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ABCG2 est un transporteur de la famille ABC impliqué dans le phénotype de résistance aux drogues développé par certaines cellules, par exemple les cellules cancéreuses. Ce transporteur a aussi un rôle physiologique de détoxication de composés endogènes, notamment les porphyrines, molécules indispensables mais qui présentent une toxicité potentielle. Cette toxicité nécessite une prise en charge particulière, évitant à ces composés d’être libres en solution. Dans ce contexte, nous avons fait l’hypothèse qu’ABCG2 pourrait participer à cette détoxication en limitant l’accumulation des porphyrines dans les cellules en les présentant à un partenaire extracellulaire. Nous montrons qu’ABCG2 transporte de l’hème ainsi que certains de ses dérivés et précurseurs et que ces porphyrines, contrairement aux autres substrats d’ABCG2, se fixent sur un domaine extracellulaire spécifique d’ABCG2, ECL3, composé d’environ 70 acides aminés. L’affinité d’ECL3 pour les porphyrines est de 0,5 à 3,5 μM, suffisamment affine pour permettre leur fixation après transport.Nous montrons aussi que l’albumine sérique humaine, impliquée dans la détoxication de l’hème, récupère les porphyrines fixées sur ECL3 par une interaction directe avec ABCG2. L’ensemble de ce travail a donc permis d’une part de mieux comprendre le rôle d’ABCG2 dans la régulation de l’homéostasie des porphyrines, notamment l’hème, et d’autre part, de façon originale, d’identifier le mécanisme moléculaire par lequel cette détoxication s’effectue. / ABCG2 belongs to the ABC-transporter family, involved in drug resistance developed by cells, notably cancer cells. This transporter has also a physiological role of endobiotic detoxification, in particular porphyrins that are essential but potentially toxic molecules. This toxicity implies a specific handle, to avoid them to remain free in solution. In that context, we hypothesized that ABCG2 participate to this detoxification, limiting the intracellular porphyrin accumulation by presenting them to an extracellular partner. We show that ABCG2 transports heme and some of its derivatives and precursors. Interestingly, these porphyrins, unlike other ABCG2 (non-porphyric) substrates, can bind to an extracellular domain, specific of ABCG2, ECL3, 70 residues-long. ECL3 displays affinities for porphyrins in the range of 0.5 to 3.5 μM, high enough to allow their binding after transport. We also show that human serum albumin, implicated in heme detoxification, releases porphyrins bound to ECL3 by a direct interaction with ABCG2. This work established a better comprehension of ABCG2 role in porphyrin and in particular heme homeostasis regulation. In addition, our results contribute to elucidate part of the molecular mechanism by which such regulation is carried out.

Thesis (Ph.D.). / Written for the Institute of Parasitology. Title from title page of PDF (viewed 2008/02/12). Includes bibliographical references.

L'obésité est une maladie multifactorielle qui constitue un facteur de risque de nombreuses pathologies, notamment les maladies cardiovasculaires, le diabète et les maladies neurodégénératives. Des études épidémiologiques récentes suggèrent un effet obésogène des contaminants environnementaux, mais peu d'informations sont disponibles sur leur effet potentiel sur le métabolisme des lipoprotéines hépatiques. L'objectif de cette étude était de déterminer l'effet de polluants environnementaux de la famille des hydrocarbures aromatiques polycycliques (HAP) sur trois récepteurs des lipoprotéines, le récepteur des LDL (LDL-R), le lipolysis-stimulated lipoprotein receptor (LSR) et le scavenger receptor B1 (SR-B1) ainsi que sur les ATP binding cassette transporter A1 (ABCA1) et G1 (ABCG1) en utilisant des modèles cellulaires et/ou animaux. Les études par immunoblots et immunofluorescence in vitro ont révélé que l'exposition de cellules Hepa1-6 au B[a]P diminue de manière significative les taux de protéine LSR, LDL-R et ABCA1, alors qu’aucune modification significative du taux et de l’activité du LSR n’a été observée lors d’une exposition au pyrène ou au phénanthrène. L’analyse en temps réel par PCR et les études avec la lactacystine ont révélé que cet effet était dû principalement à une augmentation de la dégradation par le protéasome plutôt qu’à une diminution de la transcription ou à une augmentation de la dégradation lysosomale. En outre, les ligand-blots ont révélé que les lipoprotéines exposées au B[a]P avaient une affinité réduite pour le LSR ou le LDL-R. Des souris C57Bl/6RJ ont été traitées par le B[a]P (0,5 mg / kg, i.p) toutes les 48 h pendant 15 jours. Le gain de poids observé est accompagné d’une augmentation des taux de triglycérides et de cholestérol plasmatiques, du taux de cholestérol hépatique, et d’une diminution du taux de LDL-R et ABCA1 chez animaux traités au B[a]P par rapport aux témoins. Les corrélations observées entre les taux de LSR et de LDL-R hépatiques chez les souris contrôle ne sont plus observées chez les souris traitées au B[a]P, ce qui suggère un dérèglement du métabolisme des lipoprotéines hépatiques. Ces résultats suggèrent que la prise de poids induite par le B[a]P est peut-être liée à son action inhibitrice sur le LSR et le LDL-R, ainsi que sur l’ABCA1 et le métabolisme des lipoprotéines hépatiques, ce qui conduit à un statut lipidique modifié chez les souris traitées au B[a]P, donnant ainsi un nouvel éclairage sur les mécanismes sous-jacents de la contribution des polluants tels que le B[a]P à la perturbation de l'homéostasie lipidique, susceptible de contribuer à la dyslipidémie associée à l'obésité / Obesity is a multifactorial disorder that represents a significant risk factor for many pathologies including cardiovascular diseases, diabetes and neurodegenerative diseases. Recent epidemiological studies suggest potential obesogenic effects of environmental contaminants, but little information is available on their potential effect on hepatic lipoprotein metabolism. The objective of this study was to determine the effect of the common environmental pollutants, belonging to polycyclic aromatic hydrocarbon (HAP) on three lipoprotein receptors, the LDL-receptor (LDL-R), the scavenger receptor B1 (SRB1) and the lipolysis-stimulated lipoprotein receptor (LSR) as well as the ATP binding cassette transporters A1 (ABCA1) and G1 (ABCG1) using cell and/or animal models. Immunoblot and immunofluorescence in vitro studies revealed that exposure of Hepa1-6 to benzo[a]pyrene (B[a]P) significantly decreased LSR, LDL-R and ABCA1 protein levels, whereas no significant changes in protein levels and LSR activity where observed upon cell treatment with pyrene or phenanthrene. Real-time PCR analysis, lactacystin and chloroquine studies revealed that this effect was due primarily to increased proteasome-mediated degradation rather than to decreased transcription or to increased lysosomal degradation. Furthermore, ligand blots revealed that lipoproteins exposed to B[a]P displayed markedly decreased binding to LSR or LDL-R. C57Bl/6RJ mice were treated with B[a]P (0.5 mg/kg, i.p) every 48 h for 15 days. The increased weight gain observed was accompanied by increased plasma triglycerides and cholesterol levels, increased liver cholesterol content, and decreased LDL-R, ABCA1, ABCG1 and SR-B1 protein levels in B[a]P-treated animals as compared to controls. Correlations observed between hepatic LSR and LDL-R levels in control mice were no longer observed in B[a]P treated mice, suggesting a potential dysregulation of hepatic lipoprotein metabolism.Taken together, these results suggest that B[a]P-induced weight gain may be due its inhibitory action on LSR and LDL-R, as well as ABCA1 and lipoprotein metabolism in the liver, which leads to the modified lipid status in B[a]P-treated mice, thus providing new insight into mechanisms underlying the involvement of pollutants such as B[a]P in the disruption of lipid homeostasis, potentially contributing to dyslipidemia associated with obesity

A dissertation submitted to the Faculty of Medicine University of the Witwatersrand, Johannesburg For the degree of Master of Science in Medicine / Chloroquine-resistant Plasmodium falciparum accumulate significantly less chloroquine than susceptical parasites, and this is thought to be the basis of their resistance ( Fitch, 1970 ). Martin et. al ( 1987 ), recently demonstrated that in the presence of verapamil, a calcium channel blocker, chloroquine-resistant P falciparum becomes chloroquine-sensitive, with an increase in the chloroquine accumulation.The mechanism of such reversal has yet to be elucidated / IT2018

Thesis (M.D.) -- University of Texas Southwestern Medical Center at Dallas, 2007. / Vita. Bibliography: pp.28-30

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers in Hong Kong and Southeast Asian countries. Cancer progression is often symptomless, making the early diagnosis difficult, thus leading to a high mortality rate. Treatments against HCC were often found to be less effective than other cancers. Systemic chemotherapy, which is widely used in cancer treatments, has a low response rate in HCC. New treatment regimes, such as targeted therapy, have shown partial responses in clinical trials and therefore continuous effort in searching new drug targets is warranted. Granulin-epithelin Precursor (GEP) is a pluripotent growth factor, and has been shown to be overexpressed in HCC and various cancers. Our group has demonstrated that GEP promotes tumor growth, and regulates chemoresistance in HCC. It shares a highly similar expression pattern with one of the members of ATP-binding cassette (ABC) transporter family, ABCF1. Blocking GEP, both in vitro and in vivo, showed inhibition on HCC growth. These suggest that GEP is a potential target for HCC treatment. However, there is still little information on how GEP and ABCF1 is overexpressed in HCC. This project aims to investigate the mechanisms involved. GEP and ABCF1 genes are located on chromosomes 17q and 6p, respectively, which both are frequently amplified in HCC. We used quantitative microsatellite analysis (QuMA) to detect GEP and ABCF1 amplification in HCC samples. Both GEP and ABCF1 showed about 20% of HCC cases having amplification, and their copy numbers correlated to the mRNA expression levels. The copy numbers of GEP were also found to correlate to those of ABCF1 significantly. Clinico-pathological analysis showed that GEP copy numbers correlated with gender, serum AFP levels and HBV status, while ABCF1 did not associate with any of the clinico-pathological features. Fluorescence in situ hybridization (FISH) was performed to validate the results on DNA copy number by QuMA. The cases with highest DNA copy number on GEP and ABCF1, were examined. The average difference between FISH and QuMA results ranged ± 0.3 copies, indicating QuMA and FISH results were corroborated on DNA copy number. Furthermore, the FISH results indicated that there are different degrees of aneuploidy involved in chromosome 6p and 17q in 5 out of 6 cases investigated. These suggest that the copy number variations in GEP and ABCF1 were partly caused by the abnormal number of chromosomes. In summary, we observed that GEP and ABCF1 gene copy numbers were increased in subsets of HCC cases, and the increase correlated to their respective transcript expression levels. Furthermore, these copy number variations partly could be explained by aneuploidy as demonstrated by FISH analysis. The current study may help to understand the complex genomic aberrations in HCC and allow better treatment designs in the future. / published_or_final_version / Surgery / Master / Master of Philosophy

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

Protein synthesis is a highly conserved process across all domains of life, both structurally and functionally. This cyclic process is catalyzed by numerous soluble protein factors that interact with the ribosome to facilitate efficient protein synthesis. Many canonical translation factors bind and hydrolyze GTP to induce conformational changes that facilitate translation. For example, GTP hydrolysis by EF-Tu is required for the release of aminoacyl-tRNA into the ribosomal A site; GTP hydrolysis by EF-G facilitates the movement of tRNA and mRNA from the A site to the P site of the ribosome. However, protein synthesis seems to also have a requirement for ATP; the essential yeast protein eEF-3 facilitates release of deacyl-tRNA from the ribosomal E site. In Escherichia coli, the protein product of the open reading frame yhih has been suggested to have a similar function. However, the role of this unique prokaryotic protein is not understood. Preliminary characterization of this protein suggests a nucleotide-dependent conformational change occurs in a truncated form of the protein, ΔP541 Yhih. Interestingly, this phenomenon is not observed in ΔL432 Yhih. Both ΔP541 Yhih, and to a lesser extent ΔL432 Yhih, exhibit a ribosome-dependent ATPase activity, suggesting the primary region for binding with the ribosome lies between Leu432 and Pro541. / x, 101 leaves : ill. ; 29 cm.