Lee Siu-wah. / Thesis submitted in: December 1999. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves [106]-116). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.x / Abbreviations --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.2 --- Etiology of Hepatocellular Carcinoma --- p.5 / Chapter 1.2.1 --- Viral Infection --- p.5 / Chapter 1.2.1.1 --- Hepatitis B Virus --- p.5 / Chapter 1.2.1.2 --- Hepatitis C Virus --- p.7 / Chapter 1.2.2 --- Cirrhosis and Chronic Inflammation --- p.8 / Chapter 1.2.3 --- Aflatoxin --- p.9 / Chapter 1.3 --- Genetic Studies of Hepatocellular Carcinoma --- p.9 / Chapter 1.3.1 --- Conventional Cytogenetics --- p.9 / Chapter 1.3.2 --- Molecular Cytogenetics --- p.12 / Chapter 1.3.3 --- Molecular Genetic Studies --- p.12 / Chapter 1.3.3.1 --- Proto-oncogenes --- p.12 / Chapter 1.3.3.2 --- Tumour Suppressor Genes --- p.13 / Chapter 1.3.3.3 --- Cell Cycle Genes --- p.14 / Chapter 1.4 --- Background of Study --- p.16 / Chapter 1.5 --- Objectives of Study --- p.17 / Chapter Chapter 2 --- Material and Methods --- p.18 / Chapter 2.1 --- Materials --- p.19 / Chapter 2.2 --- Analysis of Chromosomal Imbalances by Comparative Genomic Hybridization --- p.23 / Chapter 2.2.1 --- Comparative Genomic Hybridization --- p.23 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Preparation of Normal Metaphases --- p.30 / Chapter 2.2.2.2 --- Extraction of High Molecular Weight DNA --- p.30 / Chapter 2.2.2.3 --- Labeling of DNA by Nick Translation --- p.31 / Chapter 2.2.2.4 --- Labeling Efficiency --- p.31 / Chapter 2.2.2.5 --- Preparation of Probe --- p.33 / Chapter 2.2.2.6 --- Hybridization --- p.33 / Chapter 2.2.2.7 --- Washing and Detection of Signals --- p.35 / Chapter 2.2.2.8 --- Image Acquisition and Analysis --- p.35 / Chapter 2.2.2.9 --- Control Experiments --- p.36 / Chapter 2.3 --- Positional Mapping of Novel Amplicon by Interphase Cytogenetics --- p.39 / Chapter 2.3.1 --- Fluorescence in situ Hybridization --- p.39 / Chapter 2.3.2 --- Using Yeast Artificial Chromosomes (YAC) as Probe --- p.41 / Chapter 2.3.3 --- Methods --- p.48 / Chapter 2.3.3.1 --- Culture of Yeast Artificial Chromosomes --- p.48 / Chapter 2.3.3.2 --- Extraction of Total YAC DNA --- p.48 / Chapter 2.3.3.3 --- Verification of YAC Clones for Chimerism by FISH --- p.49 / Chapter 2.3.3.4 --- Inter-Alu-PCR --- p.49 / Chapter Chapter 3 --- Assessment of Genetic Changes in HCC by Comparative Genomic Hybridization (CGH) --- p.57 / Chapter 3.1 --- Introduction --- p.58 / Chapter 3.2 --- Materials and Methods --- p.58 / Chapter 3.2.1 --- Patients and Specimens --- p.58 / Chapter 3.2.2 --- Comparative Genomic Hybridization --- p.60 / Chapter 3.2.3 --- Statistical Analysis --- p.60 / Chapter 3.3 --- Results --- p.61 / Chapter 3.3.1 --- Overall Copy Number Aberrations in 67 HCC and Surrounding Cirrhotic Tissues --- p.61 / Chapter 3.3.2 --- TNM Staging --- p.61 / Chapter 3.3.3 --- Tumour Size --- p.72 / Chapter 3.3.4 --- Serum AFP Elevation --- p.72 / Chapter 3.3.5 --- Chromosomal Aberrations in HCC arising from Cirrhotic and Non- cirrhotic Livers --- p.72 / Chapter 3.4 --- Discussion --- p.73 / Chapter 3.4.1 --- Recurrent Gains --- p.73 / Chapter 3.4.2 --- Recurrent Losses --- p.75 / Chapter 3.4.3 --- Tumour Progression --- p.76 / Chapter 3.5 --- Conclusion --- p.78 / Chapter Chapter 4 --- Positional Mapping of a Novel Amplicon on Chromosome 1q21-q25 by Interphase Cytogenetics --- p.79 / Chapter 4.1 --- Introduction --- p.80 / Chapter 4.2 --- Materials --- p.82 / Chapter 4.3 --- Methods --- p.82 / Chapter 4.3.1 --- Preparation of Paraffin-embedded Tissue Sections --- p.82 / Chapter 4.3.2 --- Verification of YAC Probes for Chimerism --- p.83 / Chapter 4.3.3 --- Hybridization Efficiency of Test and Reference Probes --- p.83 / Chapter 4.3.4 --- Slide Pretreatment and FISH with YAC Probes --- p.88 / Chapter 4.3.5 --- Scoring of FISH Signals --- p.88 / Chapter 4.4 --- Results --- p.89 / Chapter 4.4.1 --- Relative Copy Number Gain --- p.89 / Chapter 4.4.2 --- Intratumour Heterogeneity --- p.90 / Chapter 4.5 --- Discussion --- p.90 / Chapter 4.6 --- Further Studies --- p.104 / Chapter 4.6.1 --- Fine Mapping of Chromosomal Region 1 q21 --- p.104 / Chapter 4.6.2 --- Isolation of Novel Genes in the Amplicon --- p.105 / Chapter 4.6.3 --- Expression Status of the EDC Genes --- p.105 / References --- p.106

Orientador: Maria Aparecida Custódio Domingues / Banca: Maria Cláudia Nogueira Zerbini / Banca: Roberto Antonio Pinto Paes / Banca: Alexandre Todorovic Fabro / Banca: Elida Paula Benquique Ojopi / Resumo: Introdução: Linfomas Double-Hit (LDH) são neoplasias de alto grau raras, caracterizadas pela translocação do gene MYC concomitante a translocação envolvendo os genes BCL2 e/ou BCL6, cujo diagnóstico depende da realização de exame citogenético, técnica de baixa disponibilidade e elevado custo. Diante disso, tem-se valorizado a identificação de fatores morfológicos e/ou imunofenotípicos que tenham associação com a detecção de tais translocações diagnósticas. Objetivos: Identificar pacientes portadores de LDH em casuística de linfomas Não-Hodgkin de alto grau e avaliar as associações entre os resultados citogenéticos imunoistoquímicos (IHQ) relativos aos marcadores MYC, BCL2 e BCL6. Material e Métodos: Procedeu-se revisão clínico-morfológica de 120 pacientes com diagnóstico de linfoma difuso de grandes células B (LDGCB) e linfoma de Burkitt (LB), com confecção de plataforma de tissue microarray (TMA) para realização IHQ (CD20, CD79a, PAX5, CD10, BCL6, BCL2, MUM1, TDT e c-MYC) e hibridação por fluorescência in situ (FISH) para detecção de translocações dos genes c-MYC, BCL6 e BCL2. Resultados: Detectaram-se três pacientes portadores de LDH: dois com translocações de MYC e BCL2 e um com translocações de MYC e BCL6, todos evoluindo para óbito em menos de seis meses. Entre 90 amostras com citogenética avaliável, houve associação entre IHQ e FISH para MYC (p=0,036) e BCL2 (p=0,001), com concordância regular indicada por valores de Kappa de 0,23 [0,0;0,49] e de 0,35 [0,13;0,56], respectivamente. No grupo de LDGCB (n=72), as translocações envolvendo individualmente os genes MYC, BCL6 e BCL2 representaram 4,1%, 12,5% e 15,2%, respectivamente, sendo a morfologia de céu estrelado fortemente associada à detecção de translocações do gene MYC (p=0,01). Conclusão: A detecção de apenas três pacientes portadores de LDH na casuística estudada, todos com evolução para óbito, corrobora... / Abstract: Background: Double-Hit Lymphomas (DHL) are rare high-grade neoplasms characterized by the translocation of gene MYC to other translocations involving genes BCL2 and/or BCL6, whose diagnosis depends on the cytogenetic examination, a technique with low availability and high cost. Therefore, it has been valued the identification of morphologic and/or immunophenotypic factors that have association with the detection of those translocations. Objectives: To identify DHL patients in group of high grade non-Hodgkin lymphoma (NHL-B) and evaluate the associations between the cytogenetic and immunohistochemical (IHC) results for the MYC, BCL2 and BCL6 markers. Design: Clinical and morphological review of 120 patients diagnosed with diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) was proceeded, with production of a tissue microarray platform (TMA) to perform IHC (CD20, CD79a, PAX5, CD10, BCL6, BCL2, MUM1, TDT and c-MYC) and fluorescence in situ hybridization (FISH) for detection of c-MYC, BCL2 and BCL6 gene translocations. Results: Three patients with DHL were detected: two with translocations of MYC and BCL2 and one with translocations of MYC and BCL6, all leading to death in less than six months. Among 90 cytogenetically evaluable samples, associations were determined between IHC and FISH for MYC (p=0.036) and BCL2 (p=0.001), though with regular compliance testing, indicated by Kappa value of 0.23 [0.0;0.49] and 0.35 [0.13;0.56], respectively. In the DLBCL group (n=72), MYC, BCL6 and BCL2 translocations were present in 4.1%, 12.5% and 15.2%, respectively. "Starry sky" morphology was strongly associated with MYC positivity (p=0.01). Conclusion: The detection of only three DHL patients in this series, all evolving to death, confirms the rarity and aggressiveness of this neoplasm. The morphological pattern "starry sky" and the expression by IHC of MYC and BCL2 can represent possible selection factors for the ... / Doutor

Submitted by JOSIANE SANTOS DE OLIVEIRA (josianeso) on 2017-10-16T11:15:43Z No. of bitstreams: 1 Alan Ricardo Dal Pizzol_.pdf: 2381946 bytes, checksum: 2f29a6e04f5f1d6b8052775644c0e64a (MD5) / Made available in DSpace on 2017-10-16T11:15:43Z (GMT). No. of bitstreams: 1 Alan Ricardo Dal Pizzol_.pdf: 2381946 bytes, checksum: 2f29a6e04f5f1d6b8052775644c0e64a (MD5) Previous issue date: 2017-08-29 / Nenhuma / Esta dissertação propõe-se a buscar a compreensão de como a Subcultura Nerd se atualiza através do consumo de referências culturais. Para isso, elencamos uma narrativa chamada Crônicas de Ghanor (2013), um produto cultural desenvolvido em várias mídias, mas que teve seu início em um jogo de tabuleiro gravado e disponibilizado pelo Grupo Jovem Nerd, a maior empresa nacional voltada a conteúdos Nerds. Para o desenvolvimento da pesquisa, utilizamos a Teoria Fundamentada (FRAGOSO; RECUERO; AMARAL, 2015), para catalogar todas as referências culturais encontradas na construção da narrativa dos Podcasts das Crônicas de Ghanor, ao mesmo tempo que utilizamos o método criado por Gelder e Thornton (1997) para compreendemos como se dá a atualização do Nerd, através da perspectiva da própria subcultura. Para o debate são convidados os autores Bourdieu (1982), Burke (2013) e García Canclini (2008). Sob a ótica do capital cultural e suas trocas simbólicas, criamos uma discussão sobre a hibridização das referências culturais que compõe o produto das Crônicas de Ghanor e de seus consumidores. Como resultados desse processo, dissertamos sobre como a atualidade da subcultura se legitima pela sua própria visão, e entendemos que se trata de uma subcultura em processo. O produto em si não parece ter hibridização, através do entendimento do capital cultural, enquanto a subcultura demonstra que sua formação é híbrida, em processo – e deve refletir em futuros produtos de consumo. / This dissertation proposes to seek the understanding of how the Subculture Nerd is updated through the consumption of cultural references. To do this, we analyze the narrative of Chronicles of Ghanor (2013), a cultural product developed in various medias, but which began in a board game recorded and made available by the Jovem Nerd Group, the largest national company focused on Nerd content. For the development of the research, we used the Grounded Theory (FRAGOSO; RECUERO; AMARAL, 2015) to catalog all the cultural references found in the Chronicles of Ghanor podcasts, while using the method created by Gelder and Thornton (1997), to understand how is updated the Nerd concept, through the perspective of the subculture itself. For the debate are invited the authors Bourdieu (1982), Burke (2013) and García Canclini (2008). From the perspective of cultural capital and its symbolic exchanges, we have created a discussion about the hybridization of the cultural references that makes up of the Chronicles of Ghanor and its consumers. As a result of this process, we have discussed how the current subculture legitimizes itself by its own vision, and we understand that it is a subculture in process. The product itself does not appear to hybridize through the understanding of cultural capital, while the subculture demonstrates that its formation is hybrid, in process – and should reflect in future Nerd products.

É crescente a busca por fontes de energia renováveis em substituição a o uso dos combustíveis fósseis, devido a grande preocupação mundial com o aquecimento global e as mudanças climáticas. O Brasil é considerado o detentor do processo de produção de etanol mais economicamente viável. Estimativas apontam para o fato de que a produção de etanol de primeira geração não será suficiente para atender a futura demanda global pelo biocombustível. Diante disto, a produção de etanol a partir da biomassa lignocelulósica se mostra como uma potencial solução. No entanto, durante o pré-tratamento e a hidrólise da biomassa, há formação de vários compostos tóxicos tais como o furfural, HMF, ácido fracos, e compostos fenólicos, os quais exercem efeitos inibitórios sobre as leveduras, tendo como consequência queda no rendimento fermentativo. Além das vantagens tecnológicas que o processo industrial brasileiro apresenta quanto à incorporação da produção de etanol 2G nas plantas já existentes, soma-se a abundância de matéria prima proveniente da própria indústria sucroalcooleira. No entanto, é de extrema necessidade o desenvolvimento de leveduras capazes de resistir as diversas condições inibitórias provenientes do novo substrato, as quais são potencializadas pelo reciclo celular. Neste sentido, o presente trabalho objetivou o desenvolvimento de novas linhagens de leveduras através das técnicas de hibridação e evolução adaptativa/seleção. Para isto, foi realizado o cruzamento massal envolvendo 5 linhagens de Saccharomyces cerevisiae, previamente selecionadas por demonstrar alta tolerância em fermentações em mosto misto a base de hidrolisado lignocelulósico e melaço de cana-de-açúcar. Inicialmente estudos foram realizados com a intenção de se obter altas taxas de esporulação, a fim de se propiciar uma grande quantidade de cruzamentos aleatórios para consequente geração de uma ampla biodiversidade, aumentando assim a possibilidade de se obter indivíduos com fenótipos melhorados. A cultura resultante do cruzamento massal foi seguida de evolução adaptativa/seleção, buscando, após cerca de 51 gerações, um enriquecimento da cultura com as linhagens mais tolerantes. Por meio de avaliação de crescimento em microplacas (DO 600nm), foram selecionadas 10 isolados evoluídos, os quais foram submetidos a ensaio de fermentação em bancada, simulando tanto quanto possível as condições industriais. Ao final, foi possível destacar uma linhagem por apresentar teor de reserva de trealose significativamente maior que as demais linhagens avaliadas, demonstrando assim a geração de um fenótipo melhorado. / Searching for renewable energy sources to substitute the fossil fuels use is growing, due to a great concern worldwide for global warming and climate change. Brazil is considered the holder of the most economically viable process of ethanol production. Estimates indicate that ethanol production of first generation will not be enough to supply future global demand for biofuel. Therefore, an ethanol production from the lignocellulosic biomass show up as a potential solution; however, during biomass pretreatment and hydrolysis, several toxic compounds such as furfural, HMF, weak acid, and phenolic compounds are formed, which exert inhibitory effects on yeasts, resulting in a fermentative yield decrease. Besides the technological advantages, presents in Brazilian industrial processes to incorporation of 2G ethanol production in existing factories, add up the abundance of feedstock comes from the own sugar and alcohol industry. However, the development of yeasts strains, resisting to inhibitory conditions from the new substrate which are potentiated by the cellular recycle, is extremely necessary. In this sense, the present work aimed the development of new yeasts strains by hybridization and adaptive evolution techniques. Mass mating was carried out involving 5 strains of Saccharomyces cerevisiae, previously selected by demonstrating high tolerance to fermentation from mixed-must composed by lignocellulosic hydrolyzate and sugarcane molasses. Previous studies were carried out to get high rates of sporulation that promote random crosses and broad biodiversity, in order to obtain individuals with improved phenotypes. The culture resulting from the mass mating was followed by an adaptation/selection, during 51 generations, generating enrichment of more tolerant strains. By means of microplate growth evaluation (DO 600nm), 10 evoluted isolates were selected, which were submitted to lab scale fermentation, simulating as much as possible as industrial conditions. At the end, it was possible to highlight a lineage demonstrating significantly higher trehalose reserve content than the other lineages evaluated, thus demonstrating a generation of an improved phenotype.

Le cancer est désormais considéré comme une maladie génomique de la cellule. Les moyens d’étude de l’oncogénome étaient basés sur les différentes modalités du caryotype, peu résolutif. L’application des techniques de micromatrices d’oligonucléotides, notamment l’aCGH, a permis une avancée majeure dans la caractérisation des génomes des cancers.La première partie de notre travail a porté sur les lymphomes de Burkitt (LB), caractérisés par une translocation entre un gène d'immunoglobuline et MYC. L’étude portait sur 12 tumeurs primaires et 15 lignées cellulaires. L’aCGH (44K et 244K), concordait avec les cytogénétiques morphologique et moléculaire (FISH) sauf pour les translocations. Plus de la moitié des variations du nombre de copies (<2Mb) étaient des polymorphismes (CNV). Les anomalies pathologiques (CNA) (n=136) intéressaient les régions suivantes : gains 1q, 13q, 7q, 8q, 2p, 11q et 15q ; pertes 3p, 4p, 4q, 9p, 6p, 17p, 6q, 11pterp13 et 14q12q21.3. Vingt régions minimales critiques (MCR) d’une taille varie entre 0.07-71.36 Mb, étaient délimitées. Trois MCR étaient identifiées sur le 1q : 1q21.1q25.2, 1q32.1 et 1q44. La région proximale de 1q21.1q25.2 était le siège d’une amplification, contenant entre autres les gènes BCA2, PIAS, BCL9. L’étude par transcriptome, sur 15 lignées, a démontré la surexpression uniquement de BCL9, remanié dans les LAL B et faisant partie de la voie de signalisation de MYC. Sur la région 11q23.1, le gain intéressait le gène POU2AF1 dont le messager était élevé. La MCR 13q31.3q32.1 était le siège d’une amplification contenant ABCC4, et le polycistron miR17-92. La corrélation des résultats d’aCGH à ceux du transcriptome et du mirnome ont démontré une surexpression du miR17-92 qui contrôle le développement des lymphocytes B et intervient dans la voie de signalisation de MYC. Sur le 9p21.3, la perte emportait le locus p16INK4A/p15INK4B. Le transcriptome avait démontré une sous expression de p15INK4B. Le locus p16INK4A/p15INK4B contrôle les 2 voies majeures, pRB et p53.La seconde partie de notre travail a consisté à étudier 17 tumeurs congelées de carcinomes adénoïdes kystiques (CAK) par aCGH 44K. Les CNA étaient validées par FISH et/ou MLPA. L’expression protéique était étudiée par immunohistochimie. Les pertes excédaient les gains (41 versus 24). La t(6;9)(q23;p22) récurrente dans les CAK était indétectable car équilibrée. Dans un seul cas, le der(6)t(6;9) est probablement présent sur le profil aCGH. Les MCR les plus fréquentes (-6q22 et -6q24) n’incluaient pas 6q23. Treize MCR étaient identifiées. La MCR délétée en 8q impliquait le miR-124A2 qui régule les gènes CDK6 et MMP2. Sur le 9p21.3, le locus p16INK4A/p15INK4B était de nouveau perdu. Des gains isolés étaient observés au niveau des locus CCND1, KIT/PDGFRA/KDR, MDM2 et JAK2. Le gène MDM2, qui était amplifié sous forme de double minutes, est un élément clé de l’axe p16INK4A-ARF-p53.Pour la troisième partie de notre travail nous avons étudié 60 tumeurs primaires d’adénocarcinomes pulmonaires (AD) de non fumeur par aCGH (244K). Dans 50/60 tumeurs, le nombre de MCR était de 14. Cinq MCR contenaient un seul gène (MOCS2, NSUN3, KHDRBS2, SNTG1 et ST18). Une MCR gagnée, 5q35, contenait le gène NSD1. Une amplification, sous forme de HSR et mise en évidence par FISH, intéressait l’oncogène FUS. Une PCR quantitative avait permis de confirmer la surexpression FUS. A notre connaissance, c’est la première étude qui incrimine le gène FUS dans la carcinogenèse de l’AD du non-fumeur. D’autres gènes étaient également impliqués : ARNT, BCL9, CDK4, p15INK4B, EGFR, ERBB2, MDM2, MDM4, MET, MYC, NKX2-1 et KRAS. Un clustering non supervisé avait permis de dégager un groupe avec un gain de MYC ; un autre groupe caractérisé par la perte des gènes suppresseurs RB et WRN et un dernier groupe caractérisé par un gain 7p et 7q, et présentait une fréquence élevée de mutations de l’EGFR. Dans 10/60, le nombre de CNA était très rare et aucune MCR n’était détectée. / Much of our current understanding of cancer is based on the hypothesis that it is a genetic disease, arising as a clone of cells that expand in an unregulated fashion because of somatically acquired mutations. High-throughput tools for nucleic acid characterization, such as array comparative genomic hybridization (aCGH), now provide the means to conduct comprehensive analyses of somatic anomalies in the oncogenome.In the first part of our work we have carried out a fine mapping of additional chromosomal anomalies in Burkitt lymphoma (BL). The hallmark of this disease is the translocation t(MYC;IG). We have applied whole-genome 244K and 44k oligonucléotides aCGH to 15 cells lines and 12 primary tumors of BL respectively. Karyotype and FISH analysis were used to validate aCGH results. As expected, all translocations remained undetectable with aCGH. More than half of the copy number alterations (CNAs) < 2 Mb were mapped to Mendelian CNVs, including GSTT1, and BIRC6. Somatic cell line-specific CNVs localized to the IG locus were consistently observed with the 244 K aCGH platform. Among 136 CNAs, gains were found in 1q, 13q, 7q, 8q, 2p, 11q and 15q. Losses were found in 3p, 4p, 4q, 9p, 13q, 6p, 17p, 6q,11pterp13 and 14q12q21.3. Twenty one minimal critical regions (MCR), (range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q: 1q21.1q25.2, 1q32.1 et 1q44. The proximal one was mapped to 1q21.1q25.2 with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2, BCL9 and PIAS3. Only BCL9 high level transcrit was noted on oligonucleotide microarray gene expression that was done on 15 cells lines. BCL9, was implicated in a LAL B translocation t(1;14)(q21;q32) and it is a member of MYC pathway. The 13q31.3q32.1, 89.58–96.81 Mb MCR contained an amplicon with several genes. The miR-17-92 cluster, upregulated on mirnome analysis that was done on 15 cells lines, is the gene driver of 13q MCR. The miR-17-92 cluster is a member of MYC pathway. The 9p21.3 MCR harbored p16INK4A/p15INK4B locus which is downregulated. MYC activates ARF,a protein encodes by p16INK4A/p15INK4B locus. . On the second part of our work, a 44k aCGH was applied on 17 frozen adenoid cystic carcinoma (ACC) to delineate with a high resolution the CNA associated with ACC. aCGH results were validated with FISH and/or MLPA. Protein expression was screened with immunohistochemistry analysis. The translocation t(6;9)(q23;p23p24)/ MYB-NFIB recurrent in ACC, was not detected with aCGH. In one case, the der(6)t(6;9) was suspected in the aCGH pattern. There were recurrent gains at 7p15.2, 17q21–25, 22q11–13, and recurrent losses at 1p35, 6q22–25, 8q12–13, 9p21, 12q12–13, and 17p11–13. Thirteen MCR were detected. The recurrent deletion at 8q12.3–13.1 contained miRN124A2 gene, whose product regulates MMP2 and CDK6. The 9p21.3 MCR harbored p16INK4A/p15INK4B locus which was deleted. On 17p11p13, the MCR contained several genes and TP53 was deleted in 2 cases. The MDM2 gene, a member of p16INK4A-ARF-p53 pathway, was amplified and overexpressed in one case. Among the other unique CNAs, gains harbored CCND1, KIT/PDGFRA/KDR, and JAK2. On the third part of this these, a high-resolution 244K aCGH was conducted on 60 frozen lung adenocarcinoma (AD) of never smokers patients in order to establish a catalog of CNA. In 50/60 tumors, fourteen new MCR of gain or loss was noted. One larger MCR of gain contained NSD1.One focal amplification and nine gains contained FUS. NSD1 and FUS are oncogenes hitherto not known to be associated with lung cancer. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. A FUS hsr was observed with FISH screening. FUS was over-expressed in 10 tumors with gain of 16p11.2 compared to 30 tumors without that gain. Other cancer genes present in aberrations included ARNT, BCL9, CDK4, p15INK4B, EGFR, ERBB2, MDM2, MDM4, MET, MYC, NKX2-1 and KRAS.

Les systèmes cyber-physiques sont une classe de systèmes complexe, de grande échelle, souvent critiques de sûreté, qui apparaissent dans des applications industrielles variées. Des approches de vérification formelle sont capable de fournir des garanties pour la performance et la sûreté de ces systèmes. Elles nécessitent trois éléments : un modèle formel, une méthode de vérification, ainsi qu’un ensemble de spécifications formelles. En revanche, les modèles industriels sont typiquement informels, ils sont analysés dans des environnements de simulation informels et leurs spécifications sont décrits dans un langage naturel informel. Dans cette thèse, nous visons à faciliter l’intégration de la vérification formelle dans le processus industriel de la conception basé sur modèle.Notre première contribution clé est une méthodologie de transformation de modèle. A partir d’un modèle de simulation standard, nous le transformons en un modèle de vérification équivalent, plus précisément en un réseau d’automates hybrides. Le processus de transformation prend en compte des différences de syntaxes, sémantique et d’autres aspects de la modélisation. Pour cette classe de modèle formel, des algorithmes d’atteignabilité peuvent être appliqués pour vérifier des propriétés de sûreté. Un obstacle est que des algorithmes d’atteignabilité se mettent à l’échelle pour des modèles affines par morceaux, mais pas pour des modèles non linéaires. Pour obtenir des surapproximations affines par morceaux des dynamiques non linéaires, nous proposons une technique compositionnelle d’hybridisation syntaxique. Le résultat est un modèle très compact qui retient la structure modulaire du modèle d’origine de simulation, tout en évitant une explosion du nombre de partitions.La seconde contribution clé est une approche pour encoder des spécifications formelles riches de façon à ce qu’elles peuvent être interprétées par des outils d’atteignabilité. Nous prenons en compte des spécifications exprimées sous forme d’un gabarit de motif (pattern template), puisqu’elles sont proche au langage naturel et peuvent être compris facilement par des utilisateurs non experts. Nous fournissons (i) des définitions formelles pour des motifs choisis, qui respectent la sémantique des automates hybrides, et (ii) des observateurs qui encodes les propriétés en tant qu’atteignabilité d’un état d’erreur. En composant ces observateurs avec le modèle formel, les propriétés peuvent être vérifiées par des outils standards de vérification qui sont automatisés.Finalement, nous présentons une chaîne d’outils semi-automatisée ainsi que des études de cas menées en collaboration avec des partenaires industriels. / Cyber-Physical Systems form a class of complex, large-scale systems of frequently safety-critical nature in various industrial applications. Formal verification approaches can provide performance and safety guarantees for these systems. They require three elements: a formal model, a formal verification method, and a set of formal specifications. However, industrial models are typically non-formal, they are analyzed in non-formal simulation environments, and their specifications are described in non-formal natural language. In this thesis, we aim to facilitate the integration of formal verification into the industrial model-based design process.Our first key contribution is a model transformation methodology. Starting with a standard simulation model, we transform it into an equivalent verification model, particularly a network of hybrid automata. The transformation process addresses differences in syntax, semantics, and other aspects of modeling. For this class of formal models, so-called reachability algorithms can be applied to verify safety properties. An obstacle is that scalable algorithms exist for piecewise affine (PWA) models, but not for nonlinear ones. To obtain PWA over-approximations of nonlinear dynamics, we propose a compositional syntactic hybridization technique. The result is a highly compact model that retains the modular structure of the original simulation model and largely avoids an explosion in the number of partitions.The second key contribution is an approach to encode rich formal specifications so that they can be interpreted by tools for reachability. Herein, we consider specifications expressed by pattern templates since they are close to natural language and can be easily understood by non-expert users. We provide (i) formal definitions for select patterns that respect the semantics of hybrid automata, and (ii) monitors which encode the properties as the reachability of an error state. By composing these monitors with the formal model under study, the properties can be checked by off-the-shelf fully automated verification tools.Furthermore, we provide a semi-automated toolchain and present results from case studies conducted in collaboration with industrial partners.

This thesis describes the development of an systems biology method to study transcriptional programs that are activated during early and late phases of cell-fusion mediated reprogramming, as well as an implementation of systems-level analysis using reverse-engineered regulatory networks to study CNS disorders like Alcohol Addiction, and neurodegenerative disorders like Alzheimer's Disease (AD), and Parkinson's Disease (PD). The results will show an unprecedented view into the mechanisms underlying complex processes and diseases, and will demonstrate the predictive power of these methodologies that extended far beyond their original contexts.

O presente trabalho visa investigar em que medida a Física, reconhecida por seu caráter paradigmático e, portanto, com leis bem definidas, está sujeita aos elementos culturais da escola. Dessa forma, pretendeu-se investigar a existência (ou não) de um conhecimento físico \"naturalizado\" e universal, para além das características de diferentes culturas nacionais. Dentro de um quadro teórico que reconhece a articulação entre os vários aspectos da complexa realidade escolar, as referências de investigação contemplaram, também, âmbitos mais gerais nos quais a disciplina escolar está inserida. Esses âmbitos foram representados pelas políticas educacionais, pelos currículos, pelas propostas pedagógicas da escola, pelo perfil da comunidade escolar e pelos materiais didáticos. Para isso, optou-se por um estudo de caso de uma escola bicultural brasileira e espanhola, e elaboraram-se três procedimentos de investigação: o primeiro, documental, teve foco na análise dos objetivos da escola em conjunto com as legislações brasileira e espanhola. O segundo, com foco nas relações da cultura escolar, centrado nos protagonistas (alunos e professora), com vivência em contextos escolares de países diferentes. Por último, o terceiro, com foco na análise da abordagem do conhecimento escolar de Física, através de sua organização curricular e dos materiais didáticos utilizados, representados por livros brasileiros e espanhóis. Do conjunto dos resultados obtidos, foi possível verificar que a Física, como disciplina escolar, está sujeita aos elementos culturais da escola, permitindo-se identificar aspectos que contribuem para sua \"desnaturalização\". Mais do que isso, foi possível reconhecer na escola estudada o surgimento de uma cultura híbrida, através de um processo não livre de tensões e movido, aparentemente, por objetivos educacionais distintos. De acordo com o estudo realizado, o produto híbrido desse processo parece ser constituído por uma cultura escolar que integra elementos complementares, sem predomínio de um ou de outro contexto cultural, e com potencial, aparentemente, para promover uma formação mais rica, devido a maior diversificação. / The goal of this research is to investigate possible national cultural elements in the introduction of Physics knowledge in high schools. This means to inquire in which extent a paradigmal science as this one, with universal laws, is presented through equivalent approaches in different cultural contexts. As school subjects are integrated in complex structures, we take into consideration theoretical references about curriculum policies, cultural schooling, and school textbooks discussions. With this purpose we developed a case study about a bicultural school, in São Paulo, attending Spanish and Brazilian students. This study was developed in three different instances: the first, with focus in documental elements extracted from school project and curriculum strategies; the second, with particular attention to student perceptions of their cultural experiences; and the third, based on the analysis of material, practices and physics textbooks used in classroom. Our results indicate that there are, in fact, significant cultural contributions in this subject, and this do not allow a \"naturalization\" of school physics content. Moreover, it was possible to identify this particular school as a hybrid cultural construction, not free of tensions and contradictions, but integrating different and complementary contributions, with potential to promote a richer scientific education.

Tanto as tradições orais apropriadas pela Literatura Infantil e Juvenil quanto pela Literatura de maneira geral contribuem para a formação da identidade cultural, porque trazem elementos da cultura através dos quais nos reconhecemos e nos identificamos pelos laços de pertencimento. Destacamos como objetivo precípuo deste trabalho a imersão no processo de construção da identidade cultural cabo-verdiana, híbrida e plural, a fim de dar visibilidade a estratégias de que lança mão a literatura para se apropriar tanto do patrimônio oral quando das vivências históricas para dinamizar o imaginário infantil e juvenil. A oralidade, resgatada em vários números da Revista Claridade (1936 e 1937), evidencia-se na narrativa oral O Lobo e o Chibinho, transposta para a linguagem literária por Baltasar Lopes, no excerto Bibia, do romance Chiquinho, e ainda em cinco capítulos dessa obra publicados naquela revista. O imaginário infantil e juvenil crioulo será ainda por nós analisado a partir da leitura de Infância, primeira parte de Chiquinho, e da obra Comandante Hussi, de Jorge Araújo, em que enfatizamos diálogos interculturais e intertextuais ensejados pela paródia a texto jornalístico que motivou a obra. As categorias teóricas em que apoiamos nossas análises foram, respectivamente, identidade cultural (Stuart Hall) e hibridação (Néstor Canclini). / The oral traditions appropriated by Childrens and Youth Literature as well as by Literature in general contributed to the cultural identity formation since they have cultural elements which we recognize and identify ourselves with through belonging ties. The main aim of this paper is the immersion in the Cape Verdean process of cultural identity formation, hybrid and plural, in order to give visibility to strategies resorted by Literature to incorporate both the oral heritage and the historical experiences to boost Children´s and juvenile imaginary. The oral tradition, rescued in many of the Claridade magazine texts (1936 and 1937), become clear in the oral narrative O Lobo e o Chibinho, transposed to the literally language by Baltasar Lopes, in the excerpt Bibia, from Chiquinho novel, including in more five chapters in the same edition. The Creole Children´s and Youth imaginary will be analysed in this paper by the readings of Infância, in the first part of Chiquinho, and of the text Comandante Hussi, by Jorge Araújo, emphasizing intercultural and intertextual dialogues occasioned by parody to journalistic text that motivated the work. The theoretical categories that support the analyses in this paper were, respectively, cultural identity (Stuart Hall) and hybridization (Néstor Canclini).

A história de domesticação da cana-de-açúcar (Saccharum spp.) é atípica. As variedades modernas derivam de um processo que inclui hibridações entre a espécie domesticada S. officinarum e a silvestre S. spontaneum, sucessivos retrocruzamentos, no sentido de recuperar o genoma de S. officinarum e a seleção de progênies superiores. Além disso, as genealogias contemplam cruzamentos entre genótipos e eventualmente espécies, todos com elevado grau de ploidia e número de cromossomos distintos, assim como aneuploidias. Frente ao exposto, este trabalho teve como objetivos estabelecer o número de cromossomos e avaliar o comportamento meiótico da cultivar IACSP93-3046, bem como, identificar as associações cromossômicas em meiose I dos genótipos IACSP93-3046, IACSP95-3018 e de um representante de S. officinarum, Caiana Fita, pela marcação dos centrômeros usando FISH. O número de cromossomos da cultivar IACSP93-3046 foi determinado a partir de preparações do meristema radicular, pré-tratado com 8-hidroxiquinolina (0,03%, 4h), e corado pelo método de Feulgen. As células metafásicas foram analisadas sob microscopia óptica, preferencialmente intactas e com o mínimo de sobreposição de cromossomos. Para a análise do comportamento meiótico utilizou-se a técnica de esmagamento, e as células foram coradas com carmim propiônico. Foram observadas as fases meióticas desde a metáfase I até a telófase II, bem como as tétrades. O pareamento cromossômico em meiose I foi analisado usando a técnica de hibridização in situ fluorescente (FISH). Para tanto, preparações dos genótipos IACSP93-3046, IACSP95-3018 e Caiana Fita foram realizadas pelo gotejamento de uma suspensão de células em diacinese. As sondas foram obtidas por PCR a partir da amplificação da região centromérica de cana-de-açúcar, marcadas com digoxigenina-11-dUTP, por nick translation, e detectadas com anti-digoxigenina-rodamina. As lâminas foram montadas em DAPI-Vectashield e analisadas sob microscopia de fluorescência. O número diplóide 2n = 112 foi observado para a cultivar IACSP93-3046, sendo caracterizado pela primeira vez neste estudo. A microsporogênese de IACSP93-3046 apresentou elevado percentual de irregularidades (68%). De modo geral, as anormalidades foram relativas à segregação dos cromossomos, e incluíram migração precoce para os polos em metáfase I e II, cromossomos retardatários em anáfase (I e II) e em telófase (I e II), cromossomos perdidos em prófase II, e micronúcleos nas tétrades. A análise dos sítios de hibridização permitiu comprovar que os cromossomos se associam predominantemente como bivalentes em IACSP93-3046, IACSP95-3018 e Caiana Fita. As irregularidades na segregação dos cromossomos conduzem a micrósporos aneuploides, como constatado em IACSP93-3046. Sugere-se que a assincronia do processo meiótico entre os genomas que compõem a cana-de-açúcar tem papel relevante na geração dessas irregularidades. / The history of the sugarcane domestication (Saccharum spp.) is atypical. Modern varieties are derived from a hybridization process between the domestic species S. officinarum and the wild species S. spontaneum, successive backcrossings to recover the genome of S. officinarum, and the selection of superior progenies. The genealogies include crossings among genotypes, and possibly Saccharum species, all with a high degree of ploidy and different numbers of chromosomes, as well as aneuploidies. The study aimed to establish the number of chromosomes and evaluate the meiotic behavior of cultivar IACSP93-3046, and identify chromosomal associations in meiosis I of genotypes IACSP93-3046, IACSP95-3018 and Caiana Fita (a representative of S. officinarum) by labeling centromeres using fluorescence in situ hybridization (FISH). The number of chromosomes in cultivar IACSP93-3046 was determined from the root meristem preparations, pretreated with 8-hydroxiquinoline and stained by the Feulgen method. Metaphasic cells, preferably intact and with minimum chromosome overlap, were analyzed under an optical microscope. Meiotic behavior was examined from the preparations by using squashing method and stained with propionic carmine. Meiotic phases were observed from metaphase I to telophase II, and tetrad stages. Chromosomal pairing in meiosis I was analyzed by using the FISH technique. The slides of genotypes IACSP93-3046, IACSP95-3018 and Caiana Fita were produced by dropping a suspension of meiocytes in diakinesis. The probes were obtained by PCR, with amplification of the centromere region, and labeled with digoxigenin-11-dUTP, by nick translation, and detected with anti-digoxigenin-rhodamine. The slides were mounted in DAPI-Vectashield and analyzed under a fluorescence microscope. The diploid number 2n = 112 was observed for cultivar IACSP93-3046 and characterized in this study for the first time. Microsporogenesis of IACSP93-3046 presented a high irregularity percentage regarding chromosome segregation, especially precocious migration to poles in metaphase I and II, laggard chromosomes in anaphase and telophase I and II, lost chromosomes in prophase II, and micronuclei in the tetrad stages. The analysis from the hybridization sites proved that the chromosomal pairing occurred predominantly as bivalents in IACSP93-3046, IACSP95-3018 and Caiana Fita. Chromosomal segregation irregularities led to aneuploid microspores, as confirmed in IACSP93-3046, suggesting the asynchrony in the meiotic process between the sugarcane genomes play an important role in producing these irregularities.