El procesamiento post cosecha en maca (Lepidium meyenii) es clave en la generación del perfil metabólico que resulta en sus propiedades nutracéuticas tan conocidas. En este artículo se describe los distintos procesos metabólicos responsables de la generación de productos bioactivos clave. / Maca’s (Lepidium  meyenii) post harvest processing is key in generating a metabolic profile that will result in its well reported nutraceutical properties. This article presents how different metabolic processes generate these changes.

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Meng Jie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references. / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / Abbreviation --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1 --- Black Mustard and Sinigrin --- p.2 / Chapter 1.2 --- Hepatocellular Carcinoma --- p.5 / Chapter 1.2.1 --- Different Stages of HCC --- p.6 / Chapter 1.2.2 --- Risk Factors --- p.8 / Chapter 1.2.3 --- Treatments of HCC --- p.10 / Chapter 1.3 --- Biomarkers Used to Evaluate Effects of Sinigrin on HCC --- p.12 / Chapter 1.3.1 --- AST & ALT --- p.12 / Chapter 1.3.2 --- Glutathione S Transferase -p (GST-p) --- p.13 / Chapter 1.4 --- Tumor Suppressor Genes and Oncogenes --- p.14 / Chapter 1.4.1 --- "p53, the Tumor Suppressor Gene" --- p.15 / Chapter 1.4.2 --- p53-dependent pathway --- p.15 / Chapter 1.4.2.1 --- Mdm2 --- p.16 / Chapter 1.4.2.2 --- Bax and Bcl-2 --- p.17 / Chapter 1.4.2.3 --- PCNA and p21wAF1/CIP1 --- p.18 / Chapter 1.5 --- Aim of the Project --- p.19 / Chapter Chapter 2: --- Materials and Methods --- p.20 / Chapter 2.1 --- In vitro Studies --- p.21 / Chapter 2.1.1 --- Neutral Red Assay --- p.21 / Chapter 2.1.1.1 --- Chemicals and Reagents --- p.21 / Chapter 2.1.1.2 --- Liver Cells --- p.23 / Chapter 2.1.1.3 --- Neutral Red Assay --- p.24 / Chapter 2.1.2 --- Flow Cytometery --- p.24 / Chapter 2.1.2.1 --- Chemicals and Reagents --- p.25 / Chapter 2.1.2.2 --- Flow Cytometery Analysis --- p.25 / Chapter 2.1.3 --- DNA Fragmentation --- p.26 / Chapter 2.1.3.1 --- Chemicals and Reagents --- p.26 / Chapter 2.1.3.2 --- DNA Extraction --- p.28 / Chapter 2.1.3.3 --- DNA Agarose Gel Electrophoresis --- p.29 / Chapter 2.1.4 --- cDNA Microarray --- p.29 / Chapter 2.1.4.1 --- Chemicals and Reagents --- p.30 / Chapter 2.1.4.2 --- RNA Extraction --- p.33 / Chapter 2.1.4.3 --- RNA Quantity and Quality Control --- p.34 / Chapter 2.1.4.4 --- RT-PCR --- p.35 / Chapter 2.1.4.5 --- cRNA Convention and Purification --- p.36 / Chapter 2.1.4.6 --- Hybridization --- p.37 / Chapter 2.1.4.7 --- Washing and Detection --- p.37 / Chapter 2.1.4.8 --- Data Analysis --- p.38 / Chapter 2.2 --- In vivo Studies --- p.39 / Chapter 2.2.1 --- Animal Treatment --- p.39 / Chapter 2.2.1.1 --- Chemicals and Reagents --- p.39 / Chapter 2.2.1.2 --- Chemical Carcinogens --- p.40 / Chapter 2.2.1.3 --- Promotion Stage --- p.41 / Chapter 2.2.1.4 --- Progression Stage --- p.44 / Chapter 2.2.2 --- Measurement of Serum ALT and AST Activities --- p.46 / Chapter 2.2.2.1 --- Chemicals and Reagents --- p.46 / Chapter 2.2.2.2 --- Activity Assay --- p.46 / Chapter 2.2.3 --- Histological Analysis --- p.47 / Chapter 2.2.3.1 --- Chemicals and Reagents --- p.47 / Chapter 2.2.3.2 --- Preparation of Slides --- p.49 / Chapter 2.2.3.3 --- H&E Staining --- p.49 / Chapter 2.2.3.4 --- GST-p Immuno-staining --- p.50 / Chapter 2.2.4 --- Semi-Quantitative RT-PCR Analysis of mRNA Expression --- p.53 / Chapter 2.2.4.1 --- Chemicals and Reagents --- p.53 / Chapter 2.2.4.2 --- Extraction of total RNA from rat liver --- p.53 / Chapter 2.2.4.3 --- Quantity and Quality Control of RNA --- p.53 / Chapter 2.2.4.4 --- RT-PCR (Reverse Transcription) --- p.54 / Chapter 2.2.4.5 --- PCR --- p.54 / Chapter 2.2.4.6 --- DNA gel electrophoresis --- p.55 / Chapter 2.2.4.7 --- Data Analysis --- p.56 / Chapter 2.2.5 --- Western Blot Analysis for Biomarkers --- p.56 / Chapter 2.2.5.1 --- Chemicals and Reagents --- p.56 / Chapter 2.2.5.2 --- Extraction of the Cytosol Protein --- p.60 / Chapter 2.2.5.3 --- Extraction of the Nuclear protein --- p.61 / Chapter 2.2.5.4 --- SDS Gel Electrophoresis --- p.61 / Chapter 2.2.5.5 --- Western Blot --- p.62 / Chapter 2.2.5.6 --- Interaction with Antibodies --- p.63 / Chapter 2.2.5.7 --- ECL Detection --- p.63 / Chapter 2.2.5.8 --- Data Analysis --- p.64 / Chapter Chapter 3: --- Results --- p.65 / Chapter 3.1 --- In vitro Studies --- p.66 / Chapter 3.1.1 --- Cell Viability test and IC50 --- p.66 / Chapter 3.1.2 --- Cell Cycle Analysis --- p.68 / Chapter 3.1.3 --- DNA Fragmentation --- p.71 / Chapter 3.1.4 --- Effects of Sinigrin on Gene Expression --- p.73 / Chapter 3.2 --- In vivo Studies --- p.77 / Chapter 3.2.1 --- Effects of Sinigrin on HCC Development (Promotion stage) in Rats --- p.77 / Chapter 3.2.1.1 --- Direct Observation --- p.77 / Chapter 3.2.1.2 --- Relative Liver / Body Weight Ratio --- p.79 / Chapter 3.2.1.3 --- AST/ALT Assay --- p.81 / Chapter 3.2.1.4 --- Basic Structure of Hepatocytes --- p.83 / Chapter 3.2.1.5 --- GST-p Foci Area --- p.85 / Chapter 3.2.1.6 --- mRNA Expression of p53 and Mdm2 --- p.88 / Chapter 3.2.1.7 --- Protein Expression of Biomarkers --- p.90 / Chapter 3.2.2 --- Effects of Sinigrin on HCC Development (Progression stage) in Rats --- p.97 / Chapter 3.2.2.1 --- Direct Observation --- p.97 / Chapter 3.2.2.2 --- Relative Liver / Body Weight Ratio --- p.99 / Chapter 3.2.2.3 --- AST/ALT Assay --- p.101 / Chapter 3.2.2.4 --- Basic Structure of Hepatocytes --- p.103 / Chapter 3.2.2.5 --- GST-p Foci Area --- p.105 / Chapter 3.2.2.6 --- mRNA Expression of p53 and Mdm2 --- p.108 / Chapter 3.2.2.7 --- Protein Expression of Biomarkers --- p.110 / Chapter Chapter 4: --- Discussion --- p.116 / Chapter 4.1 --- Protective and Therapeutic Benefits of Sinigrin --- p.117 / Chapter 4.1.1 --- Effects of SIN on Cancer and Normal Cells --- p.117 / Chapter 4.1.2 --- Effective Tumor Induction by DEN-CC14 Treatment --- p.118 / Chapter 4.1.3 --- Protective Effect of SIN in the Promotion Stage of HCC --- p.118 / Chapter 4.1.4 --- Therapeutic Effect of SIN in the Progression Stage of HCC --- p.119 / Chapter 4.2 --- Biological Activities of SIN --- p.121 / Chapter 4.3 --- Summary --- p.134 / References --- p.xiv

Les glucosinolates sont des composés thio-b-D-glucopyranosiques anioniques de structure originale présentsdans de nombreux végétaux, essentiellement dans la famille des crucifères. Les glucosinolates sont hydrolysés parune enzyme appelée myrosinase (thioglucoside glucohydrolase E.C. 3.2.3.147.) Le système myrosinase-glucosinolate est un couple enzyme-substrat que nous avons cherché à mieux comprendre et développer. Aussi l'inefficacité des méthodes classiques envers certaines molécules hétéroaromatiques et la quantité restreinte de recherches effectuées sur ce sujet nous a encouragé à vouloir développer de nouvelles voies de synthèse de glucosinolates. Les nouveaux analogues de glucosinolates avec un aglycone modifié ont été synthétisés. Un analogue hétéroaromatique soumis en test a été reconnu et hydrolysé par la myrosinase. En cette façon, une indépendance du mécanisme de reconnaissance par la myrosinase de la taille d'aglycone a été démontrée. Certains de nouveaux analogues avec un aglycone modifié, obtenus en remplaçant le thioglucose par un groupement thioéthyle, ont montré très bonne activité inhibitrice envers myrosinase. Les produits principaux de la dégradation enzymatique - des isothiocyanates et leurs thioadduits correspondants au glucosinolates obtenus auparavant ont été synthétisés. Les tests contre Plasmodium falciparum, le parasite causant le paludisme, ont montré une activité antipaludéenne de ces isothiocyanates du même rang qu'un des médicaments actuellement très largement utilisées. Une méthode de la synthèse des glucosinolates complètement nouvelle, efficace et simple a été mise au point. Cella ouvre de nouvelles possibilités pour la synthèse des glucosinolates sensibles aux conditions habituelles de la synthèse. Cette nouvelle méthode a été aussi appliquée à la synthèse des promettants et très peu étudiés. Egalement, une nouvelle voie d'approche aux glucosinolates thiofunctionalisés a été développée avec succès.

L’hypothèse selon laquelle l’accumulation des métaux lourds par les plantes a évolué comme mécanisme de défense contre les herbivores a été testée avec l’hyperaccumulatrice de zinc Thlaspi caerulescens (Brassicaceae). En utilisant l’écotype métallicole (poussant sur sols métallifères) et l’écotype non métallicole (sols normaux) de T. caerulescens, nos résultats ont conduit à rejeter l’hypothèse de défense par accumulation de métaux: les plantes ont été consommées indépendamment de leur concentration en Zn dans toutes les situations expérimentales examinées (conditions contrôlées, jardin expérimental, populations naturelles). Par contre, les herbivores ont montré une préférence systématique pour les plantes de l’écotype métallicole, quelle que soit leur concentration en Zn. Lorsque l’on mesure les concentrations en métabolites secondaires défensifs (glucosinolates) des écotypes métallicole et non métallicole de T. caerulescens, on constate que les individus d’origine métallicole produisent constitutivement moins de glucosinolates que les individus non métallicoles, tant dans les populations belges que dans les populations françaises. Par ailleurs, sur les sites métallifères où ont évolué les populations métallicoles, on constate à la fois une plus faible pression d’herbivorie sur les plantes (moins de dégâts) et une plus faible densité de gastéropodes que dans les sites normaux. La diminution des défenses chez l’écotype métallicole serait la conséquence d’un relâchement de la pression d’herbivorie sur les sites métallifères. <p>En outre, nous avons montré que la chenille spécialiste d’Issoria lathonia (Nymphalidae) est capable de se développer sur les feuilles riches en Zn de l’accumulatrice de zinc Viola calaminaria (Violaceae) en excrétant efficacement le Zn dans leurs fèces. <p> L’ensemble de nos résultats suggère donc que l’hyperaccumulation des métaux lourds n’a pas évolué en tant que mécanisme de défense contre les herbivores.<p> / Doctorat en sciences, Spécialisation biologie végétale / info:eu-repo/semantics/nonPublished

<p>All organisms live in complex habitats that shape the course of their evolution by altering the phenotype expressed by a given genotype (a phenomenon known as phenotypic plasticity) and simultaneously by determining the evolutionary fitness of that phenotype. In some cases, phenotypic evolution may alter the environment experienced by future generations. This dissertation describes how genetic and environmental variation act synergistically to affect the evolution of glucosinolate defensive chemistry and flowering time in Boechera stricta, a wild perennial herb. I focus particularly on plant-associated microbes as a part of the plant’s environment that may alter trait evolution and in turn be affected by the evolution of those traits. In the first chapter I measure glucosinolate production and reproductive fitness of over 1,500 plants grown in common gardens in four diverse natural habitats, to describe how patterns of plasticity and natural selection intersect and may influence glucosinolate evolution. I detected extensive genetic variation for glucosinolate plasticity and determined that plasticity may aid colonization of new habitats by moving phenotypes in the same direction as natural selection. In the second chapter I conduct a greenhouse experiment to test whether naturally-occurring soil microbial communities contributed to the differences in phenotype and selection that I observed in the field experiment. I found that soil microbes cause plasticity of flowering time but not glucosinolate production, and that they may contribute to natural selection on both traits; thus, non-pathogenic plant-associated microbes are an environmental feature that could shape plant evolution. In the third chapter, I combine a multi-year, multi-habitat field experiment with high-throughput amplicon sequencing to determine whether B. stricta-associated microbial communities are shaped by plant genetic variation. I found that plant genotype predicts the diversity and composition of leaf-dwelling bacterial communities, but not root-associated bacterial communities. Furthermore, patterns of host genetic control over associated bacteria were largely site-dependent, indicating an important role for genotype-by-environment interactions in microbiome assembly. Together, my results suggest that soil microbes influence the evolution of plant functional traits and, because they are sensitive to plant genetic variation, this trait evolution may alter the microbial neighborhood of future B. stricta generations. Complex patterns of plasticity, selection, and symbiosis in natural habitats may impact the evolution of glucosinolate profiles in Boechera stricta.</p> / Dissertation

Targeting the epigenome, including the use of histone deacetylase (HDAC) inhibitors, is a novel strategy for cancer chemoprevention. Sulforaphane (SFN), a compound found at high levels in broccoli and broccoli sprouts, is a potent inducer of Phase 2 detoxification enzymes and inhibits tumorigenesis in animal models. SFN also has a marked effect on cell cycle checkpoint controls and cell survival/apoptosis in various cancer cells, through mechanisms that are poorly understood. Based on the structure of known histone deacetylase inhibitors, it was hypothesized that SFN may possess HDAC inhibitory properties. Initial studies confirmed that, indeed, at physiologically-relevant concentrations, SFN inhibited HDAC activity in human colorectal cancer cells, with a concomitant increase in acetylated histones H3 and H4, induction of p21 expression, and increased acetylated histone H4 associated with the P21 promoter. A metabolite of SFN, SFN-Cysteine, was found to be the active HDAC inhibitor. Furthermore, in BPH-1, LnCaP, and PC-3 human prostate epithelial cells, SFN inhibited HDAC activity and increased acetylation of histones. SFN also induced p21 expression, with an increase in acetylated histone H4 associated with the P21 promoter in BPH-1 cells. The downstream effects of HDAC inhibition by SFN included induction of pro-apoptotic proteins and repression of anti-apoptotic proteins, and an increase in multi-caspase activity. Dietary SFN suppressed the growth of human prostate cancer PC-3 xenografts and inhibited HDAC activity in the xenografts, peripheral blood mononuclear cells (PBMC), and prostates. In time-course studies, a single oral dose of SFN induced histone acetylation at 6 and 24 h in mouse colonic mucosa, and long-term dietary SFN treatment increased histone acetylation in the ileum, colon, PBMC, and prostates. Moreover, dietary SFN suppressed intestinal tumorigenesis significantly in Apc[superscrip min] mice, with an increase in acetylated histones detected in the normal-looking ileum and polyps and polyps from the colon. Overall, the data presented in this thesis support a novel mechanism for chemoprevention by SFN in vivo, through inhibition of histone deacetylase. The findings also imply that SFN will offer significant protection against at least two of the major cancer killers in the US, namely colon and prostate cancer. / Graduation date: 2005

<p>Understanding the mechanisms that underlie the formation of, and innovation in biochemical pathways is an important goal in evolutionary biology. The following work addresses the problem of biochemical pathway evolution in two ways. In the first chapter, I combine genetic manipulations and population genetic analyses to investigate the whether flux control in the aliphatic glucosinolate pathway of <italic> Arabidopsis thaliana</italic> drives evolutionary rate heterogeneity. My results indicate that the first enzyme in the pathway, CYP79F1, has majority flux control and is the only one to show convincing evidence for positive selection. The second chapter builds on the first by asking whether flux control is stable under a variety of environmental conditions. I find that flux control remains with CYP79F1, in all my environmental treatments. In the final chapter, I address the evolution of one enzyme in this pathway from <italic>Boechera stricta</italic> that is responsible for a gain-in-function polymorphism that results in increased fitness in nature. With molecular phylogenetic analysis, site-directed mutagenesis, structural biology and enzymatic assays, I determine what residues are under selection and test their functional effects. I find that just two mutations in this enzyme are responsible for the change in function, and discuss their position within the enzyme. Strikingly, the enzyme with majority flux control in <italic>A. thaliana</italic> is homologous to the enzyme responsible for the novel function in <italic>Boechera</italic>. Together these results suggest that selection may predictably exploit the same small subset of genes to optimize biochemical pathway output and for evolutionary innovation.</p> / Dissertation

Thesis (Ph. D.)--University of Wisconsin--Madison, 2007. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--Bowling Green State University, 2009. / Document formatted into pages; contains x, 46 p. Includes bibliographical references.