Ventral body wall closure (VBW) defects are amongst the most common human congenital anomalies. They represent a wide and heterogeneous group of phenotypic defects that can present in isolation or as a component in a larger syndromic anomaly. In addition, the incidence of associated anomalies is high and reaches 75% of fetuses in some types of VBW closure defects. Nevertheless, the embryonic origin and the underlying cellular and molecular mechanisms between ventral closure defects and their associated congenital anomalies remain poorly characterised. This is in part due to the poor understanding of the physiological mechanisms that regulate the development of ventral organs and the lack of representative transgenic animal models allowing detailed in vivo analysis of defect formation. Transforming growth factor beta (TGF-ÃŽÂ2) signalling is essential for VBW closure and vascular and cardiac development. Yet, its mechanism of action and the responding cell(s) in the body wall remain largely unknown. In addition, in various cells TGF-B can induce the expression of Tagln, encoding for a cytoskeleton associated protein that enhances cell migration. No function has been ascribed to TAGLN in body wall development. I define here a role of TGF-B during a critical time window in embryonic development to fashion the ventral body wall, anterior diaphragm and parts of the circulatory system. I identify a population of TAGLN+ myofibroblasts that respond to a temporally regulated TGF-B signalling originating from the epithelium of the primary body wall. Deletion of TGF-B receptor in TAGLN+ cells leads to failure of ventral body wall closure, anterior diaphragmatic hernia, cardiac and outflow tract anomaly. Nevertheless, the descending aorta and the large aortic branches are spared. By using advanced transgenic methodology, I generated novel transgenic mouse lines that enabled me to fate map the cells that initiate the formation of important mesenchymal tissues. These studies revealed that the origin of aortic vascular smooth muscle cells can be traced back to a group of progenitor cells that reside in the wall of the dorsal aorta before the VBW closure. My studies provide intriguing evidence for spatially restricted role for TGF-B signalling in ascending but not descending aorta morphogenesis. I used a variety of techniques to characterise, analyse and quantify important mechanisms during mesenchymal and vascular development, their response to injury and repair. This thesis has been written in an alternative format, comprising the different areas which have been investigated. Collectively, the results presented here provide new insights into the role of migratory and mechanically stabilising cells in the development and maintenance of critical structures in the body and their common role in the development of concurrent congenital anomalies. A detailed understanding of the molecular signalling pathways and cells that drive VBW closure raises the hope that the related birth defects can in the future be treated by precise gene and cell therapies.

Objectives: There are three aims for this thesis: the first is to develop a field and laboratory protocol for the storage and analysis of transforming growth factor–beta 2 (TGF-β2) in human breastmilk; second, to validate this protocol and the immunoassay used to assess this new method; and lastly, to explore the ramifications of biosimilar TGF-β2 across multiple milks on human health, growth, and immunity through the review of laboratory findings and previous literature. Rational: Little anthropological research has been done on TGF-β2 in human milk. Anthropology as a discipline is well positioned to provide insight into TGF-β2, combining biocultural, evolutionary, and ecological approaches to holistically illustrate the effects this cytokine has on human immunity. This thesis provides an applied anthropological perspective and methodology on TGF-β2 in human milk. Methods: A protocol was developed for a new method of drying breastmilk on polystyrene microplates. Samples were then reconstituted using reagent diluent with 1% BSA and assayed using a Human TGF-beta 2 DuoSet enzyme-linked immunosorbent assay (ELIZA) assay kit from R&D Systems. Other mammalian milks and infant formula samples were also dried and tested for TGF-β2 concentrations. Validity of the assay and TGF-β2 concentrations were then statistically measured using linear regression analysis and Bland-Altman plots. Results: The results of the first objective in the development of a laboratory and field protocol for drying breastmilk on polystyrene plates for the extraction of TGF-β2 showed this method to hold promise for future application, but lacked statistical power in this study to confirm if this method is viable. The second objective of assay validation was unsuccessful, with the percent coefficient of variation for the intra-assay variation and inter-assay validation 38.28% and 17.70%, respectively indicating that this assay struggled to produce consistent and reliable results from the reconstituted samples. Results from the third objective suggest that biosimilar TGF-β2 in non-human milk can influence human growth and development, the extent of which, however, needs further study. Conclusions: Given these findings, more work with TGF-β2 in milk is required. TGF-β2 is a cytokine which could reveal a great deal about the developmental origins of human immunity and how it is maintained and altered across our life course and therefore an area of biology worth further research.

Epithelial ovarian cancer is the most lethal gynecologic malignancy, with a 5-year survival rate of less than 40%. This is due in part to a lack of early detection markers and lack of specific symptoms during early disease. The laying hen is the only accessible animal model which develops epithelial ovarian cancer spontaneously, with features closely resembling the human disease. It has been estimated that approximately 30% of all cancers can be prevented with diet, exercise, and maintenance of an optimal weight, and the chronic low-grade inflammation that accompanies obesity is implicated as a causal factor in the development of cancer. Flaxseed, a rich plant source of anti-inflammatory omega-3 fatty acids and lignans which act as phytoestrogens and antioxidants, exhibits chemosuppressive effects against the development and progression of ovarian cancer. We have shown that a diet of 10% flaxseed reduces the incidence and severity of ovarian cancer when fed to laying hens over 4 years, due in part to the ability of flaxseed to suppress the production of proinflammatory PGE2 in the ovary by decreasing expression of COX enzymes. To investigate other potential specific mechanisms by which flaxseed acts to suppress ovarian cancer, we examined expression and activity of pathways known to be involved in the etiology and progression of human epithelial ovarian cancer in ovarian cancer in the laying hen, and determined whether flaxseed affected these pathways during cancer development. We investigated the effect of flaxseed and its individual components upon oxidative stress in the normal ovary and in ovarian cancer by analyzing expression of target genes of the NRF2 transcription factor. The NRF2 pathway is a "master switch" that regulates expression of ROS-responsive detoxification genes. Results revealed that expression of four genes was significantly downregulated in then ovaries of hens on the defatted flaxmeal (DFM) and whole flaxseed (WF) diets compared to hens on diets that are high in pro-inflammatory omega-6 fatty acids, suggesting that flaxseed decreases oxidative stress in the ovary. Conversely, one target gene was upregulated in ovarian cancer compared to normal ovaries, and this observation was not affected by flaxseed. Additionally, nuclear accumulation Nrf2 protein was not observed in tumor cells, suggesting that flaxseed does not exert chemosuppressive effects by modulating NRF2 signaling in ovarian cancer. To further investigate pathways potentially regulated by flaxseed, we performed a microarray with 44k features and found that a set of genes involved in branching morphogenesis was upregulated in ovarian cancer and significantly decreased by flaxseed, including E-cadherin and miR-200, suggesting that flaxseed impedes the activity of an aberrantly activated developmental program that controls gland formation during ovarian cancer progression. Lack of nuclear accumulation of ZEB1 protein in tumor cells suggests that this decrease in expression is likely not due to EMT. Finally, due to its known roles in controlling developmental programs such as EMT as well as regulating cell growth and proliferation, we performed a set of experiments to examine activity of the TGF-beta pathway. PCR array analysis revealed that SMAD target genes, ligands, receptors, and co-regulatory proteins were upregulated in ovarian tumors from hens on both diet groups, suggesting TGF-beta signaling is enhanced in ovarian cancer. However, expression of SMAD6 and SMAD7 was upregulated in tumors from hens on the flaxseed diet but not control diet, with SMAD7 protein being expressed in both epithelial tumor cells and intratumoral stromal cells. Additionally, immunohistochemical staining for pSMAD2/3 was decreased in epithelial tumor cells and absent from intratumoral stromal cells in tumors from hens on the flaxseed diet compared to tumors from hens on the control diet, and these data together suggest that flaxseed may inhibit pro-oncogenic TGF-beta signaling in ovarian cancer. Finally, flaxseed prevents the downregulation of expression of p15 and the upregulation of CCNA and CCNE in ovarian tumors, suggesting that flaxseed may slow cell cycle progression. Data from these studies provides preliminary evidence that flaxseed exerts pleiotropic effects upon gene expression to negatively regulate pathways driving the progression of ovarian cancer, including aberrant TGF-beta signaling and glandular development. These studies provide groundwork for in vitro studies to test the specific effects of flaxseed upon proteins involved in TGF-beta signaling and upon the expansion of tumor epithelia.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2015-10-19T12:42:59Z No. of bitstreams: 1 Dissertação_ICS_ Patricia Seixas Auad Brandão.pdf: 3513376 bytes, checksum: 9371f172f7be035f0e53b69538f14768 (MD5) / Made available in DSpace on 2015-10-19T12:42:59Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ Patricia Seixas Auad Brandão.pdf: 3513376 bytes, checksum: 9371f172f7be035f0e53b69538f14768 (MD5) / A hepatite crônica C é um processo inflamatório persistente, de duração superior a seis meses e o seu tratamento é realizado com o uso de interferons associados à ribavirina. Fatores como o genótipo do vírus, a carga viral e características do hospedeiro estão envolvidos com o sucesso da terapia. Os fatores do hospedeiro, especialmente, os imunológicos e genéticos parecem ter um papel importante no resultado do tratamento da hepatite C. Algumas proteínas como a citocina TGF-β1 tem sido associada com as complicações decorrentes de produção excessiva de fibrose, associada com condições inflamatórias crônicas. Objetivo: O objetivo deste estudo foi avaliar a associação de polimorfismos genéticos da citocina TGF-β1 e o estágio da fibrose na hepatite crônica C. Métodos: Este é um estudo de caso-controle não pareado, onde foram avaliados pacientes monoinfectados com vírus da hepatite C (VHC) tratados com terapia combinada com interferon convencional e/ou peguilado associado a ribavirina, os quais foram divididos em dois grupos, de acordo com os estágios de fibrose: fibrose hepática leve a moderada < F2 e fibrose hepática avançada F3 e F4 e um grupo controle de uma amostra histórica de doadores de sangue. Sendo realizada a genotipagem da TGF-β1 pelo método PCR-SSP (Kit One Lambda). Resultados: Foram encontradas diferenças estatisticamente significantes entre o grupo de indivíduos com hepatite crônica C e doadores de sangue. Houve maior frequência do alelo G (p= 0,0021) nos indivíduos com hepatite crônica C como também observamos maior frequência do genótipo GG (p=0,0029) entre os indivíduos com hepatite crônica C. Conclusões: Os dados obtidos sugerem que os polimorfismos do gene TGFB1 nos códons 10 e 25 não estão associados com a fibrose hepática em indivíduos com hepatite crônica C e que o polimorfismo do gene TGFB1 no códon 25 tem maior frequência nos indivíduos com hepatite crônica C quando comparados com indivíduos sadios, podendo ser um marcador de susceptibilidade a infecção pela hepatite crônica C.

Submitted by Israel Vieira Neto (israel.vieiraneto@ufpe.br) on 2015-03-05T18:31:18Z No. of bitstreams: 2 Dissertação sergio ferreira.pdf: 495221 bytes, checksum: 34587eb69a01f6ac2b83ff92569f588d (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T18:31:18Z (GMT). No. of bitstreams: 2 Dissertação sergio ferreira.pdf: 495221 bytes, checksum: 34587eb69a01f6ac2b83ff92569f588d (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2012 / CAPES, CNPq / O câncer cervical (CC) é o segundo tipo de câncer mais comum a afetar mulheres em todo mundo. O Papillomavírus humano (HPV) é encontrado em 99% dos casos de CC e a infecção por esse vírus é considerado um fator de risco para o desenvolvimento do câncer. Muitos estudos tem demonstrado uma relação entre polimorfismos nos genes de citocinas e doenças infecciosas. Polimorfismos nos genes da Interleucina-6 (IL-6) e o Fator de Crescimento Transformador (TGF) β1, importantes mediadores do sistema imunológico, tem sido associados com níveis séricos elevados destas citocinas e no desenvolvimento de muitas doenças e tipos de cânceres. O objetivo desse estudo foi verificar se o SNP -174G/C do gene da IL-6 e T869C e G915C do gene do TGF-β1 estão relacionados com o desenvolvimento de Neoplasias Intraepiteliais Cervicais (NIC). 115 amostras de pacientes saudáveis e 115 de pacientes com lesões foram analisadas. As análises dos SNP foram realizadas através do sequenciamento automático de DNA utilizando o “MEGABACE 1000”. Os genótipos do polimorfismo -174G/C da IL-6 que possuem pelo menos um alelo C parecem estar envolvidos no desenvolvimento de NIC induzida pelo HPV (p=0.05232). Nenhuma diferença significativa foi encontrada entre as frequências alélicas e genotípicas dos polimorfismos da TGF-β1 nos dois grupos analisados. Além disso, polimorfismos nos genes da IL-6 e TGF-β1 não estão envolvidos na progressão do CIN. Este estudo sugere que o polimorfismo -174G/C do gene da IL-6 pode ser usado como um gene marcador da susceptibilidade a infecção pelo HPV, mas não como um marcador de progressão de NIC na população Pernambucana.

Abstract Dentin forms the hard tissue portion of the dentin-pulp complex, while the dental pulp is soft connective tissue that retains the vitality of the dentin. Odontoblasts form the outermost cell layer of pulp and play a central role during dentin formation by producing and mineralizing the dentin matrix. The understanding of the defensive reactions in the dentin-pulp complex is limited. Information about the transcriptome and proteome of pulp tissue and odontoblasts would facilitate understanding of their functions during health and disease. The aim of this study was to investigate the expression profiles of human pulp tissue and odontoblasts in vivo and in vitro using large-scale expression analysis methods. Also, the suitability of these methods in pulp biological research in vivo and in vitro was evaluated. cDNA microarray revealed only minor variation and 2-D electrophoresis combined with mass spectrometry revealed no differences between healthy and carious teeth pulp tissue in vivo. The effect of transforming growth factor β1 (TGF-β1) on pulp and odontoblasts was studied in vitro using oligonucleotide-based microarrays, and marked changes in the transcriptome were revealed, especially in the expression of chemokine- and cytokine-related genes. Transiently increased interleukin expression was confirmed at the protein level by antibody array. DNA microarray analysis of native pulp tissue and odontoblasts was used to search for potential odontoblast markers. Only one gene related to extracellular matrix organization and biogenesis, matrilin 4, and two expressed sequence tags (ESTs), which represent transcribed sequences encoding possibly unknown genes, were identified in odontoblasts but not in pulp. Analysis of mature native odontoblasts and cultured odontoblast-like cells by DNA microarray revealed a high similarity (84%) between native and cultured cells. Also, differential expression levels of selected neuronal proteins were observed and confirmed at the mRNA and protein levels. In conclusion, microarray is a powerful tool for pulp biology, especially for in vitro studies. TGF-β1 was revealed as a potent regulator of proinflammatory responses in the dentin–pulp complex. In addition, several potential odontoblast markers were identified by microarray, and the similarity of cultured odontoblast-like cells used in the study with native odontoblasts was confirmed.

Colorectal cancer (CRC) has a 5-year survival rate of 68% yet it still has a mortality rate of 50,000 per year. While CRC has a host of causes, one that stands out is TGFβ deficient signaling, which is disrupted in a majority of high-microsatellite-instability or inflammation-associated CRCs. Since TGFβ is a multifunctional cytokine, it has been elusive to determine whether its effect on cancer development is operating through inflammation, differentiation or developmental pathways. Additionally, it is now becoming apparent that a great number of CRC cases can be associated with and possibly caused by gut bacteria dysbiosis. Here, I present a metagenomic and metatranscriptomic study of the interactions between TGFβ deficient signaling, inflammatory signaling, and the microbiome in a CRC mouse model. TGFβ deficient mice have reduced amounts of Firmicutes as well as mRNA counts of a key butyrate enzyme. Lack of butyrate, as shown by previous literature, could be inhibiting apoptosis and promoting growth. Also, TGFβ deficient mice have increased mRNA counts of polyamine producing genes, which could act synergistically with butyrate reduction. I find that H. hepaticus inoculation, as a source of inflammatory signaling, affects another species, M. schaedleri, to produce pro- inflammatory lipopolysaccharides. Additionally, H. hepaticus itself has increased oxidative phosphorylation; reactive oxygen species from this process could be adding to cancer-promoting DNA damage. Taken together, TGFβ deficient signaling and H. hepaticus inoculation, disrupt enough pathways to cross the threshold of carcinogenicity in 40% of the mice in our study. The results of this study emphasize the importance of microbiome function and represent possible new avenues of treatment.

La voie de signalisation TGF-β joue un rôle biphasique durant la cancérogenèse. Mon laboratoire a identifié une nouvelle protéine inhibitrice de la voie TGF-β, WWP1. WWP1 est une E3 ubiquitine ligase qui induit la polyubiquitination et la dégradation du récepteur de type I au TGF-β. De plus, le gène WWP1 est amplifié dans une large proportion de cancers mammaires et prostatiques, suggérant que WWP1 pourrait jouer un rôle clé dans les processus de cancérogenèse liés au TGF-β. Mon projet de thèse était donc de caractériser la régulation de l’activité catalytique de WWP1 ainsi que son mécanisme d’action dans la cellule. Mes résultats montrent qu’à l’état basal, WWP1 est monoubiquitinée, son activité de polyubiquitination étant réduite par l’effet inhibiteur qu’exercent les domaines C2 et/ou WW sur son domaine HECT. En présence de substrats, la protéine WWP1 « s’ouvre » et peut alors induire la polyubiquitination et la dégradation de ses substrats. De plus, nous avons observé qu’un mutant de WWP1, détecté dans un cancer de la prostate, est incapable de s’autoréguler selon ce modèle. Il présente une plus forte activité ligase envers lui-même et ses substrats, ce qui entraîne une atténuation de la réponse cytostatique du TGF-β pouvant conférer une activité oncogénique à WWP1. De plus, nous avons identifié STARD13 comme un nouveau partenaire de WWP1. STARD13 est une protéine à activité RhoGAP, considérée comme un suppresseur de tumeur. Nous avons montré que STARD13 permet l’association de WWP1 avec la GTPase RhoA, entraînant ainsi la polyubiquitination et la dégradation de RhoA. De façon intéressante, le complexe WWP1/STARD13 est impliqué dans le remodelage de l’architecture du cytosquelette en dégradant préférentiellement la forme activée de RhoA. Ces résultats ont permis d’identifier un nouveau rôle de WWP1 qui pourrait jouer un rôle essentiel durant la migration des cellules cancéreuses lors du processus métastatique. La caractérisation de nouveaux mécanismes de régulation et d’action de WWP1 devrait permettre à terme d’identifier si WWP1 est un marqueur diagnostique dans le cancer et/ou une nouvelle cible thérapeutique pour le développement de médicaments anticancéreux. / The TGF-β pathway plays a biphasic role during cancerogenesis. My laboratory identified a new protein, WWP1, as a negative regulator of TGF-β signaling. WWP1 is an E3 ubiquitin ligase that triggers polyubiquitination and degradation of TGF-β type I receptor. A genomic amplification of WWP1 is found in a large portion of mammary and prostatic tumors, suggesting a key role for WWP1 during carcinogenesis related to TGF-β. My thesis project was to determine the regulation of the catalytic activity of WWP1 and a new molecular mechanism of action of WWP1 whose deregulation can be implicated in cancerogenesis. My results indicate that at steady states, WWP1 is monoubiquitinated, its polyubiquitination activity being silenced due to the inhibitory effects of C2 or/and WW domains on its Hect domain. In presence of substrates, WWP1 is « opened » and induces polyubiquitination and degradation of its substrates. Moreover, a WWP1 mutation found in prostate cancer disrupts this regulatory mechanism. It possesses an increased ligase activity towards itself and its substrates, which leads to the attenuation of TGF-β cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1. We also identified STARD13 as a novel WWP1 interacting partner. STARD13 has a RhoGAP activity, and is considered as a tumor suppressor. We have shown that STARD13 mediates the association of WWP1 with the GTPase RhoA, ultimately leading to RhoA polyubiquitination and degradation. Interestingly, the WWP1/STARD13 complex is involved in the actin cytoskeleton rearrangement by preferentially targeting the active form of RhoA for degradation. These results reveal a previously unrecognized role for WWP1, which could play a key role in the migration of cancer cells during metastasis. Characterization of new regulation and action mechanisms for WWP1 should allow identifying whether WWP1 is a diagnosis biomarker in cancer and/or a new therapeutic target for the development of anticancer drugs.

Intestinal inflammatory disorders such as Crohn’s disease contribute significantly to human mortality and morbidity. Although the cells and molecules involved in suppression of intestinal inflammation have been extensively documented in mouse models, a full understanding of how these work together in the healthy and diseased gut remains elusive. It is known, however, that tight regulation over TH17 cells and regulatory T cells (Treg) is required to maintain immune homeostasis in the intestine. Activation of the cytokine transforming growth factor-β (TGFβ), which is secreted by immune cells as an inactive complex, plays a crucial role in the induction of both Treg and TH17 cells. Recent work has shown that the αvβ8 integrin is required for activation of TGFβ by murine dendritic cells (DC). Murine integrin αvβ8 is thus of fundamental importance for Treg and TH17 induction and, subsequently, for intestinal immune homeostasis. However, little is known about the signals controlling the expression of integrin αvβ8 on intestinal DC. Furthermore, whether this system is also important for regulation of the human system is entirely unknown. Here, expression of integrin αvβ8 is shown on human intestinal CD1c+CD103+SIRPα+ DC and CD1c+CD103-SIRPα+ DC, but not on CD141+CD103+SIRPα- DC. Expression of integrin αvβ8 is increased on DC from Crohn’s disease patients, and on DC from non-IBD donors treated with LPS. Both LPS and a number of probiotic bacteria were also able to induce integrin αvβ8 expression on human monocyte-derived DC (moDC), which suggested that the microbiota may play a role in the regulation of human integrin αvβ8. Importantly, we have also shown that TGFβ is activated by integrin αvβ8 on human moDC, and that integrin αvβ8 promotes expression of forkhead box P3 (FOXP3) in naïve human T cells in vitro. Together, these data suggest that integrin αvβ8-mediated activation of TGFβ by DC may play an important role in the regulation of human T cell responses in the human intestine, and that this pathway may be perturbed during intestinal inflammation.

Este estudo in vitro teve como objetivo avaliar os efeitos moleculares do TGF-&beta;1, com diferentes períodos de suplementação, sobre a formação do fenótipo osteogênico das células MC3T3-E1, comparando-os com células tratadas com AA+&beta;-GP suplementados com Dex e/ou TGF-&beta;1, sem e com a neutralização dos receptores de TGF-&beta;1. A expressão gênica do próprio TGF-&beta;1 e Smad3 foram analisadas, bem como, a diferenciação das células osteogênicas e a biomineralização. As células tratadas com TGF-&beta;1 sem neutralização de receptores apresentam efeito inibitório nos estágios mais avançados da diferenciação dos osteoblastos e da biomineralização in vitro, mas expressarem alguns marcadores importantes envolvidos na mineralização. Observaram-se nódulos de mineral em todos os tratamentos das células que tiveram os receptores de TGF-&beta;1 neutralizados, mas houve uma diminuição na expressão de alguns genes. Os resultados confirmam a complexidade da via de sinalização do TGF-&beta;1, mostrando que existem lacunas para que seja entendido o mecanismo dessa molécula na biologia osteoblástica. / This in vitro study aimed to evaluate the molecular effects of TGF-&beta;1, with different supplementation time periods on the establishment of MC3T3-E1 cells, comparing with cells treated with AA+&beta;-GP supplemented with Dex and/or TGF-&beta;1, without or with neutralization of TGF-&beta;1 receptors. The gene expression of the TGF-&beta;1 and Smad3 were analyzed, as well as the osteoblast differentiation and biomineralization. The cells treated with TGF-&beta;1 without neutralization of receptors have had inhibitory effect on some important stages of osteoblast differentiation and biomineralization in vitro, but expressed some important mineralization markers. Mineral nodules were observed in all treatments of cells with their TGF-&beta;1 receptors neutralized, but there was a decrease in the expression of some important genes. The results confirm the complexity of the pathway signaling of TGF-&beta;1, showing that there are gaps for understand the mechanisms of this molecule in the biology of osteoblasts.