Alexis Parenté

GASP-1 et GASP-2 sont deux protéines très proches structuralement caractérisées par plusieurs modules inhibiteurs de protéases et identifiés comme des inhibiteurs de plusieurs membres de la famille TGF-ß tel que la myostatine ou GDF-11, respectivement régulateurs négatifs de la myogenèse et de l’ostéogénèse. Malgré l’organisation structurale commune des protéines GASPs, leurs profils d’expression différents laissent supposer des rôles physiologiques distincts. C’est pourquoi, nous avons généré des modèles souris Tg(Gasp-1) et Tg(Gasp-2) surexprimant Gasp-1 ou Gasp-2 afin de mieux appréhender les fonctions de ces protéines. Des analyses fonctionnelles, réalisées in vitro et in vivo dans les contextes musculaire, squelettique et anti-protéasique, nous ont permis de mettre en lumière une dualité fonctionnelle entre GASP-1 et GASP-2 dans ces différents contextes. Les deux lignées Tg(Gasp-1) et Tg(Gasp-2) présentent une augmentation de la masse musculaire due à une hypertrophie sans hyperplasie des myofibres. Cependant, les souris Tg(Gasp-1) présentent une dérégulation globale de l’homéostasie du glucose et des défauts métaboliques en vieillissant, phénotype non retrouvé chez les souris Tg(Gasp-2). Ces résultats nous a permis de proposer la protéine GASP-2 comme étant un meilleur candidat thérapeutique dans le cas de maladies musculaires. Dans le contexte squelettique, seule la surexpression de Gasp-2 entraine un phénotype osseux. L’ensemble de nos résultats permet de mettre en évidence un réseau de régulation de l’expression génique des membres de la famille TGF-ß et de leurs inhibiteurs dans le muscle.

Introduction: Breast cancer is the most frequently diagnosed cancer worldwide and a leading cause of cancer-related mortality in women. This study aimed to describe the epidemiological profile and clinical characteristics of breast cancer cases diagnosed between 2014 and 2019 among women residing in Cotonou, Benin. Patients and Method: This retrospective cohort study included 277 women with breast cancer registered in the Cotonou Population-Based Cancer Registry between 1 January 2014 and 31 December 2019. Qualitative variables were expressed as frequencies and percentages, whereas continuous variables were summarized as means with standard deviations. Overall survival was estimated using the Kaplan–Meier method. Data analyses were performed using R software (version 3.6.1) and SPSS. Results: The crude incidence of breast cancer increased from 16.8 cases per 100,000 population in 2014 to 24.7 in 2019, with a peak of 38.9 in 2018. The mean age at diagnosis was 49.6 ± 11.9 years. Most patients were diagnosed at advanced stages (III and IV), with T4 tumors being the most frequent presentation. Triple-negative breast cancer was the most prevalent molecular subtype. Overall survival was 57% at 1 year and 37% at 5 years. Conclusions: Breast cancer in Cotonou predominantly affects relatively young women and is often diagnosed at advanced stages, contributing to poor survival outcomes compared with high-income countries. Strengthening early detection and prevention strategies should be a public health priority.

Phex^L222P mouse is a new ENU mouse model for XLH disease due to Leu to Pro amino acid modification at position 222. Phex^L222P mouse is characterized by growth retardation, hypophosphatemia, hypocalcemia, reduced body bone length, and increased epiphyseal growth plate thickness and femur diameter despite the increase in PHEX^L222P expression. Actually, Phex^L222P mice show an increase in Fgf23, Dmp1, and Mepe and Slc34a1 (Na-Pi IIa cotransporter) mRNA expression similar to those observed in Hyp mice. Femoral osteocalcin and sclerostin and Slc34a1 do not show any significant variation in Phex^L222P mice. Molecular dynamics simulations support the experimental data. P222 might locally break the E217-Q224 β-sheet, which in turn might disrupt inter-β-sheet interactions. We can thus expect local protein misfolding, which might be responsible for the experimentally observed PHEX^L222P loss of function. This model could be a valuable addition to the existing XLH model for further comprehension of the disease occurrence and testing of new therapies.

Nous présentons une nouvelle stratégie instrumentale et computationnelle appelée FAMOUS (pour fast algorithm for three-dimensional (3D) multiphoton microscopy of biomedical structures) basée sur une approche de microscopie multiphotonique assistée par calcul. Le but est l’amélioration visuelle des images d'échantillons biologiques épais offrant ainsi un nouveau point de vue sur les structures biologiques. L'approche de post-traitement repose sur un algorithme de restauration d'image régularisé, alimenté par une estimation 3D précise de la fonction d'étalement du point (Point Spread Function en anglais, PSF) de l'instrument sur toute la profondeur des structures. Cette dernière étape revient à mesurer, grâce à un algorithme d'ajustement de modèle avancé, les distorsions variant en profondeur de l'image résultant de la combinaison entre la contribution instrumentale et les hétérogénéités du milieu. Les performances du pipeline FAMOUS sont évaluées pour un milieu hétérogène constitué d’un muscle entier de souris. La génération de seconde harmonique (SHG), émise par l'assemblage des chaines de myosine du muscle est enregistrée. Les artefacts optiques issus de la chaîne d'acquisition incluant des hétérogénéités dans les 3 dimensions sont estimés avec les spécificités propres à l’échantillon puis retirées numériquement. Des images brutes et restaurées sur 5 µm de l’ultrastructure fine du muscle illustrent la robustesse du pipeline FAMOUS.

Muscle atrophy is associated with many diseases including genetic disorders, sarcopenia, or cachexia syndromes. Myostatin ( Mstn ), a transforming growth factor‐beta (TGF‐β) member, plays a key role in skeletal muscle homeostasis as a powerful negative regulator. Over the last decade, about 15 clinical trials aimed at inhibiting the Mstn pathway, failed to produce conclusive results. In this context, we investigated whether growth and differentiation factor‐associated serum protein‐1 (GASP‐1) or GASP‐2, two natural inhibitors of Mstn , might represent a potential therapeutic. As we previously reported, mice overexpressing Gasp‐1 ( Tg(Gasp‐1) ) present an increase of muscle mass but develop metabolic disorders with aging. Here, we showed that overexpression of Gasp‐2 increases the muscular mass without metabolic defects. We also found that Tg(Gasp‐2) mice displayed, like Mstn ^–/– mice, a switch from slow‐ to fast‐twitch myofibers whereas Tg(Gasp‐1) mice exhibit a reverse switch. Our studies supported the fact that GASP‐2 has less affinity than GASP ‐ 1 for Mstn , leading to a constitutive Mstn upregulation only in Tg(Gasp‐1) mice, responsible for the observed phenotypic differences. Altogether, our findings highlighted a gene expression regulatory network of TGF‐β members and their inhibitors in muscle.

While GASP‐1 and GASP‐2 proteins are known to regulate myogenesis by inhibiting myostatin, their structural organization suggests a putative role as multivalent protease inhibitors controlling different protease activities. In this study, we show the non‐competitive and competitive anti‐trypsin activities of the full‐length GASP‐1 and GASP‐2 proteins, respectively, by using a bacterial system production and in vitro enzymatic experiments. The role of the second Kunitz domain in this functional duality is described by assessing the anti‐trypsin activity of GASP‐1/2 chimeric proteins. Molecular dynamics simulations support the experimental data to rationalize differences in binding modes between trypsin and the GASP‐1 and GASP‐2 second Kunitz domains. A new inhibition mechanism was evidenced for the second Kunitz domain of GASP‐2, in which the conventional cationic residue of trypsin inhibitors was substituted by the strongly‐interacting glutamine residue.

Myogenesis is a multistep process taking place during pre- and postnatal stages for muscle formation, growth, and regeneration. It is a highly regulated process involving many molecular factors which act during myoblast proliferation and differentiation. To provide new insights into the molecular mechanisms and interactions behind the regulation of these different steps, RNA interference is an efficient methodology to implement. We developed a high-throughput siRNA screen in C2C12 murine myoblast cells for identification of genes relevant to signaling pathways controlling muscle growth. The proposed protocol is based on (1) the analyses of a maximum number of cells/myotubes to detect and quantify both clear and subtle phenotypes during proliferation/fusion cells and (2) the use of two cellular fluorescent markers, DAPI and myosin, decorating nuclei and myotubes respectively. Four phenotypic criteria were quantitatively assessed: cellular density, myotubes quantity, fusion index, and size and morphology of myotubes.