Purification et caractérisation biochimique d'une nucléotidase d'origine hépatique

Ce n'est qu'au cours des deux dernières décennies que les chercheurs se sont davantage intéressés à l'importance des nucléotides extracellulaires, en particulier aux purines extracellulaires, et aux rôles qu'ils pouvaient bien jouer au niveau des divers systèmes physiologiques des animaux. Au niveau du foie, ils seraient impliqués dans la sécrétion biliaire dépendante de l'ATP, dans la glycogénolyse et la contraction/relaxation des vaisseaux sanguins irriguant le foie. Notre intérêt pour le sujet nous a amené à la purification et à la caractérisation d'une ectonucléotidase, l'ATP-diphosphohydrolase hépatique, afin de mieux comprendre le rôle de cette enzyme au niveau du foie. Le but de notre travail était de purifier l'enzyme afin d'en définir ses caractéristiques biochimiques et cinétiques. Cette étude pare la voie au clonage de son gène. Les résultats ont montré que l'ATP-diphosphohydrolase hépatique possédait des caractéristiques différentes des ATPDases déjà purifiées et caractérisées. De cette étude, nous avons pu conclure que les ions Ca [exposant] 2+ et Mg [exposant] 2+ sont nécessaires pour l'activité de l'enzyme hépatique. Ce travail contient une deuxième partie portant aussi sur une nucléotidase. Dans le cadre de la purification de nucléotidases, des résultats intéressants ont mené vers la purification d'une protéine soluble démontrant des activités phosphohydrolases. L'un des objectifs de ma maîtrise fut de caractériser une protéine soluble démontrant des activités nucléotidases et purifiée originalement par Salma Daoud. Ce peptide, appelé peptide S, est un peptide dérivé d'une protéine connue: la calponine. Ces résultats sont présentés dans l'annexe I. Ils démontrent qu'un polyphosphate est étroitement lié à la protéine. De plus, lorsque la protéine est dépourvue de cette molécule de polyphosphate, celle-ci ne possède pas d'activité nucléotidase."--Résumé abrégé par UMI

Caractérisation par imagerie en temps réel de cultures cellulaires hépatiques en biopuces microfluidiques

Le développement de méthodes alternatives à la culture in vivo pour l évaluation de la toxicité des molécules chimiques s est accéléré ces dernières années, l objectif étant de limiter l utilisation d animaux. Préconisés par l OCDE (Organisation de coopération et de développement économiques), ces modèles alternatifs visent à mimer les conditions physiologiques en employant des systèmes in vitro ou in silico. Parmi les différents systèmes développés, les biopuces microfluidiques ont prouvé leur contribution à l amélioration des fonctions cellulaires, ce qui permet des études toxicologiques pertinentes. Les travaux de ce doctorat sont basés sur l emploi de ces biopuces pour cultiver des hépatocytes (cellules du foie) et portent sur la mise au point d une méthode d analyse d images issues de ces cultures sous microscope au cours du temps. L acquisition d images tout au long de l expérience permet de suivre, après traitement, l évolution et le comportement des cellules au contact de molécules chimiques et d évaluer les réponses toxicologiques. Les premiers résultats de ces travaux ont permis l amélioration du procédé de culture microfluidique adaptée au matériel d acquisition d images, la sélection de sondes fluorescentes, et le choix d un algorithme de traitement des images sur CellProfiler. Cela nous a permis de quantifier et caractériser certaines fonctions biologiques au sein de la biopuce comme l activité mitochondriale. Le potentiel de cet outil pour évaluer la toxicité de molécule a été testé grâce à l emploi d un toxique connu : la staurosporine. Les résultats obtenus ont révélé l impact de la mise en culture en dynamique sur le comportement des hépatocytes, et la toxicité de la staurosporine visible en biopuce.The development of alternative methods of in vivo cultures for the toxicological evaluation of chemical molecules has accelerated this last years, in order to limit the use of animals. Recommended by the OECD (Organisation for Economic Cooperation and Development), these alternative models are designed to mimic the physiological conditions using in vitro or in silico systems. Among the developed systems, microfluidic biochips have proven their contribution to the improvement of cellular functions, which allows relevant toxicological studies. This PhD thesis is based on the use of these biochips for hepatocytes culture and focus on the development of an analysis method for study these cultures under microscope over time using imaging. Image acquisition throughout the experiment enables to analyze, after image processing, the evolution and the behavior of cells in contact with chemical molecules and to evaluate toxicological responses. The first results of this work led to the optimization of the microfluidic cultures under the microscope used to get the image sequences, the selection of fluorescent probes and the development of an image processing system with CellProfiler. These works allowed the quantification and the characterization of some biological functions within the biochip such as the mitochondrial activity. Staurosporine, a well-known toxic, has been used to test the potential of this tool to evaluate the toxicity of molecules. The results showed the impact of dynamic culture on the hepatocytes behavior, and the staurosporine toxicity, in biochip cultures.COMPIEGNE-BU (601592101) / SudocSudocFranceF

Quiescence status of glioblastoma stem-like cells involves remodelling of Ca 2+ signalling and mitochondrial shape

International audienceQuiescence is a reversible cell-cycle arrest which allows cancer stem-like cells to evade killing following therapies. Here, we show that proliferating glioblastoma stem-like cells (GSLCs) can be induced and maintained in a quiescent state by lowering the extracellular pH. Through RNAseq analysis we identified Ca 2+ signalling genes differentially expressed between proliferating and quiescent GSLCs. Using the bioluminescent Ca 2+ reporter EGFP-aequorin we observed that the changes in Ca 2+ homeostasis occurring during the switch from proliferation to quiescence are controlled through store-operated channels (SOC) since inhibition of SOC drives proliferating GSLCs to quiescence. We showed that this switch is characterized by an increased capacity of GSLCs' mitochondria to capture Ca 2+ and by a dramatic and reversible change of mitochondrial morphology from a tubular to a donut shape. Our data suggest that the remodelling of the Ca 2+ homeostasis and the reshaping of mitochondria might favours quiescent GSLCs' survival and their aggressiveness in glioblastoma. Multiform glioblastoma (GBM) is the most aggressive brain tumours with very poor prognosis. Despite a combination of surgical resection, radiotherapy and temozolomide (TMZ)-based chemotherapy, more than 90% of the patients show recurrence and the mean survival period rarely exceeds 2 years 1. According to the cancer stem cell model, the GBM lethality is due to a small sub-population of tumour cells with stem-like properties, called Glioblastoma Stem-Like Cells (GSLCs). The GSLCs have been further characterized as slow-cycling or relatively quiescent cells 2 , identified in vivo in a mouse model of glioblastoma 3 and in human glioblastoma tumors 4. These quiescent GSLCs are highly resistant to TMZ treatment 5. Quiescence is a cell-cycle arrest state which differs from the one observed in differentiation or senescence by the fact that it is reversible. Transcriptional profiling data reveals that quiescent stem cells are characterized by a common gene signature with the down-regulation of genes associated with cell-cycle progression (i.e. CCNA2, CCNB1 and CCNE2) and the upregulation of genes classified as tumour suppressors, including the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the G0/G1 switch gene 2 (G0S2) 6,7. These data also show that quiescence is a G 0 phase and not a prolonged G 1 phase 8. Furthermore, quiescence is actively regulated by signals provided by the stem cell microenvironment. In glioblastoma tumours, quiescent stem-like tumour cells are found close to necrotic tissues, in specific niches characterized by an hypoxic 4,5,9 and acidic microenviron-ment 10,11. The role of the microenvironment in the control of GSLCs quiescence is still poorly understood. Considering that quiescence represents a strategy for GSLCs to evade killing, it is of utmost importance to better characterize the quiescent GSLCs and to understand what governs the transition from a proliferative to a quiescence state. Here, we performed transcriptomic analysis using RNAseq to establish the RNA signatures of proliferative and quiescent GSLCs. We showed that genes involved in Ca 2+ signalling are modulated in GSLCs and we explored the causal role of Ca 2+ during this transition. Our data points out the reversible remodelling of mitochondrial morphology from tubular to donut shape, associated with an increased capacity of mitochon-dria to capture Ca 2+ and with the modification of the kinetics of Ca 2+ influx through SOC. The remodelling o

Ca2+-Dependent Transcriptional Repressors KCNIP and Regulation of Prognosis Genes in Glioblastoma

Glioblastomas (GBMs) are the most aggressive and lethal primary astrocytic tumors in adults, with very poor prognosis. Recurrence in GBM is attributed to glioblastoma stem-like cells (GSLCs). The behavior of the tumor, including proliferation, progression, invasion, and significant resistance to therapies, is a consequence of the self-renewing properties of the GSLCs, and their high resistance to chemotherapies have been attributed to their capacity to enter quiescence. Thus, targeting GSLCs may constitute one of the possible therapeutic challenges to significantly improve anti-cancer treatment regimens for GBM. Ca2+ signaling is an important regulator of tumorigenesis in GBM, and the transition from proliferation to quiescence involves the modification of the kinetics of Ca2+ influx through store-operated channels due to an increased capacity of the mitochondria of quiescent GSLC to capture Ca2+. Therefore, the identification of new therapeutic targets requires the analysis of the calcium-regulated elements at transcriptional levels. In this review, we focus onto the direct regulation of gene expression by KCNIP proteins (KCNIP1–4). These proteins constitute the class E of Ca2+ sensor family with four EF-hand Ca2+-binding motifs and control gene transcription directly by binding, via a Ca2+-dependent mechanism, to specific DNA sites on target genes, called downstream regulatory element (DRE). The presence of putative DRE sites on genes associated with unfavorable outcome for GBM patients suggests that KCNIP proteins may contribute to the alteration of the expression of these prognosis genes. Indeed, in GBM, KCNIP2 expression appears to be significantly linked to the overall survival of patients. In this review, we summarize the current knowledge regarding the quiescent GSLCs with respect to Ca2+ signaling and discuss how Ca2+via KCNIP proteins may affect prognosis genes expression in GBM. This original mechanism may constitute the basis of the development of new therapeutic strategies

Diabetic foot complications among Indigenous peoples in Canada: a scoping review through the PROGRESS-PLUS equity lens

IntroductionIndigenous peoples in Canada face a disproportionate burden of diabetes-related foot complications (DRFC), such as foot ulcers, lower extremity amputations (LEA), and peripheral arterial disease. This scoping review aimed to provide a comprehensive understanding of DRFC among First Nations, Métis, and Inuit peoples in Canada, incorporating an equity lens.MethodsA scoping review was conducted based on Arksey and O’Malley refined by the Joanna Briggs Institute. The PROGRESS-Plus framework was utilized to extract data and incorporate an equity lens. A critical appraisal was performed, and Indigenous stakeholders were consulted for feedback. We identified the incorporation of patient-oriented/centered research (POR).ResultsOf 5,323 records identified, 40 studies were included in the review. The majority of studies focused on First Nations (92%), while representation of the Inuit population was very limited populations (< 3% of studies). LEA was the most studied outcome (76%). Age, gender, ethnicity, and place of residence were the most commonly included variables. Patient-oriented/centered research was mainly included in recent studies (16%). The overall quality of the studies was average. Data synthesis showed a high burden of DRFC among Indigenous populations compared to non-Indigenous populations. Indigenous identity and rural/remote communities were associated with the worse outcomes, particularly major LEA.DiscussionThis study provides a comprehensive understanding of DRFC in Indigenous peoples in Canada of published studies in database. It not only incorporates an equity lens and patient-oriented/centered research but also demonstrates that we need to change our approach. More data is needed to fully understand the burden of DRFC among Indigenous peoples, particularly in the Northern region in Canada where no data are previously available. Western research methods are insufficient to understand the unique situation of Indigenous peoples and it is essential to promote culturally safe and quality healthcare.ConclusionEfforts have been made to manage DRFC, but continued attention and support are necessary to address this population’s needs and ensure equitable prevention, access and care that embraces their ways of knowing, being and acting.Systematic review registrationOpen Science Framework https://osf.io/j9pu7, identifier j9pu7

Clinical Study Surgery Should Complement Endocrine Therapy for Elderly Postmenopausal Women with Hormone Receptor-Positive Early-Stage Breast Cancer

Introduction. Endocrine therapy (ET) is an integral part of breast cancer (BC) treatment with surgical resection remaining the cornerstone of curative treatment. The objective of this study is to compare the survival of elderly postmenopausal women with hormone receptor-positive early-stage BC treated with ET alone, without radiation or chemotherapy, versus ET plus surgery. Materials and Methods. This is a retrospective study based on a prospective database. The medical records of postmenopausal BC patients referred to the surgical oncology service of two hospitals during an 8-year period were reviewed. All patients were to receive ET for a minimum of four months before undergoing any surgery. Results. Fifty-one patients were included and divided in two groups, ET alone and ET plus surgery. At last follow-up in exclusive ET patients (n = 28), 39% had stable disease or complete response, 22% had progressive disease, of which 18% died of breast cancer, and 39% died of other causes. In surgical patients (n = 23), 78% were disease-free, 9% died of recurrent breast cancer, and 13% died of other causes. Conclusions. These results suggest that surgical resection is beneficial in this group and should be considered, even for patients previously deemed ineligible for surgery

Standardized Whole-Blood Transcriptional Profiling Enables the Deconvolution of Complex Induced Immune Responses

SummarySystems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)