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This paper presents a new model for simulating procedural knowledge in the problem solving process with our ontological system, InfoMap. The method divides procedural knowledge into two parts: process control and action performer. By adopting InfoMap, we hope to help teachers construct curricula (declarative knowledge) and teaching strategies by capturing students' problem-solving processes (procedural knowledge) dynamically. Using the concept of declarative and procedural knowledge in intelligent tutoring systems, we can accumulate and duplicate the knowledge of the curriculum manager and student. (Contains 12 figures.)

Mycobacterium avium subsp. paratuberculosis causes an enteric infection in cattle, with a great impact on the dairy industry in the United States and worldwide. Characterizing the gene expression profile of M. avium subsp. paratuberculosis exposed to different stress conditions, or shed in cow feces, could improve our understanding of the pathogenesis of M. avium subsp, paratuberculosis. In this report, the stress response of M. avium subsp. paratuberculosis on a genome-wide level (stressome) was defined for the first time using DNA microarrays. Expression data analysis revealed unique gene groups of M. avium subsp. paratuberculosis that were regulated under in vitro stressors while additional groups were regulated in the cow samples. Interestingly, acidic pH induced the regulation of a large number of genes (n = 597), suggesting the high sensitivity of M. avium subsp.paratuberculosis to acidic environments. Generally, responses to heat shock, acidity, and oxidative stress were similar in M. avium subsp, paratuberculosis and Mycobacterium tuberculosis, suggesting common pathways for mycobacterial defense against stressors. Several sigma factors (e.g., sigH and sigE) were differentially coregulated with a large number of genes depending on the type of each stressor. Subsequently, we analyzed the virulence of six M. avium subsp, paratuberculosis mutants with inactivation of differentially regulated genes using a routine model of paratuherculosis. Both bacterial and histopathological examinations indicated the attenuation of all gene mutants, especially those selected based on their expression in the cow samples (e.g., lipN). Overall, the employed approach profiled mycobacterial genetic networks triggered by variable stressors and identified a novel set of putative virulence genes. A similar approach could he applied to analyze other intracellular pathogens.

Chronic tuberculosis represents a major health problem for one-third of the world's population today. A key question relevant to chronic tuberculosis is the physiological status of Mycobacterium tuberculosis during this important stage of infection. To examine the molecular bases of chronic tuberculosis and the role of host immunity in mycobacterial growth, we determined the mycobacterial transcriptional profiles during chronic and reactivation phases of murine tuberculosis using in vivo microarray analysis (IVMA). Following 28 days of aerosol infection, mycobacterial counts remained stable, although the bacilli were metabolically active with a 50% active transcriptome. The expression of genes involved in lipid and carbohydrate pathways was significantly enriched during the middle stage of chronic tuberculosis, suggesting a nutrient-rich microenvironment. A total of 137 genes were significantly regulated in mid-chronic tuberculosis (45 and 60 days) compared to an early stage (14 days) of infection. Additional sets of genes, including the virulence regulator virS, were up-regulated during the reactivation stage, indicating their possible roles in mycobacterial resurgence. Interestingly, a set of potential transcriptional regulators was significantly induced at the late stage of chronic tuberculosis. Bioinformatic analysis identified a large number of genes that could be regulated by one of the potential transcriptional regulators encoded by rv0348, including the sigF operon. Taken together, IVMA provided a better definition of the transcriptional machinery activated during chronic and reactivation stages of tuberculosis and identified a novel transcriptional regulator. A similar approach can be adopted to study key stages of intracellular pathogens.

Infection with Mycobacterium avium subsp. paratuberculosis causes Johne's disease in cattle and is also implicated in cases of Crohn's disease in humans. Another closely related strain, M. avium subsp. avium, is a health problem for immunocompromised patients. To understand the molecular pathogenesis of M. avium subspecies, we analyzed the genome contents of isolates collected from humans and domesticated or wildlife animals. Comparative genomic hybridizations indicated distinct lineages for each subspecies where the closest genomic relatedness existed between M. avium subsp. paratuberculosis isolates collected from human and clinical cow samples. Genomic islands (n = 24) comprising 846 kb were present in the reference M. avium subsp. avium strain but absent from 95% of M. avium subsp. paratuberculosis isolates. Additional analysis identified a group of 18 M. avium subsp. Paratuberculosis-associated islands comprising 240 kb that were absent from most of the M. avium subsp. avium isolates. Sequence analysis of DNA regions flanking the genomic islands identified three large inversions in addition to several small inversions that could play a role in regulation of gene expression. Analysis of genes encoded in the genomic islands reveals factors that are probably important for various mechanisms of virulence. Overall, M. avium subsp. avium isolates displayed a higher level of genomic diversity than M. avium subsp. paratuberculosis isolates. Among M. avium subsp. paratuberculosis isolates, those from wildlife animals displayed the highest level of genomic rearrangements that were not observed in other isolates. The presented findings will affect the future design of diagnostics and vaccines for Johne's and Crohn's diseases and provide a model for genomic analysis of closely related bacteria.