Thesis (Ph. D.)--University of Wisconsin--Madison, 1943. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 78-79).

This comprehensive review critically evaluates whether supposed health benefits propounded upon human consumption of conjugated linoleic acids (CLAs) are clinically proven or not. With a general introduction on the chemistry of CLA, major clinical evidences pertaining to intervention strategies, body composition, cardio-vascular health, immunity, asthma, cancer and diabetes are evaluated. Supposed adverse effects such as oxidative stress, insulin resistance, irritation of intestinal tract and milk fat depression are also examined. It seems that no consistent result was observed even in similar studies conducted at different laboratories, this may be due to variations in age, gender, racial and geographical disparities, coupled with type and dose of CLA supplemented. Thus, supposed promising results reported in mechanistic and pre-clinical studies cannot be extrapolated with humans, mainly due to the lack of inconsistency in analyses, prolonged intervention studies, follow-up studies and international co-ordination of concerted studies. Briefly, clinical evidences accumulated thus far show that CLA is not eliciting significantly promising and consistent health effects so as to uphold it as neither a functional nor a medical food.

Lui Oi Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves [216]-240). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABBREVIATIONS --- p.ii / ABSTRACT --- p.vii / 撮要 --- p.x / PUBLICATIONS --- p.xiii / TABLE OF CONTENTS --- p.xiv / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis and Leukemia --- p.1 / Chapter 1.1.1 --- An Overview on Hematopoietic Development --- p.1 / Chapter 1.1.2 --- Leukemia --- p.8 / Chapter 1.1.2.1 --- General Diagnostic Tests for Leukemia --- p.9 / Chapter 1.1.2.2 --- Classification and Epidemiology of Leukemia --- p.10 / Chapter 1.1.2.3 --- Conventional Approaches to Leukemia Therapy --- p.17 / Chapter 1.1.2.4 --- Novel Approaches to Leukemia Therapy --- p.20 / Chapter 1.2 --- Conjugated Linoleic Acid --- p.23 / Chapter 1.2.1 --- Introduction: Historical Development and Occurrence of Conjugated Linoleic Acid --- p.23 / Chapter 1.2.2 --- Phytochemistry and Metabolism of Conjugated Linoleic Acid --- p.24 / Chapter 1.2.2.1 --- Chemical Structures of Conjugated Linoleic Acid Isomers --- p.24 / Chapter 1.2.2.2 --- Biosynthesis of Conjugated Linoleic Acid --- p.26 / Chapter 1.2.2.3 --- Metabolism of Conjugated Linoleic Acid --- p.30 / Chapter 1.2.2.4 --- Mode of Entry and Tissue Incorporation of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.2.5 --- Toxicology of Conjugated Linoleic Acid --- p.33 / Chapter 1.2.3 --- Physiological Activities of Conjugated Linoleic Acid: Reported Health Benefits --- p.35 / Chapter 1.2.3.1 --- Anti-adipogenesis / Chapter 1.2.3.2 --- Anti-diabetogenesis --- p.36 / Chapter 1.2.3.3 --- Anti-atherosclerosis --- p.38 / Chapter 1.2.3.4 --- Anti-carcinogenesis --- p.39 / Chapter 1.2.3.5 --- Anti-tumor Activity --- p.40 / Chapter 1.2.3.6 --- Effects of Conjugated Linoleic Acid on Lipid Metabolism --- p.44 / Chapter 1.2.3.6.1 --- Actions on Phospholipids by Conjugated Linoleic Acid --- p.45 / Chapter 1.2.3.6.2 --- Conjugated Linoleic Acid as a Ligand for the PPAR System --- p.47 / Chapter 1.2.3.7 --- Immunomodulation --- p.47 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.50 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Animals --- p.52 / Chapter 2.1.2 --- Cell Lines --- p.52 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.52 / Chapter 2.1.4 --- Reagents for 3H-Thymidine Incorporation Assay --- p.54 / Chapter 2.1.5 --- Reagents and Buffers for Flow Cytometry --- p.58 / Chapter 2.1.6 --- Reagents for DNA Extraction --- p.59 / Chapter 2.1.7 --- Cell Death Detection ELISAPLUS Kit --- p.63 / Chapter 2.1.8 --- Reagents for Measuring Caspase Activity --- p.65 / Chapter 2.1.9 --- Reagents for Total RNA Isolation --- p.66 / Chapter 2.1.10 --- Reagents and Buffers for RT-PCR --- p.69 / Chapter 2.1.11 --- Reagents and Buffers for Gel Electrophoresis of Nucleic Acids --- p.74 / Chapter 2.1.12 --- "Reagents, Buffers and Materials for Western Blot Analysis" --- p.75 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Culture of the Tumor Cell Lines --- p.80 / Chapter 2.2.2 --- "Isolation, Preparation and Culture of Mouse Peritoneal Macrophages" --- p.81 / Chapter 2.2.3 --- Determination of Cell Viability --- p.82 / Chapter 2.2.4 --- Determination of Cell Proliferation by [3H]-TdR Incorporation Assay --- p.83 / Chapter 2.2.5 --- In Vivo Tumorigenicity Study --- p.83 / Chapter 2.2.6 --- Analysis of Cell Cycle Profile / DNA Content by Flow Cytometry --- p.83 / Chapter 2.2.7 --- Measurement of Apoptosis --- p.84 / Chapter 2.2.8 --- Determination of the Mitochondrial Membrane Potential --- p.86 / Chapter 2.2.9 --- Measurement of Caspase Activity --- p.87 / Chapter 2.2.10 --- Study of Intracellular Accumulation of Reactive Oxygen Species (ROS) --- p.88 / Chapter 2.2.11 --- Study of the Scavenging Activity of Antioxidants --- p.88 / Chapter 2.2.12 --- Gene Expression Study --- p.89 / Chapter 2.2.13 --- Protein Expression Study --- p.92 / Chapter 2.2.14 --- Measurement of Cell Differentiation --- p.95 / Chapter 2.2.15 --- Statistical Analysis --- p.98 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI-TUMOR ACTICITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction / Chapter 3.2 --- Results --- p.99 / Chapter 3.2.1 --- Anti-proliferative Activity of CLA-mix and CLA Isomers on Various Myeloid Leukemia Cell Lines In Vitro --- p.101 / Chapter 3.2.2 --- Cytotoxic Effect of CLA-mix on the WEHI-3B JCS Cells In Vitro --- p.109 / Chapter 3.2.3 --- Cytotoxic Effect of CLA-mix on Primary Murine Myeloid Cells In Vitro --- p.111 / Chapter 3.2.4 --- Kinetic and Reversibility Studies of the Anti-proliferative Activity of CLA-mix on the WEHI-3B JCS Cells --- p.113 / Chapter 3.2.5 --- Effect of CLA-mix and its isomers on the Cell Cycle Profiles of the WEHI-3B JCS Cells In Vitro --- p.116 / Chapter 3.2.6 --- Effect of CLA-mix and its isomer on the Expression of Cell Cycle-regulatory Genes in the WEHI-3B JCS Cells --- p.123 / Chapter 3.2.7 --- Effect of CLA-mix and its isomer on the In V Tumorigenicity of the WEHI-3B JCS Cells ivo --- p.128 / Chapter 3.3 --- Discussion --- p.131 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 4.1 --- Introduction --- p.141 / Chapter 4.2 --- Results --- p.141 / Chapter 4.2.1 --- Induction of Apoptosis in Both Murine and Human Myeloid Leukemia Cells by CLA --- p.144 / Chapter 4.2.2 --- Effect of CLA and its Isomer on the Mitochondrial Membrane Potential of the WEHI-3B JCS Cells --- p.151 / Chapter 4.2.3 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Genes of the Bcl-2 Family in the WEHI-3B JCS Cells --- p.154 / Chapter 4.2.4 --- Effect of CLA-mix and its Isomer on the Expression of Apoptosis-regulatory Proteins in the WEHI-3B JCS Cells --- p.158 / Chapter 4.2.5 --- Effect of CLA-mix and its Isomer on the Induction of Caspase Activity in the WEHI-3B JCS Cells --- p.161 / Chapter 4.2.6 --- Effect of CLA-mix and its Isomer on the Induction of ROS in the WEHI-3B JCS Cells --- p.170 / Chapter 4.2.7 --- Effect of Antioxidants on the Induction of ROS by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.173 / Chapter 4.2.8 --- Effect of Antioxidants on the Induction of Apoptosis by CLA-mix and its Isomer in the WEHI-3B JCS Cells --- p.176 / Chapter 4.2 --- Discussion / Chapter CHAPTER 5: --- STUDIES ON THE DIFFERENTIATION-INDUCING ACTIVITY OF CONJUGATED LINOLEIC ACID ON MYELOID LEUKEMIA CELLS / Chapter 5.1 --- Introduction --- p.187 / Chapter 5.2 --- Results --- p.190 / Chapter 5.2.1 --- Morphological Alterations in CLA-mix- and CLA isomer-treated WEHI-3B JCS Cells --- p.190 / Chapter 5.2.2 --- Effects of CLA-mix on the Cell Size and Granularity of WEHI-3B JCS Cells --- p.196 / Chapter 5.2.3 --- Studies of the Surface Phenotypic Changes in the CLA-mix-treated WEHI-3B JCS cells --- p.198 / Chapter 5.2.4 --- Studies on the Induction of Monocytic Serine Esterase (MSE) Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.200 / Chapter 5.2.5 --- Studies on the Induction of Endocytic Activity in the CLA-mix-treated WEHI-3B JCS Cells --- p.201 / Chapter 5.2.6 --- Studies on the Expression of the Differentiation-regulatory Cytokine Genes in the CLA-mix-treated WEHI-3B JCS Cells --- p.202 / Chapter 5.3 --- Discussion --- p.204 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES REFERENCES --- p.208 / REFERENCES --- p.217

Ho, Lai Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 213-238). / Abstract also in Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iv / Acknowledgements --- p.vii / List of abbreviations --- p.ix / Table of contents --- p.xiv / Chapter Chapter One: --- General Introduction_ --- p.1 / Chapter 1.1 --- Neuroblastoma --- p.2 / Chapter 1.1.1 --- An overview on neuroblastoma --- p.2 / Chapter 1.1.2 --- Classification of neuroblastoma --- p.4 / Chapter 1.1.3 --- Epidemiology of neuroblastoma --- p.7 / Chapter 1.1.4 --- Clinical manifestation of neuroblastoma --- p.10 / Chapter 1.1.5 --- Diagnosis of neuroblastoma --- p.10 / Chapter 1.1.6 --- Conventional therapy of neuroblastoma --- p.12 / Chapter 1.1.7 --- Novel treatments of neuroblastoma --- p.14 / Chapter 1.2 --- Conjugated linolenic acid (CLN) --- p.16 / Chapter 1.2.1 --- An overview of polyunsaturated fatty acids and conjugated fatty acids --- p.16 / Chapter 1.2.2 --- Chemical structure and physical properties of CLNs --- p.18 / Chapter 1.2.3 --- Natural occurrence of CLNs --- p.21 / Chapter 1.2.4 --- Synthesis of CLNs --- p.22 / Chapter 1.2.5 --- Metabolism and pharmacokinetics of CLNs --- p.24 / Chapter 1.2.6 --- Major biological and pharmacological activities of CLNs --- p.25 / Chapter 1.2.6.1 --- Hypolipidemic and anti-obese effects --- p.25 / Chapter 1.2.6.2 --- Anti-cancer effects --- p.27 / Chapter 1.2.6.2.1 --- Anti-proliferation --- p.27 / Chapter 1.2.6.2.2 --- Chemoprevention --- p.28 / Chapter 1.2.6.2.3 --- Apoptosis-inducing --- p.28 / Chapter 1.3 --- Aims and scope of the study --- p.31 / Chapter Chapter Two: --- Materials and Methods_ --- p.34 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Animals --- p.35 / Chapter 2.1.2 --- Cell lines --- p.35 / Chapter 2.1.3 --- "Cell culture medium, buffers and other reagents" --- p.37 / Chapter 2.1.4 --- Reagents for DNA extraction --- p.46 / Chapter 2.1.5 --- Reagents for gel electrophoresis of nucleic acids --- p.47 / Chapter 2.1.6 --- Reagents and buffers for flow cytometry --- p.49 / Chapter 2.1.7 --- Reagents and buffers for measuring caspase activity --- p.50 / Chapter 2.1.8 --- Reagents for Hoechst staining --- p.53 / Chapter 2.1.9 --- Reagents and buffers for RNA extraction --- p.53 / Chapter 2.1.10 --- Reagents and buffers for DNA digestion --- p.54 / Chapter 2.1.11 --- Reagents and buffers for reverse transcription --- p.55 / Chapter 2.1.12 --- Reagents for real-time polymerase chain reaction --- p.57 / Chapter 2.1.13 --- Reagents and buffers for Western blotting --- p.59 / Chapter 2.2 --- Methods --- p.64 / Chapter 2.2.1 --- Culture of cell lines --- p.64 / Chapter 2.2.2 --- Preparation of NIH-3T3 conditioned medium --- p.65 / Chapter 2.2.3 --- Determination of cell viability --- p.65 / Chapter 2.2.4 --- Determination of cell proliferation by tritiated thymidine incorporation assay --- p.66 / Chapter 2.2.5 --- Cytotoxicity test of CLNs on murine peritoneal macrophages --- p.67 / Chapter 2.2.6 --- Cytotoxicity test of CLNs on murine bone marrow cells --- p.68 / Chapter 2.2.7 --- Cytotoxicity test of CLNs on murine splenocytes --- p.68 / Chapter 2.2.8 --- Cytotoxicity tests of CLNs on human peripheral blood mononuclear cells --- p.69 / Chapter 2.2.9 --- Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining analyzed by flow cytometry --- p.69 / Chapter 2.2.10 --- Determination of colony forming ability --- p.70 / Chapter 2.2.11 --- Determination of cell invasiveness --- p.70 / Chapter 2.2.12 --- In vivo tumorigenicity assay --- p.71 / Chapter 2.2.13 --- Analysis of cell cycle profile/ DNA content by flow cytometry --- p.72 / Chapter 2.1.14 --- Measurements of apoptosis --- p.72 / Chapter 2.1.15 --- Measurements of differentiation --- p.77 / Chapter 2.1.16 --- Gene expression study --- p.78 / Chapter 2.2.17 --- Protein expression study --- p.81 / Chapter 2.2.18 --- Statistical Analysis --- p.84 / Chapter Chapter Three: --- Anti-proliferative Effect of CLN Isomers on Human Neuroblastoma cells --- p.86 / Chapter 3.1 --- Introduction --- p.87 / Chapter 3.2 --- Results --- p.89 / Chapter 3.2.1 --- Anti-proliferative effect of CLN isomers on various human neuroblastoma cell lines in vitro --- p.89 / Chapter 3.2.2 --- Direct cytotoxic effect of jacaric acid on neuroblastoma LA-N-1 cells in vitro --- p.100 / Chapter 3.2.3 --- Cytotoxicity of jacaric acid on primary murine cells and human normal cell lines --- p.103 / Chapter 3.2.4 --- Kinetic and reversibility studies of the anti-proliferative effect of jacaric acid on LA-N-1 cells --- p.106 / Chapter 3.2.5 --- Synergistic anti-proliferative effect of jacaric acid with daidzein and retinoic acid on LA-N-1 cells in vitro --- p.110 / Chapter 3.2.6 --- Modulatory effect of jacaric acid on the number of cell division in LA-N-1 cells --- p.113 / Chapter 3.2.7 --- Effect of jacaric acid on the cell cycle profile of LA-N-1 cells --- p.115 / Chapter 3.2.8 --- Effect of jacaric acid on the invasiveness of LA-N-1 cells --- p.118 / Chapter 3.2.9 --- Effect of jacaric acid on the colony forming ability of LA-N-1 cells in soft agar --- p.120 / Chapter 3.2.10 --- Effect of jacaric acid on the in vivo tumorigenicity of the LA-N-1 cells --- p.122 / Chapter 3.3 --- Discussion --- p.124 / Chapter Chapter Four: --- Apoptosis- and Differentiation-inducing Effects of Jacaric Acid on Human Neuroblastoma Cells --- p.133 / Chapter 4.1 --- Introduction --- p.134 / Chapter 4.2 --- Results --- p.138 / Chapter 4.2.1 --- Induction of DNA fragmentation and apoptotic ultrastructural changes in LA-N-1 cells by jacaric acid --- p.138 / Chapter 4.2.2 --- Induction of phosphatidylserine externalization by jacaric acid in human neuroblastoma cells as detected by Annexin V-GFP/ PI dual staining --- p.142 / Chapter 4.2.3 --- Effect of jacaric acid on the mitochondrial membrane potential in human neuroblastoma cells --- p.146 / Chapter 4.2.4 --- Effect of jacaric acid on the caspase-3 activity in LA-N-1 cells --- p.150 / Chapter 4.2.5 --- Effect of jacaric acid on the reactive oxygen species (ROS) generation in human neuroblastoma cells --- p.153 / Chapter 4.2.6 --- Morphological changes induced by jacaric acid in SH-SY5Y cells --- p.158 / Chapter 4.3 --- Discussion --- p.162 / Chapter Chapter Five: --- Mechanistic Studies of Anti-proliferative Effect of Jacaric Acid on Human Neuroblastoma Cells --- p.171 / Chapter 5.1 --- Introduction --- p.172 / Chapter 5.2 --- Results --- p.178 / Chapter 5.2.1 --- Effect of antioxidant a-tocopherol on the anti-proliferative effect of jacaric acid on LA-N-1 cells --- p.178 / Chapter 5.2.2 --- Effect of caspase inhibitors on the anti-proliferative effect of jacaric acid on LA-N-1 cells --- p.180 / Chapter 5.2.3 --- Jacaric acid modulated the mRNA expression of N-myc and other related transcription factors in LA-N-1 cells --- p.182 / Chapter 5.2.4 --- Jacaric acid modulated the protein expression of N-myc --- p.186 / Chapter 5.2.5 --- Jacaric acid modulated the mRNA expression of apoptosis-associated genes --- p.188 / Chapter 5.3 --- Discussion --- p.191 / Chapter Chapter Six: --- Conclusions and Future Perspectives --- p.202 / References --- p.212

Yip, Wai Ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 203-228). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.i / ABBREVIATIONS --- p.iii / ABSTRACT --- p.x / 撮要 --- p.xiv / TABLE OF CONTENTS --- p.xvii / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Hematopoiesis and Leukemia / Chapter 1.1.1 --- An Overview on Hematopoiesis Development --- p.1 / Chapter 1.1.1.1 --- Hematopoietic Growth Factors --- p.4 / Chapter 1.1.1.2 --- Site Switching of Hematopoiesis --- p.5 / Chapter 1.1.2 --- An Overview on Leukemia --- p.7 / Chapter 1.1.2.1 --- Classification of Leukemia --- p.7 / Chapter 1.1.2.2 --- Conventional Therapy of Leukemia --- p.10 / Chapter 1.1.2.3 --- Novel Approaches to Leukemia Therapy: Apoptosis and Differentiation Induction --- p.13 / Chapter 1.2 --- Polysaturated Fatty Acids / Chapter 1.2.1 --- An Overview on Polyunsaturated Fatty Acids --- p.16 / Chapter 1.2.2 --- An Overview on Essential Fatty Acids --- p.17 / Chapter 1.2.2.1 --- Alpha Linolenic Acids (ALA) --- p.17 / Chapter 1.2.2.2 --- Gamma Linolenic Acid (GLA) --- p.18 / Chapter 1.2.3 --- "An Overview on Conjugated Fatty Acids: Conjugated Linoleic Acid (CLA), Conjugated EPA and Conjugated DHA" --- p.20 / Chapter 1.2.4 --- Conjugated Linolenic Acid (CLN) --- p.24 / Chapter 1.2.4.1 --- Identification and Production of CLN --- p.28 / Chapter 1.2.4.2. --- Metabolism of CLN --- p.29 / Chapter 1.2.4.3 --- Anti-Obese and Hypolipidemic Effect of CLN --- p.30 / Chapter 1.2.4.4 --- Anti-Proliferative Effect of CLN --- p.30 / Chapter 1.2.4.5 --- Other Novel Effects of CLN --- p.32 / Chapter 1.3 --- Aims and Scopes of This Investigation --- p.34 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.36 / Chapter 2.1.1 --- Animals --- p.36 / Chapter 2.1.2 --- Human Cell Lines --- p.36 / Chapter 2.1.3 --- "Cell Culture Medium, Buffers and Other Reagents" --- p.38 / Chapter 2.1.4 --- Reagents and Buffer for Flow Cytometry --- p.44 / Chapter 2.1.5 --- Reagents for DNA Extraction --- p.47 / Chapter 2.1.6 --- Cell Death Detection ELISApLus --- p.48 / Chapter 2.1.7 --- Reagents for Measuring Caspase Activity --- p.50 / Chapter 2.1.8 --- Reagents for FACE´ёØ ELISA Kit --- p.53 / Chapter 2.1.9 --- Reagents for Western Blotting --- p.55 / Chapter 2.2 --- Methods --- p.65 / Chapter 2.2.1 --- Culturing the Tumor Cell Lines --- p.65 / Chapter 2.2.2 --- "Isolation, Preparation and Culturing of Murine Peritoneal Macrophages and Bone Marrow Cells" --- p.66 / Chapter 2.2.3 --- Anti-proliferation Assays --- p.67 / Chapter 2.2.4 --- Cell Viability Determination --- p.68 / Chapter 2.2.5 --- Determination of Anti-leukemia Activity In Vivo (In Vivo Tumorigenicity Assay) --- p.69 / Chapter 2.2.6 --- Cell Cycle Analysis by Flow Cytometry --- p.69 / Chapter 2.2.7 --- Detection of Apoptosis --- p.70 / Chapter 2.2.8 --- Assessment of Differentiation-associated Characteristics --- p.74 / Chapter 2.2.9 --- Measurement of Caspase Activities --- p.76 / Chapter 2.2.10 --- Protein Expression Study --- p.78 / Chapter 2.2.11 --- Detection of Phosphorylation of JNK by FACE´ёØ JNK ELISA Kit --- p.83 / Chapter 2.2.12 --- Detection of Phosphorylation of NF-kB by FACE´ёØ NF-kB p65 Profiler --- p.85 / Chapter 2.2.13 --- Statistical Analysis --- p.85 / Chapter CHAPTER 3: --- STUDIES ON THE ANTI PROLIFERATIVE EFFECTS OF CONJUGATED LINOLENIC ACIDS ON THE HUMAN MYELOID LEUKEMIA CELLS / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Results / Chapter 3.2.1 --- Anti-proliferative Activity of CLN Isomers on Various Myeloid Leukemia and Lymphoma Cell Lines In Vitro --- p.88 / Chapter 3.2.2 --- Direct Cytotoxic Effect of Jacaric Acid on HL-60 Cells In Vitro --- p.95 / Chapter 3.2.3 --- Cytotoxic Effect of Jacaric Acid on Primary Murine Cells and Human Normal Cell Lines In Vitro --- p.98 / Chapter 3.2.4 --- Kinetics and Reversibility Studies of the Anti-proliferative Effect of Four CLN Isomers on the Human Promyelocytic Leukemia HL-60 Cells --- p.101 / Chapter 3.2.5 --- Synergistic Anti-proliferative Effect of Jacaric Acid with Vitamin D3 and All Trans-Retinoic Acid (ATRA) on the Human Promyelocytic Leukemia HL-60 Cells In Vitro --- p.114 / Chapter 3.2.6 --- Effect of Jacaric Acid on the Cell Cycle Profile of the HL-60 Cells In Vitro --- p.116 / Chapter 3.2.7 --- Effect of Jacaric Acid on the In Vivo Tumorigenicity of the HL-60 Cells --- p.119 / Chapter 3.3 --- Discussion --- p.121 / Chapter CHAPTER 4: --- STUDIES ON THE APOPTOSIS-INDUCING AND DIFFERENTIATION-INDUCING EFFECTS OF CONJUGATED LINOLENIC ACIDS ON THE HUMAN MYELOID LEUKEMIA CELLS / Chapter 4.1.1 --- Introduction --- p.128 / Chapter 4.2 --- Results / Chapter 4.2.1 --- Induction of Apoptosis in the Human Promyelocytic Leukemia HL-60 Cells by Jacaric Acid --- p.134 / Chapter 4.2.2 --- Apoptosis-Inducing Effect of Jacaric Acid on the Human Promyelocytic Leukemia HL-60 Cells as Detected by Annexin V-GFP PI Double Staining Method --- p.138 / Chapter 4.2.3 --- Effect of Jacaric Acid on the Mitochondrial Membrane Potential in the Human Promyelocytic Leukemia HL-60 Cells --- p.140 / Chapter 4.2.4 --- Effects of Jacaric Acid on the Caspase Activities in the Human Promyelocytic Leukemia HL-60 Cells --- p.142 / Chapter 4.2.5 --- Effects of Jacaric Acid and Antioxidants on the ROS Induction in the Human Promyelocyic Leukemia hl-6 Cells --- p.147 / Chapter 4.2.6 --- Effect of N-acetyl-L-Cysteine on the Apoptosis-Inducing Activity of Jacaric Acid in the Human Promyelocytic Leukemia HL-60 Cells --- p.149 / Chapter 4.2.7 --- Morphological Studies on the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.151 / Chapter 4.2.8 --- Cell Size and Granularity of the Human Promyelocytic Leukemia HL-60 Cells after Treatment with Different CLN Isomers --- p.153 / Chapter 4.2.9 --- Expression of Differentiation-Related Cell Surface Markers in the Human Promyelocytic Leukemia HL-60 Cells after Treatment with Jacaric Acid --- p.155 / Chapter 4.3 --- Discussion --- p.158 / Chapter CHAPTER 5: --- STUDIES ON THE APOPTOSIS-ASSOCIATED PROTEINS AND SIGNALING PATHWAYS IN CONJUGATED LINOLENIC ACID-INDUCED APOPTOSIS OF THE HUMAN MYELOID LEUKEMIA CELLS / Chapter 5.1 --- Introduction --- p.165 / Chapter 5.2 --- Results / Chapter 5.2.1 --- Expression of Fas and Fas Ligand Proteins in the Jacaric Acid- treated Human Promyelocytic Leukemia HL-60 Cells --- p.171 / Chapter 5.2.2 --- Expression of Bcl-2 Family Member Proteins in the Jacaric Acid- treated Human Promyelocytic Leukemia HL-60 Cells --- p.173 / Chapter 5.2.3 --- Cytochrome c Release in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.175 / Chapter 5.2.4 --- Cleavage of Poly(ADP-ribose) Polymerase (PARP) in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.177 / Chapter 5.2.5 --- Phosphorylation of ERK in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.179 / Chapter 5.2.6 --- Phosphorylation of JNK in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.181 / Chapter 5.2.7 --- Phosphorylation of NF-kB Protein in the Jacaric Acid-treated Human Promyelocytic Leukemia HL-60 Cells --- p.183 / Chapter 5.3 --- Discussion --- p.185 / Chapter CHAPTER 6: --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.195 / REFERENCES --- p.203

Consumer awareness of the link between dietary fats and health outcomes has led to increased demand for food products enriched with bioactive fatty acids (FA). Ruminant-derived fats, such as dairy fats, contribute significantly to the American diet and contain many unique beneficial FA, such as short- and medium-chain FA, n-3 FA, conjugated linoleic acids (CLA), vaccenic acid (VA), as well as odd-and branched-chain FA (OBCFA). Increasing these FA in dairy products by altering farm management practices, such as breed, lactation stage, and nutrition, may improve human health without a change to the diet. The overarching goal of this dissertation was to evaluate on-farm strategies to increase the content of bioactive FA in bovine milk. The first objective was to enrich milk fat with bioactive FA via supplementation with echium oil, a terrestrial oil rich in n-3 FA. Treatments were 1.5% and 3.0% dry matter as lipid encapsulated echium oil (EEO) which were compared to a control (no EEO). Milk fat contents of n-3 FA increased with EEO supplementation but the transfer of n-3 FA from EEO into milk fat was rather low (< 5%). In a subsequent trial, ruminal protection of EEO and post-ruminal release of EEO-derived FA was examined. EEO-derived FA were preferentially incorporated into plasma lipid fractions unavailable to the mammary gland. Moreover, fecal excretion of EEO-derived FA ranged from 7-14% of intake, and VA and CLA, the biohydrogenation and metabolism products of n-3 FA, increased in milk and feces with EEO supplementation. Therefore, lipid-encapsulation provided inadequate digestibility and low transfer efficiency of n-3 FA into milk. The second objective was to compare the bacterial community structure and unique bioactive FA in bacterial membranes and milk fat between Holstein (HO), Jersey (JE), and HO x JE crossbreeds (CB) across a lactation. Lactation stage had a prominent effect on rumen bacterial taxa, with Firmicutes being most abundant during early lactation. The FA composition of bacterial cells was affected by both lactation stage and genetics, and OBCFA in bacterial cells were positively correlated with several bacteria of the Firmicutes phylum. HO and CB exhibited greater contents of various bioactive FA in milk than JE. The highest content of all bioactive FA occurred at early lactation, while OBCFA were highest at late lactation. The third objective was to determine the effects of grazing a monoculture vs. a diverse pasture on rumen bacterial and protozoal taxa, their membrane FA composition, and milk FA. Microbial communities shifted in response to grazing regime accompanied with changes in their membrane FA profiles. Rumen microbiota from cows grazing a diverse pasture had higher contents of n-3 FA and VA, but lower contents of OBCFA. Microbial membrane FA correlated with microbial taxa, the contents of ALA and n-3 FA were positively correlated with the bacterial genus Butyrivibrio and the protozoal genus Eudioplodinium. Milk contents of CLA and n-3 FA increased when cows grazed a diverse pasture, while grazing a monoculture led to greater milk contents of OBCFA. In conclusion, grazing cows on a diverse pasture, when compared to genetic effects and lipid supplementation, was the most efficacious strategy to increase the content of bioactive FA in milk.

Le tissu adipeux blanc (TA) joue un rôle central dans la physiopathologie de l'obésité et la résistance à l'insuline. Durant ma thèse, j’ai étudié la réponse cellulaire et moléculaire du TA au stress métabolique induit chez la souris par l'administration d'un isomère de l'acide linoléique (CLA). Nous avons utilisé l’isomère trans10,cis12 de l'acide linoléique (t10,c12-CLA) pour promouvoir un syndrome lipoatrophique chez la souris C57BL/6J. La dynamique de cette réponse a été explorée dans le TA, l'intestin et au niveau systémique, sur deux périodes consécutives de 7 jours d’administration de t10,c12-CLA puis d’interruption du traitement. Nos résultats montrent que t10,c12-CLA induit une dérégulation des gènes métaboliques et un dépôt de matrice extracellulaire (ECM) dans le TA. Le profil immunologique est perturbé, avec l'accumulation de macrophages M2 anti-inflammatoires. Ces altérations métaboliques, immunitaires et structurelles sont inversées, mais pas toujours normalisées, après 7 jours de récupération. La réversibilité des modifications observées dans le TA indique que cette réponse est plus adaptative que pathologique. Cependant, les altérations induites par t10, c12-CLA dans le TA sont temporellement liées à l'hyperinsulinémie, ce qui renforce l’idée que l'intégrité du TA est cruciale pour l'homéostasie glucidique. Bien qu’aucune réponse immunitaire n’ait été observée dans l'intestin, l’expression des gènes codant pour des protéines de jonctions serrées (claudine et occludine) est diminuée. Une partie des effets de t10,c12-CLA pourrait être relayée par l’augmentation de la perméabilité intestinale. Cette étude montre que, en réponse à un stress nutritionnel de courte durée, le TA regagne rapidement son homéostasie. Lorsque la contrainte est plus chronique, comme dans l'obésité, la plupart des interventions ne parviennent pas à inverser l'état dysfonctionnel du TA. Le grand défi dans les années à venir reste de trouver des stratégies thérapeutiques à long terme dans les maladies métaboliques. / White adipose tissue (WAT) plays a central role in the physiopathology of obesity and insulin resistance. During my PhD, I studied the cellular and molecular response of WAT to metabolic stress induced in mice by the administration of an isomer of linoleic acid (CLA). We used trans10,cis12 isomer of linoleic acid (t10,c12-CLA) as a tool to promote a lipoatrophic syndrome in C57BL/6J lean female mice. The short-term dynamics of this response was explored in WAT, gut and at systemic level, over two consecutive 7-day periods of t10,c12-CLA administration and withdrawal. Our results show that t10,C12-CLA-induced alterations in WAT included metabolic gene deregulation and extracellular matrix deposition. The immunological profile of WAT was markedly disturbed, with anti-inflammatory M2-polarized macrophage accumulation. Metabolic, immune and structural alteration were fully revered, but not always normalized after 7 days without treatment. The reversibility of WAT alterations indicates a more adaptive than pathological response to t10,C12-CLA. However, t10,c12-CLA-induced WAT deregulation is temporally linked to hyperinsulinemia supporting the primacy of WAT integrity for glucose homeostasis. No immune alteration was observed in response to t10,C12-CLA in the gut, where genes encoding tight junction proteins (claudin and occludin) were down regulated. Thus, part of the effects of t10,c12-CLA could be mediated by increased intestinal permeability. This study shows that in response to an acute nutritional stressor, WAT and glucose homeostasis could be rapidly regained. When the stress is more chronic, as in obesity, most of the interventions fail to reverse WAT dysfunctional state, underlining the big challenge in the years ahead of finding long-term therapeutic strategies in metabolic diseases.

Stearoyl-CoA desaturase1 (SCD1) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) in the mammary gland of ruminant animals. We hypothesized that single nucleotide polymorphisms (SNPs) in the coding region, 5' and 3' untranslted regions (UTRs) of the SCD1 gene would influence the activity of SCD1 enzyme and consequently account for some within-breed variations in milk CLA and MUFA. Sequence analysis of the coding region of the SCD1 gene of Jerseys and Holsteins revealed c.702A&rarr;G, c.762T&rarr;C and c.878C&rarr;T SNPs in exon 5 in both breeds and c.435G&rarr;A in exon 3 in Holsteins. The SNPs resulted in: A (G435A702T 762C878), A1 (A435A702T 762C878), B (G435G702C 762T878) and B1 (A435G702C 762T878) coding variants in Holsteins and only variants A and B in Jerseys. Only SNP 878C&rarr;T resulted in a non-synonymous codon change resulting in p.293Ala and p.293Val protein variants or alleles at the SCD1 locus. Subsequent association studies found significantly higher C10 index, C12 index and C14 index and consequently higher concentrations of C10:1 and C12:1 in p.293AA cows compared to the p.293VV cows in both breeds. The SCD1 genotype had no influence on concentrations of C141, C16:1, C18:1 and CLA in both breeds. / Sequence analysis of the 5' and 3' UTRs revealed no SNPs in the 5'UTR and a total of 14 SNPs in the 3'UTR of both breeds. The SNPs were in complete linkage disequilibrium resulting in 3 haplotypes or regulatory variants: H1 (G1571G1644C1763C2053A2584 A3007C3107G3208 T3290G 3497G3682A4399C4533G4881), H2 (G1571G1644A1763C2053A 2584G3007 C3107G3208T3290G3497G 3682A4399C4533G4881) and H3 (T 1571C1644A1763 T2053G2584G3007T 3107A3208C3290A3497A3682T 4399T4533A4881) in Holsteins and only H1 and H3 variants in Jerseys. A subsequent association study involving 862 Holstein cows, found the H1 regulatory variant to be associated with higher C10 and C12 desaturase indices and consequently with higher concentrations of C10:1 and C12:1 compared with the H3 variant. The effects of the H2 variant were intermediate to those of H1 and H3. 3'UTR genotype had no influence on the concentrations of C14:1, C16:1, C18:1 and CLA. The concentrations of C10:1 and C12:1 in milk fat could therefore be due to effects of SNPs in the open reading frame and the 3'UTR regions of the SCD1 gene. These results indicate that SNPs in the coding and 3'UTR regions of the SCD1 gene could be used as markers for genetic selection for increased C10:1 and C12:1 contents of milk.

Die vorliegende Arbeit befasst sich mit der Einflussnahme von konjugierten Linolsäuren (CLA) und strukturverwandten Fettsäuren auf Pansenepithelzellen (PEZ) in Kultur. Die in-vitro Untersuchungen mit primär kultivierten PEZ vom Schaf haben gezeigt, dass eine 48-stündige Inkubation mit CLA C18:2 cis-9, trans-11 (c9t11) und CLA C18:2 trans-10, cis-12 (t10c12) sowie Linolsäure, Ölsäure und trans-Vaccensäure (TVA) zu Veränderungen im Fettsäuremuster der Zellen sowie in der Expression der mRNA verschiedener Proteine führte. Aus den Veränderungen im Fettsäuremuster wurde die Aufnahme sowie die Metabolisierung der supplementierten Fettsäuren abgeleitet. Als wesentlicher Metabolit der TVA wurde die c9t11 identifiziert. Mit dem Nachweis der Stearoyl-CoA-Desaturase (SCD) -mRNA in den PEZ sowie im nativen Gewebe konnte so die endogene CLA-Synthese in den PEZ bestätigt werden. Analysiert wurden außerdem mittels quantitativer rt-PCR die Expression der SCD-mRNA und die mRNA von zwei Transportproteinen, den Monocarboxylat-Transportern (MCT) 1 und 4. Von beiden ist bekannt, dass sie in den Transport kurzkettiger Fettsäuren (SCFA) involviert sind. Die Inkubation mit den einzelnen Fettsäuren führte einheitlich zur Abnahme der SCD-mRNA Expression sowie zu einem verminderten Gehalt der Hauptprodukte der SCD, der C16:1 cis-9 und der C18:1 cis-9. Bezüglich der MCT1 und 4 mRNA-Expression wurde in der Regel eine Heraufregulierung nach Zugabe der Fettsäuren beobachtet. Des Weiteren wurden regulative Einflüsse von PPARα auf die MCTs und die SCD und von PPARγ auf den MCT1 und die SCD nach c9t11-Inkubation mit entsprechenden Antagonisten-Versuchen festgestellt. Die Studien beleuchten somit grundlegende Mechanismen des Stoffwechsels von langkettigen ungesättigten Fettsäuren in PEZ und deren Einflussnahme auf Transkriptionsfaktoren und auf spezifische, für den Wiederkäuer bedeutende Transportproteine für SCFA. Die funktionelle Relevanz dieser Ergebnisse für den Pansen und den Wiederkäuer aber auch für andere Gewebe und Species muss in weiteren Studien geklärt werden.:Inhaltsverzeichnis Abkürzungsverzeichnis 3 1 Einleitung 5 Allgemein 5 1.1 CLA-Nomenklatur 6 1.2 Bildung von CLAs und deren Vorkommen 7 1.3 CLA-Supplementierung beim Rind 9 1.4 Zu CLAs strukturverwandte Fettsäuren im Pansen 10 1.5 Potenzielle Wirkungen von CLAs und strukturverwandten Fettsäuren auf das Pansenepithel 11 1.5.1 Aufbau und Bedeutung des Pansenepithels 11 1.5.2 Resorption von langkettigen Fettsäuren über das Pansenepithel 11 1.5.3 Veränderungen der Fettsäurezusammensetzung nach Supplementierung 11 1.5.3.1 Metabolisierung 12 1.5.3.2 SCD und die endogene Synthese von CLA c9t11 13 1.5.4 CLA-Targets im Pansenepithel 15 1.5.4.1 SCD 15 1.5.4.2 Monocarboxylattransporter 16 1.5.4.3 PPAR als Transkriptionsfaktor für die SCD und die MCTs 19 2 Zielstellung 21 3 Originalarbeit 22 4 Ergänzung zur Originalarbeit 43 4.1 Nachweis von SCD-mRNA im nativen Pansenepithel des Schafes 43 Zusammenfassung der Arbeit 44 Literaturverzeichnis 49 Anhang (Supplemental Material) 59 Erklärung zum Eigenanteil der Dissertationsschrift 67 Erklärung über die eigenständige Abfassung der Arbeit 69 Publikationen und Vorträge im Rahmen der Dissertation 70

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