Cell behavior is influenced to a large extent by the surrounding microenvironment. Therefore, in the body, the cellular microenvironment is highly controlled with cells growing within well-defined tissue architectures. However, traditional culture techniques allow only for the random placement of cells onto culture dishes and biomaterials. Cell micropatterning strategies aim to control the spatial localization of cells on their underlying material and in relation to other cells. Developing such strategies provides us with tools necessary to eventually fabricate the highly-controlled microenvironments found in multicellular organisms. Employing natural extracellular matrix (ECM) materials in patterning techniques can increase biocompatibility. In the future, with such technologies, we can hope to conduct novel studies in cell biology or optimize cell behavior and function towards the development of new cell-based devices and tissue engineering constructs. Herein, a novel cell patterning platform was developed on a hydrogel base of crosslinked hyaluronic acid (HA). Hydrogels are often employed in tissue engineering due to their ability to mimic the physicochemical properties of natural tissues. HA is a polymer present in all connective tissues. Cell-adhesive regions on the hydrogel were created using the RGDS peptide sequence, found within the cell-adhesive ECM protein, fibronectin. The peptide was bound to a 2-nitrobenzyl “caging group” via a photolabile bond to render the peptide light-responsive. Finally, this “caged” peptide was covalently bound to the hydrogel to form a novel HA hydrogel with a cell non-adhesive surface which could be activated with near-UV light to become adhesive. In this way, we successfully formed chemically patterned cell-adhesive regions on a HA hydrogel using light as a stimulus to form controlled cell patterns. While the majority of cell patterning strategies to date are limited to patterning one cell population and cannot be changed with time, our strategy was novel in using small, adhesive, caged peptides combined with multiple, aligned light exposure steps to allow for dynamic chemical cell patterning on a hydrogel. Multiple cell populations, even held apart from one another, were successfully patterned on the same hydrogel. Furthermore, cell patterns were deliberately modified with time to direct cell growth and/or migration on the hydrogel base.

Cell behavior is influenced to a large extent by the surrounding microenvironment. Therefore, in the body, the cellular microenvironment is highly controlled with cells growing within well-defined tissue architectures. However, traditional culture techniques allow only for the random placement of cells onto culture dishes and biomaterials. Cell micropatterning strategies aim to control the spatial localization of cells on their underlying material and in relation to other cells. Developing such strategies provides us with tools necessary to eventually fabricate the highly-controlled microenvironments found in multicellular organisms. Employing natural extracellular matrix (ECM) materials in patterning techniques can increase biocompatibility. In the future, with such technologies, we can hope to conduct novel studies in cell biology or optimize cell behavior and function towards the development of new cell-based devices and tissue engineering constructs. Herein, a novel cell patterning platform was developed on a hydrogel base of crosslinked hyaluronic acid (HA). Hydrogels are often employed in tissue engineering due to their ability to mimic the physicochemical properties of natural tissues. HA is a polymer present in all connective tissues. Cell-adhesive regions on the hydrogel were created using the RGDS peptide sequence, found within the cell-adhesive ECM protein, fibronectin. The peptide was bound to a 2-nitrobenzyl “caging group” via a photolabile bond to render the peptide light-responsive. Finally, this “caged” peptide was covalently bound to the hydrogel to form a novel HA hydrogel with a cell non-adhesive surface which could be activated with near-UV light to become adhesive. In this way, we successfully formed chemically patterned cell-adhesive regions on a HA hydrogel using light as a stimulus to form controlled cell patterns. While the majority of cell patterning strategies to date are limited to patterning one cell population and cannot be changed with time, our strategy was novel in using small, adhesive, caged peptides combined with multiple, aligned light exposure steps to allow for dynamic chemical cell patterning on a hydrogel. Multiple cell populations, even held apart from one another, were successfully patterned on the same hydrogel. Furthermore, cell patterns were deliberately modified with time to direct cell growth and/or migration on the hydrogel base.

No description available.

Os vegetais apresentam variados mecanismos de defesa, os quais conferem tolerância a elementos considerados tóxicos, como o alumínio (Al). Em primeiro experimento, objetivou-se avaliar a tolerância diferencial ao Al em quatro plantas forrageiras do gênero Brachiaria (B. decumbens cv. Basilisk, B. brizantha cv. Marandu, B. brizantha cv. Piatã e B. brizantha cv. Xaraés), por meio da quantificação da área foliar; aspectos morfológicos do sistema radicular (comprimento total e superfície total de raízes); produção de biomassa de raízes e parte aérea; concentração, acúmulo e transporte de Al à longa distância; peroxidação lipídica em tecidos de folhas e raízes e concentração de H2O2 nas folhas. As concentrações de Al empregadas na solução nutritiva foram de 0; 0,44; 0,89 e 1,33 mmol L-1, as quais foram distribuídas conforme delineamento experimental de blocos completos ao acaso, utilizando-se esquema fatorial 4 x 4 (quatro doses de Al x quatro genótipos de Brachiaria), com quatro repetições. A atividade do Al3+ livre na solução nutritiva foi estimada utilizando o software GeoChem-EZ®, o qual evidenciou que cerca de 81% do Al estava disponível, considerando a variação nos valores de pH de 3,0 a 4,0. A adição de Al na solução nutritiva resultou na redução de parâmetros produtivos da parte aérea e do sistema radicular, além de aumentar a concentração e o acúmulo do metal nas raízes. Por intermédio de tais parâmetros, permitiu-se a seguinte classificação, quanto à tolerância diferencial ao Al: B. brizantha cv. Xaraés > B. decumbens cv. Basilisk >= B. brizantha cv. Piatã > B. brizantha cv. Marandu. No segundo experimento a B. brizantha cv. Marandu (menor tolerância) e a B. brizantha cv. Xaraés (maior tolerância) foram cultivadas em solução nutritiva e, em seguida, foram efetuadas avaliações referentes à morfologia e anatomia do sistema radicular (pêlos radiculares), por meio de microscopia de luz e microscopia eletrônica de varredura, determinação do Al no apoplasto e simplasto das raízes, bem como a quantificação da atividade de enzimas antioxidantes catalase (CAT), ascorbato peroxidase (APX), guaiacol peroxidase (GPOX) e glutationa redutase (GR), em folhas e raízes. Utilizaram-se as concentrações de Al na solução de 0 e 1,33 mmol L-1, as quais foram distribuídas conforme delineamento experimental de blocos completos ao acaso, utilizando-se esquema fatorial 2 x 2 (duas concentrações de Al x dois genótipos de Brachiaria), com oito repetições. As atividades das enzimas CAT, APX, GPOX e GR foram mais expressas em tecidos radiculares. O excesso de Al reduziu a atividade da CAT e da GPOX nas raízes de B. brizantha cv. Xaraés e da APX e GR nas raízes de B. brizantha cv. Marandu. Quanto à compartimentação do Al no sistema radicular, constatou-se que a maior parte do metal concentrou-se no simplasto radicular, para ambos os genótipos. Por sua vez, na condição de excesso do metal, a maior concentração de Al trocável no apoplasto radicular foi verificada no cultivar Xaraés, sendo 49% superior ao cultivar Marandu. Foram verificadas maiores injúrias na epiderme radicular, como microfissuras e descamação, no cultivar Marandu. Os resultados fornecem evidências de que os genótipos de Brachiaria apresentam distintas respostas ao excesso de Al, com maior ou menor eficiência, caracterizando a tolerância diferencial / A variety of plant defense mechanisms have been shown, which confer tolerance to elements considered toxics, such as aluminum (Al). The aim of the first experiment was to evaluate the differential aluminum tolerance in four forage plants of Brachiaria genus (B. decumbens cv. Basilisk, B. brizantha cv. Marandu, B. brizantha cv. Piatã and B. brizantha cv. Xaraés), by measuring leaf area; root system morphology (total root length and total root surface); quantifying roots and plant top biomass yield; the Al-concentration, uptake and Al-long distance transport; evaluating lipid peroxidation in roots and leaves tissues, as well as the H2O2 content in leaves. Aluminum rates used were 0; 0.44; 0.89 and 1.33 mmol L-1, which were distributed as randomized block design, using a factorial 4 x 4 (four Al rates x four Brachiaria genotypes), with four replications. The free Al3+ activity in the nutrient solution was estimated using the software GeoChem-EZ®, reveling that around 81% of Al was available, considering the pH range between 3.0 and 4.0. Al addition in the nutrient solution decreased the plant top and root dry matter yield, increased Al-concentration and uptake in the roots. Though all these parameters, this following rank - as related to differential Al tolerance - was done: B. brizantha cv. Xaraés > B. decumbens cv. Basilisk >= B. brizantha cv. Piatã > B. brizantha cv. Marandu. In the second experiment, B. brizantha cv. Marandu (lower Al tolerance) and B. brizantha cv. Xaraés (higher Al tolerance) were grown in nutrient solution, with 0 and 1.33 mmol L-1 Al-concentrations, which were distributed as randomized block design, using a factorial 2 x 2 (two Al rates x two Brachiaria genotypes), with eight replications. Root system morphology and anatomy (root hairs) evaluations by using light and scanning electron microscopy, the Al concentration in the apoplast and symplast of roots, as well as the antioxidant enzymes activities such as catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX) and glutathione reductase (GR) were taken in the leaves and roots tissues. The CAT, APX, GPOX and GR activities were more expressed in root tissues than leaves tissues. Al toxicity decreased CAT and GPOX activities in roots of B. brizantha cv. Xaraés on the one hand; and the other the APX and GR activity in B. brizantha cv. Marandu roots. As regards to Al partition in root system compartments, it was found that most of metal was accumulated in symplast, to both genotypes. On the other hand, in metal excess condition, the highest Al concentration on the root apoplast was verified to Xaraés cultivar, being 49% higher than those quantified on the Marandu cultivar. Major injuries were found in the root epidermis, as ruptures and small clefts, which in turn have induced significant structural changes on the root surface of Marandu genotype. Taken together, the results provide evidences that Brachiaria genotypes have distinct responses to Al excess, with greater or lesser efficiency mechanism, featuring differential Al-tolerance

No description available.

Os vegetais apresentam variados mecanismos de defesa, os quais conferem tolerância a elementos considerados tóxicos, como o alumínio (Al). Em primeiro experimento, objetivou-se avaliar a tolerância diferencial ao Al em quatro plantas forrageiras do gênero Brachiaria (B. decumbens cv. Basilisk, B. brizantha cv. Marandu, B. brizantha cv. Piatã e B. brizantha cv. Xaraés), por meio da quantificação da área foliar; aspectos morfológicos do sistema radicular (comprimento total e superfície total de raízes); produção de biomassa de raízes e parte aérea; concentração, acúmulo e transporte de Al à longa distância; peroxidação lipídica em tecidos de folhas e raízes e concentração de H2O2 nas folhas. As concentrações de Al empregadas na solução nutritiva foram de 0; 0,44; 0,89 e 1,33 mmol L-1, as quais foram distribuídas conforme delineamento experimental de blocos completos ao acaso, utilizando-se esquema fatorial 4 x 4 (quatro doses de Al x quatro genótipos de Brachiaria), com quatro repetições. A atividade do Al3+ livre na solução nutritiva foi estimada utilizando o software GeoChem-EZ®, o qual evidenciou que cerca de 81% do Al estava disponível, considerando a variação nos valores de pH de 3,0 a 4,0. A adição de Al na solução nutritiva resultou na redução de parâmetros produtivos da parte aérea e do sistema radicular, além de aumentar a concentração e o acúmulo do metal nas raízes. Por intermédio de tais parâmetros, permitiu-se a seguinte classificação, quanto à tolerância diferencial ao Al: B. brizantha cv. Xaraés > B. decumbens cv. Basilisk >= B. brizantha cv. Piatã > B. brizantha cv. Marandu. No segundo experimento a B. brizantha cv. Marandu (menor tolerância) e a B. brizantha cv. Xaraés (maior tolerância) foram cultivadas em solução nutritiva e, em seguida, foram efetuadas avaliações referentes à morfologia e anatomia do sistema radicular (pêlos radiculares), por meio de microscopia de luz e microscopia eletrônica de varredura, determinação do Al no apoplasto e simplasto das raízes, bem como a quantificação da atividade de enzimas antioxidantes catalase (CAT), ascorbato peroxidase (APX), guaiacol peroxidase (GPOX) e glutationa redutase (GR), em folhas e raízes. Utilizaram-se as concentrações de Al na solução de 0 e 1,33 mmol L-1, as quais foram distribuídas conforme delineamento experimental de blocos completos ao acaso, utilizando-se esquema fatorial 2 x 2 (duas concentrações de Al x dois genótipos de Brachiaria), com oito repetições. As atividades das enzimas CAT, APX, GPOX e GR foram mais expressas em tecidos radiculares. O excesso de Al reduziu a atividade da CAT e da GPOX nas raízes de B. brizantha cv. Xaraés e da APX e GR nas raízes de B. brizantha cv. Marandu. Quanto à compartimentação do Al no sistema radicular, constatou-se que a maior parte do metal concentrou-se no simplasto radicular, para ambos os genótipos. Por sua vez, na condição de excesso do metal, a maior concentração de Al trocável no apoplasto radicular foi verificada no cultivar Xaraés, sendo 49% superior ao cultivar Marandu. Foram verificadas maiores injúrias na epiderme radicular, como microfissuras e descamação, no cultivar Marandu. Os resultados fornecem evidências de que os genótipos de Brachiaria apresentam distintas respostas ao excesso de Al, com maior ou menor eficiência, caracterizando a tolerância diferencial / A variety of plant defense mechanisms have been shown, which confer tolerance to elements considered toxics, such as aluminum (Al). The aim of the first experiment was to evaluate the differential aluminum tolerance in four forage plants of Brachiaria genus (B. decumbens cv. Basilisk, B. brizantha cv. Marandu, B. brizantha cv. Piatã and B. brizantha cv. Xaraés), by measuring leaf area; root system morphology (total root length and total root surface); quantifying roots and plant top biomass yield; the Al-concentration, uptake and Al-long distance transport; evaluating lipid peroxidation in roots and leaves tissues, as well as the H2O2 content in leaves. Aluminum rates used were 0; 0.44; 0.89 and 1.33 mmol L-1, which were distributed as randomized block design, using a factorial 4 x 4 (four Al rates x four Brachiaria genotypes), with four replications. The free Al3+ activity in the nutrient solution was estimated using the software GeoChem-EZ®, reveling that around 81% of Al was available, considering the pH range between 3.0 and 4.0. Al addition in the nutrient solution decreased the plant top and root dry matter yield, increased Al-concentration and uptake in the roots. Though all these parameters, this following rank - as related to differential Al tolerance - was done: B. brizantha cv. Xaraés > B. decumbens cv. Basilisk >= B. brizantha cv. Piatã > B. brizantha cv. Marandu. In the second experiment, B. brizantha cv. Marandu (lower Al tolerance) and B. brizantha cv. Xaraés (higher Al tolerance) were grown in nutrient solution, with 0 and 1.33 mmol L-1 Al-concentrations, which were distributed as randomized block design, using a factorial 2 x 2 (two Al rates x two Brachiaria genotypes), with eight replications. Root system morphology and anatomy (root hairs) evaluations by using light and scanning electron microscopy, the Al concentration in the apoplast and symplast of roots, as well as the antioxidant enzymes activities such as catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX) and glutathione reductase (GR) were taken in the leaves and roots tissues. The CAT, APX, GPOX and GR activities were more expressed in root tissues than leaves tissues. Al toxicity decreased CAT and GPOX activities in roots of B. brizantha cv. Xaraés on the one hand; and the other the APX and GR activity in B. brizantha cv. Marandu roots. As regards to Al partition in root system compartments, it was found that most of metal was accumulated in symplast, to both genotypes. On the other hand, in metal excess condition, the highest Al concentration on the root apoplast was verified to Xaraés cultivar, being 49% higher than those quantified on the Marandu cultivar. Major injuries were found in the root epidermis, as ruptures and small clefts, which in turn have induced significant structural changes on the root surface of Marandu genotype. Taken together, the results provide evidences that Brachiaria genotypes have distinct responses to Al excess, with greater or lesser efficiency mechanism, featuring differential Al-tolerance

The inability of the myocardium to regenerate after injury plus the inadequate number of available hearts for transplantation have drawn attention to the creation of functional tissue constructs for implantation within the injured heart. In addition, there is an increasing interest in developing in vitro models to study heart physiology and pathology as well as to evaluate drug efficacy. Formation of these in vitro models and tissue constructs requires highly specific conditions to mimic the normal environment of cells in the body. Firstly, in this study, plasma lithography patterning of elastomeric substrates is exploited for creating microtissues composed of neonatal cardiomyocytes, and investigating their development in different mechanical microenvironments. Immunofluorescence microscopy and force spectroscopy show that the size and shape of the cardiomyocyte clusters, as well as the sarcomere length, fiber alignment, and beating amplitude and frequency of the cardiomyocytes, are regulated by microenvironmental cues. Computational analysis reveals that the mechanical stress at the cluster-substrate interface strongly correlates with the aforementioned characteristics of the cardiomyocytes. Taken together, our results underscore a collective mechanoadaptation scheme in cardiac development. Secondly, a silicone substrate with tunable elasticity is characterized for biological studies. Uniaxial tensile testing and microindentation show that these substrates could cover the biological range of stiffness for normal and pathological conditions. Spectrophotometry demonstrates that the transmittance of these substrates is comparable to those of glass and Sylgard 184. Atomic force microscopy shows that the surface roughness of samples is lower than that of widely-used Sylgard 184. Contact angle measurements before and after exposure to air plasma indicate that these samples are compatible with plasma lithography patterning. Thirdly, a new technique for cell patterning is developed which utilizes selective plasma lithography to modify protein adhesion on the substrate. This approach is based on controlling the conformation of Pluronic F-127 layer adsorbed on the surface by modifying surface wettability. Contact angle measurements show that both PDMS and plastic petri dish are compatible with this technique. X-ray photoelectron spectroscopy and atomic force microscopy confirm the adsorption of PF-127 layers with controlled conformation. Fluorescent and bright-field microscopy demonstrate selective adhesion of proteins and attachment of cells merely on plasma-treated areas. Finally, micropillar arrays are employed to determine the effects of two proteins associated with regulation of thin filament length, i.e. Lmod2 and Tmod1, on contractile force generation at the cellular level. Our results demonstrate that the contractile force of single isolated Lmod2-KO cardiomyocytes decreases compared to the wildtype control. Transduction of Lmod2 in the knockout cardiomyocytes restores their contractile force to the level of their WT counterparts, verifying that the observed contractile dysfunction is specific to the loss of Lmod2. Our data demonstrate that overexpression of Tmod1 in cardiomyocytes decreases their contractile force compared to the WT cells and confirm the effects of Lmod2 knockout on contractile force generation.

Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer-by-layer self-assembly technique and photolithography offers a simple, versatile and silicon compatible approach that overcomes chemical surface patterning limitations, such as short-term stability and low protein adsorption resistance. In this study, direct photolithographic patterning of PAA/PAAm and PAA/PAH polyelectrolyte multilayers was developed to pattern mammalian neuronal, skeletal and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a negative surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cell-selective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108-15 cells) to a greater extent, while providing a little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or in the medium for attachment or differentiation, and could not be patterned using the traditional methods.

Tumorigenesis is often caused by the dysregulation of developmental pathways that are activated during repair, a process that recapitulates development. The Hedgehog (Hh) pathway is a signalling pathway essential for cell patterning and identity during embryogenesis. Activation of Hh signalling has been reported in small cell lung cancer progression, but the role of the Hh receptor, Patched1 (Ptch1), remains poorly understood. Therefore, it is imperative that we understand how Ptch1 is involved in development and tissue repair in order to understand its roles in cancer. This project aimed to study the role of Ptch1 during the branching process of lung development and in the regeneration of airway epithelial cells. A conditional knockout approach was utilised to excise Ptch1 by crossing Ptch1 conditional mice with Dermo1-Cre mice (Dermo1Cre+/-;Ptch1lox/lox), thereby activating the Hh pathway in the mesenchyme, independent of ligand. Dermo1Cre+/-;Ptch1lox/lox embryos died at E12.0 and showed secondary lung branching arrest leading to lobe formation defects. Expression of Ptch1, Gli1 and Foxf1 were shown to be upregulated in both proximal and distal lung mesenchyme, indicating inappropriate pathway activation and disruption of the Hh gradient. Fgf10 expression was spatially reduced in Dermo1Cre+/-;Ptch1lox/lox lungs and the addition of Fgf10 to these lungs in culture showed partial restoration of branching, thus Hh signalling was shown to regulate branching via Fgf10. Due to the patterning defect associated with our in vivo model, we took an in vitro approach to delete Ptch1 in lung explants cultures. This also showed reduced branching and validated that mesenchymal proliferation was enhanced after Ptch1 deletion, consistent with the previously reported role of Hh signalling in mesenchymal cell survival. Small cell lung cancer originates in the proximal lung and has been linked to aberrant repair processes. Therefore, Hh signalling in proximal airway repair was investigated. Ptch1 expressing cells were detected in the bronchial epithelium and stroma during homeostasis. But these cells were not detected following polidocanol-induced injury in the murine nasal septum and lung. However during naphthalene-induced repair, Ptch1 expressing cells were detected in the regenerating bronchial epithelium, suggesting that Hh dependent progenitors respond specifically to naphthalene-induced damage and perhaps are pulmonary neuroendocrine or variant Clara cells. Therefore, this project has provided insight into how Ptch1 patterns lung branching and lobe specification during development and also highlights the importance of Ptch1 in pulmonary epithelial regeneration.

Le développement des Interventions Médicales Assistées par ordinateur (CAMI) est le résultat d'évolutions convergeantes dans les domaines de la médecine, physique, biomatériaux, électronique, informatique et robotique. CAMI visent à fournir les outils qui permettent au clinicien d'utiliser des données multi-modales de manière rationnelle et quantitative pour planifier, simuler et exécuter des interventions médicales mini-invasives avec précision et sans risque. Parallèlement, les avancées technologiques dans les domaines de l’automatisation, la miniaturisation, la conception assistée par ordinateur et l'usinage ont aussi mené au développement des technologies telles que la bioimpression assistée par ordinateur permettant une impression couche par couche de biomatériaux avec une géométrie contrôlée dans l’espace. Ces résultats ouvrent la voie pour l’utilisation des technologies de bioimpression pour des Interventions Médicales Assistées par ordinateur plus précises et sans risque. Dans ce travail, nous montrons que des constructions tissulaires 3D peuvent être imprimées in vivo et in situ et adaptées à la morphologie d’un défaut. Les résultats ont montré que l'impression de cellules in situ avec une résolution à l’échelle cellulaire a tendance à orienter la réparation tissulaire. / The development of Computer-Assisted Medical Interventions (CAMI) results from converging evolutions in medicine, physics, materials, electronics, informatics and robotics. CAMI aim at providing tools that allow the clinician to use multi-modal data in a rational and quantitative way in order to plan, simulate and execute mini-invasive medical interventions accurately and safely. In parallel, technological advances in the fields of automation, miniaturization and computer aided design and machining have also led to the development of bioprinting technologies which could be defined as the computer-aided, layer-by-layer deposition, transfer and patterning of biologically relevant materials. These results pave the way of using bioprinting technologies for Computer-Assisted Medical Interventions. More precisely, we show that 3D tissue constructs can be printed in vivo and in situ in relation with defect morphology. Interestingly, we demonstrate that printing cells in situ with a cell-level resolution tends to orientate tissue repair.