Films biomimétiques multicouches pour les applications dans l'ingénierie tissulaire musculosquelettique.

Tissue engineering approach consists in combining cells, engineering and biomaterials to improve the biological functions of damaged tissues or to replace them. Production of “artificial tissues” is still challenging and requires collaboration of scientists from different domains like cell biology, chemistry, materials and polymer science. Skeletal muscle tissue engineering holds promise for the replacement of muscle due to an injury and for the treatment of muscle diseases, such as muscle dystrophies or paralysis, but is also required for pharmaceutical assays. To this end, materials with tunable mechanical and biochemical properties for myoblast expansion and differentiation in vitro, as well as for the studies of myogenesis on controlled 2D microenvironments or in 3D scaffolds, are crucially needed. In this work, we use layer-by-layer (LbL) assemblies for two goals. The first consisted in the development of multifunctional biomimetic thin films for the control of skeletal muscle cell fate on 2D substrates. We use LbL films made of polypeptides, which can be stiffened by chemical cross-linking and can be specifically functionalized by grafting of biomimetic peptides onto their surface. In addition, we combined the peptide-grafted films with substrate microtopography. Such approach is promising for the development or multifunctional materials that combine the different stimuli present in in vivo ECM, among them physical and biochemical cues, but also microtopography. In the second part, we use LbL assemblies for the construction of 3D skeletal muscle microtissues. This allows to rapidly build 3D muscle tissues and is promising for the in vitro construction of physiologically relevant skeletal muscle tissue models.L'ingénierie tissulaire consiste à assembler de façon intelligente des cellules et des matériaux biocompatibles dans le but de créer des tissus artificiels. Pour la construction de tissus en laboratoire, il est indispensable d'élaborer des matériaux qui miment cet environnement. Dans ce cadre, la collaboration entre les scientifiques de différents domaines (matériaux, chimie, biologie, biochimie) s'avère nécessaire. L'ingénierie du muscle squelettique est prometteuse pour remplacer le tissu musculaire endommagé et pour le traitement des maladies du muscle, mais aussi pour les essais pharmaceutiques. Dans ce but, les matériaux avec les propriétés mécaniques et chimiques contrôlées sont requis -- pour l'amplification et la différenciation in vitro de cellules souches musculaires, mais aussi pour l'étude de la myogenèse sur des microenvironnements contrôlés 2D et dans les matrices 3D. Dans ce travail, nous avons utilisé la technique d'assemblage couche par couche (LbL, layer-by-layer) pour deux buts. Le premier a été de développer de nouveaux films biomimétiques possédant des propriétés biochimiques et mécaniques parfaitement contrôlées, pour étudier les interrelations entre ces deux paramètres sur les processus cellulaires. En plus, nous avons associé ces films biomimetiques aux substrats avec la topographie contrôlée, afin de guider la formation du tissu. Dans un second temps, nous avons utilisé la technique LbL pour organiser les cellules en structures 3D. Nous avons ainsi crée des microtissus d'épaisseur contrôlée, qui pourraient être utilisés en tant que modèles de tissus artificiels pour les applications thérapeutiques ou pour les évaluations de médicament en industrie pharmaceutique

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Inativação fotodinâmica de bactérias por porfirinas catiónicas : os seus alvos celulares e potenciais aplicações ambientais

Doutoramento em BiologiaPhotodynamic inactivation (PDI) is defined as the process of cell destruction by oxidative stress resulting from the interaction between light and a photosensitizer (PS), in the presence of molecular oxygen. PDI of bacteria has been extensively studied in recent years, proving to be a promising alternative to conventional antimicrobial agents for the treatment of superficial and localized infections. Moreover, the applicability of PDI goes far beyond the clinical field, as its potential use in water disinfection, using PS immobilized on solid supports, is currently under study. The aim of the first part of this work was to study the oxidative modifications in phospholipids, nucleic acids and proteins of Escherichia coli and Staphylococcus warneri, subjected to photodynamic treatment with cationic porphyrins. The aims of the second part of the work were to study the efficiency of PDI in aquaculture water and the influence of different physicalchemical parameters in this process, using the Gram-negative bioluminescent bacterium Vibrio fischeri, and to evaluate the possibility of recycling cationic PS immobilized on magnetic nanoparticles. To study the oxidative changes in membrane phospholipids, a lipidomic approach has been used, combining chromatographic techniques and mass spectrometry. The FOX2 assay was used to determine the concentration of lipid hydroperoxides generated after treatment. The oxidative modifications in the proteins were analyzed by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Changes in the intracellular nucleic acids were analyzed by agarose gel electrophoresis and the concentration of doublestranded DNA was determined by fluorimetry. The oxidative changes of bacterial PDI at the molecular level were analyzed by infrared spectroscopy. In laboratory tests, bacteria (108 CFU mL-1) were irradiated with white light (4.0 mW cm-2) after incubation with the PS (Tri-Py+-Me-PF or Tetra-Py+-Me) at concentrations of 0.5 and 5.0 μM for S. warneri and E. coli, respectively. Bacteria were irradiated with different light doses (up to 9.6 J cm-2 for S. warneri and up to 64.8 J cm-2 for E. coli) and the changes were evaluated throughout the irradiation time. In the study of phospholipids, only the porphyrin Tri-Py+-Me-PF and a light dose of 64.8 J cm-2 were tested. The efficiency of PDI in aquaculture has been evaluated in two different conditions: in buffer solution, varying temperature, pH, salinity and oxygen concentration, and in aquaculture water samples, to reproduce the conditions of PDI in situ. The kinetics of the process was determined in realtime during the experiments by measuring the bioluminescence of V. fischeri (107 CFU mL-1, corresponding to a level of bioluminescence of 105 relative light units). A concentration of 5.0 μM of Tri-Py+-Me-PF was used in the experiments with buffer solution, and 10 to 50 μM in the experiments with aquaculture water. Artificial white light (4.0 mW cm-2) and solar irradiation (40 mW cm-2) were used as light sources.Os resultados deste trabalho mostraram que E. coli foi totalmente inativada com ambas as porfirinas, enquanto S. warneri foi completamente inativado apenas com a Tri-Py+-Me-PF, ao fim do tempo de irradiação previamente estabelecido. A IF induziu alterações no perfil fosfolipídico bacteriano, com aumento da abundância relativa de algumas das classes maioritárias de fosfolípidos, decréscimo de ácidos gordos insaturados, formação de espécies moleculares oxidadas a partir de ácidos gordos insaturados, nomeadamente nas cardiolipinas de S. warneri e nas fosfatidiletanolaminas de E. coli. Estas espécies oxidadas foram identificadas como derivados hidroxi e hidroperoxi (observados em E. coli) e também grupos carbonilo (em S. warneri). A formação de hidroperóxidos lipídicos confirmou os danos oxidativos nos fosfolípidos. A IF causou redução do conteúdo intracelular dos ácidos nucleicos bacterianos. Em E. coli observou-se a seguinte hierarquia de modificações: rRNA 23S > rRNA 16S > DNA genómico. Os ácidos nucleicos de S. warneri foram extensivamente reduzidos com a Tri-Py+-Me-PF após 5 min de irradiação, mas menos reduzidos com a Tetra-Py+-Me, após 40 min de irradiação. Esta degradação dos ácidos nucleicos ocorreu paralelamente à inativação e quando as células já estavam inativadas mais do que 99.9%. A IF induziu uma diminuição geral do conteúdo proteico de ambas as bactérias, sugerindo degradação em larga escala, ocorrendo as alterações de forma mais rápida e evidente com a porfirina Tri-Py+-Me-PF. Observou-se o aumento da expressão de algumas proteínas, alterações no peso molecular, desaparecimento após tratamento e formação de novas proteínas. As alterações foram associadas a mecanismos de resposta ao stress oxidativo. A espetroscopia de infravermelho mostrou ser um método rápido e económico de avaliar as alterações induzidas pela IF ao nível molecular. Evidenciou os resultados obtidos pelos métodos convencionais com maior detalhe, nomeadamente ao nível das ligações, grupos funcionais e conformações moleculares. As variações de pH (6.5 - 8.5), temperatura (10 - 25 ºC), salinidade (20 - 40 g L-1) e concentração de oxigénio não afetaram significativamente a IF de V. fischeri, uma vez que em todas as condições testadas o sinal bioluminescente diminuiu até ao limite de deteção do método (redução ≈ 7 log10). Os ensaios com água de aquacultura mostraram que a eficiência do processo é afetada pela presença de matéria em suspensão. A IF total de V. fischeri em água de aquacultura foi conseguida com luz solar na presença de 20 μM de Tri-Py+-Me-PF. Os híbridos nanomagnete-porfirina puderam ser reutilizados em 6 ciclos de IF e reciclados em 3 ciclos. Na reciclagem, houve perda de atividade de ciclo para ciclo, atribuída à perda de nanopartículas durante a recuperação. A acumulação de matéria orgânica causou uma redução da eficiência do processo durante a reutilização, contudo foi observada a eliminação de 38 a 42 log10 de bactérias ao fim de 21h30 a 27h de tratamento. O FS não foi fotodegradado e a magnetite das nanopartículas não foi afetada pela irradiação ou pela oxidação inerente ao processo fotodinâmico. O presente trabalho demonstrou o caráter multi-alvo da inativação fotodinâmica, pela elucidação dos mecanismos oxidativos que ocorrem ao nível dos principais constituintes moleculares das bactérias. Também demonstrou que a inativação fotodinâmica é uma metodologia com potencial para ser implementada na desinfeção de águas de aquacultura utilizando fotossensibilizadores imobilizados, permitindo a sua reutilização e reciclagem, com a possibilidade de reduzir os custos associados a este tipo de tratamento

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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