Le mélanome est le cancer cutané le plus invasif. Au cours de l’invasion tumorale et de la dissémination métastatique, les cellules tumorales sont capables de dégrader la matrice extracellulaire par la sécrétion de protéases telles que les MMPs. Lors de cette protéolyse matricielle, différents fragments de la matrice extracellulaire exerçant des activités anti-tumorale et/ou anti-angiogénique, nommés matrikines, sont libérés et modulent la croissance tumorale. De nombreuses matrikines dérivées des collagènes de membrane basale sont capables de limiter la progression tumorale. Nous avons étudié les propriétés anti-tumorales du domaine NC1 α4(IV), nommé tétrastatine, à la fois in vitro et in vivo dans un modèle de mélanome humain. La tétrastatine induit une inhibition de la prolifération et de l’invasion des cellules de mélanome in vitro. L’inhibition de prolifération est corrélée à un retard en phase G1/S du cycle cellulaire en présence de tétrastatine. L’inhibtion de l’invasion peut notamment s’expliquer par une inhibition de l’activation de la MMP-14 et une modification de sa répartition cellulaire, avec perte du phénotype migratoire en présence de tétrastatine. In vivo, la surexpression de la tétrastatine induit une forte inhibition de la croissance tumorale, dans un modèle de xénogreffe de mélanome humain chez la souris nude. Nous avons également pu identifier l’intégrine αvβ3 comme un récepteur potentiel de la tétrastatine. Enfin, l’étude des capacités anti-prolifératives et anti-invasives des cellules UACC 903 en présence de différents peptides permet aujourd’hui de mieux préciser la séquence responsable de l’activité anti-tumorale de ce domaine. En conclusion, la tétrastatine est une nouvelle matrikine à fort potentiel anti-tumoral capable de limiter la progression du mélanome. / Melanoma is the most invasive cutaneous cancer. During tumor invasion and metastatic dissemination, tumor cells degrade the extracellular matrix by secretion of proteases such as MMPs. During matrix proteolysis, fragments of the extracellular matrix with anti-tumor and/or anti-angiogenic activities, called matrikines, are released and modulate tumor growth. Many matrikines derived from basement membrane collagens are able to inhibit tumor progression. We studied the anti-tumor properties of the domain NC1 α4 (IV), named tetrastatin, both in vitro and in vivo in a human melanoma model. Tetrastatin induces inhibition of proliferation and invasion of melanoma cells in vitro. This inhibition of proliferation is correlated to a cell cycle delay in G1/S phase when cells are incubated with tetrastatin. The inhibition of invasion could be due, at least partly, to the inhibition of MMP-14 active form and modification of its cellular distribution, with a loss of the migratory phenotype in the presence of tetrastatin. In vivo, tetrastatin overexpression induces a strong inhibition of tumor growth, in a human melanoma xenograft model in nude mice. We also identified integrin αvβ3 as a potential receptor of tetrastatin. Finally, the study of the anti-proliferative and anti-invasive properties of the UACC 903 cells in the presence of different peptides allows us to better identify the sequence responsible of the anti-tumor activity. In conclusion, tetrastatin is a new potent anti-tumor matrikine capable of limiting melanoma progression.

Le vieillissement cutané s'accompagne d'altérations des composants de la matrice extracellulaire. Des études ont montré que le contenu en collagène IV diminuait dans la peau à partir de 35 ans. Le collagène IV, constituant majeur des membranes basales, est formé de l'association, en triple hélice, de 3 chaînes alpha parmi 6 possibles: alpha1(IV) à alpha6(IV). Au niveau de la membrane basale cutanée, encore appelée jonction dermo-épidermique, seulement deux isoformes de collagène IV ont été mises en évidence : [alpha1(IV)2; alpha2(IV)], isoforme majoritaire, et [alpha5(IV)2; alpha6(IV)], isoforme minoritaire, synthétisées par les fibroblastes et les kératinocytes. Nous avons vérifié, par western-blot sur des extraits de peaux, cette diminution de collagène IV. Nous avons ensuite analysé la répartition du collagène IV au niveau de la JDE sur des coupes transversales de peau et n'avons pu mettre en évidence de discontinuité dans le réseau de collagène IV avec l'âge, du fait des fortes variations inter-individuelles. Parallèlement, sur coupe de peau, nous avons tenté de mettre en évidence des différences spectrales du collagène IV avec l'âge par spectroscopie Raman mais la résolution s'est avérée insuffisante. Nous avons isolé des fibroblastes de patients d'âges différents et montré une diminution de l'expression génique de la chaîne alpha1(IV) malgré de fortes variations inter-individuelles. Afin de s'affranchir de ces variations inter-individuelles pour étudier les mécanismes impliqués dans la diminution d'expression du collagène IV, nous avons mis au point un modèle de vieillissement accéléré de fibroblastes traités à l'H2O2 et vérifié le phénotype sénescent des cellules (morphologie modifiée, augmentation de l'activité SA-beta-galactosidase, augmentation de p21WAF-1…). Nous nous sommes intéressé à la voie du TGF-beta1. Nous avons montré que l'expression du récepteur au TGF-beta1, TbetaRII, diminuait dans le modèle de vieillissement accéléré. Nous avons également montré qu'un anticorps bloquant anti-TGF-beta1 reproduisait la diminution d'expression de collagène IV observée au cours de la sénescence. La détermination du mécanisme impliqué pourrait permettre, à terme, de proposer de nouvelles stratégies pour maintenir l'intégrité de la membrane basale lors du vieillissement cutané. / During aging skin there are extracellular matrix alterations. Preliminary studies showed that type IV collagen content decreased in skin with age after 35 years. Type IV collagen is a major component of basement membranes. It's constituted by the association of 3 alpha chains among 6 possible (alpha1 to alpha6). In the cutaneous basement membrane also called dermo-epidermal junction, only two isoform of type IV collagen were found: [alpha1(IV)2; alpha2(IV)], which is majoritary isoform, and [alpha5(IV)2; alpha6(IV)], which is minoritary, both synthesized by fibroblasts and keratinocytes. We checked the decrease in type IV collagen by western-blot on skin extracts. We then analyzed the distribution of this collagen in the DEJ on skin sections but we were not able to highlight a discontinuity in the network of type IV collagen during aging. At the same time, we tried to highlight spectral differences of the collagen IV with aging by Raman spectroscopy on skin sections but the resolution was insufficient. 35 years. We showed a decrease of type IV collagen expression by dermal fibroblasts in spite of strong variation between patients. In order to study the mechanism involved in type IV collagen variation during aging in dermal fibroblasts avoiding inter-individual variations, we develop an accelerated aging model of fibroblasts by treatment with H2O2. We checked the senescent phenotype of the cells (modified morphology, increase of SA-beta-galactosidase activity, increase of p21WAF-1…). We were interested on the TGF-1 pathway and we showed that TGF-beta1 receptor, called TbetaRII, was decreased in our accelerated aging model. We also showed that a blocking antibody against TGF-beta1 reproduced the decrease of type IV collagen expression observed during the senescence. The determination of the involved mechanism could lead to propose new strategies to maintain the integrity of the basal membrane during skin aging.

The extracellular matrix (ECM) offers a protective shelter for cells and provides signaling paths important for cell to cell communication. ECM consists of basement membranes (BM) and interstitial matrix. BMs provide mechanical support for parenchymal cells, influence cell proliferation, survival, migration and differentiation. They are also important for tissue integrity. Laminins (LM) are the major non-collagenous component of BMs. Cell-ECM interactions, mediated by receptors, are indispensable during embryonic development, wound healing, remodeling and homeostasis of tissues. The integrins are the major cell-adhesion receptors. The expression of alpha11 integrin chain in the cornea is of great interest, as it is part of the alpha11beta1 integrin receptor for collagen type I, the predominant component of the corneal stroma. The aims were to thoroughly characterize the ECM in the developing and adult human eye, with particular focus on the cornea, LM and alpha11 integrin chains, and to examine alpha11 integrin chain in an animal model of corneal wound healing and remodeling. Human fetal eyes, 9-20 weeks of gestation (wg), and adult human corneas with different diagnosis were treated for immunohistochemistry with specific antibodies against LM and alpha11 integrin chains. Normal and knockout (ko) mice were treated with laser surgery to create a deep wound in the corneal stroma. The wound healing process was followed at different time points. The cellular source of alpha11 integrin chain was studied in cell cultures. In the fetal eyes, the BM of the corneal epithelium, the Descemet’s membrane (DM) and the Bruch’s membrane each had their specific combinations of LM chains and time line of development, whereas the lens capsule and the internal limiting membrane showed constant LM chain patterns. The epithelial BMs of normal and diseased adult corneas contained similar LM chains. The normal morphology of the epithelial BM was altered in the different diseases, particularly when scarring was present. In the scarred keratoconus corneas there were excessive LM chains. The majority of keratoconus corneas also expressed extra LM chains in the DM. At 10-17 wg alpha11 integrin chain was present in the human corneal stroma, especially in the anterior portion, but it was scarce at 20 wg, in normal adult corneas and in Fuchs’ endothelial dystrophy. In contrast, it was increased in the anterior portion of the stroma in keratoconus corneas with scarring. Alpha11 integrin ko mice had a defective healing with subsequent thinner corneas. Alpha11 integrin expression correlated to the presence of alpha-smooth muscle actin in vivo as well as in vitro. The distinct spatial and temporal patterns of distribution for alpha11 integrin and each of the LM chains suggest that they play an important role in human ocular differentiation. The selectively affected LM composition and the novel expression of alpha11 integrin chain in scarred keratoconus corneas as well as the pathologic healing in ko mice, indicate that alpha11 integrin and LM chains also play an important role in the process of corneal healing, remodeling and scarring and might participate in the pathogenesis of corneal disease. This knowledge is of practical importance for future topical therapeutic agents capable of modulating the corneal wound healing processes.

A sinalização da matriz extracelular (MEC) é essencial para a determinação do destino e comportamento de células epiteliais da glândula mamária. Entretanto, pouco é conhecido sobre os mecanismos moleculares envolvidos nesse processo. A via Hippo, uma cascata de sinalização que participa da regulação de diversos comportamentos celulares, incluindo o tamanho de órgãos, parece ser uma importante candidata a mediadora sinalização da MEC. Resultados preliminares do laboratório indicam que a arquitetura tecidual e a membrana basal, componente da MEC de epitélios e outros tecidos, influenciam a localização, concentração e atividade de YAP, uma proteína efetora da via Hippo, em células epiteliais mamárias. Neste contexto, o objetivo deste trabalho foi identificar as proteínas que interagem com Yap (ortólogo de YAP em camundongo) nas células epiteliais da glândula mamária em resposta à membrana basal. Foram utilizadas células EpH4, uma linhagem mamária não-tumoral murina, como modelo de diferenciação funcional e formação de ácinos em um ensaio de cultura tridimensional (3D). O tratamento de estruturas multicelulares 3D pré-formadas em placas nãoadesiva com uma matriz rica em laminina (lrECM) alterou a localização e o padrão subcelular de Yap, assim como a expressão gênica de membros da via Hippo e dos alvos de Yap, mas não alterou a expressão das proteínas da via em nível de proteína. O ensaio de co-imunoprecipitação (CoIP) seguida de análise por espectrometria de massas identificou um conjunto diferencial de proteínas que interagem com Yap na fração citoplasmática de células EpH4 cultivadas na ausência ou na presença de lrECM em um modelo de ECM-overlay. Uma análise realizada junto à database KEGG Pathways revelou que os possíveis interagentes Yap nas células cultivadas não-tratadas com lrECM participam de processos relacionados à proteólise mediada por ubiquitina, enquanto nas células expostas à lrECM os possíveis interagentes estão associados a processos metabólicos e são especialmente proteínas-chave do metabolismo de lipídios. A busca na plataforma de redes de interação STRING não identificou trabalhos que destaquem a interação de Yap com estas proteínas. A plataforma Vizit indica a participação de Yap em processos relacionados à síntese e atividade de lipídios e hormônios, o que reforça as evidências de que está pode ser uma nova função de Yap ainda não explorada em detalhes. A fim de se obter resultados complementares à CoIP, padronizamos o ensaio de identificação por biotinilação dependente de proximidade (BioID) em células embrionárias de rim humano da linhagem 293FT. As proteínas isoladas por pulldown foram identificadas por espectrometria de massas e uma análise junto à database Gene Ontology indicou que os possíveis interagentes de Yap nestas células são em sua maioria proteínas relacionadas à via Hippo, o que reforça a robustez do ensaio. Nós pretendemos transpor este sistema para as células EpH4. A expectativa é que, em conjunto, estes resultados nos orientem em projetos futuros para compreender os mecanismos de sinalização da MEC na morfogênese e diferenciação da glândula mamária. / Extracellular matrix (ECM)-signaling is crucial for determination of epithelial cell fate and behavior in the mammary gland. However, little is known about the molecular mechanisms involved in these processes. The Hippo pathway, a signaling cascade involved in the regulation of several cellular processes, including organ size, seems to be an important candidate as a mediator of this signaling. Our preliminary results indicate that the tissue architecture and the basement membrane, an ECM component of epithelia and other tissues, influence the location, level and activity of YAP, an effector of the Hippo pathway. In this context, the goal of this work was to identify the proteins that interact with Yap (ortholog of YAP in mouse) in mammary epithelial cells in response to the basement membrane. We used EpH4 cells, a nontumoral murine mammary cell, in a functional differentiation and acini-forming in tridimensional (3D) culture assay. Treatment of 3D multicellular structures pre-formed on nonadhesive plates with a laminin-rich extracellular matrix (lrECM) altered the subcellular localization and pattern of Yap, as well as gene expression of Hippo pathway proteins and Yap targets, but did not altered the expression of the pathway members at the protein level. Coimmunoprecipitation (CoIP) followed by mass spectrometry analysis identified a differential set of proteins interacting with Yap in cytoplasmic fractions of EpH4 cells in the absence or presence of lrECM in an ECM-overlay culture model. An analysis performed with the KEGG Pathways database revealed that putative Yap interactors in non-treated cells participate in processes related to ubiquitin-mediated proteolysis, whereas in cells exposed to lrECM Yap interactors are associated to metabolic processes and are mainly key-proteins of metabolism of lipids and carbohydrates. A search in interaction networks platform STRING did not identify previous works that showing the interaction of Yap with these proteins. Vizit platform indicated the participation of Yap in processes related to the synthesis and activity of lipids and hormones, which reinforces the evidences that Yap can play a novel poorly explored role. To obtain complementary results to CoIP, we devised the proximity-dependent biotinylation identification (BioID) assay on embryonic renal cells of 293FT cell line. Pulldown-isolated proteins were identified by mass spectrometry and an analysis performed with Gene Ontology database revealed that putative Yap interactors are Hippo pathway-related proteins, which reinforces the robustness of the assay. We intend to transpose this system to the EpH4 cells. We expect that, together, these results will guide us in future projects to understand the signaling mechanisms of ECM in mammary gland morphogenesis and differentiation.

O presente estudo foi realizado para esclarecer o surgimento de vesículas subepidérmicas em enxertos de pele comumente descritos como áreas enxertadas. Devido à discrepância existente entre a literatura, que afirma surgirem vesículas nessas áreas, e a nossa experiência clínica, onde não observamos tal fato, decidimos investigar o problema. Para isso, estudamos a transição dermoepidérmica (TDE), em 23 pacientes submetidos à enxertia de pele, para verificar se há ou não alteração dessa estrutura que pudesse justificar a formação de vesículas. Nos 23 pacientes estudados foram feitas duas biópsias: a primeira, imediatamente antes da excisão do enxerto, na área doadora - pele sã, considerada como amostra padrão normal da TDE - Amostra Padrão AD. Após 10 dias, realizou-se uma segunda biópsia, com o mesmo vazador, próxima à área da primeira biópsia - Amostra Teste - ENX. Cada amostra foi dividida em 2 partes iguais (46 amostras) e estudadas por microscopia de luz e por imunofluorescência direta (imunomapeamento), pesquisando-se a possível alteração da zona da membrana basal (ZMB) na TDE através dos antígenos penfigóide bolhoso, laminina, colágeno IV e colágeno VII. Na microscopia de luz estudou-se, em cada biópsia, a relação entre a medida linear do relevo da trasição dermoepidérmica e a medida linear do relevo da superfície da camada granulosa, logo abaixo da camada córnea, equivalente a medida linear da superfície da pele. Nas 46 amostras as análises por microscopia de luz e de imunomapeamento para os quatro antígenos evidenciou-se a manutenção do mesmo padrão morfológico. Não houve diferença no imunomapeamento. Observou-se relações lineares das medidas com médias de 1,17 para a amostra AD e 1,44 para a amostra ENX, diferença que foi estatisticamente significativa, porém conservando a manutenção do padrão da TDE em relação à pele normal. Foi observada a manutenção do padrão do relevo da TDE no enxerto, em relação à pele sã, doadora. / SUMMARY: The present study has been done to elucidate the onset of subepidermal blisters in skin grafts, usually mistaken as grafted sites. Due to the discrepancy between literature - assigning this onset of blisters in grafts and our experience opposite we have decided to carry out this study. To do so we have studied the dermal-epidermal junction in 23 burned patients who underwent skin grafting so that we could verify whether or not there could be any alteration in the dermal-epidermal junction structure that may explain this fact. Among the 23 studied patients, two biopsies were carried out: the first one just before harvesting the skin graft from donor site healthy skin. The so called sample was regarded as an ordinary standard one of the dermal-epidermal junction STANDARD SAMPLE - DS. After graft take, by ten days, a second biopsy was performed with the same punch, close to the first biopsy TEST SAMPLE GS. Both samples were split into two equal parts (46 samples) and studied using light microscopy and direct immunofluorescence (immune mapping), searching for possible alterations in basement membrane zone in the dermal-epidermal junction through bullous pemphigoid, laminin and types IV and VII collagen antigens. On light microscopy, relation between the linear measure of dermal-epidermal junction projection and that of stratum granulosum surface, just underneath the stratum corneum, corresponding to skin surface, was studied in each biopsy. The four analyses of the antigens by light microscopy and direct immunofluorescence in the 46 samples clearly showed the keeping of the same pattern, either for STANDARD SAMPLE DS or TEST SAMPLE GS. There were no differences on the immune mapping. Regarding the relation of the linear measures it was noted a mean of 1.17 for STANDARD SAMPLE DS and a mean of 1.44 for TEST SAMPLE GS. Such difference was statistically significant. Nevertheless, it maintained the keeping of the same pattern of dermal-epidermal junction when compared to healthy skin.

A presente dissertação aborda o estudo dos efeitos de escavações realizadas acima de túneis já executados. Os trabalhos mais relevantes envolvendo a interação entre escavações e túneis já existentes são apresentados e discutidos. São realizadas simulações numéricas de casos hipotéticos, em estado plano de deformações utilizando o modelo de Mohr-Coulomb, permitindo a identificação dos parâmetros mais relevantes para esse tipo de problema e suas influências nos resultados. É discutido o comportamento mais rígido do maciço durante as trajetórias de descarregamento, bem como qual o módulo de elasticidade mais adequado para representar o fenômeno. Modelagens numéricas de um caso real em São Paulo (a escavação dos subsolos do edifício Stan Paulista, acima de túneis do Metrô) são realizadas, utilizando o modelo e os conceitos estudados. Os resultados obtidos das simulações são discutidos e comparados com os valores de instrumentação. Constata-se que a metodologia utilizada para a análise dos efeitos de escavações acima de túneis já existentes pode ser utilizada na avaliação de outros casos. / The presented research approaches the study of excavations effects above existing tunnels. Some of the most relevant papers related to interaction between excavation and tunnels are presented and discussed. Numerical simulations of hypothetic cases assuming plane strain condition and Mohr-Coulomb model for soil behavior are performed, allowing the study of influence of input parameters in the results. It is discussed behavior of the soil in stress paths that indicates reduction of the mean stress, wich is more rigid than the triaxial compression path. It is also discussed wich is an appropriate elastic modulus to be used in this situation. A real case (basement excavation of Stan Paulista building, above Metrô tunnels) is analyzed with numerical models, utilizing the concepts studied. The results of the simulations are compared with field instrumentation data. It is concluded that the methodology used for the analysis of the effect of excavation above existing tunnels can be utilized in other cases.

The morphogenetic events that give rise to the early post-implantation mouse embryo (egg cylinder) have not been thoroughly studied and our knowledge is restricted to "snap-shot" descriptions of embryos recovered at different stages of implantation from the mother. A central feature of the egg cylinder is the pro-amniotic cavity, which spans the embryo and participates in formation of the extraembryonic membranes. The major aims of my PhD studies have been to reveal how this cavity is formed (Aim 1) and then how the egg cylinder grows (Aim 2). In order to address how the pro-amniotic cavity forms (Aim 1), I first characterised in detail development of the architecture of the extra-embryonic ectoderm (ExE), which has to be remodelled to permit cavity formation. My findings indicate that the ExE comprises cells in direct contact with a basement membrane and cells that lie deeper in the tissue. The ExE originates in the polar trophectoderm, a monolayer covering the epiblast of the blastocyst, which expands and undergoes invagination to form a slit-like cavity. By carrying out analyses of fixed specimens and live imaging of cultured embryos, I have found that the epiblast and ExE cavity extend towards each other through the formation and resolution of multiple rosette structures. This leads to the fusion of the ExE and epiblast cavities to form the unified pro-amniotic cavity. I show that this process is dependent on signalling cues stemming from the underlying basement membrane that activate the b1-integrin signalling pathway to regulate cell polarity, ExE tissue architecture and rosette formation. In addition to the basement membrane's role in b1-integrin signalling, it also has physical functions that I characterise in the second part of my study (Aim 2). High resolution imaging revealed that the basement membrane underlying the epiblast is highly perforated during the implantation stages. These perforations are initially evenly distributed and then accumulate asymmetrically at the future posterior part of the embryo, just prior to gastrulation. Finally, I demonstrate that remodelling of the basement membrane requires the expression of matrix metalloproteinases (MMPs) in the epiblast under the control of Nodal. The anterior visceral endoderm inhibits Nodal signalling and hence MMP inhibition in the anterior. I demonstrate that activity of the MMPs and perforations in the basement membrane are essential for embryo growth. The domain of posterior basement membrane perforations persists beyond gastrulation suggesting a potential role for these perforations in primitive streak formation and extension. Together, my studies bring new important insights into the understanding of early mouse embryo morphogenesis.

Gravel bed braided rivers are distinctive natural environments that provide a wide range of key environmental, economic and recreational services. There is, however, a growing concern that over the twentieth century, an increasing number of braided rivers have metamorphosed into wandering or single thread channels, representing a loss of key habitats, geodiversity and amenity. While in some situations, shifts in channel pattern may be unambiguously linked to abrupt changes in flow or sediment supply, the lack of a theoretical basis underpinning the development and maintenance of braiding makes identification of the cause and effect of channel metamorphosis hazardous. A growing body of research has suggested that the transition between channel patterns may depend on the poorly understood interaction between the flow regime, sediment supply and vegetation colonisation. Such interactions are governed by critical thresholds, due to changes in flow resistance and bank strength associated with the distribution, form and intensity of vegetation colonisation. Subtle changes in flow or sediment supply that promote vegetation growth or indeed remove it through inundation or attrition. This can lead to complex non-linear shifts in the balance of forces that govern sediment transport and bedform morphodynamics, ultimately resulting in one-way changes in channel morphology. There is, therefore, a critical need to develop a quantitative understanding of these feedbacks in order to design sustainable river management programmes that seek to optimize the ecological and socio-economic benefits these rivers offer. During the last three decades, significant advances in the understanding of the morphodynamics of braided rivers have been made through a combination of field and physical experimentation. More recently, the emerging field of numerical modelling has created a new avenue to investigate the processes that govern channel dynamics. While this methodology offers significant promise through the construction of virtual experiments that examine the spectrum of drivers and responses of river systems, such models require careful and critical evaluation before they can be used to guide management practice. The wider goal of this research is to explore the application of a numerical modelling to investigate the feedbacks associated with the development and maintenance of braiding. Specifically, the state-of-the-art numerical model, BASEMENT, was used to examine channel responses to steady, and unsteady flow regimes, with and without the representation of vegetation. The research focuses on four main contributions: 1. The development of a systematic framework to quantify the evolving form and processes of braided rivers that can be used as part of a comprehensive approach to model validation. 2. Simulation of braiding development and maintenance using BASEMENT under steady flow conditions. Model simulations based on the natural prototype of the braided River Feshie were used to examine the sensitivity of emergent channel morphologies to the model parameterisation, focusing in particular on the representation of bank erosion and gravity-driven sediment transport. A novel multi6metric framework for model validation is presented and the results demonstrate the critical importance of lateral bank migration processes in order to maintain braided morphologies under steady flow. 3. A critical evaluation of the simulation of braiding under different form of steady and unsteady flow regimes is presented. These experiments investigate how the morphodynamics of braiding vary under energetically-normalised flow regimes characterized by differences in hydrograph form (peak discharge and duration). This experiment provides a novel insight into the role of flow variation in the maintenance of braiding. 4. Finally, the feedback between flow regimes, sediment transport and vegetation growth are examined using a novel model of vegetation colonisation and die- back. Four scenarios are presented, a no-vegetation model, one based on low growth rate, one based on an intermediate growth rate, and finally a high growth rate parameterisation. These simulations provide a clear insight into the non-linear processes driving channel evolution and demonstrate how subtle changes in the balance between flow frequency and vegetation growth can lead to divergent channel patterns. In summary, this thesis aims to advance our understanding of the morphodynamics of braided rivers and the role numerical models may have in helping to interrogate their behaviour and governing controls. It is hoped that this work may contribute, albeit in a small way, to advancing the science that promotes the sustainability of these fascinating and valuable environments.

In the developing embryo, the timely fusion of opposing epithelial sheets into one uniform layer denotes the completion of several developmental events. Failure of this epithelial sheet fusion event (ESF) within the choroid fissure (CF) is associated with the congenital disorder Ocular Coloboma, and is one of the leading causes of pediatric blindness. A requirement for a highly coordinated dismantling of the basement membrane (BM) to allow for fusion to occur is undoubted, however the underlying mechanisms of this process are poorly understood. Due to its BM crosslinking capabilities, I have hypothesized that the regulation of nidogen plays a crucial role in the disassembly of the BM prior to ESF. Whole mount in situ hybridization for all four BM components has revealed that expression of nidogen decreases prior to that of other BM components. Additionally, preliminary IHC data has revealed nidogen and collagenIV deposition within the CF. Further, knock-down of nidogen1a and 1b, or the expression of dominant negative nidogen1b resulted in gross morphological, as well as BM organization defects in developing eyes. Together, these data suggest that nidogen plays a role in regulating the integrity of the BM of the eye and may play a role in its disassembly prior to ESF.

The Cartoogechaye terrane (CT) is an enigmatic migmatite terrane within the Central Blue Ridge province of the southern Appalachians. Previous work identified exotic Pb isotope compositions within the CT (Quinn, 2012). More recent studies that mapped the extent of potentially exotic metaigneous lithologies yield U-Pb zircon ages consistent with a native Laurentian margin metasedimentary origin (Larkin, 2016). This study focused on the possible extent of similar lithologies in the Clyde quadrangle and provides further constraints on the crustal affinity of the CT. The Clyde quadrangle consists of four distinct lithologic packages: the CT, Ashe metamorphic suite, Great Smoky Group, and Grenville basement. Five samples within the Clyde quadrangle and one sample from Wayah Bald quadrangle were collected for detrital zircon (DZ) U-Pb geochronology and whole rock geochemistry for comparison similar anlayses from other bedrock units in the region. Dominant DZ age modes consist of the Grenville doublet (1050 Ma and 1150 Ma) or a modified version of it. Minor age modes exist at ~450 Ma, 600-750 Ma, and 1300-1550 Ma. Zircons for all but one sample display heterogeneous external and internal cathodoluminescence morphologies, consistent with a sedimentary protolith for the paragneisses. Whole rock compositions are consistent with weathering of and derivation from a local basement source. U-Pb age data are most consistent with an eastern Laurentian sedimentary provenance for five samples. The presence of 450-460 Ma grains is most consistent with high-grade Taconian regional metamorphism. The lack of a major Shawinigan age mode and zircon morphology for ca. 980-1050 Ma metamorphic zircons indicate that sample CLY16-1 is a syn-orogenic metasediment within the Grenville basement underlying the CT.