The development of hard real-time systems using a formal approach

Hard real-time systems are a class of computer control systems that must react to demands of their environment by providing `correct' and timely responses. Since these systems are increasingly being used in systems with safety implications, it is crucial that they are designed and developed to operate in a correct manner. This thesis is concerned with developing formal techniques that allow the specification, verification and design of hard real-time systems. Formal techniques for hard real-time systems must be capable of capturing the system's functional and performance requirements, and previous work has proposed a number of techniques which range from the mathematically intensive to those with some mathematical content. This thesis develops formal techniques that contain both an informal and a formal component because it is considered that the informality provides ease of understanding and the formality allows precise specification and verification. Specifically, the combination of Petri nets and temporal logic is considered for the specification and verification of hard real-time systems. Approaches that combine Petri nets and temporal logic by allowing a consistent translation between each formalism are examined. Previously, such techniques have been applied to the formal analysis of concurrent systems. This thesis adapts these techniques for use in the modelling, design and formal analysis of hard real-time systems. The techniques are applied to the problem of specifying a controller for a high-speed manufacturing system. It is shown that they can be used to prove liveness and safety properties, including qualitative aspects of system performance. The problem of verifying quantitative real-time properties is addressed by developing a further technique which combines the formalisms of timed Petri nets and real-time temporal logic. A unifying feature of these techniques is the common temporal description of the Petri net. A common problem with Petri net based techniques is the complexity problems associated with generating the reachability graph. This thesis addresses this problem by using concurrency sets to generate a partial reachability graph pertaining to a particular state. These sets also allows each state to be checked for the presence of inconsistencies and hazards. The problem of designing a controller for the high-speed manufacturing system is also considered. The approach adopted mvolves the use of a model-based controller: This type of controller uses the Petri net models developed, thus preservIng the properties already proven of the controller. It. also contains a model of the physical system which is synchronised to the real application to provide timely responses. The various way of forming the synchronization between these processes is considered and the resulting nets are analysed using concurrency sets.

Species-specific differences in the expression of the HNF1A, HNF1B and HNF4A genes

Background: The HNF1A, HNF1B and HNF4A genes are part of an autoregulatory network in mammalian pancreas, liver, kidney and gut. The layout of this network appears to be similar in rodents and humans, but inactivation of HNF1A, HNF1B or HNF4A genes in animal models cause divergent phenotypes to those seen in man. We hypothesised that some differences may arise from variation in the expression profile of alternatively processed isoforms between species. Methodology/Principal Findings: We measured the expression of the major isoforms of the HNF1A, HNF1B and HNF4A genes in human and rodent pancreas, islet, liver and kidney by isoform-specific quantitative real-time PCR and compared their expression by the comparative Ct (??Ct) method. We found major changes in the expression profiles of the HNF genes between humans and rodents. The principal difference lies in the expression of the HNF1A gene, which exists as three isoforms in man, but as a single isoform only in rodents. More subtle changes were to the balance of HNF1B and HNF4A isoforms between species; the repressor isoform HNF1B(C) comprised only 6% in human islets compared with 24–26% in rodents (p = 0.006) whereas HNF4A9 comprised 22% of HNF4A expression in human pancreas but only 11% in rodents (p = 0.001). Conclusions/Significance: The differences we note in the isoform-specific expression of the human and rodent HNF1A, HNF1B and HNF4A genes may impact on the absolute activity of these genes, and therefore on the activity of the pancreatic transcription factor network as a whole. We conclude that alterations to expression of HNF isoforms may underlie some of the phenotypic variation caused by mutations in these genes.

Telomere length attrition, a marker of biological senescence, is inversely correlated with triglycerides and cholesterol in South Asian males with type 2 diabetes mellitus

South Asians have a higher risk of type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) than white Caucasians, for a given BMI. Premature biological ageing, assessed by reduction in telomere length (TL), may be mediated by factors resulting from altered metabolic profiles associated with obesity. We hypothesise that ethnicity and metabolic status represent detrimental factors contributing to premature biological ageing. Therefore we assessed TL in two South Asian, age and BMI-matched cohorts [T2DM (n = 142) versus non-T2DM (n = 76)] to determine the effects of BMI, gender, lipid and CVD profile on biological ageing. Genomic DNA was obtained from the UKADS cohort; biochemical and anthropometric data was collected and TL was measured by quantitative real-time PCR. Our findings indicated a gender-specific effect with reduced TL in T2DM men compared with non-T2DM men (P = 0.006). Additionally, in T2DM men, TL was inversely correlated with triglycerides and total cholesterol (r = -0.419, P <0.01; r = -0.443, P <0.01). In summary, TL was reduced amongst South Asian T2DM men and correlated with triglycerides and total cholesterol. This study highlights enhanced biological ageing among South Asian, T2DM men, which appears to be tracked by changes in lipids and BMI, suggesting that raised lipids and BMI may directly contribute to premature ageing.

Unique reporter-based sensor platforms to monitor signalling in cells

Introduction - In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. Methods - Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. Findings - We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. Conclusions - We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters.