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The multiple experimental approaches outlined in this proposal should lead to a better understanding of the mechanism(s) involved in development of biofilm-specific antibiotic resistance, and potentially to new therapeutic strategies for modulating these properties. Specific Aim I. Studies of known genes in biofilm-specific antibiotic resistance. Specific Aim II. Determine the role of a glucan synthetase enzyme in the development of biofilm-specific antibiotic resistance. Specific Aim III. Studies of the role of a putative new efflux pump in biofilm-specific antibiotic resistance.

2a) Development of peptide nucleic acid (PNA) molecules that inhibit expression of AAC(6')-Ib. 2b) Bioavailability studies of PNA molecules. A novel strategy to inhibit gene expression using antisense technology is now available with the synthesis of peptide nucleic acids (PNAs). PNAs may be developed as antimicrobial agents in prokaryotic systems. The investigators will test the in vitro and in vivo activity of several PNA molecules to interfere with the expression of aac(6')-Ib. The investigators will then study the bioavailability of naked and liposome encapsulated PNA molecules.

Based on these observations and our own preliminary data, we hypothesize that vertebrate TLR4, which is expressed by cardiac myocytes, plays a pivotal role in the response to injury in the heart. Specifically, we will examine: 1) the regulation of TLR4 expression in cardiac myocytes in vitro in response to injury induced by UV light, by anthracycline antibiotics and by cyclic biaxial strain coupled with electric field pacing, as well as in cardiac myocytes in situ in remodeling murine ventricular muscle following experimental myocardial infarction; 2) the signal transduction pathways leading to NFkappaB and MAP kinase activation by activated TLR4 in cardiac myocytes and their possible localization to caveolar microdomains; and 3) the functional role(s) of TLR4 in the response to myocyte injury in vitro and in vivo; specifically, we will test the hypothesis that: i) a constitutively activated TLR4 construct conveys a survival signal in vitro; ii) that TLR4 participates in the recognition and removal of apoptotic cells; and iii) that animals with targeted disruption of TLR4, or of its immediate downstream signaling target MyD88, exhibit altered rates of ventricular remodeling in response to myocardial infarction.

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