Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Tuberculosis (TB) is an epidemic disease characterised by wet, persistent coughing, weight loss, fever, fatigue, and blood in the sputum. It has been reported that one in every three individuals is currently infected with Mycobacterium tuberculosis, the causative agent of TB, and that 10% of them will develop the active disease. The high prevalence and low penetrance of this disease has resulted in increased research performed to ascertain what factors play a role in susceptibility to M. tuberculosis infection. Some factors known to play a role in a minority of cases may include: HIV infection, diabetes, alcohol abuse and old age, but racial differences in susceptibility have also been observed. However, the influence of genetic factors is gaining popularity in current research. M. tuberculosis requires iron to proliferate, which it must appropriate from its host. For this reason the genes involved in the metabolism of iron in the human host are of particular interest when considering susceptibility to M. tuberculosis infection. In order to determine whether the expression of these genes may influence disease susceptibility, the promoter region of the CYBRD1, HAMP, HFE, LTF and SLC40A1 genes have been targeted for investigation. The aim of this study was to determine whether DNA variation in the promoter region of these genes involved in iron metabolism is associated with M. tuberculosis susceptibility. The study cohort consisted of 49 TB patients and 51 healthy, unrelated, population-matched controls, all of whom were Black, Xhosa-speaking individuals. Initially, 15 patient samples were randomly selected for exploratory screening, utilising semi-automated bi-directional sequencing analysis. In this manner, 40 variants were identified of which 30 were previously described. The novel variants included CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; and LTF: -1377 T/G, -457 T/C, -437 C/G. A number of loci demonstrated a statistically significant difference in allele and genotype frequencies, and in iron parameter levels when comparing the patient and control groups and for each variant separately. In silico analyses revealed the creation and/or abolishment of several transcription factor binding sites (TFBSs) due to the presence or absence of certain identified variants. The change in the composition of TFBSs in the promoter region may lead to differential expression of the gene. This study served as a pilot investigation to identify promoter region variants in the candidate genes involved in iron metabolism, in TB patients. The results presented here indicate that the identified variants (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) could possibly contribute to the increased absorption of iron in the TB patient group, which could subsequently increase the occurrence of pathogenic infection. The findings of this study could further aid in the elucidation of the exact mechanism(s) by which iron, its metabolism, and the genes involved affect disease susceptibility, more specifically, M. tuberculosis susceptibility. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is „n epidemiese siekte gekarakteriseer deur nat, aanhoudende hoes, gewigsverlies, moegheid, en bloed in die speeksel. Dit is gerapporteer dat een in elke drie individue tans geïnfekteer is met Mycobacterium tuberculosis, die veroorsakende agent van TB, en dat 10% van dié individue die aktiewe vorm van die siekte sal ontwikkel. Die hoë voorkoms en lae effek van hierdie siekte het daartoe gelei dat meer navorsing mettertyd gedoen is om die faktore wat „n rol mag speel in M. tuberculosis infeksie, te bepaal. Sommige faktore bekend vir hul rol in „n minderheid van gevalle sluit in: MIV infeksie, diabetes, alkoholmisbruik en bejaardheid, maar etniese verskille in vatbaarheid vir die siekte is ook al waargeneem. Die waarskynlikheid van genetiese invloed op die ontwikkeling van TB word ook meer deur navorsers ondersoek. M. tuberculosis benodig yster om te vermeerder, wat dit moet bekom vanaf die gasheer. Vir hierdie rede is die gene betrokke by yster metabolisme in die menslike gasheer veral van belang vir die oorweging van vatbaarheid vir M. tuberculosis. Om te bepaal of die uitdrukking van hierdie gene moontlik „n invloed het op vatbaarheid vir die siekte, was die promoter areas van die CYBRD1, HAMP, HFE, LTF en SLC40A1 gene geteiken. Die doel van hierdie studie was om te bepaal of DNS variasie in die promoter area van hierdie gene betrokke in yster metabolisme moontlik verband kan hou met vatbaarheid vir M. tuberculosis. Die studie kohort het uit 49 TB pasiënte en 51 gesonde, onverwante, populasie-gepaarde kontroles, waarvan almal Swart, Xhosa-sprekende individue was, bestaan. Aanvanklik was 15 pasiënt monsters lukraak gekies vir ondersoekende sifting, deur die gebruik van semi-outomatiese twee-rigting volgordebepalings. Op hierdie manier is 40 variante geïdentifiseer waarvan 30 voorheen beskryf is. Die nuwe variante sluit in CYBRD1: -849 C/G, -492 A/G, -454 C/T, -397 A/C; HAMP: -323 C/T; HFE: -561 A/G, -331 A/C; en LTF: -1377 T/G, -457 T/C, -437 C/G. „n Aantal loci het statisties betekenisvolle verskille getoon in alleel en genotipe frekwensies, en in yster parameter vlakke met die vergelyking van die pasiënt groep met die kontrole groep. In silico analise het verder die skepping en/of afskaffing van verskeie transkripsiefaktor bindingsetels (TFBSs), as gevolg van die teenwoordigheid of afwesigheid van sekere variante, getoon. Die verandering in die samestelling van TFBSs in die promoter area kan lei tot differensiële uitdrukking van die geen. Dié studie het gedien as „n voorlopige ondersoek om te bepaal of promoter area variante, geïdentifiseer in kandidaat gene betrokke by yster metabolisme, „n invloed het in die ontwikkeling van TB. Die resultate wat hier gewys word dui aan dat die geïdentifiseerde variante (-1813 C/T, -1459 T/C, -238 A/G, -166 C/G [CYBRD1]; -561 A/G [HFE]; -1470 C/T, -1355 G/C and -1098 G/A [SLC40A1]) moontlik die verhoogde opname van yster kan veroorsaak, wat later die toename van die patogeniese infeksie kan veroorsaak. Die bevindinge van hierdie studie kan moontlik bydra tot die toeligting van die presiese meganisme(s) waardeur yster, yster metabolisme, en die betrokke gene vatbaarheid vir siekte, meer spesifiek M. tuberculosis vatbaarheid, beïnvloed.

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

Software testing is an important process for ensuring the quality of the software. As the complexity of the software increases, traditional means of manual testing becomes increasingly more complex and time consuming. In most embedded systems, designing software with as few errors as possible is often critical. Resource usage is also of concern for proper behavior because of the very nature of embedded systems.  To design reliable and energy-efficient systems, methods are needed to detect hot points of consumption and correct them prior to deployment. To reduce testing effort, Model-based testing can be used which is one testing method that allows for automatic testing of model based systems. Model-based testing has not been investigated extensively for revealing resource usage anomalies in embedded systems. UPPAAL SMC is a statistical model checking tool which can be used to model the system’s resource usage. Currently UPPAAL SMC lacks the support for performing automatic test generation and test selection. In this thesis we provide this support with a framework for automatic test generation and test selection using mutation analysis, a method for minimizing the generated test suite while maximizing the fault coverage and a tool implementing the framework on top of the UPPAAL SMC tool. The thesis also evaluates the framework on a Brake by Wire industrial system. Our results show that we could for a Brake-by-wire system, simulated on a consumer processor with five mutants, in best case find a test case that achieved 100% mutation score within one minute and confidently identify at least one test case that achieved full mutation score within five minutes. The evaluation shows that this framework is applicable and relatively efficient on an industrial system for reducing continues resource usage target testing effort.

Test coverage criteria introduces metrics to measure the adequacy of a test suite as well as defined rules for performing software testing. This makes it possibly to formally define requirements for testing in various industries where software quality is essential, such as in aviation. This bachelor thesis aims to study the effectiveness of 11 different coverage criteria in two ways. Firstly how effective they are at finding faults and secondly their cost effectiveness. Test cases were created for each individual criteria for three different programs. The effectiveness of these test cases was then measured using mutation analysis. The results revealed that the scale of the experiment was too small to truly draw any conclusions regarding effectiveness. It was however shown that due to the “test noise” effect, the effectiveness of the test criteria can differ notably. It was also shown that test coverage criteria alone may not be sufficient in order to perform efficient testing.

Software testing is an important process for ensuring the quality of the software. As the complexity of the software increases, traditional means of manual testing becomes increasingly more complex and time consuming. In most embedded systems, designing software with as few errors as possible is often critical. Resource usage is also of concern for proper behavior because of the very nature of embedded systems.  To design reliable and energy-efficient systems, methods are needed to detect hot points of consumption and correct them prior to deployment. To reduce testing effort, Model-based testing can be used which is one testing method that allows for automatic testing of model based systems. Model-based testing has not been investigated extensively for revealing resource usage anomalies in embedded systems. UPPAAL SMC is a statistical model checking tool which can be used to model the system’s resource usage. Currently UPPAAL SMC lacks the support for performing automatic test generation and test selection. In this thesis we provide this support with a framework for automatic test generation and test selection using mutation analysis, a method for minimizing the generated test suite while maximizing the fault coverage and a tool implementing the framework on top of the UPPAAL SMC tool. The thesis also evaluates the framework on a Brake by Wire industrial system. Our results show that we could for a Brake-by-wire system, simulated on a consumer processor with five mutants, in best case find a test case that achieved 100% mutation score within one minute and confidently identify at least one test case that achieved full mutation score within five minutes. The evaluation shows that this framework is applicable and relatively efficient on an industrial system for reducing continues resource usage target testing effort.

Abstract Sensorineural hearing impairment (SNHI) is a well-recognized manifestation of mitochondrial diseases and occurs either in a non-syndromic form or as a part of a syndrome. Mitochondrial deafness is bilateral, usually progressive and is inherited maternally. Approximately 70% of patients with the most common syndromes, Kearns-Sayre, MELAS or MERRF, have SNHI. Several mutations in mitochondrial DNA (mtDNA) have been found to cause non-syndromic SNHI, including 1555A>G, 7445T>C, 7472insC and 7511T>C. In order to estimate prevalences of pathogenic mtDNA mutations in population-based cohorts of patients with SNHI, we obtained samples from 133 patients with SNHI, reportedly representing 117 separate maternal lineages. We found five patients with the 3243A>G mutation and three with the 1555A>G mutation, whereas the other point mutations associated with SNHI were absent. The frequencies of the mutations in the cohort were thus 4.3 % for 3243A>G and 2.6 % for 1555A>G, suggesting a total frequency of 6.9 % for mtDNA mutations known to be associated with hearing impairment. We found a mutation 10044A>G, which has been reported as pathogenic, in our patients with SNHI, but we also found it among the controls. Our results show it to be a homoplasmic polymorphism associated with a fairly rare haplotype within mtDNA haplogroup H which has recently been confirmed as subcluster H4. These results highlight the difficulty in determining the pathogenicity of a mtDNA mutation when it is identified only in one family. Therefore, in addition to the previously published criteria, we suggest that a sufficient number of haplotype-specific controls should be screened before the pathogenic nature of a mtDNA mutation can be verified. We determined the complete mtDNA sequences for 121 Finns, and after complementing our recent data, for a total of 192 Finns, and were able to construct a phylogenetic network based on complete mtDNA sequences, the largest set of complete sequences available at that time. These mtDNAs provide a rich source of information for studies in population genetics and a potential tool for analysing new substitutions and genotypes that entail a risk of mitochondrial disease. We used the phylogenetic network to find new pathogenic mutations or risk genotypes for SNHI. The entire coding region sequences of mtDNA were determined in 32 patients with SNHI and compared with the network. The patients were found to harbour more rare polymorphisms and haplotypes than the controls and to show increased variation in their mtDNA sequences, suggesting mildly deleterious effects for these substitutions. Two of the new mutations were suggested as putatively pathogenic.

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.

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.

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.

No description available.