Modulation of lung development by In utero gene transfer

Tese de doutoramento em Ciências da Saúde (ramo de conhecimento em Ciências Biológicas e Biomédicas)Advances in prenatal diagnosis of genetic and congenital disorders with progressively more sensitive techniques may increase opportunities for consideration of prenatal gene therapy. There are a number of genetic and acquired disorders with peri or postnatal pulmonary manifestations. These include monogenetic diseases like cystic fibrosis or surfactant protein B deficiency that would presumably require long-term expression of the deficient or defective gene. However, there are also abnormalities of lung growth, such as congenital diaphragmatic hernia, or lung maturation, such as respiratory distress syndrome of prematurity, that could potentially benefit from strategies that achieve transient gene expression in specific pulmonary distributions. Considered an attractive target organ for fetal gene transfer, the developing lung, poses also some obstacles that would only be overcomed with the development of a variety of gene transfer methodologies: different types of vector, optimal site, route and timing of gene delivery. The fundament of this dissertation was to modulate lung growth/maturation by in utero gene transfer, aiming to unveil underlying mechanisms of normal and abnormal lung development. The first objective of this dissertation was to develop a new approach of gene transfer targeting the fetal lung in early stages of lung development. We developed a new method for direct injection of viral vectors into the rat fetal lung as early as the pseudoglandular phase of lung development using ultrasound guided microinjections. The pseudoglandular stage, characterized by intense branching morphogenesis, is the period of greatest overall growth of the airways and vasculature of the fetal lung and corresponds to a stage of immunologic immaturity. Therefore, gene transfer during this period has the potential to have major effects on key elements of lung growth with minimal potential for detrimental immune responses. We aimed to compare two distinct types of vectors: an adenoviral vector and a lentiviral vector (equine infectious anemia virus-based), both expressing the enhanced green fluorescent protein reporter gene. This study confirmed that adenoviral vectors are more suitable when rapid, high-level and transient expression of the transgene is required; whereas lentiviral vectors are more appropriate to induce sustained and long-term expression. One of the concerns in gene transfer protocols is to target a specific cellular compartment of a determined organ/system. Interestingly, interstitial compartment rather than epithelial cells were transduced in opposition to previous studies describing intrapulmonary, intraamniotic and intratracheal administrations of viral vectors. The observation of transduction of distinct cell populations within the lung with different routes of transduction raises the possibility of manipulating gene expression in specific and separate cell populations within the developing lung. We then decided to use this model system to perform an in vivo study of dynamic lung morphogenesis, involving a major player in branching morphogenesis, fibroblast growth factor 10 (FGF10). We observed that FGF10 mesenchymal overexpression, on the fetal rat lung, resulted in the induction of consistent patterns of malformation, the appearance of which were developmental stage and location dependent. These malformations, in total, appear to closely recapitulate the morphology and histology of the entire spectrum of human Congenital Cystic Adenomatoid Malformation (CCAM).Os avanços no diagnóstico pré-natal de patologias genéticas e congénitas devido a técnicas progressivamente mais sensíveis, aumenta as oportunidades de aplicação de terapias génicas prénatais. Existem várias patologias pulmonares genéticas e adquiridas que se manifestam no período peri e pós-natal. Estas incluem doenças monogenéticas como a Fibrose Cística ou a Deficiência em Proteína Surfactante B, que requerem expressão a longo-termo do gene em causa. No entanto, existem também patologias relacionadas com crescimento pulmonar, como a Hérnia Diafragmática Congénita, ou maturação pulmonar, como o Síndrome Prematuro de Distress Respiratório, que podem beneficiar de estratégias de indução de expressão genica de forma transiente. Considerado um aliciante órgão-alvo para a terapia génica fetal, o pulmão fetal, coloca alguns obstáculos que só poderão ser ultrapassados com o desenvolvimento de várias metodologias: tipos de vectores, optimização de local, via e período de transferência génica. O fundamento desta dissertação foi modular crescimento/maturação pulmonar através de transferência génica in utero, pretendendo esclarecer os mecanismos moleculares reguladores no desenvolvimento pulmonar normal e anormal. O primeiro objectivo desta dissertação foi desenvolver uma nova abordagem de transferência génica para o pulmão nas fases inicias do seu desenvolvimento. Desenvolveu-se um novo método de injecção directa de vectores víricos no pulmão fetal de rato, durante a fase pseudoglandular, utilizando microinjecções guiadas por ultrasonografia. A fase pseudoglandular, caracterizada por intensa ramificação e crescimento global das vias aéreas e vasculatura do pulmão fetal, corresponde a um estadio de imaturidade imunológica. Sendo assim, a transferência génica durante este período terá, um maior efeito em elementos fundamentais do crescimento pulmonar com menor hipótese de desencadear respostas imunes. Dois tipos distintos de vectores víricos foram utilizados: um vector adenovírico e um vector lentivírico (Equine Infectious Anemia Virus), ambos expressam Enhanced Green Fluorescent Protein como gene marcador. Este estudo confirmou que os vectores adenovíricos são mais adequados quando se pretende obter expressão rápida, elevada e transiente do transgene; enquanto que os vectores lentivíricos induzem uma expressão sustentada no tempo. Uma das preocupações nos protocolos de transferência génica é atingir especificamente um determinado compartimento celular de um determinado órgão ou sistema. É de salientar que, ambos os vectores, transfectaram células do compartimento interstitial em oposição ao epitelial, descrito em estudos anteriores em que as vias de administração foram a intrapulmonar, a intra-amniótica e a intra-traqueal. A transdução de populações celulares distintas em estreita relação com o tipo de via de administração, aumenta as possibilidades de manipulação genética do pulmão em desenvolvimento. A etapa seguinte consiste em utilizar este modelo para efectuar um estudo in vivo da morfogénese pulmonar, manipulando um dos factores fundamentais no processo de ramificação, o fibroblast growth factor 10 (FGF10). A sobreexpressão mesenquimatosa de FGF10, no pulmão fetal de rato, resultou na indução de malformações císticas, cujo fenótipo era dependente do local e período em que se induzia essa mesma sobreexpressão. O fenótipo de todos os tipos de malformações induzidas, parece recapitular todo o espectro da Malformação Cística Adenomatoide Congénita do humano.Fundação para a Ciência e Tecnologia e FSE através do programa POCTI 2010, sobre a forma de bolsa (referência SFRH/BD/15260/2004) e sob a forma de projecto (POCI/SAL-OBS/56248/2004)

Capacity and coverage trade-off in WCDMA environments with repeaters deployment

radio planning, WCDMA, repeaters, capacity and coverageThis work derives the analytic expression of the feasibility condition for the uplink of a WCDMA mobile communications system with repeaters deployment in a multiservice environment with a general heterogeneous layout. In particular, a compact closed expression for the admission region is presented, suitable for a system where the users belong to an arbitrary number of different service classes. A tradeoff between capacity and coverage arises and it has been analysed both theoretically and by means of simulations. Different parameters are shown to have a major impact and their adjustment is discussed.Peer ReviewedPostprint (published version

Capacity and coverage tradeoff in WCDMA environments with repeaters deployment.

Postprint (published version

La prueba piloto de adaptación al EEES en la Escuela Politécnica Superior de Castelldefels

La prueba piloto de adaptación al EEES que se ha llevado a cabo en la Escuela Politécnica Superior de Castelldefels entre 2004 y 2007 ha motivado una importante actividad innovadora que nos ha permitido extraer numerosas conclusiones interesantes de cara al diseño e implantación de los nuevos títulos de grado. El elemento principal de la prueba piloto fue el establecimiento de cinco criterios que debían seguir las asignaturas como guía en el procedo de adaptación. Los cinco criterios son: (1) formulación correcta de los objetivos formativos específicos (qué es lo que los estudiantes deben haber aprendido al final del curso), (2) preparación de un programa de actividades y entregas que los alumnos deben realizar para conseguir los objetivos del curso y al que deben dedicar tantas horas como corresponda en función de la asignación de créditos ECTS, (3) desarrollo de un plan de recogida sistemática de datos sobre la marcha del curso (tiempos reales de dedicación, resultados académicos, opiniones de los alumnos, etc.), (4) articulación de un proceso de mejora continuada que se base en los datos recogidos durante el curso, y (5) desarrollo de mecanismos de comunicación eficaz con el estudiante (distribución de información y documentación del curso, gestión de las entregas de los alumnos, retroalimentación por parte de los profesores, etc.). En esta ponencia se describen en detalle estos cinco criterios y se realiza una valoración de las dificultades de su implantación así como de los resultados obtenidosPostprint (published version

Enhanced analysis of WCDMA networks with repeaters deployment

This paper addresses the analysis of WCDMA systems with repeaters deployment. A generic and compact expression for up- and downlinks evaluation has been mathematically derived so that transmission powers and other radio resource management parameters can be calculated without simplifications. In particular, the real different path delays, taking into account the repeaters presence and the finite nature of the time window of Rake receivers are considered. This allows an enhanced analysis with respect to classical approaches from a system level viewpoint. Furthermore, higher reliable and accurate predictions on network performance can be obtained, which can be remarkably useful for network planning and management. By using these expressions, relevant network parameters have been evaluated and compared with the ones obtained using the classical approximations. The differences in the obtained metrics are highlighted, putting in evidence the improvement provided by the proposed analysis.Peer Reviewe

Desdobramentos da Atividade Orientadora de Ensino para a organização do ensino e para a investigação sobre a atividade pedagógica

The objective of this work is to discuss the consequences that the concept of Teaching-orienteering Activity (TOE) has for both the teaching organization and the development of educational research. This bibliographic work analyzes 19 doctoral theses produced within the scope of the Group of Studies and Research on Pedagogical Activity (GEPAPe), from 2003 to 2016. The analysis shows how the TOE was initially linked to the teaching organization and that, later, it is also understood as a theoretical-methodological instrument for the development of research in education, which has as its object the pedagogical activity.Este trabajo tiene como objetivo discutir los desdoblamientos que el concepto de Actividad Orientadora de la enseñanza (AOE) tiene tanto para la organización de la enseñanza y para el desarrollo de investigaciones educativas. Este trabajo bibliográfico analiza 19 tesis de doctorado producidas en el ámbito del Grupo de Estudios e Investigación sobre Actividad Pedagógica (GEPAPe), en el período de 2003 a 2016. El análisis muestra cómo la AOE estaba inicialmente, ligada a la organización de la enseñanza, y que, posteriormente se pasa a ser comprendida también, como instrumento teórico-metodológico para el desarrollo de las investigaciones en educación, la cual tiene como objeto la actividad pedagógica.Ce texte a pour objectif de discuter des développements du concept d'activité d'orientation l'enseignement (AOE) pour l'organisation de l'enseignement et pour le développement de la recherche en éducation. Une recherche documentaire a été réalisée pour analyser dix-neuf thèses produites dans le cadre du Groupe d’études et de recherches sur l’activité pédagogique (GEPAPe), de 2003 à 2016. L'analyse montre comment l'AOE était initialement lié à l'organisation de l'enseignement et que, par la suite, il est également compris comme un instrument théorique et méthodologique pour le développement de la recherche en éducation, qui a pour objet l'activité pédagogiqueEste texto tem como objetivo discutir os desdobramentos do conceito de Atividade Orientadora de ensino (AOE) para a organização do ensino e para o desenvolvimento de pesquisas educacionais. Foi realizada uma investigação de caráter documental em que se analisa dezenove teses de doutorado produzidas no âmbito do Grupo de Estudos e Pesquisa sobre Atividade Pedagógica (GEPAPe), no período de 2003 a 2016. A análise mostra como a AOE estava inicialmente, ligada à organização do ensino, e que, posteriormente ela passa a ser compreendida, também, como instrumento teórico-metodológico para o desenvolvimento das pesquisas em educação, a qual tem como objeto a atividade pedagógica

Cystic adenomatoid malformations are induced by localized FGF10 overexpression in fetal rat lung

Fibroblast growth factor-10 (FGF10) is a mesenchymal growth factor, involved in epithelial and mesenchymal interactions during lung branching morphogenesis. In the present work, FGF10 overexpression was transiently induced in a temporally and spatially restricted manner, during the pseudoglandular or canalicular stages of rat lung development, by trans-uterine ultrasound-guided intraparenchymal microinjections of adenoviral vector encoding the rfgf10 transgene. The morphologic and histologic classification of the resulting malformations were dependent upon developmental stage and location. Overexpression of FGF10 restricted to the proximal tracheobronchial tree during the pseudoglandular phase resulted in large cysts lined by tall columnar epithelium composed primarily of Clara cells with a paucity of Type II pneumocytes, resembling bronchiolar type epithelium. In contrast, FGF10 overexpression in the distal lung parenchyma during the canalicular phase resulted in small cysts lined by cuboidal epithelial cells composed of primarily Type II pneumocytes resembling acinar epithelial differentiation. The cystic malformations induced by FGF10 overexpression appear to closely recapitulate the morphology and histology of the spectrum of human congenital cystic adenomatoid malformation (CCAM). These findings support a role for FGF10 in the induction of human CCAM and provide further mechanistic insight into the role of FGF10 in normal and abnormal lung development.This project was in part funded by proceeds from the Ruth and Tristram C. Colket Jr. Chair in Pediatric Surgery (A.W.F.), and the Fundação para a Ciência e Tecnologia (POCI/SAUOBS/56428/2004). S.G. was supported by FCT grant ref. SFRH/BD/15260/2004

Targeted gene transfer to fetal rat lung interstitium by ultrasound-guided intrapulmonary injection

In utero gene transfer to the developing lung may have clinical or research applications. In this study, we developed a new method for specifically targeting the fetal rat lung with adeno and lentiviral vectors encoding the enhanced green fluorescence protein (EGFP) marker gene at E15.5 using ultrasound biomicroscopy (UBM). Survival rate, morphometric parameters, viral biodistribution, and lung transduction efficiency were analyzed and compared to the intra-amniotic route of administration. Expression of EGFP started as early as 24 and 72 h after the injection of adenoviral and lentiviral vectors, respectively. Both vectors transduced lung parenchyma with gene expression limited to interstitial cells of the injected region, in contrast to intra-amniotic injection, which targeted the pulmonary epithelium. Expression of EGFP was most intense at E18.5 and E21.5 for adenoviral and lentiviral vectors, respectively. In contrast to lentivirus, adenoviral expression significantly declined until final analysis at 1 week of age. This study demonstrates the feasibility of targeting the fetal rat lung interstitium with viral vectors under UBM guidance during the pseudoglandular stage. This model system may facilitate in vivo studies of dynamic lung morphogenesis and could provide insight into the efficacy of prenatal gene transfer strategies for treatment of specific lung disorders.FCT Grant (SFRH/BD/15260/2004) on behalf of the FCT Grant POCI/SAU-OBS/56428/200

An intensity recovery algorithm (IRA) for minimizing the edge effect of LIDAR data

The terrestrial laser scanner is an equipment developed for surveying applications and is also used for many other purposes due to its ability to acquire 3D data quickly. However, before intensity data can be analyzed, it must be processed in order to minimize the edge or border effect, one of the most serious problems of LIDAR’s intensity data. Our research has focused on characterizing the edge effect behavior as well as to develop an algorithm to minimize edge effect distortion automatically (IRA). The IRA showed to be effective recovering 35.71% of points distorted by the edge effect, providing significant improvements and promising results for the development of applications based on TLS data intensity to many studies

Congenital lung lesions-underlying molecular mechanisms

Congenital lung lesions comprise a broad spectrum of rare but clinically significant developmental abnormalities, including congenital cystic adenomatoid malformation, bronchopulmonary sequestrations, congenital lobar emphysema, and bronchogenic cysts, which are commonly surgically treated. Although the terms congenital cystic adenomatoid malformation, bronchopulmonary sequestrations, congenital lobar emphysema, and bronchogenic cysts are entrenched in clinical usage and comfortably correspond to rigid pathologic definitions, there is a considerable overlap in the findings. Disregarding the controversy about lesion nomenclature and classification, it is widely accepted that congenital lung lesions result from perturbations in lung and airway embryogenesis. It is generally accepted that both place (level in the tracheobronchial tree) and timing (gestational age) of the embryologic insult correlates with the type of lesion and histopathology that is manifested. The objective of this review is to briefly review normal lung development and to analyze the known molecular mechanisms underlying those diseases.S G was supported by FCT grant ref SFRH/BD/15.260/2004, Y H is supported by the Sophia Foundation (SSWO project 482