Sistemas Reativos caracterizam-se por reagir continuamente a estímulos externos e internos e controlar atividades humanas. A ocorrência de falhas nesses sistemas pode resultar em grandes prejuízos. Dessa forma, o uso de métodos e técnicas rigorosas para a especificação do comportamento desse tipo de sistema é essencial, buscando-se evitar inconsistências e ambigüidades no modelo. Redes de Petri é uma das técnicas que têm sido usadas para a especificação de sistemas reativos. Teste e validação são atividades essenciais na produção dessa classe de sistemas. Por isso, o critério Análise de Mutantes, um critério de teste baseado em erros normalmente aplicado ao teste de programas, tem sido explorado no contexto de teste de especificações de sistemas reativos. É necessário o desenvolvimento de ferramentas que apóiem sua utilização, visto que a aplicação manual do critério é impraticável. O objetivo deste trabalho é a implementação da ferramenta Proteum-RS/PN, que apóia a aplicação do critério Análise de Mutantes para validar especificações baseadas em Redes de Petri. / Reactive Systems are characterized by continuously reacting to external as well as internal stimuli and controlling human activities. In these systems, faults can result in large losses. The use of rigorous methods and techniques for the specification of their behavior is essential to avoid inconsistencies and ambiguities. Petri Nets have been used for reactive-system specification. The test and validation of the underlying model are essential activities for the production of such systems. Thus, the Mutant Analysis -- a fault-based criterion usually used for program testing -- has been explored in the context of specification testing. The development of tools to support its application is necessary, since its manual application is unrealistic. The objective of this work is the implementation of Proteum-RS/PN, a testing tool which supports the application of Mutant Analysis criterion to validate Petri-Nets based specifications.

<p>Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The <i>VMD2</i> gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD.</p><p>In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a <i>C. elegans</i> protein family, expressed in retina, was identified as the <i>VMD2</i> gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the <i>VMD2</i> gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. <i>VMD2</i> expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation.</p><p>Analysis of mouse <i>Vmd2</i> and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. <i>Vmd2</i> expression levels were highest around birth.</p>

<p>Glaucoma is one of the leading causes of visual impairment in the world. In glaucoma, the patient’s peripheral vision is lost due to progressive and irreversible deterioration of the retinal ganglion cells and atrophy of the optic nerve. The effect on the visual field is gradual and painless, and the progression so slow, that the patient may not notice until a substantial part of the visual field is lost. If left untreated, glaucoma can lead to blindness.</p><p>In this thesis, genes associated to glaucoma have been analysed in Swedish patients with primary open angle and exfoliative glaucoma. The genes studied were <i>MYOC</i>, <i>oculomedin,</i> <i>GSTM1</i> and <i>OPTN</i>.</p><p>The coding sequence of <i>MYOC</i> was analysed and mutations were found in 1% of the primary open angle glaucoma patients. Additionally, a predisposing variant was found in 1% of the patients as well as in 0.5% of the controls. No disease-associated variation was found in the exfoliative glaucoma cases. Mutations were also found in two families affected by glaucoma. The coding sequence of <i>oculomedin</i> was analysed, but none of the variants found were classified as disease causing in either patient group. <i>GSTM1</i> was analysed for its presence in the patients. No association could be found for either hetero- or homozygous deletions. The coding sequence and haplotype distribution of <i>OPTN</i> was analysed. None of the variants found were classified as disease causing and none of the haplotypes were associated to the disease in either patient group.</p><p>There are just a few per cent of the Swedish primary open angle glaucoma patients with genetic variation associated to disease, in the genes analysed in this study. No association to exfoliative glaucoma was found. This indicates heterogeneity in the genetics of glaucoma when different subtypes and different populations are compared. Likely, there are genes still to be identified.</p>

Best macular dystrophy (BMD) is an autosomal dominant inherited eye disease with a juvenile onset. Accumulation of the pigment lipofuscin in the retinal pigment epithelium can later cause macular degeneration and loss of vision. BMD have histopathologic similarities with age-related macular degeneration, the most common cause of blindness among elderly. BMD diagnosis is made with fundus examination and electrophysiology. The VMD2 gene, causing BMD, has previously been localized to 11q13 using linkage and recombination of a 12 generation family with BMD. In this study the genetic region has been further narrowed using polymorphic markers in the BMD family. A human homolog for a C. elegans protein family, expressed in retina, was identified as the VMD2 gene. It has a 1755 bp open reading frame with 11 exons and encodes a 585 amino acid protein called bestrophin. Mutation analysis of the VMD2 gene in BMD families from Sweden, Denmark and Netherlands revealed 15 missense mutations, altering single amino acids in bestrophin, accumulating in the N-terminal half of the protein. VMD2 expression analysis with in situ hybridization revealed specific localization in the retinal pigment epithelium and Northern blot showed expression in retina and brain. Clinical and genetic analysis of a BMD family with generally late onset revealed a novel bestrophin mutation. Analysis of mouse Vmd2 and bestrophin during development showed presence of mouse bestrophin in retinal pigment epithelium at postnatal day 10 and in photoreceptor outer segments during the entire postnatal period. Vmd2 expression levels were highest around birth.

Glaucoma is one of the leading causes of visual impairment in the world. In glaucoma, the patient’s peripheral vision is lost due to progressive and irreversible deterioration of the retinal ganglion cells and atrophy of the optic nerve. The effect on the visual field is gradual and painless, and the progression so slow, that the patient may not notice until a substantial part of the visual field is lost. If left untreated, glaucoma can lead to blindness. In this thesis, genes associated to glaucoma have been analysed in Swedish patients with primary open angle and exfoliative glaucoma. The genes studied were MYOC, oculomedin, GSTM1 and OPTN. The coding sequence of MYOC was analysed and mutations were found in 1% of the primary open angle glaucoma patients. Additionally, a predisposing variant was found in 1% of the patients as well as in 0.5% of the controls. No disease-associated variation was found in the exfoliative glaucoma cases. Mutations were also found in two families affected by glaucoma. The coding sequence of oculomedin was analysed, but none of the variants found were classified as disease causing in either patient group. GSTM1 was analysed for its presence in the patients. No association could be found for either hetero- or homozygous deletions. The coding sequence and haplotype distribution of OPTN was analysed. None of the variants found were classified as disease causing and none of the haplotypes were associated to the disease in either patient group. There are just a few per cent of the Swedish primary open angle glaucoma patients with genetic variation associated to disease, in the genes analysed in this study. No association to exfoliative glaucoma was found. This indicates heterogeneity in the genetics of glaucoma when different subtypes and different populations are compared. Likely, there are genes still to be identified.

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.

The main objective of this dissertation is to assist in reducing the chaotic state of the software engineering discipline by providing insights into both the effectiveness of software defect reduction methods and ways these methods can be improved. The dissertation is divided into two main parts. The first is a quasi-experiment comparing the software defect rates and initial development costs of two methods of software defect reduction: software inspection and test-driven development (TDD). Participants, consisting of computer science students at the University of Arizona, were divided into four treatment groups and were asked to complete the same programming assignment using either TDD, software inspection, both, or neither. Resulting defect counts and initial development costs were compared across groups. The study found that software inspection is more effective than TDD at reducing defects, but that it also has a higher initial cost of development. The study establishes the existence of a defect-reduction gap between software inspection and TDD and highlights the need to improve TDD because of its other benefits.The second part of the dissertation explores a method of applying mutation analysis to TDD to reduce the defect reduction gap between the two methods and to make TDD more reliable and predictable. A new change impact analysis algorithm (CHA-AS) based on CHA is presented and evaluated for applications of software change impact analysis where a predetermined set of program entry points is not available or is not known. An estimated average case complexity analysis indicates that the algorithm's time and space complexity is linear in the size of the program under analysis, and a simulation experiment indicates that the algorithm can capitalize on the iterative nature of TDD to produce a cost savings in mutation analysis applied to TDD projects. The algorithm should also be useful for other change impact analysis situations with undefined program entry points such as code library and framework development.An enhanced TDD method is proposed that incorporates mutation analysis, and a set of future research directions are proposed for developing tools to support mutation analysis enhanced TDD and to continue to improve the TDD method.

Comprehensive characterisation of the mutational landscapes of solid tumours is a multistep process involving the collection of suitable samples, the extraction of nucleic acids and the preparation of these materials for mutational analyses. In this thesis, I aimed to develop a streamlined process for the analysis of colorectal cancer (CRC) patient samples in order to identify novel mutations that hallmark the development of advanced disease. Papers I and II outline a technique for serial extraction of nucleic acids from frozen tissue that we developed and subsequently implemented on a robotic platform to enable high-throughput processing. The extracted nucleic acids were validated in downstream processes relevant for genetic analyses, including traditional Sanger and next generation sequencing  techniques. In Paper III, we developed a genotyping method based on multiplex ligation-dependent genome amplification. The method was designed such that InDel polymorphisms of between 30 and 70 % prevalence in a European population were selected and amplified in a multiplex PCR assay. DNA from 24 patient-matched colorectal tumour and normal tissues was genotyped and paired with a high match probability. In Paper IV, we performed targeted resequencing of 107 primary CRCs, of which approximately half developed metastatic disease or had distant metastases at the time of diagnosis. We chose to analyse 676 genes based on their involvement in key signalling pathways in CRC. We found an enrichment of mutations in the Eph receptor tyrosine kinase gene family in metastatic patients, indicating a potential role for these genes in CRC metastasis. This thesis outlines a series of procedures that can be employed in a high-throughput setting for the analysis of solid tumours. We applied these methods to the analysis of colorectal tumours and propose a link between novel somatic mutations and metastatic disease.

In der Diagnostik der gastrointestinalen Stromatumoren (GIST) spielt neben der Histologie die Immunhistochemie eine zentrale Rolle. Die vorliegende Arbeit befasst sich mit der Fragestellung, welche Wertigkeit der Mutationsanalyse im diagnostischen Kontext zukommt und wie stabil Immunphänotyp und Mutationsstatus im Verlauf der Erkrankung tatsächlich sind. In drei Fällen rezidivierter GIST war die Histomorphologie, die Immunhistochemie und der Mutationsstatus im Vergleich zum Primärtumor stabil. Bei den untersuchten synchron auftretenden Tumoren von drei Patienten waren in der Mutationsanalyse unterschiedliche Ergebnisse zu erheben. Bei zwei Patienten unterstützte das unterschiedliche Mutationsmuster das Vorliegen synchroner Tumoren, bei einem Patienten ist das Vorliegen eines Primärtumors und einer Metastase statt einem synchronen GIST wahrscheinlich. Die Untersuchung metastasierter GIST wurde an verschiedenen Tumoren von neun Patienten durchgeführt. Acht der neun Fälle zeigten sich bezüglich der Metastasen genotypisch stabil, einer der acht Fälle wies zusätzlich einen Zugewinn einer Punktmutation auf, die als Möglichkeit eines Tumormosaiks oder als neu erworbene zusätzliche Mutation zu werten sein könnte. Zudem wurden 28 Fälle unklarer spindelzelliger Tumoren mit uneinheitlichem immunhistochemischen Profil untersucht. In Zusammenschau mit der Mutationsanalyse war eine eindeutige Bestimmung der Tumorentität möglich. Abschließend zeigt sich die Kombination aus Histomorphologie, immunhistochemischer Untersuchung und Mutationsanalyse als gutes diagnostisches Mittel zur Sicherung der Tumorentität und Entdeckung eventuell neu aufgetretener prognostisch relevanter Mutationen mit therapeutischer Konsequenz.

Diss. Linköping : Univ., 2003.