TM6SF2 rs58542926 influences hepatic fibrosis progression in patients with non-alcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) is an increasingly common condition, strongly associated with the metabolic syndrome, that can lead to progressive hepatic fibrosis, cirrhosis and hepatic failure. Subtle inter-patient genetic variation and environmental factors combine to determine variation in disease progression. A common non-synonymous polymorphism in TM6SF2 (rs58542926 c.449 C>T, p.Glu167Lys) was recently associated with increased hepatic triglyceride content, but whether this variant promotes clinically relevant hepatic fibrosis is unknown. Here we confirm that TM6SF2 minor allele carriage is associated with NAFLD and is causally related to a previously reported chromosome 19 GWAS signal that was ascribed to the gene NCAN. Furthermore, using two histologically characterized cohorts encompassing steatosis, steatohepatitis, fibrosis and cirrhosis (combined n=1,074), we demonstrate a new association, independent of potential confounding factors (age, BMI, type 2 diabetes mellitus and PNPLA3 rs738409 genotype), with advanced hepatic fibrosis/cirrhosis. These findings establish new and important clinical relevance to TM6SF2 in NAFLD.

Oncogenic activation of c-Abl tyrosine kinase in non-small cell lung cancer: FUS1 tumor suppressor down-regulates c-Abl tyrosine kinase

The FUS1 tumor suppressor gene (TSG) has been found to be deficient in many human non-small cell lung cancer (NSCLC) tissue samples and cell lines (1,2,3). Studies have shown potent anti-tumor activity of FUS1 in animal models where FUS1 was delivered through a liposomal vector (4) and the use of FUS1 as a therapeutic agent is currently being studied in clinical human trials (5). Currently, the mechanisms of FUS1 activity are being investigated and my studies have shown that c-Abl tyrosine kinase is inhibited by the FUS1 TSG.^ Considering that many NSCLC cell lines are FUS1 deficient, my studies further identified that FUS1 deficient NSCLC cells have an activated c-Abl tyrosine kinase. C-Abl is a known proto-oncogene and while c-Abl kinase is tightly regulated in normal cells, constitutively active Abl kinase is known to contribute to the oncogenic phenotype in some types of hematopoietic cancers. My studies show that the active c-Abl kinase contributes to the oncogenicity of NSCLC cells, particularly in tumors that are deficient in FUS1, and that c-Abl may prove to be a viable target in NSCLC therapy.^ Current studies have shown that growth factor receptors play a role in NSCLC. Over-expression of the epidermal growth factor receptor (EGFR) plays a significant role in aggressiveness of NSCLC. Current late stage treatments include EFGR tyrosine kinase inhibitors or EGFR antibodies. Platelet-derived growth factor receptor (PDGFR) also has been shown to play a role in NSCLC. Of note, both growth factor receptors are known upstream activators of c-Abl kinase. My studies indicate that growth factor receptor simulation along deficiency in FUS1 expression contributes to the activation of c-Abl kinase in NSCLC cells. ^

Post-transcriptional Regulation of Mammalian Gene Expression in Non-coding Region of Target RNA

Tumor Suppressor Candidate 2 (TUSC2) is a novel tumor suppressor gene located in the human chromosome 3p21.3 region. TUSC2 mRNA transcripts could be detected on Northern blots in both normal lung and some lung cancer cell lines, but no endogenous TUSC2 protein could be detected in a majority of lung cancer cell lines. Mechanisms regulating TUSC2 protein expression and its inactivation in primary lung cancer cells are largely unknown. We investigated the role of the 5’- and 3’-untranslated regions (UTRs) of the TUSC2 gene in the regulation of TUSC2 protein expression. We found that two small upstream open-reading frames (uORFs) in the 5’UTR of TUSC2 could markedly inhibit the translational initiation of TUSC2 protein by interfering with the “scanning” of the ribosome initiation complexes. Site-specific stem-loop array reverse transcription-polymerase chain reaction (SLA-RT-PCR) verified several micoRNAs (miRNAs) targeted at 3’UTR and directed TUSC2 cleavage and degradation. In addition, we used the established let-7-targeted high mobility group A2 (Hmga2) mRNA as a model system to study the mechanism of regulation of target mRNA by miRNAs in mammalian cells under physiological conditions. There have been no evidence of direct link between mRNA downregulation and mRNA cleavages mediated by miRNAs. Here we showed that the endonucleolytic cleavages on mRNAs were initiated by mammalian miRNA in seed pairing style. Let-7 directed cleavage activities among the eight predicted potential target sites have varied efficiency, which are influenced by the positional and the structural contexts in the UTR. The 5’ cleaved RNA fragments were mostly oligouridylated at their 3’-termini and accumulated for delayed 5’–3’ degradation. RNA fragment oligouridylation played important roles in marking RNA fragments for delayed bulk degradation and in converting RNA degradation mode from 3’–5’ to 5’–3’ with cooperative efforts from both endonucleolytic and non-catalytic miRNA-induced silencing complex (miRISC). Our findings point to a mammalian miRNA-mediated mechanism for the regulation of mRNA that miRNA can decrease target mRNA through target mRNA cleavage and uridine addition

Advances in the Simultaneous Multiple Surface optical design method for imaging and non-imaging applications

Classical imaging optics has been developed over centuries in many areas, such as its paraxial imaging theory and practical design methods like multi-parametric optimization techniques. Although these imaging optical design methods can provide elegant solutions to many traditional optical problems, there are more and more new design problems, like solar concentrator, illumination system, ultra-compact camera, etc., that require maximum energy transfer efficiency, or ultra-compact optical structure. These problems do not have simple solutions from classical imaging design methods, because not only paraxial rays, but also non-paraxial rays should be well considered in the design process. Non-imaging optics is a newly developed optical discipline, which does not aim to form images, but to maximize energy transfer efficiency. One important concept developed from non-imaging optics is the “edge-ray principle”, which states that the energy flow contained in a bundle of rays will be transferred to the target, if all its edge rays are transferred to the target. Based on that concept, many CPC solar concentrators have been developed with efficiency close to the thermodynamic limit. When more than one bundle of edge-rays needs to be considered in the design, one way to obtain solutions is to use SMS method. SMS stands for Simultaneous Multiple Surface, which means several optical surfaces are constructed simultaneously. The SMS method was developed as a design method in Non-imaging optics during the 90s. The method can be considered as an extension to the Cartesian Oval calculation. In the traditional Cartesian Oval calculation, one optical surface is built to transform an input wave-front to an out-put wave-front. The SMS method however, is dedicated to solve more than 1 wave-fronts transformation problem. In the beginning, only 2 input wave-fronts and 2 output wave-fronts transformation problem was considered in the SMS design process for rotational optical systems or free-form optical systems. Usually “SMS 2D” method stands for the SMS procedure developed for rotational optical system, and “SMS 3D” method for the procedure for free-form optical system. Although the SMS method was originally employed in non-imaging optical system designs, it has been found during this thesis that with the improved capability to design more surfaces and control more input and output wave-fronts, the SMS method can also be applied to imaging system designs and possesses great advantage over traditional design methods. In this thesis, one of the main goals to achieve is to further develop the existing SMS-2D method to design with more surfaces and improve the stability of the SMS-2D and SMS-3D algorithms, so that further optimization process can be combined with SMS algorithms. The benefits of SMS plus optimization strategy over traditional optimization strategy will be explained in details for both rotational and free-form imaging optical system designs. Another main goal is to develop novel design concepts and methods suitable for challenging non-imaging applications, e.g. solar concentrator and solar tracker. This thesis comprises 9 chapters and can be grouped into two parts: the first part (chapter 2-5) contains research works in the imaging field, and the second part (chapter 6-8) contains works in the non-imaging field. In the first chapter, an introduction to basic imaging and non-imaging design concepts and theories is given. Chapter 2 presents a basic SMS-2D imaging design procedure using meridian rays. In this chapter, we will set the imaging design problem from the SMS point of view, and try to solve the problem numerically. The stability of this SMS-2D design procedure will also be discussed. The design concepts and procedures developed in this chapter lay the path for further improvement. Chapter 3 presents two improved SMS 3 surfaces’ design procedures using meridian rays (SMS-3M) and skew rays (SMS-1M2S) respectively. The major improvement has been made to the central segments selections, so that the whole SMS procedures become more stable compared to procedures described in Chapter 2. Since these two algorithms represent two types of phase space sampling, their image forming capabilities are compared in a simple objective design. Chapter 4 deals with an ultra-compact SWIR camera design with the SMS-3M method. The difficulties in this wide band camera design is how to maintain high image quality meanwhile reduce the overall system length. This interesting camera design provides a playground for the classical design method and SMS design methods. We will show designs and optical performance from both classical design method and the SMS design method. Tolerance study is also given as the end of the chapter. Chapter 5 develops a two-stage SMS-3D based optimization strategy for a 2 freeform mirrors imaging system. In the first optimization phase, the SMS-3D method is integrated into the optimization process to construct the two mirrors in an accurate way, drastically reducing the unknown parameters to only few system configuration parameters. In the second optimization phase, previous optimized mirrors are parameterized into Qbfs type polynomials and set up in code V. Code V optimization results demonstrates the effectiveness of this design strategy in this 2-mirror system design. Chapter 6 shows an etendue-squeezing condenser optics, which were prepared for the 2010 IODC illumination contest. This interesting design employs many non-imaging techniques such as the SMS method, etendue-squeezing tessellation, and groove surface design. This device has theoretical efficiency limit as high as 91.9%. Chapter 7 presents a freeform mirror-type solar concentrator with uniform irradiance on the solar cell. Traditional parabolic mirror concentrator has many drawbacks like hot-pot irradiance on the center of the cell, insufficient use of active cell area due to its rotational irradiance pattern and small acceptance angle. In order to conquer these limitations, a novel irradiance homogenization concept is developed, which lead to a free-form mirror design. Simulation results show that the free-form mirror reflector has rectangular irradiance pattern, uniform irradiance distribution and large acceptance angle, which confirm the viability of the design concept. Chapter 8 presents a novel beam-steering array optics design strategy. The goal of the design is to track large angle parallel rays by only moving optical arrays laterally, and convert it to small angle parallel output rays. The design concept is developed as an extended SMS method. Potential applications of this beam-steering device are: skylights to provide steerable natural illumination, building integrated CPV systems, and steerable LED illumination. Conclusion and future lines of work are given in Chapter 9. Resumen La óptica de formación de imagen clásica se ha ido desarrollando durante siglos, dando lugar tanto a la teoría de óptica paraxial y los métodos de diseño prácticos como a técnicas de optimización multiparamétricas. Aunque estos métodos de diseño óptico para formación de imagen puede aportar soluciones elegantes a muchos problemas convencionales, siguen apareciendo nuevos problemas de diseño óptico, concentradores solares, sistemas de iluminación, cámaras ultracompactas, etc. que requieren máxima transferencia de energía o dimensiones ultracompactas. Este tipo de problemas no se pueden resolver fácilmente con métodos clásicos de diseño porque durante el proceso de diseño no solamente se deben considerar los rayos paraxiales sino también los rayos no paraxiales. La óptica anidólica o no formadora de imagen es una disciplina que ha evolucionado en gran medida recientemente. Su objetivo no es formar imagen, es maximazar la eficiencia de transferencia de energía. Un concepto importante de la óptica anidólica son los “rayos marginales”, que se pueden utilizar para el diseño de sistemas ya que si todos los rayos marginales llegan a nuestra área del receptor, todos los rayos interiores también llegarán al receptor. Haciendo uso de este principio, se han diseñado muchos concentradores solares que funcionan cerca del límite teórico que marca la termodinámica. Cuando consideramos más de un haz de rayos marginales en nuestro diseño, una posible solución es usar el método SMS (Simultaneous Multiple Surface), el cuál diseña simultáneamente varias superficies ópticas. El SMS nació como un método de diseño para óptica anidólica durante los años 90. El método puede ser considerado como una extensión del cálculo del óvalo cartesiano. En el método del óvalo cartesiano convencional, se calcula una superficie para transformar un frente de onda entrante a otro frente de onda saliente. El método SMS permite transformar varios frentes de onda de entrada en frentes de onda de salida. Inicialmente, sólo era posible transformar dos frentes de onda con dos superficies con simetría de rotación y sin simetría de rotación, pero esta limitación ha sido superada recientemente. Nos referimos a “SMS 2D” como el método orientado a construir superficies con simetría de rotación y llamamos “SMS 3D” al método para construir superficies sin simetría de rotación o free-form. Aunque el método originalmente fue aplicado en el diseño de sistemas anidólicos, se ha observado que gracias a su capacidad para diseñar más superficies y controlar más frentes de onda de entrada y de salida, el SMS también es posible aplicarlo a sistemas de formación de imagen proporcionando una gran ventaja sobre los métodos de diseño tradicionales. Uno de los principales objetivos de la presente tesis es extender el método SMS-2D para permitir el diseño de sistemas con mayor número de superficies y mejorar la estabilidad de los algoritmos del SMS-2D y SMS-3D, haciendo posible combinar la optimización con los algoritmos. Los beneficios de combinar SMS y optimización comparado con el proceso de optimización tradicional se explican en detalle para sistemas con simetría de rotación y sin simetría de rotación. Otro objetivo importante de la tesis es el desarrollo de nuevos conceptos de diseño y nuevos métodos en el área de la concentración solar fotovoltaica. La tesis está estructurada en 9 capítulos que están agrupados en dos partes: la primera de ellas (capítulos 2-5) se centra en la óptica formadora de imagen mientras que en la segunda parte (capítulos 6-8) se presenta el trabajo del área de la óptica anidólica. El primer capítulo consta de una breve introducción de los conceptos básicos de la óptica anidólica y la óptica en formación de imagen. El capítulo 2 describe un proceso de diseño SMS-2D sencillo basado en los rayos meridianos. En este capítulo se presenta el problema de diseñar un sistema formador de imagen desde el punto de vista del SMS y se intenta obtener una solución de manera numérica. La estabilidad de este proceso se analiza con detalle. Los conceptos de diseño y los algoritmos desarrollados en este capítulo sientan la base sobre la cual se realizarán mejoras. El capítulo 3 presenta dos procedimientos para el diseño de un sistema con 3 superficies SMS, el primero basado en rayos meridianos (SMS-3M) y el segundo basado en rayos oblicuos (SMS-1M2S). La mejora más destacable recae en la selección de los segmentos centrales, que hacen más estable todo el proceso de diseño comparado con el presentado en el capítulo 2. Estos dos algoritmos representan dos tipos de muestreo del espacio de fases, su capacidad para formar imagen se compara diseñando un objetivo simple con cada uno de ellos. En el capítulo 4 se presenta un diseño ultra-compacto de una cámara SWIR diseñada usando el método SMS-3M. La dificultad del diseño de esta cámara de espectro ancho radica en mantener una alta calidad de imagen y al mismo tiempo reducir drásticamente sus dimensiones. Esta cámara es muy interesante para comparar el método de diseño clásico y el método de SMS. En este capítulo se presentan ambos diseños y se analizan sus características ópticas. En el capítulo 5 se describe la estrategia de optimización basada en el método SMS-3D. El método SMS-3D calcula las superficies ópticas de manera precisa, dejando sólo unos pocos parámetros libres para decidir la configuración del sistema. Modificando el valor de estos parámetros se genera cada vez mediante SMS-3D un sistema completo diferente. La optimización se lleva a cabo variando los mencionados parámetros y analizando el sistema generado. Los resultados muestran que esta estrategia de diseño es muy eficaz y eficiente para un sistema formado por dos espejos. En el capítulo 6 se describe un sistema de compresión de la Etendue, que fue presentado en el concurso de iluminación del IODC en 2010. Este interesante diseño hace uso de técnicas propias de la óptica anidólica, como el método SMS, el teselado de las lentes y el diseño mediante grooves. Este dispositivo tiene un límite teórica en la eficiencia del 91.9%. El capítulo 7 presenta un concentrador solar basado en un espejo free-form con irradiancia uniforme sobre la célula. Los concentradores parabólicos tienen numerosas desventajas como los puntos calientes en la zona central de la célula, uso no eficiente del área de la célula al ser ésta cuadrada y además tienen ángulos de aceptancia de reducido. Para poder superar estas limitaciones se propone un novedoso concepto de homogeneización de la irrandancia que se materializa en un diseño con espejo free-form. El análisis mediante simulación demuestra que la irradiancia es homogénea en una región rectangular y con mayor ángulo de aceptancia, lo que confirma la viabilidad del concepto de diseño. En el capítulo 8 se presenta un novedoso concepto para el diseño de sistemas afocales dinámicos. El objetivo del diseño es realizar un sistema cuyo haz de rayos de entrada pueda llegar con ángulos entre ±45º mientras que el haz de rayos a la salida sea siempre perpendicular al sistema, variando únicamente la posición de los elementos ópticos lateralmente. Las aplicaciones potenciales de este dispositivo son varias: tragaluces que proporcionan iluminación natural, sistemas de concentración fotovoltaica integrados en los edificios o iluminación direccionable con LEDs. Finalmente, el último capítulo contiene las conclusiones y las líneas de investigación futura.

Source terms for calculations of vaporizing and burning fuel sprays with non-unity Lewis numbers in gases with temperature-dependent thermal conductivities

Liquid-fueled burners are used in a number of propulsion devices ranging from internal combustion engines to gas turbines. The structure of spray flames is quite complex and involves a wide range of time and spatial scales in both premixed and non-premixed modes (Williams 1965; Luo et al. 2011). A number of spray-combustion regimes can be observed experimentally in canonical scenarios of practical relevance such as counterflow diffusion flames (Li 1997), as sketched in figure 1, and for which different microscalemodelling strategies are needed. In this study, source terms for the conservation equations are calculated for heating, vaporizing and burning sprays in the single-droplet combustion regime. The present analysis provides extended formulation for source terms, which include non-unity Lewis numbers and variable thermal conductivities.

Experimental measurement of switched reluctance motor non-linear characteristics

For a Switched Reluctance Motor (SRM), the flux linkage characteristic is the most basic magnetic characteristic, and many other quantities, including the incremental inductance, back emf, and electromagnetic torque can be determined indirectly from it. In this paper, two methods of measuring the flux linkage profile of an SRM from the phase winding voltage and current measurements, with and without rotor locking devices, are presented. Torque, incremental inductance and back emf characteristics of the SRM are then obtained from the flux linkage measurements. The torque of the SRM is also measured directly as a comparison, and the closeness of the calculated and directly measured torque curves suggests the validity of the method to obtain the SRM torque, incremental inductance and back emf profiles from the flux linkage measurements. © 2013 IEEE.

Rotation invariant iris feature extraction using Gaussian Markov random fields with non-separable wavelet

Rotation invariance is important for an iris recognition system since changes of head orientation and binocular vergence may cause eye rotation. The conventional methods of iris recognition cannot achieve true rotation invariance. They only achieve approximate rotation invariance by rotating the feature vector before matching or unwrapping the iris ring at different initial angles. In these methods, the complexity of the method is increased, and when the rotation scale is beyond the certain scope, the error rates of these methods may substantially increase. In order to solve this problem, a new rotation invariant approach for iris feature extraction based on the non-separable wavelet is proposed in this paper. Firstly, a bank of non-separable orthogonal wavelet filters is used to capture characteristics of the iris. Secondly, a method of Markov random fields is used to capture rotation invariant iris feature. Finally, two-class kernel Fisher classifiers are adopted for classification. Experimental results on public iris databases show that the proposed approach has a low error rate and achieves true rotation invariance. © 2010.

Prétraitement d'un isolat de protéines de lin par haute pression hydrostatique : impacts sur la structure protéique, l'hydrolyse enzymatique et les capacités antioxydantes des hydrolysats finaux

Les graines de lin sont des oléagineux largement cultivés au Canada. Cependant, les résidus générés suite au processus d’extraction de l’huile contiennent une importante quantité de protéines et peuvent être valorisées dans l’alimentation humaine en raison, principalement, de certaines fractions peptidiques possédant des propriétés bioactives. Dans le cadre de ce travail, l’influence des hautes pressions hydrostatiques (HPH) sur un isolat de protéines de lin a été étudiée concernant les modifications de la structure protéique, l’hydrolyse enzymatique ainsi que l’activité antioxydante des hydrolysats. Ainsi, des solutions protéiques de lin (1% m/v) ont été soumises à un traitement de HPH à 600 MPa pendant 5 et 20 minutes, à 20°C et comparés à des échantillons non-pressurisés. Deux traitements subséquents d’hydrolyse ont été effectués suite au traitement ou non de pressurisation : une première hydrolyse trypsique suivie d’une deuxième par la pronase. Dans un premier temps, la caractérisation de l’isolat protéique de lin pressurisé et non pressurisé a été réalisée par spectrofluorimétrie et par une analyse de la taille des particules afin d’étudier l’effet de la pressurisation sur les HPH la matrice protéique végétale. Par la suite, les hydrolysats protéiques ont été caractérisés par HPLC-MS et leur capacité antioxydante a été déterminée par ORAC. Les résultats ont démontré que le niveau de pressurisation et la durée du traitement ont un impact sur la structure protéique en induisant la dissociation des protéines, et la formation d’agrégats. Ceux-ci seraient occasionnés par la décompression ou créés durant l’entreposage des isolats. Suite à l’hydrolyse enzymatique des solutions protéiques pressurisées ou non par la trypsine seule et par la trypsine-pronase, les analyses chromatographiques ont révélé que la concentration de certains peptides a été modifiée lorsque la trypsine seule était utilisée après un traitement à HPH. Enfin, les HPH ont amélioré la capacité antioxydante des hydrolysats obtenus lors de l’hydrolyse trypsine-pronase comparativement au contrôle non-pressurisé.

Commande optimale d'un satellite à propulsion électrique utilisant les perturbations pour l'élimination des débris orbitaux en basse altitude

Depuis le lancement du premier satellite Spoutnik 1 en 1957, l’environnement spatial est de plus en plus utilisé et le nombre de débris orbitaux se multiplie naturellement, soit par des explosions, des collisions ou tout simplement par les opérations normales des satellites. Au-delà d'un certain seuil, la densité des débris orbitaux risque de créer une réaction en chaîne incontrôlée : l’effet Kessler. L’élimination des débris orbitaux en basse altitude permettrait de limiter cette réaction et ainsi de préserver l’environnement spatial afin de pouvoir l’utiliser de façon sécuritaire. L’élimination des débris orbitaux est une opération complexe et coûteuse. Elle consiste à déplacer des objets spatiaux inactifs vers une orbite basse pour mener à leur désintégration dans la basse atmosphère terrestre. En utilisant les perturbations orbitales, il est possible de réduire le coût du carburant requis pour effectuer les manœuvres orbitales nécessaires à l’élimination de ces débris. L'objectif principal de cette étude consiste à développer une procédure et une stratégie de commande autonome afin de modifier l'orbite des satellites non opérationnels (débris) pour mener à leur désintégration naturelle tout en optimisant les facteurs carburant et temps. Pour ce faire, un modèle d’atmosphère basé sur le modèle de Jacchia (1977) est développé. Un modèle de la dynamique du satellite inclut aussi les perturbations principales, soit : traînée atmosphérique, non sphéricité et distribution non uniforme de la masse de la Terre. Ces modèles ainsi qu'un algorithme de commande optimale pour un propulseur électrique sont développés et le tout est validé par simulations numériques sur Matlab/Simulink. Au terme de cette étude, les conditions optimales dans lesquelles il faut laisser un débris afin qu'il se désintègre dans la basse atmosphère de la Terre en quelques semaines seront données (type d'orbite : altitude, inclinaison, etc.) ainsi que le coût en carburant pour une telle mission. Cette étude permettra de prouver qu'il est possible de réaliser des missions d'élimination des débris orbitaux tout en réduisant les coûts associés aux manœuvres orbitales par l'utilisation des perturbations naturelles de l'environnement.

Oligodeoxynucleotide synthesis using protecting groups and a linker cleavable under non-nucleophilic conditions

Oligodeoxynucleotides (ODNs) containing latent electrophilic groups can be highly useful in antisense drug development and many other applications such as chemical biology and medicine, where covalent cross-linking of ODNs with mRNA, protein and ODN is required. However, such ODN analogues cannot be synthesized using traditional technologies due to the strongly nucleophilic conditions used in traditional deprotection/cleavage process. To solve this long lasting and highly challenging problem in nucleic acid chemistry, I used the 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) function to protect the exo-amino groups on the nucleobases dA, dC and dG, and to design the linker between the nascent ODN and solid support. These protecting groups and linker are completely stable under all ODN synthesis conditions, but can be readily cleaved under non-nucleophilic and nearly neutral conditions. As a result, the new ODN synthesis technology is universally useful for the synthesis of electrophilic ODNs. The dissertation is mainly comprised of two portions. In the first portion, the development of the Dmoc-based linker for ODN synthesis will be described. The construction of the dT-Dmoc-linker required a total of seven steps to synthesize. The linker was then anchored to the solid support―controlled pore glass (CPG). In the second portion, the syntheses of Dmoc-protected phosphoramidites ODN synthesis monomers including Dmoc-dC-amidite, Dmoc-dA-amidite, Dmoc-dG-amidite are described. The protection of dC and dA with 1,3-dithian-2-yl-methyl 4-nitrophenyl carbonate proceeded smoothly giving Dmoc-dC and Dmoc-dA in good yields. However, when the same acylation procedure was applied for the synthesis of Dmoc-dG, very low yield was obtained. This problem was later solved using a highly innovative and environmentally benign procedure, which is expected to be widely useful for the acylation of the exo-amino groups on nucleoside bases. The reactions to convert the Dmoc-protected nucleosides to phosphoramidite monomers proceeded smoothly with high yields. Using the Dmoc phosphoramidite monomers dA, dC, dG and the commercially available dT, and the Dmoc linker, four ODN sequences were synthesized. In all cases, excellent coupling yields were obtained. ODN deprotection/cleavage was achieved by using non-nucleophilic oxidative conditions. The new technology is predicted to be universally useful for the synthesis of ODNs containing one or more electrophilic functionalities.

The role of non-coding RNA in the development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH.

Visualising In/Equalities through Contemporary Documentary Cinema. A Diffractive Reading of Feminist Practices in Spanish and Italian Non-Fiction Films

This thesis brings together feminist documentary film theory and feminist new materialism(s) to describe how feminist material-discursive practices in a sample of Spanish and Italian documentary cinema made between 2013-2018 (can) visualise gender in/equalities. The accomplished objectives have been: 1. Building a bridge between feminist documentary film theory and Karen Barad’s diffractive methodology by approaching non-fiction cinema that deals with social inequalities as a diffraction apparatus. 2. Developing a feminist toolbox for a response-able gaze by gathering different insights from feminist film theory. 3. Identifying feminist material-discursive practices in a sample of documentary films produced in Spain and Italy over the last six years (2013-2018). 4. Analysing the effects that these feminist material-discursive practices in documentary cinema have, particularly in terms of visualising gender in/equalities on both sides of the camera and on both sides of the screen. 5. Revealing patterns between the ten case studies by reading through one another (i.e. diffractively) insights raised in each one of them. In ten documentary films/case studies, I identify patterns of continuities and differences concerning feminist material-discursive practices at four levels: content, form, production and reception. In terms of contents, I detect two patterns in which feminist material-discursive practices may operate: enacting the right to appear or enacting the right to look back and/or against the grain. As for the forms, I exemplify how feminism politicises Bill Nichols’s six modes of representation. My analysis of production practices is elaborated along the filmmakers’ self-positions/situatedness, tensions/obstructions, and effects/affects/emotions regarding four key concepts: documentary cinema, equality, gender and feminism(s). And in the case of reception practices, I identify patterns of affective identification and/or intellectual reflections.

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.