3^rd International Conference on Lipid Science & Technology December 11-12, 2017 Rome, Italy

Jingwen Liu is a Principal Investigator at the Palo Alto VA Hospital where she directs a research program seeking new therapeutic options to treat cardiometabolic diseases through studies conducted in liver cells and various animal models. Among numerous accomplishments, her team has characterized a new cellular pathway that upregulates the liver LDL receptor transcription through a cholesterol-independent mechanism and identified a novel post-transcriptional mechanism of LDLR gene that mediates the cholesterol-lowering effects of herbal medicine berberine. In the last decade, PCSK9 has emerged as a new therapeutic target for treating hypercholesterolemia. Her research in characterizing the transcriptional mechanism of PCSK9 has brought new insight for developing novel therapeutic approach to inhibit PCSK9 biosynthesis with a potential to improve the efficacy of current cholesterol- lowering drug statins. Her recent study in obeticholic acid has led to new understandings of lipid regulatory effects of FXR agonists in hyperlipidemic patients.

The farnesoid X receptor (FXR) and liver X receptor (LXR) are known to regulate distinct biological pathways in BA synthesis and cholesterol metabolism. Obeticholic acid (OCA) is an FXR agonist being developed for treating various chronic liver diseases. Previous studies reported inconsistent effects of OCA on regulating plasma cholesterol levels in different animal models and in different patient populations. The mechanisms underlying its divergent effects yet have not been thoroughly investigated. The scavenger receptor class B type I (SR-BI) is an FXR modulated gene and the major receptor for HDL-C. We recently showed that OCA treatment effectively lowered plasma HDL-C levels and increased hepatic SR-BI expression in hypercholesterolemic hamsters but not in normolipidemic hamsters, suggesting that hepatic cholesterol might play a role in OCA-induced SR-BI transcription. In this current study, by conducting genomic sequence analysis, reporter assays and direct DNA binding assays, we have identified a highly conserved regulatory region in the first intron of hamster SR-BI gene that contains a functional FXRE motif and an LXRE site separated by 57 base pairs. Promoter reporter activity assays demonstrated the functional involvement of this critical regulatory region in SR-BI gene transcription upon activations of FXR and LXR in a synergistic fashion. In vivo studies of normolipidemic hamsters showed that hepatic SR-BI gene expression is not affected by separate treatment of OCA or LXR agonist GW3965 but the co-activation of FXR and LXR by the combined treatment of OCA and GW3965 results in significant increases in hepatic SR-BI mRNA and protein levels. Taken together, we have discovered an unprecedented cross-talk between FXR and LXR that leads to a synergistic activation of SR-BI gene transcription in liver tissue and consequently affected HDL-C metabolism. Our novel findings shed new light for a better understanding of lipid regulatory effects of OCA in hyperlipidemic patients.

 

Charles Chalfant received his PhD from the University of South Florida, College of Medicine and was an NRSA Postdoctoral fellow at both Duke Medical Center and The Medical University of South Carolina under Dr Yusuf Hannun. He is currently a GS15 Research Career Scientist with the Richmond Veterans Administration Medical Center. He is also a Tenured Professor and Vice Chair of the Department of Biochemistry and Molecular Biology at Virginia Commonwealth University, School of Medicine. He currently holds the Paul M Corman, MD Endowed Chair in Cancer Research for the VCU Massey Cancer Center and has published more than 100 peer-reviewed manuscripts. The Chalfant laboratory has more than 20 years of experience in Lipid Biology, Cell Signaling, and RNA Splicing.

New roles for sphingolipids such as ceramide, ceramide-1-phosphate (C1P), and sphingosine-1-phosphate continue to emerge. My research, for example, has implicated C1P as a major regulator of eicosanoid synthesis, and despite the importance of eicosanoids in the inflammatory process, the regulation of eicosanoid synthesis proximal to the activation of Group IVA phospholipase A₂ (cPLA₂α) is still an enigma. In this regard, my laboratory demonstrated that C1P is a direct and required lipid co-factor for cPLA₂α activation in cellular models. In further studies, one interaction site for C1P was localized to the calcium-lipid binding domain (C2 domain) of the enzyme allowing for the genetic ablation of the site in vivo via the generation of a cPLA₂α knock-in (KI) mouse. In this lecture, the characterization of this new mouse model in comparison to the full genetic ablation of the enzyme will be presented. Specifically, the loss of the C1P/cPLA₂α interaction induced a class-switch in the production specific eicosanoids and specialized lipid mediators driving accelerated wound repair and regeneration, both in acute and chronic murine models. Cellular studies demonstrated that loss of this lipid, protein interaction led to enhanced dermal fibroblast and neutrophil migration, which was mimicked in vivo. In further mechanistic studies, C1P was found to modulate the substrate specificity of cPLA₂α in opposition to another lipid mediator of the enzyme, PIP2, explaining the class switch as to bioactive lipid mediators observed in the cPLA₂α KI mouse. Using lipidomic analyses, these specific lipid fingerprints were linked to human wound healing outcomes, which suggests that modulation of specific lipid mediators could be explored to promote wound healing and regeneration in a number of contexts.

Figure 1.The current mechanistic hypothesis, “shifting function hypothesis, to explain the loss of pro-inflammatory lipid mediators and concomitant increase in pro-resolution lipid mediators in response to loss of the C1P/cPLA₂α interaction.

Giuseppe Maulucci attended La Sapienza University in Rome, where he obtained his Bachelor’s degree in Biophysics. He gained his PhD degree at Roma Tre University and worked as Teacher and Researcher at the Institute of Physics and at the center of light and electron microscopy (LABCEMI) of the Catholic University of the Sacred Heart (UCSC). He is an expert in microscopy techniques (University of Florence, University of Genoa, Hebrew University of Jerusalem, University of Patras). His research activity is focused on Metabolic Imaging, a discipline that unites Molecular Biology and In vivo Imaging. It enables the visualization of endogenous molecules and supramolecular properties of major importance to maintain energy homeostasis in the cells, and provides a window to several important metabolic processes essential to cell survival.

Although numerous investigators have studied lipid droplets formation through several imaging techniques, they can’t draw conclusions regarding the overall process of lipid metabolism in live cells. Here, a method for studying lipid metabolism with high spatial and temporal resolution is presented, based on the measurement of intracellular polarity through the solvatochromic and lipophilic probe Nile red, which undergoes to a red shift upon a change of polarity of the neighboring lipids. A confocal spectral imaging approach captures in detail polarity variations by acquiring the fluorescence emission spectra at pixel resolution. The analysis of the spectra trough the technique of spectral phasors allowed semi-blind spectral unmixing of the contribution of different classes of lipids in the image, namely hyper polar, polar and non-polar lipids. The method allows a fine-tuned, real-time monitoring of fatty acid metabolism in live cells with submicrometric resolution.

Figure 1: Overview of fatty acid metabolism, and relative change of polarity of the Nile Red probe.

Chryssostomos Chatgilialoglu is a Research Director at the Italian National Research Council (CNR) in Bologna, Italy. He is also a Co-founder and President of the spin-off company Lipinutragen. His research interests lie in free radical reactivity applied to biosciences and biomarker discovery. He is an author of more than 250 publications in peer-reviewed journals, 34 book chapters and six patents and author or editor of several books, including Membrane Lipidomics for Personalized Health, Wiley 2015.

Lipid research in life sciences has renewed its attractiveness in the beginning of the 21st century linking chemical, analytical, and biological subjects, with health and nutrition, as well as with the increasing societal relevance of diseases related to inflammation and lipid metabolism. Fatty acid-based membrane lipidomics examines phospholipid components related to individual metabolic and nutritional status. Indeed, fatty acid-based nutrition and nutraceuticals are essential to life and occupy a leader positions in the healthcare market. Unsaturated fatty acids are involved in oxidative and free radical processes, which also naturally occur during metabolic functions and signaling activities. The transformations due to peroxidation processes have been considered for many decades to be by far the most crucial events to natural lipids. In the last decade, the cis to trans isomerization of the double bond, due to the reversible addition of sulfur-centered radicals, has attracted considerable attention. On the top of these transformations, fatty acids are affected by stress conditions that activate the remodeling by phospholipase enzymes (PLA2 response), starting the cascade of lipid signaling, so decisive for inflammatory and apoptotic consequences. Fatty acid-based membrane lipidomics represents a powerful diagnostic tool for assessing the quantity and quality of fatty acid constituents of the lipid pool in individuals, and also for the follow-up of the membrane fatty acid remodeling, that are associated with different physiological and pathological conditions. We contributed in this area of lipidomics studying the chemical and biological responses to various metabolic and environmental conditions of free radical and metabolic stress, modeling the effects of a liposome, cellular and animal models. Our analytical methodology is able to evidence positional and geometrical fatty acid isomers. For example: sapienic acid, a member of the hexadecenoic family, has been recognized as biomarker of obesity and; the reference membrane lipidomic cluster, formed by the cohort of ten fatty acids in phospholipids of red blood cell membranes, has been clearly connected to metabolic and nutritional status in healthy and diseased subjects.

Figure 1: Fatty acid-based membrane lipidomics (membrane profile) at the crossroad of personalized health.

Calvano Cosima Damiana is currently working as Assistant Professor at University of Bari in Department of Chemistry and she received the eligibility to become Associate Professor in Analytical Chemistry. She graduated in Chemistry at the University of Bari in 2003. In 2007, she accomplished her PhD studies in Microbiology, Health and Food Chemistry. In 2012, she received the Best Young Researcher Award from the Analytical Chemistry Division of the Italian Chemical Society (SCI). Her research interests focus on the development of new analytical protocols for biomolecules study, on the use of mass spectrometry in proteomics and lipidomics fields for applications in molecular science, food, clinical and cultural heritage. Her scientific activity is documented by 75 papers divided as peerto- peer reviewing publications in international journals, book chapters and conference proceedings. She is the author/co-author of more than 100 communications to national/international conferences.

Parkinson’s disease (PD) is a progressive neurodegenerative disease involving the nigrostriatal pathway; patients’ manifest motor symptoms dysfunction when more than 50% of neurons are lost. Though it is well recognized that alterations of lipid signaling, and metabolism plays a significant role in many human diseases, little is known about the role of lipids during this specific disease. Recently, it has been reported that altered lipid pathways in the primary visual cortex and the anterior cingulate are possible in neurological disorders such as PD by analyzing post-mortem tissues from patients in advanced neuronal degeneration stage. Such an approach, however, hinders the identification of the first neuronal changes. Thus, understanding the mechanisms of PD and recognizing neuronal changes in the early phase of PD represents a crucial task. According to their polygenic predisposition and environmental etiopathology skin fibroblasts are today widely recognized as a useful model of primary human cells, capable of reflecting the chronological and biological aging of the patients. A lipidomics study of easily accessible primary human fibroblasts is presented here based on hydrophilic interaction liquid chromatography coupled to electrospray ionization-Fourier transform mass spectrometry, using both positive and negative polarities. Phospholipids (PL) from dermal fibroblasts of two unrelated PD patients with different parkin mutations and two controls were characterized by recurring to single and tandem MS measurements on a hybrid quadrupole-Orbitrap mass spectrometer. This untargeted approach enabled the identification of various PL classes as phosphatidylethanolamines (PE), phosphatidylcholines (PC), sphingomyelins, lysoPC, lysoPE, phosphatidylinositols, phosphatidylserines, mono-, di- and tri-hexosylceramides  and ganglioside GM1, GM2 and GM3. To identify the main lipids involved in the pathological condition of PD, lipidomics data on a higher number of samples need to be collected and processed by multivariate statistical analyses. In this communication, an interesting set of preliminary findings will be reported and discussed.

 

Figure 1. HILIC-ESI (+) MS total ion current chromatogram of a lipid extract from human fibroblasts.

Elena A Goun has been appointed tenure track Assistant Professor of Bio- Organic Chemistry at the School of Basic Sciences (FSB). She received her MS degree from University of Central Florida under supervision of D Howard Miles in the field of Medicinal Chemistry of Natural Products. She then continued her PhD studies in the field of Medicinal Chemistry and Drug Delivery in the group of Professor Paul Wender at Stanford University. After graduation with a PhD degree in 2008, she did her Postdoctoral studies in the field of Chemical Biology at the group of Carolyn Bertozzi University of California at Berkeley. She is an Advocate of an Interdisciplinary Approach, Combining Synthetic Chemistry, Optical Imaging, and an understanding of cellular functions at molecular level to find solutions to fundamental problems in biology and medicine. She has developed several new non-invasive imaging techniques that allow studies of molecular signatures of cancer and metabolic diseases. She will perform her research work in the context of the new Chair in Biological Chemistry, at EPFL’s Institute of Chemical Sciences and Engineering.

Triglycerides (TG) are the main form of fat in the human diet, but increased consumption of TG may lead to the development of obesity and diseases such as type 2 diabetes, cardiac lipotoxicity, hepatic steatosis and cancer. Despite the important role of TG in human health and nutrition the role of this important metabolite still remains elusive due to the lack of real-time noninvasive imaging tools. To address this unmet need we developed a novel optical probe that is based on sensitive bioluminescent readout. This reagent allows quantification and imaging of TG uptake in live cells and living mice non- invasively in real time. Using this new reagent, we have investigated the role of  gut microbiota on the rate/amount of absorption of TG in live mice.

 

Sergey Korolev has his expertise in Protein Crystallography and Biochemistry. He is an Associate Professor of Biochemistry and Molecular Biology at Saint Louis University School of Medicine. He has published structural and functional studies of medically relevant proteins including ubiquitination systems, DNA recombination and repair proteins and enzymes. His current projects in lab include structure-function studies of tumor suppressors and proteins involved in neurodegeneration.

Statement of the Problem: Calcium-independent phospholipase PLA₂G6A (also known as iPLA₂ β or PNPLA9) is a signaling enzyme which hydrolyzes phospholipids to generate potent lipid second messengers in response to stress or injury1,2. The enzyme is a product of the PARK14 gene with strong genetic link to a spectrum of neurodegenerative disorders including Parkinson’s disease (PD)3,4,5. It is also linked to idiopathic PD and represents one of the major phospholipase activities in the brain. Alterations in iPLA₂β function have demonstrated its role in other human pathologies including cardiovascular disease, cancer and diabetes. Correspondingly, novel inhibitors of PLA₂G6A have been sought for therapeutic applications. Mechanisms of its activation and tissue-specific functions remain poorly understood. This contrasts with known enzymatic activity and several well-characterized downstream signaling cascades implicated in agonist-induced arachidonic acid release, insulin secretion, vascular constriction/relaxation, store-operated calcium-entry, cellular proliferation, migration and autophagy.

Methodology & Theoretical Orientation: We have solved a crystal structure of the full-length mammalian PLA₂G6A and investigated mechanisms of the protein activity and interaction with calmodulin.

Findings: The first crystal structure of PLA₂G6A significantly revises existing mechanistic models6. It demonstrated unexpected oligomeric structure and the conformation of catalytic and auxiliary protein-interaction domains. The structure suggests the mechanisms of inhibition by calmodulin, activation through the autoacylation reaction and the potential role of ATP in stabilizing ankyrin repeats.

Conclusion & Significance: The novel crystal structure together with biochemical studies has immediate implications for the mechanisms of the phospholipase activity, of the inhibition and activation as well as of the potential mechanism of tissue specific cellular localization. It provides a well-defined framework to investigate the role of neurodegenerative mutations and the function of PLA₂G6A in the brain as well as its role in other diseases.

Figure 1: The mechanism of PLA₂G6 activation and interaction with membrane and membrane proteins.

Ida Perrotta obtained her degree in Biological Sciences and a Doctorate in Animal Biology from the University of Calabria. Currently, she is Chief Technician of Transmission Electron Microscopy Unit at the Centre for Microscopy and Microanalysis (CM2), University of Calabria. She has published more than 60 scientific papers and is actively involved in research as well as in a number of international projects in the atherosclerosis field.

Introduction: Lipid droplets (LDs) are lipid-rich organelles found nearly ubiquitously in cells where they perform diverse functions such as lipid storage for energy production and membrane synthesis, intracellular trafficking, and viral replication. Long perceived as a simple reservoir of neutral lipids, LDs have recently attracted great interest in biomedical research being recognized as dynamic structures with a complex and interesting biology. In many non-adipocyte cells, LDs have been shown to be highly motile and have been often found to interact with the endoplasmic reticulum (ER), mitochondria, endosomes, and peroxisomes. To date, however, much remains unknown regarding the topographical connections between LDs and other cell organelles in the cardiovascular system, especially in atherosclerosis. In order to fill this gap, an electron microscopic analysis has been conducted on human atherosclerotic plaques and the results are presented herein.

Materials & Methods: Specimens of atherosclerotic human aorta were obtained from patients undergoing surgical repair of the aortic atherosclerotic aneurysm. Tissues were fixed in glutaraldehyde, post-fixed with osmium tetroxide, dehydrated through a graded acetone series, and embedded in araldite. Ultra-thin sections were examined with a JEOL JEM-1400 PLUS transmission electron microscope operating at 80 kV.

Findings: LDs have been found to be closely associated with the convoluted rough-surfaced ER membranes and even with the lumen of these structures probably to facilitate a non-vesicular lipid transport between the ER and the droplets. TEM examination also revealed the presence of membrane-like structures in the LD core.

Conclusion & Significance: The selective physical interaction between LDs and ER membranes in the foam cells of human atherosclerotic plaques may function as a lipid-buffer system that allows the sequestration of free fatty acid, therefore, reducing the risk of lipotoxicity. The ultrastructural anatomy of these organelles might also account for some of their specific functional properties such as the synthesis of inflammatory lipid mediators by specific membrane-spanning enzymes which might reside in their internal membranous structures.

Figure 1: Viable foam cells contain large Lipid Droplets (LDs), intact mitochondria, well-developed rER, and numerous electrondense ribosomes. LDs frequently interact with the ER (arrow) throught an extended membrane stalk.

Guido Galliani has graduated in Chemistry at Milano University in 1974 where he has spent ten years as a Research fellow and an Assistant Professor. His main research interests were physical organic chemistry, oxidation mechanism, and model reactions for oxygen reactive intermediates. He served as a R&D Director and then as a factory manager in companies producing Active Principle Ingredients (Labochim, Honeywell). Partner for ten years of a company (Chorisis) eventually sold to Euticals group, he developed API impurities characterization, design of experiments, and continuous process development. In the same years, he held an assignment as adjunct Professor at Bicocca Milano University. He has spent more than six years in Norwegian factories (Lipro, PharmaMarine), introducing new techniques and processes for omega-3 products, including technology for the industrial production of squid oil as a DHA source. Since January 2017 he is acting as a Consultant for Gleaner srls.

Until not so long ago, wax esters had an overall bad reputation. Spermaceti, jojoba oil, orange roughy oil are typical examples of wax esters, well renown insofar that they are used topically, mostly for cosmetic purposes. However, it is equally well known that these products should never be ingested, under penalty of very unpleasant consequences, such as nausea, diarrhoea, and steatorrhea. Some years ago, we started a program to understand some rather contradictory data. It is known that roe (fish eggs) is consumed as a food since time immemorial. This occurs either on roe as is (caviar, lumpfish roe) or after some treatment (Italian bottarga, Greek taramosalata, Japanese karasumi, Scandinavian Lysekils kaviar). Moreover, it was known that wax esters are an important constituent of roe, in some instances the majority component. In spite of this, no concern had ever been raised as to edibility or side effects of such products. The first results in analyzing wax esters from several roe-based foods showed that polyunsaturated fatty acids were a very important component of the acidic moiety. Successive results from other research groups confirmed our original results. These findings partially supported substantiating and launching a commercial product, Calanus® oil, extracted from copepod Calanus finmarchicus, rich in wax esters and in omega-3 polyunsaturated acids. From all this, it is clear that the term wax ester is just a rough chemical definition. Within this class of lipids different molecules are included. Some of them are essentially saturated molecules, primarily intended to assist buoyancy and to store energy. Other molecules represent a more complex metabolism, not yet fully understood. Differences are reflected in the metabolic fate when ingested. Competitive kinetics results will be presented from several wax esters and lipases, revealing suggestions about wax esters as a delivering form for omega- 3.

Jorge Maldonado-Hernández has his expertise in the development of non-invasive methods and biological markers for the diagnosis of metabolic disorders as hyperhomocysteinemia, insulin resistance and non-alcoholic fatty liver disease. He has extensive experience in the management of analytical techniques such as isotope ratio mass spectrometry, gas chromatography and liquid-mass chromatography. Recently, he has been interested in the effect of dietary lipids on the biosynthesis of ceramides in human muscle tissue.

Objective: To assess the association between plasma ceramides and hepatic steatosis (HS) in adolescents, independently of obesity.

Material & Methods: Ninety-four adolescents from two previous studies conducted and published by our crew were included. Study subjects were stratified in three groups: normal weight (n=18), obesity (n=34) and obesity + HS (n=42). The presence of HS was defined when AST/ALT ratio was <1. Ceramides subspecies (C14:0, C16:0, C18:0, C24:0 and C24:1) were determined by LC/MS.

Results: All ceramides correlated inversely with the AST/ALT ratio and directly with ALT levels in plasma; the strongest correlation was observed among C14:0 ceramide (r=-0.54 and r=0.41, respectively; P<0.001). Furthermore, significant correlations were observed between cholesterol and all ceramides except for C24:1 ceramide. Fasting insulin and HOMAIR correlated directly with ceramides C14:0, C18:0 and C24:1. For assessing HS, a cut-off points of 10.3 nmol/L for C14:0 Ceramide reported a sensitivity of 92.7% and a specificity of 73.5% when normal weight and obesity groups (n=52) were compared against obesity + HS group (n=42). Positive and negative predictive values were 77.5% and 90.2%, respectively.

Conclusions: plasma ceramides are closely associated with hepatic steatosis in adolescents. C14:0 ceramide could be a novel biomarker of HS independently of obesity.

Figure 1. Obesity and insulin resistance increase the production of pro-inflammatory cytokines and free fatty acids delivery to the liver that promotes ceramides production. In the hepatocyte ceramides increase apoptosis through ROS mechanism. The increase of ceramides in the liver facilitate its release into the circulation, which in turn affects insulin sensitivity in muscle tissue. IR= insulin resistance, AT= adipose tissue, FFA= free fatty acids, ROS= reactive oxygen species. 

Irina A Pikuleva studies cholesterol metabolism and cytochrome P450 enzymes whose role is to generate more polar forms of cholesterol for cellular elimination or regulation. Her laboratory focuses on the role of cholesterol-related proteins in retinal structure and function; and potential of CYP46A1 as a pharmacologic target for Alzheimer's disease. The ultimate goal of these studies is to identify new strategies for treatment of diseases of the eye (age-related macular degeneration and diabetic retinopathy) and the brain (Alzheimer’s disease). In search for these new strategies, the Pikuleva laboratory evaluates existing medications with a goal of drug re-purposing.

Statement of the Problem: Alzheimer’s disease (AD) is a progressive brain disorder and the most common cause of dementia among older adults. Currently, there is no cure for AD with all anti-AD treatments being only symptomatic. Cytochrome P450 46A1 (CYP46A1) converts cholesterol to 24-hydroxycholesterol and thereby initiates the major pathway for cholesterol elimination from the brain. CYP46A1 also controls the rate of cholesterol biosynthesis in the brain and the rate of cerebral cholesterol turnover. Studies of mouse models of AD showed that increasing CYP46A1 expression in the brain by genetic means ameliorates the manifestations of AD such as amyloid burden and cognitive deficits. We were able to enhance CYP46A1 activity pharmacologically in vitro and in mice by discovering that CYP46A1 has an allosteric site, binding to which of some non-cholesterol related drugs (e.g., the anti-HIV medication efavirenz) increases the rate of CYP46A1-mediated cholesterol 24-hydroxylation. The purpose of this study was to evaluate the effect of efavirenz, activating CYP46A1 at a very small dose, on 5XFAD mice, a model of rapid amyloidogenesis.

Methodology & Theoretical Orientation: Mice were treated with efavirenz for four and nine months and assessed for cholesterol homeostasis in the brain, amyloid pathology, cognition, and brain genome.

Findings: Efavirenz treatment: stably activated CYP46A1 and enhanced cholesterol turnover in the 5XFAD brain; reduced amyloid-β burden and microglia activation in the brain cortex and hippocampus; and affected mouse behavior in a taskand treatment time-specific manner with ultimately improving mouse performance in Morris water maze. The data obtained enabled generation of a mechanistic model, which unified EFV effects.

Conclusion & Significance: CYP46A1-mediated enhancement of cholesterol metabolism and turnover in the brain has a potential as a therapeutic strategy for AD and should be evaluated in a clinical trial.

Figure 1: Cholesterol metabolism in the brain.

Suzanne A Al-Bustan has completed her PhD in Human Genetics from the Duncan Guthrie Institute in Medical Genetics at Glasgow University in 1992. She is an Associate Professor of Human and Molecular Genetics in the Department of Biological Sciences and the Director of the joint MSc degree in Molecular Biology at Kuwait University. She has published numerous papers in reputed journals and has been active in both scientific research and supervision of several graduate students in the areas of genetics and molecular biology. Her main line of research expertise and emphasis is on genetic association of candidate genes in the lipid metabolism and transport with dyslipidemia in the Kuwaiti population.

Plasma lipids have been well documented to be influenced by lifestyle factors, such as diet, obesity, and physical activity, as well as genetic factors. Persistent fluctuation in levels of cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL), as well as decreased high-density-lipoprotein (HDL), often lead to clinical dyslipidemia which may consequently manifest into diabetes mellitus and/or coronary heart disease. Genetic association studies including genome wide association studies (GWAS) have attempted to elucidate the genetic and molecular mechanisms of dyslipidemia and have identified a limited number of candidate genes and pathways relevant to lipid metabolism and/or transport. However, conflicting results across different populations and ethnic groups have been reported. Variation in DNA sequences and candidate genes for blood lipid levels, therefore, remain unresolved. To demonstrate the importance of identifying cofounding gene variants in candidate gene loci relevant to specific ethnic groups and their role in dyslipidemia results from different studies conducted on the Kuwaiti population will be presented. Genetic association of the APOA promoter sequence, APOB and APOE common variants, and other related gene loci have been studied and positive association results from these will be presented. These include the association of the I -75G>A with increased levels of LDL-C and TC, APOB signal peptide and 3611 MspI polymorphisms with variation in TG levels and the APOE2 with LDL-C levels in the Kuwaiti population sampled. The nutrition of Kuwaitis is relatively high in fat and the lifestyle adapted by many in the Kuwaiti population often lead to an increase in many of the risk factors leading to heart disease especially dyslipidemia making this population ideal for genetic association studies. Important implications and conclusions drawn from these studies will be discussed.

Kawthar Alghamdi is pursuing her final year PhD in the School of Biomolecular and Biomedical Science, University College Dublin, Ireland under the supervision of Associate Professor Orina Belton. She received her MSc in Biotechnology from University College Dublin in 2013. Her previous work in the atherosclerosis research group at University College Dublin has shown that dietary administration of conjugated linoleic acid induces the regression of pre-established atherosclerosis via altering monocyte function. Her recent research has focused on the identification of distinct pathways regulated by conjugated linoleic acid that may yield further information as to how atherosclerosis can be reversed. To do this she has employed a comprehensive proteomic approach, to construct networks and identify key signaling pathways associated with altered monocyte/macrophage fate and function. Through this work she has identified and validated that TGFβ signaling mediates the atheroprotective effect of CLA.

The chronic recruitment of monocytes and their subsequent migration through the endothelium contribute to atherosclerotic plaque development, the underlying cause of heart attacks and stroke. A specific blend of conjugated linoleic acid (80:20 cis-9, trans-11: trans-10, cis-12-CLA) has the unique property of inducing regression of pre-established atherosclerosis in vivo via modulation of monocyte function. Currently, there are no therapeutic targets which induce regression of pre-established atherosclerosis. Therefore, understanding the mechanisms through which CLA 80:20 mediates its atheroprotective effect is important for more effective management of this disease. This study aimed to identify novel pathways regulated by CLA, which inhibits monocyte function using a proteomic approach. THP-1 monocytes were treated for 18 h with CLA blend, a lipid control Oleic acid (OA) or DMSO (n=3 per treatment group). Proteins were trypsin-digested prior to analysis by liquid chromatography coupled to high resolution, high mass accuracy Orbitrap mass spectrometry. Global proteomic protein identities and relative quantitation were determined in a label-free approach, using the MaxQuant, Perseus and IPA suite of programs. A total of more than 1500 proteins were identified by mass spectrometry across the experimental groups using Perseus. Following statistical analysis using the t-test, 121 proteins were found to be significantly altered following treatment with CLA 80:20 compared to the control (DMSO). 103 proteins were unique to CLA blend and not altered by OA. Subsequent bioinformatics analysis of the regulated proteins showed enrichment of the TGFβ signalling pathway. Validation of proteomic analysis was performed by Western blot analysis of THP-1 monocytes. Our data revealed that CLA 80:20 blend regulates the TGFβ signaling pathway in monocytes. This work contributes to our understanding of the atheroprotective pathways regulated by CLA 80:20 which impacts on monocyte/macrophage fate.

Figure 1: Global Proteomic Analysis: Significant proteins which were altered by CLA blend compared to control were determined by Student’s t-test, where considered significant a p-value < 0.05 (A) Hierarchical clustering global proteomic analysis exhibited only the 121 proteins which were statistically different with CLA relative to the control DMSO, 63 proteins down- regulated (green) and 85 proteins were upregulated(red), it was determined by Student’s t-test, where p< 0.05 flowing with z-score normalization of the median value of logarithmized intensities (Log 2) of each protein profile. Statistical analysis was performed in Perseus software (version 1.5.0.15). (B) Expression profile of the two hierarchical clusters, which were statistically different compare to the control group DMSO. (C) Common and unique number of proteins regulated by CLA blend and OA which were significantly changed compared to the control DMSO for both groups.