The relevance of bacterial isoprenoid biosynthetic pathways for host-microbe interactions

This thesis was undertaken to investigate the relevance of two bacterial isoprenoid biosynthetic pathways (Mevalonate (MVAL) and 2-C-methyl-D-erythritol 4-phosphate (MEP)) for host-microbe interactions. We determined a significant reduction in microbial diversity in the murine gut microbiota (by next generation sequencing) following oral administration of a common anti-cholesterol drug Rosuvastatin (RSV) that targets mammalian and bacterial HMG-CoA reductase (HMG-R) for inhibition of MVAL formation. In tandem we identified significant hepatic and intestinal off-target alterations to the murine metabolome indicating alterations in inflammation, bile acid profiles and antimicrobial peptide synthesis with implications on community structure of the gastrointestinal microbiota in statin-treated animals. However we found no effect on local Short Chain Fatty Acid biosynthesis (metabolic health marker in our model). We demonstrated direct inhibition of bacterial growth in-vitro by RSV which correlated with reductions in bacterial MVAL formation. However this was only at high doses of RSV. Our observations demonstrate a significant RSV-associated impact on the gut microbiota prompting similar human analysis. Successful deletion of another MVAL pathway enzyme (HMG-CoA synthase (mvaS)) involved in Listeria monocytogenes EGDe isoprenoid biosynthesis determined that the enzyme is non-essential for normal growth and in-vivo pathogenesis of this pathogen. We highlight potential evidence for alternative means of synthesis of the HMG-CoA substrate that could render mvaS activity redundant under our test conditions. Finally, we showed by global gene expression analysis (Massive Analysis of cDNA Ends (MACE RNA-seq) a significant role for the penultimate MEP pathway metabolite (E)-4-hydroxy-3-methyl-2-but-2-enyl pyrophosphate (HMBPP) in significant up regulation of genes of immunity and antigen presentation in THP-1 cells at nanomolar levels. We infected THP-1 cells with wild type or HMBPP under/over-producing L. monoctyogenes EGDe mutants and determined subtle effects of HMBPP upon overall host responses to Listeria infection. Overall our findings provide greater insights regarding bacterial isoprenoid biosynthetic pathways for host-microbe/microbe-host dialogue.

Experimental detection of sudden stiffness change in a structural system employing Laser Doppler Vibrometry

Sudden changes in the stiffness of a structure are often indicators of structural damage. Detection of such sudden stiffness change from the vibrations of structures is important for Structural Health Monitoring (SHM) and damage detection. Non-contact measurement of these vibrations is a quick and efficient way for successful detection of sudden stiffness change of a structure. In this paper, we demonstrate the capability of Laser Doppler Vibrometry to detect sudden stiffness change in a Single Degree Of Freedom (SDOF) oscillator within a laboratory environment. The dynamic response of the SDOF system was measured using a Polytec RSV-150 Remote Sensing Vibrometer. This instrument employs Laser Doppler Vibrometry for measuring dynamic response. Additionally, the vibration response of the SDOF system was measured through a MicroStrain G-Link Wireless Accelerometer mounted on the SDOF system. The stiffness of the SDOF system was experimentally determined through calibrated linear springs. The sudden change of stiffness was simulated by introducing the failure of a spring at a certain instant in time during a given period of forced vibration. The forced vibration on the SDOF system was in the form of a white noise input. The sudden change in stiffness was successfully detected through the measurements using Laser Doppler Vibrometry. This detection from optically obtained data was compared with a detection using data obtained from the wireless accelerometer. The potential of this technique is deemed important for a wide range of applications. The method is observed to be particularly suitable for rapid damage detection and health monitoring of structures under a model-free condition or where information related to the structure is not sufficient.