O objetivo do presente estudo foi avaliar os níveis das metaloproteinases da matriz MMP-9, MMP-3 e MMP-13, e das quimiocinas MCP-1, MIP-1β e RANTES, no fluido gengival de dentes sob força ortodôntica. Foram recrutados 14 pacientes (3 homens e 11 mulheres) que foram submetidos à movimentação ortodôntica dos caninos superiores. Amostras do fluido gengival foram coletadas com tiras de Periopaper em diferentes tempos. O volume do FG foi determinado com o uso do Periotron e os niveis das MMPs e das quimiocinas quantificados usando-se uma multianálise imunoenzimática com microesferas. Os resultados mostraram que existe um aumento significativo no volume do FG na área de pressão em quase todos os tempos analisados, quando comparados às áreas de tensão. Os níveis da MMP-9 foram muito superiores aos das MMP-13 e MMP-3. Na análise da evolução do tempo pode ser observada uma alteração significativa nos níveis da quantidade total de MMP-9, MMP-13 e MMP-3 e na concentração de MMP-13 e MMP-3 durante o período de movimentação dentária no lado de pressão. A elevação dos níveis de expressão destas MMPs 1 hora após a aplicação da força ortodôntica sugere que estas enzimas estejam envolvidas na remodelação periodontal induzida. As quimiocinas MCP-1, MIP-1β e RANTES foram detectadas no sulco gengival em todos os diferentes intervalos de tempo analisados, nas áreas de tensão e de pressão. Porém, diversas amostras estavam abaixo do nível de detecção do ensaio e, os níveis dessas quimiocinas no fluido gengival não parecem ser alterados pelas forças ortodônticas. / The goal of the present study was to evaluate the levels of the matrix metalloproteinases MMP-9, MMP-3 and MMP-13 and of the chemokines MCP-1, MIP-1β and RANTES in the gingival crevicular fluid of teeth under orthodontic forces. Fourteen subjects (3 males and 11 females) were enrolled and subjected to orthodontic tooth movement of their maxillary canines. Samples of gingival crevicular fluid were collected from both tension and pressure sides using Periopaper strips at different time points. The volume of GCF was determined using a Periotron and the levels of MMPs and chemokines quantified using a multiplex microbead immunoassay. The results demonstrated that the levels of MMP-9 were higher than the levels of MMP-13 and MMP-3. Statistically significant fluctuations during the orthodontic tooth movement could be detected for the total amount of MMP-9, MMP-13 and MMP-3 and for the concentration of MMP-13 and MMP-3 at the pressure side. The elevation in the levels of these MMPs, 1 hour after the application of the orthodontic force, suggests that these enzymes are involved in the induced periodontal remodeling. The chemokines MCP-1, MIP-1β and RANTES were detected in the GCF in both tension and pressure sides at different time points. However, several samples were below the level of detection of the assay and the levels of theses mediators in GCF did not seem to be altered by the orthodontic forces.

A method of predicting delamination in fibre-reinforced composite materials including several previously disregarded strength issues is presented. Thermal residual stresses, volume of stressed material, in-plane stresses and the hydrostatic stress in the polymer matrix are introduced and their influence on composite material strength discussed. These factors are then applied in a stress based method for predicting delamination which can deal with both unidirectional and general laminates. The results from a series of scaled unidirectional specimens designed to produce interlaminar tensile strength data are used to determine the strength parameters for the method. The method is shown to be effective in predicting failure in the fill-in region of two 'T'- piece specimen designs to within 14%. The failures were dominated by tension acting between fibres in large blocks of unidirectional material which had high thermal residual stresses and tensile hydrostatic stress due to constraint from the surrounding material. The method is also applied to a series of test pieces which used general laminates. The designs are based on sandwich panel sections and a tapered I-beam specimen. In the sandwich panel specimens, the edge closure sections were constructed using 0,90 and ±45° plies. Delamination occurred in a region of dropped plies and curvature making all the stress components important in producing accurate predictions, which are within 16% of the failure loads in testing. The tapered I-beam specimens were designed to delaminate in a doubly-curved laminate region of 90 and ±45° plies. The delamination predictions were within 13% of the first delamination loads found in testing. The method produced failure predictions which were all within 16% of the failure loads of the tested specimens. It is found that the local geometry of the delamination region is critical in determining the stress levels in the specimens and therefore their strength. Variations in the manufacture of such specimens and components is therefore clearly important in establishing the delamination loads of composite structures.

We have shown herein that β1 integrin stably depleted PC3 sub-clonal cells confer a trend towards increased survival of mice compared to β1 integrin expressing counterparts when tested in an intracardial bone metastasis model. Therefore, we sought to investigate novel factors that mediate β1 integrin-dependent cellular migration and three dimensional growth of prostate cancer PC3 cells in vitro. We show herein that depletion of β1 integrin using siRNA directed techniques results in increased SPARC protein expression. We further show that suppression of SPARC by β1 integrin appears to occur through a JNK dependent mechanism. Moreover, siRNA mediated depletion of β1 integrin results in impaired sphere formation in 3D BME assays. This was mediated in part by the increased production of SPARC. β1 integrin-depleted cells also diminished the enhanced migration of cells on the predominant bone matrix, collagen I. Concomitant SPARC depletion in β1 integrin-depleted cells did not rescue this enhanced migration. These findings suggests that the role of β1 integrin in mediating 3D growth of PC3 cells occurs at least in part through the suppression of SPARC protein expression.

[pt] Programas de computador já vem sendo utilizados há muito tempo no projeto de circuitos integrados, principalmente para verificação e simulação. Os anos 80 viram surgir os compiladores de silício, novas ferramentas para a geração automática de estruturas e leiautes. O sistema apresentado nesse trabalho permite a geração automática do leiaute de uma via de dados (conjunto de registradores e ALUs) a partir de uma descrição de seu tamanho e componentes. É também apresentado um gerador de leiautes gate matrix com facilidades para posicionamento de terminais, utilizando para a criação das células da biblioteca do gerador de vias. São discutidos também os problemas envolvidos nas diversas etapas de geração, assim como as soluções encontradas. / [en] Computer programs have been used for a long time in the design of integrated circuits, specially for verification and simulation. In the 80s silicon compilers appeared, a newkind of tool used for circuit synthesis and layout generation. The system here presented permits the automatic generation of the layout of a datapath (a collection of registers and ALUs) from a specification containing the number of bits and functions desired. A gate matrix layout generator with facilities for placing terminals is also presented, as a tool for creating the necessary cells for the datapath library. The several problems involved on layout generation are discussed, as well as the solutions employed.

The tail dependence coefficient (TDC) is a natural tool to describe extremal dependence. Estimation of the tail dependence coefficient can be performed via empirical process theory. In case of extremal independence, the limit degenerates and hence one cannot construct a test for extremal independence. In order to deal with this issue, we consider an analog of the covariance matrix, namely the extremogram matrix, whose entries depend only on extremal observations. We show that under the null hypothesis of extremal independence and for finite dimension d ≥ 2, the largest eigenvalue of the sample extremogram matrix converges to the maximum of d independent normal random variables. This allows us to conduct an hypothesis testing for extremal independence by means of the asymptotic distribution of the largest eigenvalue. Simulation studies are performed to further illustrate this approach.

The aerospace industry is steadily increasing its use of polymer-matrix composites (PMCs) in airframe structures as it seeks to benefit from the high specific in-plane strength of laminated structural PMCs. However, PMC laminates suffer from low interlaminar shear strength due to their weaker polymer-matrix. Minimising risks of delamination is of paramount importance towards improving the safety of PMC structures. Multi-scale composites that are reinforced by both continuous fibres and nano-particles were identified as a potential solution for improving toughness and reducing risks of delamination in PMCs. An important challenge in the fabrication of multi-scale PMCs is to ensure that nano-particles are dispersed uniformly within the matrix. This is only achieved through minimal filtration of nano-particles during processing. The short resin flow lengths enabled by the resin film infusion (RFI) process make this process a prime candidate for the fabrication of multi-scale PMCs. The main objective of this thesis is to validate the possibility of using out-of-autoclave RFI for fabricating multi-scale carbon fibre composites featuring epoxy resins modified with carbon nanotubes (CNTs). The work is accomplished in 5 phases. In phase 1, preliminary work investigates the fabrication of PMCs with and without CNTs, using out-of-autoclave RFI. Results show that the types of reinforcement and matrix have strong effects on the porosity and interlaminar strength of PMCs. These results ushered the need for more thorough investigation and understanding of the RFI process, beyond what is available in the literature. Phases 2 to 4 focus on understanding how the choices of materials and types of stacking configuration can affect parts made using RFI. Phase 2, the in-situ characterisation of resin saturation during RFI is performed. Results enable a detailed analysis of the way in which resin flows around and inside yarns. Phase 3 consists in the characterisation of void formation during RFI. Two types of voids are observed: flow-induced voids resulting from either the merging of resin flow fronts or the drainage from capillary action; and gas-induced voids resulting from resin volatiles going out of solution and remaining in the resin matrix. In this work, the greatest source of porosity was caused by volatiles. In phase 4, the distribution and filtration of CNTs during RFI processing is characterised. Results show that processing choices can limit filtration and that clustering of CNTs prevents a uniform dispersion of CNTs in PMCs. Finally, the possibility of using RFI for making a multi-scale PMC demonstrator part is investigated. The work culminated with the successful fabrication of a delta-stringer panel. This thesis makes several important contributions to the knowledge pertaining to multi-scale PMC processing and performance, and to RFI. Firstly, it provides a robust description of RFI processing beyond was it available in literature, through in-situ observations of resin flow and void formation. Secondly, it assesses the viability of RFI for producing multi-scale PMCs featuring CNTs. In-situ observations of RFI processing enabled the identification of mechanisms leading to a loss of CNT dispersion during processing, partly explaining the minimal improvements in the interlaminar properties of composites observed when adding CNTs to the matrix. Thirdly, the fabrication of a delta-stringer panel made of a multi-scale PMC was successful, making it the first validation of the scalability of out-of-autoclave RFI processing for manufacturing multi-scale PMCs. The work presented herein contributed to the dissemination of knowledge; one conference paper was presented at ICCM20 (20th International Conference on Composite Materials), and another was presented at CANCOM2017 (10th Canadian-International Conference on Composites), and one journal article written in collaboration with project partners was submitted to Composites Science and Technology.

Abstract Type XIII collagen belongs to the group of transmembrane collagens. In this thesis the plasma membrane localisation and function of type XIII collagen have been studied using cell biological methods. Type XIII collagen was found to reside in focal adhesions. It appeared in these structures at a very early stage of their assembly and disappeared from them concurrently with focal adhesion proteins talin and vinculin. Insect cells expressing type XIII collagen showed an enhanced adhesion to certain matrix components. These localisation and adhesion data suggested that the function of type XIII collagen is related to cell adhesion. Supporting this, in tissues type XIII collagen was found to localise to cell-matrix and cell-cell adhesion structures. Type XIII collagen was found to be partly present in cholesterol-enriched membrane microdomains. With other membrane proteins this localisation has been shown to be linked to ectodomain shedding. The connection between the membrane microdomain localisation and the ectodomain shedding of type XIII collagen was also characterised, and it was demonstrated that manipulation of the cellular cholesterol level affected the efficiency of the ectodomain shedding. Additionally, insights into intracellular shedding of type XIII collagen in the Golgi apparatus were obtained. The study of type XIII collagen expression in human cancers revealed that it was enhanced especially in the desmoplastic cancer stroma. Since the increased expression of type XIII collagen was detected during the dysplastic stages, type XIII collagen may be involved in the early pathogenesis of cancer. The result indicated that type XIII collagen is involved in the matrix remodelling. In support of this, the cell culture experiments showed that the soluble type XIII collagen ectodomain altered the vitronectin-rich matrix unfavourable for cell adhesion and spreading. This may enhance cancer metastasis. Type XIII collagen expression was also induced in the remodelled stroma of keratoconus and corneal wounds. Data suggested that myofibroblasts were responsible for the increased expression of type XIII collagen in these situations. Therefore both in cancer and in the corneal pathologies studied, type XIII collagen expression was induced by the activated stromal cells.

Chronic wounds represent a severe socio-economic burden and a key area of unmet clinical need. PKCα is ubiquitous in the skin, particularly the epidermis and functions in numerous pathways that are fundamental to wound repair. By utilising a global PKCα-/- mouse we have identified PKCα-regulated processes both in unwounded skin and during wound healing. PKCα-/- mice display considerably delayed wound healing with a dramatic reduction in re-epithelialisation. By analysing the ultrastructure of the epidermis, I have shown that this delay directly correlates with a failure of wound edge desmosomes to switch to a their adhesive properties. A major risk factor for the development of chronic wounds is age. Crucially, this delay in modulating cell adhesion is conserved in human chronic wounds and aged murine skin. Furthermore, manipulation of PKCα using an inducible bitransgenic mouse containing epidermal specific constitutively active PKCα can accelerate the modulation of desmosome adhesion and subsequently improve re-epithelialisation. Global gene expression analysis of PKCα-/- skin and wounds revealed further defects. Upon wounding, we observed a failure to correctly regulate expression of key collagen and Wnt signalling genes that are essential for correct and timely wound healing. Finally, intrinsic gene expression changes were identified in the skin of PKCα-/- mice, specifically a downregulation of multiple extracellular matrix genes. Of note was the downregulation of small leucine-rich proteoglycans which led to alterations to dermal collagen structure and skin tensile strength. These changes render the PKCα-/- skin susceptible to breaking and wound development. To conclude, we have identified multiple roles for PKCα intrinsically in the skin and also during cutaneous wound healing. Importantly, these intrinsic changes appear to predispose PKCα-/- skin to the development of cutaneous wounds and altered wound-specific processes that manifest in a delayed healing phenotype.

Soft tissue sarcomas (STS) are rare human malignant neoplasms, arising mostly from stem cells within non-skeletal connective tissues. They account for approximately 1% of all human malignancies. Matrix metalloproteinases (MMPs) are enzymes involved in degradation of the extracellular matrix and their expression by cancer cells allows the cells to penetrate basement membranes and tissue matrix, thereby invading and metastasising. The most studied malignant tumours from the perspective of MMP expression and its relationship to malignant behaviour are epithelial-derived carcinomas. MMPs role in invasion and metastasis of sarcomas has been very little investigated. This is in part because of the difficulty in accumulating sufficient tumour tissue to enable statistically relevant analysis of sufficient tumours. The purpose of this thesis was to examine the expression of key MMPs - MMP-2, MMP-7, MMP-9, and MMP-14 and their inhibitors (TIMP-1 and TIMP-2) at the invasive/subcapsular edge of human malignant and benign connective tissue tumours using immunohistochemistry, a technique that allows a very high level of reaction product localisation within tumours. In three different STS types and appropriate benign equivalents, the expression of MMPs -2, -7, -9, and -14 and their inhibitors (TIMPs -1 and -2) were measured using intensity of staining and the percentage area of staining by image analysis. The results were compared between tumour types and against histological grading that is widely used as a prognostic factor. The findings from this research indicated that metalloproteinases were commonly expressed in STS and benign equivalents. There were differences in expression of some benign versus malignant neoplasms of the same group. No uniform pattern of expression of any of MMPs was observed across the tumours, but some of the data, most notably that for expression of MMP-2 and -9 indicate, a role for MMPs in malignant behaviour and some showed (e.g. MMPs -7 and -14) change in expression with the grade of malignant tumours in the same broad category. There is some evidence of an inverse relationship between MMP and appropriate TIMP expression suggesting that a failure of inhibition, as much as increased expression, is a feature of malignancy.

This thesis is devoted to a number of structural and behavioural problems in linear multivariable control system theory. The first problem addresses the subject of determination of the finite and infinite frequency structure of a rational matrix. A novel method is proposed that determines the finite and infinite frequency structure of any rational matrix. Some neat and numerically stable algorithms are developed to implement this method. The second problem concerns the resol vent decompositions of a regular polynomial matrix and solutions of regular polynomial matrix descriptions (PMDs). Regarding these fundamental is'sues, three contributions are made therein. Firstly, based on a general resolvent decomposition a complete solution of regular PMDs is presented that takes into account both the non-zero initial conditions of the pseudo state and the non-zero initial conditions of the input. Secondly, two special resolvent decompositions are proposed, both of which are applied to formulate the solution of the regular PMDs. The first one is formulated in terms of the finite, infinite, and the generalised infinite Jordan pairs, which is a refinement of the results given by Gohberg et al. [74] and Vardulakis [25]. The second resolvent decomposition is proposed on the Weierstrass canonical form of the generalised companion matrix of the polynomial matrix. Thirdly, a new characterization of the impulsive free initial conditions of regular PMDs is given and the relationship between the finite and infinite frequency structure of a regular polynomial matrix and its generalised companion matrix is determined. In the third problem a generalization of the chain-scattering representation for general plants is presented. Through the notion of input-output consistency, the conditions under which the generalised chain-scattering representation and the dual generalised chain-scattering representation exist are proposed. Some algebraic system properties of the GCSRs and DGCSRs are studied. The fourth problem is devoted to a new notion of realization of behaviour. We introduce a notion realization of behavior which is shown to be a generalization of the classical concept of a realization of transfer function. By using this approach, the input-output structures of the generalized chain-scattering representations and the dual generalized chain-scattering representations are investigated in a behavioral theory context. The last problem is devoted to the subjects of system wellposedness and internal stability. We present certain generalisations to the classical concepts of wellposedness and internal stability. The input consistency and output uniqueness of the closed-loop system in the standard control feedback configurations are discussed. Based on this, a number of notions are introduced such as fully internal wellposedness, externally internal wellposedness, and externally internal stability, which characterize the rich input-output and stability features of the general control systems in a general setting. On the basis of these notions the extended JL control problem is defined in a general setting.