The fatty acid amides, a structurally diverse endogenous congener of molecules active in cell signaling, may prove to have diverse activity due to their interface with a number of receptor systems, including, but not limited to cannabinoid receptor 2 (CB2) and PPARγ. Select extracts of Echinacea spp. contain the fatty acid amides known as alkylamides. These extracts were a previously popular remedy relied on by U.S. physicians, one of the top sellers in the natural products industry and are currently a frequently physician prescribed remedy in Germany. In the series of experiments contained within, Galenic ethanolic extracts of Echinacea spp. root were used for the quantification, identification, degradation and bioactivity studies. On extraction, depending on the ratio of plant to solvent and fresh or dry, the data indicate that there is variability in the alkylamide classes extracted. For example the acetylene alkylamides appear to extract under different concentrations, as well as degrade faster than the olefinic alkylamides. In addition, the alkylamides are found to degrade significantly in both cut/sift and powdered forms of echinacea root. Human liver microsome oxidation of the major alkylamide dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide generate hydroxylated, caboxylated and epoxidized metabolites. The carboxylated metabolite has, thus far, shown different immune activity than the native tetraene isobutylamide. Bioactivity studies, based on cytokine modulation of the alkylamides have been assumed to be due to a classic CB2 response. However, the results of experiments contained herein suggest that IL-2 inhibition by the alkylamide undeca-2E-ene-8,10-diynoic acid isobutylamide, which does not bind to CB2, is due to PPARγ activation. These data, combined with data generated by other groups, suggest that the alkylamides of Echinacea spp. are polyvalent in effect, in that they modulate multiple biochemical pathways.

Le carcinome rénal à cellules claires (cRCC) est le principal type histologique de carcinome rénal et l'une des tumeurs les plus résistantes à la chimio et à la radiothérapie. L'absence de biomarqueurs pour la détection précoce et pour le suivi des patients est responsable d'un mauvais pronostic. Il est nécessaire d'identifier de nouveaux biomarqueurs et des cibles thérapeutiques pour améliorer la prise en charge des patients. Les microARNs, des petits ARN non codants de 22 nucléotides, qui ont été précédemment montrés comme favorisant l'initiation et la progression tumoral, semblent être de bons candidats. Nous avons focalisé notre étude sur (i) miR-21 qui est le principal oncomiR surexprimé dans le cRCC et (ii) le récepteur nucléaire PPARα (Peroxisome Proliferator Activated Receptor), l'une des cibles de miR-21.D'une part, sur une cohorte de 99 échantillons de cRCC primaires, nous avons montré que l'expression de miR-21 était plus élevée dans les tissus cancéreux que dans les tissus non tumoraux adjacents. In vitro, miR-21 est également surexprimé dans les lignées cellulaires de carcinomes rénaux comparées à la lignée cellulaire épithéliale HK-2 provenant de tubes proximaux humains. De plus, nous avons également montré que la surexpression de miR-21 augmente les propriétés de migration et d'invasion des cellules cancéreuses rénales ainsi que les voies de signalisation prolifératives et anti-apoptotiques, alors que des résultats opposés ont été observés en utilisant une stratégie d'inhibition anti-miR-21. Enfin, nous avons évalué le rôle du miR-21 dans la chimiorésistance du cRCC et montré, en outre, que l'inhibition de miR-21 augmentait significativement la chimiosensibilité au paclitaxel, au 5-fluorouracile, à l'oxaliplatine et au dovitinib, diminuait l'expression des transporteurs à efflux MRP1-6/ABCC1-6 et augmentait l'expression des transporteurs à influx SLC22A1/OCT1, SLC22A2/OCT2 et SLC31A1/CTR1. Ces résultats ont permis la publication d'un article dans Tumor Biology se trouvant en annexe.D'autre part, dans les tissus de patients atteints de cRCC, nous avons montré pour la première fois que la surexpression de miR-21 est en corrélation avec une perte d'expression de PPARα. In vitro, nous avons montré que miR-21 cible le 3'-UTR de PPARα et diminue son expression protéique et que la surexpression de miR-21 diminue l'activité transcriptionnelle de PPARα. En outre, la surexpression et l'activation de PPARα diminuent l'expression de miR-21. En effet, PPARα interagit avec les facteurs de transcription AP-1 et NF-κB et empêche ainsi leur liaison au promoteur de miR-21 diminuant ainsi sa transcription.En conclusion, nous avons montré que (i) miR-21 est un acteur clé de la progression du cancer du rein et joue un rôle important dans la résistance aux chimiothérapies et (ii) qu'il existe une boucle de régulation négative entre miR-21 et PPARα dans le cRCC. / Renal clear cell carcinoma (cRCC) is the major histological type of renal carcinoma and one of the most chemo- and radio-resistant tumors. The absence of biomarkers for early detection and for monitoring patients is responsible of a poor prognosis. It is necessary to identify new biomarkers and therapeutic targets to improve patient care. MicroRNAs, small noncoding RNAs of 22 nucleotides, which have been previously shown to promote malignant initiation and progression, appear to be good candidates.We focused our study on (i) miR-21 which is the main overexpressed oncomirs in cRCC and (ii) the nuclear receptor PPARα (Peroxisome Proliferator Activated Receptor), one of miR-21 targets.In one hand, by using a cohort of 99 primary cRCC samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating cRCC chemoresistance and further showed that miR-21 silencing significantly increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin and dovitinib, decreased expression of multi-drug resistance genes and increased SLC22A1/OCT1, SLC22A2/OCT2 and SLC31A1/CTR1 platinum influx transporter expression. These results led to the publication of an article in Tumor Biology in annex.In other hand, in cRCC tissue patients, we showed for the first time that miR-21 overexpression correlates with a loss of expression of PPARα. In vitro, we showed that miR-21 targets PPARα 3'-UTR and decreases its protein expression and miR-21 overexpression decreases the transcriptional activity of PPARα. Furthermore, PPARα overexpression and activation decrease miR-21 expression. In fact, PPARα interacts with AP-1 and NF-kappaB transcription factors and thus prevents their binding to the miR-21 promoter thus decreasing its transcription.In conclusion, we have shown that (i) miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs and (ii) there is a negative regulatory loop between miR-21 and PPARα in cRCC.

Li, Sui Mui. / On t.p. "beta" appears as the Greek letter. / Thesis submitted in: December 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 182-189). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vi / List of figures --- p.xii / List of appendices --- p.xix / Abbreviations --- p.xx / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter Chapter 2 --- Role of PPARP in adipocyte differentiation - an in vitro study --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.23 / Chapter 2.2.1 --- Preparation ofPPARβ (+/+) and PPARβ (-/-) MEFs --- p.23 / Chapter 2.2.1.1 --- Materials --- p.23 / Chapter 2.2.1.2 --- Methods --- p.23 / Chapter 2.2.1.2.1 --- Isolation of MEFs --- p.23 / Chapter 2.2.1.2.2 --- Passage ofMEF culture --- p.25 / Chapter 2.2.2 --- Genotyping of PPARβ (+/+) and PPARβ (-/-) MEFs --- p.25 / Chapter 2.2.2.1 --- Materials --- p.26 / Chapter 2.2.2.2 --- Methods --- p.26 / Chapter 2.2.2.2.1 --- Primer design --- p.26 / Chapter 2.2.2.2.2 --- Genomic DNA extraction --- p.27 / Chapter 2.2.2.2.3 --- PCR reaction --- p.29 / Chapter 2.2.3 --- Western blotting of PPARβ(+/+) and PPARβ (-/-) MEFs --- p.30 / Chapter 2.2.3.1 --- Materials --- p.30 / Chapter 2.2.3.2 --- Methods --- p.31 / Chapter 2.2.3.2.1 --- Preparation of nuclear extracts --- p.31 / Chapter 2.2.3.2.2 --- Western blot --- p.32 / Chapter 2.2.4 --- Induction of adipocyte differentiation of PPARβ (+/+) and PPARβ(-/-) MEFs --- p.33 / Chapter 2.2.4.1 --- Materials --- p.34 / Chapter 2.2.4.2 --- Methods --- p.34 / Chapter 2.2.4.2.1 --- Seeding ofMEFs --- p.34 / Chapter 2.2.4.2.2 --- Adipocyte differentiation --- p.35 / Chapter 2.2.5 --- Oil Red O staining of differentiated PPARβ(+/+) and PPARβ(-/-) MEFs --- p.36 / Chapter 2.2.5.1 --- Materials --- p.36 / Chapter 2.2.5.2 --- Method --- p.37 / Chapter 2.2.5.2.1 --- Oil Red O staining --- p.37 / Chapter 2.2.6 --- Determination of triglyceride-protein assay of differentiated PPARβ (+/+) and PPARβ (-/-) MEFs --- p.37 / Chapter 2.2.6.1 --- Materials --- p.39 / Chapter 2.2.6.2 --- Methods --- p.39 / Chapter 2.2.6.2.1 --- Lysis of differentiated MEFs --- p.39 / Chapter 2.2.6.2.2 --- Measurement of triglyceride concentration in cell lysate --- p.40 / Chapter 2.2.6.2.3 --- Measurement of protein concentration in cell lysate --- p.41 / Chapter 2.2.7 --- Preparation of PPARβ(+/+) and PPARβ (-/-) MEF RNA for RT-PCR and Northern blot analysis --- p.42 / Chapter 2.2.7.1 --- Materials --- p.42 / Chapter 2.2.7.2 --- Method --- p.42 / Chapter 2.2.7.2.1 --- RNA isolation --- p.42 / Chapter 2.2.8 --- RT-PCR analysis of differentiated PPARβ(+/+) and PPARβ (-/-) MEFs --- p.44 / Chapter 2.2.8.1 --- Materials --- p.45 / Chapter 2.2.8.2 --- Methods --- p.45 / Chapter 2.2.8.2.1 --- Primer design --- p.45 / Chapter 2.2.8.2.2 --- RT-PCR --- p.46 / Chapter 2.2.9 --- Northern blot analysis of differentiated PPARβ(+/+) and PPARβ (-/-) MEFs --- p.47 / Chapter 2.2.9.1 --- Materials --- p.48 / Chapter 2.2.9.2 --- Methods --- p.49 / Chapter 2.2.9.2.1 --- Preparation of cDNA probes for Northern blotting --- p.49 / Chapter 2.2.9.2.1.1 --- RNA extraction --- p.49 / Chapter 2.2.9.2.1.2 --- Primer design --- p.49 / Chapter 2.2.9.2.1.3 --- RT-PCR of extracted mRNA --- p.50 / Chapter 2.2.9.2.1.4 --- Subcloning of amplified cDNA products --- p.50 / Chapter 2.2.9.2.1.5 --- Screening of recombinant clones by phenol-chloroform extraction --- p.51 / Chapter 2.2.9.2.1.6 --- Confirmation of the recombinant clones by restriction enzyme site mapping --- p.52 / Chapter 2.2.9.2.1.7 --- Confirmation of the recombinant clones by PCR method --- p.52 / Chapter 2.2.9.2.1.8 --- Mini-preparation of plasmid DNA from the selected recombinant clones --- p.54 / Chapter 2.2.9.2.1.9 --- Preparation of cDNA probes --- p.54 / Chapter 2.2.9.2.1.10 --- Formaldehyde agarose gel electrophoresis of RNA --- p.55 / Chapter 2.2.9.2.1.11 --- Hybridization and color development --- p.56 / Chapter 2.3 --- Results --- p.58 / Chapter 2.3.1 --- Confirmation of PPARβ(+/+) and PPARβ (-/-) MEFs genotypes --- p.58 / Chapter 2.3.2 --- PPARβ (-/-) MEFs differentiated similarly to PPARβ(+/+) MEFs as measured by Oil Red O staining --- p.61 / Chapter 2.3.3 --- PPARβ (-/-) MEFs differentiated similarly to PPARβ(+/+) MEFs as reflected by their intracellular triglyceride contents --- p.64 / Chapter 2.3.4 --- PPARβ(-/-) MEFs expressed the adipocyte differentiation marker genes similarly to PPARβ (+/+) MEFs --- p.66 / Chapter 2.4 --- Discussion --- p.77 / Chapter Chapter 3 --- Role of PPARβ in adipocyte differentiation and lipid homeostasis - an in vivo study --- p.82 / Chapter 3.1 --- Introduction --- p.83 / Chapter 3.2 --- Materials and Methods --- p.85 / Chapter 3.2.1 --- Animal and high fat diet treatment --- p.85 / Chapter 3.2.1.1 --- Materials --- p.85 / Chapter 3.2.1.2 --- Method --- p.86 / Chapter 3.2.1.2.1 --- Animal treatment --- p.86 / Chapter 3.2.2 --- Tail-genotyping of PPARβ (+/+) and PPARβ (-/-) mice --- p.87 / Chapter 3.2.2.1 --- Materials --- p.87 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- DNA extraction from tail --- p.88 / Chapter 3.2.2.2.2 --- PCR tail-genotyping --- p.89 / Chapter 3.2.3 --- "Measurement of serum triglyceride, cholesterol and glucose levels by enzymatic and spectrophometric methods" --- p.89 / Chapter 3.2.3.1 --- Materials --- p.90 / Chapter 3.2.3.2 --- Methods --- p.91 / Chapter 3.2.3.2.1 --- Serum preparation --- p.91 / Chapter 3.2.3.2.2 --- Measurement of serum triglycerides --- p.91 / Chapter 3.2.3.2.3 --- Measurement of serum cholesterol --- p.92 / Chapter 3.2.3.2.3 --- Measurement of serum glucose --- p.93 / Chapter 3.2.4 --- Measurement of serum insulin and leptin levels by ELISA --- p.94 / Chapter 3.2.4.1 --- Materials --- p.95 / Chapter 3.2.4.2 --- Methods --- p.95 / Chapter 3.2.4.2.1 --- Measurement of serum insulin --- p.95 / Chapter 3.2.4.2.2 --- Measurement of serum leptin --- p.97 / Chapter 3.2.5 --- "Histological studies of liver, interscapular BF and gonadal WF pads" --- p.99 / Chapter 3.2.5.1 --- Materials --- p.100 / Chapter 3.2.5.2 --- Methods --- p.100 / Chapter 3.2.5.2.1 --- "Fixation, dehydration, embedding in paraffin and sectioning" --- p.100 / Chapter 3.2.5.2.2 --- H&E staining --- p.101 / Chapter 3.2.6 --- Analyses of fecal lipid contents --- p.102 / Chapter 3.2.6.1 --- Materials --- p.102 / Chapter 3.2.6.2 --- Method --- p.103 / Chapter 3.2.6.2.1 --- Extraction of lipid contents from stools --- p.103 / Chapter 3.2.7 --- Statistical analysis --- p.104 / Chapter 3.3 --- Results --- p.105 / Chapter 3.3.1 --- Confirmation of genotypes by PCR --- p.105 / Chapter 3.3.2 --- PPARβ (-/-) mice were more resistant to high fat diet-induced obesity --- p.105 / Chapter 3.3.3 --- PPARβ (-/-) mice consumed similarly as to PPARβ (+/+) counterparts… --- p.122 / Chapter 3.3.4 --- Effect of high fat diet on organ weights --- p.128 / Chapter 3.3.4.1 --- PPARβ (-/-) mice were more resistant to high fat diet-induced liver hepatomegaly --- p.134 / Chapter 3.3.4.2 --- PPARβ (-/-) mice were resistant to high fat diet-induced increased white fat depots --- p.134 / Chapter 3.3.4.3 --- PPARβ (-/-) mice were resistant to high fat diet-induced increased brown fat mass --- p.137 / Chapter 3.3.5 --- Effect of high fat diet on organ histology --- p.142 / Chapter 3.3.5.1 --- PPARβ(-/-) mice were more resistant to high fat diet-induced liver steatosis --- p.143 / Chapter 3.3.5.2 --- No defect in white adipocyte expansion in PPARβ(-/-) mice upon high fat diet feeding --- p.153 / Chapter 3.3.5.3 --- No defect in brown adipocyte expansion in PPARβ (-/-) mice upon high fat diet feeding --- p.159 / Chapter 3.3.6 --- "Effect on high fat diet on serum cholesterol, triglyceride, glucose, insulin and leptin levels" --- p.164 / Chapter 3.3.6.1 --- "PPARβ (-/-) mice had a lower serum cholesterol level, but a similar triglyceride level as compared to PPARβ (+/+) mice upon high fat diet feeding" --- p.165 / Chapter 3.3.6.2 --- PPARβ (-/-) mice were resistant to high fat diet-induced insulin resistance --- p.167 / Chapter 3.3.6.3 --- PPARβ (-/-) mice had a similar serum leptin level as PPARβ (+/+) mice --- p.170 / Chapter 3.3.7 --- No decision made in fecal lipid content of PPARβ (+/+) and PPARβ (-/-) mice --- p.173 / Chapter 3.4 --- Discussion --- p.176 / References --- p.182 / Appendices --- p.190

Yuen, Yee Lok. / On t.p. "alpha" appears as the Greek letter. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 165-177). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vii / Table of Contents --- p.viii / List of Abbreviations --- p.xiii / List of Figures --- p.xv / List of Tables --- p.xx / Chapter Chapter 1 --- Literature review --- p.1 / Chapter 1.1 --- Peroxisome proliferator-activated receptor (PPAR) --- p.1 / Chapter 1.1.1 --- PPARα isoforms --- p.1 / Chapter 1.2 --- PPARα ligands --- p.2 / Chapter 1.3 --- Biological roles of PPARα --- p.3 / Chapter 1.3.1 --- Lipid metabolism --- p.3 / Chapter 1.3.2 --- Bile acid metabolism --- p.4 / Chapter 1.3.3 --- Biotransformation --- p.6 / Chapter 1.4 --- Roles of PPARα in hepatocarcinogenesis --- p.7 / Chapter 1.4.1 --- Cell proliferation and apoptosis --- p.7 / Chapter 1.4.2 --- Oxidative stress --- p.8 / Chapter 1.5 --- Discovery of novel PPARα target genes --- p.9 / Chapter 1.5.1 --- Identification of a novel PPARα-regulated gene L5#55 by fluorescent differential mRNA display (FDD) analysis --- p.9 / Chapter 1.6 --- Sulfotransferase (SULT) --- p.15 / Chapter 1.7 --- Objective of the present study --- p.16 / Chapter Chapter 2 --- Molecular cloning and characterization of mouse liver sulfotransferase-like (mL-STL) gene --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and methods --- p.17 / Chapter 2.2.1 --- Animals --- p.17 / Chapter 2.2.2 --- Treatments --- p.18 / Chapter 2.2.3 --- Total RNA extraction --- p.18 / Chapter 2.2.3.1 --- Materials --- p.18 / Chapter 2.2.3.2 --- Methods --- p.19 / Chapter 2.2.4 --- Rapid amplification of cDNA ends (RACE) --- p.19 / Chapter 2.2.4.1 --- Materials --- p.19 / Chapter 2.2.4.2 --- Methods --- p.20 / Chapter 2.2.4.2.1 --- Primer design --- p.20 / Chapter 2.2.4.2.2 --- Rapid amplification of 5'- and 3'-cDNA ends --- p.20 / Chapter 2.2.5 --- Cloning of the 5'- and 3' RACE products --- p.25 / Chapter 2.2.5.1 --- Materials --- p.25 / Chapter 2.2.5.2 --- Methods --- p.25 / Chapter 2.2.6 --- Northern blot analysis --- p.28 / Chapter 2.2.6.1 --- Materials --- p.28 / Chapter 2.2.6.2 --- Methods --- p.28 / Chapter 2.2.6.2.1 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.31 / Chapter 2.2.6.2.2 --- PCR DIG-labeling --- p.31 / Chapter 2.2.6.2.3 --- Hybridization and signal detection --- p.32 / Chapter 2.2.7 --- Reverse transcription (RT)-PCR --- p.34 / Chapter 2.2.7.1 --- Materials --- p.34 / Chapter 2.2.7.2 --- Methods --- p.34 / Chapter 2.3 --- Results and discussion --- p.37 / Chapter 2.3.1 --- Cloning of the full-length mL-STL cDNA --- p.37 / Chapter 2.3.2 --- In silico analysis of the mL-STL cDNAs --- p.50 / Chapter 2.3.3 --- Genomic organization of the mL-STL gene --- p.61 / Chapter 2.3.4 --- Tissue distribution of mL-STL mRNA transcript --- p.68 / Chapter 2.3.5 --- "PPARα-dependent regulation of mL-STL mRNA expression by fasting and Wy-14,643 treatment" --- p.74 / Chapter Chapter 3 --- Identification of the native mL-STL protein in mouse liver --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Materials and methods --- p.87 / Chapter 3.2.1 --- Animal and treatments --- p.87 / Chapter 3.2.2 --- Cloning of the mL-STL cDNA into a modified pRSET (mpRSET) expression vector --- p.88 / Chapter 3.2.2.1 --- Materials --- p.88 / Chapter 3.2.2.2 --- Methods --- p.88 / Chapter 3.2.2.2.1 --- Amplification of mL-STL cDNA fragments --- p.88 / Chapter 3.2.2.2.2 --- Preparation of mpRSET expression vector --- p.92 / Chapter 3.2.2.2.3 --- "Ligation, transformation, and screening of recombinants" --- p.92 / Chapter 3.2.3 --- Over-expression of the mL-STL recombinant proteins in E coli strains --- p.94 / Chapter 3.2.3.1 --- Materials --- p.94 / Chapter 3.2.3.2 --- Methods --- p.94 / Chapter 3.2.4 --- Mass spectrometry analysis of the mL-STL recombinant proteins --- p.95 / Chapter 3.2.4.1 --- Materials --- p.96 / Chapter 3.2.4.2 --- Methods --- p.96 / Chapter 3.2.4.2.1 --- Trypsin digestion and peptide extraction --- p.96 / Chapter 3.2.4.2.2 --- Matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometry --- p.97 / Chapter 3.2.5 --- Purification of the mL-STL recombinant proteins --- p.98 / Chapter 3.2.5.1 --- Materials --- p.98 / Chapter 3.2.5.2 --- Methods --- p.98 / Chapter 3.2.5.2.1 --- Semi-purification of the mL-STL recombinant proteins by preparative SDS-PAGE --- p.98 / Chapter 3.2.5.2.2 --- Purification of mL-STL recombinant proteins by column chromatography --- p.99 / Chapter 3.2.6 --- Rabbit immunization using purified mL-STL recombinant proteins --- p.101 / Chapter 3.2.7 --- Subcellular fractionation of mouse liver by ultracentrifugation --- p.101 / Chapter 3.2.7.1 --- Materials --- p.101 / Chapter 3.2.7.2 --- Methods --- p.102 / Chapter 3.2.8 --- Western blot analysis of the native mL-STL protein --- p.104 / Chapter 3.2.8.1 --- Materials --- p.104 / Chapter 3.2.8.2 --- Methods --- p.104 / Chapter 3.2.8.2.1 --- SDS-PAGE and electro-blotting of proteins --- p.104 / Chapter 3.2.8.2.2 --- Immunostaining and signal detection --- p.105 / Chapter 3.3 --- Results and discussion --- p.106 / Chapter 3.3.1 --- Cloning of the mL-STLl and mL-STL2 cDNAs into a modified pRSET (mpRSET) vector --- p.106 / Chapter 3.3.2 --- IPTG induction of the mpRSET-mL-STL protein expression --- p.106 / Chapter 3.3.3 --- Confirmation of mL-STL recombinant proteins by mass spectrometry --- p.118 / Chapter 3.3.4 --- Purification of mL-STL recombinant proteins for rabbit immunization and polyclonal antisera production --- p.130 / Chapter 3.3.5 --- Antigenicity of mL-STL antisera --- p.134 / Chapter 3.3.6 --- Identification of mL-STL native protein and its induction pattern in mouse liver --- p.139 / Chapter 3.3.7 --- "Time-course of fasting and Wy-14,643 treatment on the mL- STLl native protein expression" --- p.147 / Chapter Chapter 4 --- Overall discussion --- p.153 / Future study --- p.163 / References --- p.165 / "Appendix A. Alignment of nucleotide sequences of mouse chromosome 7，Riken2810007J24, mL-STLl, and mL-STL2 cDNA sequences" --- p.178 / Appendix Bl. Theoretical tryptic peptide masses of mpRSET- mL-STLl protein --- p.217 / Appendix B2. Raw data from mass spectrometry analysis of mpRSET-mL-STLl protein --- p.218 / Appendix C1. Residue molecular mass of amino acids --- p.219 / Appendix C2. Di-peptide table --- p.220 / Appendix D1. Theoretical tryptic peptide masses of mpRSET- mL-STL2 protein --- p.221 / Appendix D2. Raw data from mass spectrometry analysis of mpRSET-mL-STL2 protein --- p.222

Xu, Jian. / Thesis Ph.D. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 238-255). / Abstracts also in Chinese. / Title from PDF title page (viewed on 24, October, 2016).

Neuropathic pain affects up to 50% of the 29 million diabetic patients in the United States. Neuropathic pain in diabetes manifests as a disease of the peripheral and central nervous systems. The prevalence of type 2 diabetes is far greater than type 1 (90%), yet the overwhelming focus on type 1 models this has left the mechanisms of pain in type 2 diabetes largely unknown. Therefore I aimed to improve the current mechanistic understanding of pain associated with type 2 diabetes using two preclinical rodent models: Zucker Diabetic Fatty rats and db/db mice. In addition, I highlight the translational importance of simultaneous measurement of evoked/sensory and non-evoked/affective pain-related behaviors in preclinical models. This work is the first to show a measure of motivational-affective pain in a model of type 2 diabetes. I used methodological approaches including: (1) immunohistochemical and calcium imaging to assess stimulus-evoked sensitization; (2) measurement nociceptive behaviors and evoked sensory thresholds as well as pain affect using novel mechanical conflict avoidance and conditioned place preference/aversion assays; (3) pharmacological and genetic manipulation of methylglyoxal, TRPA1, AC1, and PPARγ. I hypothesized that the thiazolidinedione class of peroxisome proliferator-activated receptor gamma (PPARγ) agonists would reduce neuropathic pain-like behavior and spinal neuron sensitization in traumatic nerve injury and type 2 diabetes. As PPARγ is a nuclear receptor, and already targeted clinically to promote cellular insulin sensitization to reduce hyperglycemia, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. In two separate research aims, I challenged this view and tested whether the PPARγ agonist pioglitazone would (1) rapidly alleviate neuropathic pain through a non-genomic mechanism and (2) reduce painful sensitization in nociceptive and neuropathic pain models independent from lowering blood glucose. I aimed to investigate the contribution of the glucose metabolite methylglyoxal to painful type 2 diabetes. I tested the hypothesis that methylglyoxal produces nociceptive, evoked, and affective pain that is dependent on activation of the sensory neuron cation channel TRPA1 and the secondary messenger enzyme AC1. I also tested whether pioglitazone or the novel methylglyoxal scavenging peptide GERP10 could alleviate painful type 2 diabetes.

A la phase aiguë des accidents vasculaires cérébraux, les stratégies thérapeutiques reposent sur l'utilisation d'agents fibrinolytiques dont l'utilisation est limitée par une fenêtre thérapeutique étroite en raison du risque d'hémorragies intra-cérébrales lié à des altérations vasculaires et parenchymateuses. Cette approche fibrinolytique nécessiterait d'être complétée par une double protection de la paroi vasculaire et du parenchyme cérébral. Au sein de l'unité neurovasculaire, les leucocytes, et particulièrement les polynucléaires neutrophiles, jouent un rôle important dans l'inflammation, tant vasculaire que parenchymateuse qui conduit aux lésions cérébrales.<br />Ce travail a pour objectif de déterminer si dans un modèle d'ischémie cérébrale la modulation des interactions leucocytes-endothélium pourrait représenter une cible pertinente : (i) pour diminuer les conséquences lésionnelles de l'ischémie ; (ii) limiter le risque de complications hémorragiques de la thrombolyse. Ceci s'est fait par une double approche pharmacologique : (i) une modulation directe des polynucléaires neutrophiles ; (ii) une modulation des interactions par l'activation des récepteurs nucléaires PPAR-alpha.<br />Dans un premier temps, nos résultats suggèrent une contribution importante des leucocytes dans les altérations vasculaires post-ischémiques ainsi que dans la survenue des complications hémorragiques induites par la fibrinolyse. Dans la deuxième partie de notre travail, l'activation des récepteurs nucléaires PPAR-alpha prévient les altérations endothéliales vasculaires post-ischémiques parallèlement à un effet neuroprotecteur. Ces effets étaient associés à une diminution des complications hémorragiques et du recrutement leucocytaire au sein du foyer ischémique.<br />En conclusion, la protection neurovasculaire après une ischémie cérébrale suivie ou non d'une fibrinolyse apparaît comme une cible intéressante susceptible de prévenir la maturation de l'ischémie cérébrale et les complications hémorragiques. Les récepteurs nucléaires PPAR-alpha représentent expérimentalement une stratégie thérapeutique potentielle de protection de la paroi vasculaire et du tissu cérébral en modulant les interactions leucocytes-endothélium

L'épilepsie affecte environ 1% de la population. Il s'agit d'une maladie où les crises d'épilepsie surviennent de façon spontanée et imprévue. La prise en charge de ces crises peut se faire par des antiépileptiques, mais dans environ 20 à 30% des cas, les épilepsies ne répondent pas ou peu aux médicaments. Il s'agit alors d'épilepsies pharmacorésistantes. Un des traitements de recours utilisé pour la prise en charge de ces épilepsies est le régime cétogène. Le régime cétogène consiste à apporter une large quantité de lipides, pour une faible quantité de protéines et de glucides. Le régime possède des propriétés anticonvulsivantes, décrites chez l'Homme et le rongeur, mais les mécanismes d'action restent inconnus à ce jour. Plusieurs mécanismes ont été suggérés tels que : les propriétés anticonvulsivantes des corps cétoniques, la modulation de la neurotransmission, la modulation de l'excitabilité cérébrale via la modulation de canaux ioniques tels que les KATP. L'implication des acides gras poly-insaturés (AGPI) a également été suggérée. Ceux-ci pourraient moduler la fluidité membranaire, l'activité des canaux ioniques ou encore les voies de l'inflammation. Notre hypothèse de travail a été d'explorer des voies supposées être impliquées dans les propriétés anticonvulsivantes du régime cétogène. Nous nous sommes intéressés aux AGPI et à l'activation des récepteurs nucléaires PPAR-alpha (peroxisome proliferator-activated receptor alpha) en comparaison au régime cétogène. Nous avons utilisé un modèle murin d'état de mal épileptique (lithium-pilocarpine) et l'induction de crises épileptiques avec mesure du seuil au pentylènetétrazole.<br />Dans un premier temps, nous avons administré per-os, pendant 4 semaines un mélange d'AGPI contenant 70% d'oméga-3 et 25% d'oméga-6 à des rats Wistar. Les animaux ayant reçu la complémentation alimentaire par des AGPI présentaient une augmentation du seuil au PTZ comparable à celle obtenue chez les animaux ayant reçu un régime cétogène. Les animaux supplémentés par les AGPI ou ayant reçu le régime cétogène présentaient des variations plasmatiques en AGPI concernant l'acide arachidonique, l'acide alpha linolénique et l'acide eicosapentaenoïque. Aucune modification du statut nutritionnel ou des phospholipides cérébraux membranaires n'était retrouvée. Dans un second temps, nous avons administré pendant 14 jours de la nourriture contenant 0,2% de fénofibrate (agoniste des récepteurs PPAR-alpha) à des rats Wistar. Le traitement par 0,2% de fénofibrate conduisait à augmenter le seuil au PTZ et retarder le début de l'état de mal épileptique dans le modèle lithium-pilocarpine. Ces résultats étaient comparables à ceux obtenus avec le régime cétogène. En revanche le traitement associant le régime cétogène et le fénofibrate ne conduisait pas à moduler le seuil au PTZ chez les animaux.<br />Ces travaux ont permis de montrer que les AGPI ont des propriétés anticonvulsivantes, comparables à celles du régime cétogène. Ces propriétés anticonvulsivantes ont également été retrouvées suite à l'activation des récepteurs nucléaires PPAR-alpha par le fénofibrate. Les propriétés anticonvulsivantes portées par les AGPI ne sont pas liées à une variation de la composition des membranes cellulaires cérébrales en phospholipides. Les récepteurs nucléaires PPAR-alpha modulent quant à eux de nombreuses voies (métaboliques, inflammatoires, stress oxydant) via des variations d'expression génique et peuvent être activés par les AGPI. L'implication de ces différentes voies dans l'efficacité anticonvulsivante du fénofibrate, reste à explorer. Ces résultats, s'ils sont confirmés par des études complémentaires dans d'autres modèles, laissent penser qu'une simplification du régime cétogène pourrait être envisagée via l'utilisation des AGPI et/ou via l'activation des récepteurs nucléaires PPAR-alpha

A central aim in drug development is to ensure that the new drug is efficacious and safe in the intended patient population. Mathematical models describing the pharmacokinetic-pharmacodynamic (PK-PD) properties of a drug are valuable to increase the knowledge about drug effects and disease and can be used to inform decisions. The aim of this thesis was to develop mechanism-based PK-PD-disease models for important safety and efficacy biomarkers used in anti-diabetic drug development. Population PK, PK-PD and disease models were developed, based on data from clinical studies in subjects with varying degrees of renal function, non-diabetic subjects with insulin resistance and patients with type 2 diabetes mellitus (T2DM), receiving a peroxisome proliferator-activated receptor (PPAR) α/γ agonist, tesaglitazar. The PK model showed that a decreased renal elimination of the metabolite in renally impaired subjects leads to increased levels of metabolite undergoing interconversion and subsequent accumulation of tesaglitazar. Tesaglitazar negatively affects the glomerular filtration rate (GFR), and since renal function affects tesaglitazar exposure, a PK-PD model was developed to simultaneously describe this interrelationship. The model and data showed that all patients had decreases in GFR, which were reversible when discontinuing treatment. The PK-PD model described the interplay between fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c) and haemoglobin in T2DM patients. It provided a mechanistically plausible description of the release and aging of red blood cells (RBC), and the glucose dependent glycosylation of RBC to HbA1c. The PK-PD model for FPG and fasting insulin, incorporating components for β-cell mass, insulin sensitivity and impact of disease and drug treatment, realistically described the complex glucose homeostasis in the heterogeneous patient population. The mechanism-based PK, PK-PD and disease models increase the understanding about T2DM and important biomarkers, and can be used to improve decision making in the development of future anti-diabetic drugs.

Colorectal cancer is the 4th most widespread cause of cancer mortality. One of the pathways that are involved in the development of colorectal cancer is the arachidonic acid metabolizing lipoxygenase (LOX) pathway. Inflammatory molecules formed from this pathway exert profound effects that may exacerbate the development and progression of colon and other cancers. 15 lipoxygenase-1 (15-LOX-1) is a member of LOX protein family that metabolizes primarily linoleic acid to 13-(S)-HODE. Several lines of evidence support an antiangiogenic role for 15-LOX-1, especially through 13-(S)-HODE. The expression of 15-LOX-1 is lost in colon cancer cells. Our aim in this thesis was to study whether 15-LOX-1 expression has an anticarcinogenic role, particularly on the metastatic and angiogenic potential of colon cancer cells. For this purpose, 15-LOX-1 was introduced into HCT-116 colon cancer cell lines. Having confirmed 15-LOX-1 expression and activity it was observed that expression of 15-LOX-1 significantly decreased cell proliferation, cell motility, anchorage-independent growth, migration and invasion across Matrigel, the expression of the metastasis-related MTA-1 protein, neoangiogenesis and induced apoptosis. Mechanistically, most of these effects were arbitrated by the 15-LOX-1 mediated inhibition of the inflammatory transcription factor NF-&kappa / B via the orphan nuclear receptor PPAR&gamma / . In conclusion, we propose that 15-LOX-1 has anti-tumorigenic properties and can be exploited for therapeutic benefits.