Tese de mestrado. Reabilitação do Património Edificado. Faculdade de Engenharia. Universidade do Porto. 2005

Tese de mestrado. Estruturas de Engenharia Civil. Faculdade de Engenharia. Universidade do Porto. 2004

Gli stress abiotici determinando modificazioni a livello fisiologico, biochimico e molecolare delle piante, costituiscono una delle principali limitazioni per la produzione agricola mondiale. Nel 2007 la FAO ha stimato come solamente il 3,5% della superficie mondiale non sia sottoposta a stress abiotici. Il modello agro-industriale degli ultimi cinquant'anni, oltre ad avere contribuito allo sviluppo economico dell'Europa, è stato anche causa di inquinamento di acqua, aria e suolo, mediante uno sfruttamento indiscriminato delle risorse naturali. L'arsenico in particolare, naturalmente presente nell'ambiente e rilasciato dalle attività antropiche, desta particolare preoccupazione a causa dell'ampia distribuzione come contaminante ambientale e per gli effetti di fitotossicità provocati. In tale contesto, la diffusione di sistemi agricoli a basso impatto rappresenta una importante risorsa per rispondere all'emergenza del cambiamento climatico che negli anni a venire sottoporrà una superficie agricola sempre maggiore a stress di natura abiotica. Nello studio condotto è stato utilizzato uno stabile modello di crescita in vitro per valutare l'efficacia di preparati ultra diluiti (PUD), che non contenendo molecole chimiche di sintesi ben si adattano a sistemi agricoli sostenibili, su semi di frumento preventivamente sottoposti a stress sub-letale da arsenico. Sono state quindi condotte valutazioni sia a livello morfometrico (germinazione, lunghezza di germogli e radici) che molecolare (espressione genica valutata mediante analisi microarray, con validazione tramite Real-Time PCR) arricchendo la letteratura esistente di interessanti risultati. In particolare è stato osservato come lo stress da arsenico, determini una minore vigoria di coleptile e radici e a livello molecolare induca l'attivazione di pathways metabolici per proteggere e difendere le cellule vegetali dai danni derivanti dallo stress; mentre il PUD in esame (As 45x), nel sistema stressato ha indotto un recupero nella vigoria di germoglio e radici e livelli di espressione genica simili a quelli riscontrati nel controllo suggerendo un effetto "riequilibrante" del metabolismo vegetale. / The unquestionable importance of abiotic stresses to world agriculture is demonstrated by the fact that altogether abiotic factors provide the major limitation to crop production worldwide. A 2007 FAO report stated that only 3.5% of the global land area is not affected by environmental stresses and that climate change will increase areas characterized by abiotic stresses. The agro-industrial model that enriched European population in the last fifty years, however has resulted in irreversible contamination of water, air and soil. In particular arsenic is a toxic metalloid widely disseminated in the environmental as a consequence of natural processes and anthropic activities, that causes global concern for health and environmental hazards. Within this context, sustainable farming systems and the use of organic treatments such as ultrahigh diluted treatments (PUD) could be important tools of mitigating the effects of climate change. The objective of the present study was to test the biological effect of PUD As45x on wheat seedling poisoned with a sub-letal dose of arsenic, using an in vitro plant system. The evaluation of the arsenic toxicity and the effectiveness of As45x was conducted through biometric measurements and molecular analyses (microarray and Real-Time PCR). The results provided evidence for a strong gene modulating effect of arsenic, upregulating molecular pathways involved in cellular protection and defense from oxidative stress. It was also observed a reduction in the length of shoots and roots. Inversely in seedlings grown from poisoned seeds and treated with PUD, a recovery of coleoptiles and roots vigor was detected together with a massive reduction of gene expression levels to values comparable to those of the control group. A plausible hypothesis is that PUD induced a reequilibration of those genes that were upregulated during arsenic stress by bringing the expression levels closer to the basal levels normally occurring in control plants.

La inflamación y el estrés oxidativo son mecanismos moleculares que contribuyen al daño y a la disfunción muscular. Existen procesos crónicos con fenómenos inflamatorios de bajo grado pero de larga duración (EPOC, con o sin pérdida de peso), así como situaciones clínicas con liberación aguda de gran cantidad de moléculas proinflamatorias (Sepsis). Los mecanismos de regeneración del músculo en ambas entidades son esenciales para la correcta recuperación funcional y pueden condicionar cambios fenotípicos. Objetivo: En la EPOC, evaluar el estrés oxidativo, inflamación y estructura antes y después de un programa de rehabilitación en el vasto lateral (VL) del cuádriceps e intercostal. En dos grupos adicionales estudiar cambios regenerativos en el VL y realizar un análisis de cambios fibrilares y supervivencia. En pacientes con sepsis, investigar los eventos de estrés oxidativo e inflamación en los mismos músculos. Métodos: Estudio 1: Se analizaron cambios fibrilares (inmunohistoquímica) y estrés oxidativo (western-blot) en el músculo VL e intercostal en 25 pacientes con EPOC, antes y después de un entrenamiento de alta intensidad (8-10 semanas). En todos los pacientes se determinaron la capacidad de ejercicio (test de marcha de 6’ y cicloergómetro) y calidad de vida relacionada con la salud (cuestionarios SGRQ y SF36). Estudio 2: En un grupo adicional de 392 pacientes EPOC se analizó el fenotipo fibrilar del VL y su supervivencia. Estudio 3: Se obtuvieron muestras del VL en 15 pacientes con EPOC sin bajo peso, 15 pacientes EPOC con bajo peso y 10 sujetos control. Se analizaron cambios fibrilares, daño y número de células satélite (inmunohistoquímica), expresión de ARN de genes clave en la regeneración, mediadores de inflamación y mantenimiento de la masa muscular (PCR en tiempo real), y niveles de proteínas clave relacionadas con la regeneración (western-blot). Se obtuvieron cultivos primarios de mioblastos de dichas muestras musculares, en las que se evaluaron propiedades de migración (microscopía de tracción), capacidad de proliferación (Population doubling time y ELISA) y diferenciación (ELISA y PCR en tiempo real). Estudio 4: En el VL e intercostal de 6 pacientes con sepsis grave y 6 sujetos control se analizó la estructura muscular, células inflamatorias, estrés oxidativo y expresión de mediadores inflamatorios (técnicas mencionadas anteriormente). Resultados: Estudio 1: Situación basal, en el VL respecto del intercostal se observaron niveles de estrés oxidativo y antioxidantes (SOD2) aumentados. Tras 10 semanas de entrenamiento, se observaron mejorías en la capacidad de ejercicio y calidad de vida sin aumentos en los niveles de estrés oxidativo en ninguno de los músculos. Estudio 2: En el VL de pacientes EPOC el aumento en la proporción de fibras rápidas se asoció a una mayor mortalidad, especialmente en aquéllos con enfermedad grave. Estudio 3: En el VL de pacientes EPOC respecto de los sujetos control se observó mayor limitación al ejercicio, aumento de fibras rápidas, signos de daño, núcleos internalizados y células satélite. En los mismos pacientes se observó un incremento en la expresión de genes involucrados en fases precoces de la regeneración muscular (PAX7, MYOG y MYF6), pero descenso en los que regulan fases tardías (MYH8). Además en los pacientes con bajo peso se observó un aumento en los niveles de IL-6 y una sobreregulación en la expresión de IGF-1 y MGF en el VL. En los cultivos primarios de mioblastos no se observaron diferencias entre pacientes y controles en la capacidad de migración, proliferación, diferenciación ni en la expresión de genes clave de regeneración muscular. Estudio 4: En el VL de pacientes con sepsis los niveles de diversos marcadores de estrés oxidativo e inflamación (TNF-alfa y sus receptores, IL-1β, IL-6 y CD18) estaban aumentados, mientras que no se observaron diferencias en el músculo intercostal. Conclusiones: Se han encontrado alteraciones musculares en la EPOC y la sepsis, aunque con un patrón diferenciado entre la musculatura respiratoria y periférica en esta última. El entrenamiento de alta intensidad induce una mejoría clínica en los pacientes EPOC, sin conllevar aumento en los niveles de estrés oxidativo o inflamación. Los mecanismos de regeneración/reparación parecen hallarse alterados en el VL de pacientes con EPOC, probablemente debido a la interacción de factores locales in vivo. El fenotipo fibrilar en este músculo predice la mortalidad de dichos pacientes. / Both inflammation and oxidative stress are molecular mechanisms that contribute to muscle damage and dysfunction. Although different chronic disorders share inflammatory phenomena, some of them are characterized by a low-grade but long duration of these events (COPD, with or without weight loss) and others are more acute with a dramatic release of many proinflammatory molecules (Sepsis). The mechanisms of muscle regeneration in both entities are essential for a proper structural and functional recovery, and can condition phenotypic changes. Objective: To assess oxidative stress, inflammation and muscle structure before and after a rehabilitation program in the vastus lateralis (VL) and intercostal muscles of COPD patients. Moreover, to evaluate regenerative changes in the VL and to carry out a survival analysis. In parallel, to investigate the events of oxidative stress and inflammation in the muscles of septic patients. Methods: Study 1: fiber changes (immunohistochemistry) and oxidative stress (western-blot) were analyzed in the intercostal and VL muscles of 25 COPD patients before and after a high intensity training (8-10 weeks). Exercise capacity (6’ walking test and cycloergometry) and health-related quality of life (SGRQ and SF36) were also determined. Study 2: fiber phenotype of VL and survival was analyzed in an additional group of 392 COPD patients. Study 3: fiber changes, damage and number of satellite cells (immunohistochemistry), RNA expression of key genes involved in regeneration, mediators of inflammation and maintenance of muscle mass (real time PCR), and levels of key proteins related to regeneration (western-blot) were analyzed in the VL of COPD patients (15 underweight and 15 with normal weight) and control subjects (n=10). Primary myoblast cultures of these muscle samples were also performed and cell mechanical properties (linked to migration, tensile microscopy), proliferation (population doubling time and ELISA) and differentiation (ELISA and PCR) were quantified. Study 4: samples of intercostal and VL of 6 patients with severe sepsis and 6 control subjects were analyzed for muscle structure, oxidative stress and inflammatory mediators (above mentioned techniques). Results: Study 1: Basal levels of oxidative stress and antioxidants (SOD2) were increased in the VL versus the intercostal muscle of COPD patients. After 10 weeks of training, significant improvements were observed in exercise capacity and quality of life with no increases in oxidative stress in either of the two muscles. Study 2: The increase in the proportion of fast fibers in the VL of COPD patients was associated with increased mortality, especially in those with a severe disease. Study 3: Exercise limitation as well as increases in the percentage of fast fibers, signs of muscle damage, internalized nuclei and satellite cells was observed in the VL of COPD patients compared to controls. Furthermore, increased expression of genes involved in early stages of muscle regeneration (PAX7, MYOG and Myf6) but a decrease in those regulating late phases (MYH8) was also observed in the muscle of COPD patients when compared to controls. Patients with COPD and low weight also showed upregulation of the genes encoding IL-6 and muscle growth factors (IGF-1 and MGF). We saw no difference on primary myoblast cultures in either the ability of migration, proliferation, differentiation or the expression of key muscle regeneration genes between patients and controls. Study 4: Different oxidative stress and inflammatory (TNF- and its receptors, IL-1β, IL-6 and CD18) markers were increased in the VL of septic patients compared with controls, with no differences in the intercostal muscle. Conclusions: We found significant alterations in the muscles of both COPD and septic patients, although the latter had a distinct pattern between respiratory and peripheral muscles. High intensity training induces clinical improvement in COPD patients, with no increases in the levels of oxidative stress and inflammation. The mechanisms of muscle regeneration appear to be altered in the VL of COPD patients, which is probably due to the interaction of local factors in vivo. Muscle fiber phenotype predicts mortality of these patients.

Skeletal muscle dysfunction and wasting are major comorbidities of chronic obstructive pulmonary disease (COPD) and lung cancer (LC). Despite that the lower limb muscles are usually more severely affected, the respiratory muscles may also experience structural and functional abnormalities in COPD. Muscle dysfunction negatively impacts on the patients’ quality of life by impairing their exercise tolerance even of daily life activities. Several molecular mechanisms are involved in the etiology of COPD muscle dysfunction. In this regard, we hypothesized that oxidative stress may be a trigger of enhanced muscle proteolysis and dysfunction in the limb muscles of cachectic patients bearing two different respiratory conditions such as COPD and LC, and that epigenetic events may be involved in the pathophysiology of COPD muscle dysfunction of both respiratory and limb muscles. / La disfunció muscular esquelètica i la pèrdua de massa muscular són dues manifestacions sistèmiques freqüents en malalties com la Malaltia Pulmonar Obstructiva Crònica (MPOC) i el Càncer de Pulmó (CP). Malgrat que en els malalts amb MPOC, els músculs perifèrics sovint es veuen més greument afectats, els músculs respiratoris també mostren alteracions estructurals i funcionals. La disfunció muscular que pateixen aquests pacients afecta negativament la seva qualitat de vida, no només reduint la seva tolerància a l’exercici físic sinó també a les seves activitats quotidianes. Diferents mecanismes moleculars estan implicats en la etiologia de la disfunció muscular en la MPOC. La nostra hipòtesi és que l’estrès oxidatiu podria ser un desencadenant de l’augment de proteòlisi i disfunció muscular en els músculs perifèrics de pacients amb caquèxia associada a processos respiratoris com ara la MPOC i el CP. Els mecanismes epigenètics podrien estar també implicats en la fisiopatologia de la disfunció muscular en la MPOC.

Animal models have been relevant to study the molecular mechanisms of cancer and to develop new antitumor agents. Anyway, the huge divergence in mouse and human evolution made difficult the translation of the gained achievements in preclinical mouse based studies. The generation of clinically relevant murine models requires their humanization both concerning the creation of transgenic models and the generation of humanized mice in which to engraft a functional human immune system, and reproduce the physiological effects and molecular mechanisms of growth and metastasization of human tumors. In particular, the availability of genotypically stable immunodepressed mice able to accept tumor injection and allow human tumor growth and metastasization would be important to develop anti-tumor and anti-metastatic strategies. Recently, Rag2-/-;gammac-/- mice, double knockout for genes involved in lymphocyte differentiation, had been developed (CIEA, Central Institute for Experimental Animals, Kawasaki, Japan). Studies of human sarcoma metastasization in Rag2-/-; gammac-/- mice (lacking B, T and NK functionality) revealed their high metastatic efficiency and allowed the expression of human metastatic phenotypes not detectable in the conventionally used nude murine model. In vitro analysis to investigate the molecular mechanisms involved in the specific pattern of human sarcomas metastasization revealed the importance of liver-produced growth and motility factors, in particular the insulin-like growth factors (IGFs). The involvement of this growth factor was then demonstrated in vivo through inhibition of IGF signalling pathway. Due to the high growth and metastatic propensity of tumor cells, Rag2-/-;gammac-/- mice were used as model to investigate the metastatic behavior of rhabdomyosarcoma cells engineered to improve the differentiation. It has been recently shown that this immunodeficient model can be reconstituted with a human immune system through the injection of human cord blood progenitor cells. The work illustrated in this thesis revealed that the injection of different human progenitor cells (CD34+ or CD133+) showed peculiar engraftment and differentiation abilities. Experiments of cell vaccination were performed to investigate the functionality of the engrafted human immune system and the induction of specific human immune responses. Results from such experiments will allow to collect informations about human immune responses activated during cell vaccination and to define the best reconstitution and experimental conditions to create a humanized model in which to study, in a preclinical setting, immunological antitumor strategies.

Calcium works as a second intracellular messenger in all cell types and its downstream signalling is a key pathway for many systemic functions. In the lungs, the majority of activating stimuli trigger intracellular calcium increase, which is indispensable for the normal functioning of the airways; thus, deregulation of this pathway leads to pathological conditions. This Thesis aims to understand the relationship of intracellular calcium homeostasis and chronic respiratory pathologies such as asthma. I have studied three different processes involved in calcium homeostasis and their role in asthma pathophysiology: 1) I have shown the genetic association of a defect in calcium entry via TRPV1 with wheezing and cough, which is one feature of asthma pathophysiology; 2) I have also demonstrated the product of the asthma associated ORMDL3 gene is a Ca++ homeostasis and UPR modulator; and 3) I have provided a new Ca++ dependent sorting mechanism for secretory cargoes that bind calcium. / El Calci és un segon missatger intracel·lular en tots els tipus cel·lulars i la cascada de senyalització generada pel calci és una via de senyalització cel·lular clau per moltes funcions sistèmiques. En els pulmons, la majoria d’estímuls activadors produeixen un increment del calci intracel·lular, el qual és indispensable pel funcionament correcte de les vies respiratòries; i, per tant, una desregulació d’aquesta via de senyalització porta a diferents situacions patològiques. Aquesta Tesi té com a objectiu entendre la relació entre l’homeòstasi del calci intracel·lular i les malalties respiratòries cròniques, com per exemple, l’asma. Hem estudiat tres processos diferents implicats en l’homeòstasi del calci i el seu rol en la fisiopatologia de l’asma: 1) Hem demostrat que hi ha una associació genètica entre un defecte en l’entrada de calci via TRPV1 i un dels trets característics de l’asma, la tos; 2) també hem trobat que l’ORMDL3, que havia estat associat amb l’asma, és un modulador de l’homeòstasi del calci i de la UPR; i 3) hem aportat un nou mecanisme de classificació en el Golgi depenent de Ca++ per a proteïnes que uneixen calci i que seran secretades.

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