Characterization of structure and dynamics of submicrometric systems via fluorescence correlation spectroscopy

The presented thesis revolves around the study of thermally-responsive PNIPAAm-based hydrogels in water/based environments, as studied by Fluorescence Correlation Spectroscopy (FCS).rnThe goal of the project was the engineering of PNIPAAm gels into biosensors. Specifically, a gamma of such gels were both investigated concerning their dynamics and structure at the nanometer scale, and their performance in retaining bound bodies upon thermal collapse (which PNIPAAm undergoes upon heating above 32 ºC).rnFCS’s requirements, as a technique, match the limitations imposed by the system. Namely, the need to intimately probe a system in a solvent, which was also fragile and easy to alter. FCS, on the other hand, both requires a fluid environment to work, and is based on the observation of diffusion of fluorescents at nanomolar concentrations. FCS was applied to probe the hydrogels on the nanometer size with minimal invasivity.rnVariables in the gels were addressed in the project including crosslinking degree; structural changes during thermal collapse; behavior in different buffers; the possibility of decreasing the degree of inhomogeneity; behavior of differently sized probes; and the effectiveness of antibody functionalization upon thermal collapse.rnThe evidenced results included the heightening of structural inhomogeneities during thermal collapse and under different buffer conditions; the use of annealing to decrease the inhomogeneity degree; the use of differently sized probes to address different length scale of the gel; and the successful functionalization before and after collapse.rnThe thesis also addresses two side projects, also carried forward via FCS. One, diffusion in inverse opals, produced a predictive simulation model for diffusion of bodies in confined systems as dependent on the bodies’ size versus the characteristic sizes of the system. The other was the observation of interaction of bodies of opposite charge in a water solution, resulting in a phenomenological theory and an evaluation method for both the average residence time of the different bodies together, and their attachment likelihood.

Functional hydrogels

Hydrogels are used in a variety of applications in daily life, such as super absorbers, contact lenses and in drug delivery. Functional hydrogels that allow the incorporation of additional functionalities have enormous potential for future development. The properties of such hydrogels can be diversified by introducing responsiveness to external stimuli. These crosslinked polymers are known to respond to changes in temperature, pH and pressure, as well as chemical and electrical stimuli, magnetic fields and irradiation. From this responsive behavior possible applications arise in many fields like drug delivery, tissue engineering, purification and implementation as actuators, biosensors or for medical coatings. However, their interaction with biomaterial and way of functioning are yet not fully understood. Therefore, thorough investigations regarding their optical, mechanical and chemical nature have to be conducted. A UV-crosslinkable polymer, consisting of N-isopropylacrylamide, methacrylic acid and the UV-crosslinker 4-benzoylphenyl methacrylate was synthesized. Its composition, determined by a comprehensive NMR study, is equivalent to the composition of the monomer mixture. The chemical characteristics were preserved during the subsequently formation of hydrogel films by photo-crosslinking as proved by XPS. For the optical characterization, e.g. the degree of swelling of very thin films, the spectroscopy of coupled long range surface plasmons is introduced. Thicker films, able to guide light waves were analyzed with combined surface plasmon and optical waveguide mode spectroscopy (SPR/OWS). The evaluation of the data was facilitated by the reverse Wentzel-Kramers-Brillouin (WKB) approximation. The meshsize and proper motion of the surface anchored hydrogels were investigated by fluorescence correlation spectroscopy (FCS), micro photon correlation spectroscopy (µPCS) and SPR/OWS. The studied gels exhibit a meshsize that allowed for the diffusion of small biomolecules inside their network. For future enhancement of probing diffusants, a dye that enables FRET in FCS was immobilized in the gel and the diffusion of gold-nanoparticles embedded in the polymer solution was studied by PCS. These properties can be conveniently tuned by the crosslinking density, which depends on the irradiation dose. Additionally, protocols and components for polymer analogous reactions based on active ester chemistry of the hydrogel were developed. Based on these syntheses and investigations, the hydrogel films are applied in the fields of medical coatings as well as in biosensing as matrix and biomimetic cushion. Their non-adhesive properties were proved in cell experiments, SPR/OWS and ToF-SIMS studies. The functionality and non-fouling property of the prepared hydrogels allowed for adaption to the needs of the respective application.

Complex tracer mobilities in polymer networks revealed by fluorescence correlation spectroscopy

Gels are elastic porous polymer networks that are accompanied by pronounced mechanical properties. Due to their biocompatibility, ‘responsive hydrogels’ (HG) have many biomedical applications ranging from biosensors and drug delivery to tissue engineering. They respond to external stimuli such as temperature and salt by changing their dimensions. Of paramount importance is the ability to engineer penetrability and diffusion of interacting molecules in the crowded HG environment, as this would enable one to optimize a specific functionality. Even though the conditions under which biomedical devices operate are rather complex, a bottom-up approach could reduce the complexity of mutually coupled parameters influencing tracer mobility. The present thesis focuses on the interaction-induced tracer diffusion in polymer solutions and their homologous gels, probed by means of Fluorescence Correlation Spectroscopy (FCS). This is a single-molecule-sensitive technique having the advantage of optimal performance under ultralow tracer concentrations, typically employed in biosensors. Two different types of hydrogels have been investigated, a conventional one with broad polydispersity in the distance between crosslink points and a so-called ‘ideal’, with uniform mesh size distribution. The former is based on a thermoresponsive polymer, exhibiting phase separation in water at temperatures close to the human body temperature. The latter represents an optimal platform to study tracer diffusion. Mobilities of different tracers have been investigated in each network, varying in size, geometry and in terms of tracer-polymer attractive strength, as perturbed by different stimuli. The thesis constitutes a systematic effort towards elucidating the role of the strength and nature of different tracer-polymer interactions, on tracer mobilities; it outlines that interactions can still be very important even in the simplified case of dilute polymer solutions; it also demonstrates that the presence of permanent crosslinks exerts distinct tracer slowdown, depending on the tracer type and the nature of the tracer-polymer interactions, expressed differently by each tracer with regard to the selected stimulus. In aqueous polymer solutions, the tracer slowdown is found to be system-dependent and no universal trend seems to hold, in contrast to predictions from scaling theory for non-interacting nanoparticle mobility and empirical relations concerning the mesh size in polymer solutions. Complex tracer dynamics in polymer networks may be distinctly expressed by FCS, depending on the specific synergy among-at least some of - the following parameters: nature of interactions, external stimuli employed, tracer size and type, crosslink density and swelling ratio.

Estudio de la homodimerización del dominio PH de la quinasa PDK1 por espectroscopía por correlación de la emisión fluorescente (FCS)

El objetivo de este trabajo es comprobar la homodimerización del dominio PH de la PDK1 in vitro.

Estudo da prevalência das alterações biológicas e mecânicas associadas à reabilitação com prótese parcial removível nas clínicas pedagógicas de Medicina Dentária da FCS-UFP

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária

A Fast BCS/FCS Algorithm for Image Segmentation

A fast and efficient segmentation algorithm based on the Boundary Contour System/Feature Contour System (BCS/FCS) of Grossberg and Mingolla [3] is presented. This implementation is based on the FFT algorithm and the parallelism of the system.

Qualidade, sustentabilidade e FCS: o caso de uma IES privada no Brasil

Este trabalho de investigação foi realizado com o objetivo geral de se descobrir quais são os Fatores Críticos de Sucesso e os aspectos chaves de qualidade e sustentabilidade, a serem considerados no ensino de Engenharia de Produção em um IES privada no Brasil. No presente estudo de caso executou-se uma revisão teórica sistemática nas áreas temáticas da pesquisa, que propiciou a criação de um framework que define os 7 Fatores Críticos de Sucesso ao ensino de Engenharia de Produção, apontando sua divisão nas áreas de qualidade e sustentabilidade, e, por fim, a subdivisão em 6 subáreas de ações operacionais a serem consideradas pela gestão de IES para alcançar melhorias de desempenho em suas atividades. Posteriormente ao levantamento teórico, foi realizado o estudo de caso propriamente dito, que dividiu-se em duas etapas, uma qualitativa e uma quantitativa. Na primeira elaborou-se um roteiro de entrevista em profundidade aplicada a um terço do corpo docente do curso em estudo, captando a impressão destes sobre as temáticas em estudo voltadas à execução de suas atividades. Os dados obtidos foram organizados com o auxílio do software WebQDA, gerando ideias do que deve ser trabalhado para melhorar as condições e resultados do referido curso. A etapa quantitativa foi iniciada com a elaboração de um questionário on-line, desenvolvido a partir do enquadramento teórico e da informação obtida na parte qualitativa do estudo, que envolveu respostas de cerca de dois terços dos discentes de Engenharia de Produção. A informação foi recolhida através de um formulário disponibilizado pelo googledocs, e os resultados foram analisados com o auxílio do software SPSS, permitindo conhecer as expectativas e percepções dos alunos e, consequentemente, os gaps provenientes a serem trabalhados. Finalmente, o estudo revelou que para aspectos de qualidade, os pontos de tangibilidade, como infraestrutura geral, laboratórios técnicos específicos, cantina e biblioteca, precisam ser melhorados, e, para aspectos de sustentabilidade, surge a necessidade de melhorar as formas de divulgação do curso, captação de alunos e propostas para a retenção dos mesmos para outros cursos e projetos da IES. Deste modo, o trabalho desenvolvido atingiu seu objetivo e se mostrou válido para aplicações na IES e em outros cursos da mesma (com as devidas adaptações). Fica como recomendação final aplicar o estudo periodicamente para se captar as mudanças situacionais que ocorrerem, manter um controle eficiente dos aspectos estudados e gerar melhorias contínuas no curso estudado e na própria IES.

Diffusion dans un hydrogel : applications aux biocapteurs et optimisation de la technique de spectroscopie par corrélation de fluorescence (FCS)

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

N-Vinylamidderivate zur Synthese neuartiger Kern-Schale-Partikel und Hydrogele

N-Vinylamidderivate sind eine toxikologische unbedenkliche Monomerklasse. Mit diesen Monomeren wurden verschiedene technische Anwendungsgebiete im Bereich der Kern-Schale-Partikel und der fließfähigen und vernetzten Hydrogele untersucht. Kern-Schale-Partikel Für die Synthese von Kern-Schale-Partikeln wurden die N-Vinylamidderivate als Schalenpolymere auf kommerziellen Poly(styrol-stat.-butadien)-Kernpartikeln aufpolymerisiert. Mit Hilfe verschiedener Untersuchungsmethoden (DLS, SEM, FFF, Ultrazentrifuge) wurde die Kern-Schale-Strukturbildung und die Effizienz der Pfropfungsreaktion untersucht und eine erfolgreiche Synthese der Kern-Schale-Partikel belegt. Durch die gezielte Modifizierung des Schalenpolymers wurde ein kationisches, organisches Mikropartikelsystem entwickelt, charakterisiert und auf die Eignung als „Duales Flockungsmittel“ untersucht. Diese Versuche belegten die Eignung der modifizierten Kern-Schale-Partikel als „Duales Flockungsmittel“ und bieten eine Alternative zu kommerziell verwendeten Retentionsmitteln. Außerdem wurden die filmbildenden Eigenschaften der Poly(N﷓vinylformamid)-Kern-Schale-Dispersionen untersucht. Nach der Verfilmung der Dispersionen wurden transparente und harte Filme erhalten. Die Auswirkungen auf die mechanischen Eigenschaften der Filme wurden durch die Variation verschiedener Parameter eingehend studiert. Auf der Basis dieser Partikel wurden selbstvernetzende Dispersionssysteme entwickelt. Das P﷓(VFA)-Schalenpolymer wurde teilweise hydrolysiert und die generierten freien Aminogruppen des Poly(N-vinylamins) durch eine Michael-Addition mit einem divinylfunktionalisierten Acrylat (Tetraethylenglykoldiacrylat) vernetzt. Untersuchungen zur mechanischen Beständigkeit der Filme zeigten bei geringen Vernetzungsgraden eine deutliche Optimierung der maximalen Zugbelastungen. Die Untersuchungen belegten, dass die Verwendung des selbstvernetzenden Dispersionssystems als Dispersion für eine Polymerbeschichtung möglich ist. Hydrogele Die Synthese von fließfähigen und quervernetzten Hydrogelen erfolgte auf der Basis verschiedener N﷓Vinylamide. Mit Hilfe geeigneter Vernetzer wurden feste Hydrogelplatten synthetisiert und für die Auftrennung von DNA-Sequenzen mit Hilfe der Gelelektrophorese verwendet. Scharfe und gute Auftrennung der verschiedenen „DNA-Ladder Standards“ wurden durch die Variation des Vernetzeranteils, der Polymerzusammensetzung, der angelegten Spannung und der Verweilzeit in der Gelelektrophoresekammer mit P﷓(MNVA)-Hydrogelplatten erreicht. Fließfähige und quervernetzte Elektrolytgele auf Poly-(N-vinylamid)-Basis wurden in wartungsfreien pH﷓Elektroden eingesetzt. Die Eignung dieser Hydrogele wurden in Bezug auf die Anwendung eingehend charakterisiert. Elektroden befüllt mit Poly(N-vinylamid)-Gelen wurden in Dauerbelastungsexperimenten, direkt mit kommerziellen pH﷓Elektroden verglichen. Es konnte gezeigt werden, dass die fließfähigen und quervernetzten Poly-(N-vinylamid)-Elektrolytgele in ihren Messeigenschaften gleichwertige bzw. bessere Potentialstabilitäten aufweisen als kommerzielle Referenzelektroden. Die Hydrogele auf Basis von Poly(N-vinylamidderivaten) boten für die beiden getesteten Anwendungen eine toxikologisch unbedenkliche Alternative zu Poly(acrylamid)-Gelen. In dieser Arbeit konnten die durchgeführten Untersuchungen belegen, dass N﷓Vinylamide eine attraktive Monomerklasse ist, die erfolgreich in vielen technischen Anwendungen einsetzbar ist.

FCS educational circular.

Some vols. issued in revised editions

FCS information.

Title varies slightly.

The difficulty of measuring culture and diversity. Comparison of three international models of cultural measurement: FCS-2009, ESSnet-2012 and CAB-2015

The growing interest in quantifying the cultural and creative industries, visualize the economic contribution of activities related to culture demands first of all the construction of internationally comparable analysis frameworks. Currently there are three major bodies which address this issue and whose comparative study is the focus of this article: the UNESCO Framework for Cultural Statistics (FCS-2009), the European Framework for Cultural Statistics (ESSnet-Culture 2012) and the methodological resource of the “Convenio Andrés Bello” group for working with the Satellite Accounts on Culture in Ibero-America (CAB-2015). Cultural sector measurements provide the information necessary for correct planning of cultural policies which in turn leads to sustaining industries and promoting cultural diversity. The text identifies the existing differences in the three models and three levels of analysis, the sectors, the cultural activities and the criteria that each one uses in order to determine the distribution of the activities by sector. The end result leaves the impossibility of comparing cultural statistics of countries that implement different frameworks.

Aferição das dificuldades/competências na área da Endodontia percepcionadas pelos alunos do 4º e 5º anos da FCS-UFP

Introdução: Com uma consciencialização cada vez maior das populações para a preservação dos dentes naturais, a Endodontia tem assumido uma importância crescente, tendo por objectivo principal a manutenção de dentes funcionais, sem prejudicar a saúde dos pacientes. Estes devem beneficiar de um tratamento segundo o “standard of care”, proporcionado por profissionais competentes. Vários estudos demonstram que a Endodontia é considerada uma área difícil e stressante para os estudantes, exibindo um sentimento de menor confiança na prática clínica, sobretudo no que diz respeito a procedimentos mais complexos, nomeadamente, no tratamento de dentes posteriores. Em 2010, na tentativa de homogeneizar as competências adquiridas pelos Médicos Dentistas (MD), a Associação para a Educação Dentária Europeia (AEDE) definiu critérios para a sua formação pré-graduada, indicando que o MD recém-graduado deve ter adquirido competências e capacidades técnicas que lhe permita começar a sua prática clínica de forma independente. Objectivos: Analisar os conhecimentos adquiridos e as dificuldades sentidas na área da Endodontia pelos alunos do 4º e 5º anos do Mestrado Integrado em Medicina Dentária da Faculdade de Ciências de Saúde (FCS) da Universidade Fernando Pessoa (UFP), fazendo, igualmente, uma abordagem ao ensino ministrado na área da Endodontia face aos resultados obtidos. Materiais e Métodos: Este trabalho está dividido em duas partes: revisão bibliográfica e investigação científica. A revisão bibliográfica do tema engloba o ensino graduado em Medicina Dentária, o uso de isolamento absoluto (IA) na prática clínica de Endodontia, a instrumentação manual versus rotatória, a influência no sucesso do tratamento endodôntico (TE) da sua execução por estudantes do 4º e 5º ano, as Guidelines para o TE e o ensino de Endodontia na UFP. A pesquisa bibliográfica foi efectuada através da base de dados PubMed, tendo sido utilizadas, em diferentes combinações, as seguintes palavras-chave: ”Endodontics”, “teaching”; “pre-clinical”; “undergraduate”; “Clinical”; “treatment” e “Europe”. O trabalho de investigação consistiu na elaboração, aprovação pela Comissão de Ética da UFP e posterior aplicação de um questionário destinado aos alunos do 4º e 5º ano do Mestrado Integrado em Medicina Dentária da FCS-UFP. A população estimada de alunos do 4º ano foi de 190 alunos e a de 5º ano de 150 alunos, o que perfaz um total de 340 alunos. Dos inquéritos respondidos, apenas foram considerados 338, uma vez que dois foram anulados pois continham respostas inválidas. Resultados: A “condição socioeconómica do paciente” foi considerada a causa menos relevante para avaliar um caso endodôntico, tendo sido apontada por 34,3% dos alunos. Assinala-se que 92% dos alunos de 4º ano e 93,3% dos alunos de 5º ano consideram que os molares superiores são os dentes mais difíceis de tratar, sendo a visibilidade uma das principais razões para esta opinião. O grau de dificuldade para a colocação do IA é definido como “Elevado” para a maioria dos alunos de 4º ano. A maior parte dos alunos de 5º ano considera que o grau de dificuldade para a “Determinação do tipo de reconstrução/prótese fixa mais indicada” é “Elevado”. O passo do TE onde os alunos do 4º ano se sentem mais confiantes é no “Diagnóstico de cárie”, sendo que os alunos do 5º ano se sentem mais confiantes ao realizar o “TE em dentes com 1 ou 2 canais”. O “Conteúdo leccionado nas aulas teóricas” foi considerado o principal aspecto positivo do ensino endodôntico. Por outro lado, o “Número de actos clínicos realizados” foi considerado o principal aspecto negativo, tanto para alunos de 4º como de 5º ano. Em ambos os anos, os principais pontos que os alunos acham que devem ser melhorados são o “Número de pacientes nas aulas clínicas” e a “Aprendizagem da técnica de instrumentação mecanizada/obturação termoplástica”. As principais preocupações referidas, tanto por alunos de 4º como de 5º anos, foram a “Insegurança na prática clínica” e a “Dificuldade em encontrar ofertas profissionais”. Relativamente à qualidade dos seus TE existe um número significativo de alunos do 5º ano que já se auto-avaliam como “Bons” (33,6%), por comparação com os do 4º ano (21,2%), sendo a auto-avaliação de “Razoável” dominante em ambos os anos de formação. Conclusões: Conclui-se que na disciplina de Endodontia na FCS-UFP, são seguidas as Guidelines da European Society of Endodontology (ESE), sendo que estas indicam o protocolo mais correcto a seguir durante o Tratamento Endodôntico Não-Cirúrgico (TENC). Contudo, o reduzido número de actos clínicos e a consequente falta de prática faz com que os alunos se sintam pouco confiantes ao iniciar a sua actividade profissional.

Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure.