Stress Activated Protein Kinase Regulation of Gene Expression in Apoptotic Neurons: A Dissertation

Summary Basic biological processes require gene expression. Tightly regulated molecules known as transcription factors mediate the expression of genes in development and disease. Signal transduction pathways, which respond to environmental cues or stressors are major regulators of the transcription factors. Use of macromolecular synthesis inhibitors in models of normal neurodevelopment and neurodegenerative cell death has led to the discovery that gene expression is required for these processes to occur (Martin et. al.,(1988), J Cell Biol 106p829). To date, however, the identities of very few of the genes required in these events have been revealed. Hence, the activation or requirement of specific signaling pathways leading to the expression of known apoptotic genes is not well established. Utilizing the neurothrophic factor deprivation and neurotoxin models of programmed cell death we address these gaps in our understanding of the molecular mechanism of apoptosis as it occurs in neuronal cell death. Nerve growth factor (NGF) withdrawal from PC12 cells leads to the activation of p38 and apoptosis. The functional significance of 38 activation in this paradigm of cell death is not known. To increase our understanding of apoptosis I examined the requirement for p38 activity in pro-apoptotic gene expression in PC12 cells. I performed a subtractive hybridization that led to the identification of the monoamine oxidase (MAG) gene as induced in response to NGF withdrawal. Using the p38 inhibitor PD169316 I showed that the NGF withdrawal stimulated induction of the MAG gene and apoptosis is blocked by inhibition of the p38 MAP kinase pathway. I also determined that the MAG inhibitor clorgyline blocked cell death indicating that MAG activity contributes to the cell death caused by NGF withdrawal. Together, these data indicate that the p38 MAP kinase pathway targets the MAG gene in response to apoptotic stimuli. To study the requirement for the JNK signaling pathway in neurodegeneration I stimulated primary cortical neurons with the neurotoxin arsenite. Arsenite treatment of primary neurons leads to both JNK and p38 activation and subsequently apoptosis. Utilizing transgenic mice lacking the JNK3 gene I demonstrated that JNK3 specifically contributes to the effects of arsenite in these cells. Ribonuclease protection assays were used to identify Fas ligand as a molecule whose arsenite-induced expression is dependent on the JNK3 signal transduction pathway. Furthermore, I have shown that neurons deficient in signaling mediated by the receptor for Fas ligand are resistant to cell death due to arsenite treatment. These results in total have established that the JNK3 mediated expression of Fas ligand contributes to the arsenite induced death of cortical neurons. In summary, the work presented in these studies identifies the JNK and p38 MAP kinase signal transduction pathways as mediators of apoptosis in neuronal cells. Importantly, I have provided evidence that these stress activated pathways are responsible for the expression of specific genes in apoptotic neuronal cells

Simulation and experimental verification of W-band finite frequency selective surfaces on infinite background with 3D full wave solver NSPWMLFMA

We present the design, processing and testing of a W-band finite by infinite and a finite by finite Grounded Frequency Selective Surfaces (FSSs) on infinite background. The 3D full wave solver Nondirective Stable Plane Wave Multilevel Fast Multipole Algorithm (NSPWMLFMA) is used to simulate the FSSs. As NSPWMLFMA solver improves the complexity matrix-vector product in an iterative solver from O(N(2)) to O(N log N) which enables the solver to simulate finite arrays with faster execution time and manageable memory requirements. The simulation results were verified by comparing them with the experimental results. The comparisons demonstrate the accuracy of the NSPWMLFMA solver. We fabricated the corresponding FSS arrays on quartz substrate with photolithographic etching techniques and characterized the vector S-parameters with a free space Millimeter Wave Vector Network Analyzer (MVNA)

A qualitative risk assessment for human salmonellosis due to the consumption of fresh pork in Belgium

Although pigs contaminated with Salmonella rarely show clinical symptoms, control is important because of the public health concern. Both producers and consumers are interested in procedures for minimizing the risk of Salmonella infections. This study outlines the entire production path for fresh pork in Belgium, from farm to fork. Additionally, it describes the different critical points for Salmonella contamination, with emphasis on those steps that need extra attention and/or improvement. The data was collected by means of questionnaires at the different steps of the process. In total, 3658 questionnaires were collected, which made it possible to draw up a nationwide image of the pork production process. In the primary production phase, there are several points relating to biosecurity that can be improved in order to minimize the risk for Salmonella in fattening pigs that are sent to slaughter. In the slaughterhouse, there has been an increase in the number of pigs or carcasses that become infected with Salmonella. Attention should be paid to avoiding contact of the feces and tonsils of contaminated pigs with the carcass, and strict hygienic measures should be taken to avoid cross-contamination. During the transformation and distribution of the carcasses, there is a low risk of further spreading of Salmonella spp. Finally, during the consumer phase, the risk for Salmonella contamination increases because of inappropriate temperature conditions during storage, manipulation of the meat and possible cross-contamination with other food products, and the consumption of insufficiently heated and/or raw meat. The present study illustrates that the risk of Salmonella infection by consumption of fresh pork is relatively low under Belgian conditions. Nevertheless, it can be further decreased by implementing additional control measures, mainly in the slaughterhouse and in the domestic kitchen

Virulence profiling and quantification of verocytotoxin-producing Escherichia coli O145:H28 and O26:H11 isolated during an ice cream-related hemolytic uremic syndrome outbreak

In September-October 2007, a mixed-serotype outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145:H28 and O26:H11 occurred in the province of Antwerp, Belgium. Five girls aged between 2 and 11 years developed hemolytic uremic syndrome, and seven other coexposed persons with bloody diarrhea were identified. Laboratory confirmation of O145:H28 infection was obtained for three hemolytic uremic syndrome patients, one of whom was coinfected with O26:H11. The epidemiological and laboratory investigations revealed ice cream as the most likely source of the outbreak. The ice cream was produced at a local dairy farm using pasteurized milk. VTEC of both serotypes with indistinguishable pulsed-field gel electrophoresis patterns were isolated from patients, ice cream, and environmental samples. Quantitative analysis of the ice cream indicated concentrations of 2.4 and 0.03 CFU/g for VTEC O145 and O26, respectively. Virulence typing revealed that the repertoire of virulence genes carried by the O145:H28 outbreak strain was comparable to that of O157 VTEC and more exhaustive as compared to the O26:H11 outbreak strain and nonrelated clinical strains belonging to these serotypes. Taken together, these data suggest that O145:H28 played the most important role in this outbreak

Ambient-aware continuous care through semantic context dissemination

Background: The ultimate ambient-intelligent care room contains numerous sensors and devices to monitor the patient, sense and adjust the environment and support the staff. This sensor-based approach results in a large amount of data, which can be processed by current and future applications, e. g., task management and alerting systems. Today, nurses are responsible for coordinating all these applications and supplied information, which reduces the added value and slows down the adoption rate. The aim of the presented research is the design of a pervasive and scalable framework that is able to optimize continuous care processes by intelligently reasoning on the large amount of heterogeneous care data. Methods: The developed Ontology-based Care Platform (OCarePlatform) consists of modular components that perform a specific reasoning task. Consequently, they can easily be replicated and distributed. Complex reasoning is achieved by combining the results of different components. To ensure that the components only receive information, which is of interest to them at that time, they are able to dynamically generate and register filter rules with a Semantic Communication Bus (SCB). This SCB semantically filters all the heterogeneous care data according to the registered rules by using a continuous care ontology. The SCB can be distributed and a cache can be employed to ensure scalability. Results: A prototype implementation is presented consisting of a new-generation nurse call system supported by a localization and a home automation component. The amount of data that is filtered and the performance of the SCB are evaluated by testing the prototype in a living lab. The delay introduced by processing the filter rules is negligible when 10 or fewer rules are registered. Conclusions: The OCarePlatform allows disseminating relevant care data for the different applications and additionally supports composing complex applications from a set of smaller independent components. This way, the platform significantly reduces the amount of information that needs to be processed by the nurses. The delay resulting from processing the filter rules is linear in the amount of rules. Distributed deployment of the SCB and using a cache allows further improvement of these performance results

Using Soil and Water Conservation Contests for Extension: Experiences from the Bolivian Mountain Valleys

Soil and water conservation (SWC) contests among farmer groups were organized in five rural villages in the Bolivian mountain valleys. The contests were aimed at quickly achieving widespread sustainable results. This article analyzes the effectiveness of these contests as an extension tool. Mixed results were obtained. In three villages, participation rates in the SWC activities introduced in the contests were still high even 2 years after project withdrawal. These were all villages where a solid foundation for sustainable development had been laid before the contests were held. Two years later, most families were still involved in maintenance of the SWC practices introduced in the contests, and many farmers had started to experiment with different soil management practices. However, replications of these SWC practices were not widespread, Conservation Leaders did not continue with their training activities, and the quality of maintenance of the practices was often not satisfactory. In order to become a more effective extension tool and achieve widespread impact, SWC contests must receive continued support by a catalyst agency. Moreover, other SWC contests should also be organized in which practices are not predefined. Given that SWC contests are a low-budget extension tool, local municipalities could become more actively involved

Validation of EN ISO method 10273-Detection of pathogenic Yersinia enterocolitica in foods

EN ISO 10273 method for the detection of pathogenic Yersinia enterocolitica in foods was validated in the project Mandate M/381 funded by European Commission. A total of 14 laboratories from five European countries participated in the interlaboratory study (ILS) organized during 2013 and 2014. Before the ITS, the method was revised by an international group of experts and the performance of the revised method was assessed in an ILS study. The results are published as a part of the standard EN ISO 10273 revision. The study included three rounds with different sample types; raw milk, iceberg lettuce and minced meat, inoculated with a low and high level of pathogenic Y. enterocolitica strains representing major pathogenic bioserotypes 4/O:3 and 2/O:9. The homogeneity and stability of the samples were verified before dispatching them to the laboratories. The results demonstrated the method sensitivity of 96% in raw milk, 97% in minced meat, and 98% in lettuce at high inoculation level of pathogenic Y. enterocolitica. The specificity was 100% in raw milk, 96% in minced meat, and 98% in lettuce. The level of detection, LOD50, varied between study rounds, being 9.4 CFU/25 ml in raw milk, 9.9 CFU/25 g in minced meat and 63 CFU/25 g in lettuce samples. During the study, confirmation by using real-time PCR method ISO/TS 18867 together with pyrazinamidase testing was also validated, as alternative to conventional biochemical confirmation. When comparing different isolation steps used in the revised method during the study rounds, PSB enrichment and plating on CIN after alkaline (KOH) treatment showed the highest sensitivity (52-92%) in raw milk and minced meat samples. In lettuce samples, however, ITC with KOH treatment before plating on CIN showed higher sensitivity (64% at low level; 82% at high level) than plating on CIN from PSB with KOH treatment (44% at low level; 74% at high level). Statistical analysis of different isolation steps supported the use of two enrichment media, PSB and ITC, in the revised method. Recovery of pathogenic Y. enterocolitica on ON was most efficient after KOH treatment and, based on the analysis, plating on CIN agar without KOH treatment could be left as optional procedure in the method.Peer reviewe

Regulation of mammary gland branching morphogenesis by the extracellular matrix and its remodeling enzymes.

A considerable body of research indicates that mammary gland branching morphogenesis is dependent, in part, on the extracellular matrix (ECM), ECM-receptors, such as integrins and other ECM receptors, and ECM-degrading enzymes, including matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). There is some evidence that these ECM cues affect one or more of the following processes: cell survival, polarity, proliferation, differentiation, adhesion, and migration. Both three-dimensional culture models and genetic manipulations of the mouse mammary gland have been used to study the signaling pathways that affect these processes. However, the precise mechanisms of ECM-directed mammary morphogenesis are not well understood. Mammary morphogenesis involves epithelial 'invasion' of adipose tissue, a process akin to invasion by breast cancer cells, although the former is a highly regulated developmental process. How these morphogenic pathways are integrated in the normal gland and how they become dysregulated and subverted in the progression of breast cancer also remain largely unanswered questions

Diet-Induced Muscle Insulin Resistance Is Associated With Extracellular Matrix Remodeling and Interaction With Integrin α2β1 in Mice

OBJECTIVE: The hypothesis that high-fat (HF) feeding causes skeletal muscle extracellular matrix (ECM) remodeling in C57BL/6J mice and that this remodeling contributes to diet-induced muscle insulin resistance (IR) through the collagen receptor integrin α(2)β(1) was tested. RESEARCH DESIGN AND METHODS: The association between IR and ECM remodeling was studied in mice fed chow or HF diet. Specific genetic and pharmacological murine models were used to study effects of HF feeding on ECM in the absence of IR. The role of ECM-integrin interaction in IR was studied using hyperinsulinemic-euglycemic clamps on integrin α(2)β(1)-null (itga2(-/-)), integrin α(1)β(1)-null (itga1(-/-)), and wild-type littermate mice fed chow or HF. Integrin α(2)β(1) and integrin α(1)β(1) signaling pathways have opposing actions. RESULTS: HF-fed mice had IR and increased muscle collagen (Col) III and ColIV protein; the former was associated with increased transcript, whereas the latter was associated with reduced matrix metalloproteinase 9 activity. Rescue of muscle IR by genetic muscle-specific mitochondria-targeted catalase overexpression or by the phosphodiesterase 5a inhibitor, sildenafil, reversed HF feeding effects on ECM remodeling and increased muscle vascularity. Collagen remained elevated in HF-fed itga2(-/-) mice. Nevertheless, muscle insulin action and vascularity were increased. Muscle IR in HF-fed itga1(-/-) mice was unchanged. Insulin sensitivity in chow-fed itga1(-/-) and itga2(-/-) mice was not different from wild-type littermates. CONCLUSIONS: ECM collagen expansion is tightly associated with muscle IR. Studies with itga2(-/-) mice provide mechanistic insight for this association by showing that the link between muscle IR and increased collagen can be uncoupled by the absence of collagen-integrin α(2)β(1) interaction