Biomatériaux dérivés d’une matrice extracellulaire (MEC) pour l’ingénierie tissulaire et les dispositifs médicaux

Abstract: Tissue engineering involves the production of whole organ or a part of it in vitro or in vivo. Decellularized organs as scaffolds for reconstructing organs have been emerging due to the potential of the ExtraCellular Matrix (ECM). ECM is a complex structure primarily composed of proteins and glycosaminoglycans (GAGs). Most common proteins include collagens, laminins, fibronectins and elastins. Several commercial products have been derived from ECM including tissue papers, 3D-printed scaffolds, and wound dressings. Bioadhesives are currently employed alone or as adjuncts to sutures to seal leaks of air or blood from organs following surgical interventions. ECM-incorporated bioadhesives could be hypothesized to not only seal leaks, but also to regenerate tissues. This thesis aims to investigate the composition and properties of ECMs derived from different porcine organs (bladders, kidneys, livers, lungs, and pancreas) using detergent-based and detergent-free methods. The first experimental work includes the design of a cell culture system to study the effect of detergent-based and detergent-free decellularized bladders on insulin-secreting rat pancreatic cell (INS-1) proliferation and functionality. ECMs were characterized initially for conservation of ultrastructure and removal of dsDNA. CyQUANT proliferation assay indicated cell proliferation following 7 days of culture on detergent-free decellularized bladders. Glucose-stimulated insulin secretion (GSIS) and immunostaining confirmed that cells were functional. The second experimental work involved decellularization of the five porcine organs using the detergent-based and detergent-free methods. Two additional steps were added to the detergent-free approach (pH adjustment and ethylenediaminetetraacetic acid (EDTA) treatment) to aid in the removal of residual hemoglobin from the organs. ECMs were characterized by staining for the removal of cellular content and conservation of ultrastructure. Further, mass spectrometry revealed better conservation of a greater number of key proteins such as collagen IV, laminins, fibronectin, and elastin in the ECM resulting from the detergent-free methods, as compared to that produced using the detergent-based one. Collagen fibers orientation measurement indicated preservation of the fibers orientation in the ECMs as compared to that measured in the native organs. The third experimental work initially screened the INS-1 cell response on different organ ECMs. INS-1 cells were functional on certain detergent-free decellularized organs following 7 days of cell culture. Finally, mouse primary pancreatic islets were seeded on the detergent-free decellularized bladders, revealing functional islets following 48 hours of culture.Le génie tissulaire consiste à construire un organe entier ou une partie de celui-ci in vitro ou in vivo. Les organes décellularisés utilisés comme échafaudages pour la reconstruction d'organes sont de plus en plus populaires en raison, entre autres, du potentiel de la matrice extracellulaire (MEC). La MEC consiste en un ensemble complexe composé principalement de protéines et de glycosaminoglycanes (GAG). Les protéines les plus courantes comprennent les collagènes, les laminines, les fibronectines et l’élastine. Plusieurs produits commerciaux sont composés de MEC, notamment des papiers tissulaires, des encres pour l’impression 3D et des pansements pour le traitement de plaies. Les bio-adhésifs sont actuellement utilisés seuls ou en complément des sutures pour sceller les fuites d'air ou de sang à la suite d’interventions chirurgicales. On pourrait supposer, par exemple, qu’un bio-adhésif incorporant la MEC permettrait non seulement de sceller une fuite, mais qu'il contribuerait également à la régénération tissulaire. Cette thèse a pour objectif général d’évaluer la composition et les propriétés de la MEC dérivée de différents organes porcins (vessie, rein, foie, poumon et pancréas) décellularisés à l'aide de méthodes utilisant un détergent et sans détergent. Également, le projet vise à développer une nouvelle famille de biomatériaux à base de MEC pour des applications en médecine. Le premier travail expérimental comprend la conception d'un système de culture cellulaire pour étudier l'effet des vessies décellularisées, avec ou sans détergent, sur la prolifération et la fonctionnalité des cellules pancréatiques (INS-1 cellules) de rat sécrétant de l'insuline en réponse à des gradients de glucose. Les MECs ont été initialement caractérisées pour la conservation de l'ultrastructure et l'élimination de l'ADN double brin. L'analyse utilisant un test de prolifération CyQUANT a indiqué une prolifération cellulaire après 7 jours de culture sur les vessies décellularisées sans détergent. La sécrétion d'insuline stimulée par le glucose (GSIS) et l'immunomarquage ont confirmé que les cellules étaient également fonctionnelles. Le deuxième travail expérimental visait la décellularisation des cinq organes porcins à l'aide d’une méthode utilisant un détergent et de méthodes sans détergent. Deux étapes supplémentaires ont été ajoutées à la technique sans détergent (ajustement du pH et traitement par éthylènediaminetétraacétate (EDTA)) afin de réduire la présence d'hémoglobine résiduelle dans les organes décellularisés. Les MECs ont été caractérisées en histologie par différentes colorations pour investiguer l'élimination du contenu cellulaire et la conservation de l'ultrastructure. De plus, la spectrométrie de masse a révélé la conservation d'un plus grand nombre de protéines clés telles que le collagène IV, les laminines, la fibronectine et l'élastine dans les MECs produites avec des méthodes sans détergent par rapport à celles résultantes de la méthode utilisant un détergent. Les mesures de l’orientation du collagène ont indiqué une conservation de l'orientation dans les MECs par rapport à la structure native. Le troisième travail expérimental a initialement investigué la réponse des cellules INS-1 exposées aux différentes MEC d'organes. Les cellules INS-1 demeuraient fonctionnelles sur certains organes décellularisés sans détergent après 7 jours de culture. Enfin, des îlots pancréatiques primaires de souris ont été ensemencés sur des vessies décellularisées sans détergent, révélant ainsi que les îlots étaient fonctionnels après 48 heures de culture

Transgenerational response to early spring warming in Daphnia

Abstract Temperature and photoperiod regulate key fitness traits in plants and animals. However, with temperature increase due to global warming, temperature cue thresholds are experienced at shorter photoperiods, disrupting the optimal seasonal timing of physiological, developmental and reproductive events in many species. Understanding the mechanisms of adaptation to the asynchrony between temperature and photoperiod is key to inform our understanding of how species will respond to global warming. Here, we studied the transgenerational mechanisms of responses of the cyclical parthenogen Daphnia magna to different photoperiod lengths co-occurring with warm temperature thereby assessing the impact of earlier spring warming on its fitness. Daphnia uses temperature and photoperiod cues to time dormancy, and to switch between sexual and asexual reproduction. Daphnia life cycle offers the opportunity to measure the relative contribution of plastic and genetic responses to environmental change across generations and over evolutionary time. We use transgenerational common garden experiments on three populations ‘resurrected’ from a biological archive experiencing temperature increase over five decades. Our results suggest that response to early spring warming evolved underpinned by a complex interaction between plastic and genetic mechanisms while a positive maternal contribution at matching environments between parental and offspring generation was also observed

Refining the evolutionary time machine:an assessment of whole genome amplification using single historical Daphnia eggs

This dataset contains the original raw sequence files used in the associated publication "Refining the evolutionary time machine: an assessment of whole genome amplification using single historical Daphnia eggs" by O'Grady, Dhandapani, Colbourne & Frisch in Molecular Ecology Resources. DOI:10.1111/1755-0998.13524 (filename > name used in associated publication) DF1_ATTACTC-GGCTCTG_L008_R1_001.fastq.gz -> DM1 DF2_TCCGGAG-GGCTCTG_L008_R1_001.fastq.gz -> DM2 DF3_CGCTCAT-GGCTCTG_L008_R1_001.fastq.gz -> DM3 DF4_GAGATTC-GGCTCTG_L008_R1_001.fastq.gz -> DM4 DF5_CTGAAGC-GGCTCTG_L008_R1_001.fastq.gz -> DM5 DF11_CTGAAGC-AGGCGAA_L008_R1_001.fastq.gz -> DM6 DF12_TAATGCG-AGGCGAA_L008_R1_001.fastq.gz -> DM7 DF6_TAATGCG-GGCTCTG_L008_R1_001.fastq.gz -> DP1 DF7_ATTACTC-AGGCGAA_L008_R1_001.fastq.gz -> DP4 DF8_TCCGGAG-AGGCGAA_L008_R1_001.fastq.gz -> DP5 DF9_CGCTCAT-AGGCGAA_L008_R1_001.fastq.gz -> DP2 DF10_GAGATTC-AGGCGAA_L008_R1_001.fastq.gz -> DP3 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-004_1.fastq.gz -> DP6 (fw) 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-004_2.fastq.gz -> DP6 (rv) 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-010_1.fastq.gz -> DP7 (fw) 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-010_2.fastq.gz -> DP7 (rv) 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-005_1.fastq.gz -> DP8 (fw) 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-005_2.fastq.gz -> DP8 (rv) 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-006_1.fastq.gz -> DP9 (fw) 170426_E00397_0064_BHJ2JWALXX_2_TP-D7-006_2.fastq.gz -> DP9 (rv

Historical exposure to chemicals reduces tolerance to novel chemical stress in Daphnia (waterflea)

Until the last few decades, anthropogenic chemicals used in most production processes have not been comprehensively assessed for their risk and impact on wildlife and humans. They are transported globally and usually end up in the environment as unintentional pollutants, causing long‐term adverse effects. Modern toxicology practices typically use acute toxicity tests of unrealistic concentrations of chemicals to determine their safe use, missing pathological effects arising from long‐term exposures to environmentally relevant concentrations. Here, we study the transgenerational effect of environmentally relevant concentrations of five chemicals on the priority list of international regulatory frameworks on the keystone species Daphnia magna. We expose Daphnia genotypes resurrected from the sedimentary archive of a lake with a known history of chemical pollution to the five chemicals to understand how historical exposure to chemicals influences adaptive responses to novel chemical stress. We measure within‐ and transgenerational plasticity in fitness‐linked life history traits following exposure of “experienced” and “naive” genotypes to novel chemical stress. As the revived Daphnia originate from the same genetic pool sampled at different times in the past, we are able to quantify the long‐term evolutionary impact of chemical pollution by studying genome‐wide diversity and identifying functional pathways affected by historical chemical stress. Our results suggest that historical exposure to chemical stress causes reduced genome‐wide diversity, leading to lower cross‐generational tolerance to novel chemical stress. Lower tolerance is underpinned by reduced gene diversity at detoxification, catabolism and endocrine genes in experienced genotypes. We show that these genes sit within pathways that are conserved and potential chemical targets in other species, including humans

Transgenerational response to early spring warming in Daphnia

Temperature and photoperiod regulate key fitness traits in plants and animals. However, with temperature increase due to global warming, temperature cue thresholds are experienced at shorter photoperiods, disrupting the optimal seasonal timing of physiological, developmental and reproductive events in many species. Understanding the mechanisms of adaptation to the asynchrony between temperature and photoperiod is key to inform our understanding of how species will respond to rapid environmental change. Here, we studied the transgenerational mechanisms of responses of the cyclical parthenogen Daphnia magna to different photoperiod lengths co-occurring with warm temperature, thereby assessing the impact of earlier spring warming on fitness. Daphnia uses temperature and photoperiod cues to time dormancy and to switch between sexual and asexual reproduction. Moreover, its life cycle offers the opportunity to measure the relative contribution of plastic and genetic responses to environmental change across generations and over evolutionary time. We use transgenerational common garden experiments on three populations ‘resurrected’ from a biological archive experiencing temperature increase over five decades. Our results suggest that D. magna has the potential to evolve in response to early spring warming via a complex interaction between plastic and genetic mechanisms. The results also suggest a positive maternal effect in presence of matching environments between parental and offspring generation

Genomic and Post-Translational Modification Analysis of Leucine-Rich-Repeat Receptor-Like Kinases in Brassica rapa.

Among several receptor-like kinases (RLKs), leucine-rich-repeat receptor-like kinases (LRR-RLKs) are a major group of genes that play crucial roles in growth, development and stress responses in plant systems. Given that they have several functional roles, it is important to investigate their roles in Brassica rapa. In the present study, 303 LRR-RLKs were identified in the genome of B. rapa and comparative phylogenetic analysis of 1213 combined LRR-RLKs of B. rapa, Arabidopsis thaliana, Oryza sativa and Populus trichocarpa helped us to categorize the gene family into 15 subfamilies based on their sequence and structural similarities. The chromosome localizations of 293 genes allowed the prediction of duplicates, and motif conservation and intron/exon patterns showed differences among the B. rapa LRR-RLK (BrLRR-RLK) genes. Additionally, computational function annotation and expression analysis was used to predict their possible functional roles in the plant system. Biochemical results for 11 selected genes showed variations in phosphorylation activity. Interestingly, BrBAK1 showed strong auto-phosphorylation and trans-phosphorylation on its tyrosine and threonine residues compared with AtBAK1 in previous studies. The AtBAK1 receptor kinase is involved in plant growth and development, plant innate immunity, and programmed cell death, and our results suggest that BrBAK1 might also be involved in the same functions. Another interesting result was that BrBAK1, BrBRI1, BrPEPR1 and BrPEPR2 showed activity with both anti-phosphotyrosine and anti-phosphothreonine antibodies, indicating that they might have dual-specificity kinase activity. This study provides comprehensive results for the BrLRR-RLKs, revealing expansion of the gene family through gene duplications, structural similarities and variations among the genes, and potential functional roles according to gene ontology, transcriptome profiling and biochemical analysis