It is currently believed that the splicing of most pre-mRNAs occurs, at least in part, co-transcriptionally. In order to validate this principle in yeast and establish an experimental system for monitoring spliceosome assembly in vivo, I have employed the chromatin immunoprecipitation (ChIP) assay to study co-transcriptional splicing events. Here, I use ChIP to examine key questions with respect to the recent proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. In my thesis, I address: 1) whether the U1 snRNP, which binds to the 5¡¦ splice site of each intron, is recruited co-transcriptionally in vivo and 2) if so, where along the length of active genes the U1 snRNP is concentrated. U1 snRNP accumulates on downstream positions of genes containing introns but not within promoter regions or along intronless genes. More specifically, accumulation correlated with the presence and position of the intron, indicating that the intron is necessary for co-transcriptional U1 snRNP recruitment and/or retention (Kotovic et al., 2003). In contrast to capping enzymes, which bind directly to Pol II (Komarnitsky et al., 2000; Schroeder et al., 2000), the U1 snRNP is poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP is dependent on RNA synthesis and is abolished by intron removal. Microarray data reveals that intron-containing genes are preferentially selected by ChIP with the U1 snRNP furthermore indicating recruitment specificity to introns. Because U1 snRNP levels decrease on downstream regions of intron-containing genes with long second exons, our lab is expanding the study to 3¡¦ splice site factors in hopes to address co-transcriptional splicing. In my thesis, I also focus on questions pertaining to the requirements for recruitment of the U1 snRNP to sites of transcription. To test the proposal that the cap-binding complex (CBC) promotes U1 snRNP recognition of the 5¡¦ splice site (Colot et al., 1996), I use a ?´CBC mutant strain and determine U1 snRNP accumulation by ChIP. Surprisingly, lack of the CBC has no effect on U1 snRNP recruitment. The U1 snRNP component Prp40p has been identified as playing a pivotal role in not only cross-intron bridging (Abovich and Rosbash, 1997), but also as a link between Pol II transcription and splicing factor recruitment (Morris and Greenleaf, 2000). My data shows that Prp40p recruitment mirrors that of other U1 snRNP proteins, in that it is not detected on promoter regions, suggesting that Prp40p does not constitutively bind the phosphorylated C-terminal domain (CTD) of Pol II as previously proposed. This physical link between Pol II transcription and splicing factor recruitment is further tested in Prp40p mutant strains, in which U1 snRNP is detected at normal levels. Therefore, U1 snRNP recruitment to transcription units is not dependent on Prp40p activity. My data indicates that co-transcriptional U1 snRNP recruitment is not dependent on the CBC or Prp40p and that any effects of these players on spliceosome assembly must be reflected in later spliceosome events. My data contrasts the proposed transcription factory model in which Pol II plays a central role in the recruitment of mRNA processing factors to TUs. According to my data, splicing factor recruitment acts differently than capping enzyme and 3¡¦ end processing factor recruitment; U1 snRNP does not accumulate at promoter regions of intron-containing genes or on intronless genes rather, accumulation is based on the synthesis of the intron. These experiments have lead me to propose a kinetic model with respect to the recruitment of splicing factors to active genes. In this model, U1 snRNP accumulation at the 5¡¦ splice site requires a highly dynamic web of protein-protein and protein-RNA interactions to occur, ultimately leading to the recruitment and/or stabilization of the U1 snRNP.

Les ribosomes sont des macrocomplexes ribonucléoprotéiques sophistiqués, essentiels pour décoder l’information génétique et la traduire en protéines fonctionnelles. Chez les organismes eucaryotes, le ribosome est constitué de deux sous-unités, la petite (40S) et la grande (60S). Leur biogenèse est un processus fondamental, très complexe, qui mène à la synthèse et l’assemblage de 4 ARNr et 80 protéines ribosomiques (79 chez la levure). La biogenèse du ribosome a longtemps été étudiée chez Saccharomyces cerevisiae. Près de 20 ans de recherches ont été nécessaires à la communauté scientifique pour identifier les quelques 200 facteurs de synthèse du ribosome levurien. Alors que le schéma global de cette voie de biosynthèse semble conservé chez les organismes eucaryotes, de nombreux éléments suggèrent qu’elle serait plus élaborée chez l’homme et nécessiterait un plus grand nombre de facteurs que chez la levure. De plus, la caractérisation de nombreuses ribosomopathies, ou maladies du ribosome prédisposant aux cancers, a suscité un intérêt accru pour l’étude de la voie de biosynthèse du ribosome dans le paradigme expérimental le plus approprié, la cellule humaine.<p><p>Au cours de ma thèse de doctorat, j’ai contribué à un projet systématique d’identification de facteurs d’assemblage (FA) du ribosome chez l’homme. Pratiquement, nous avons identifié 286 FA humains, dont beaucoup sont homologues aux facteurs levuriens connus, et 74 sont sans équivalent chez la levure. Par ailleurs, j’ai caractérisé en détail certains facteurs. En particulier, Trm112 pour lequel j’ai montré qu’il agit comme un stabilisateur de la méthyltransférase (MTase) Bud23, spécifique à l’ARNr 18S de la sous-unité levurienne 40S. J’ai également participé à la caractérisation de mutations à l’interface du complexe Bud23-Trm112. Enfin, j’ai contribué à l’étude de trois FA que nous avons identifiés chez l’homme, DIMT1L et WBSCR22-TRMT112. J’ai montré que ces protéines sont les orthologues des MTases levuriennes Dim1 et Bud23-Trm112, qu’elles sont requises pour la synthèse et la modification de l’ARNr mature de la petite sous-unité ribosomique, et qu’elles seraient impliquées dans un mécanisme conservé contrôlant la qualité de la voie de biosynthèse du ribosome.<p><p>La totalité des FA que nous avons identifiés en cellule humaine sont à la disposition de la communauté scientifique dans une base de données en ligne accessible sur la page www.RibosomeSynthesis.com. Nous espérons que cette ressource contribuera à une meilleure compréhension des mécanismes moléculaires sous-jacents au développement des ribosomopathies et à l’élaboration d’agents thérapeutiques efficaces.<p> / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

It is currently believed that the splicing of most pre-mRNAs occurs, at least in part, co-transcriptionally. In order to validate this principle in yeast and establish an experimental system for monitoring spliceosome assembly in vivo, I have employed the chromatin immunoprecipitation (ChIP) assay to study co-transcriptional splicing events. Here, I use ChIP to examine key questions with respect to the recent proposal that RNA polymerase II (Pol II) recruits pre-mRNA splicing factors to active genes. In my thesis, I address: 1) whether the U1 snRNP, which binds to the 5¡¦ splice site of each intron, is recruited co-transcriptionally in vivo and 2) if so, where along the length of active genes the U1 snRNP is concentrated. U1 snRNP accumulates on downstream positions of genes containing introns but not within promoter regions or along intronless genes. More specifically, accumulation correlated with the presence and position of the intron, indicating that the intron is necessary for co-transcriptional U1 snRNP recruitment and/or retention (Kotovic et al., 2003). In contrast to capping enzymes, which bind directly to Pol II (Komarnitsky et al., 2000; Schroeder et al., 2000), the U1 snRNP is poorly detected in promoter regions, except in genes harboring promoter-proximal introns. Detection of the U1 snRNP is dependent on RNA synthesis and is abolished by intron removal. Microarray data reveals that intron-containing genes are preferentially selected by ChIP with the U1 snRNP furthermore indicating recruitment specificity to introns. Because U1 snRNP levels decrease on downstream regions of intron-containing genes with long second exons, our lab is expanding the study to 3¡¦ splice site factors in hopes to address co-transcriptional splicing. In my thesis, I also focus on questions pertaining to the requirements for recruitment of the U1 snRNP to sites of transcription. To test the proposal that the cap-binding complex (CBC) promotes U1 snRNP recognition of the 5¡¦ splice site (Colot et al., 1996), I use a ?´CBC mutant strain and determine U1 snRNP accumulation by ChIP. Surprisingly, lack of the CBC has no effect on U1 snRNP recruitment. The U1 snRNP component Prp40p has been identified as playing a pivotal role in not only cross-intron bridging (Abovich and Rosbash, 1997), but also as a link between Pol II transcription and splicing factor recruitment (Morris and Greenleaf, 2000). My data shows that Prp40p recruitment mirrors that of other U1 snRNP proteins, in that it is not detected on promoter regions, suggesting that Prp40p does not constitutively bind the phosphorylated C-terminal domain (CTD) of Pol II as previously proposed. This physical link between Pol II transcription and splicing factor recruitment is further tested in Prp40p mutant strains, in which U1 snRNP is detected at normal levels. Therefore, U1 snRNP recruitment to transcription units is not dependent on Prp40p activity. My data indicates that co-transcriptional U1 snRNP recruitment is not dependent on the CBC or Prp40p and that any effects of these players on spliceosome assembly must be reflected in later spliceosome events. My data contrasts the proposed transcription factory model in which Pol II plays a central role in the recruitment of mRNA processing factors to TUs. According to my data, splicing factor recruitment acts differently than capping enzyme and 3¡¦ end processing factor recruitment; U1 snRNP does not accumulate at promoter regions of intron-containing genes or on intronless genes rather, accumulation is based on the synthesis of the intron. These experiments have lead me to propose a kinetic model with respect to the recruitment of splicing factors to active genes. In this model, U1 snRNP accumulation at the 5¡¦ splice site requires a highly dynamic web of protein-protein and protein-RNA interactions to occur, ultimately leading to the recruitment and/or stabilization of the U1 snRNP.

Orientador: Jose Sanches Mantovani / Resumo: Neste trabalho propõem-se metodologias para realizar o planejamento de sistemas de distribuição de energia elétrica de média tensão (MT), baixa tensão (BT) e o planejamento integrado de sistemas de média e baixa tensão (MT/BT). Nos modelos de funções objetivos considerados minimizam-se os custos associados à construção, expansão, operação e confiabilidade das redes de MT, BT e MT/BT, considerando a presença de geradores distribuídos e variáveis de natureza estocástica. A geração distribuída presente no planejamento de sistemas de distribuição é avaliada através de metodologias de geração de cenários e análise de risco. Como método de busca de soluções para o problema de planejamento dos sistemas de distribuição é proposta a meta-heurística de busca em vizinhança variável GVNS (General Variable Neighborhood Search). A meta-heurística GVNS trabalha com uma série de estruturas de vizinhanças que permitem explorar o espaço de busca de forma eficiente através dos critérios de diversificação e intensificação, aumentando a probabilidade de obter soluções que não sejam ótimos locais. No planejamento integrado MT/BT, as soluções obtidas são analisadas através de um fluxo de potência integrado entre as redes de MT e BT, considerando as relações de conexão dos transformadores entre estes sistemas. Os algoritmos propostos relacionados ao planejamento de sistemas MT, BT e MT/BT são implementados em linguagem de programação C++ e testado em diferentes sistemas testes de MT (54 barras e 182... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Submitted by DIOGO RUPOLO null (rupolo.diogo@gmail.com) on 2017-09-08T14:50:14Z No. of bitstreams: 1 Tese Final Diogo Rupolo.pdf: 4125842 bytes, checksum: 60d451cc4d13afb90cca7e443d7436b1 (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-11T20:51:49Z (GMT) No. of bitstreams: 1 rupolo_d_dr_ilha.pdf: 4125842 bytes, checksum: 60d451cc4d13afb90cca7e443d7436b1 (MD5) / Made available in DSpace on 2017-09-11T20:51:49Z (GMT). No. of bitstreams: 1 rupolo_d_dr_ilha.pdf: 4125842 bytes, checksum: 60d451cc4d13afb90cca7e443d7436b1 (MD5) Previous issue date: 2017-08-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Neste trabalho propõem-se metodologias para realizar o planejamento de sistemas de distribuição de energia elétrica de média tensão (MT), baixa tensão (BT) e o planejamento integrado de sistemas de média e baixa tensão (MT/BT). Nos modelos de funções objetivos considerados minimizam-se os custos associados à construção, expansão, operação e confiabilidade das redes de MT, BT e MT/BT, considerando a presença de geradores distribuídos e variáveis de natureza estocástica. A geração distribuída presente no planejamento de sistemas de distribuição é avaliada através de metodologias de geração de cenários e análise de risco. Como método de busca de soluções para o problema de planejamento dos sistemas de distribuição é proposta a meta-heurística de busca em vizinhança variável GVNS (General Variable Neighborhood Search). A meta-heurística GVNS trabalha com uma série de estruturas de vizinhanças que permitem explorar o espaço de busca de forma eficiente através dos critérios de diversificação e intensificação, aumentando a probabilidade de obter soluções que não sejam ótimos locais. No planejamento integrado MT/BT, as soluções obtidas são analisadas através de um fluxo de potência integrado entre as redes de MT e BT, considerando as relações de conexão dos transformadores entre estes sistemas. Os algoritmos propostos relacionados ao planejamento de sistemas MT, BT e MT/BT são implementados em linguagem de programação C++ e testado em diferentes sistemas testes de MT (54 barras e 182 barras), BT (76 barras) e MT/BT (172 e 412 barras), sob diferentes cenários operacionais. / This work proposes methodologies for the planning of medium voltage (MV), low voltage (LV), and integrated planning of medium and low voltage (MV/LV) systems. In the objective function models are considered the costs associated with the construction, expansion, operation and reliability of MV, LV and integrated MV/LV networks. Distributed generators and stochastic variables are also considered in the models. The distributed generation present in the planning of distribution systems is evaluated through methodologies of scenario generation and risk analysis. As a method of finding solutions to the problem of distribution system planning, the GVNS metaheuristic (General Variable Neighborhood Search) is proposed. The metaheuristic GVNS works with a series of neighborhood structures that allow to explore the search space efficiently through diversification and intensification criteria, increasing the probability of obtaining solutions that are not local optimum. In integrated MV/LV planning, the solutions obtained are analyzed through an integrated power flow between the MV and LV networks, considering the connection ratios of the transformers between these systems. The proposed algorithms related to the planning of MV, LV and integrated MV/LV systems are implemented in C ++ programming language and tested in different distribution systems, MV (54 and 182 bus), LV (76 bus) and MV/LV (172 and 412 bus), under different operating scenarios.

Derived from and inspired by the adaptive immune system of bacteria, CRISPR has gone from basic biology knowledge to a revolutionizing biotechnological tool, applicable in many research areas such as medicine, industry and agriculture. The full mechanism of CRISPR-Cas9 was first published in 2012 and various CRISPR-Cas systems have already passed the first stages of clinical trials as new gene therapies. The immense research has resulted in continuously growing knowledge of CRISPR systems and the technique seems to have the potential to greatly impact all life on our planet. Therefore, this literature study aims to thoroughly describe the CRISPR-Cas system, and further suggest an undergraduate laboratory exercise involving gene editing with the CRISPR-Cas9 tool. In this paper, we describe the fundamental technical background of the CRISPR-Cas system, especially emphasizing the most studied CRISPR-Cas9 system, its development and applications areas, as well as highlighting its current limitations and ethical concerns. The history of genetic engineering and the discovery of the CRISPR system is also described, along with a comparison with other established gene editing techniques.  This study concludes that a deeper knowledge about CRISPR is important and required since the technique is applicable in many research areas. A laboratory exercise will not only inspire but also provide extended theoretical and practical knowledge for undergraduate students.