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.

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.

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.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

<p>Basement membranes are a dense, sheet-like form of extracellular matrix that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stresses. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instruct cell behavior and morphogenetic processes. In this thesis, I discuss how changes to basement membrane affect anchor cell (AC) invasion during C. elegans uterine vulval attachment. During AC invasion, the uterine AC breaches two juxtaposed basement membranes to contact the underlying vulval epithelium. Using live-cell imaging, genetics, molecular biology and electron microscopy I identify three modifications to the BM that affect AC invasion. In Chapter 2, I describe a system for linking juxtaposed basement membranes to stably align or connect adjacent tissues. This adhesion system promotes rapid AC invasion and also regulates a more long-term connection between the uterine tissue and the hypodermal seam cell in the adult worm. Chapter 3 elucidates how the BM component SPARC promotes cell invasion. As SPARC overexpression is correlated with cancer metastasis, this aims to understand how SPARC overexpression promote invasion in a pathological situation. In Chapter 4, I discuss preliminary data showing that the AC actively secretes laminin into the basement membrane targeted for invasion. I outline how future studies could elucidate the mechanism by which AC-derived laminin might promote cell invasion. Finally, Chapter 5 discusses conclusions and future directions for these studies.</p> / Dissertation

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

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.

Abstract In this thesis study, the primary structure of the human α1(XVIII) polypeptide was elucidated, its tissue distribution was studied, and the phenotypic changes in the mouse eye due to lack of type XVIII collagen in a knock-out mouse model were studied further. In addition, the consequences of simultaneous lack of both type XVIII and XV collagen were studied in a mouse model lacking both of these proteins. Two variant forms of human α1(XVIII) polypeptide were identified in this study, although, to date, a third form has also been characterized. The analysis of tissue distribution of the two polypeptide forms revealed differences in their tissue distribution, since the longest variant occurs prominently in the liver, while the short form is the major transcript in other tissues studied, e.g. in the kidney. The study of the type XVIII single null mouse eyes revealed abnormalities in the anterior eye segment in addition to the previously reported defects in the posterior eye part. In the type XVIII single null mice the iris was fragmented, pigment deposits could be seen in the pupil, and the pupillary ruff in the edge of a normal mouse iris was missing in these mice. The ciliary body was also abnormal, since the ciliary processes start to show regression in adult animals and eventually the basal infoldings of the non-pigmented ciliary body epithelia become flattened in the null mice. The intraocular pressure stabilizes to a lower level in adult mutant mice compared to controls, most likely reflecting the atrophied ciliary epithelia. The BM zones were also defective in the type XVIII null mouse eyes. The absence of an immunosignal with one of the antibodies detecting laminin γ2 chain in the type XVIII null mouse eyes may implicate conformational changes in the laminin γ2 chain due to lack of type XVIII collagen, and subsequently interaction between type XVIII collagen and laminin γ2 chain in normal mouse eye BMs. The study of the type XVIII and XV double null mice revealed that these mice were viable and fertile and had no major additional abnormalities compared to both single null mice. However, the regression of hyaloid capillaries (vasa hyaloidea propria, VHP) was studied in these mice, and a slight delay in the detachment of these vessels from the retina was noticed. Thus, the two collagens do not function entirely independently from each other. The studies with type XVIII collagen single null mice indicate that in addition to the posterior eye phenotype, this collagen is needed for the normal structural integrity of the anterior eye segment and basement membranes of the eye. The mouse model lacking both type XVIII and type XV collagen indicates that the roles of the two collagens are essentially diverse, although a slight compensatory effect was observed in the detachment of the hyaloid capillaries from the retina.

Abstract Lysyl hydroxylase (E.C. 1.14.11.4) catalyzes the formation of hydroxylysine in collagens and other proteins with collagenous domains. The resulting hydroxylysine residues participate in the formation of collagen crosslinks, and serve as attachment sites for carbohydrate units. They have been regarded as non-essential, since the absence of lysyl hydroxylase 1 activity is not lethal, although it leads to the kyphoscoliotic type of Ehlers-Danlos syndrome, and since recombinant collagens I and III lacking any hydroxylysine form native-type fibrils in vitro. A novel human lysyl hydroxylase isoenzyme, lysyl hydroxylase 3, was identified, cloned and characterized here. The novel isoenzyme was expressed as a recombinant protein in insect cells, and the protein was shown to catalyze hydroxylation of lysine residues in vitro. No differences were found in the catalytic properties between the recombinant lysyl hydroxylases 3 and 1. The human lysyl hydroxylase 3 gene was shown to be 11.6 kb in size and to contain 19 exons. The introns contain 15 full-length or partial Alu retroposons, which are known to be involved in most human gene rearrangements that occur by homologous recombination. The three recombinant human lysyl hydroxylase isoenzymes were isolated here for the first time as homogenous proteins. Limited proteolysis data suggested that the lysyl hydroxylase polypeptides might consist of at least three distinct domains, A-C. The N-terminal domain A was found to play no role in lysyl hydroxylase activity as a recombinant B-C polypeptide was a fully active hydroxylase. This work also confirmed that lysyl hydroxylase 3 has collagen glucosyltransferase activity as well as trace amounts of collagen galactosyltransferase activity. However, the levels of these activities were so low that their biological significance remains to be determined. In the last part of this work, lysyl hydroxylase 3 knock-out mice were produced and analyzed. The homozygous null embryos were found to die at a very early stage of development due to lack of type IV collagen in the basement membranes. The data demonstrated that hydroxylysine formed by lysyl hydroxylase 3 is essential for early mouse development and that lysyl hydroxylase 1 or 2 cannot compensate for the lack of its function.