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Ischaemia causes irreversible tissue damage in cardiovascular disease. Since regenerative angiogenesis fails to consistently induce sufficient reperfusion to facilitate repair, targeted manipulation of angiogenesis is clinically desirable. The Wilms' tumour suppressor (Wt1) is a transcription factor which regulates numerous genes and cellular processes, including many intrinsic to angiogenesis. We hypothesise that WT1 in the endothelium influences the angiogenic function of endothelial cells. WT1 was identified in endothelial and non-endothelial cells comprising vessel outgrowths generated by cultured aortic rings from WT1-GFP reporter mice. Inducible deletion of WT1 from the endothelium (VE-Wt1 KO) significantly delayed angiogenesis in this assay (p < 0.05 relative to controls). In vivo, WT1 expression was evident in vascular endothelial and perivascular cells of the hindlimb as early as 3 days following femoral artery ligation to induce ischaemia, often in cells expressing epithelial and mesenchymal markers simultaneously. However, VE-Wt1 KO had no effect on hindlimb reperfusion (laser Doppler; days 0-28) or on vessel density (day 28). Similarly, VE-Wt1 KO had no effect on vessel density or expression of angiogenic factors (qRT-PCR) in sponges inserted subcutaneously in mice (20 days). To further understand the role of WT1 in angiogenesis, transcriptomic RNA expression analysis was performed in WT1+ and WT1- cells isolated (FACs) from sponges after implantation in WT1-GFP mice. WT1+ cells exhibited higher expression of genes involved in a number of processes relevant to tissue repair, including angiogenesis (p=3.11x10-8), wound healing (p=3.45x10-7) and epithelial-to-mesenchymal transition (EMT) (p=5.86x10-4). These results shed new light on the role of WT1 in ischaemic angiogenesis. In concurrence with previously published work, we show that deletion of endothelial WT1 can delay angiogenesis however, WT1 is not just instrumental in endothelial cells in this context. WT1 has a broader role in tissue repair in ischaemia, in part through regulation of cell transition (EMT). This work has improved our understanding of the regulatory role of WT1 in angiogenesis and repair, while revealing a number of novel insights into the function of WT1. This highlights WT1 as a potentially beneficial therapeutic target to facilitate regeneration in cardiovascular disease.

HACE1 is a tumour suppressor gene located at human chromosome 6q21. HACE1 is downregulated in Wilms’ tumour as well as several other human cancers. Its role in normal development remains unknown. The zebrafish has established itself as a robust model for studying vertebrate development and human cancers. A zebrafish hace1 homologue has been identified. Whole mount in situ hybridization (WISH) assays and colocalization studies demonstrate conserved hace1 expression. Moreover, morpholino knockdown of hace1 reveals perturbed cardiac development and function. Transgenic zebrafish harboring either wild type or dominant negative mutated C876S (C876S DN) human HACE1 genes have been generated. DN zebrafish display increased apoptosis, both untreated and following irradiation-induced cellular damage. There was no difference in cell cycle progression between wild type embryos and C876S DN. Further characterization of the HACE1 transgenic zebrafish model will serve to better our understanding of the role of human HACE1 in normal development and tumourigenesis.

In 1899, the German surgeon Max Wilms hypothesised that different cell types in a variety of childhood kidney cancers were all derived from the mesodermal layer during embryonic development. Nearly a century later, the WT1 gene was identified on the short arm of chromosome 11, and was thought to be inactive in ~20% of nephroblastomas (Wilms’ tumours). The expression of WT1 after birth appears to be restricted to a finite number of tissues, namely, the glomerular podocytes, mesothelium and ~1% of bone marrow cells. Emerging evidence suggests WT1 is required not only for development, but also for tissue homeostasis, regeneration, repair and angiogenesis. Interestingly, WT1 has been implicated in the response to myocardial infarction and tumour angiogenesis, yet its precise role remains unclear. This thesis aims to address the hypothesis that activation of the WT1 gene in the vascular endothelium is essential for physiological and pathophysiological angiogenesis in the adult. In order to assess whether Wt1 was expressed in quiescent endothelial cells (ECs) immunofluorescence was used to analyse a variety of tissues in the adult mouse. Whilst Wt1 was detected in renal podocytes, no endothelial Wt1 expression was discovered in the lung, heart, kidney, spleen and gastrocnemius muscle. In contrast, tissues known to undergo physiological angiogenesis (endometrium and breast) did exhibit Wt1 expression in the vascular endothelium. Moreover, tubular EC outgrowths generated by aortic rings embedded in collagen ex vivo were positive for Wt1. The role of Wt1 in ischaemic angiogenesis was assessed using models of hind-limb and coronary ischaemia in the mouse. Wt1 was detected in ECs and non-vascular cells following ischaemic injury by a combination of immunofluorescence and qualitative real-time polymerase chain reaction (qRT-PCR). Using a time course analysis of these experimental models the chronology of this relationship was demonstrated, alongside the association with key angiogenic factors, such as Vegf. Given the findings in ischaemic tissue the C3(1)/Tag transgenic mammary cancer model was used to test the hypothesis that Wt1 would be upregulated in the tumour vasculature. Endothelial Wt1 was up regulated in these tumours compared to healthy control tissue. This finding was mirrored in a sub-set of aggressive breast cancers, confirming that the results obtained in mice can be translated to humans. Quantitative PCR revealed no association between histopathological grade of the tumours, oestrogen receptor status, and WT1 expression. In order to delineate the cell types involved in vessel formation, Wt1+ cells were sorted using fluorescent activated cell sorting (FACS) from transgenic mice with a green fluorescent protein knocked into the Wt1 locus following sponge implantation. Distinct sub-populations of Wt1+ cells were identified, some of which expressed EC and pericyte markers. Moreover, these Wt1+ sub-populations changed in composition and number over time. These findings were confirmed by genetic fate mapping of Wt1+ cells in this model. Finally, a conditional knockout mouse was generated to allow the selective deletion of Wt1 from vascular ECs in the sponge model of angiogenesis. The results demonstrated that deletion of Wt1 from this cellular compartment led to a dramatic reduction in vessel formation supporting a potential role in regulating angiogenesis. These results support the hypothesis that expression of WT1 in the vascular endothelium contributes to the regulation of angiogenesis in tumours and ischaemic tissue, and provides evidence that selective deletion of the gene inhibits new vessel formation. This suggests that targeting WT1 may have a therapeutic benefit in cancer and could aid regeneration of ischaemic tissues following injury in conditions such as myocardial infarction and critical limb ischaemia.

Thesis--Köln. / Cover title: Johann Wilhelm Wilms und das Amsterdamer Musik leben (1772-1847). Includes bibliographical references (p. 169-181) and index.

Orientadores: Laurecir Gomes, Maria Tereza Cartaxo Muniz / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T03:40:26Z (GMT). No. of bitstreams: 1 Arruda_IzabelBarrosde_D.pdf: 1365851 bytes, checksum: 2a1a28a0a33d73a75e48051e8b746627 (MD5) Previous issue date: 2014 / Resumo: O tumor de Wilms é uma neoplasia embrionária, originada de células do blastema metanéfrico, sendo histologicamente caracterizado como tumor trifásico, pois as células blastematosas, estromais e epiteliais estão presentes em proporções variáveis, com grande diversidade de arranjo celular e graus de diferenciação com ou sem anaplasia. Este tumor está associado a síndromes genéticas e a malformações do trato geniturinário. O objetivo desta tese foi avaliar as alterações nos genes WT1 e WT2 e a relação do índice apoptótico com o estadiamento tumoral. Para investigação das alterações nos genes WT1 e WT2 foi utilizada o método FISH, a imunorreatividade das proteínas WT1, p53 e MMP-2 foi empregada imunoistoquímica e para detectar células em apoptose, utilizou-se o método TUNEL. Os resultados evidenciaram cópias extras dos genes WT1 e WT2, com citogenética molecular nuc ish (WT1x3),(WT2x3) [100%], sugerindo que a evidencia da cópia-extra do gene WT2 pode ser explicada por trissomia ou por isodissomia uniparental, porém a presença da cópia-extra do WT1, até o momento, não foi relatada em qualquer caso clínico na literatura, sendo necessários mais estudos que a justifiquem. Por imunohistoquímica, constatou-se intensa imunomarcação no tumor para WT1 (80%), p53 (96,4%), assim como no estroma e ao redor do tumor, para MMP-2 (97%). Possivelmente essa alta expressão de WT1 pode estar refletindo a perda da regulação desse gene no desenvolvimento normal celular. A detecção da p53 neste estudo pode indicar a mutação do gene TP53, sugerindo que os tumores de Wilms poderiam progredir para anaplasia, após adquirem mutação do TP53, porém não como um evento isolado. Outros eventos seriam necessários para esta progressão, como a perda do alelo normal do TP53. A alta expressão da MMP-2 no estroma e ao redor do tumor não implica em sua atividade proteolítica, já que essa proteína pode estar em sua forma latente como também ativa e a técnica imunohistoquímica utilizada neste trabalho não diferencia essas formas. Não se identificou diferença significativa do índice apoptótico segundo estadio tumoral (p=0,937), mas os tumores em estadio III apresentaram índice seis vezes menor o daqueles em estadio II, em ausência de neoadjuvância, proporção que aumentou para 29 vezes, na presença de quimioterapia neoadjuvante, parecendo indicar que a neoadjuvância pode interferir sobre os mecanismos envolvidos na apoptose / Abstract: Wilms' tumor is an embryonic neoplasm arising from cells of the metanephric blastema, being histologically characterized as three-phase tumor, because blastematosas, stromal and epithelial cells are present in varying proportions, with great diversity of cell arrangement and degree of differentiation with or without anaplasia. This tumor is associated with genetic syndromes and malformations of the genitourinary tract. The aim of this thesis was to evaluate changes in WT1 and WT2 genes and the ratio of apoptotic cells to tumor staging. For investigation of changes in both WT1 and WT2 genes, the FISH method was used for immunoreactivity of the WT1 p53 and MMP-2 protein was employed and immunohistochemistry to detect cells undergoing apoptosis, we used the TUNEL. The results showed extra copies of WT1 and WT2 genes, with molecular cytogenetic nuc ish (WT1x3), (WT2x3) [100%], suggesting that the evidence of extra copy of WT2 gene can be explained by trisomy or uniparental isodisomy, but the presence of extra copy of WT1, so far, has not been reported in any clinical case in the literature, being necessary more studies to justify it. By immunohistochemistry, intense immunostaining was found in the tumor WT1 (80%), p53 (96.4%) as well as in the stroma surrounding the tumor, MMP-2 (97%). Possibly this high expression of WT1 may reflect the loss of regulation of this gene in the normal cell development. The detection of p53 in this study may indicate the mutation of the TP53 gene, suggesting that the Wilms' tumors may progress to anaplasia, after acquiring mutation of TP53, but not as an isolated event. Other events are needed for this progression, as loss of the normal allele of TP53. The high expression of MMP-2 in the stroma and around the tumor does not imply its proteolytic activity, since this protein may be at its latent form as well as active and the immunohistochemical technique used in this study does not differentiate these forms. There was no significant difference in the apoptotic index according to the tumor stage (p = 0.937), but in stage III tumors showed rate six times smaller than those in the stage II, in the absence of neoadjuvant therapy, rising to 29 times in the presence of chemotherapy neoadjuvant, seeming to indicate that neoadjuvant therapy may interfere with the mechanisms involved in apoptosis / Doutorado / Biologia Celular / Doutora em Biologia Celular e Estrutural

La tumeur de Wilms est la tumeur du rein la plus courante chez les enfants. Actuellement, près de 80 % à 90 % des patients survivent, mais quelques patients ne répondront pas aux traitements ou auront une rechute. L’objectif de ce projet est de caractériser la nouvelle mutation P904L dans le gène DNMT3A retrouvée chez un patient atteint de la tumeur de Wilms qui était en rechute. L’impact de cette nouvelle mutation est encore inconnu, mais des mutations dans le gène de DNMT3A sont fréquemment retrouvées dans le cancer. Le contexte de la mutation dans le patient et sa récurrence dans d’autres cancers et syndrome nous portent à croire que cette mutation affecte la fonction de DNMT3A. La perte de fonction de DNMT3A ainsi que certains de ses mutants sont connus pour altérer des caractéristiques du cancer comme la différenciation et l’immortalisation des cellules. On sait aussi que les tumeurs de Wilms ont des cellules qui gardent un aspect non différencié ou embryonnaire. Nous supposons que l’impact de la mutation P904L sur DNMT3A contribue au développement de la tumeur de Wilms. Pour démontrer l’impact de cette nouvelle mutation sur la protéine et sur le développement tumoral, nous avons utilisé à la fois des tests fonctionnels classiques et le séquençage de nouvelle génération. Puisque DNMT3A est un gène qui affecte la méthylation de l’ADN et régule l’expression, nous avons évalué le profil d’expression de la mutation P904L pour voir son impact sur la fonction du gène DNMT3A. Nos travaux démontrent que la nouvelle mutation P904L cause une perte de fonction de l’enzyme DNMT3A. Cette mutation altère le renouvellement cellulaire et la migration en plus de moduler la réponse à des agents thérapeutiques. Nous avons aussi constaté que la mutation module l’expression génique et que cette modulation est cohérente avec le profil d’expression du patient. En conclusion, nous suggérons trois mécanismes par lesquels le mutant contribue à la progression et au développement de la tumeur de Wilms. Nous démontrons aussi la nécessité d’approfondir nos connaissances sur cette tumeur afin de pouvoir proposer de nouvelles options thérapeutiques aux patients en rechute ou qui ne répondent pas aux traitements classiques. / Wilms tumours are the most common kidney tumour in children. As of now, almost 80 % to 90 % of the patients survive but there is still some who do not respond. This project objective is to study the novel mutation P904L in the DNMT3A gene discovered in a relapse Wilms tumour’s patient. The impact of this variant is unknown, but mutations in DNMT3A are frequently found in cancer. The context of the mutation in the patient and the recurrence of this mutation in other cancers and syndrome make us believe that it is affecting the function of the DNMT3A protein. Loss of function and some mutations of DNMT3A are known to affect crucial characteristics of cancers such as differentiation and immortalization. It is also known that Wilms tumours are made of undifferentiated or embryonic looking cells. We supposed that the impact of the mutation on DNMT3A protein contribute to the development of the tumour. To prove that this new mutation is affecting the protein and the development of the tumour, we used both functional assays and next generation sequencing technologies. Because DNMT3A is a gene affecting the methylation of the DNA and thus regulating gene expression, we used expression profile to assess the impact of the mutation on the enzyme DNMT3A. We demonstrate that the new mutation P904L causes a loss of function of the DNMT3A protein. This mutation affects the self-renew and the migration of the cells. Moreover, it modulates the response to drugs. We also found that the mutation modulates the gene expression in the cell line and this modulation is coherent with the expression pattern of the patient. In conclusion, we suggest three mechanisms by which this new mutant contributes to the development and progression of Wilms tumours. We also show that there is a need to further our knowledge of this tumour in order to propose new therapeutics options to non-responsive patient.

Made available in DSpace on 2013-08-07T19:04:26Z (GMT). No. of bitstreams: 1 000444782-Texto+Completo-0.pdf: 1327290 bytes, checksum: 51c0bcf62b76809d6ed98fd7aca7d6b9 (MD5) Previous issue date: 2011 / The Wilms Tumors (WT) is the most frequent renal tumors of children, and although curable, fatal outcomes may occur. A number a genetic alterations have been suggested as associated factors but still, the exact pathogenesis of WT remains to be fully characterized. Tissue factor (TF) is a glycoprotein which happens to be a key receptor for factor VII/VIIa and is the primary initiator of blood coagulation. Also important, TF has been associated with processes that lead to angiogenesis. It is widely expressed among cells and tissues and recent evidences pointed out an important role for TF in cancer progression and metastasis. Recent evidences suggested that TF may have a role in WT since its immunodetection was associated with poor prognosis. In the present investigation the differential expression of TF was assessed in WT using real-time PCR of RNA retrieved from paraffin sections using microdissection. Different histological components of WT - (blastema, epithelial and stromal) were analysed and the results revealed that TF in blastema and epithelial components was upregulated (14. 38 and 16. 02-fold respectively, P<0. 001). Stroma and control non neoplasic tissues expressed similar levels of expression (P>0. 05). TF expression in metastatic lesions from WT was also singificantly upregulated compared to non metastatic lesions. Microvessel density was positively correlated with TF expression (r=0. 721). As described for other tumors, TF seems to play a role in malignancy or WT. Further investigations are warranted to better understand the pathways by which TF exerts its effects on tumor progression. Noteworthy, pharmacological strategies that aim at controlling angiogenesis through regulation of TF may be very promising. / Esta pesquisa teve como objetivo demonstrar a expressão diferencial do fator tecidual (FT) em tumor de Wilms (TW), através de um estudo do tipo transversal. O TW é a neoplasia renal maligna mais comum na infância. Os estudos sobre angiogênese em neoplasias malignas pediátricas apresentam possíveis vias de terapias antiangiogênicas, com menor agressividade e melhor especificidade tumoral. E, entre os fatores angiogênicos possíveis, foi estudo o Fator Tecidual (FT), proteína transmebrana com sua principal função no processo de hemostasia, mas que demonstra ter importante papel nos processos patológicos de tumorigênese, angiogênese e microambiente tumoral favorável à disseminação neoplásica. Sua expressão vem sendo associada às metástases e piora no prognóstico. Contuto, só a partir da pesquisa de Maciel et al (1) que a associação do FT com TW foi observada, onde se encontrou, utilizando imunoistoquímica, a correlação positiva entre a expressão do FT, recidiva tumoral e óbito. Então, a partir desses dados iniciais, a presente pesquisa analisou uma amostra com 27 espécimes fixadas em parafina de TW e 26 controles (área sem TW), para demonstrar a expressão diferencial do FT em TW, através da técnica de quantificação de ácidos nucléicos (RNAm) por Reação Cadeia de DNA Polimerase em Tempo Real (RT-PCR), avaliar a expressão do FT em diferentes componentes tumorais, com a densidade microvascular (DMV) do TW e a ocorrência de metástases. Foi observado aumento da expressão diferencial do FT no TW, sua maior variação do FT foi encontrada nos componentes blastematoso e epitelial, enquanto no componente estromal apresentou variação mínima em relação ao tecido não tumoral, em lesões metastáticas o FT se mostrou significativamente mais elevado, sugerindo um papel importante para essa proteína no processo de disseminação dessas células malignas, o aumento da DMV apresentou associação positiva com a expressão do FT. Os resultados apresentados corroboram os achados de Maciel et al (1) de forma mais concisa e quantitativa, enfatizando a importância do FT na biologia do TW.

Made available in DSpace on 2014-06-12T23:13:20Z (GMT). No. of bitstreams: 2 arquivo1478_1.pdf: 1125386 bytes, checksum: c5bf67313992d932cf87971da2d0851e (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Universidade Federal de Pernambuco / Métodos complementares de diagnóstico por imagem são ferramentas de apoio à prática clínica, sendo utilizados em contextos variados, o que inclui tanto o diagnóstico de doenças, como sua avaliação. São métodos desenvolvidos por engenheiros e físicos, que procuram melhorar aspectos da qualidade da imagem, tempo de exame, segurança para técnicos e usuários. Sua aplicação clínica, entretanto, é terreno de outra área de pesquisa, a de saúde. Estabelecer os parâmetros que separam doença de variações do normal é uma das dificuldades em pesquisas na área, em parte pela importância da padronização dos critérios de interpretação. Este trabalho procurou avaliar as dificuldades encontradas nos estudos que definem parâmetros em dois cenários clínicos distintos, sendo uma neoplasia e a outra uma doença caracterizada por má digestão/absorção. Os fundamentos da formação e interpretação dos exames de imagem são apresentados em uma revisão da literatura, constando de três artigos, com ênfase nos aspectos associados à correlação das representações gráficas obtidas com os conhecimentos de fisiopatologia sobre as doenças. Foram desenvolvidos dois estudos empíricos avaliando os parâmetros de detecção ultrassonográfica abdominal da doença celíaca e de estadiamento local de tumor de Wilms por tomografia computadorizada. Os resultados mostraram que poucos parâmetros ultrassonográficos abdominais foram associados à doença celíaca, sendo relacionados à má digestão/absorção e alterações mucosas, como aumento do peristaltismo, do conteúdo líquido, do calibre e da espessura parietal das alças intestinais. O processo inflamatório não teve expressão na aparência dos linfonodos abdominais. No segundo estudo, a tomografia computadorizada em pacientes com tumor de Wilms mostrou baixas especificidade e valor preditivo positivo para detecção de disseminação ganglionar. A ausência de gânglios detectáveis e de sinais de invasão local torna improvável a sua presença. Nos dois trabalhos, a definição de parâmetros de fácil reprodução foi difícil. Baseando-se nas discussões apresentadas, verifica-se que, embora as pesquisas básicas que originam os métodos de diagnóstico por imagem sejam apoiadas na física, química e biologia, sua aplicação final é na área clínica. As pesquisas que envolvem esta aplicação devem seguir padrões próprios para este fim. Por serem representações gráficas do corpo humano, devem ser apoiadas pela fisiopatologia. Mesmo diante do conhecimento de que parâmetros definidos de interpretação dos exames são necessários, sua aplicação muitas vezes é difícil, em função do caráter subjetivo dos aspectos de imagem. Especificamente em relação aos resultados dos estudos empíricos, os achados ultrassonográficos associados à má digestão/absorção podem ser úteis na sugestão da doença celíaca, sendo necessários estudos com maior número de pacientes para avaliar melhor outros parâmetros. O estadiamento ganglionar e local do tumor de Wilms por tomografia computadorizada apresentou melhor desempenho na exclusão de comprometimento linfonodal e invasão de órgãos adjacentes

The notochord is the defining structure of all chordates. A semi-­‐flexible elongated tube of cells, it forms along the central axis of the embryo and provides axial support during development. It also acts as a signalling centre during early embryogenesis, controlling the patterning of a number of tissues and establishing the early body axis of the embryo. In vertebrates, the function of the notochord expands beyond early development. It creates morphogenic gradients for the patterned formation of the vertebral bodies and, in adults, the remnants of the notochord form the nucleus pulposus, a gel-­‐like structure with an integral role in the distribution of vertebral pressure in the intervertebral disc. Little is known about how the notochord copes with damage during embryogenesis, but degeneration of the nucleus pulposus can lead to debilitating spinal disorders. In this thesis, I use a zebrafish model system to present new data that describes the cellular behaviours associated with how the notochord copes with external damage and how this damage can influence the future development of the vertebrae. I have uncovered a novel damage response in the notochord of zebrafish larvae and characterised the morphogenetic changes involved in the process using transgenic fluorescent lines. I have explored the damage in the context of the Wilms’ Tumour 1 (Wt1) gene, a vertebrate-­‐conserved transcription factor, which has recently been associated with several regenerative responses, and discovered that one of its zebrafish orthologues, wt1b, becomes upregulated in the notochord damage response. I have used fluorescent confocal imaging and immunohistochemistry to present new evidence that shows that upon injury, the outer notochord sheath cells upregulate the expression of wt1b. Additionally, I have used time-­‐lapse microscopy to show that damage to the notochord induces novel morphological changes in the injured organ, which include the loss of cellularity of the inner vacuolated cells and the movement of the wt1b-­‐positive outer sheath cells into the injured lumen. Long-­‐term imaging experiments have also demonstrated the capacity of the notochord to heal the damage over time, which ultimately leads to the formation of an extra, smaller vertebra in the wounded area. Skeletal staining of these fish has revealed a previously unknown putative cartilage switch at the site of damage, which leads to the formation of the new vertebral body. This finding has been supported by the microarray analysis of the injured area, which shows the unexpected de-­‐novo expression of cartilage markers at the site of damage The work in this thesis identifies for the first time an endogenous repair mechanism in the notochord of zebrafish larvae and describes the cellular, genetic and molecular processes cotrolling this novel wt1b-­‐associated damage response.