Chemical differences of atherosclerotic plaques in native and bypass human coronary arteries and diseased and non-diseased human aortas

ABSTRACT Cardiovascular disease, primarily atherosclerosis involves a number of distinct processes that are associated with plaque development. The use of differential gel electrophoresis to determine differences between proteins produced in native and bypass coronary arteries from the same heart has been studied. The analytical techniques presented in this study to characterize plaque samples include two dimensional gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, mass spectrometry, differential gel electrophoresis, and high performance liquid chromatography. An overview of the stages involved in atherosclerosis and the theories implicated in the manifestation of atherosclerosis was presented. A design study using the Box Behnken method was used to optimize the components in lysis buffer to solubilize the membrane proteins present in the intima in atherosclerotic plaques. The number of proteins located as spots in the gel were optimized in this study. The isolation and characterization of proteins in intima and media extracts of diseased aortas were separated using two dimensional gel electrophoresis followed by excision of gel spots and tryptic digestion to peptides. Mass analysis (MS and MS/MS) of the digested peptides with a linear quadrupole trap Fourier transform ion cyclotron resonance mass spectrometer was used to determine the amino acid sequences. Protein extracts of normal and diseased aortas and native and bypass arteries were analyzed using differential gel electrophoresis system. Cydye 3 (1-(5-carboxypentyl)-1`-propylindocarbocyanine halide N-hydroxy-succinimidyl ester) and Cydye 5 (1-(5-carboxypentyl)-1´-methylindodicarbocyanine halide N-hydroxy-succinimidyl ester) are fluorescent dyes used in our study to identify differentially expressed proteins of interest in the same gel using matrix assisted laser desorption ionization time-of-flight mass spectrometry. Several proteins in normal and diseased tissues were identified A study of the separation and quantification of cholesterol and cholesteryl esters was performed using reverse phase high performance liquid chromatograph interfaced with atmospheric pressure chemical ionization probe that was introduced into a quadrupole mass analyzer. Thirty-five samples were analyzed by principal component analysis that produced two distinct age groups based on age. In addition, a disease severity index was generated from the data. This study concluded that age and low disease severity index were indicators of the atherogenic states of the extracts analyzed

Development and characterization of molecular tools for microbial forensics

Scope and Method of Study:The anthrax attacks of 2001 prompted the rapid establishment and growth of the fields of microbial and plant pathogen forensics. A complete forensic capability includes the ability to discriminate between a natural and an intentional disease outbreak, collection of forensic evidence, generation of genetic profiles for use during attribution and storage of samples. This document describes (i) the molecular characterization of plant virus populations derived from plants that were naturally and mechanically-inoculated with a model plant virus, (ii) the application of microarray-based technologies to genetically fingerprint plant viruses, (iii) the characterization of a previously-designed microarray platform for the identification and diagnosis of known and novel plant viruses, and (iv) the use of FAME profiles to aid in the discrimination of media components used to prepare Bacillus cereus T-strain spores. Though all of these projects are not directly related, they all fall within the scope of microbial forensics.Findings and Conclusions:The molecular characterization of plant virus populations derived from a natural inoculation event and those from mechanically-inoculated plants displayed minor differences in haplotype and pair-wise nucleotide diversities. Additionally, the number of recombination events was found to be lower in the mechanically-inoculated plants than those collected from the natural disease outbreak. These results indicate that differences between the two types of inoculation events exist and may be a direct function of the infection time, source(s) of inoculum(a) or environmental effects.The solution-based minisequencing and capture array technique demonstrated reproducibility at the same concentration of targets, but was less accurate using variable amounts of synthetic targets. The use of solution-based minisequencing followed by tag-array capture appears to be a promising approach to genotyping plant viruses.A significant amount of cross-hybridization was observed using the universal plant virus microarray (Virochip). The microarray platform failed to strongly hybridize to most of the known plant viruses that were applied to the array. Hybridization with Wheat streak mosaic virus-infected material indicated that the system strongly hybridized with the negative-sense strand, but not the corresponding positive-sense strands.Discrimination of individual media components was achieved by analyzing fatty acids derived from Bacillus cereus T-strain spores prepared in different media. One FAME biomarker, oleic acid, was found to be exclusively associated with media supplemented with blood

Designing Thermal Modulators for Portable GC x GC Systems.

Microelectromechanical systems (MEMS) have the advantage of scale and can be manufactured in bulk. One of the active areas of MEMS research is the development of micro-scale comprehensive two-dimensional gas chromatography (μGC×μGC). Our previous work demonstrated the development of the first microscale thermal modulator(µTM) for use in GC×GC. However, our demonstration was limited to very simple mixtures. Rapid, GC×GC separations by use of a mid-point µTM are demonstrated and the effects of various µTM design and operating parameters on performance are characterized here. A 9 compound structured chromatogram and a 21-component separation was achieved in < 3 min. Next we demonstrate GC × GC with all microfabricated components. The first dimension consists of two series coupled μcolumn chips with etched channels, with a PDMS stationary phase. The second dimension consists of a μcolumn chip with either a trigonal tricationic room-temperature ionic liquid (RTIL) or a commercial poly(trifluoropropylmethyl siloxane) (OV-215) stationary phase. Conventional injection methods and flame ionization detection were used. Current conventional thermal modulators can achieve FWHH of modulated peaks of ~ 10 ms, which is necessary to obtain optimum peak capacity in GC×GC by using cryogenic consumables or high amounts of power. However, since we are limited in the amount of cooling power we can use, we need to understand the fundamental physics governing the thermal modulation, and optimize our modulators. Hence we developed a theoretical model of single-stage TM with the aim to elucidate factors leading to improvements in GC×GC analyses. Model predictions were compared with experimental data obtained using our μTM operating as a single-stage TM and excellent match is obtained. To make a more realistic model, we demonstrated the physics behind the operation of a two-stage modulator. We show that parameters such as the time constant of modulation can be used to reduce the FWHH, breakthrough and hence improve the peak capacity of the GC×GC significantly. Going forward, this theory can be used to optimize the performance of the thermal modulator and coupled with thermal simulations to design the next generation of thermal modulators.PhDMechanical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/116744/1/dibya_1.pd

PCF-Based Cavity Enhanced Spectroscopic Sensors for Simultaneous Multicomponent Trace Gas Analysis

A multiwavelength, multicomponent CRDS gas sensor operating on the basis of a compact photonic crystal fibre supercontinuum light source has been constructed. It features a simple design encompassing one radiation source, one cavity and one detection unit (a spectrograph with a fitted ICCD camera) that are common for all wavelengths. Multicomponent detection capability of the device is demonstrated by simultaneous measurements of the absorption spectra of molecular oxygen (spin-forbidden b-X branch) and water vapor (polyads 4v, 4v + δ) in ambient atmospheric air. Issues related to multimodal cavity excitation, as well as to obtaining the best signal-to-noise ratio are discussed together with methods for their practical resolution based on operating the cavity in a “quasi continuum” mode and setting long camera gate widths, respectively. A comprehensive review of multiwavelength CRDS techniques is also given

Laser diagnostics and minor species detection in combustion using resonant four-wave mixing

Peer reviewedPostprin

Semiconductor Nanocrystals: From Quantum Dots to Quantum Disks

The bottom-up colloidal synthesis opened up the possibility of finely tuning and tailoring the semiconductor nanocrystals. Numerous recipes were developed for the preparation of colloidal semiconductor nanocrystals, especially the traditional quantum dots. However, due to the lack of thorough understanding to those systems, the synthesis chemistry is still on the empirical level. CdS quantum dots synthesis in non-coordinating solvent were taken as a model system to investigate its molecular mechanism and formation process, ODE was identified as the reducing agent for the preparation of CdS nanocrystals, non-injection and low-temperature synthesis methods developed. In this model system, we not only proved it\u27s possible to systematically study the formation procedure of semiconductor nanocrystals, the insight learned during the research but also enhanced our understanding to this delicate system and promoted the development of synthetic chemistry. Although quantum dots could be routinely prepared in the lab with mature recipes, the colloidal semiconductor quantum well type materials are still hard to fathom. CdSe quantum disks structure was thoroughly analyzed with polar axes as the growth direction along the thickness direction, with both basal planes ended with Cd atom layer, which was coordinated with carboxylate ligands. Besides, four different thickness CdS quantum disks were prepared, its size-dependent lattice dilation, extremely sharp band-edge emission, and two-order of magnitude faster photoluminescence decay compared to quantum dots was investigated

Characterisation of two quorum sensing systems in the endophytic Serratia plymuthica strain G3: differential control of motility and biofilm formation according to life-style

<p>Abstract</p> <p>Background</p> <p><it>N</it>-acylhomoserine lactone (AHL)-based quorum sensing (QS) systems have been described in many plant-associated Gram-negative bacteria to control certain beneficial phenotypic traits, such as production of biocontrol factors and plant growth promotion. However, the role of AHL-mediated signalling in the endophytic strains of plant-associated <it>Serratia </it>is still poorly understood. An endophytic <it>Serratia </it>sp. G3 with biocontrol potential and high levels of AHL signal production was isolated from the stems of wheat and the role of QS in this isolate was determined.</p> <p>Results</p> <p>Strain G3 classified as <it>Serratia plymuthica </it>based on 16S rRNA was subjected to phylogenetic analysis. Using primers to conserved sequences of <it>luxIR </it>homologues from the <it>Serratia </it>genus, <it>splIR </it>and <it>spsIR </it>from the chromosome of strain G3 were cloned and sequenced. AHL profiles from strain G3 and <it>Escherichia coli </it>DH5α expressing <it>splI </it>or <it>spsI </it>from recombinant plasmids were identified by liquid chromatography-tandem mass spectrometry. This revealed that the most abundant AHL signals produced by SplI in <it>E. coli </it>were <it>N</it>-3-oxo-hexanoylhomoserine lactone (3-oxo-C6-HSL), <it>N</it>-3-oxo-heptanoylhomoserine lactone (3-oxo-C7-HSL), <it>N</it>-3-hydroxy-hexanoylhomoserine lactone (3-hydroxy-C6-HSL), <it>N</it>-hexanoylhomoserine lactone (C6-HSL), and <it>N</it>-heptanoyl homoserine lactone (C7-HSL); whereas SpsI was primarily responsible for the synthesis of <it>N</it>-butyrylhomoserine lactone (C4-HSL) and <it>N</it>-pentanoylhomoserine lactone (C5-HSL). Furthermore, a quorum quenching analysis by heterologous expression of the <it>Bacillus </it>A24 AiiA lactonase in strain G3 enabled the identification of the AHL-regulated biocontrol-related traits. Depletion of AHLs with this lactonase resulted in altered adhesion and biofilm formation using a microtiter plate assay and flow cells coupled with confocal laser scanning microscopy respectively. This was different from the closely related <it>S. plymuthica </it>strains HRO-C48 and RVH1, where biofilm formation for both strains is AHL-independent. In addition, QS in G3 positively regulated antifungal activity, production of exoenzymes, but negatively regulated production of indol-3-acetic acid (IAA), which is in agreement with previous reports in strain HRO-C48. However, in contrast to HRO-C48, swimming motility was not controlled by AHL-mediated QS.</p> <p>Conclusions</p> <p>This is the first report of the characterisation of two AHL-based quorum sensing systems in the same isolate of the genus <it>Serratia</it>. Our results show that the QS network is involved in the global regulation of biocontrol-related traits in the endophytic strain G3. However, although free-living and endophytic <it>S. plymuthica </it>share some conservation on QS phenotypic regulation, the control of motility and biofilm formation seems to be strain-specific and possible linked to the life-style of this organism.</p

Chemometric tools for automated method-development and data interpretation in liquid chromatography

The thesis explores the challenges and advancements in the field of liquid chromatography (LC), particularly focusing on complex sample analysis using high-resolution mass spectrometry (MS) and two-dimensional (2D) LC techniques. The research addresses the need for efficient optimization and data-handling strategies in modern LC practice. The thesis is divided into several chapters, each addressing specific aspects of LC and polymer analysis. Chapter 2 provides an overview of the need for chemometric tools in LC practice, discussing methods for processing and analyzing data from 1D and 2D-LC systems and how chemometrics can be utilized for method development and optimization. Chapter 3 introduces a novel approach for interpreting the molecular-weight distribution and intrinsic viscosity of polymers, allowing quantitative analysis of polymer properties without prior knowledge of their interactions. This method correlates the curvature parameter of the Mark-Houwink plot with the polymer's structural and chemical properties. Chapters 4 and 5 focus on the analysis of cellulose ethers (CEs), essential in various industrial applications. A new method is presented for mapping the substitution degree and composition of CE samples, providing detailed compositional distributions. Another method involves a comprehensive 2D LC-MS/MS approach for analyzing hydroxypropyl methyl cellulose (HPMC) monomers, revealing subtle differences in composition between industrial HPMC samples. Chapter 6 introduces AutoLC, an algorithm for automated and interpretive development of 1D-LC separations. It uses retention modeling and Bayesian optimization to achieve optimal separation within a few iterations, significantly improving the efficiency of gradient LC separations. Chapter 7 focuses on the development of an open-source algorithm for automated method development in 2D-LC-MS systems. This algorithm improves separation performance by refining gradient profiles and accurately predicting peak widths, enhancing the reliability of complex gradient LC separations. Chapter 8 addresses the challenge of gradient deformation in LC instruments. An algorithm based on the stable function corrects instrument-specific gradient deformations, enabling accurate determination of analyte retention parameters and improving data comparability between different sources. Chapter 9 introduces a novel approach using capacitively-coupled-contactless-conductivity detection (C4D) to measure gradient profiles without adding tracer components. This method enhances inter-system transferability of retention models for polymers, overcoming the limitations of UV-absorbance detectable tracer components. Chapter 10 discusses practical choices and challenges faced in the thesis chapters, highlighting the need for well-defined standard samples in industrial polymer analysis and emphasizing the importance of generalized problem-solving approaches. The thesis identifies future research directions, emphasizing the importance of computational-assisted methods for polymer analysis, the utilization of online reaction modulation techniques, and exploring continuous distributions obtained through size-exclusion chromatography (SEC) in conjunction with triple detection. Chemometric tools are recognized as essential for gaining deeper insights into polymer chemistry and improving data interpretation in the field of LC