No / Despite promising early data, the natural product dolastatin 10 has not been successful as a single agent in phase II clinical trials. Herein the mechanism of action and efficacy of a synthetic analogue, auristatin PYE, was investigated in 2 human colon adenocarcinoma models, DLD-1 and COLO 205. In vivo efficacy was assessed in subcutaneous xenografts following intravenous administration. Mechanistic studies investigated effects of auristatin PYE on microtubule disruption using immunocytochemistry, whilst cell cycle effects were studied using flow cytometry. Possible effects on tumour functional blood vasculature were assessed in tumour-bearing mice. Auristatin PYE was less potent in vitro than dolastatin 10, but was significantly more effective (p<0.01) in vivo against both tumours. Significant effects on tumour blood vasculature were seen, with optimal shutdown at 6-h post-treatment. Extensive necrosis became more evident over time after treatment. Auristatin PYE caused severe disruption of normal microtubule structure at concentrations and times comparable with the IC50 data, and also instigated a G2/M cell cycle block. Auristatin PYE was more effective in the DLD-1 and COLO 205 models than dolastatin 10, with anti-tumour effects mediated through vascular shutdown. These data suggest that auristatin PYE has good potential as an anti-cancer agent.

As imagens de angiografia por ressonância magnética (angio-RM) ou por tomografia computadorizada (angio-TC) permitem uma análise minuciosa das redes vasculares. A segmentação de redes vasculares a partir de tais imagens é uma das tarefas iniciais no diagnóstico de doenças vasculares como estenoses ou aneurismas. Porém, a grande diversidade de arquiteturas dos vasos dificulta a validação dos algoritmos de segmentação. Assim, a construção de redes vasculares sintéticas realistas permitem validar novas metodologias de segmentação de vasos. Este trabalho descreve uma metodologia de geração de redes vasculares sintéticas em três dimensões utilizando sistemas de Lindenmayer (L-systems) estocásticos. Para atingir esse objetivo, foram implementados um analisador léxico, um analisador sintático e um gerador de L-systems para a criação de vasos sintéticos baseado em gramáticas. A parametrização destas gramáticas possibilita a simulação de características naturais de vasos reais como o ângulo de bifurcação, comprimento, diâmetro médio e possibilita a simulação de anomalias vasculares. As expressões resultantes são utilizadas para criar imagens angiográficas sintéticas que simulam a distribuição de intensidades dos vasos em imagens angio-RM e angio-TC reais. As redes vasculares sintéticas podem também ser delimitadas por superfícies 3D arbitrárias de forma similar à geometria de órgãos. A flexibilidade de parametrização e natureza estocástica desta metodologia faz com que ela se torne uma ferramenta ideal para a validação de algoritmos de segmentação de vasos em imagens angiográficas. / Magnetic resonance angiography (MRA) or computed tomography angiography (CTA) images allow for a thorough analysis of the blood vessels. Vessel segmentation from MRA or CTA is thus the primary task in the diagnosis of vascular diseases such as stenosis and aneurysms. The wide architectural variability of the blood vessels, however, hinders the validation of vascular segmentation methods. The construction of synthetic realistic vascular architecture trees will aid in the validation of new vessel segmentation methodologies. This thesis describes a three-dimensional synthetic blood vessel generation methodology that employs stochastic Lindenmayer systems (L-systems). For this purpose, we implemented a parser and a generator of L-systems to create grammars that represent blood vessel architectures. The parameterization of the grammar allows one to simulate natural features of real vessels such as bifurcation angle, average length and diameter, and also accounts for vascular anomalies. The resulting expressions are used to create synthetic angiographic images that mimic real vessel intensity distributions in MRA and CTA. Blood vessel growth can also be delimited by arbitrary 3D surfaces that may represent organ geometries. The flexibility in the parameterization and stochastic nature of this methodology makes it an ideal tool for the validation of blood vessel segmentation algorithms from angiographic images.

Le cil primaire est un organite présent à la surface de la plupart des cellules de Vertébrés. Il participe à l’organogénèse en régulant l’activité de voies de signalisation comme la voie Hedgehog. Une dysfonction du cil primaire mène à des maladies rares, sévères et pléiotropiques, les ciliopathies, qui peuvent inclure des défauts cardiaques. Cependant, le rôle que le cil primaire joue dans la morphogénèse cardiaque est encore mal compris. Le projet principal de la thèse porte sur l’étude du rôle du cil primaire des cellules cardiaques dans le développement du cœur. Dans ce but, nous avons utilisé un modèle murin de délétion conditionnelle de Ift20, un gène essentiel pour la ciliogénèse. La délétion est contrôlée par l’allèle Mesp1Cre exprimé dans la plupart des précurseurs précoces cardiaques. A la naissance, les mutants conditionnels présentent des défauts importants de septation des voies efférentes et des chambres cardiaques, les oreillettes et les ventricules. Ces défauts sont similaires à ceux caractérisés dans les mutants de la voie Hedgehog. Nous avons également identifié de nouveaux phénotypes associés à la suppression du cil. Les mutants présentent une augmentation significative de la taille du ventricule droit et des malformations du réseau de vascularisation coronaire. Pour mieux comprendre la cause des défauts de croissance observés à la naissance, nous avons analysé les comportements cellulaires sous-jacents. Aucune différence significative des taux de prolifération, de la taille et de la proportion des types cellulaires n’a été détectée au stade prénatal, suggérant que ces défauts ont une origine développementale plus précoce. Des expériences sont en cours pour déterminer les mécanismes moléculaires des défauts observés. Dans le cadre d’une collaboration avec le laboratoire de Julien Vermot, à Strasbourg, nous avons étudié le rôle du cil primaire dans le développement du proépicarde, un organe précurseur de l’épicarde du cœur mature. Nous avons montré que les embryons mutants Ift20 constitutifs présentent une augmentation significative du volume du proépicarde. Des analyses sont en cours pour identifier les voies de signalisation impliquées dans ce phénotype. Les travaux effectués durant ce projet de thèse ont permis de caractériser de nouveaux rôles du cil primaire dans le développement cardiaque. Nos résultats participent à une meilleure compréhension des ciliopathies et des défauts cardiaques qui leur sont associés. / The primary cilium is an organelle present at the surface of most of Vertebrate cells. It is involved in organogenesis by regulating signalling pathways such as Hedgehog signalling. Primary cilium dysfunction leads to severe, rare and pleiotropic diseases, ciliopathies, which can include cardiac defects. Howevr, the role that the primary cilia plays in cardiac morphogenesis is still poorly understood. The main project of the PhD focuses on the study of the role of primary cilia in cardiac cells during heart development. We have used a mouse mode of conditional deletion of Ift20, a gene essential for ciliogenesis. The deletion is controlled by the Mesp1Cre allele, expressed in the majority of cardiac precursors. At birth, conditional mutants display severe defects in septation of the outflow tract, the atria and the ventricles. These defects are similar to the ones characterized in Hedgehog signalling mutants. We also have identified novel phenotypes linked to cilium suppression. The mutants display a significant increase in the size of the right ventricle and defective coronary vasculature development. To better understand the growh defects observed at birth, we analysed the underlying cell behaviour. No significant differences in the proliferation rates, nor in the size and proportions of different cell types were detected at prenatal stages, suggesting that these defects have an earlier developmental origin. Experiments are underway to determine the molecular mechanisms of the observed defects. In collaboration with the laboratory of Julien Vermot, in Strasbourg, we studied the role of the primary cilium in the development of the proepicardium, a precursor organ of the mature epicardium. We have shown that Ift20 constitutive mutants show a significant increase in proepicardial volume. Analyses are ongoing to identify the signalling pathways involved in this phenotype. The works performed during this PhD project allowed the characterization of new roles for the primary cilium in cardiac development. Our results participate in a better understanding of ciliopathies and their associated cardiac defects.

The Euphorbiaceae are notable for floral and inflorescence diversity and evolutionary complexity. Croton, is the second largest genus in the family and exhibits particular diversity in its flowers, especially regarding perianth and number of stamens, besides the inflorescences, which are also very diverse. Considering Croton\'s great variability in the reproductive structures, the aim of this thesis was to study flowers and inflorescences with an evolutionary approach, including morphology, ontogeny, vasculature, auxin regulation and genetic expression. Flowers in several stages of development were analyzed using light microscopy and scanning electron microscopy. Inflorescences were analyzed in stereomicroscope and the traits were plotted on the most recent phylogeny of the genus. The genetic expression was tested using RNAseq. In the first chapter the flowers showed similarity in the initiation of sepals and the presence of filamentous, petaloid structures in Croton lundianus (Didr.) Müll. Arg., interpreted here as staminodes. In Croton sphaerogynus Baill., staminodes were described for the first time. The staminodes reported here could be interpreted as transitional structures that we considered as evolutionary reductions. In the second chapter, the staminate flowers showed polystemonous androecium and the delay in petals\' initiation and the antesepalous nectaries development interfered in the development of the stamens, characterizing obdiplostemony. Vasculature corroborated obdiplostemony and revealed a central stamen in C. fuscescens with carpelar features, interpreted here as a homeosis case. Glandular staminodes were registered and interpreted as a heterotopy case. The obdiplostemony may be related to modulation of the free IAA concentrations during floral developmental steps and Croton flowers can be used as good models for obdiplostemony, homeosis and heterotopy. In the third and fourth chapter we studied Croton inflorescences, which showed 17 patterns with differences on the organization and distribution of pistillate flowers. The inflorescence traits analyzed were very homoplastic, most likely determined by convergent evolution in distantly related lineages distributed in similar habitats. The genetic expression of C. fuscescens was particularly analyzed and the transcriptome showed that the different zones have their development guided through the same transcripts set. Each zone has different expression level and these variations and gradient could be interpreted as the boundary between each inflorescence zone. The floral developmental novelties and evolutionary links identified here raise the importance of future floral studies with the genus, what would bring a better understanding on how the reproductive structures evolved in the history of the group / Euphorbiaceae é uma família que recebe destaque quanto à diversidade de flores e inflorescências, além de sua complexidade evolutiva. Croton L. é o segundo maior gênero da família e exibe particular diversidade floral, em especial quanto a o perianto e número de estames, além das inflorescências, que também se apresentam muito diversas. Considerando a grande variação nas estruturas reprodutivas de Croton, o objetivo desta tese foi estudar as flores e inflorescências com abordagem evolutiva, incluindo morfologia, ontogênese, vascularização, regulação hormonal e expressão gênica. Flores em diversos estágios de desenvolvimento foram analisadas em microscopia der luz e varredura. Inflorescências foram estudadas em estereomicroscópio e os caracteres observados foram analisados nas filogenias mais recentes do grupo. A expressão gênica foi analisada com a técnica RNAseq. No primeiro capítulo as flores apresentaram semelhanças na iniciação das sépalas e presença de filamentos, estruturas petaloides em Croton lundianus (Didr.) Müll. Arg., interpretadas como estaminódios. Em Croton sphaerogynus Baill., estaminódios foram descritos pela primeira vez. Estas estruturas podem ser interpretadas como estruturas de transição evolutiva e reduções florais. No segundo capítulo as flores estaminadas apresentaram androceu polistêmone e o retardo na iniciação das pétalas e o desenvolvimento antessépalo dos nectários foram considerados como fatores chave para o desenvolvimento do androceu como obdiplostêmone. A vascularização corroborou a obdiplostemonia e revelou um estame central com características carpelares em C. fuscescens, interpretado aqui como um caso de homeose. Nectários glandulares foram registrados e interpretados como uma mudança heterotópica. A obdiplostemonia pode estar relacionada com as diferentes concentrações de auxina ao longo das etapas de desenvolvimento e as flores de Croton podem ser consideradas como bons modelos de obdiplostemonia, homeose e heterotopia. No terceiro e quarto capítulo nós investigamos as inflorescências de Croton, que apresentaram 17 padrões com diferenças na organização e distribuição das flores pistiladas especialmente. Os caracteres das inflorescências se mostraram homoplásticos e provavelmente determinados por evolução convergente em linhagens distantes distribuídas em habitats semelhantes. A expressão gênica de C. fuscescens foi particularmente analisada e o transcriptoma demonstrou que o desenvolvimento das diferentes zonas é regulado pelo mesmo conjunto gênico. Cada zona, pistilada ou estaminada, apresenta níveis distintos de expressão diferencial e o gradiente na expressão pode ser o delimitador entre as zonas. Os novos relatos quanto ao desenvolvimento floral em Croton e os links evolutivos identificados nesta tese levanta a importância de estudos para uma melhor compreensão sobre a evolução das estruturas reprodutivas neste grupo tão importante

Os objetivos deste estudo foram desenvolver um modelo experimental e avaliar o período bem como a viabilidade de retalhos cutâneos pré-fabricados com área cutânea de 100cm2 em parede abdominal lateral em coelhos. Os retalhos cutâneos foram confeccionados através da implantação direta de vasos gastroepiplóicos em arco com fluxo contínuo abaixo do subcutâneo da parede lateral em coelhos. Foram utilizados 18 animais da linhagem de coelhos New Zealand divididos em três Grupos: Grupo I (grupo controle), sem implantação de vasos gastroepiplóicos, e Grupo II e Grupo III, nos quais foram realizados os implantes de vasos gastroepiplóicos em arco no subcutâneo da parede abdominal lateral dos animais, os quais foram submetidos posteriormente a cirurgia para confecção de retalho axial, pediculado exclusivamente nos vasos gastroepiplóicos transpostos. O período entre a primeira cirurgia e a segunda foi de duas semanas no Grupo II e seis semanas no Grupo III. Quatorze dias após a segunda cirurgia os animais foram sacrificados e os retalhos avaliados. Nos animais do Grupo I, controle, foi observado 100% de área de necrose no tecido descolado, não preservando conexão vascular com o mesmo, e suturado novamente no leito original; nos animais do Grupo II foi observada área de necrose média de 56,83% e nos animais do Grupo III, ausência total de necrose nos retalhos. Amostras dos retalhos dos animais dos Grupos II e III foram avaliados histologicamente através de protocolo padrão para coloração por hamatoxilina-eosina e através de estudo imunohistoquímico para avaliação de viabilidade do tecido através da quantificação da atividade de divisão celular, considerando índice de células marcadas para o Antígeno Nuclear de Células em Ploriferação (iPCNA) como parâmetro. Foi observado aumento significante no iPCNA (p<0,01, teste bicaudal de Mann-Whitney) entre os Grupos II e III, com índice mais alto no Grupo III; a avaliação da coloração por hematoxilina-eosina confirmam a maior viabilidade dos retalhos do Grupo III. O estudo demonstra a possibilidade de criar e transferir um pedículo vascular em arco para o subcutâneo e, depois de certo tempo, confeccionar e transpor um retalho com dimensões consideráveis cuja circulação seja exclusiva deste novo pedículo. Este estudo traz informações adicionais para elaboração de retalhos pré-fabricados microcirúrgicos ou em ilha para o reparo de defeitos complexos que precisam de grandes áreas de cobertura cutânea e pedículos longos, com mínima morbidade à área doadora. / The aim of this study was to develop an experimental model and to evaluate the period as well as the viability of prefabricated cutaneous flaps with a cutaneous area of 100cm2 in lateral abdominal wall in rabbits. The cutaneous flaps were made through the direct implantation of gastroepiploic vessels in arch with flow-through below the subcutaneous of the lateral wall in rabbits. Eighteen animals of the New Zealand rabbits lineage were used divided in three Groups: Group I (control group), without implantation of gastroepiploic vessels, and Group II and Group III, which the implant of gastroepiploic vessels was accomplished in arch in the subcutaneous of the lateral abdominal wall of the animals. These animals were submitted to surgery later for making of axial flap, exclusively based in the transposed gastroepiploic vessels. The period between the first surgery and the second one was of two weeks in the Group II and six weeks in the Group III. Fourteen days after the second surgery the animals were sacrificed and the flaps evaluated. In Group I animals, control, it was observed 100% of necrosis area in the detached skin portion that was sutured again at the original bed, not preserving vascular connection with the local tissue; in the animals of the Group II an average necrotic area of 56,83% was observed and in the animals of the Group III, total absence of necrosis in the flaps. Samples of flaps tissue from the animals of the Groups II and III were evaluated through standard histological hamatoxilin-eosin protocols and imunohistochemical protocol for evaluation of tissue viability through the activity of cellular division, considering the index of Proliferating cell nuclear antigen (iPCNA) marked cells. It was observed significant increase on the iPCNA (p<0,01, two-tailed Mann-Whitney test) between the Groups II and III, with higher index in the Group III; the standard hematoxilin-eosin evaluation confirm the better viability of the flaps from the Group III. The study demonstrates the possibility to create and transfer an arch vascular pedicle to subcutaneous and, after certain time, to make and to transpose a flap with considerable dimensions which circulation comes from this new pedicle. This study brings additional information for elaboration of prefabricated microsurgical or island flaps for repair of complex defects that need great areas of skin covering and long pedicles, with low morbosity to the donor area.

Os objetivos deste estudo foram desenvolver um modelo experimental e avaliar o período bem como a viabilidade de retalhos cutâneos pré-fabricados com área cutânea de 100cm2 em parede abdominal lateral em coelhos. Os retalhos cutâneos foram confeccionados através da implantação direta de vasos gastroepiplóicos em arco com fluxo contínuo abaixo do subcutâneo da parede lateral em coelhos. Foram utilizados 18 animais da linhagem de coelhos New Zealand divididos em três Grupos: Grupo I (grupo controle), sem implantação de vasos gastroepiplóicos, e Grupo II e Grupo III, nos quais foram realizados os implantes de vasos gastroepiplóicos em arco no subcutâneo da parede abdominal lateral dos animais, os quais foram submetidos posteriormente a cirurgia para confecção de retalho axial, pediculado exclusivamente nos vasos gastroepiplóicos transpostos. O período entre a primeira cirurgia e a segunda foi de duas semanas no Grupo II e seis semanas no Grupo III. Quatorze dias após a segunda cirurgia os animais foram sacrificados e os retalhos avaliados. Nos animais do Grupo I, controle, foi observado 100% de área de necrose no tecido descolado, não preservando conexão vascular com o mesmo, e suturado novamente no leito original; nos animais do Grupo II foi observada área de necrose média de 56,83% e nos animais do Grupo III, ausência total de necrose nos retalhos. Amostras dos retalhos dos animais dos Grupos II e III foram avaliados histologicamente através de protocolo padrão para coloração por hamatoxilina-eosina e através de estudo imunohistoquímico para avaliação de viabilidade do tecido através da quantificação da atividade de divisão celular, considerando índice de células marcadas para o Antígeno Nuclear de Células em Ploriferação (iPCNA) como parâmetro. Foi observado aumento significante no iPCNA (p<0,01, teste bicaudal de Mann-Whitney) entre os Grupos II e III, com índice mais alto no Grupo III; a avaliação da coloração por hematoxilina-eosina confirmam a maior viabilidade dos retalhos do Grupo III. O estudo demonstra a possibilidade de criar e transferir um pedículo vascular em arco para o subcutâneo e, depois de certo tempo, confeccionar e transpor um retalho com dimensões consideráveis cuja circulação seja exclusiva deste novo pedículo. Este estudo traz informações adicionais para elaboração de retalhos pré-fabricados microcirúrgicos ou em ilha para o reparo de defeitos complexos que precisam de grandes áreas de cobertura cutânea e pedículos longos, com mínima morbidade à área doadora. / The aim of this study was to develop an experimental model and to evaluate the period as well as the viability of prefabricated cutaneous flaps with a cutaneous area of 100cm2 in lateral abdominal wall in rabbits. The cutaneous flaps were made through the direct implantation of gastroepiploic vessels in arch with flow-through below the subcutaneous of the lateral wall in rabbits. Eighteen animals of the New Zealand rabbits lineage were used divided in three Groups: Group I (control group), without implantation of gastroepiploic vessels, and Group II and Group III, which the implant of gastroepiploic vessels was accomplished in arch in the subcutaneous of the lateral abdominal wall of the animals. These animals were submitted to surgery later for making of axial flap, exclusively based in the transposed gastroepiploic vessels. The period between the first surgery and the second one was of two weeks in the Group II and six weeks in the Group III. Fourteen days after the second surgery the animals were sacrificed and the flaps evaluated. In Group I animals, control, it was observed 100% of necrosis area in the detached skin portion that was sutured again at the original bed, not preserving vascular connection with the local tissue; in the animals of the Group II an average necrotic area of 56,83% was observed and in the animals of the Group III, total absence of necrosis in the flaps. Samples of flaps tissue from the animals of the Groups II and III were evaluated through standard histological hamatoxilin-eosin protocols and imunohistochemical protocol for evaluation of tissue viability through the activity of cellular division, considering the index of Proliferating cell nuclear antigen (iPCNA) marked cells. It was observed significant increase on the iPCNA (p<0,01, two-tailed Mann-Whitney test) between the Groups II and III, with higher index in the Group III; the standard hematoxilin-eosin evaluation confirm the better viability of the flaps from the Group III. The study demonstrates the possibility to create and transfer an arch vascular pedicle to subcutaneous and, after certain time, to make and to transpose a flap with considerable dimensions which circulation comes from this new pedicle. This study brings additional information for elaboration of prefabricated microsurgical or island flaps for repair of complex defects that need great areas of skin covering and long pedicles, with low morbosity to the donor area.

BACKGROUND: Prostate cancer is the most common cancer among Swedish men. For patients with metastatic prostate cancer the standard therapy is castration, a treatment that initially provides symptomatic relief but unfortunately is not curative. New therapeutic targets for advanced prostate cancer are therefore needed.  Prostate cancers are composed of tumor epithelial cells as well as many non-epithelial cells such as cancer associated fibroblasts, blood vessels and inflammatory cells.  Many components of the tumor microenvironment such as tumor associated macrophages and angiogenesis have been shown to stimulate tumor progression. This thesis aims to explore mechanisms by which the local environment influences prostate tumor growth and how such mechanisms could be targeted for treatment. MATERIALS AND METHODS: We have used animal models of prostate cancer, in vitro cell culture systems and clinical materials from untreated prostate cancer patients with long follow up. Experiments were evaluated with stereological techniques, immunohistochemistry, western blotting, quantitative real-time PCR, PCR arrays and laser micro dissection. RESULTS: We found that the presence of a tumor induces adaptive changes in the surrounding non-malignant prostate tissue, and that androgen receptor negative prostate tumor cells respond to castration treatment with temporarily reduced growth when surrounded by normal castration-responsive prostate tissue. Further, we show that macrophages are important for prostate tumor growth and angiogenesis in the tumor and in the surrounding non-malignant tissue. In addition, the angiogenesis inhibitor Pigment epithelium-derived factor (PEDF) was found  to be down-regulated in metastatic rat and human prostate tumors. Over-expression of PEDF inhibited experimental prostate tumor growth, angiogenesis and metastatic growth and stimulated macrophage tumor infiltration and lymphangiogenesis. PEDF was found to be down-regulated by the prostate microenvironment and tumor necrosis factor (TNF) α. CONCLUSIONS: Our studies indicate that not only the nearby tumor microenvironment but also the surrounding non-malignant prostate tissue are important for prostate tumor growth. Both the tumor and the surrounding non-malignant prostate were characterized by increased angiogenesis and inflammatory cell infiltration. Targeting the surrounding prostate tissue with castration, targeting tumor associated macrophages, or targeting the vasculature directly using inhibitors like PEDF were all shown to repress prostate tumor growth and could prove beneficial for patients with advanced prostate cancer.

Radiation therapy is administered to more than 50% of patients diagnosed with cancer. Mechanisms of interaction between radiation and tumour cells are relatively well understood on a molecular level, but much remains uncertain regarding how radiation interacts with the tumour as a whole. Recent studies have suggested that tumour response to radiation may in fact be regulated by endothelial cell response, consequently stressing the role of tumour blood vessels in radiation treatment response. As a result, various treatment regimens have been proposed to strategically combine radiation with vascular targeting agents. A great deal of effort has been aimed towards developing efficient vascular targeting agents. Nonetheless, no optimal method has yet been devised to strategically deliver such agents. Recent evidence suggesting that these drugs may “normalize” tumour blood vessels and enhance radiosensitivity, is supporting experiments where anti-angiogenic drugs are combined with cytotoxic therapies such as radiotherapy. In contrast, ultrasound-stimulated microbubbles have recently been demonstrated to enhance radiation therapy by biophysically interacting with endothelial cells. When combined with single radiation doses, these microbubbles are believed to cause localized vascular destruction followed by tumour cell death. Finally, a new form of ‘pro-angiogenics’ has also been demonstrated to induce a therapeutic tumour response. The overall aim of this thesis is to study the role of tumour blood vessels in treatment responses to single-dose radiation therapy and to investigate radiation-based vascular targeting strategies. Using pharmacological and biophysical agents, blood vessels were altered to determine how they influence tumour cell death, clonogenicity, and tumour growth, and to study how these may be optimally combined with radiation. Three-dimensional high-frequency power Doppler ultrasound was used throughout these studies to investigate vascular response to therapy.

As imagens de angiografia por ressonância magnética (angio-RM) ou por tomografia computadorizada (angio-TC) permitem uma análise minuciosa das redes vasculares. A segmentação de redes vasculares a partir de tais imagens é uma das tarefas iniciais no diagnóstico de doenças vasculares como estenoses ou aneurismas. Porém, a grande diversidade de arquiteturas dos vasos dificulta a validação dos algoritmos de segmentação. Assim, a construção de redes vasculares sintéticas realistas permitem validar novas metodologias de segmentação de vasos. Este trabalho descreve uma metodologia de geração de redes vasculares sintéticas em três dimensões utilizando sistemas de Lindenmayer (L-systems) estocásticos. Para atingir esse objetivo, foram implementados um analisador léxico, um analisador sintático e um gerador de L-systems para a criação de vasos sintéticos baseado em gramáticas. A parametrização destas gramáticas possibilita a simulação de características naturais de vasos reais como o ângulo de bifurcação, comprimento, diâmetro médio e possibilita a simulação de anomalias vasculares. As expressões resultantes são utilizadas para criar imagens angiográficas sintéticas que simulam a distribuição de intensidades dos vasos em imagens angio-RM e angio-TC reais. As redes vasculares sintéticas podem também ser delimitadas por superfícies 3D arbitrárias de forma similar à geometria de órgãos. A flexibilidade de parametrização e natureza estocástica desta metodologia faz com que ela se torne uma ferramenta ideal para a validação de algoritmos de segmentação de vasos em imagens angiográficas. / Magnetic resonance angiography (MRA) or computed tomography angiography (CTA) images allow for a thorough analysis of the blood vessels. Vessel segmentation from MRA or CTA is thus the primary task in the diagnosis of vascular diseases such as stenosis and aneurysms. The wide architectural variability of the blood vessels, however, hinders the validation of vascular segmentation methods. The construction of synthetic realistic vascular architecture trees will aid in the validation of new vessel segmentation methodologies. This thesis describes a three-dimensional synthetic blood vessel generation methodology that employs stochastic Lindenmayer systems (L-systems). For this purpose, we implemented a parser and a generator of L-systems to create grammars that represent blood vessel architectures. The parameterization of the grammar allows one to simulate natural features of real vessels such as bifurcation angle, average length and diameter, and also accounts for vascular anomalies. The resulting expressions are used to create synthetic angiographic images that mimic real vessel intensity distributions in MRA and CTA. Blood vessel growth can also be delimited by arbitrary 3D surfaces that may represent organ geometries. The flexibility in the parameterization and stochastic nature of this methodology makes it an ideal tool for the validation of blood vessel segmentation algorithms from angiographic images.

The surfactant protein-G (SP-G) has recently been discovered in the brain and linked to fluid balance regulations. Stroke is characterized by impaired vessel integrity, promoting water influx and edema formation. The neurovascular unit concept (NVU) has been generated to cover not only ischemic affections of neurons or vessels but also other regionally associated cells. This study provides the first spatio-temporal characterization of SP-G and NVU elements after experimental stroke. Immunofluorescence labeling was applied to explore SP-G, vascular and cellular markers in mice (4, 24, and 72 h of ischemia), rats (24 h of ischemia), and sheep (two weeks of ischemia). Extravasated albumin indicated vascular damage within ischemic areas. Quantifications revealed decreasing SP-G signals in the ischemia-affected neocortex and subcortex. Inverse immunosignals of SP-G and vascular elements existed throughout all models. Despite local associations between SP-G and the vasculature, a definite co-localization was not seen. Along with a decreased SP- G-immunoreactivity in ischemic areas, signals originating from neurons, glial elements, and the extracellular matrix exhibited morphological alterations or changed intensities. Collectively, this study revealed regional alterations of SP-G, vascular, and non-vascular NVU elements after ischemia, and may thus stimulate the discussion about the role of SP-G during stroke.