The early gene region, EIA, of Adenovirus is responsible for two mRNAs that appear to be, along with their protein products, necessary for oncogenic transformation. The two proteins differ only in that the larger 289R protein has an extra internal sequence of 46 amino acids. This single difference must account for the functional differences between the two proteins. One function associated with this unique sequence is transactivation, the ability to transcriptionally activate the other early viral genes. In this thesis the construction and analysis of three in-frame deletion mutants are described. These three deletions, along with a fourth previously made, span the entire unique region. All three mutants had lost their transactivation ability, suggesting that the entire domain is necessary for transactivation. Transformation assays with these mutants also suggest that this function blocks transformation. Thus, the unique domain must encode another as yet unidentified function necessary for full transformation. Further evidence for another function in the unique domain comes from the differently reduced abilities of the mutants to grow on HeLa cells. Each mutant has differentially affected some function that is also necessary for lytic infection. / Thesis / Master of Science (MS)

Correlated mutation analysis is an effective approach for predicting functional and structural residue interactions from protein multiple sequence alignments. A prediction pipeline over the Pfam database was developed to predict residue contacts within protein domains. Cross- reference with the PDB showed these contacts are spatially close. Furthermore, we found our predictions to be biochemically reasonable and correspond closely with known contact matrices. This large-scale search for coevolving regions within protein domains revealed that if two sites in an alignment covary, then neighboring sites in the alignment would also typically covary, resulting in clusters of covarying residues. The program PatchD was developed to measure the covariation between disconnected sequence clusters to reveal patch covariation. Patches that exhibited strong covariation identified multiple residues that were generally nearby in the protein structures, suggesting that the detection of covarying patches can be used in addition to traditional CMA approaches to reveal functional interaction partners.

Correlated mutation analysis is an effective approach for predicting functional and structural residue interactions from protein multiple sequence alignments. A prediction pipeline over the Pfam database was developed to predict residue contacts within protein domains. Cross- reference with the PDB showed these contacts are spatially close. Furthermore, we found our predictions to be biochemically reasonable and correspond closely with known contact matrices. This large-scale search for coevolving regions within protein domains revealed that if two sites in an alignment covary, then neighboring sites in the alignment would also typically covary, resulting in clusters of covarying residues. The program PatchD was developed to measure the covariation between disconnected sequence clusters to reveal patch covariation. Patches that exhibited strong covariation identified multiple residues that were generally nearby in the protein structures, suggesting that the detection of covarying patches can be used in addition to traditional CMA approaches to reveal functional interaction partners.

Abstract A mitochondrial disorder may be one of the rare aetiologies of occipital stroke. Clinical and molecular analysis has suggested that 10% of young patients with occipital stroke have a mitochondrial disorder and 6% harbour the mutation 3243A>G in mitochondrial DNA (mtDNA), causing the MELAS syndrome. To identify other possible mtDNA mutations involved, we studied mtDNA genotypes in patients who had suffered an occipital stroke and in whom the common pathogenic mutations in mtDNA had been excluded. Since one systematic way of comparing mtDNA sequences is through phylogenetic analysis, a phylogenetic network for the Finnish mtDNA haplogroup U was constructed and used to identify differences in mtDNA between patients and controls. The usefulness of conformation sensitive gel electrophoresis (CSGE) for analysing differences within the coding sequence of mtDNA was also estimated. We studied mtDNA genotypes of 29 patients with occipital stroke. The aetiology of the stroke was assessed using the criteria of the Baltimore-Washington Cooperative Young Stroke Study, and migraine was diagnosed in 18 patients according to the International Headache Society criteria. Moreover, we studied the mtDNA genotypes of 42 patients with migraine and a total of 480 population controls who reported that they themselves and their mothers were healthy with respect to common clinical manifestations of mtDNA disease. The mtDNA haplogroups were detected by restriction fragment analysis and the mtDNA structures of 14 patients with occipital stroke and 43 subjects belonging to haplogroup U were examined by CSGE. The data acquired by CSGE were then used to construct a phylogenetic network for the Finnish mtDNA haplogroup U. We found CSGE to be a highly sensitive and specific method for screening mutations and polymorphisms in mtDNA. The sequence data on the 43 subjects belonging to the mtDNA haplogroup U were used to construct a phylogenetic network, which was found to be an unambiguous tree with few homoplasies that pointed to several previously unidentified common polymorphisms. The major branch of the network was U5, which seemed to be quite specific to the Finns. Branches representing haplogroups U2, U4, U7 and K could also be detected. Restriction fragment analysis of the patients with occipital stroke revealed that all those with migraine as a probable aetiology belonged to the mtDNA haplogroup U, suggesting that this genotype confers a risk of occipital stroke. In addition to the five patients with migrainous stroke, we analyzed the complete mtDNA coding sequences of nine other patients with occipital stroke belonging to haplogroup U by CSGE. Analysis of the phylogenetic network revealed an association of migrainous stroke with mtDNA haplogroup U5. Furthermore, the distribution of the mtDNA genotypes in the patients with stroke differed from that found in the controls. Four patients harboured potentially pathogenic mutations. CSGE proved to be an effective method for use in mitochondrial genetics, enabling us to construct an unambiguous network for the Finnish haplogroup U. Similar phylogenetic networks are required for the purposes of both medical genetics and population genetics. Such networks were found to be helpful in deciding between a rare polymorphism and a pathogenic mutation in patients with occipital stroke. Likewise, they enabled more detailed comparisons to be made between and within populations and allowed more accurate phylogenetic relationships to be determined.

Abstract Fragile X syndrome, the most common inherited form of mental retardation syndrome, is caused by an expansion of the CGG trinucleotide repeat in the 5' UTR of the FMR1 gene, with concurrent hypermethylation of the region, which represses FMR1 expression. The syndrome is associated with the folate-sensitive chromosomal fragile site at Xq27.3 (FRAXA), where the gene responsible for the syndrome was first localized by linkage analysis using RFLP markers. In this study the linkage relationships of the RFLP markersat Xq27-28 and the characteristics of the CGG repeat expansion were investigated in northern Finnish fragile X families and molecular diagnostic methods were applied in order to improve diagnosis of the syndrome. Furthermore, the origin of fragile X mutations in the northern part of Finland was studied by haplotype analysis. Linkage studies were performed in 34 northern Finnish fragile X families/pedigrees using a total of 15 RFLPs (defining 11 loci). A refined genetic map around FRAXA including five RFLP markers having recombination fractions of 0.04 or less with FRAXA was obtained in an international study of 112 affected families, containing linkage data on twelve northern Finnish families. Linkage analysis significantly improved carrier detection in fragile X families compared with previous cytogenetic methods used in diagnosis. The most efficient RFLP-based protocol for carrier detection was proposed, which is based on use of the most adjacent markers and a minimum number of restriction enzymes. CGG repeat expansion of the FMR1 gene was investigated in original families collected for linkage studies and additional new ones. Large CGG repeat expansions (Δ > 500 bp) with concomitant methylation of the adjacent CpG island, i.e. full mutations, were found to be associated with mental retardation completely in males, but only 50% of the females having a full mutation were mentally impaired. Premutations (Δ < 700 bp) were found in healthy carriers. There was a size range of Δ = 500 to 700 bp, where the expansions could be either abnormally methylated or non-methylated, and it appeared that methylation is more important in determining the phenotype than the exact size of an expansion. Instability of the enlarged CGG repeats was detected, leading preferentially to size increases in successive generations. The instability of premutations was found to be stronger and the size increases larger in maternal than in paternal transmissions, and transition to a full mutation occurred only in female transmissions. In addition, the size of a maternal premutation was shown to have an important influence on the risk of its transition to a full mutation when transmitted. The critical premutation size leading invariably to full mutation in the offspring was found to be between Δ = 175 to 200 bp. In one of the studied families a rare contraction of a paternal premutation to a normal CGG repeat number in one of the daughters and further in her son was detected. Direct mutation analysis including measurement of the CGG repeat size and hypermethylation allowed unambiguous diagnosis of carriers and affected individuals in most cases. Haplotype analysis using two tightly linked microsatellite markers flanking the CGG repeat mutation was performed in 60 unrelated northern and eastern Finnish fragile X families. A significant difference was found in allelic and haplotypic distributions between normal X and fragile X chromosomes. A single haplotype, which was present only in 8% of the normal X chromosomes, accounted for 80% of the fragile X chromosomes. This enrichment of one fra(X) mutation in the Finnish population suggests founder effect.

The ability to test software is invaluable in all areas of computer science, but it is often neglected in computer science curricula. Test adequacy criteria (TAC), tools that measure the effectiveness of a test suite, have been used as aids to improve software testing teaching practices, but little is known about how students respond to them. Studies have examined the cognitive processes of students programming and professional developers writing tests, but none have investigated how student testers test with TAC. If we are to improve how they are used in the classroom, we must start by understanding the different ways that they affect students’ thought processes as they write tests. In this thesis, we take a grounded theory approach to reveal the underlying cognitive processes that students utilize as they test under no feedback, condition coverage, and mutation analysis. We recorded 12 students as they thought aloud while creating test suites under these feedback mechanisms, and then we analyzed these recordings to identify the thought processes they used. We present our findings in the form of the phenomena we identified, which can be further investigated to shed more light on how different TAC affect students as they write tests. i

Myoclonus-Dystonia (MD) is an inherited, rare, autosomal dominant movement disorder characterized by quick, involuntary muscle jerking or twitching (myoclonus) and involuntary muscle contractions that cause twisting and pulling movements, resulting in abnormal postures (dystonia). The first MD locus was mapped to 7q21-q31 and called DYT11; this locus corresponds to the SGCE gene. Our group previously identified a second MD locus (DYT15) which maps to a 3.18 Mb region on 18p11. Two patients were chosen to undergo next-generation sequencing, which identified 2,292 shared novel variants within the critical region. Analysis of these variants revealed a 3 bp duplication in a transcript referred to as CD108131, which is believed to be a long non-coding RNA. Characterization of this transcript determined that it is 863 bp in size, it is ubiquitously expressed, with high expression in the cerebellum, and it accounts for ~3% of MD cases.

Hypochondroplasia (MIM 146000) is a skeletal dysplasia characterized by disproportional dwarfism with rhizomelic or mesomelic shortening of the upper and lower extremities, with variable severity. Patients often have macrocephaly with normal facial features. Hypochondroplasia is a disease with autosomal dominant inheritance. In some patients it is caused by germline mutations in the FGFR3 gene, in others the cause of the disease remains unknown . The FGFR3 gene encodes a tyrosine kinase receptor. This receptor negatively regulates the conversion of cartilage to bone. FGFR3 gene mutations that cause hypochondroplasia lead to constitutive activation of the receptor and inhibit the growth of long bones. In this study, we analysed selected regions (exons) of the FGFR3 gene in 98 patients with disproportional dwarfism and clinical diagnosis of hypochondroplasia. Eighteen patients from 12 families had familial and 80 patients had sporadic form of the disease. All patients were previously tested negative for frequent germline mutations in exon 13 (codon 540) and exon 15 (codon 650). Genomic DNA was isolated from patient's peripheral blood leukocytes. The examination was conducted with the informed consent of the patient or his legal representative. We performed mutational analysis by direct sequencing of...

Este trabalho propõe a extensão do critério Análise de Mutantes, originalmente desenvolvido para o teste de programas, para sua aplicação no teste de especificações do aspecto comportamental de Sistemas Reativos. Esses sistemas constituem hoje um componente fundamental em várias atividades humanas e, em geral, falhas nos mesmos podem envolver grandes riscos a vida ou ao patrimônio. Isso toma imprescindível um maior rigor no processo de desenvolvimento e, em particular, na atividade de teste, que é fundamentalmente baseada em simulação, não fornecendo critério que avalie essa atividade de forma quantitativa. A proposta aborda a aplicação da Análise de Mutantes na validação de especificações de Sistemas Reativos baseadas em três técnicas formais, que possuem apoio gráfico, mais utilizadas para este fim: Máquinas de Estados Finitos, Redes de Petri e Statecharts. Para a aplicação do critério nesse contexto, estabeleceu-se um paralelo entre os níveis de programa e de especificação, quanto as suas hipóteses básicas do programador competente e do efeito de acoplamento. Foram definidos os operadores de mutação para cada uma das três técnicas consideradas, além de critérios de mutação alternativa que visam a minimização no custo de aplicação do critério. Foram realizados, manualmente, dois experimentos com o objetivo de validar os mecanismos propostos. Um deles foi aplicado em Máquinas de Estados Finitos e o outro, em Redes de Petri. Os resultados mostram evidências do aspecto complementar do critério Análise de Mutantes em relação as formas disponíveis de teste de especificações existentes na literatura. Apresenta-se também um protótipo da ferramenta Proteum-RSIFSM que apóia a aplicação da Análise de Mutantes em Máquinas de Estados Finitos e discute-se a instanciação dessa ferramenta para apoiar a aplicação do critério nos contextos de Statecharts e Redes de Petri / Reactive Systems are a fundamental component in severa1 activities and failures in these systems may cause risks to life or financia1 losses. Thus, the development process and particularly the test activity must be carried on with extreme care. This work proposes the use of Mutation Analysis - a technique originally proposed at program level testing - in the context of testing and validation of specifications of Reactive Systems; three graphical techniques are considered: Finite State Machines, Petri Nets and Statecharts. Currently, the test of such specifications is mainly based on simulation. The relevance of a test adequacy assessment criterion for such activity has been recognized by many researchers and practitioners and constitutes the objective of this work. The application of Mutation Analysis in this context is based in the assumption that the basic hypothesis valid to the program level - the hypothesis of the competent programmer and coupling effect also hold to the specification level. Mutation operators are defined for the three specification techniques. Also, alternative criteria were defined aiming at reducing the cost of application of Mutation Analysis in this context. Two experiments were manually conducted in order to validate the proposed ideas. The results show evidences of the complementary aspects among existent methods and Mutation Analysis for testing specifications of Reactive Systems. A prototype of Proteum-RSIFSM, a to01 to support Mutation Analysis for Finite State Machines is presented and its extensions to Statecharts and to Petri Nets are discussed

Estudos empíricos têm mostrado que a Análise de Mutantes – um dos critérios de teste baseado em erros – é bastante eficaz em revelar a presença de erros. Entretanto, seu alto custo, decorrente principalmente do grande número de mutantes gerados, tem motivado a proposição de diversas abordagens alternativas para a sua aplicação. Um estudo relevante nesse sentido resultou na determinação de um conjunto essencial de operadores de mutação para a linguagem Fortran, mostrando-se que é possível reduzir o custo de aplicação do critério, preservando um alto grau de adequação em relação à Análise de Mutantes. Alguns estudos também têm demonstrado que a redução da eficácia não é significativa. Este trabalho tem como objetivo investigar alternativas pragmáticas para a aplicação do critério Análise de Mutantes e, nesse contexto, é proposto um procedimento para a determinação de um conjunto essencial de operadores de mutação para a linguagem C, a partir dos operadores implementados na ferramenta Proteum. Procurando aplicar e validar o procedimento proposto, dois grupos distintos de programas são utilizados. Para ambos os grupos, o conjunto essencial obtido apresenta resultados bastante significativos quanto à redução de custo, com um decréscimo muito pequeno no grau de adequação em relação à Análise de Mutantes. Estratégias para evoluir e refinar um conjunto essencial para diferentes domínios de aplicação também são investigadas. / Mutation Analysis – one of the error based criteria – has been found to be effective on revealing faults. However, its high cost, due to the high number of mutants created, has motivated the proposition of many alternative approaches for its application. In this perspective, a relevant study resulted on the determination of a sufficient mutant operator set for Fortran, indicating that it is possible to have a large cost reduction of mutation testing, preserving a high mutation score. Some studies have also shown that the reduction on the effectiveness is not significant. This work aims to investigate pragmatic alternatives for mutation analysis application and, in this context, a procedure for the determination of a sufficient mutant operators set for C is proposed, using Proteum testing tool. Aiming to apply and validate the proposed procedure, two different groups of programs are used. For both of them, the sufficient mutant operator set presents very significant results in terms of cost reduction, with a very small reduction on the mutation score. Strategies to evolve and refine an essential mutant operator set to different application domains are also investigated.