Michael Joseph Smith - US grants

Septic shock is the thirteenth leading cause of death in the United States and sepsis due to gram negative bacteria accounts for over 50% of those cases. Bacterial lipopolysaccharide from gram negative bacteria stimulates monocytes and macrophages to release a variety of soluble immune system mediators and when introduced systemically, to trigger the pathological events observed during endotoxic shock. The long-term objective of the proposed research is to define the biochemical mechanisms through which bacterial lipopolysaccharide (LPS) acts to modulate monocyte and macrophage functions. Specifically, this project proposes to define the LPS-induced nuclearsignalling events which lead to the induction of the interleukin-1 receptor antagonist (IL-1ra) gene. Previously, we have identified regions of the human secretory IL-1ra promoter which contain three positive acting and one inhibitory LPS responsive elements. The proposed studies will expand this work to conclusively identified the cis-acting DNA sequences and the trans- acting factors which regulate the LPS-induced expression of the IL-1ra gene. Specific aim 1 will define the LPS-responsive cis-acting DNA elements in the human IL-1ra promoter. An IL-1ra promoter/luciferase reporter chimeric gene construct will be used in transient transfection studies. The sequences of important transcriptional elements in the promoter will be determined through the use of heterologous promoter constructs, DNase I protection assays, and electrophoretic mobility shift assays (EMSA). Confirmation of the importance of an element in the LPS-induced activation will be made through site-directed mutagenesis of each element alone and in combination with other identified elements. Specific aim 2 will characterize the factors which bind to the LPS responsive elements defined in specific aim 1. We will determine the specific translational and posttranslational requirements for factor binding and activation as well as its molecular weight. The goal of these studies is to determine if the LPS responsive factors are newly identified transcription factor or previously characterized ones. It is expected that these studies will provide significant new insights into the mechanisms through which LPS acts to regulate gene expression in monocytes and macrophages.

DESCRIPTION (Adapted from applicant's summary):Lipopolysaccharide (LPS; also known as endotoxin), derived from the outer membrane of gram negative bacteria, is one of the principal activators of the sepsis cascade. Although we have known of endotoxin's powerful effects on the immune system for over 100 years, a clear understanding of how this bacterial constituent can elicit host responses is still lacking. A better understanding of the molecular mechanisms through which endotoxin regulates inflammatory and anti-inflammatory gene expression will provide new targets for the development of therapies aimed at controlling the cascade of events induced during a gram negative bacterial infection. It is the long-term goal of this research to provide information on these new targets by characterizing both the nuclear and cytoplasmic signaling mechanisms through which LPS acts to induce inflammatory and anti-inflammatory gene expression in monocytes and macrophages. The proposed studies seek to define the nuclear events, elicited by LPS, which lead to the expression of the anti-inflammatory secretory IL-1Ra (sIL-1Ra) gene in macrophages. At least four distinct LPS response elements (LREs) are involved in inducing sIL-1Ra gene expression in response to LPS. Our previous studies have identified the transcription factors which bind to two of these (LRE1 and 3) as the myeloid-specific factor PU.1. Specific Aim 1 will characterize the mechanisms through which the PU.1 transcription factor regulates LPS-inducible gene expression in macrophages. Specific Aim 2 will characterize a newly-discovered major LPS responsive promoter element located between B3.4 and B1.7 kb upstream of the transcription start site of the sIL-1Ra gene and develop a transgenic mouse model in order to study the role of the distal enhancer element in the regulation of gene expression in vivo.

[unreadable] DESCRIPTION (provided by applicant): The ability to induce or ablate gene expression in a tissue-specific manner has proven to be a hugely important tool for biologists in many different disciplines. By mating a transgenic mouse in which the Cre recombinase gene is expressed under the control of a tissue-specific promoter to a mouse containing the gene of interest flanked by Cre recognition sites (loxP) an investigator can rapidly and efficiently knock out or induce the gene in a tissue- and/or temporal-specific manner. This conditional transgenesis circumvents some problems associated with whole animal gene knockouts that arise due to embryonic lethality or, involvement of the gene in the biology of multiple tissues. The limiting factor in conditional transgenesis studies is the derivation of mouse strains in which the Cre recombinase is expressed in the appropriate tissue-specific manner. Currently there are no mouse strains available which express the Cre recombinase specifically, or even predominantly, in the gastric epithelium. The lack of this important resource has impaired our ability to clearly understand the physiology and pathophysiology of the gastric mucosa. The goal of this proposal is to develop the technology to target transgene expression to the two predominant cell lineages within the gastric epithelium: the mucous-producing pit cells at the apical surface of the gastric gland and the zymogenic chief cells located towards the base of the gland. This aim will be achieved by "knocking-in" a Cre-recombinase cassette into the loci for the trefoil factor 1 and pepsinogen C loci. These mice can then be mated to any mouse strain containing a gene of interest which has been modified to be flanked by loxP recombinase signals. Once derived and characterized, these mice will be highly beneficial to numerous groups working on projects that cross several disciplines including immunology and inflammation, cancer, nutrition, and developmental biology. Thus, development of this unique resource will be of use to investigators supported by multiple NIH Institutes and Centers including NIDDK, NIAID, NCI, and NIGMS. [unreadable] [unreadable]

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5)

This engineering education award to the University of Virginia will develop an innovative model for an accelerated masters degree in systems engineering that is customized to the needs and strengths of veterans. A particular emphasis will be including research experiences and industrial internships within the degree program. The results will also be presented on the web, at technical conferences and published in professional journals. The project is expected to assist the transition of veterans to civilian life and to enhance the number of students who complete engineering degrees and are ready to fill engineering jobs or start new high tech businesses.

It is widely accepted that systems engineering is a key element of all large engineering projects; however, one estimate is that the Department of Defense alone loses about $200 million per day as a result of poor systems engineering, and other Federal agencies acknowledge that they cannot meet their projected missions using current systems engineering practices. There has been a proliferation of calls for systems engineering, including the Secretary of Defense and the National Academies. Simultaneously over the past 25 years, elements of a theory of systems engineering have emerged. These elements point to an opportunity to formulate systems engineering as a rigorous engineering discipline. The elements span mathematics, economics, business, and psychology, as well as significant elements of engineering and the sciences. Numerous Universities and professional societies offer diverse educational programs and certifications in systems engineering. This workshop on systems engineering education will assist in the unification of these disparate perspectives and help the systems engineering community move forward via improving the overall systems engineering education function.

A survey of systems engineering related programs identified a diversity of systems engineering educations programs, without a consensus on how to create better systems engineering processes and the associated underlying educational foundations. The primary purpose of the workshop is to investigate the future of systems engineering education and not conduct a review of the history nor the current state of systems engineering education, but a look forward. The workshop will bring the leaders of the systems engineering community in the US together to investigate the following issues: 1) the needs of government and industry in terms of the tasks and roles that belong to systems engineering, 2) the elements of the skill and knowledge sets required to meet the systems engineering needs, 3) the organization of these elements into coherent systems engineering curricula at all levels, 4) the underlying theories that provide the needed skill and knowledge sets and that will form the basis of systems engineering education, and 5) the knowledge gaps that need to be researched that would enhance systems engineering education. Results of the workshop will be disseminated via a report and an archival publication.