This thesis describes experiments investigating the translational recoding activities and the novel dual signalling properties of eukaryotic ribosome skipping 2A sequences. Over twenty years ago, the 19 amino acid 2A region of a Picornavirus; namely, Foot-and-Mouth Disease Virus (FMDV) polypeptide was shown to possess apparent “self-cleaving” abilities, cutting at its own C-terminus during translation (Ryan et al., 1991). Active FMDV 2A-like sequences were subsequently found in a number of related viruses (Luke et al., 2008), with several now utilised as essential biotechnology multi-gene transfer tools (Luke et al., 2010b). Then, in 2006, eukaryotic 2A-like sequences were identified from trypanosome non-LTR sequences. These were found to be functional in vitro (Heras et al., 2006). I have been able to identify over 400 putative eukaryotic 2A-like sequences through searching the freely available online proteomic and genomic databases. Data is presented to show that these 2As were encoded in frame with non-LTRs, or metabolic, or immune function genes, from a wide range of eukaryotic organisms; but I could not discern any obvious phylogenetic distribution for 2A. I have discovered that the majority of eukaryotic 2A sequences tested can mediate ribosome skipping in vitro. Modelling in silico indicated that active 2A-like sequences possessed the propensity to form a central alpha-helical region, whereas the models suggested that inactive 2A-like sequences would be essentially unstructured. I also report that some of these eukaryotic 2A peptides constitute a novel form of dual protein targeting as they play a dual role as exocytic pathway signal peptides mediating extracellular protein trafficking. I have shown that this protein trafficking ability is evolutionarily conserved, with an echinoderm sequence able to direct protein targeting in both plant and mammalian cells. I therefore propose that these novel eukaryotic 2A sequences could potentially become extremely valuable in biotechnological engineering.

Doctor of Philosophy / Department of Psychological Sciences / Laura A. Brannon / Two common types of advertisement avoidance behaviors in digital domains are skipping and zipping. Skipping involves pressing a “skip ad” button when viewing television content online, and zipping involves pressing a fast-forward button when viewing the same content through some type of recording device (e.g. Digital Video Recording device). The purpose of these studies was to examine if specific factors regarding the content of the advertisement, the persuasion context, and characteristics of the viewer reduce occurrences of skipping and zipping behavior. Participants in these two studies saw a combination of television shows and advertisements. One target advertisement marketed a fictional MP3 player while another discussed the dangers of binge drinking. One version each of the MP3 and binge drinking advertisements contained average-looking (i.e. normal) actors, and the other half contained above-average-looking (i.e. attractive) actors. Half of the viewers were allowed to choose which type of advertisements they would watch while the other half were forced to watch a particular type. The results of one study showed that participants were more likely to skip the MP3 advertisement than the binge drinking advertisement after making an advertisement choice when both contained normally attractive actors. These findings demonstrate that the effect of advertisement choice may be more complicated than previously found. Instead of acting as a means to improve avoidance rates, advertisement choice may make the content more salient to participants. Thus, viewers’ perceptions of the advertisement after making an advertisement choice may determine whether avoidance occurs.

O sistema complemento participa da imunidade natural e da imunidade adquirida. Este sistema pode ser ativado por três diferentes vias: a via clássica, a via alternativa e a via das lectinas, desencadeando uma cascata proteolítica que irá resultar na possível eliminação de microorganismos e, por sua vez, no restabelecimento da homeostasia do indivíduo. A proteína C3 possui um papel central participando das três vias deste sistema. Sua clivagem gera fragmentos que estão associados a várias funções biológicas como opsonização, quimiotaxia, anafilatoxinas, além da formação da C3-convertases, que amplificam a via de ativação efetora contra patógenos. A completa deficiência de C3 é rara, está comumente associada a repetidas infecções, ao desenvolvimento de doenças mediadas por imunocomplexos e glomerulonefrite. Neste trabalho nós investigamos as bases moleculares desta deficiência em uma paciente com histórico de consanguinidade. Nosso estudo identificou uma mutação genética responsável pela completa deleção do éxon 27, implicando na perda de 99 nucleotídeos (região 3450 até 3549 correspondente ao cDNA do C3). O sequenciamento do gene C3 da região 6690313 até 6690961 mostrou uma troca de nucleotídeos T por um C (T → C) na posição 6690626, causando a exclusão do o éxon 27 e deficiência da proteína C3 do complemento humano nessa paciente. Também confirmamos o padrão de herança autossômica recessiva desta deficiência. Verificamos ainda que os fibloblastos da paciente estimulados com LPS por 24 h foram incapazes de secretar a proteína C3, o que nos leva a suspeitar que esta proteína mutante seja rapidamente degradada ainda dentro da célula, impedindo sua liberação para o meio extracelular. / The complement system participates in the natural immunity and acquired immunity. This system can be activated by three different pathways: the classical pathway, the alternative pathway and the lectin pathway, triggering a proteolytic cascade that will possibly result in the elimination of microorganisms and, in turn, in restoring the homeostasis of the individual. The C3 protein has a central role in the activation of all pathways. Its cleavage produces fragments that are associated with various biological functions such as opsonization, chemotaxis, and production of anaphylatoxins. In addition, the C3 participates in formation of C3 convertase of the alternative pathway which amplifies the activation of effector against pathogens. The complete C3 deficiency is rare and commonly associated with recurrent infections, development of diseases mediated by immune complexes and glomerulonephritis. In this work, we investigated the molecular basis of this deficiency in a patient family with a history of consanguinity. Our study has identified a genetic mutation responsible for the complete deletion of exon 27, resulting in the loss of 99 nucleotides (region 3450 to 3549 of the cDNA corresponding to C3). The sequencing of the C3 region 6690313 to 6690961 showed a T nucleotide exchange to C (T → C) at position 6690626 causing deletion of exon 27 leading to deficiency of the human complement protein C3 in this patient. We also confirmed the autosomal recessive patters of this patient deficiency in the family. We observed that patient fibroblasts stimulated with LPS for 24 h were unable to secrete C3, which leads us to suspect that this protein is degraded within the cell preventing its release into the extracellular.

Le but de cette thèse est d'améliorer la qualité des systèmes de recommandation pour la navigation Web en utilisant la séquentialité des actions de navigation des utilisateurs. La notion de séquentialité a déjà été étudiée dans ce contexte. De telles études tentent habituellement de trouver un bon compromis entre précision, complexité en temps et en mémoire, et couverture. De plus, le Web a cela de particulier que du bruit peut être contenu au sein des navigations (erreurs de navigation, apparition de pop-ups, etc.), et que les utilisateurs peuvent effectuer des navigations parallèles. La plupart des modèles qui ont été proposés dans la littérature exploitent soit des suites contiguës de ressources et ne sont pas résistants au bruit, soit des suites discontiguës de ressources et induisent une complexité en temps et en mémoire importantes. Cette complexité peut être réduite en effectuant une sélection sur les séquences, mais cela engendre alors des problèmes de couverture. Enfin à notre connaissance, le fait que les utilisateurs puissent effectuer des navigations parallèles n'a jamais été étudié du point de vue de la recommandation. La problématique de cette thèse est donc de proposer un nouveau modèle séquentiel ayant les cinq caractéristiques suivantes : (1) une bonne précision de recommandation, (2) une bonne résistance au bruit, (3) la prise en compte des navigations parallèles, (4) une bonne couverture (5) et une faible complexité en temps et en mémoire. Afin de répondre à cette problématique, nous nous inspirons de la Modélisation Statistique du Langage (MSL), qui a des caractéristiques très proches de celles de la navigation Web. La MSL est étudiée depuis beaucoup plus longtemps que les systèmes de recommandation et a largement prouvé sa précision et son efficacité. De plus, la plupart des modèles statistiques de langage qui ont été proposés prennent en compte des séquences. Nous avons donc étudié la possibilité d'exploiter les modèles utilisés en MSL et leur adaptation aux contraintes spécifiques de la navigation Web.

This dissertation studies three public transit operations control strategies with automatic vehicle location (AVL) data available. Specifically, holding control, stop-skipping control and vehicle dispatching with swapping are investigated. Moreover, AVL data from Tucson, Arizona are employed to investigate the methodologies for deriving vehicle operating parameters.The problem of holding vehicles at multiple holding stations can be modeled as a convex mathematical programming problem which can be solved to near optimality by a proposed heuristic. A simulation study on the holding problem suggests that holding control based on the proposed problem formulation can effectively reduce the total passenger cost. Also, multiple holding stations may offer more opportunities to regularize vehicle headways so that holding vehicles at multiple stations can further reduce the passenger cost compared to holding vehicles only at a single station.Stop-skipping is investigated to respond more rapidly to vehicle disruptions occurring in the middle of a route. Based on a preliminary analysis of the basic stop-skipping policy, a policy alternative is constructed. The stop-skipping strategy is formulated separately for both policies as a nonlinear integer programming problem. The problem solution relies on an exhaustive search method. Another simulation study is conducted to examine how the performance of the two policies change with the passenger distribution pattern, the vehicle disruption location and length, and the vehicle travel time variability. The simulation result suggests selective superiority of the two policies.The vehicle dispatching problem investigates the potential of integrating real-time swapping into the vehicle dispatching strategies at a transit transfer terminal. With a hypothetical study design, simulation is employed again to evaluate the significance of real-time swapping by comparing the performance of a swapping-holding combined strategy with the holding-only strategy. A sensitivity analysis is also employed to compare these two strategies among key transit operating factors.Finally, using three different understandings (assumptions) of vehicle operating behavior, regression methods are proposed for using AVL data to derive the vehicle running speeds and passenger boarding rates, which serve as inputs to the operations control models. The regression results show that the day-specific operating behavior may not be appropriate, and that operating behavior combining both trip-specific and day-specific effects seems to be slightly superior to the trip-specific behavior overall.

This thesis proposes an efficient method to accelerate ray based volume rendering with texture slabs using programmable graphics hardware. In this method, empty space skipping and early ray termination are utilized without performing any preprocessing on CPU side. The acceleration structure is created on the fly by making use of depth buffer efficiently on Graphics Processing Unit (GPU) side. In the proposed method, texture slices are grouped together to form a texture slab. Rendering all the slabs from front to back viewing order in multiple rendering passes generates the resulting volume image. Slab silhouette maps (SSM) are created to identify and skip empty spaces along the ray direction at pixel level. These maps are created from the alpha component of the slab and stored in the depth buffer. In addition to the empty region information, SSM also contains information about the terminated rays. The method relies on hardware z-occlusion culling that is realized by means of SSMs to accelerate ray traversals. The cost of generating this acceleration data structure is very small compared to the total rendering time.

This paper analyzes acceleration in education as a practice for meeting the educational needs of gifted students, and points out favorable and unfavorable arguments on the use of this practice. Acceleration is an educational practice consisting of several teaching strategies designed to encourage academically gifted students and reduce their time spent in school. It promotes faster learning by matching the curriculum to the student’s level of knowledge, interest and motivation. There are several arguments in favor of acceleration, such as the improvement of academic performance, self-esteem and student’s social adjustment. However, educators are reluctant to implement this practice, arguing that students may be immature or lose part of the content of the regular curriculum. / Se analiza la aceleración de la enseñanza como práctica de atención a las necesidades educacionales de alumnos superdotados y se presentan argumentos favorables y contrarios. La aceleración de la enseñanza es una práctica educacional compuesta por diversas estrategias para estimular al alumno académicamente superdotado y reducir su tiempo de permanencia en la escuela. Promueve un aprendizaje más rápido al equiparar el currículum al nivel de conocimiento, interés y motivación. Son varios los argumentos a favor de la aceleración, como mejora del desempeño académico, la autoestima y el ajuste social del alumno. Sin embargo, educadores se resisten a implementar esta práctica alegando que los alumnos pueden ser inmaduros o perder parte del contenido del currículum regular.

O sistema complemento participa da imunidade natural e da imunidade adquirida. Este sistema pode ser ativado por três diferentes vias: a via clássica, a via alternativa e a via das lectinas, desencadeando uma cascata proteolítica que irá resultar na possível eliminação de microorganismos e, por sua vez, no restabelecimento da homeostasia do indivíduo. A proteína C3 possui um papel central participando das três vias deste sistema. Sua clivagem gera fragmentos que estão associados a várias funções biológicas como opsonização, quimiotaxia, anafilatoxinas, além da formação da C3-convertases, que amplificam a via de ativação efetora contra patógenos. A completa deficiência de C3 é rara, está comumente associada a repetidas infecções, ao desenvolvimento de doenças mediadas por imunocomplexos e glomerulonefrite. Neste trabalho nós investigamos as bases moleculares desta deficiência em uma paciente com histórico de consanguinidade. Nosso estudo identificou uma mutação genética responsável pela completa deleção do éxon 27, implicando na perda de 99 nucleotídeos (região 3450 até 3549 correspondente ao cDNA do C3). O sequenciamento do gene C3 da região 6690313 até 6690961 mostrou uma troca de nucleotídeos T por um C (T → C) na posição 6690626, causando a exclusão do o éxon 27 e deficiência da proteína C3 do complemento humano nessa paciente. Também confirmamos o padrão de herança autossômica recessiva desta deficiência. Verificamos ainda que os fibloblastos da paciente estimulados com LPS por 24 h foram incapazes de secretar a proteína C3, o que nos leva a suspeitar que esta proteína mutante seja rapidamente degradada ainda dentro da célula, impedindo sua liberação para o meio extracelular. / The complement system participates in the natural immunity and acquired immunity. This system can be activated by three different pathways: the classical pathway, the alternative pathway and the lectin pathway, triggering a proteolytic cascade that will possibly result in the elimination of microorganisms and, in turn, in restoring the homeostasis of the individual. The C3 protein has a central role in the activation of all pathways. Its cleavage produces fragments that are associated with various biological functions such as opsonization, chemotaxis, and production of anaphylatoxins. In addition, the C3 participates in formation of C3 convertase of the alternative pathway which amplifies the activation of effector against pathogens. The complete C3 deficiency is rare and commonly associated with recurrent infections, development of diseases mediated by immune complexes and glomerulonephritis. In this work, we investigated the molecular basis of this deficiency in a patient family with a history of consanguinity. Our study has identified a genetic mutation responsible for the complete deletion of exon 27, resulting in the loss of 99 nucleotides (region 3450 to 3549 of the cDNA corresponding to C3). The sequencing of the C3 region 6690313 to 6690961 showed a T nucleotide exchange to C (T → C) at position 6690626 causing deletion of exon 27 leading to deficiency of the human complement protein C3 in this patient. We also confirmed the autosomal recessive patters of this patient deficiency in the family. We observed that patient fibroblasts stimulated with LPS for 24 h were unable to secrete C3, which leads us to suspect that this protein is degraded within the cell preventing its release into the extracellular.

Video streaming is an important field of global communications and data processing. It is divided into server and client sides connected via network. Video streaming is concerned with delivering video data from server to client over the network as fast and with as little loss as possible. In this study the possibilities to minimize the amount of data transferred over the network in video streaming are investigated and a video streaming technique comprised of server and client sides is proposed. To expand the flexibility and adaptability of the proposed video streaming technique an operational parameter system was constructed and the parameter value ranges were defined. The proposed video streaming technique was then applied to three sample videos. Before streaming the server side of the proposed technique reduced the frame count of input videos based on operational parameter values while the client side reconstructed the skipped frames. Then the quality of the resulting videos was measured and evaluated. To evaluate the reconstructed frames and videos the PSNR measurement method was used. The study concludes that by using the proposed video streaming technique it is possible to reduce the amount of transfer data by dropping frames on the server side and reconstructing them on the client side.

Les Epidermolyses Bulleuses Dystrophiques (EBD) sont des génodermatoses rares et sévères transmises sur un mode autosomique récessif (EBDR) ou dominant (EBDD), dues à une perte de l’adhésion dermo-épidermique. Les patients atteints d’EBD souffrent de décollements bulleux cutanéo-muqueux qui menacent le pronostic fonctionnel et vital dans les formes les plus graves. Toutes les formes d’EBD sont dues à des mutations du gène COL7A1 codant pour le collagène VII qui est le constituant des fibres d’ancrage assurant l’adhésion de l’épiderme au derme. Il n’existe actuellement pas de thérapie satisfaisante des EBD. La première partie de ma thèse visait à démontrer la faisabilité d’une approche thérapeutique par saut d’exon des EBDR. Cette stratégie consiste à exciser l’exon porteur de la mutation durant le processus d’épissage, afin de restaurer l’expression d’une protéine fonctionnelle. Les exons 73 et 80 de COL7A1 sont particulièrement intéressants car ils sont le siège de mutations récurrentes et que leur excision préserve le cadre ouvert de lecture. Nous avons dans un premier temps démontré le caractère non indispensable des séquences codées par ces exons in vivo en utilisant un modèle de xénogreffe de peau humaine EBDR reconstruite, génétiquement modifiée à l’aide de vecteurs rétroviraux exprimant l'ADNc de COL7A1 délété des séquences des exons 73 ou 80. Puis, j’ai pu établir que la transfection d’oligoribonucléotides antisens (AONs) dirigés contre certaines séquences régulatrices de l’épissage permettait d’induire le saut en phase de ces exons dans des cellules primaires de patients EBDR, avec une efficacité atteignant 90% de saut d’exon. Les analyses par western blot et immunocytofluorescence après transfection ont permis de mettre en évidence une réexpression significative du collagène VII (jusqu’à 25%) dans les cellules de trois patients EBDR. Enfin, j’ai pu démontrer la réexpression du collagène VII in vivo, après injection de différentes doses d’AONs dans des peaux équivalentes générées avec des cellules de patients et greffées sur des souris immunodéficientes. Dans la seconde partie de ma thèse, j’ai étudié une famille EBD particulière, dont les deux enfants atteints présentaient une EBD beaucoup plus sévère que leur mère et leur grand père maternel, atteints d’une forme modérée d’EBDD. Le séquençage des 118 exons de COL7A1 et des régions d’épissage adjacentes a permis d’identifier une seule mutation dominante c.6698G>A (p.Gly2233Asp) dans l’exon 84, à l’état hétérozygote chez les quatre sujets. A partir de l’étude des transcrits paternels, j’ai pu identifier une nouvelle mutation c.2587+40G>A dans l’intron 19 de COL7A1, qui active un site donneur cryptique dans l’intron 19, entraînant sa rétention partielle et la formation d’un codon stop prématuré. La confirmation de la présence de cette seconde mutation, récessive, dans l’ADN des deux enfants a ainsi permis d’expliquer les différences phénotypiques observées, les deux enfants atteints étant hétérozygotes composites pour une mutation dominante et une mutation récessive de COL7A1. Cette mutation récessive constitue la mutation intronique la plus distante des sites consensus d’épissage de COL7A1 et souligne l’importance de l’étude des ARNm pour la recherche de mutations dans le cadre des EBD. Dans une dernière partie de ma thèse, j’ai débuté la caractérisation d’un modèle murin knock-in d’EBDR développé par notre laboratoire, qui mime certaines des caractéristiques phénotypiques des patients EBDR. Mon travail a permis de démontrer in vivo la faisabilité de l’approche par saut d’exons pour COL7A1. Cette première étape importante conduit à développer des études de preuve de principe et de toxicologie dans des modèles animaux, dans la perspective d’une transition vers la clinique. Il illustre également les variations pathologiques d’épissage pouvant faire l’objet d’approches thérapeutiques similaires. / Dystrophic Epidermolysis Bullosa (DEB) is a group of rare and severe genetic skin disorders, inherited in a dominant (DDEB) or recessive (RDEB) manner, and characterised by loss of adhesion between the epidermis and the underlying dermis. DEB patients suffer from severe blistering of the skin and mucosae after mild traumas, and in the most severe forms, DEB can be life-threatening. DEB is caused by mutations in the COL7A1 gene encoding type VII collagen that assembles into anchoring fibrils forming key dermo-epidermal adhesion structures. To date, there is no specific treatment for DEB. The first part of my thesis was to develop exon skipping as a therapeutic approach for RDEB. In this work, exon skipping strategy consists in modulating the splicing of a premessenger RNA to induce the skipping of a mutated exon and lead to the synthesis of a shorter but functional protein. Exons 73 and 80 of COL7A1 are of particular interest since they carry many recurrent mutations and their excision preserves the open reading frame. In first instance, we have demonstrated the dispensability of these exons for type VII collagen function in an in vivo xenograft model using RDEB cells transduced with retroviral vectors containing COL7A1 cDNAs, deleted of the sequences of exon 73 or 80. I have subsequently transfected primary RDEB keratinocytes and fibroblasts with antisense oligoribonucleotides (AONs) targeting key splicing regulatory elements of these exons, and achieved efficient skipping of these exons (up to 90%). Western blot and immunocytofluorescence experiments demonstrated significant type VII collagen re-expression (up to 25% of the normal amount) in cells from three RDEB patients. Finally, I have generated skin equivalents with cells of these patients, grafted them on immunodeficient mice and injected different doses of AONs in the grafts, and I have demonstrated type VII collagen re-expression in vivo. In the second part of my thesis, I have studied the case of a particular DEB family, in which two affected children presented a DEB much more severe than their mother and maternal grandfather, suffering from a mild form of DDEB. Sequencing of the 118 exons ofCOL7A1 and of their flanking splice sites, lead to the identification of a single dominant mutation c.6698G>A (p.Gly2233Asp) in exon 84, at the heterozygous state in the four individuals. By carrying out analyses on the paternal transcripts, I have identified a novel c.2587+40G>A recessive mutation in intron 19, which activates a cryptic donor splice site in this intron, leading to its partial retention and to the formation of a premature termination codon. I confirmed the presence of this second, recessive, mutation in the DNA of the two affected children, thus providing an explanation for the observed intrafamilial phenotypic variability: the two affected offsprings being compound heterozygotes for a dominant mutation and a recessive mutation in COL7A1. This novel mutation is the deepest intronic mutation found in COL7A1 so far, and emphasizes the importance of studying COL7A1 at the transcripts level to unravel intronic mutations, understand their molecular consequences and their involvement in the development of the disease. In the last part of my thesis, I have started the characterisation of a knock-in murine model of RDEB generated by our laboratory, which mimics some of the phenotypic characteristics of RDEB patients. My thesis work provided the in vivo demonstration of the feasibility of an exon skipping therapeutic approach for COL7A1. This first important step leads to development of proof of concept studies and toxicological studies in different animal models, with the aim of a clinical translation. It also illustrates the pathological splicing alterations that could benefit from similar approaches.