L'épissage des ARN pré-messagers est une étape essentielle pour l'expression des gènes chez les eucaryotes supérieurs. La reconnaissance des exons par la machinerie d'épissage est réalisée grâce à différents éléments cis-régulateurs incluant les séquences consensus d'épissage et les séquences auxiliaires activatrices ou inhibitrices d'épissage. Le pré-ARNm représente une nouvelle cible thérapeutique pour le traitement des maladies génétiques. L'approche du saut d'exon thérapeutique, destinée à restaurer l'expression d'une protéine totalement ou partiellement fonctionnelle en interférant avec le processus d'épissage, suscite un grand intérêt notamment pour la dystrophie musculaire de Duchenne où la modification du transcrit permettrait d'obtenir une forme modérée de la maladie, la Dystrophie musculaire de Becker. Des oligonucléotides antisens sont utilisés pour masquer les signaux d'épissage de reconnaissance d'un exon par le spliceosome, et induire son excision (ou saut) du transcrit mature. La détermination de la meilleure séquence cible des AONs est une difficulté majeure de cette approche. Pour le gène DMD, nous avons pu établir grâce à des analyses bioinformatiques et statistiques combinées avec des tests fonctionnels utilisant des minigènes rapporteurs d'épissage, que le ciblage de motifs exoniques qui fixent le facteur d'épissage SF2/ASF permettait d'obtenir la meilleure efficacité des AONs. Par ailleurs, nous avons exploré la régulation de l'épissage des pseudoexons dans le gène DMD, et notamment les mécanismes conduisant à l'inclusion de ces séquences introniques dans le transcrit mature en condition pathologique. L'étude de deux cas exceptionnels d'activation de pseudoexons associée à des remaniements introniques rares (double délétion, inversion) élargit le spectre des mutations à l'origine de ces défauts d'épissage, et illustre le rôle encore mal connu des remaniements introniques en pathologie humaine. / Splicing of pre-messenger RNAs to mature transcripts is a crucial step in eukaryotic gene expression. The recognition of exon by the splicing machinery involves different cis-regulatory elements, including the splice site motifs and auxiliary sequences, which can act by stimulating or repressing splicing. The pre-mRNA represents a new therapeutic target for the treatment of genetic diseases. Notably, the exon skipping strategy is currently one of the most promising therapeutic approaches for the Duchenne muscular dystrophy. It intends to restore the expression of a partially functional protein by interfering with the splicing process, and converts the severe DMD phenotype into the moderate form of the disease, Becker muscular Dystrophy (BMD). Antisense oligonucleotides are used to mask the splicing signals involved in exon recognition by the spliceosome to induce its skipping from the mature transcript and restore an open reading frame. The determination of the best target sequence of the AONs is one of the major hurdles to overcome. For the DMD gene, a bioinformatic and statistical analysis combined with minigenes studies allowed us to establish that targeting binding sites for the splicing factor SF2/ASF maximizes the AONs efficiency. In a second part of this work, we investigated the splicing regulation of pseudoexons in the DMD gene, in particular the mechanisms leading to the inclusion of these intronic sequences in the mature transcript in pathological conditions. The study of two exceptional cases of pseudoexons activation associated with rare intronic rearrangements (double-deletions, inversion) expands the spectrum of missplicing mutations, and demonstrates the potential role of pure intronic rearrangements in human pathology.

Nesta tese, uma família de memórias associativas bidirecionais caóticas (família C-BAM) é proposta, implementada e testada com o objetivo de estender a relevância da presença e do estudo do fenômeno caótico a modelos de redes associativas. Na modelagem da família C-BAM, todos os neurônios da memória associativa bidirecional caótica (BAM), BAM com atraso e BAM exponencial (eBAM) foram substituídos por neurônios caóticos. Cada parâmetro do neurônio caótico na família C-BAM tem sua influência estimada através do planejamento de experimentos, em diferentes dinâmicas. Com base no planejamento de experimentos, valores de parâmetros são selecionados a fim de ilustrar a emergência de comportamentos dinâmicos como bifurcação, caos determinístico e crise. A existência de dinâmicas caóticas é confirmada pelo cálculo dos expoentes de Lyapunov. Experimentos empíricos mostraram que a dinâmica caótica modifica a acessibilidade à memória da família C-BAM. Ao invés de recuperar um único par, como a família BAM fazia, a versão caótica é capaz de gerar uma grande diversidade de padrões recuperados, envolvendo complexas transições entre os padrões armazenados, para algumas variações paramétricas. Tal comportamento permite à família C-BAM acessar padrões inacessíveis às redes BAMs originais. Além disso, a nova acessibilidade à memória, na qual seqüências de recuperação (com diferentes tamanhos) compostas de padrões treinados e não treinados têm emergido, pode ser usada para modelar a habilidade de um indivíduo saltar passos na solução de uma tarefa. Esta tese seleciona a rede C-BAM para ilustrar que a seqüência de recuperação da rede pode modelar a habilidade de um noviço ou a habilidade de um especialista executar uma tarefa. Embora a família C-BAM possa alcançar todos os padrões armazenados durante o comportamento caótico, ela não consegue convergir para um padrão específico. Duas estratégias de controle são propostas para permitir que as redes caóticas convirjam para a memória desejada: o método de controle por pinagem e um método de controle adaptativo. Conseqüentemente, os modelos C-BAM podem, de fato, realizar a hetero-associação de memórias antes inacessíveis, e a rede C-BAM pode estabilizar-se no estado final de uma tarefa, dado o primeiro estado / In this thesis, a family of bidirectional associative memories (C-BAM family) is proposed, implemented and tested to extend the study of chaotic phenomenon in associative models. In the C-BAM model, all the original neurons of bidirectional associative memory (BAM), BAM with delay and exponenetial BAM (eBAM) were substituted for chaotic neurons. Based on the experimental design, values of C-BAM family parameters are set to illustrate the emergence of a diversity of dynamic behavior, such as bifurcation, deterministic chaos and crisis. The existence of the chaotic dynamics is confirmed by calculation of Lyapunov exponents. Empiric experiments showed that the chaotic dynamics modifies the behavior of memory accessibility. Instead of recalling a single pair, as BAM did, its chaotic version yielded a wide diversity of recalled patterns, involving complex transitions via memorized patterns for some parametric variations. Hence, C-BAM family can access patterns that original BAM family cannot. Moreover, the new way of memory accessibility, in which several recall sequences (with distinct sizes) composed of trained and nontrained patterns have emerged, can be used to model the ability of skipping steps by an individual in a task solution. This thesis selected C-BAM network to illustrate that the retrieval sequence can model the ability of a novice or the ability of an expert to execute a task. There are also illustrated cases in which a novice recall can be transformed into an expert recall through parametric variation. Although C-BAM family can reach all stored patterns during the chaotic behavior, it can not converge towards a specific pattern, consequently a desired output is not produced. In this thesis, two control strategies are proposed in order to make the chaotic networks to converge towards the desired memory: the pinning control method and the adaptive control method. Consequently, the C-BAM models can effectively realize the correct heteroassociation to former non-accessible memories and the C-BAM network can quickly be stabilized in the final state of a task, given the first state

Reading is a complex cognitive task based on the analyses of visual stimuli. Due to the physiology of the eye, only a small number of letters around the fixation position can be extracted with high visual acuity, while the visibility of words and letters outside this so-called foveal region quickly drops with increasing eccentricity. As a consequence, saccadic eye movements are needed to repeatedly shift the fovea to new words for visual word identification during reading. Moreover, even within a foveated word fixation positions near the word center are superior to other fixation positions for efficient word recognition (O’Regan, 1981; Brysbaert, Vitu, and Schroyens, 1996). Thus, most reading theories assume that readers aim specifically at word centers during reading (for a review see Reichle, Rayner, & Pollatsek, 2003). However, saccades’ landing positions within words during reading are in fact systematically modulated by the distance of the launch site from the word center (McConkie, Kerr, Reddix, & Zola, 1988). In general, it is largely unknown how readers identify the center of upcoming target words and there is no computational model of the sensorimotor translation of the decision for a target word into spatial word center coordinates. Here we present a series of three studies which aim at advancing the current knowledge about the computation of saccade target coordinates during saccade planning in reading. Based on a large corpus analyses, we firstly identified word skipping as a further factor beyond the launch-site distance with a likewise systematic and surprisingly large effect on within-word landing positions. Most importantly, we found that the end points of saccades after skipped word are shifted two and more letters to the left as compared to one-step saccades (i.e., from word N to word N+1) with equal launch-site distances. Then we present evidence from a single saccade experiment suggesting that the word-skipping effect results from highly automatic low-level perceptual processes, which are essentially based on the localization of blank spaces between words. Finally, in the third part, we present a Bayesian model of the computation of the word center from primary sensory measurements of inter-word spaces. We demonstrate that the model simultaneously accounts for launch-site and saccade-type contingent modulations of within-word landing positions in reading. Our results show that the spatial saccade target during reading is the result of complex estimations of the word center based on incomplete sensory information, which also leads to specific systematic deviations of saccades’ landing positions from the word center. Our results have important implications for current reading models and experimental reading research. / Lesen ist eine komplexe kognitive Aufgabe, die auf der Analyse visueller Reize beruht. Aufgrund der Physiologie des Auges kann jedoch nur eine kleine Anzahl von Buchstaben um den Fixationsort mit hoher visueller Genauigkeit wahrgenommen werden, während die Sichtbarkeit der Buchstaben und Wörter außerhalb der sogenannten fovealen Zone mit zunehmender Entfernung stark abnimmt. Während des Lesens sind deshalb sakkadische Augenbewegungen erforderlich, um die Fovea zur visuellen Identifikation neuer Wörter wiederholt innerhalb des Textes zu verschieben. Auch innerhalb eines direkt betrachteten Wortes erlauben mittige Fixationsorte eine effizientere Wortverarbeitung als randnahe Blickpositionen (O’Regan, 1981; Brysbaert, Vitu, and Schroyens, 1996). Die meisten Lesemodelle nehmen deshalb an, dass Leser auf die Mitte von Worten zielen (für eine Übersicht siehe Reichle, Rayner, & Pollatsek, 2003). Es zeigt sich aber, dass Landepositionen innerhalb von Wörtern im Lesen von der Distanz der Startposition einer Sakkade zur Mitte des Zielwortes moduliert werden (McConkie, Kerr, Reddix, & Zola, 1988). Noch ist weitgehend unklar, wie Leser die Mitte eines Zielwortes identifizieren. Es fehlt an computationalen Modellen die die sensumotorische Umwandlung der Auswahl eines Zielwortes in eine räumliche Koordinate der Wortmitte beschreiben. Wir präsentieren hier eine Reihe von drei Studien, die darauf abzielen, das Wissen über die Berechnung von Sakkadenzielkoordinaten im Lesen zu erweitern. In einer umfangreichen Korpusanalyse identifizerten wir zunächst das Überspringen von Wörtern als weiteren wichtigen Faktor bei der Sakkadenprogrammierung, der einen ähnlich systematischen und großen Effekt auf die Landepositionen hat wie die Startpositionen der Sakkaden. Anschließend zeigen wir Ergebnisse eines einfachen Sakkadenexperiments, welche nahelegen, dass der Effekt übersprungener Wörter das Ergebnis hoch automatisierter perzeptueller Prozesse ist, die wesentlich auf der Bestimmung von Leerzeichen zwischen Wörtern basieren. Schließlich präsentieren wir ein Bayesianisches Modell der Berechnung von Wortmitten auf der Grundlage der primären sensorischen Erfassungen von Leerzeichen zwischen Wörtern. Wir zeigen, dass das Modell gleichzeitig Effekte der Startposition und des Sakkadentyps erklärt. Unsere Arbeiten zeigen, dass die Berechnung räumlicher Koordinaten für die Sakkadenprogrammierung im Lesen auf einer komplexen Schätzung der Wortmitte anhand unvollständiger sensorischer Informationen beruht, die zu systematischen Abweichungen von der tatsächlichen Wortmitte führt. Unsere Ergebnisse haben wichtige Folgen für gegenwärtige Lesemodelle und für die experimentelle Leseforschung.

Après la transcription, le pré ARNm subit des étapes de maturation avant de sortir du noyau pour être traduit. Une des étapes de maturation est l'épissage. Il permet de souder les séquences codantes de l'ARNm entre elles (les exons) et d'exclure les régions non codantes (les introns).Des mutations génétiques sont à l'origine de défaut d'épissage. Elles peuvent conduire à des rétentions d'intron, des sauts d'exon et des inclusions de séquences introniques appelées pseudo exons.Ma thèse a porté sur l'utilisation du saut d'exon pour corriger ces inclusions. Je me suis intéressé à deux pathologies : la myopathie à cores et le syndrome de LowePour le premier cas, je me suis intéressé à une mutation dans l'intron 101 du gène RyR1. Cette mutation est à l'origine de la création d'un site donneur d'épissage qui active un site accepteur provoquant l'inclusion d'un pseudo exon de 99 nucléotides. Cette inclusion induit une baisse de la quantité du canal calcique RyR1 dans les cellules du patient. Ce canal permet le couplage entre l'excitation et la contraction musculaire. Ses défauts conduisent à diverses myopathies dont la myopathie à cores. Le patient présentait une hypotonie néonatale, une scoliose et des défauts respiratoires, et n'a jamais acquis la marche. J'ai dessiné des oligonucléotides, je les ai transfectés dans les cellules du patient en culture et ainsi montré par RT PCR que le saut du pseudo exon était possible. Afin d'optimiser l'efficacité pour pouvoir évaluer la restauration au niveau protéique et fonctionnel, j'ai développé un lentivirus exprimant une cassette U7 SmOPT avec les AON choisis. Après transduction des cellules, j'ai pu montrer que le saut du pseudo exon permettait le retour de la protéine et de sa fonctionnalité, cette approche pourrait donc permettre une correction chez le patient.Pour le deuxième cas, j'ai tenté de corriger une mutation du gène OCRL. Cette mutation crée un site donneur d'épissage dans l'intron 4 du gène OCRL et active un site accepteur d'épissage 66 nucléotides en amont. L'inclusion du pseudo exon induit la chute du taux de transcrit OCRL par un mécanisme de "Non sense mediated mRNA Decay". OCRL est une phosphatidyl inositol 5 Phosphatase permettant de réguler la quantité de Ptd Ins(4,5)P2 dans la cellule. Les défauts dans OCRL sont responsables d'une pathologie multisystémique, le syndrome de Lowe. J'ai pu obtenir des fibroblastes cutanés du patient. J'ai transfecté ces cellules avec des AONs choisis pour permettre un saut de l'exon pathogène. J'ai ensuite intégré la séquence des AONs efficaces dans un lentivirus U7. J'ai transduit les cellules du patient en culture et observé un retour de la protéine et un retour de l'activité enzymatique, cette approche pourrait donc théoriquement permettre une correction chez le patient.Ces deux travaux sont les premières preuves de principes de thérapie par modulation de l'épissage pour les myopathies congénitales et pour le syndrome de Lowe. Ils ouvrent la voie à des perspectives de traitement pour ces maladies génétiques. / After transcription, the pre mRNA will undergo different maturation step before getting out of the nucleus for translation. One of these step of maturation is the splicing. It allows to concatenate the coding sequences of the mRNA (the exons) and induces the exclusion of the non coding sequences (the introns).Genetics mutations can lead to splicing defects. These defects could be intron retention, exon skipping and exonisation of intronic sequences called pseudo exons.My thesis work was to evaluate the exon skipping therapy to correct these exonisation. I focuses on two diseases: core myopathy and Lowe syndrome.For the first one, my interest was on a mutation in the 101 th intron of RYR1 gene. This mutation creates a splicing donor site wich unveils a cryptic acceptor site. This leads to the inclusion of a 99 nucleotides pseudo exon. This inclusion induces a decrease of the quantity of the calcium channel RyR1 in the patient cells. This channel allows the excitation-contraction coupling, and therefore the muscular contraction. Defects in this channel lead to different myopathies (eg. core myopathy). The patient present at birth a major neonatal hypotonia, scoliosis and respiratory defects. He has never walked. I designed oligonucleotides (AON), transfected them in the cultured patient cells and showed by RT PCR that exon skipping was possible. In order to optimise the efficiency and to evaluate the rescue at a protein level and at a fonctionnal level, I devellopped a lentivirus which express a U7 Sm OPT cassette with the choosen AONs. After cell transduction, I have shown that exon skipping allowed the rescue of the protein and of its functionnality. This approach could permit a genetic correction for the patientFor the second case, I have tried to correct an OCRL mutation. This upstream creates a splicing donor site and unveils an acceptor site 66 nucleotides before. This leads to the inclusion of a pseudo exon which induces a severe decrease of OCRL transcripts level due to a "non sense mediated mRNA Decay". OCRL is a phosphatidyl inositol 5 Phosphatase, which regulates the Ptd Ins(4,5)P2 pool in the cell. OCRL defects induces a multi systemic disease the Lowe syndrome. I obtained patient cutaneous fibroblasts. I transfected these cells with choosen AONs to correct the splicing defect. I integrated the AONs sequence into a U7 lentiviral cassette. I transduced the cultured patient cells and observed a rescue of the protein with a rescue of its activity. This approach could, theoritically permit a correction in the patient.These two studies are the first proof of concept of splicing modulation therapy for congenital myopathy and for Lowe syndrome. This work offers a lot of perspective for the tratment of these genetic illness

Nesta tese, uma família de memórias associativas bidirecionais caóticas (família C-BAM) é proposta, implementada e testada com o objetivo de estender a relevância da presença e do estudo do fenômeno caótico a modelos de redes associativas. Na modelagem da família C-BAM, todos os neurônios da memória associativa bidirecional caótica (BAM), BAM com atraso e BAM exponencial (eBAM) foram substituídos por neurônios caóticos. Cada parâmetro do neurônio caótico na família C-BAM tem sua influência estimada através do planejamento de experimentos, em diferentes dinâmicas. Com base no planejamento de experimentos, valores de parâmetros são selecionados a fim de ilustrar a emergência de comportamentos dinâmicos como bifurcação, caos determinístico e crise. A existência de dinâmicas caóticas é confirmada pelo cálculo dos expoentes de Lyapunov. Experimentos empíricos mostraram que a dinâmica caótica modifica a acessibilidade à memória da família C-BAM. Ao invés de recuperar um único par, como a família BAM fazia, a versão caótica é capaz de gerar uma grande diversidade de padrões recuperados, envolvendo complexas transições entre os padrões armazenados, para algumas variações paramétricas. Tal comportamento permite à família C-BAM acessar padrões inacessíveis às redes BAMs originais. Além disso, a nova acessibilidade à memória, na qual seqüências de recuperação (com diferentes tamanhos) compostas de padrões treinados e não treinados têm emergido, pode ser usada para modelar a habilidade de um indivíduo saltar passos na solução de uma tarefa. Esta tese seleciona a rede C-BAM para ilustrar que a seqüência de recuperação da rede pode modelar a habilidade de um noviço ou a habilidade de um especialista executar uma tarefa. Embora a família C-BAM possa alcançar todos os padrões armazenados durante o comportamento caótico, ela não consegue convergir para um padrão específico. Duas estratégias de controle são propostas para permitir que as redes caóticas convirjam para a memória desejada: o método de controle por pinagem e um método de controle adaptativo. Conseqüentemente, os modelos C-BAM podem, de fato, realizar a hetero-associação de memórias antes inacessíveis, e a rede C-BAM pode estabilizar-se no estado final de uma tarefa, dado o primeiro estado / In this thesis, a family of bidirectional associative memories (C-BAM family) is proposed, implemented and tested to extend the study of chaotic phenomenon in associative models. In the C-BAM model, all the original neurons of bidirectional associative memory (BAM), BAM with delay and exponenetial BAM (eBAM) were substituted for chaotic neurons. Based on the experimental design, values of C-BAM family parameters are set to illustrate the emergence of a diversity of dynamic behavior, such as bifurcation, deterministic chaos and crisis. The existence of the chaotic dynamics is confirmed by calculation of Lyapunov exponents. Empiric experiments showed that the chaotic dynamics modifies the behavior of memory accessibility. Instead of recalling a single pair, as BAM did, its chaotic version yielded a wide diversity of recalled patterns, involving complex transitions via memorized patterns for some parametric variations. Hence, C-BAM family can access patterns that original BAM family cannot. Moreover, the new way of memory accessibility, in which several recall sequences (with distinct sizes) composed of trained and nontrained patterns have emerged, can be used to model the ability of skipping steps by an individual in a task solution. This thesis selected C-BAM network to illustrate that the retrieval sequence can model the ability of a novice or the ability of an expert to execute a task. There are also illustrated cases in which a novice recall can be transformed into an expert recall through parametric variation. Although C-BAM family can reach all stored patterns during the chaotic behavior, it can not converge towards a specific pattern, consequently a desired output is not produced. In this thesis, two control strategies are proposed in order to make the chaotic networks to converge towards the desired memory: the pinning control method and the adaptive control method. Consequently, the C-BAM models can effectively realize the correct heteroassociation to former non-accessible memories and the C-BAM network can quickly be stabilized in the final state of a task, given the first state

Environ 6000 à 8000 maladies rares différentes existent aujourd’hui, affectant environ 6 à 8% de la population mondiale. La grande majorité d’entre elles correspond à des maladies génétiques, pour lesquelles il n'existe pas de traitement curatif. La révolution génomique a augmenté l’espoir d’obtenir des traitements spécifiques du défaut génétique pour de nombreuses maladies. Dans ces conditions, le traitement et l’analyse des données ne sont pas triviaux et s’éloignent de la simple routine.Cette thèse rapporte la création de Saut d'exon, capables d’aider les chercheurs et les cliniciens à identifier les mutations responsables de certaines maladies et développer de nouvelles stratégies thérapeutiques. Ainsi, les systèmes Human Splicing Finder et UMD-Predictor permettent respectivement de prédire l’effet d’une mutation sur l’épissage et la fonction de la protéine. Ils ont été validés grâce à des jeux de données de référence et/ou issus de la littérature, et peuvent aider les cliniciens à annoter correctement les variations de signification inconnue. De plus, cette thèse propose deux outils à visées thérapeutiques : Skip-E, un outil d’identification des AON candidats à la thérapie par saut d’exon, et NR-Analyser, un système de prédiction des codons de terminaison prématurés candidats à la thérapie par translecture des codons stop.Ces différents systèmes s'intègrent dans un projet plus global dédié à la recherche translationnelle. Par ces deux volets, prédictif et thérapeutique, cette thèse s’inscrit dans une stratégie de recherche en adéquation avec les objectifs du Consortium International pour la Recherche contre les Maladies Rares, l’IRDiRC. / About 6,000 to 8,000 distinct rare diseases exist today and are estimated to affect 6-8% of the world population. The vast majority of them are genetic and for most of them there is no cure. The genomic revolution has increased the hope of specific treatments based on the gene for many diseases. New technologies have emerged, changing drastically data scale produced in biomedical research. In these conditions, treatment and analysis of data are far from trivial and mere routine, despite spectacular advances in computer technology.This thesis reports the creation of bioinformatics systems, capable of helping researchers and clinicians to identify mutations responsible for certain diseases and to develop new therapies. Thus, the Human Splicing Finder and UMD-Predictor systems predict the effect of a mutation on splicing and protein, respectively. Both bioinformatics systems have been validated through high quality reference datasets, and may help clinicians to properly annotate variations of unknown significance. In addition, this thesis offers two new systems for therapeutic purposes: the Skip-E system identifies optimal candidates AONs for exon skipping therapies, and NR-Analyser, a system that predicts premature termination codons potentially candidates to nonsense readthrough therapies.These different systems are part of a larger project dedicated to translational research. With its predictive and therapeutic aspects, this thesis is part of a research strategy matching with the objectives of the IRDiRC (International Rare Diseases Research Consortium).

Les dysferlinopathies sont des dystrophies musculaires qui se manifestent par la dystrophie musculaire des ceintures de type 2B (LGMD2B) ou la myopathie de Miyoshi (MM). Elles sont causées par des mutations dans le gène dysferline. La dysferline est une protéine membranaire exprimée dans le muscle squelettique, responsable de la réparation des microlésions du sarcolemme. L’absence d’une telle réparation de la membrane entraîne une atrophie musculaire progressive. Ce travail de thèse explore le potentiel thérapeutique d'une stratégie de modulation d'épissage pour le traitement de la LGMD2B causée par la mutation faux-sens c4022T>C dans l'exon 38 du gène dysferline. Des oligonucléotides et des petits ARN U7 délivrés par un vecteur viral de type adéno-associé ont été utilisés comme outils antisens pour induire un saut d'exon in vitro et in vivo. Ce projet de thèse étudie également la capacité de la dysferline tronquée à se localiser de façon appropriée à la membrane et ainsi la réparer. / Dysferlinopathy is a muscular dystrophy that manifests as two major phenotypes: limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi myopathy (MM). It is caused by mutations in the dysferlin gene. Dysferlin is a membrane protein expressed in skeletal muscle. It is responsible for the repair of sarcolemma microlesions produced by muscle contractions. A compromised membrane repair leads to slowly progressing muscle wasting. This thesis explores the therapeutic potential of an antisense mediated splice switching strategy in LGMD2B caused by the missense mutation c4022T>C in the exon 38 of the dysferlin gene. Antisense oligonucleotides and U7 snRNAs delivered by an adeno-associated viral vector were used as antisense tools to trigger exon skipping in vitro and in vivo. The thesis investigates also if the truncated dysferlin maintainsa proper membrane localization and its membrane repair ability.

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The world is heading towards an energy crisis and desperate efforts are being made to find an alternative, reliable and clean source of energy. Solar Energy is one of the most clean and reliable source of renewable energy on earth. Conventionally, extraction of solar power for electricity generation was limited to PV farms, however lately Distributed Generation form of Solar Power has emerged in the form of residential and commercial Grid Tied Micro-Inverters. Grid Tied Micro-Inverters are costly when compared to their string type counterparts because one inverter module is required for every single or every two PV panels whereas a string type micro-inverter utilizes a single inverter module over a string of PV panels. Since in micro-inverter every panel has a dedicated inverter module, more power per panel can be extracted by performing optimal maximum power tracking over single panel rather than over an entire string of panels. Power per panel extracted by string inverters may be lower than its maximum value as few of the panels in the string may or may not be shaded and thereby forming the weaker links of the system. In order to justify the higher costs of Micro-Inverters, it is of utmost importance to convert the available power with maximum possible efficiency. Typically, a microinverter consists of two important blocks; a Front End DC-DC Converter and Output DCAC Inverter. This thesis proposes efficiency optimization techniques for both the blocks of the micro-inverter. iv Efficiency Optimization of Front End DC-DC Converter This thesis aims to optimize the efficiency of the front end stage by proposing optimal design procedure for resonant parameters of LLC Topology as a Front End DC-DC Converter for PV Applications. It exploits the I-V characteristics of a solar panel to design the resonant parameters such that resonant LLC topology operates near its resonant frequency operating point which is the highest efficiency operating point of LLC Converter. Efficiency Optimization of Output DC-AC Inverter Due to continuously variable irradiance levels of solar energy, available power for extraction is constantly varying which causes the PV Inverter operates at its peak load capacity for less than 15% of the day time. Every typical power converter suffers through poor light load efficiency performance because of the load independent losses present in a power converter. In order to improve the light load efficiency performance of Three Phase Inverters, this thesis proposes Phase Skipping Control technique for Three Phase Grid Tied Micro-Inverters. The proposed technique is a generic control technique and can be applied to any inverter topology, however, in order to establish the proof of concept this control technique has been implemented on Three Phase Half Bridge PWM Inverter and its analysis is provided. Improving light load efficiency helps to improve the CEC efficiency of the inverter.

真核生物在遺傳資訊核糖核酸實際轉譯成蛋白質之前，可能受環境、序列上的特定二級結構、特定部分序列樣式……等影響，而製造出目的、功能不同的蛋白質，這項生物機制稱為選擇性接合。目前對於選擇性接合機制的形成原因、根據何項資訊作選擇性調控，尚未有全面性的研究足以判斷。本研究嘗試透過發展適當的資料探勘技術，分析大量核糖核酸序列，找出可能影響選擇性接合的序列樣式。 選擇性接合可分為七種類型，我們針對其中一類稱為跳接式選擇性接合的基因資料，根據分析該資料的特性，提出兩類型的容錯資料探勘方法與流程，分別是全序列樣式探勘與轉化重複結構樣式探勘。前者對發生跳接式選擇性接合的整段intron序列，找出所有容錯頻繁樣式。再利用Kum[18]等人提出的一致性序列樣式的近似探勘方法，找出足以代表同一群聚中所有頻繁容錯樣式的一致性序列樣式。 轉化重複結構樣式探勘的作法則是先找出intron序列的前後部分區段中，可能具有容錯轉化重複樣式的序列集合。再進行容錯頻繁樣式探勘與一致性序列樣式的近似探勘方法。由於轉化重複樣式是生物序列中常見的一種序列結構，可能透過該類型結構，影響跳接式選擇性接合的發生方式。因此利用這樣的探勘方法，我們可以找到可能的具重要決定性轉化重複結構樣式。 最後，我們對兩個選擇性接合資料集合Avatar-120和ISIS-54，進行全序列樣式探勘與轉化重複結構樣式探勘實驗，討論發掘出序列樣式的支持度及平均錯誤率。並進一步與Miriami[24]等人研究發表的兩個樣式比較，利用整體序列最佳並列排比，評估樣式間的差異性，以發掘出“新穎”的樣式。 / Before RNA sequences are translated into proteins, eukaryotes may produce different functional proteins from the same RNA sequences. It is due to influence of environment, second structure, specific substring pattern, etc. This mechanism is named alternative splicing. At present, there are still not enough research to judge causes and critical information of alternative splicing. We try to develop suitable data mining technologies to analyze large number of RNA sequences, and find out possible patterns affecting alternative splicing. Basically, there are seven possible types of alternative splicing. We focus on “exon skipping” type. According to the analysis of exon skipping data, we propose two fault-tolerant data mining methods and procedures: “Full Sequence Pattern Mining (FSPM)” and “Inverted Repeat Pattern Mining (IRPM).” Full sequence pattern mining method can be applied to mine all fault-tolerant frequent substrings in the whole intron sequences, and then get consensus sequential patterns using ApproxMap method proposed by Kum[18]. Inverted repeat pattern mining method can be used to look for consenesus patterns with structure of inverted repeat. Because inverted repeat patterns are often appeared in biological sequences and such structural patterns may result in exon skipping. We could discover some important patterns by this method. Finally, we mined patterns from two alternative splicing databsets “Avatar-120” and “ISIS-54”by above two proposed methods. The support and average fault number of mined patterns were discussed. These patterns were also used global alignment method as compared with two patterns (C / G-rich) discovered by Miriami[24]. Novel patterns measured by discrimination were reported.