La technique d'une DPC pour adénocarcinome a évolué ces dernières années tant au niveau sécurité qu'au niveau carcinologique mais cela n'a pas suffit à faire progresser la survie. On peut se demander si la modification de la technique chirurgicale pourrait avoir un impact significatif sur la survie.A) Nous avons recherché, sur 23 pièces de DPC encrées, la présence de la mutation K-ras au niveau de la marge veineuse affirmée R0 en analyse histologique : 13 spécimens (groupe kras+) exprimaient une mutation K-ras au sein de la marge veineuse versus 10 spécimens (groupe kras-) ne l'exprimant pas. Les tumeurs des 2 groupes étaient comparables (taille, envahissement ganglionnaire, engainement périnerveux…). La survie globale à 1 an et 3 ans des groupes kras- versus kras+ étaient de 80% versus 84,6% et 16,7% versus 0% (p=0,03), respectivement. Les médianes de survie des groupes kras- versus kras+ étaient de 24 mois versus 16 mois (p=0,04), respectivement.B) Nous avons comparé, après appariement, 19 patients ayant eu une DPC avec résection « par excès » de la veine porte (groupe VP) avec 19 patients ayant eu une DPC sans résection de la veine porte (groupe contrôle). Les survies médianes et à 3 ans du groupe VP versus groupe contrôle étaient 42 mois versus 22 mois (p=0,04) et 60% versus 31% (p=0,03), respectivement.En conclusion, notre travail a montré qu'au-delà de la marge déterminée par le chirurgien pendant la chirurgie, de celle de l'anatomopathologiste déterminée par l'analyse microscopique, il existait une marge génique. La résection systématique de la veine porte semblait bénéfique car elle permettait sans doute de passer au-delà de cette marge génique. / Pancreticoduodenectomy (PD) for adenocarcinoma was safer during the last decades but did not improve survival. We sought to determine if technical changes during PD could improve survival.A) In a first study, we determine the presence of K-ras mutation in the venous margin of 23 PD's specimens. Thirteen specimens had K-ras mutation (kras+ group) and 10 specimens did not (kras- group). Except K-ras mutation status, tumors of the 2 groups were not different when comparing major histological findings (margin status, lymph node invasion, perineural invasion…). Overall 1- and 3-years survival of patients of kras- group versus kras+ group were 80% versus 84,6% and 16,7% versus 0% (p=0,03), respectively. Median survival of patients of kras- group versus kras+ group were 24 months versus 16 months (p=0,04), respectively.B) In a second study, we compared 19 patients with “excessive” portal vein resection during PD (PV group) with 19 matched patients who underwent PD without venous resection (control group). Median survival of patients of PV group versus control group were 42 months versus 22 months (p=0,04), respectively.In conclusion, we showed that the « genic margin » concept was consistent. Systematic portal vein resection could avoid positive genic margin and might be benefic for patient who underwent PD for resecable adenocarcinoma.

Les gliomes de bas-grade représentent la tumeur cérébrale la plus fréquente chez l’enfant. Elles sont caractérisées par un large spectre de sous-types tumoraux, très hétérogènes. Leur définition actuelle est principalement basée sur des critères histologiques ce qui représente une limite importante car ces classifications souffrent d’un manque de précision. Les progrès récents de la génomique nous permettent d’approfondir considérablement les connaissances sur la biologie de ces tumeurs afin d’enrichir leur classification actuelle. Ce travail présente une analyse approfondie des altérations génomiques de l’ADN et l’ARN des gliomes de bas-grade pédiatriques. Le premier niveau d’analyse se base sur l’analyse du séquençage à haut débit de 169 gliomes de bas-grade de l’enfant. Bien que les mutations des gènes BRAF et FGFR1 sont les plus fréquemment décrites dans ces tumeurs, nous avons identifié pour la première fois le réarrangement chromosomique MYB-QKI majoritairement associé aux gliomes angiocentriques. Dans un deuxième temps ce travail décrit l’analyse du transcriptome de 151 gliomes de bas grade extraits à partir de tissu conservé en paraffine. Nous avons observé des différences moléculaires en fonction de leur sous-type histologique, de la localisation tumorale et de leur statut BRAF. Dans le dernier volet de ce travail, nous avons testé la faisabilité d’isoler par cytométrie en flux une cellule unique en les distinguant selon un marqueur de différenciation glial (A2B5+ et A2B5-) et d’effectuer une analyse transcriptomique à haut-débit en séquençant l’ARN à l’échelle d’une cellule unique. Cette technique nous a permis de décrire des différences moléculaires intéressantes entre des cellules A2B5+ et A2B5-. Ces résultats soulignent l’intérêt d’exploiter des nouvelles technologies de pointe pour servir de base à l’étude des caractéristiques biologiques des cellules tumorales. / Low-grade gliomas represent the most frequent brain tumor arising during childhood. They are characterized by a broad spectrum of tumor types.The definition of low-grade gliomas has been mainly based on morphology. This histological classification of pediatric low-grade gliomas (PLGG), suffers from the lack of reproducibility. The recent progress in molecular biology and genetics has brought new insights in the biology of those tumors and allows better understanding of their biology. This work provides a comprehensive analysis of two different genetic approaches in PLGGs. The first part is based on the description of somatic genetic alterations of the DNA. Using a large PLGG cohort, we have dissect the genome of those tumors and draw the landscape of their genetic alteration. Although BRAF and FGFR1 alterations are predominantly altered, we have discovered a new translocation, MYB-QKI, that is almost exclusively present in a specific histological subgroup; angiocentric gliomasThe second part of the thesis describes transcriptomic analysis of bulk PLGGs. This work describes molecular differences between PLGGs from distinct histologies and arising from different locations in the brain as well as different BRAF mutation status.We were also able to test single-cell expression analyses in three pilocytic astrocytomas (PAs) using RNA-sequencing. In this experimental work we have successfully tested the hypothesis that we can isolate single-cells from fresh PLGG tumors in order to analyze the trasncriptome at a large scale. We observed that single-cells expressing A2B5, a glial progenitor marker, isolated in pediatric PAs are characterized as a distinct biological population. These results underline the importance to improve the precision of the transcriptomic studies to capture the molecular signal of tumor cells and further understand the different pattern between normal cells and tumor cells.

No desenvolvimento de software, a qualidade do produto está diretamente relacionada à qualidade do processo de desenvolvimento. Diante disso, atividades de Verificação, Validação & Teste (VV&T) realizadas por meio de métodos, técnicas e ferramentas são de extrema necessidade para o aumento da produtividade, qualidade e diminuição de custos no desenvolvimento de software. Do mesmo modo, técnicas e critérios contribuem para a produtividade das atividades de teste. Um ponto crucial para o teste de software é sua automatização, tornando as atividades mais confiáveis e diminuindo significativamente os custos de desenvolvimento. Na automatização dos testes, os oráculos são essenciais, representando um mecanismo (programa, processo ou dados) que indica se a saída obtida para um caso de teste está correta. Este trabalho de mestrado utiliza a ideia de mutação para criar implementações alternativas de oráculos de teste e, assim, avaliar a sua qualidade. O teste de mutação se refere à criação de versões do sistema em desenvolvimento com pequenas alterações sintáticas de código. A mutação possui alta eficácia na detecção de defeitos e é bastante flexível na sua aplicação, podendo ser utilizada em diversos tipos de artefatos. Adicionalmente, este trabalho propõe operadores de mutação específicos para oráculos, implementa uma ferramenta de apoio à utilização desses operadores para oráculos e também descreve um estudo empírico dos operadores, destacando benefícios e desafios associados ao seu uso. / In software development, product quality is directly related to the quality of the development process. Therefore, activities of Verification, Validation & Testing (VV&T) performed by methods, techniques and tools are urgently required to increase productivity, quality and cost reduction in software development. Similarly, testing technique and criteria contribute to the productivity of test activities. A crucial point for the software testing automation is making the most reliable activities and significantly reducing development costs. Regarding software testing automation, test oracles are essential, representing an mechanism (program, process or data) to indicate whether the actual output for a given test case is correct. This masters thesis aims to explore concepts of mutation testing to create alternative implementations of the oracle procedure and thus assess their quality. Mutation testing refers to the creation of system development versions with minor syntactic code changes. It has high efficiency on defects detecting and it is very flexible in its application and it is being used in various types of artifacts. This work also proposes specific mutation operators for oracles, implements an useful support tool for using these oracle mutation operators and conducts an empirical study of operators, highlighting benefits and challenges associated with their use.

Um dos pontos fundamentais na atividade de teste de software é o projeto de casos de teste. Diversos critérios de adequação têm sido propostos com o objetivo de fornecer meios que permitam que a avaliação e elaboração de casos de teste sejam feitas de maneira sistemática e fundamentadas teoricamente. Infelizmente, a maioria dos critérios de adequação de casos de teste definidos tem seu uso restrito ao teste de unidade. Para fases posteriores da atividade de teste, em particular para o teste de integração, nota-se a ausência de critérios de adequação, principalmente porque os critérios propostos definem requisitos de teste que se restringem aos limites de uma única unidade, não exercitando de maneira efetiva as interações entre as unidades, que devem ser alvo principal no teste de integração. Com exceção de alguns poucos trabalhos que procuram estender critérios estruturais para o nível interprocedimental, tem-se utilizado nessa fase de teste, quase que exclusivamente, critérios funcionais. Dada essa ausência de critérios e salientando ainda o caráter complementar entre as diferentes técnicas de teste, esta tese apresenta um critério de teste interprocedimental baseado em defeitos chamado de Mutação de Interface. Esse critério busca exercitar as interações entre as unidades através da seleção de casos de teste que distingam mutantes criados pela introdução de defeitos típicos e que, de acordo com um modo definido, caracterizamos erros de integração. Definiu-se um conjunto de operadores de Mutação de Interface que concentram sua aplicação em pontos do programa relacionados com as interações entre as unidades, como, por exemplo, chamadas de subprogramas e seus parâmetros. Dados o alto custo de aplicação, inerente de critérios baseados em mutação, e pelas próprias características do conjunto de operadores de Mutação de Interface, torna-se necessário definirem-se abordagens para reduzir esse custo. Assim, foram estabelecidas maneiras de se parametrizar a aplicação dos operadores de mutação, definindo-se critérios de Mutação de Interface alternativos, estendendo-se abordagem sutilizadas no teste de mutação convencional como mutação restrita. A aplicação de um critério de teste está fortemente condicionada à sua automatização. A definição de um critério de teste sem que pelo menos se apontem soluções para sua automatização tem pouca utilidade prática. Por isso, especificou-se e implementou-se a ferramenta PROTEUM/IM para apoiar a aplicação do critério Mutação de Interface. Essa ferramenta torna-se essencial neste trabalho à medida que permite que estudos empíricos possam ser realizados, avaliando o critério proposto. Dois estudos de caso são apresentados. Esses estudos aplicam o critério Mutação de interface em programas reais e buscam avaliar seu custo e sua eficácia em revelar erros. Estes estudos aplicam ainda critérios alternativos, mostrando que a Mutação de interface é bastante efetiva em revelar erros o de ter custo de aplicação bastante reduzido, quando aplicada de maneira incremental, utilizando-se as parametrizações que os operadores de mutação oferecem. / The project of test cases is one of the most important topics in the software testing activity. Several criteria have been proposed aiming at allowing the evaluation and selection of test cases in a systematic and theoretically well founded way. Unfortunately, the use of most of these criteria is restricted to the unit testing phase. For other testing phases, in particular for integration testing. there is a lack of such criteria, mainly because the existing criteria define test requirements only in the scope of a single unit. They arc not able to effectively exercise the interactions between units, what should be the focus of integration testing. Excepting some few works that extend structural criteria to the interprocedural level, only functional testing has been used at integration testing phase. Given this lack of criteria and the complementary characteristics of different testing techniques, this thesis presents an interprocedural fault based criterion named Interface Mutation. This criterion exercises the interactions between units through the selection of test cases that distinguish mutants created by introducing typical faults that characterize integration errors. A set of Interface Mutation operators was defined. The focus of these operators are the points of the program related to the unit interactions, for instance, subprogram calls and their parameters. Given the high cost associated to mutation testing in general and particularly to the Interface Mutation operators, it is necessary to define some approaches to reduce its application cost. Thus, some parameterizations were defined to the mutation operators, allowing to establish alternative Interface Mutation criteria, extending approaches already used in conventional mutation testing, as random mutation and constrained mutation. The application of any testing criterion strongly depends on its automatization. The definition of a criterion, without pointing out ways to its implementation has little practical utility. So, a tool named PROTEUM/IM was specified and implemented to support the application of Interface Mutation. This tool is an essential point in the present work because it allows the conduction of empirical studies aiming at evaluating the proposed criterion. Two case studies arc presented. In these studies the criterion Interface Mutation is applied to real programs and the cost of its application as well as its errors revealing effectiveness are evaluated. Alternative criteria are also used. Showing that Interface Mutation is very effective to reveal errors and can be applied with a reduced cost if used in an incremental way, taking advantage of the parameterization characteristics provided by the Interface Mutation operators set.

Tsang Po Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 135-152). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Abstract 摘要 --- p.iv / List of Figures --- p.vi / List of Tables --- p.viii / List of Abbreviations --- p.ix / Table of Contents --- p.xii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- The Role of Glucose in Biological System --- p.1 / Chapter 1.2 --- Glucose Transporter Families --- p.1 / Chapter 1.2.1 --- Na+-Dependent Glucose Transporters --- p.2 / Chapter 1.2.2 --- Facilitative Glucose Transporters --- p.3 / Chapter 1.3 --- Glucose Transporter Type1 --- p.7 / Chapter 1.3.1 --- Primary Structure of the Glutl Protein --- p.7 / Chapter 1.3.2 --- Secondary Structure --- p.8 / Chapter 1.3.3 --- Tertiary Structure --- p.8 / Chapter 1.3.4 --- Kinetics Properties --- p.11 / Chapter 1.3.5 --- Tissue Distribution --- p.12 / Chapter 1.3.6 --- Multifunctional Property --- p.13 / Chapter 1.3.7 --- Characterization of GLUT1 Gene --- p.13 / Chapter 1.3.8 --- Regulation of GLUT1 Expression --- p.14 / Chapter 1.4 --- Glucose Transporter Type 1 and the Brain --- p.16 / Chapter 1.5 --- Glucose Transporter Type 1 Deficiency Syndrome (GIutlDS) --- p.19 / Chapter 1.5.1 --- Backgronnd of GIutlDS --- p.19 / Chapter 1.5.2 --- Clinical Features of GIutlDS --- p.23 / Chapter 1.5.3 --- Genotype-Phenotype Correlations --- p.24 / Chapter 1.5.4 --- Diagnosis --- p.26 / Chapter 1.5.5 --- Manage nent --- p.27 / Chapter 1.5.5.1 --- Ketogenic Diet --- p.27 / Chapter 1.6 --- Hypothesis and Objectives --- p.29 / Chapter Chapter 2: --- Biochemical and Molecular Analysis of GLUT1 in a Suspected GlutlDS Case --- p.31 / Chapter 2.1 --- Materials --- p.32 / Chapter 2.1.1 --- Clinical History of Suspected GlutlDS Patient --- p.32 / Chapter 2.1.2 --- Blood Samples --- p.32 / Chapter 2.1.3 --- Reagents and Buffers for Reverse Transcription --- p.32 / Chapter 2.1.4 --- Reagents and Buffers for TA Cloning --- p.34 / Chapter 2.1.5 --- Reagents for Genomic DNA Extraction --- p.34 / Chapter 2.1.6 --- Reagents and Buffers for Polymerase Chain Reaction (PCR) --- p.34 / Chapter 2.1.7 --- Reagents and Buffers for Agarose Gel Electrophoresis --- p.35 / Chapter 2.1.8 --- Reagents for Zero-trans 3-OMG Influx in Erythrocytes --- p.37 / Chapter 2.1.9 --- Reagents for Zero-trans 3-OMG Efflux from Erythrocytes --- p.38 / Chapter 2.1.10 --- Reagents for Erythrocytes Membrane Extraction and Detection --- p.39 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- GLUT1 Gene Analysis --- p.44 / Chapter 2.2.2 --- Zero-trans 3-OMG Influx into Erythrocytes --- p.51 / Chapter 2.2.3 --- Zero-trans 3-OMG Efflux from Erythrocytes --- p.52 / Chapter 2.2.4 --- Glutl Protein Expression --- p.54 / Chapter 2.2.5 --- Statistics --- p.57 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Molecular Analysis of the GLUT1 Gene of a Suspected GlutlDS Patient --- p.58 / Chapter 2.3.2 --- Functional Analysis of the GlutlDS Patient's Glutl Protein --- p.61 / Chapter 2.3.3 --- Glutl Protein Expression in the GlutlDS Patient --- p.64 / Chapter 2.4 --- Discussion --- p.66 / Chapter Chapter 3: --- Pathogenicity Studies of GLUT1 Mutations --- p.71 / Chapter 3.1 --- Materials --- p.72 / Chapter 3.1.1 --- Construction of Glutl-Encoding Vectors --- p.72 / Chapter 3.1.2 --- Cell Lire --- p.73 / Chapter 3.1.3 --- "Cell Culture Media, Buffers and Other Reagents" --- p.73 / Chapter 3.1.4 --- Cell Culture Wares --- p.75 / Chapter 3.1.5 --- Reagents for Transfection --- p.75 / Chapter 3.1.6 --- Reagents for Protein Determination and Western Blot Analysis --- p.76 / Chapter 3.1.7 --- Consumables for Confocal Microscopy --- p.77 / Chapter 3.1.8 --- Reagents and Buffers for Flow Cytometry --- p.77 / Chapter 3.1.9 --- Reagents for 2-DOG Uptake in CHO-K1 Cells --- p.77 / Chapter 3.2 --- Methods --- p.79 / Chapter 3.2.1 --- Cell Culture Methodology --- p.79 / Chapter 3.2.2 --- Construction of GLUT1 Mutants --- p.80 / Chapter 3.2.3 --- Establishment of Wild Type and Mutant Glutl Expressing Cell Lines --- p.84 / Chapter 3.2.4 --- Protein Expression Study --- p.85 / Chapter 3.2.5 --- 2-DOG Influx Assay in CHO-K1 Cells --- p.87 / Chapter 3.2.6 --- Confocal Microscopy Studies on Glutl Cellular Localization --- p.89 / Chapter 3.2.7 --- Statistics --- p.90 / Chapter 3.3 --- Results --- p.91 / Chapter 3.3.1 --- Molecular Analysis of 1034-1035Insl2 Mutation --- p.91 / Chapter 3.3.2 --- Expression of the Wild Type and Mutant GFP-Glutl Fusion Proteins --- p.92 / Chapter 3.3.3 --- Functional Analysis of the 1034-1035Insl2 Mutant --- p.95 / Chapter 3.4 --- Discussion --- p.97 / Chapter Chapter 4: --- GLUT1 Promoter Study --- p.100 / Chapter 4.1 --- Materials --- p.101 / Chapter 4.1.1 --- Construction of GLUT1 Promoter Vectors --- p.101 / Chapter 4.1.2 --- Cell Lines --- p.102 / Chapter 4.1.3 --- Cell Culture Media and Other Reagents --- p.103 / Chapter 4.1.4 --- Dual Luciferase Reporter Assay System --- p.103 / Chapter 4.2 --- Methods --- p.105 / Chapter 4.2.1 --- Bioinformatics --- p.105 / Chapter 4.2.2 --- Cell Culture --- p.105 / Chapter 4.2.3 --- Construetion of GLUT1 Promoter Vectors --- p.105 / Chapter 4.2.4 --- 5'-Deletion Analysis of GLUT1 Promoter --- p.108 / Chapter 4.2.5 --- Determination of the Activities of GLUT1 Promoter Fragments --- p.110 / Chapter 4.2.6 --- Statistics --- p.113 / Chapter 4.3 --- Results --- p.114 / Chapter 4.3.1 --- Determination of the Promoter Activity of the 5'-deletion Fragments --- p.114 / Chapter 4.3.2 --- Prediction of Transcription Factors in the 5'-deletion Fragments --- p.119 / Chapter 4.4 --- Discussion --- p.121 / Chapter Chapter 5: --- General Conclusion and Future Perspectives --- p.133 / References --- p.135

HBV DNA replicates through a genomic RNA intermediate. The HBV reverse transcriptase lacks proof-reading activity, resulting in a much higher mutation rate for the HBV genome compared with other DNA viruses. HBV DNA thus is often present in quasi-species in an individual. One or more species may be favorably selected by factors like host immune clearance and use of antiviral drugs. / Hepatitis B virus (HBV) infected millions of people worldwide. Chronic HBV infection is the leading cause of liver cirrhosis and hepatocellular carcinoma (HCC). / In summary, this study developed and validated two platforms for (1) HBV mutation discovery; and (2) HBV mutation detection in viral quasi-species. These tools may be useful for research on HBV drug resistant mutations, clinical instructing and monitoring of antiviral treatment. / In this study, I have developed high-throughput methods for (1) discovery of novel HBV mutations; and (2) highly multiplexed detection of known HBV mutations, both in the background of HBV quasi-species. Patients undergoing long-term lamivudine treatment were used for mutation discovery. For mutation discovery in quasi-species, the MassCLEAVE(TM) technology, a method based on base-specific RNA cleavage and automated Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS), was used. I found that MassCLEAVE(TM) can be used to discover mutations present as minorities. Additionally, a synergistic effect was found between direct sequencing and MassCLEAVE(TM) in identifying minority mutations. Multi-PLEX, a method based on single nucleotide extension and automated MALDI-TOF MS, was used to develop a highly multiplexed assay for simultaneous detection of 60 HBV mutations including all functionally known HBV mutations and other frequently observed mutations during antiviral treatment with unknown functions. This multiplex assay was tested on a large cohort of single and multiple drug-resistant patients and was shown to be highly accurate in detecting HBV viral mutations in quasi-species. / Nucleotide and nucleoside analogues (NAs) are widely used for antiviral therapy by effectively suppressing viral DNA replication. However, long-term administration may select for drug-resistant mutant strains, leading to treatment failure and liver disease progression. A number of HBV mutations such as rtM204V/I, rtN236T and rtL180M within the HBV reverse transcriptase are known to confer drug resistance. Detection of these known mutations is useful genotypic markers for monitoring antiviral treatment. In addition, novel drug resistant mutations continue to be discovered. / by Luan, Ju. / Adviser: Chunming Ding. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 136-149). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

by Kwok Fung, Lo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 89-97). / Acknowledgement --- p.i / Table of Content --- p.ii / Abstract --- p.vi / List of Abbreviation --- p.viii / List of Tables and figures --- p.ix / Chapter Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- The immunoglobulin --- p.1 / Chapter 1.1.1 --- Immunoglobulin structure --- p.1 / Chapter 1.1.2 --- Immunogloblulin genes --- p.1 / Chapter 1.1.3 --- Immunogloblulin gene recombination --- p.3 / Chapter 1.1.4 --- Antibody diversity --- p.4 / Chapter 1.1.4.1 --- Imprecise joining --- p.5 / Chapter 1.1.4.2 --- N region addition --- p.5 / Chapter 1.1.4.3 --- Somatic mutation --- p.6 / Chapter 1.2 --- Hypermutation --- p.6 / Chapter 1.2.1 --- Features of hypermutation --- p.6 / Chapter 1.2.2 --- Germinal centre & Affinity maturation --- p.8 / Chapter 1.2.3 --- Mutational Hotspots --- p.9 / Chapter 1.2.4 --- Intrinsic characteristics of hypermutation --- p.10 / Chapter 1.2.5 --- Models for the mechanism of hypermutation --- p.11 / Chapter 1.2.5.1 --- DNA replication --- p.11 / Chapter 1.2.5.2 --- DNA repair --- p.12 / Chapter 1.2.5.3 --- Gene conversion --- p.13 / Chapter 1.2.5.4 --- Transcription --- p.15 / Chapter 1.2.5.5 --- Homologous recombination of reverse transcribed mRNA --- p.16 / Chapter 1.2.5.6 --- Transcription-coupled repair --- p.17 / Chapter 1.3 --- Scope of investigation --- p.17 / Chapter Chapter 2 --- Material and Method --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Methods (first generation of LMPCR) --- p.21 / Chapter 2.2.1 --- Animal and cell lines --- p.21 / Chapter 2.2.2 --- Oxazolone antigen immunization --- p.21 / Chapter 2.2.2.1 --- Preparation of Bordetella pertussis --- p.21 / Chapter 2.2.2.2 --- Coupling of phenyloxazolone with CSA or BSA --- p.22 / Chapter 2.2.2.3 --- Preparation of aluminium hydroxide adjuvant --- p.23 / Chapter 2.2.2.4 --- Mice immunization --- p.23 / Chapter 2.2.3 --- Detection of anti-phOx antibody by enzyme-linked immunosorbent assay (ELISA) --- p.24 / Chapter 2.2.3.1 --- Reagents --- p.24 / Chapter 2.2.3.2 --- Assay procedure --- p.24 / Chapter 2.2.4 --- Extraction of genomic DNA (mice/cell line) --- p.25 / Chapter 2.2.4.1 --- Reagents --- p.25 / Chapter 2.2.4.2 --- Isolation of DNA from cell line (NQ2.12.4 & NQ5.4.3) --- p.25 / Chapter 2.2.4.3. --- DNA extraction from mice --- p.26 / Chapter 2.2.5 --- Ligation-mediated polymerase chain reaction (LMPCR) --- p.26 / Chapter 2.2.5.1 --- Procedure --- p.26 / Chapter 2.2.5.1.1 --- First primer extension --- p.29 / Chapter 2.2.5.1.2 --- Ligation --- p.29 / Chapter 2.2.5.1.3 --- PCR amplification --- p.30 / Chapter 2.2.5.1.4 --- Labelling of LMPCR product --- p.30 / Chapter 2.2.6 --- Marker preparation --- p.31 / Chapter 2.2.7 --- Polyacrylamide gel electrophoresis --- p.32 / Chapter 2.2.7.1 --- Reagents --- p.32 / Chapter 2.2.7.2 --- Procedure --- p.32 / Chapter 2.2.8 --- Southern blot hybridization --- p.33 / Chapter 2.2.8.1 --- Reagents --- p.33 / Chapter 2.2.8.2 --- DNA blotting --- p.34 / Chapter 2.2.8.3 --- Preparation of 32P labelling DNA probe --- p.34 / Chapter 2.2.8.4 --- Prehybridization and Hybridization --- p.35 / Chapter 2.2.9 --- Simplified protocol for the first generation of LMPCR --- p.36 / Chapter 2.3 --- Method (second generation of LMPCR) --- p.37 / Chapter 2.3.1 --- Excess linker removal --- p.37 / Chapter 2.3.1.1 --- Exonuclease III Treatment --- p.37 / Chapter 2.3.1.2 --- Mung bean nuclease Treatment --- p.37 / Chapter 2.3.1.3 --- Chroma Spin Treatment --- p.37 / Chapter 2.3.2 --- HindIII digestion after LMPCR --- p.38 / Chapter 2.3.3 --- DNA sequencing --- p.38 / Chapter 2.3.3.1 --- Cloning of amplified sequences to M13mpl9 plasmid --- p.38 / Chapter 2.3.3.2 --- Plaque hybridization --- p.39 / Chapter 2.3.3.3 --- Preparation of single-stranded templates --- p.39 / Chapter 2.3.3.4 --- Sanger dideoxy sequencing of single-stranded DNA --- p.40 / Chapter 2.3.4 --- Simplified protocol for second generation of LMPCR --- p.41 / Chapter Chapter 3 --- First generation of LMPCR --- p.42 / Chapter 3.1 --- General design --- p.42 / Chapter 3.1.1 --- LMPCR protocol and its modification --- p.42 / Chapter 3.1.2 --- Oligonucleotide design --- p.44 / Chapter 3.1.3 --- Experimental design --- p.48 / Chapter 3.2 --- Result --- p.50 / Chapter 3.2.1 --- Anti-phOx Ig level in normal and immunized mice --- p.50 / Chapter 3.2.2 --- LMPCR analysis of the sense strand of VkOxl --- p.50 / Chapter 3.2.2.1 --- Overall patterns of the LMPCR signals --- p.50 / Chapter 3.2.2.2 --- Southern hybridization --- p.50 / Chapter 3.2.2.3 --- Distribution of signals --- p.57 / Chapter 3.2.2.4 --- LMPCR analysis of the VkOxl-Jk5 anti-phOx transgene --- p.61 / Chapter 3.2.2.5 --- Effect of the number of cells carrying the VkOxl-Jk5 gene on LMPCR --- p.61 / Chapter 3.2.3 --- LMPCR analysis of the antisense strand of VkOxl --- p.64 / Chapter 3.3 --- Discussion --- p.64 / Chapter Chapter 4 --- Second generation of LMPCR --- p.72 / Chapter 4.1 --- Introduction(experi mental modification) --- p.72 / Chapter 4.1.1 --- Tagging the specific LMPCR products by addition of a Hin dIII site in the linker --- p.72 / Chapter 4.1.2 --- "Removal of excess linker, OXUH" --- p.72 / Chapter 4.1.2.1 --- Exonuclease III treatment --- p.73 / Chapter 4.1.2.2 --- Chroma spin treatment --- p.73 / Chapter 4.1.2.3 --- Mung Bean Nuclease treatment --- p.75 / Chapter 4.1.3 --- Other modifications in LMPCR --- p.75 / Chapter 4.2 --- Results --- p.75 / Chapter 4.2.1 --- Effect of including Exonuclease III treatment --- p.75 / Chapter 4.2.2 --- Effect of including Mung Bean Nuclease treatment --- p.76 / Chapter 4.2.3 --- Effect of including Chroma spin treatment --- p.76 / Chapter 4.2.4 --- Strand break positions detected at the sense strand --- p.76 / Chapter 4.2.5 --- DNA sequence analysis of the antisense strand LMPCR products --- p.82 / Chapter 4.3 --- Discussion --- p.84 / References --- p.89 / Appendix I --- p.98

Le syndrome de Usher (USH) est une maladie transmise selon le mode autosomique récessif caractérisée par l’association d’une surdité congénitale (HL) et d’une rétinite pigmentaire (RP), et dans certains cas, d’une aréflexie vestibulaire. Une hétérogénéité clinique et génétique est reconnue. Environ 10 % des cas USH restent non résolus après analyse moléculaire exhaustive des différents gènes. Ces cas incluent les patients qui ne portent aucune mutation dans un des gènes USH connus ainsi que les patients porteurs d’une seule mutation dans un gène USH. Au cours de cette thèse, nous nous sommes intéressés à l’étude des patients porteurs d’une seule mutation dans les gènes USH2A et PCDH15.Dans la première partie de la thèse, nous avons analysé une cohorte de patients avec un phénotype USH2A bien défini : 5 patients pour lesquels une seule mutation à l’état hétérozygote avait été identifiée dans le gène USH2A et un patient porteur d’un variant silencieux en trans d’une mutation non-sens.Pour les 5 patients, nous avons émis l’hypothèse que la seconde mutation, restant à être identifiée, pourrait se trouver dans des régions introniques profondes. Pour cela, nous avons développé une approche de séquençage à haut débit (NGS) de l’ADN pour identifier les variants introniques profonds dans le gène USH2A et évaluer leurs conséquences sur l’épissage. Comme preuve de concept et pour valider l’approche, y compris le pipeline bio-informatique et l’évaluation des outils de prédiction de l’épissage, nous avons analysé un patient porteur d’un pseudoexon (PE) connu dans le gène USH2A. Ensuite, les 5 patients ont été étudiés en utilisant le pipeline défini, ce qui a conduit à l’identification de 3 nouveaux variants introniques profonds chez 4 d’entre eux. Tous les variants ont été prédits comme pouvant avoir un impact sur l’épissage et aboutir à l’insertion de PE. Ces prédictions ont été validées par les essais minigènes. Grâce à cette étude, nous présentons une stratégie innovante pour identifier les mutations introniques profondes, lorsque l’analyse des transcrits n’est pas possible. Par ailleurs, le pipeline bio-informatique développé fonctionne indépendamment de la taille du gène analysé, ce qui permet l’application possible de cette approche à n’importe quel gène. Par ailleurs, un oligonucléotide antisens de type morpholino (AMO) a été évalué in vitro afin de rétablir l’altération d’épissage induite par une des mutations identifiées. Les résultats ont montré un taux d’exclusion élevé du transcrit aberrant et suggèrent une application possible en thérapie moléculaire. Nous avons ensuite effectué des études sur le variant USH2A c.1377T>A, un variant silencieux afin d’évaluer son effet sur l’épissage. L’analyse de l’ARN issu de cellules nasales du patient a montré que ce variant conduit au saut de l’exon 8 dans les transcrits USH2A. Ceci a été confirmé par un essai minigène. En outre, des études préliminaires ont été réalisées en utilisant des outils de prédictions et des essais minigènes pour évaluer l’implication des éléments cis-régulateurs dans le défaut d’épissage observé chez le patient. Dans la deuxième partie de la thèse, nous avons analysé une patiente USH1, pour laquelle une seule mutation avait été identifiée dans le gène PCDH15. Dans ce cas, nous avons combiné la culture des cellules épithéliales nasales avec l’analyse des transcrits PCDH15. Celle-ci a été réalisée par séquençage de cinq RT-PCR chevauchantes. Grâce à cette analyse, nous avons réussi à délimiter une région d’intérêt dans le transcrit, dont l’amplification a échoué exclusivement pour l’allèle porteur de la mutation non identifiée. D’autres analyses ont été effectuées dans la région génomique correspondante par capture ciblée couplée au séquençage NGS et LongRange PCR suivi de séquençage Sanger. Cependant, aucun variant candidat n’a été identifié à ce jour. Nous suggérons l’implication de mécanismes moléculaires complexes qui restent à être caractérisés. / Usher syndrome (USH) is an autosomal recessive disorder characterized by the association of sensorineural hearing loss (HL) and retinitis pigmentosa (RP), and in some cases, vestibular areflexia. Clinical and genetic heterogeneity are recognised. Indeed, three clinical types can be caused by mutations in one of the 10 known genes and USH2A represents the most frequently involved gene.Approximately 10 % of the USH cases remain genetically unsolved after extensive molecular analysis of the different genes, which includes sequencing of the exons and their intronic boundaries, combined to large rearrangements screening by array CGH. These unsolved cases include patients who do not carry any mutation in any of the known USH genes and patients who carry a single USH mutation. During this thesis we focalised on the study of patients carrying a single mutation in USH2A and PCDH15 gene.First, we have analysed a cohort of well-defined USH2A patients: five patients, for whom a single USH2A heterozygous mutation had been identified and one patient carrying a silent variant in trans to a nonsense mutation. For the 5 patients, we supposed that the second mutation remaining to be found could be localised deep in the introns. Indeed, a deep intronic mutation resulting in the inclusion of a pseudoexon (PE 40) in USH2A transcripts had been identified, following RNA analysis from nasal cells. Unfortunately, analysing USH2A transcripts still represent a challenging approach in a diagnostic settings and it is not always possible. To circumvent this issue, we have developed a DNA-Next Generation Sequencing (NGS) approach to identify deep intronic variants in USH2A and evaluate their consequences on splicing. As a proof of concept and to validate this approach, including the bioinformatics pipeline and the assessment of splicing predictor tools, the patient carrying the PE 40 was analysed at first. Then, the 5 patients were studied using the defined pipeline, which led to the identification of 3 distinct novel deep intronic variants in 4 of them. All were predicted to affect splicing and resulted in the insertion of PEs, as shown by minigene assays. Through this study, we present a new and attractive strategy to identify deep intronic mutations, when RNA analyses are not possible. In addition, the bioinformatics pipeline developed is independent of the gene size, implying the possible application of this approach to any disease-linked gene. Moreover, an antisense morpholino oligonucleotide (AMO) tested in vitro for its ability to restore the splicing alterations caused by one of the identified mutation provided high inhibition rates. These results are indicative of a potential application for molecular therapy.In the second case, we have performed studies on the USH2A c.1377T>A silent variant to investigate its effect on splicing. Analysis of RNA from nasal cells of patients showed that this variant led to the skipping of exon 8 in USH2A transcripts. This was confirmed by minigene assay. Moreover, preliminary studies have been performed using prediction tools and minigene assays to assess the involvement of cis-acting elements in causing the aberrant splicing.In the second part of the thesis, we have analysed an USH1 patient, for whom only one mutation had been identified in the PCDH15 gene. In this case, we combined nasal epithelial cells culture with the analysis of the PCDH15 transcripts. This was performed by sequencing five overlapping RT-PCRs. Through this analysis, we were able to delimit a region within the transcript, which failed to be amplified exclusively in the allele carrying the unidentified mutation. Further analyses have been performed in the corresponding genomic region by NGS-target capture and LongRange PCR associated with Sanger sequencing. However, no evident mutation has been identified so far. Therefore, we suggest the involvement of complex molecular mechanisms that remain to be characterised.

Wong, Ah Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (p. 125-130). / Abstracts in English and Chinese. / ABSTRACT --- p.V / 摘要 --- p.VIII / TABLE OF CONTENTS --- p.X / ACKNOWLEDGEMENTS --- p.XII / INTRODUCTION --- p.13 / Adaptive Mutation versus Spontaneous Mutation --- p.13 / Fluctuation Test --- p.13 / Adaptive Reversion of lacI - lacZ Fusion Mutant --- p.17 / Putative Models of Adaptive Mutation Mechanisms --- p.18 / Point Mutagenesis --- p.18 / Hypermutation --- p.19 / Gene Amplification --- p.21 / Controversy over the Mechanism of Adaptive Mutation --- p.21 / Induced Mutagenesis under Other Stresses --- p.23 / The General Stress Response --- p.23 / The SOS Response --- p.24 / Reduced Mismatch Repair --- p.24 / Adaptive Mutation in Other Micro-organisms --- p.25 / Mutation Generation under Antibiotic Stress --- p.26 / Fluoroquinolones --- p.26 / Beta-Lactams --- p.27 / Aminoglycosides --- p.27 / Justification and Objectives of this Study --- p.28 / MATERIAL AND METHODS --- p.30 / Bacterial Strains --- p.30 / Culture Media --- p.30 / Antibiotics --- p.30 / Resistance Induction Assay --- p.31 / Rationale of Experimental Design --- p.31 / Agar Selection Method --- p.32 / Broth Selection Method --- p.34 / Isolation of Organisms which Exhibited Reduced Drug Susceptibility and Determination the Minimal Inhibitory Concentration (MIC) --- p.36 / Indole Test --- p.37 / DNA Extraction --- p.37 / Polymerase Chain Reaction (PCR) on the gyrA and rpoB Genes --- p.37 / PCR Product Purification and Nucleotide Sequencing --- p.39 / RESULTS --- p.40 / Solid Agar Selection Approach --- p.41 / Broth Selection Approach --- p.48 / Strain MG1655 --- p.49 / Strain BW25113 --- p.53 / recA Deletion Mutant --- p.54 / mutS Deletion Mutant --- p.56 / DISCUSSION --- p.59 / Development of Resistance Induction Assay --- p.59 / Background Resistance to Gentamicin and Rifampicin --- p.62 / Resistance Induction Effect of Ciprofloxacin --- p.64 / Relative Effects of recA and mutS Deletion --- p.66 / Putative Origins of Antibiotic Resistance Gene Mutations --- p.69 / TABLES AND FIGURES --- p.71 / REFERENCES --- p.125

Mutation of repetitive DNA by repeat-induced point mutation (RIP) is a process that occurs in many filamentous fungi of the Ascomycota during the sexual cycle. Concurrently, direct DNA repeats are often deleted by homologous recombination at high frequency during the sexual cycle. Thus, the processes of RIP and deletion compete to either mutate or remove repetitive DNA from the genome of filamentous fungi during sexual cycles. Both processes contribute to genome streamlining by controlling proliferation of transposable elements and by limiting expansion of gene families. While the genetic requirements for deletion by homologous recombination are well known, the mechanism behind the specific detection and mutation of repetitive DNA by RIP has yet to be elucidated as only a single gene essential for RIP, rid, has been identified. We have developed Fusarium graminearum as a model organism for the study of RIP by showing that it mutates repetitive DNA frequently during the sexual cycle and that the mutations due to RIP are dependent on rid. Further, we have sequenced a genetic mapping strain of F. graminearum (00-676-2) and identified 62,310 single nucleotide polymorphisms (SNPs) compared to the reference strain (PH-1). The SNP map will be useful for quickly mapping new mutants by bulk segregant analysis and high-throughput sequencing for which bioinformatic tools were specifically developed. The groundwork has thus been laid for identification of novel RIP mutants in F. graminearum, which being homothallic has a major advantage for identification of recessive mutations. We used a forward genetics approach to shed light on the mechanism of RIP in Neurospora crassa. Two rrr mutants that dominantly r��educe R��IP and r��ecombination were characterized and identified as different mutated alleles of the same gene, rrr-1[superscript L496P] and rrr-1[superscript G325N] by bulk segregant analysis and high-throughput sequencing. Bioinformatic characterization suggests RRR-1 belongs to a previously uncharacterized group of dynamin-like proteins, which are generally involved in membrane fission and fusion. RRR-1-GFP localizes to the nuclear membrane, but not DNA, suggesting it affects RIP and recombination frequency indirectly by altering nuclear membrane dynamics during sexual development and thereby altering temporal aspects of RIP and recombination. We used a reverse genetics approach to determine whether high frequency RIP and homologous recombination of repetitive DNA during the sexual cycle are linked mechanistically or spatio-temporally. We tested strains where genes important for deletion by homologous recombination were knocked out and found all to be completely RIP competent except mre11, which, while sterile in homozygous deletion crosses, displayed lower RIP frequency in heterozygous crosses. This suggests that mre11 has roles in homologous recombination as well as non-homologous end joining may be important for RIP. Collectively, this work developed methods for efficiently mapping mutations and identified a novel protein that reduces RIP and recombination frequency but did not identify any mechanistic link between the two processes. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Dec. 10, 2012 - Dec. 10, 2014