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Bioactive Glycerophospholipids and Their Role in Modulating Neuronal Vulnerability Following Cerebral IschemiaSyrett, Andrew J. 11 January 2012 (has links)
Stroke is a devastating and debilitating condition resulting from a blockage or
hemorrhage in the vasculature of the brain. Despite extensive research, the etiology
and pathophysiology of the disease at the level of the cell membrane are poorly
understood, and effective treatment has been elusive. Though much research has
shown marked increases in lipid metabolism following stroke, the impact of these
changes have often been overlooked given the technical challenges associated with
identifying regionally specific changes in degenerating tissue. The advent of
lipidomics – a systems biology approach to the large-scale profiling of individual
lipid species in tissues – has renewed interest in understanding the role of
membrane lipids and their metabolites in the cell and in ischemic injury. In this
thesis, I have used an unbiased LC-ESI-MS-based lipidomic approach to profile the
small molecular weight glycerophosphocholine second messenger lipidome in
anterior and posterior regions of cortex and striatum in the forebrain of wild-type and
platelet activating factor receptor (PAFR) null-mutant mice before and after middle
cerebral artery occlusion (MCAO). From these profiles, I have outlined the potential
use of lipid second messenger distribution as topographic landmarks to identify
functional subdomains within neural tissue. Further, I have demonstrated that
ischemia does not simply disrupt lipid second messenger metabolism globally but
produces regionally specific changes in discrete species and that these changes are
altered by the loss of lipid regulatory effectors (i.e., PAFR null mutation). Based on
the lipid species identified in this profile of healthy and ischemic tissue, I proposed
that tight regulation of PC(O-22:6/2:0) homeostasis by PAFR-expressing microglia is
ii
required for proper dopaminergic signaling in prefrontal cortex. Finally, I have
outlined a model whereby increased PAF synthesis following ischemia contributes
the inflammatory response by promoting blood-brain barrier permeability, microglial
activation and immune cell infiltration in a PAFR-dependent manner.
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Developing Mass Spectrometry-Based Analytical Methodologies for Analyzing Complex Protein and Lipid SamplesHou, Weimin 18 September 2013 (has links)
Mass spectrometry has increasingly become the method of choice for the analysis of
complex biological samples, including proteins and lipids. This thesis describes the
development of MS-based analytical methodologies for the analysis of complex proteomic
and lipidomic samples.
Chapter 3 describes the development of microfluidic proteomic reactors, in the
formats of SCX reactor, SCX 96-well plate reactor, and SAX reactor, for the enzymatic
digestion of complex proteomic samples for subsequent LC-MS/MS analysis. These
microfluidic proteomic reactors greatly simplified the enzymatic digestion of complex
proteomic samples by combining multiple processing steps, such as rapid extraction and
enrichment of proteins. Furthermore, chemical and enzymatic treatments of proteins were all
performed in a few nanoliters effective volume, resulting in an increased protein digestion
efficacy. After the protein digestion process, the resulting peptides were eluted in buffers that
were compatible with HPLC-MS/MS analysis.
In chapter 4, a methodology based on nano-HPLC-ESI-MS/MS for the analysis of
PAF and LPC lipid species is described. In this method, lipid extracts from biological
samples were separated by nano-flow HPLC prior to being introduced into a Q-TRAP 2000
mass spectrometer, where the lipid species of interest were detected using a precursor ion
scan at m/z 184. Absolute quantitation of PAF family lipid species were performed with
standard addition method, where 5 standard solutions containing 0.2-1 ng each of C16:0,
C18:0 PAF and C16:0, C18:0 lyso-PAF were used in the experiment. Further, the spiking of
identical amount of non-endogenous C13:0 LPC at time of extraction allow the relative
comparisons of other LPC lipid species of interest between different samples. The developed
methods were employed to analyze the changes of PAF and LPC lipid species in NGFdifferentiated
PC12 cells, in the posterior/entorhinal cortex of AD patients and TgCRND8
transgenic mice, and over the course of 24 hour exposure of human hNT neurons to Aβ42
treatment, respectively, in comparison to controls.
iii
Chapter 5 describes the development of a novel shotgun lipidomic methodology for the determination of stereospecificity of diacyl glycerophospholipids including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols(PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized under negative ion mode. The stereospecificity of diacyl glycerophospholipids was determined based on the relative abundance of the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2]-) and as ketenes ([M-(Sn2-H2O)]-) exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1]- and [M-(Sn1-H2O)]-). We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined.
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Bioactive Glycerophospholipids and Their Role in Modulating Neuronal Vulnerability Following Cerebral IschemiaSyrett, Andrew J. January 2011 (has links)
Stroke is a devastating and debilitating condition resulting from a blockage or
hemorrhage in the vasculature of the brain. Despite extensive research, the etiology
and pathophysiology of the disease at the level of the cell membrane are poorly
understood, and effective treatment has been elusive. Though much research has
shown marked increases in lipid metabolism following stroke, the impact of these
changes have often been overlooked given the technical challenges associated with
identifying regionally specific changes in degenerating tissue. The advent of
lipidomics – a systems biology approach to the large-scale profiling of individual
lipid species in tissues – has renewed interest in understanding the role of
membrane lipids and their metabolites in the cell and in ischemic injury. In this
thesis, I have used an unbiased LC-ESI-MS-based lipidomic approach to profile the
small molecular weight glycerophosphocholine second messenger lipidome in
anterior and posterior regions of cortex and striatum in the forebrain of wild-type and
platelet activating factor receptor (PAFR) null-mutant mice before and after middle
cerebral artery occlusion (MCAO). From these profiles, I have outlined the potential
use of lipid second messenger distribution as topographic landmarks to identify
functional subdomains within neural tissue. Further, I have demonstrated that
ischemia does not simply disrupt lipid second messenger metabolism globally but
produces regionally specific changes in discrete species and that these changes are
altered by the loss of lipid regulatory effectors (i.e., PAFR null mutation). Based on
the lipid species identified in this profile of healthy and ischemic tissue, I proposed
that tight regulation of PC(O-22:6/2:0) homeostasis by PAFR-expressing microglia is
ii
required for proper dopaminergic signaling in prefrontal cortex. Finally, I have
outlined a model whereby increased PAF synthesis following ischemia contributes
the inflammatory response by promoting blood-brain barrier permeability, microglial
activation and immune cell infiltration in a PAFR-dependent manner.
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Developing Mass Spectrometry-Based Analytical Methodologies for Analyzing Complex Protein and Lipid SamplesHou, Weimin January 2013 (has links)
Mass spectrometry has increasingly become the method of choice for the analysis of
complex biological samples, including proteins and lipids. This thesis describes the
development of MS-based analytical methodologies for the analysis of complex proteomic
and lipidomic samples.
Chapter 3 describes the development of microfluidic proteomic reactors, in the
formats of SCX reactor, SCX 96-well plate reactor, and SAX reactor, for the enzymatic
digestion of complex proteomic samples for subsequent LC-MS/MS analysis. These
microfluidic proteomic reactors greatly simplified the enzymatic digestion of complex
proteomic samples by combining multiple processing steps, such as rapid extraction and
enrichment of proteins. Furthermore, chemical and enzymatic treatments of proteins were all
performed in a few nanoliters effective volume, resulting in an increased protein digestion
efficacy. After the protein digestion process, the resulting peptides were eluted in buffers that
were compatible with HPLC-MS/MS analysis.
In chapter 4, a methodology based on nano-HPLC-ESI-MS/MS for the analysis of
PAF and LPC lipid species is described. In this method, lipid extracts from biological
samples were separated by nano-flow HPLC prior to being introduced into a Q-TRAP 2000
mass spectrometer, where the lipid species of interest were detected using a precursor ion
scan at m/z 184. Absolute quantitation of PAF family lipid species were performed with
standard addition method, where 5 standard solutions containing 0.2-1 ng each of C16:0,
C18:0 PAF and C16:0, C18:0 lyso-PAF were used in the experiment. Further, the spiking of
identical amount of non-endogenous C13:0 LPC at time of extraction allow the relative
comparisons of other LPC lipid species of interest between different samples. The developed
methods were employed to analyze the changes of PAF and LPC lipid species in NGFdifferentiated
PC12 cells, in the posterior/entorhinal cortex of AD patients and TgCRND8
transgenic mice, and over the course of 24 hour exposure of human hNT neurons to Aβ42
treatment, respectively, in comparison to controls.
iii
Chapter 5 describes the development of a novel shotgun lipidomic methodology for the determination of stereospecificity of diacyl glycerophospholipids including glycerophosphatidic acids (PA), glycerophosphoserines (PS), glycerophosphoglycerols (PG), glycerophosphoinositols(PI), and glycerophosphoethanolamines (PE), which can be conventionally ionized under negative ion mode. The stereospecificity of diacyl glycerophospholipids was determined based on the relative abundance of the lyso-form fragment ions, attributed to the neutral loss of fatty acyl moieties. The fragmentation patterns of a variety of diacyl glycerophospholipid standards were first fully examined over a wide range of collision energy. We observed that lyso-form fragment ions corresponding to the neutral loss of fatty acyl moieties attached to the sn2 position as free fatty acids ([M-Sn2]-) and as ketenes ([M-(Sn2-H2O)]-) exhibited consistently higher intensity than their counter part ions due to the neutral loss of fatty acyl moieties attached to the sn1 position ([M-Sn1]- and [M-(Sn1-H2O)]-). We then examined the product ion spectra of diacyl glycerophospholipids recorded from lipid extracts of rat hepatoma cells, where the stereospecific information of these lipids was conclusively determined.
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Cell Membrane Lipid Alterations In Blood Plasma At Pre-Conception And During Pregnancy Are Associated With Gestational Diabetes DevelopmentLuevano, Jennifer J 01 October 2023 (has links) (PDF)
Introduction: Gestational diabetes mellitus (GDM) is a metabolic disorder that has been defined as glucose intolerance that is first identified during pregnancy. The etiology of GDM is not yet fully understood, but there are several risk factors that appear to contribute to its development such as advanced age at pregnancy, family history of type 2 diabetes mellitus, and a previous history of GDM. The discovery of predictive GDM biomarkers has the potential to enable early GDM detection and lead to earlier diagnosis and preventative interventions.
Objective: Perform metabolomics analysis on plasma samples collected at pre-conception and at 26-weeks gestation to investigate metabolic differences between participants of the gestational diabetes prevention (GDP) clinical trial who developed GDM and those who did not.
Methods: Targeted metabolomics, comprised of primary metabolomics, biogenic amines, and lipidomics assays, was performed using UPLC-MS on plasma samples collected from a subset of 30 participants that completed the GDP study at preconception and 26 weeks gestation. The samples used for this analysis were from participants who developed GDM (n=19) and those who did not (n=11) in their pregnancy following their participation in the GDP study.
Results: Multivariate analysis revealed indoxyl sulfate as significantly higher in the GDM group at both preconception and at 26 weeks gestation (VIP scores > 2.9). Preconception samples collected at the end of the GDP intervention study PC 38:0 was higher in the GDM group versus the non-GDM group (p < 0.05) whereas thymidine was lower in the GDM group (p < 0.05), in addition to numerous cell membrane lipids (VIP > 2.0). At 26 weeks gestation, D-glucuronic acid was higher in the GDM group versus the non-GDM group (p < 0.03), while LPE 22:6, SM 18:1 (22:4), PE 38:6, PE 40:6, PE 40:7, and PE (O-38:0) were lower in the GDM group (p < 0.04), in addition to numerous cell membrane lipids (VIP > 2.0).
Discussion: The differences observed between the GDM and non-GDM groups at the two plasma collection time points may suggest metabolic alterations associated with GDM-induced metabolic dysregulation. These findings may help direct future research to focus on changes in lipid metabolism during pre-pregnancy for possible biomarkers of GDM. Repeat studies with diverse cohorts are needed to help identify a panel of metabolites that may serve as early biomarkers of GDM.
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Etude fonctionnelle de deux marqueurs régionaux du cerveau chez la souris / A functional study of two regional markers of the mouse brainCaudy, Nada 09 September 2011 (has links)
Ce travail porte sur l’étude fonctionnelle de deux gènes préférentiellement exprimés dans deux régions du cerveau touchées par des pathologies neurodégénératives : Capucine, un marqueur du striatum, structure qui dégénère au cours de la maladie de Huntington et Agpat4, un marqueur de l’aire tegmentaire ventrale et de la substance noire compacte, dont les neurones dopaminergiques sont sélectivement atteints lors de la maladie de Parkinson. Des lignées de souris invalidées pour ces gènes ont été générées au laboratoire et au cours de ma thèse j’ai procédé à leur caractérisation. L’expression striatale du gène de la Capucine étant significativement diminuée dans des modèles murins de la maladie de Huntington, nous avons souhaité évaluer son rôle éventuel dans la pathogenèse de cette maladie. Pour ce faire, nous avons examiné, dans le cadre d’une collaboration, l’effet du knock-out et de la surexpression du gène de la Capucine sur la vulnérabilité des neurones striataux à un fragment de la Huntingtine mutée dans un modèle murin de la maladie de Huntington. Les données montrent que la Capucine n’a pas d’effet significatif sur la toxicité du fragment de la Huntingtine mutée dans le modèle étudié.La protéine Agpat4 présente des homologies de séquence avec des acyltransférases impliquées dans le métabolisme des phosphoglycérides. J’ai réalisé des études d’expression par différentes techniques de biologie moléculaire qui montrent que le gène d’Agpat4 est exprimé dans la plupart des tissus catécholaminergiques. Pour déterminer l’activité endogène d’Agpat4 et son rôle physiologique dans les tissus où elle est exprimée, j’ai comparé le métabolome de tissus de souris invalidées pour le gène d’Agpat4 et sauvages par chromatographie en phase liquide couplée à la spectrométrie de masse. Mes résultats indiquent que l’invalidation du gène d’Agpat4 perturbe le métabolisme non seulement de différentes classes de lipides, notamment les lysophosphatidyléthanolamines, mais aussi celui des catécholamines. / This work concerns the functional study of two genes preferentially expressed in two brain regions affected by neurodegenerative diseases: Capucine, a marker of the striatum, a structure that degenerates in Huntington's disease and Agpat4, a marker of the ventral tegmental area and the substantia nigra pars compacta, whose dopaminergic neurons are selectively affected in Parkinson's disease. Mouse lines deficient for Capucine and Agpat4 have been generated in the laboratory and during my PhD thesis I carried out their characterization.As the striatal gene expression of Capucine is significantly reduced in mouse models of Huntington's disease, we wished to evaluate its possible role in the pathogenesis of this disease. In a collaborative work, we examined the effect of the knockout and overexpression of the Capucine gene on the vulnerability of striatal neurons to a mutant Huntingtin fragment in a mouse model of Huntington’s disease. The data show that Capucine has no significant effect on the toxicity of the mutant Huntingtin fragment in the considered model.The Agpat4 protein has sequence homologies with acyltransferases involved in the metabolism of phosphoglycerides. I conducted expression studies using different molecular biology techniques, which showed that the Agpat4 gene is expressed in most catecholaminergic tissues. To determine the endogenous activity of Agpat4 and its physiological role in the tissues where it is expressed, I compared the metabolomes of Agpat4-deficient and wild-type mice tissues by liquid chromatography coupled with mass spectrometry. My results indicate that Agpat4 deficiency alters not only the metabolism of different lipid classes, in particular lysophosphatidylethanolamines, but also the metabolism of catecholamines.
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Epidemiology of metabolite profile and prostate cancer riskSchmidt, Julie Andersen January 2017 (has links)
Insulin-like growth factor-I (IGF-I) is the only known potentially modifiable risk factor for prostate cancer. Intake of dietary protein, especially from dairy products, might also be associated with risk and with circulating IGF-I, but it is not clear if amino acids play a role in these relationships. Moreover, investigations of circulating concentrations of metabolites might reveal novel risk factors for prostate cancer. This thesis investigates plasma concentrations of amino acids and other metabolites in relation to protein intake, IGF-I, and prostate cancer risk using data from the European Prospective Investigation into Cancer and Nutrition (EPIC). To characterise plasma metabolite profile in men consuming markedly different amounts and types of animal products (meat-eaters, fish-eaters, vegetarians and vegans), cross-sectional analyses of 392 men in the EPIC-Oxford sub-cohort were conducted. Of 21 amino acids, six varied significantly by diet group, and the metabolite profile of vegans was different from those of other diet groups owing to lower concentrations of several glycerophospholipids and sphingolipids. In a case-control study nested within EPIC, with a mean follow-up time of seven years, the relationship of plasma metabolites with risk of prostate cancer overall, by time to diagnosis, by tumour characteristics, and with risk of prostate cancer death, was investigated. Data from 1,077 matched sets suggested that seven metabolites, from various classes, were associated with risk of prostate cancer overall (p < 0.05). After correction for multiple testing, 12 glycerophospholipids were inversely associated with risk of advanced prostate cancer (the strongest OR<sub>1SD</sub> = 0.54; 95%CI: 0.40-0.72). In multivariate analyses, including data from 1,593 matched sets, principal component analysis (PCA) and treelet transform (TT) were used to identify patterns in metabolite profile, five of which were associated with risk of more aggressive tumour sub-types (high grade, advanced and aggressive disease) and/or prostate cancer death. There was a ≈ 50% lower risk of advanced and high grade prostate cancer in men with metabolite profiles characterised by high glycerophospholipids and sphingolipids (for advanced OR<sub>TT, top vs bottom third</sub> = 0:48; 95%CI: 0:31-0:74), with similar results for high grade and PCA). To investigate if associations between protein intake and circulating IGF-I may be mediated by plasma amino acid concentrations, cross-sectional analyses of amino acid concentrations with protein intake and IGF-I concentrations were carried out in 1,697 and 1,142 control participants, respectively, from the nested case-control study. Dairy protein intake was positively associated with concentrations of branched-chain amino acids and several other essential amino acids, while plant protein intake was strongly associated only with histidine. Serum IGF-I was positively associated with arginine and inversely with ornithine and certain amino acid ratios. In conclusion, men with different dietary habits with respect to the consumption of protein types have different amino acid and metabolite profiles, and metabolite concentrations may be associated with risk of more high-risk prostate cancer sub-types (high grade, advanced and aggressive disease) and prostate cancer death. Further large-scale studies are needed to determine if metabolites play a role in aetiology or are markers of sub-clinical prostate cancer.
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Analysis of Glycerophospholipids and Sphingolipids in Murine Brain Using Liquid Chromatography – Electrospray Ionization - Tandem Mass Spectrometry and Matrix-Assisted Laser Desorption Ionization – Imaging Mass SpectrometryNguyen, Thao January 2017 (has links)
Mass spectrometry is an indispensable tool in lipidomics research. Current advances and progress in the technology of mass spectrometry have allowed for the identification, quantification and characterization of lipid molecular species to further our understanding of their biological roles. In this thesis, I assessed the influence post-mortem times have on quantitative lipidomics. Using liquid chromatography - electrospray ionization tandem mass spectrometry (LC-ESIMS/MS) on a triple-quadrupole mass spectrometer and multiple-reaction-monitoring (MRM) mode, the glycerophosphocholine (GPC) metabolites and second messengers in the hippocampus of N3 & N4 C57BL/6 x 129/SV were profiled at various post-mortem interval (PMI). I found that disruption to the GPC metabolite and second messengers lipidome occured as early as 1 hour postmortem and fluctuate up till at least 12 hours post-mortem. Therefore, PMI is a variable in lipidomic studies that must be controlled for, and brain samples which are collected with PMI variations must be matched to avoid misinterpretation. Subsequently, I developed a working protocol to visualize the location and distribution of different classes of glycerophospholipids, ceramides, and sphingomyelin in whole mouse brain sections. This visualization technique is novel because it does not require tissue staining or immunohistochemistry; instead, it was performed using an atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) source coupled to an orbitrap mass spectrometer. As part of this lipid visualization technique, I also developed a protocol for sublimation as a simple, effective and reproducible matrix application
method for brain tissue. The lipid-compatible matrix, 2,5-dihydroxybenzoic acid (DHB), was assessed and optimized for imaging lipid targets. The high mass-resolution and accuracy characteristics of the orbitrap mass spectrometer and its capability to perform tandem mass spectrometry via high-collision dissociation allowed for the identification of approximately 200 different lipid species directly from brain tissue using the visualization technique I developed.
Altogether, the work in this thesis has showed that post-mortem changes in the lipidome are quantifiable and has provided a novel avenue to further assess these changes by means of imaging mass spectrometry.
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