition de la prolifération et à une réduction des interactions fonctionnelles entre les mitochondries et le réticulum endoplasmique. Cependant, l’analyse des fonctions d’ATAD3A et d’ATAD3B dans différentes lignées cancéreuses humaines montre au contraire que ces protéines peuvent avoir des propriétés antiprolifératives et augmenter la chimiorésistance. ATAD3 semble donc impliquée dans une fonction mitochondriale encore inconnue, essentielle à la croissance des organismes pluricellulaires et impliquée dans la tumorigenèse

Iron-Based Hollow Nanoplatforms for Cancer Imaging and Theranostics

Over the past decade, iron (Fe)-based hollow nanoplatforms (Fe-HNPs) have attracted increasing attention for cancer theranostics, due to their high safety and superior diagnostic/therapeutic features. Specifically, Fe-involved components can serve as magnetic resonance imaging (MRI) contrast agents (CAs) and Fenton-like/photothermal/magnetic hyperthermia (MTH) therapy agents, while the cavities are able to load various small molecules (e.g., fluorescent dyes, chemotherapeutic drugs, photosensitizers, etc.) to allow multifunctional all-in-one theranostics. In this review, the recent advances of Fe-HNPs for cancer imaging and treatment are summarized. Firstly, the use of Fe-HNPs in single T1-weighted MRI and T2-weighted MRI, T1-/T2-weighted dual-modal MRI as well as other dual-modal imaging modalities are presented. Secondly, diverse Fe-HNPs, including hollow iron oxide (IO) nanoparticles (NPs), hollow matrix-supported IO NPs, hollow Fe-complex NPs and hollow Prussian blue (PB) NPs are described for MRI-guided therapies. Lastly, the potential clinical obstacles and implications for future research of these hollow Fe-based nanotheranostics are discussed

SIRT1 mediates the inhibitory effect of Dapagliflozin on EndMT by inhibiting the acetylation of endothelium Notch1

Abstract Background Endothelial–mesenchymal transition (EndMT) plays a crucial role in promoting myocardial fibrosis and exacerbating cardiac dysfunction. Dapagliflozin (DAPA) is a sodium–glucose-linked transporter 2 (SGLT-2) inhibitor that has been shown to improve cardiac function in non-diabetic patients with heart failure (HF). However, the precise mechanisms by which DAPA exerts its beneficial effects are yet to be fully elucidated. Methods Isoproterenol (ISO) was used to generate a HF model in mice. For in vitro experiments, we used TGF-β1-stimulated human umbilical vein endothelial cells (HUVECs) and mouse aortic endothelial cells (MAECs). Results Both our in vivo and in vitro results showed that EndMT occurred with decreased SIRT1 (NAD+-dependent deacetylase) protein expression, which could be reversed by DAPA therapy. We found that the protective effect of DAPA was significantly impaired upon SIRT1 inhibition. Mechanistically, we observed that SIRT1 phosphorylation, a required modification for its ubiquitination and degradation, was reduced by DAPA treatment, which induces the nucleus translocation of SIRT1 and promotes its binding to the active intracellular domain of Notch1 (NICD). This interaction led to the deacetylation and degradation of NICD, and the subsequent inactivation of the Notch1 signaling pathway which contributes to ameliorating EndMT. Conclusions Our study revealed that DAPA can attenuate EndMT induced by ISO in non-diabetic HF mice. This beneficial effect is achieved through SIRT1-mediated deacetylation and degradation of NICD. Our findings provide greater insight into the underlying mechanisms of the therapeutic effects of DAPA in non-diabetic HF. Graphical Abstrac

Rock Mechanics Characteristics Test and Optimization of High-Efficiency Mining in Dajishan Tungsten Mine

Rock mechanics test is not only the basis for obtaining the mechanical parameters of rock but also an important means for studying rock mechanics and engineering. In this paper, the uniaxial compression deformation test, Brazilian splitting test, and cornea pressure shear test are carried out for rocks in the Dajishan tungsten mine. The basic mechanical parameters such as uniaxial compressive strength, tensile strength, elastic modulus, Poisson’s ratio, and internal friction angle of ore rock and surrounding rock are obtained. Meanwhile, damage characteristics of rock are deeply studied and analyzed under different experimental conditions. According to rock mechanics parameters which are obtained from indoor rock mechanics tests, three design schemes of stope structure parameters are optimized by using the FLAC3D numerical simulation software. On the premise of ensuring the stability of the stope structure, the recovery rate of ore and the production capacity of the stope are taken into consideration. It is suggested that the second scheme should be adopted for mines (18 m for ore room and 7 m for ore pillar), which provides scientific guidance for the safe and efficient mining of mines

Numerical Calculation Analysis of the Structural Stability of Cemented Fill under Different Cement-Sand Ratios and Concentration Conditions

The effect of lime-sand ratio and slurry concentration on the mechanical properties of backfills is important. To achieve green and high-efficiency mining, accurately determining the optimum ratio of cemented tailings for certain tungsten tailings and ensuring the safety and stability of the mine stope structure are important. The cement-sand ratios used in this research were 1 : 6 and 1 : 8. The mechanical properties were evaluated by using 68%, 72%, and 78% of tailing cemented filling materials. The corresponding physical and mechanical parameters were obtained through uniaxial compression, splitting, and shearing mechanical experiments on the backfill specimens. FLAC3D was used to investigate the mechanical properties of cement-filled pillars and the stability of supporting surrounding rocks on the basis of the mine’s current room pillar structure size parameters and mining sequence. The key factors that affect the stability of the goaf were analyzed by evaluating the plastic zone area of the stope, maximum and minimum principal stresses, and displacement change. The structural characteristics of stope structures and changes of rock mass damage were obtained under different cement-tailing ratios and concentrations. A cemented backfill with a cement-tailing ratio of 1 : 8 and a concentration of 68% was selected as the best solution for the mine in terms of safety and economic considerations