Lightly cross-linked polyelectrolyte microgels are materials with interesting properties for a range of applications. For instance, the volume of these particles can be drastically changed in response to pH, ionic strength, temperature, or the concentration of specific ions and metabolites. In addition, microgel particles can bind substantial amounts of oppositely charged substances, such as proteins and peptides, and release them upon changes in the external environment. Consequently, microgels have potential in catalysis, photonics, biomaterials, and not at least, as protective and stimuli-sensitive carriers for protein and peptide drugs. In this thesis, the interaction between anionic microgels and cationic peptides was investigated by monitoring microgel deswelling and reswelling in response to peptide binding and release using micromanipulator-assisted light microscopy. In addition, peptide distribution in microgels was analyzed with confocal laser scanning microscopy and peptide uptake determined with solution depletion measurements. The aim of the thesis was to clarify how parameters such as peptide size, charge density, pH, ionic strength and hydrophobicity influences the peptide binding to, distribution in and release from, polyelectrolyte microgels. Results obtained in this thesis show that electrostatic attraction is a prerequisite for interaction to occur although non-electrostatic contributions are responsible the finer details of the interactions. The size and charge density of the interacting peptides play a major role, as large and highly charged peptides are restricted to enter and interact with the microgel core, thus displaying a surface-confined distribution. The peptide-microgel interaction strength is highly reflected in the probability of peptides to be detached from the gel network. For instance, reducing the electrostatic interactions by adding salt induces significant peptide release of sufficiently small and moderately charged peptides, whereas longer and more highly charged peptides is retained in the microgel network due to the strong interaction, insufficient salt screening, and gel network pore size restriction. Decreasing the charge density of microgel network and/or peptides increases the probability for peptide detachment tremendously. To summarize, interactions occurring in oppositely charged microgel-peptide systems can be tuned by varying parameters such as charge density and peptide size and through this, the peptide uptake, distribution and release can be controlled to alter the performance of microgels in peptide drug delivery.

Small proline-rich protein-2 (SPRR2) functions as a determinant of flexibility and permeability in the mature cornified envelope of the skin. SPRR2 is strongly upregulated by the commensal flora and may mediate signaling to differentiated epithelia of the small intestine and colon. Yet, SPRR2 function in the GI tract is largely unexplored. Using the Caco-2 model of intestinal epithelial differentiation along the crypt-villus axis, we hypothesized that SPRR2 would be preferentially expressed in post-confluent differentiated Caco-2 cells and examined SPRR2 regulation by the protein kinase A pathway (PKA) and short chain fatty acids (SCFAs). Differentiation-dependent SPRR2 expression was examined in cytoskeletal-, membrane-, and nuclear-enriched fractions by immunoblotting and confocal immunofluorescence. We studied the effect of SCFAs, known inducers of differentiation, on SPRR2 expression in pre-confluent undifferentiated Caco-2 cells and explored potential mechanisms involved in this induction using MAP kinase inhibitors. SPRR2 expression was also compared between HIEC crypt cells and 16 to 20 week primary fetal villus cells as well as in different segments in mouse small intestine and colon. We determined if SPRR2 is increased by gram negative bacteria such as S. typhimurium. SPRR2 expression increased in a differentiation-dependent manner in Caco-2 cells and was present in human fetal epithelial villus cells but absent in HIEC crypt cells. Differentiation-induced SPRR2 was down-regulated by 8-Br-cAMP as well as by forskolin/IBMX co-treatment. SPRR2 was predominantly cytoplasmic and did not accumulate in Triton X-100-insoluble cytoskeletal fractions. SPRR2 was present in the membrane- and nuclear-enriched fractions and demonstrated co-localization with F-actin at the apical actin ring. No induction was seen with the specific HDAC inhibitor trichostatin A, while SCFAs and the HDAC inhibitor SBHA all induced SPRR2. SCFA responses were inhibited by MAP kinase inhibitors SB203580 and U0126, thus suggesting that the SCFA effect may be mediated by orphan G-protein receptors GPR41 and GPR43. S. typhimurium induced SPRR2 in undifferentiated cells. We conclude that SPRR2 protein expression is associated with differentiated epithelia and is regulated by PKA signaling and by by-products of the bowel flora. This is the first report to establish an in vitro model to study the physiology and regulation of SPRR2. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-04-25 12:39:06.427 / This work was funded by the CIHR GIDRU Training Grant and Aid in Research from Crohn's and Colitis Foundation of Canada

Radiata pine (Pinus radiata) is the most important plantation tree in New Zealand forestry, and factors that reduce the quality of wood cause significant economic loss. Two of the most important of these issues are compression wood and spiral grain. Compression wood is a type of reaction wood, formed when a tree moves away from the vertical, and is characterised by biochemical and structural changes within the wood that reduce its quality and value. Spiral grain, however, is the alignment of the wood grain in a helix around the tree’s axis and away from the vertical. Again, this reduces the structural qualities of the wood and thus its value. Spiral grain and compression wood are notorious for their deleterious effect on the quality of wood produced and are very important for the forest industry due to the huge economic loss they cause. The demand for reliable tools to evaluate these wood quality issues in clonal planting material at an early stage, within 3 years of germination rather than at 8 to 15 years as in current practise, is of ever increasing importance from plant breeders and other industry stake holders. Therefore this research was undertaken with an overall aim to develop quick, easy and reproducible techniques to evaluate young radiata pine clones (up to 3 years old) based on compression wood content and presence of spiral grain. This is important because a shortened breeding cycle could provide significant economic benefits to the forest industry. The incidence of these commercially important wood quality parameters has been studied in this thesis in research conducted on young trees (1 to 3 years old). The research described in this thesis used a variety of different imaging approaches to investigate wood structure, including polarised light and confocal microscopy, and X-ray tomography and circular polarised light scanning. The images achieved have been analysed using a range of different software, including Photoshop, ImageJ and Matlab bringing a quantification approach to the imaging. Compression wood was quantified in young clonal material using images collected with a commercial document scanner, and processed using image analysis tools available in Photoshop. An easy, reliable and robust, automatic image analysis protocol was successfully developed and tested for the detection and quantification of compression wood in these young trees. This new technique to detect and quantify compression wood was based on the thresholding of the blue channel of the scanned RGB image as this was demonstrated to contain the greatest image contrast. Development of this new technique may reduce the waiting time for screening clonal planting materials based on compression wood content. To understand the organisation of the grain at a cellular level within these young trees, confocal microscopy techniques were utilised. The cell wall characteristics and fluorescence properties of compression wood in comparison with normal wood were investigated using a new cellulose specific dye, pontamine fast scarlet 4B. Staining protocols for this dye for confocal microscopy were optimised, and the potential of measuring the microfibril angle of the S1 and S3 layers of the pontamine treated opposite wood was demonstrated through either direct observations of these layers, or through the property of bifluorescence where the dye is excited only when aligned parallel to the polarisation of the incident light. Despite extensive work with confocal microscopy, this technique proved to be unsuitable for investigations of spiral grain because although it provided cellular detail, imaging was limited to the surface layers of sections, and the area over which observations were required was prohibitive. Instead of confocal microscopy, the incidence of spiral grain in young stems was investigated in two completely new ways. Resin canals, which are formed from the same cambial initials as the tracheids and which align with the grain, were used as a proxy to demonstrate the grain changes. A novel technique, using circular polarised light and a professional flatbed scanner, was developed to image whole serial transverse sections of the young stems to detect the resin canals. Using ImageJ, the number and location of resin canals was measured on vertical controls, and trees that had been rocked and leaned. The number and frequency of resin canals were less in tilted trees, especially in compression wood, compared to the higher number of canals formed in the rocked trees. More importantly, a combination of serial sectioning and this approach allowed a 3-dimensional view of the orientation of resin canals inside a stem to be generated with ImageJ, and the angles of these canals could be measured using Matlab. The resin canals were oriented with a left-handed spiralling near the stem surface whereas the canals near to the pith were nearly straight, consistent with previous observations of the development of spiral grain in radiata pine. However, it was observed that while vertical trees had a symmetric pattern of grain and grain changes around the stem, this was not the case in tilted trees. In these, the opposite wood often had severe spiral grain visible through formation of twist whereas the compression wood formed on the lower side had bending. Consistent with this, grain associated with compression wood was significantly straighter than in opposite wood. This hitherto unknown link between the incidence of compression wood and spiral grain was investigated and explained on the basis of the characteristics of resin canals in these types of wood. X-ray micro-tomography was also used to investigate resin canals in the stubs from which serial sections were collected. The 3D reconstructions of the resin canals showed exactly the same patterns as observed by polarised light scanning.

Optical Confocal Holography is a combination of two well known concepts: confocal microscopy and optical (laser) holography. Confocal microscopy places an aperture at a conjugate focus to the specimen focus. This filters any rays that are not on the focus plane, allowing a 3-dimensional image of the specimen to be built up over a set of planes. Holography is the measurement of both the amplitude and phase characteristics of light. Typically most methods only measure the amplitude of the image. The phenomenon of interference allows the determination of the phase shift for a coherent source as well. The phase information is directly related to the index of refraction of a material, which in turn is a function of the temperature and composition. As a technique, confocal holography holds promise to better characterize many physical processes in materials science, such as combustion and convection. It also may contribute to the biological sciences by imaging low-contrast, weak-phase objects. Thanks to the ongoing, continued improvement in computer processing speed, it has recently become practical to interpret data from confocal holography microscopy with a computer. The objective of the microscope is to non-invasively measure the three-dimensional, internal temperatures and compositions (e.g. solute/solvent gradient) of a specimen. My contributions over the course of two years to the project were: generation and optimization of an optical design with a software package known as Zemax; sourcing and purchasing all components; formation of a CAD model of the microscope; experiments to characterize building vibrations and air currents; and the development of software in Visual Basic to simulate holograms and execute reconstruction algorithms for the specific application of confocal holography.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 8 uppsatser.

Thesis (Ph.D.)--University of Wollongong, 2005. / Typescript. Includes bibliographical references: p. 198-203.

Mucosal associated invariant T (MAIT) cells are a population of innate-like lymphocytes, with an emerging role in tuberculosis (TB). They are characterised by the expression of high levels of CD161 and IL-18Rα, possession of a Vα7.2⁺ T cell receptor (TCR), and restriction by the MHC class I-related protein (MR1). MAIT cells can be activated by MR1 presenting microbe-derived riboflavin metabolites; or, by the cytokines IL-12 and IL-18 in a TCR-independent fashion. How human MAIT cells integrate these signals for their activation in response to Mtb is unclear. Lymphatic TB (LNTB) is a common extra-pulmonary manifestation of TB; however, little is known about the status of MAIT cells in LNTB - or in other granulomatous diseases, such as sarcoidosis. In this study, an in vitro approach was used to probe MAIT cell activation by Mtb, and the roles of IL-12/-18, the TCR, cell-cell contact and the immunological synapse (IS). Following TCR ligation, TNFα expression was rapid and transient, and was enhanced following sustained IL-12/-18 exposure. IFNγ expression occurred following sustained exposure to ng/ml concentrations of IL-12/-18; however, alongside TCR stimulation, pg/ml concentrations were sufficient. Using an artificial bilayer system, CD161 was excluded from the central regions of the MAIT cell IS, whilst the distribution of IL-18Rα remained unaffected. In response to Mtb and BCG, MR1 was necessary for rapid activation and TNFα expression, IL-12/-18 were necessary for robust and sustained IFNy expression, whilst an anti-Mtb effect was indicated in an intracellular infection model. Assessment of patients with TB or sarcoid lymphadenopathy revealed a depletion of MAIT cells in the blood in sarcoidosis, but not LNTB. In both groups, MAIT cells could be detected within a proportion of sampled lymph nodes. Overall, these findings indicate the importance of inflammatory cytokine signals in the induction of high-intensity and sustained MAIT cell effector function, including in response to Mtb. The observation of a numerical deficiency of MAIT cells in sarcoidosis requires further investigation.

[pt] Em um mundo globalizado, a demanda por energia está sempre crescendo. Uma vez que a indústria de óleo e gás é responsável pela entrega da maior parte desta demanda, isso faz dos hidrocarbonetos componentes cada vez mais importantes no mercado mundial. Entretanto tais recursos são finitos, portanto, uma exploração consciente, buscando sempre o máximo desempenho se faz necessária. Como os reservatórios de petróleo, logo após a aplicação das técnicas de recuperação primária e secundária, geralmente ainda possuem cerca de 65 por cento do volume de óleo originalmente contido em seus poros, métodos que visam a redução dessa porcentagem estão ganhando um papel cada vez mais importante na indústria energética. Nesse contexto, esse trabalho apresenta um micromodelo tridimensional representativo de um meio poroso que será utilizado para a análise do escoamento de fluidos na escala de poro. A microscopia confocal será adotada para visualizar os diferentes fenômenos que ocorrem em microescala, permitindo a obtenção de informações específicas sobre a dinâmica dos gânglios de óleo, em relação a sua formação, mobilização e aprisionamento, e assim, ao final do experimento quantificar a saturação residual de óleo em diferentes condições de escoamento. Os resultados obtidos mostram que o uso das suspensões de microcápsulas flexíveis como agente de controle de mobilidade, modifica a distribuição dos fluidos no meio poroso, o que melhora a eficiência de deslocamento do fluido deslocante na escala de poro, e consequentemente diminui a saturação de óleo residual. / [en] In a globalized world, the demand for energy is always growing. Since the oil and gas industry is responsible for delivering most of this demand, this makes hydrocarbon components increasingly important in the worldwide economy. However, such resources are finite, so a conscious exploration always seeking the maximum performance is required. As oil reservoirs after the application of primary and secondary recovery techniques usually still have about 65 percent of the original oil volume contained in their pores, methods that aim its reduction are gaining an increasingly important role in the energy industry. In this context, this work presents a three-dimensional micromodel representative of a porous medium that is used for pore-scale flow analysis. Confocal microscopy is used to visualize the microscale phenomena, leading to specific information about ganglia dynamics, related to its formation, mobilization and entrapment. The residual oil saturation, an important value to measure the amount of oil produced in a given reservoir is determined for different flow conditions. The results show that the suspensions composed by flexible microcapsules could be used as a mobility control agent, since it modifies the fluid distribution in the porous media, improving the pore-scale displacement efficiency, and consequently reducing the residual oil saturation.

Objetivos: O objetivo desse estudo foi avaliar a influência da presença do cimento endodôntico e agente cimentante na retenção de pinos de fibra. Metodologia: Cento e oitenta dentes extraídos foram divididos em dois grupos (n=90) de acordo com a forma de obturação: condensação lateral ou obturação apenas do terço apical. Cada um destes grupos foi subdividido em três grupos (n=10) de acordo com o cimento utilizado para a obturação (AH Plus, Endofil e MTAFillapex). Os cimentos foram manipulados e acrescidos de corante Rodamina B na proporção de 0,1% para possibilitar visualização através da microscopia confocal a laser. Quinze dias após a obturação, estes dentes foram preparados para a cimentação do pino de fibra (Reforpost Angelus) com os cimentos RelyX ARC (ARC), U200 e Gold Label Cement Lining (GL). Os dentes foram seccionados transversalmente para obtenção de fatias com 1mm de espessura e submetidos à microscopia confocal a laser para verificar a penetração do cimento endodôntico nos túbulos dentinários, posteriormente ao teste de push-out. O padrão de falha foi analisado em esteromicroscópio e imagens representativas foram feitas em microscopia eletrônica de varredura. Os valores de resistência de união em MPa de acordo com a técnica de obturação foram analisados pelo teste de Mann-Whitney. Os valores de resistência de união dos grupos experimentais foram analisados pelo teste de Kruskal-Wallis e post hoc de Dunn, com nível de significância de 5%. Resultados: Não houve diferença significativa entre as formas de obturação (P>0,05). O tipo de cimento obturador influenciou a resistência de união, e o cimento MTA-Fillapex apresentou valores de resistência de união significativamente inferiores ao AH Plus (P<0,05). Os grupos que utilizaram GL para cimentação dos pinos de fibra de vidro apresentaram valores para o teste de push-out superiores aos cimentos resinosos (P < 0,05). Não houve diferença significativa entre o ARC e o U200 (P > 0,05). Conclusões: Valores de resistência de união mais altos foram observados quando os pinos de fibra foram cimentados com cimento GL . Os padrões de falha mistas foram predominantes e ocorreram em todos os grupos experimentais. / Aim: Evaluate the influence of the endodontic sealer and luting cements on the fiber post bond strength. Methodology: One hundred and eighty extracted teeth were assigned to two groups, considering the root filling technique: lateral condensation or root filling of the apical portion only. Each group were divided into three groups (n=10) according the sealer applied (AH Plus, Endofill or MTA-Fillapex). Rhodamine B was mixed to the sealer in a ratio of 0,1% in order to provide adequate fluorescence assessed by confocal microscopy. Fifty days after root canal filling, post-space preparation were performed with luting cements: RelyX ARC (ARC), U200 and Gold Label Cement Lining (GL). One thick slices were produced and submitted to confocal miscroscopy to assessed sealer penetration into dentinal tubules, and than submitted to push-out test. The failure modes were analysed in stereomicroscope and representative images were obtained in a scanning electron microscope. Push-out strength values of the root filling technique were analyzed using Mann-Whitney test. Push-out strength values of experimental groups were analysed using Kruskal-Wallis and Dunn´s post hoc test at significance level 5%. Results: No significant difference was observed (P>0.05) between the root filling technique. The type of endodontic sealer influenced the push-out bond strength, and MTA-Fillapex sealers presents lower bond strength values than AH Plus (P<0.05). Fiber posts cemented with GL presented higher push-out bond strength than resin cements (P<0.05). There was no significant difference between ARC and U200 (P>0.05). Conclusions: Fiber posts showed higher bond strength values when cemented with GL. Mixed failures were predominant and occurred in all the experimental groups.

Ce projet concerne l’analyse de l’organisation de populations microbiennes au sein de structures complexescomme des dépôts ou des biofilms.Si différentes méthodes sont couramment utilisées pour étudier la structure globale ou l’organisation localed’agrégats microbiens, peu d’entre elles, permettent de réaliser simultanément ces deux analyses et nécessitentsouvent des étapes de préparation qui pénalisent un approche in-vivo et en dynamique.La stratégie proposée repose sur la mise en oeuvre de micro-organismes modèles autofluorescents (levures etbactéries) qui peuvent sans aucun traitement être directement observés en microscopie. La similitude ducomportement physiologique de ces micro-organismes avec celui des souches sauvages a été démontrée. Lesconditions d’acquisition des images en microscopie confocale ont été optimisées. Des dispositifs spécifiques ontété conçus pour générer des dépôts ou des biofilms dans des conditions de contraintes physico-mécaniques etbiochimiques maîtrisées afin d’analyser simultanément leurs caractéristiques et les performances du bioprocédé.Ainsi les dépôts ont pu être observés in-vivo et in-situ grâce à une cellule de filtration équipée d’une fenêtred’observation. Le développement d’un biofilm mixte composé de levures et bactéries modèles autofluorescentes,dans un réacteur continu spécifique, a également été analysé par microscopie confocale.Le traitement et les analyses des images acquises au cours des expériences ont été effectués et ont permisd‘étudier la structure globale des agrégats biologiques et l’organisation tridimensionnelle des micro-organismesdans ces structures, en mettant par exemple en évidence une répartition hétérogène de deux populationsmicrobiennes dans des dépôts de filtration ou en comparant la capacité de deux espèces microbiennes à formerdes biofilms en étudiant in-vivo la dynamique de croissance de chacune des espèces.Cette étude a en outre permis de démontrer la pertinence de la méthode proposée, de définir ses limites et sonchamp d’application / The aim of this project deals with the analysis of both the local localization and organization of microbialpopulations in complex structures such as deposits or biofilms. Different methods are currently used to study theglobal structure or the local organization of biological aggregates but only few ones allow a combined approachand require ex-vivo analyses.The proposed strategy uses home-designed model auto-fluorescent microorganisms (yeasts and bacteria) whichcan be observed directly by microscopy without any dying treatment. Same kinetic behaviours between the wildstrains and their recombinant ones were demonstrated. The confocal microscopy conditions were optimised.Specific devices were developed to generate deposits or biofilms under controlled and known hydrodynamic orbiochemical environment conditions to analyse their structure characteristics linked to the bioprocessperformances.Based on the proposed strategy, microbial deposits modifications due to pressure constraints were observed invivo in a specifically designed flow cell equipped with a microscope glass coverslip. A mixed biofilm composedby our auto-fluorescent yeasts and bacteria was carried out in a specific bioreactor allowing the sampling ofbiofilms during their development to be analysed by confocal microscopy. Both studies have shown specificorganisations between yeasts and bacteria mainly depending on their size and on the environment conditions(pressure or dilution rate).These studies of both local and global structure of biological aggregates and 3D-organisation of themicroorganisms within theses structures demonstrated the relevance of the proposed strategy defining the limitsof the method and proposing various perspectives for further characterizations and applications