Aplicação da citometria de fluxo ao estudo do genoma vegetal

Doutoramento em BiologiaHoje em dia, a aplicação da citometria de fluxo (CMF) no estudo do genoma vegetal consiste maioritariamente em análises do nível de ploidia e em estimativas do conteúdo em ADN nuclear. Esta Tese de Doutoramento tem como objectivo aplicar a CMF e outras técnicas citológicas, como a microscopia de fluorescência e a hibridização in situ, no estudo do genoma vegetal. O primeiro Capítulo introduz a CMF, nomeadamente o seu funcionamento, aspectos metodológicos da análise do conteúdo em ADN nuclear em plantas, e aplicações da CMF na biologia vegetal. No Capítulo II são apresentados dois estudos sobre a estabilidade do nível de ploidia durante o processo de cultura in vitro de sobreiro (Capítulo II.1) e zimbro (Capítulo II.2), duas espécies lenhosas de elevada importância económica e ecológica. No Capítulo III são apresentados os resultados das análises de conteúdo em ADN nuclear efectuadas em três grupos de espécies vegetais. No Capítulo III.1 a CMF foi utilizada para estimar pela primeira vez o tamanho do genoma nuclear de cultivares de oliveira e zambujeiro; a variação intraspecífica como tópico de elevado interesse é aqui discutida tendo em consideração as boas práticas. No Capítulo III.2 foi analisado o conteúdo em ADN nuclear de três espécies de Ulmaceae da Península Ibérica que contêm compostos mucilaginosos, conhecidos por interferirem com as análises por CMF; é também apresentado um protocolo eficiente que previne o efeito negativo deste compostos e permite uma correcta análise do tamanho do genoma de espécies com problemas similares. No Capítulo III.3 é apresentado um estudo extensivo de CMF e citogenética molecular realizado em espécies de festuca da Península Ibérica. Considerando os resultados obtidos nos Capítulos II e III, no Capítulo IV foram efectuados estudos inovadores de índole metodológica. No Capítulo IV.1 é apresentada a primeira comparação sistemática de tampões de isolamento nuclear, enquanto no Capítulo IV.2 é estudado o efeito negativo do ácido tânico, um composto fenólico conhecido por interferir com os núcleos vegetais e com as estimativas de conteúdo em ADN. De acordo com os resultados obtidos nestes estudos, no Capítulo IV.3 foram testados dois novos tampões de lise nuclear em 37 espécies vegetais. No Capítulo V é introduzida a base de dados “Plant DNA Flow Cytometry (FLOWER)”. Esta base de dados contém informações retiradas de mais de 700 publicações na área da CMF vegetal, disponibilizando-as de uma forma atraente e permitindo a realização de análises quantitativas respeitantes a muitos tópicos importantes. Finalmente, no Capítulo VI são apresentadas as conclusões da presente Tese de Doutoramento, com especial incidência nas boas práticas necessárias para estimar o conteúdo em ADN nuclear em plantas e nas direcções futuras da CMF no estudo do genoma vegetal.Nowadays, the application of flow cytometry (FCM) to study plant genomes is mostly focused on DNA ploidy level analyses and nuclear DNA content estimations. The objective of this PhD Thesis is to applly FCM and related techniques, as fluorescence microscopy and in situ hybridization, to study plant genomes. The first chapter introduces FCM, namely the functioning of this technique, the methodological aspects of the analysis of nuclear DNA content in plants, and the applications of FCM in plant sciences. In Chapter II, studies of ploidy stability of the in vitro culture process of two economically important woody plant species, Quercus suber (Chapter II.1) and Juniperus phoenicea (Chapter II.2), are presented. In Chapter III, three studies regarding nuclear DNA content analyses in plant species are given. In Chapter III.1 the genome size of olive cultivars and wild olive was estimated for the first time using FCM; the hot topic of intraspecific variation is discussed here with respect to best practices. In Chapter III.2, the nuclear DNA content of three Ulmaceae species, containing mucilaginous compounds that interfere with FCM analysis, is analysed; an efficient protocol that circumvented this problem and enabled reliable genome size estimations in these species is presented. A through flow cytometric and molecular cytogenetic study on fescue species of the Iberian Peninsula is performed in Chapter III.3. In light with the results obtained in Chapters II and III, innovative methodological studies were performed in Chapter IV. In Chapter IV.1 the first systematic comparison of lysis buffers is presented, while in Chapter IV.2 the negative effect of tannic acid, a common phenolic compound known to interfere with plant nuclei and DNA content estimations, is analysed. According with the results obtained in these studies, in Chapter IV.3 two new lysis buffers were tested with a set of 37 plant species. In chapter V the Plant DNA Flow Cytometry Database (FLOWER) is introduced. The FLOWER database collects information from more than 700 publications in this area of FCM, and makes it accessible in one user-friendly design that enables quantitative analysis of many important topics. Finally in Chapter VI the conclusions of the present PhD Thesis are presented with special focus on a compilation of best practices for nuclear DNA estimation using FCM and the future directions on the use of this technique to study plant genomes

Nanostructured thin coatings from chitosan and an elastin-like recombinamer with acute stimuli-responsive behavior

Publicado em: Materials Science Forum, vols. 730-732; Online available since 2012/Nov/12In the present work, chitosan (CHI) and elastin-like recombinamers (ELRs) were used to conceive nanostructured thin films driven by sequential electrostatic layer-by-layer (LbL), a simple and versatile technique that discards the use of harmful reagents. Two similar ELRs were engineered to contain negatively charged aminoacids and organized and a single monoblock or a triblock. The buildup of the films was monitored in real time using a quartz-crystal microbalance with dissipation monitoring (QCM-D). Wettability transitions were observed from a moderate hydrophobic surface to an extremely wettable upon increasing the temperature to 50 ºC, accompanied by topography changes at the nanoscale as assessed by atomic force microscopy (AFM). Furthermore, the dependence on time for the surface molecular rearrangement was studied for the films with each ELR. The potential of this technology may stimulate the development of devices and biomaterials for biomedical applications in the near future, such as surfaces with tunable and patterned cell adhesion, while the use of ELRs will allow developing polypeptides with biological significance.The authors acknowledge the financial support through Fundação para a Ciência e Tecnologia (FCT, PhD grant SFRH/BD/61126/2009, ‘‘Junta de Castilla y Leon’’ (VA034A09), the MICINN (MAT 2009-14195-C03-03, ACI2009-0890, IT2009-0089, MAT2010-15310, and MAT2010- 15982), the CIBER-BBN and the ‘‘Network Center of Regenerative Medicine and Cellular Therapy of Castilla y León’’.MICINNFundação para a Ciência e a Tecnologia (FCT)Junta de Castilla y LeónCIBER-BBNNetwork Center of Regenerative Medicine and Cellular Therapy of Castilla y Leó

Effect of poling on the mechanical properties of β-poly(vinylidene fluoride)

In this work, mechanical and thermal experimental techniques have been applied in order to relate the mechanical response with the microscopic variations of the material. Stress-strain results along the main directions of β-poly(vinylidene fluoride), β-PVDF, in poled and non-poled samples enables to investigate the influence of the poling process on the mechanical response of the material. Further, differential scanning calorimetry experiments allow the investigation of the effect of poling in the degree of crystallinity of the material as well as on the stability of the crystalline phase. Thermogravimetric analysis was used to investigate the kinetics of the thermal degradation of poled and non-poled β-PVDF samples. The differences observed between the two materials suggest that the poling affects the mechanical properties of the material especially in the direction parallel to the polymeric chains and originates changes at a molecular level that remain beyond the melting of the material.(undefined

Layer-by-layer assembly of chitosan and recombinant biopolymers into biomimetic coatings with multiple stimuli-responsive properties

In this work, biomimetic smart thin coatings using chitosan and a recombinant elastin-like recombinamer (ELR) containing the cell attachment sequence arginine–glycine–(aspartic acid) (RGD) are fabricated through a layer-by-layer approach. The synthetic polymer is characterized for its molecular mass and composition using mass spectroscopy and peptide sequencing. The adsorption of each polymeric layer is followed in situ at room temperature and pH 5.5 using a quartz-crystal microbalance with dissipation monitoring, showing that both polymers can be successfully combined to conceive nanostructured, multilayered coatings. The smart properties of the coatings are tested for their wettability by contact angle (CA) measurements as a function of external stimuli, namely temperature, pH, and ionic strength. Wettability transitions are observed from a moderate hydrophobic surface (CAs approximately from 62° to 71°) to an extremely wettable one (CA considered as 0°) as the temperature, pH, and ionic strength are raised above 50 °C, 11, and 1.25 m, respectively. Atomic force microscopy is performed at pH 7.4 and pH 11 to assess the coating topography. In the latter, the results reveal the formation of large and compact structures upon the aggregation of ELRs at the surface, which increase water affinity. Cell adhesion tests are conducted using a SaOs-2 cell line. Enhanced cell adhesion is observed in the coatings, as compared to a coating with a chitosan-ending film and a scrambled arginine–(aspartic acid)–glycine (RDG) biopolymer. The results suggest that such films could be used in the future as smart biomimetic coatings of biomaterials for different biomedical applications, including those in tissue engineering or in controlled delivery systems.EUJCyL - VA034A09, VA030A08Fundação para a Ciência e Tecnologia (FCT) - SFRH/BD/61126/2009, SFRH/BD/61390/2009MICINN - MAT 2007-66275-C02-01, MAT 2007-61604, MAT 2009-14195-C03-03, PSE-300100-2006-1European regional development fund (ERDF)Junta de Castilla y LeonNetwork Center of Regenerative Medicine and Cellular Therapy of Castilla and LeónCIBER-BBN (project CB06-01-0003

Nanostructured multilayer compartments : towards multifunctionality and ‘‘cell-like’’ hierarchical complexity

In living organisms, there are phenomena that require the presence of specific biomolecules with distinct function and in variable concentrations at a given time, such as the healing and regeneration of tissue and organ lesions. In this work, we propose the use of a compartmented drug delivery device for the multiple release of bioactive agents. It consists of nanostructured microcapsules confined within a millimetric container that can be easily handled, mimicking the concept of cells which possess organelles with specialized functions. Each hierarchical structure was conceived using the layer-by-layer (LbL) method to form micro and macrocapsules that could individually carry either molecules and release them with distinct kinetics or magnetic nanoparticles (MNPs) to be used in targeted therapies. Furthermore, the internal microcontainers were constructed with a temperature-responsive elastin-like recombinamer (ELR) to further add smart properties to the proposed system. Sacrificial CaCO3 microparticles empty or entrapping either rhodamine or Fe3O4 MNPs were incubated in chitosan and ELR solutions using LbL for the conception of the microcapsules. Then, the microcapsules were suspended in alginate which was ionically crosslinked in CaCl2 drop-wise. Rhodamine could be encapsulated at this point in the alginate. The bead was coated with chitosan and alginate to build the external macrocapsule compartment. All structures were coated with 3 bilayers. The CaCO3 cores were chelated and the alginate beads liquefied using EDTA. Fluorescence microscopy using FITC and rhodamine markers showed a uniform distribution of the microcapsules within the macroreservoir. The release of rhodamine from either in the micro or macrocapsule was assessed at 25 and 37 °C in PBS. While the release from the macrocapsule follows a profile similar to that of traditional drug delivery systems, it is more sustained and delayed when released from the internal compartments. Such retention is more pronounced at 37 ºC (65% of release in comparison to 90%). This is due to the temperature responsive behavior of ELRs, which undergo a phase transition and make the LbL shell less permeable. For the magnetic response, the incorporation of the MNPs was observed by transmitted light microscopy. The attraction of the devices was observed by applying an external magnetic field along a defined trajectory. The results let foresee the use of such multilayer devices as compartmented structures to encapsulate growth factors, MNPs and stem cells for their controlled differentiation and maintenance or for guided regeneration of tissues and organs.Fundo Social Europeu (FSE)Fundação para a Ciência e a Tecnologia (FCT)Programa Diferencial de Potencial Humano (POPH

Functional nanostructured chitosan–siloxane hybrids

New organic–inorganic hybrids were prepared by a sol–gel method from the biopolymer chitosan and a silane coupling agent, 3-isocyanatopropyltriethoxysilane (ICPTES), in which covalent bridges, essentially composed of urea, bond the chitosan to the poly(siloxane) network. The structural characterization of the advanced chitosan–siloxane hybrids was performed by Fourier transform infrared spectroscopy, X-ray diffraction and 29Si and 13C nuclear magnetic resonance. The presence of siloxane nanodomains was detected by small angle X-ray diffraction. The chitosan–siloxane hybrids are bifunctional materials with interesting photoluminescent features and bioactive behaviour. The photoluminescence spectra display an additional high-energy band with longer lifetime, relatively to the characteristic emission of pure chitosan. This band is associated with electron–hole recombinations arising from silicon-related defects at the surface of the siliceous nanodomains. The bioactive behaviour of these materials was also evaluated; the apatite formation was shown to depend on the amount and arrangement of silanol groups

Reversible imine crosslinking in waterborne self-healing polymer coatings

Waterborne polymer coatings have the potential to address the environmental concerns associated with solvent based systems. To improve their performance without using volatile organic compounds, we propose a new approach based on reconfigurable covalent crosslinking that provides mechanical resistance and self-healing properties. The new waterborne polymer coatings are based on mixtures of aldehyde- and amine-functionalized polymer nanoparticles (PNPs) that take advantage of the reversibility of imine bonds in the presence of water. Different degrees of functional monomer incorporation (10 % to 40 %) allowed us to balance crosslinking and interdiffusion during film formation, to obtain mechanically robust and solvent resistant films. A clear structure-properties relation was assessed by following the formation of water resulting from amine-aldehyde condensation crosslinking, measured by differential scanning calorimetry. The resulting polymer coatings further show self-healing properties at room temperature, triggered with residual amounts of water and featuring high recovery rates of the mechanical properties. Our mechanically robust waterborne polymer coatings based in imine reversible crosslinking, featuring self-healing in mild conditions, offer excellent prospects for application in smart coating materials.publishe

Poling of ß-poly(vinylidene fluoride): dielectric and IR spectroscopy studies

ß-Poly(vinylidene fluoride) (ß-PVDF) exhibits ferroelectric properties due to the special arrangement of the chain units in the crystalline phase. The piezoelectric properties of the material can be optimised by poling the original stretched film. The main effect of the poling process is the alignment of the randomly organised dipolar moments against the applied field. In this work, poled and non-poled ß-PVDF from the same batch are characterised by dielectric spectroscopy. The origin of the electrical and mechanical response of poled and non-poled ß-PVDF were further explored by far IR spectroscopy and discussed on a molecular level. The main effect of the poling process on-the dielectric response of the-material is a small increase of the dielectric constant due to the 'preferential alignment of the main dipolar contribution and a slight decrease of the dielectric loss, due to the more organized amorphous structure. The conductivity is strongly increased by poling, especially the high-temperature conductivity, ascribed mainly to hopping conductivity due to free charges induced during poling. FTIR experiments indicate that the origin of these effects and also of the variations in the thermo-mechanical response of the material can be found in the reorientation of the crystalline dipoles along the poling field, together with a partial reduction of the amount of alpha phase and an increase of the amount of ß phase. The alpha to beta transformation, mainly due to the stretching process, seems to be optimized by the poling process.Fundação para a Ciência e Tecnologia (FCT) - Programa Operacional "Ciência, Tecnologia, Inovação" (POCTI) - POCTI/CTM/33501/99

Development of an injectable system based on elastin-like recombinamer particles for tissue engineering applications

An elastin-like recombinamer (ELR) containing the RGD cell adhesion domain was used to fabricate microparticles by an innovative and affordable process based on the use of superhydrophobic surfaces. Two microparticles types with different crosslinking extents were prepared. The biological response was tested using an osteoblast-like cell line (SaOs-2) performing proliferation and alkaline phosphatase (ALP) quantification tests, as well as assessing cytotoxicity, morphology and cell distribution on the particles. The main goal of the work was the assessment of the in vitro formation of cell-induced microparticle aggregates that could provide indications for the possible formation of an in situ-forming scaffold upon implantation. ELR microparticles have been successfully obtained by deposition of a polymeric solution on bioinspired polystyrene superhydrophobic surfaces and two different crosslinking extents were achieved by controlling the time of exposure to the crosslinker. The crosslinking extent affected swelling behavior and the dynamic mechanical properties of the particles. SaOs-2 morphology, ALP expression, spatial distribution and ability to bind the microparticles together were dependent on the physicochemical properties of the microparticles: the more crosslinked condition was the most favorable for cell proliferation and to form a cell-induced aggregation scaffold, making these particles suitable to be applied in bone tissue engineering

Comparação da técnica de microdiluição em caldo e ETEST para o cetoconazol frente à Malassezia pachydermatis

Malassezia pachydermatis is recognized as a normal inhabitant and an opportunistic pathogen of the external ear canal and skin of dogs and cats. In special clinical conditions, and mainly in the cases of therapeutic failure related to external otitis and dermatitis complicated by this yeast, is recommended testing susceptibility to antifungal drugs. Different approaches of evaluating the susceptibility of yeasts faced to antifungals in laboratory exist, some of them are commercial approaches and others previously standardized by the CLSI (NCCLS, 2002). The purpose of this study was to evaluate the susceptibility of 17 samples of M. pachydermatis from canine external otitis using two different in vitro antifungal susceptibility methods: the Etest® and the broth Microdilution Method (MD) with ketoconazole. The mean MIC observed between the 17 samples were 0.103mg/mL to ETEST and 0.0012mg/mL to MD ranging from 0.004 to 0.75mg/mL in ETEST and 0.0019 and 0.03mg/mL in MD using the same samples. By ETEST, two (11.8%) samples were resistant, eight (47.1%) susceptible and seven (41.1%) showed intermediate susceptibility. Through the MD it was observed four (23.5%) resistant samples, seven (41.2%) susceptible and six (35.3%) samples with intermediate susceptibility. Despite of the percentages being equivalent in each rank of susceptibility through the two techniques, the results do not correspond to the same sample. These results showed that there is an urgent need to standardize those values considered as parameters for growth inhibition of this yeast. Then a simple and efficient method could be used routinely in the laboratory practice.Malassezia pachydermatis é reconhecida como um habitante normal e patógeno oportunista do meato acústico externo e da pele de cães e gatos. Em condições clínicas especiais e em casos de falha terapêutica relatada em otite externa e dermatite complicada por esta levedura, é recomendado o teste de suscetibilidade antifúngica. Existem diferentes métodos de avaliação da suscetibilidade da levedura frente a antifúngicos em laboratório, alguns métodos comerciais e outros previamente padronizados pelo CLSI (NCCLS, 2002). O objetivo deste estudo foi o de avaliar a suscetibilidade de 17 amostras de M. pachydermatis proveniente de otite externa canina por meio de duas técnicas in vitro de antifungigramas: o Etest® e a microdiluição em caldo (MC) com o cetoconazol. A media da Concentração Inibitória Mínima (CIM) observada entre as 17 amostras foram 0.103mg/mL para o ETEST e 0.0012mg/mL para a MC variando de 0.004 a 0.75mg/mL no ETEST e entre 0.0019 e 0.03mg/mL na MC usando as mesmas amostras. Pelo ETEST, duas (11.8%) amostras foram resistentes, oito (47.1%) sensíveis e sete (41.1%) mostraram sensibilidade intermediária. Na MC foram observadas quatro (23.5%) amostras resistentes, sete (41.2%) sensíveis e seis (35.3%) amostras com sensibilidade intermediária. Apesar das porcentagens de sensibilidade serem semelhantes pelas duas técnicas, os resultados de CIM não correspondem na uma mesma amostra. Estes resultados mostraram que há uma urgente necessidade de padronização dos valores considerados como parâmetros para inibição do crescimento da levedura. Portanto, um método simples e eficiente deveria ser usado rotina na prática de laboratório