Progress 10/13/99 to 10/12/05
Outputs
Copper (Cu) is an essential mineral/nutrient in human and animal health. Interruptions in Cu tranport or excretion are the basis for many chronic and spontaneous human and animal diseases, such as Alzheimer's diseae, Mad Cow disease, and Sway back. Wilson and Menkes diseases in humans arise through faulty Cu homeostasis and both have been linked to the expression of Cu-dependent ATPase enzymes that reguate the flow of Cu into the system and out of cells. Our studies of the homeostasis mechansim has revealed unexpected modifications to the mRNA that codes for the Cu ATPase enzyme. Modification include smaller transcripts that may play a role in intracellualr Cu movement as well as stop codons that cancel expression entirely. Through compartive assessments we concluded that the promoter region of the Cu ATPase gene in higher animals has structural elements that respond to Cu. Such elements allow genetic expression to be linked with the Cu status of the cell or
individual. Overall, the research has revealed that the synthesis and processing of the ATPase mRNA should be a focus for gaining insight into changes that occur with aging and why it is necessary to have a functioning ATPase enzyme to protect against Cu toxicity and to assure safe use of Cu in normal metabolism.
Impacts
This researh recognizes the importance of Cu as an essential nutrient and provides insight into how a living system can use this element safely, avoiding toxicity. The research has also helped in the understanding of the role of Cu in preventing diseases and debilitating conditions we associate with faulty nutrition and aging.
Publications
- Harris, E.D. (2001) Copper homeostasis: the role of cellular transporters. Nutr. Rev. 59, 281-285.
- Harris, E.D.(2001) Cellular transporters for zinc. Nutr. Rev. 60, 121-124.
- Harris, E.D. (2003). Basic and clinical aspects of copper. Rev. Clin. Lab. Sci. 40, 1-40.
- Harris, E.D. and Majumdar, S. (2003). Pre- and post-transcriptional regulation of the Menkes Disease gene. J. Tr. Ele. Exp. Med. 16, 1-9.
- Harris, E.D. (2003). A requirement for copper in angiogenesis. Nutr. Rev. 62:60-64.
|
Progress 01/01/04 to 12/31/04
Outputs
Copper (Cu)is both toxic and essential to human health. Its safe use defines Cu nutrition and challeges one to consider how cells are protected from Cu toxicity. Our program studies the role of membrane-bound Cu-ATPases in modulating cellular Cu levels. These proteins use the energy of ATP to export Cu from cells. In studying the genetic expression of Cu-ATPases, we have found segments in the promoter that respond to high and low Cu. We have also isolated proteins from the cell nucleus that appear to recognize the elements. Our immediate goal is to purify and identify these proteins and study their Cu-binding properties and eventually to learn how they control the expression the Cu-ATPase genes.
Impacts
To date only yeast are know to have genes that sense a low and high cellular environment of Cu. The finding of structurally similar elements in the promoter of the human Cu-ATPases is evidence that Cu may regulate the expression of these important homeostatic proteins. From a nutritional view point, responding to low (as opposed to high) Cu enhances cellular retention when Cu is in short supply and protects the system when Cu is in excess. Nutritional studies in humans have shown that the fractional absorption of Cu across the intestine increases as the amount of Cu in the diet decreases. This research has provided insight into a possible explanation for this behavior.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
A previous report detailed the finding of metal-response elements (MREs) in the promoter region of the Menkes disease gene. Cloning the specific promoter element that senses Cu has now been achieved and we are poised to learn the role of bases in the interaction. The sequence TTTCATGCTG, has been the subject of further investigation. Using a luciferase reporter gene with the putative MRE elements downstream of an SV40 promoter element, it was found that luciferase expression in SH-SY5Y cells in culture was modulated by the Cu content of the medium. Cu levels amplified by the addition of 100 mM CuCl2 showed less reporter gene activity compared to cells grown in an ambient Cu medium. In contrast, cells grown in medium supplemented with 100 mM bathocuproine sulfonate, a Cu(I) chelator, were nearly twice as strong in their expression. These findings basically are consistent with earlier results obtained from HepG2 cells and provide further support that the Menkes disease
gene, which controls Cu homeostasis via an export-driven mechanism, is activated by low and as opposed to high cellular Cu levels.
Impacts
To date only yeast genes that encode Cu transport proteins and their translation regulators are known to have promoter elements that sense a low cellular environment of Cu. The finding of structurally similar elements in Menkes disease gene provided evidence, and now direct experimental proof, that this gene responds to a low Cu status. From a nutritional view point, responding to low (as opposed to high) amounts of Cu may be a means to regulate Cu absorbing proteins in humans when dietary Cu is in short supply. Fractional absorption of Cu across the intestine increases as the amount in the diet decreases. This research has provided insight into a possible mechanism.
Publications
- No publications reported this period
|
Progress 01/01/02 to 12/31/02
Outputs
The research has investigated, with the hope of identifying, transcription factors that control the expression of genes that code for Cu-dependent proteins. A gene coding for a Cu-transporting ATPase is the focus. Previous data has shown that the gene is active in embryonic liver, but silenced in the adult. Two potential Cu-sensitive elements have been identified in the promoter region of the gene. Expression of a luciferase reporter gene driven by promoter elements was found to depend on the Cu status of HepG2 cells in culture. Cu levels in the cells were controlled by bathocuproine sulfonate. Cells deprived of Cu through the action of the chelator were more prone to show greater expression of the Cu-ATPase gene. The enhanced expression correlated with the presence or absence of a Cu-sensing element in the promoter that had the sequence TTTCATGCTG. Using PCR generated DNA fragments containing the elements, we have succeeded in obtaining gel-shift and Southwester
blotting evidence for two nuclear proteins that recognize the promoter elements. Sequence data on these protein is currently underway as well as studies to learn the precise bases in the promoter that engage one or both proteins.
Impacts
Knowing how Cu regulates genes that control the expression of Cu-binding proteins will open new insights into the essentiality of Cu in human and animal health. It seems certain that Cu has the capacity to control its own metabolism in cells. This research will determine the nature of the factors that engage Cu and take part in the homeostasis mechanisms.
Publications
- Harris, E.D., Reddy, M.C.M., and Majumdar, S. (2002). The meaning of alternative transcripts of the Menkes disease gene. In: Handbook of Copper Pharmacology and Toxicology (Massaro, E., ed) The Humana Press, Inc., pp. 333-341.
- Reddy, M.C.M., Majumdar, S., Cantera, M., and Harris, E.D. (2002) Pretranslational control of Menkes disease gene expression. Biometals 16, 55-61.
- Garrick, M.D., Nunez, M.T., Olivares, M. and Harris, E.D. (2002). Parallels and contrasts between iron and copper metabolism. Biometals 16, 1-8.
|
Progress 01/01/01 to 12/31/01
Outputs
Our research focuses on the expression of the gene for a Cu-ATPase in specific cell types and during development. We have cloned a 2.2 kb DNA fragment of the promoter and have conducted reporter gene (luciferase) assays to identify active groups that regulate promoter activity. Sequence analysis of the distal region revealed two potential elements. One has the sequence motif of the binding site (Hb) for the Hunchback gene in Drosophila. The other resembled the binding site (Fh) for the forkhead gene. Both Hb and Fh are known to regulate gene expression of higher organisms including humans. Removing a promoter fragment with the Hb and Fh sites stimulated promoter activity in two human cell lines, suggesting a suppressive function when active. Mutating the Hb site, however, abolished promoter activity. Fragments containing wild type sequences strongly enhanced SV40 promoter activity, whereas a mutated fragment was either functionless or slightly suppressive. The
analysis revealed that occupancy of the Hb site emulated an active state, whereas Fh occupancy suggested a suppressed state. Further involvement of the distal promoter region was seen in gel shift analysis (EMSA) in which proteins from two humans cell line were shown to interact with a distal fragment containing the Hb and Fh sites. The data also suggested that proteins from liver were qualitatively or quantitatively different from proteins from brain cells, which is consistent with cell specific regulation. Our studies, therefore, point to positive and negative control elements in the distal promoter region of the gene. They have contributed to a better understanding of the factors that regulate gene expression.
Impacts
While 0.9 mg has been established as a safe and adequate amount of copper for the average human to consume in the diet each day, there is still an urgent need to learn more about the metabolism of this essential mineral element as well as its complete scope of biological functions. Copper is known to interact with components at the genetic level of cell functions and in so doing regulate the expression of genes, including those involved in its metabolism. One such gene codes for a protein that controls the absorption of copper by the intestine and brain. A defect in the gene leads to Menkes disease, a fatal X-linked disorder affecting male infants which terminates life on or before the third year. Our research is aimed at learning how the gene controlling copper homeostasis is regulated during development and in specific cell types. We have turned to examining the promoter of the gene and have identified nucleotide bases in the distal region that may play a role in
activating or suppressing gene expression. By mutating the suspected elements we have been able to deter expression of the gene in two human cell lines, showing that these elements activate or suppress promoter activity and that a disproportionate combination of the two will ultimately determine the extent to which the gene is expressed (or inhibited).
Publications
- HARRIS, E.D. (2000) Differential PCR and DNA microarrays, the era of modern nutrition. Nutrition 16, 174-175
- HARRIS, E.D. Copper and iron: a landmark connection of two essential metals (2001). J. Tr. Elem. Exp. Med. 14, 207-210
- HARRIS, E.D. (2001) Zinc and copper: evidence for interdependence, not antagonism. Nutrition 19, 734.
|
Progress 01/01/00 to 12/31/00
Outputs
Studies to learn the mechanism of Cu transport in human nutrition were carried out in 5 cell types (HepG2, BeWo, Caco-2, SY5Y, and normal fibroblasts). The studies used mRNA that coded for a Cu-transporting membrane ATPase (ATP7A) and PCR to amplify the coding region. Unexpectedly, PCR application gave rise to 4 transcripts that appeared to code for abridged proteins that could play a role in the transport. All 5 cell types showed the same pattern, which supports a uniform mechanism across all cells. One transcript coded for a protein that was missing exons 3-15, which effectively eliminated 2 of the 6 Cu-binding sites, 2 of the 8 transmembrane domains, and the CPC channel motif. A second transcript, however, coded for a nuclear localization sequence at the N-terminus. The product of the second transcript, when fused to Green Fluorescent protein localized in the cell nucleus. The data suggest that mRNA that codes for a membrane-bound Cu transporting protein could have
a dual purpose in coding for other protein involved in Cu transport. A select mRNA splicing mechanism is suggested. The discovery of a Cu-binding peptide with a nuclear localization signal is the first report of its kind to show a mRNA coding for a protein that selectively transports Cu into the nucleus of mammalian cells. The protein appears to be a nuclear chaperone.
Impacts
(N/A)
Publications
- Harris, E.D. (2000) Transport and cellular metabolism of copper. Ann. Rev. Nutr. 20,291-310.
- Reddy, M.C.M., Majumdar, S., and Harris, E.D. (2000) Evidence for a Menkes-like protein with a nuclear targeting sequence. Biochem. J. 350, 855-863.
|
Progress 01/01/99 to 12/31/99
Outputs
The overall objective of this project is to learn the molecular events in the movement of metals through placental cells by identifying the key components that take part in the transition. Specifically we will: 1)identify cellular Cu-binding proteins to learn if ceruloplasmin (Cp) enters or Cu passes the membrane as a Cp-free ion Cu-binding ligands in the membrane 3)isolate the membrane proteins that receive Cu from Cp4 4)determine if placental Cp translocates Cu between maternal and fetal circulations. These objectives will be achieved using a variety of approaches. These will include: 1)establishing an epithelial membrane barrier (syncytiotrophoblast) model 2)determining the status of copper delivered to the cells 3)isolation and purification of ceruploplasmin receptors and Cu-binding proteins from placentas 4)characterization of the intracellular transport mechanism 5)determining if BeWo cells express Cp mRNA and are capable of synthesizing an assembled protein
that incorporates 67Cu. Menkes disease and Wilson disease represent two conditions where a defect in copper uptake and distribution can be pathogenic. The purpose of this project is to learn the molecular events in the movement of metals through placental cells.
Impacts
(N/A)
Publications
- Harris, E.D. Reddy M.C.M., Qian, Y., Majumdar, S., and Nelson, J. (1999) Multiple forms of the Menkes CuATPase. Adv. Exp. Med. Biol. 448, 39-51.
- Harris, E.D., Reddy, M.C.M., Majumdar, S. (1999) Menkes Gene, A potential locus for copper metabolism. J. Tr. Ele. Exp. Med. 12, 331-335.
- Harris, E.D. (1999) Ceruloplasmin and iron, vindication after 30 years. Nutrition 15, 72-73.
|
Progress 01/01/98 to 12/31/98
Outputs
Our research efforts continue to focus on the Cu-transporting ATPase enzyme that has been established as a key factor for achieving copper homeostasis in tissues and organs. The Cu-ATPase holds the key to learning how cells regardless of species obtain and use copper, an essential micronutrient. Our current focus is on the Cu-ATPase gene expression. We use human cells to study the synthesis and location of the gene product. Recently, we showed that the 5' end of the some of the mRNAs that code for the Cu-ATPase is modified. We further determined that there are also internal modifications resulting in spliced variants that have the potential to give rise to Menkes-like proteins with presumably important functions in copper transport. The alternative forms could code for copper chaperones, small proteins that deliver copper to organelles. These alternate forms arise by an as yet uncharacterized splicing mechanism that selectively removes exons from the full-length mRNA.
One alternative mRNA lacks exons 3-15 and is unable to localize in the Golgi apparatus. Our hypothesis is to consider that the gene coding for the copper-transporting ATPase may code for more a multitude of proteins that take part in copper movement in the cell. The different forms may be specific for recognizing organelles and delivering copper to key enzymes within these organelles.
Impacts
(N/A)
Publications
- REDDY, MCM and HARRIS, ED. 1998. Multiple transcripts coding for the Menkes gene. Evidence for alternative splicing of Menkes mRNA. Biochem. J. 334, 71-77.
- HARRIS ED, QIAN Y, and REDDY, MCM.. 1998. Genes regulating copper metabolism. Molec. Cell. Biochem. 188, 57-62.
- QIAN Y, TIFFANY-CASTIGLIONI E, and HARRIS, ED. 1995. Copper transport and kinetics in cultured astroglial cells. Am. J. Physiol. 269 (Cell Physiol. 38), C892-C898.
- TIFFANY-CASTIGLIONI, LEGARE ME, SCHNEIDER, LA, HARRIS ED, BARTHOUMI R, ZMUDKI J, QIAN Y, and BURGHARDT, RC. 1996. Heavy Metal Effects on Glia. Methods in Neurosciences 30:135-166.
- HARRIS, ED. 1996. Copper. In: Handbook of Nutritionally Essential Mineral Elements, O'Dell, B.L. and Sunde R. Eds. Marcel Dekker, pp. 231-273.
- QIAN Y, TIFFANY-CASTIGLIONI E, WELSH J, and HARRIS ED. 1998. Copper entry into murine CNS is controlled by a Menkes Cu-ATPase. J. Nutr. 128, 1276-1282
- QIAN Y., TIFFANY-CASTIGLIONI E and HARRIS ED. 1998. Sequence of a Menkes-type Cu-transporting ATPase from rat C6 glioma cells: comparison o f the rat protein with other mammalian Cu-transporting ATPases. Molec. Cell. Biochem. 181, 49-61.
- HARRIS E.D., QIAN Y., TIFFANY-CASTIGLIONI, E, LACY AR and REDDY MCM. 1998. A functional analysis of copper homeostasis in cell culture models. A new perspective on internal copper transport. Am. J. Clin. Nutr. 67, 988S-995S.
|
Progress 01/01/97 to 12/31/97
Outputs
Research in this laboratory continues to focus on the Cu-transporting ATPase enzyme that has been shown to be a major factor in achieving copper homeostasis in tissues and organs. The Cu-ATPase is fast becoming to a focus of attention for learning the precise mechanism by which cells obtain and retain copper, an essential micronutrient. We have been studying the expression of the Cu-ATPase gene in human cells as a means to learn the function of the Cu-ATPase and factors that regulate its expression. In our most recent studies we have identified sequences at the 5' end of the gene that lead us to suspect there may be more than one Cu-ATPase expressed by cells. We have followed these sequences and have deduced that the alternative forms of the protein are likely to play a role in deciding cellular location. We have confirmed the presence of the sequences in genomic DNA which leads us to suspect that alternative forms arise by a splicing mechanism that is selective for
the exons that will be included in the mRNA. One of the alternative forms is a truncated version of the original Cu-ATPase that appears to serve as a transport protein for copper. Our feeling at this time is what has been considered a gene for the copper-transporting ATPase is in reality a copper locus capable of generating many different forms of the protein each with a specific function to perform and each involved in the transport of copper into the cell, out of the cell or within the cell.
Impacts
(N/A)
Publications
- QIAN, Y., TIFFANY-CASTIGLIONI, E., AND HARRIS, E.D. 1997. A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat. Mole. Brain Res. 48:60-66.
|
Progress 01/01/96 to 12/30/96
Outputs
We have studied a newly discovered Cu-transporting ATPase enzyme known to be a lesion site of Menkes disease &, to date, the most solid biochemical component for explaining copper homeostasis in cells. The ATPase is known to be associated with the transport of nutrients from maternal blood to fetal blood. Our goal is to learn how transport is accomplished. We have used a human cell line (BeWo) to study the mechanism. Copper transport is severly disrupted in Menkes disease. We have determined that the Menkes gene is under close regulation in placental cells. Cells grown on plastic surfaces fail to show any sign of the mRNA for the ATPase. On the other hand, cells grown on filters or exposed to 2.5 mM butyrate show the mRNA as detected by RT-PCR. We have correlated gene expression with Cu efflux. In other studies we have determined that the Menkes ATPase is also expressed in two brain cells, astroglia and neurons. We have completed the sequence of the rat gene protein
(Atp7a) & found it to have a 94% identity with human. Our findings of the Atp7a in brain cells could not fully explain why brain from Menkes infants are low in Cu. Thus, we have undertaken a search of Menkes-like gene in blood brain barrier cells to see if these cells express the Atp7a mRNA. The initial attempts were successful in showing the gene in mouse microvascular epithelial cells that serve as the barrier cells. At present efforts are being to sequence the gene to confirm its identity.
Impacts
(N/A)
Publications
- QIAN Y., TIFFANY-CASTIGLIONI E., and HARRIS, E.D. 1996. Functional ahalysis of genetic defect of copper transport (Menkes disease) in different cell lines. Am.J. Physiol. (Cell Physiol. 40) C378-C384.
- QIAN Y., TIFFANY-CASTIGLIONI, E., and HARRIS, E.D. 1996. Coincident Expression of Menkes gene with copper efflux in human placental cells. Am. J. Physiol. (Cell Physiol. 39) C1880-C1884.
- QIAN Y., TIFFANY-CASTIGLIONI, E., and HARRIS, E.D. 1996. A Menkes P-type ATPase involved in copper homeostasis in the central nervous system of the rat. Mole. Brain Res. (In Press).
|
Progress 01/01/95 to 12/30/95
Outputs
The transport of essential trace elements from mother to fetus during pregnancy has very little understanding as to mechanism. We have employed a human placental cell line (BeWo) to anwer basic questions. The recent discovery of a Cu-transporting ATPase in the membrane of placental tissue has put the project in clearer focus. We have determined that BeWo cells express the genes for two Cu-transporting ATPase. One ATPase is the product of a gene (MNK)associated with Menkes disease in human infants, the other with the gene for Wilson's disease (WND)in mature adults. Tissue- and cell-specific faults in copper distribution can be traced to failure to express either gene product. We have determined that only BeWo cells grown on porous filter surfaces as opposed to solid plastic surfaces express the MNK gene. These cell take on a Menkes-type character in being unable to efflux Cu that has been absorbed from the medium. The expression of the gene seems to correlate with the
establishment of Cu efflux behavior in the cells. The data support the hypothesis that cells that do not efflux absorbed Cu do not express MNK whereas competent effluxing cells give the MNK signal as determined by RT-PCR. It remains to be determined if the release of Cu from the cell via the Cu-transporting ATPase in the membrane is one way Cu ions are forced across the placental barrier separating mother from fetus.
Impacts
(N/A)
Publications
- Brenner A.J. and Harris E.D. 1995. A quantitative test for copper using bicinchoninic acid. Anal. Biochem. 226, 80-84.
- Harris E.D. 1995. The iron-copper connection: the link to ceruloplasmin grows stronger. Nutr. Rev. 53:170-173.
- Qian Y., Tiffany-Castiglioni E. and Harris E.D. 1995. Copper transport and kinetics in cultured C6 rat glioma cells. Am.J.Physiol. (Cell Physiol.38):C892-C898.
- Qian Y., Tiffany-Castiglioni E. and Harris E.D. 1996. Functional analysis of a genetic defect of copper transport (Menkes disease) in different cell lines. Am.J. Physiol. (Cell Physiol.) in press.
|
Progress 01/01/94 to 12/30/94
Outputs
The overall goal is to give mechanistic insights into the transport of essentialmetals from mother to fetus. The project uses BeWo cells, a human choriocarcinoma cell line, grown on bicameral filters, to emulate the morphology of the placental syncytium. The studies have focused on three essential metals: copper, zinc, and manganese. Previously, we reported that a cDNA probe for ceruloplasmin (Cp) hybridized with two mRNA transcripts, 3.9 and a 4.4 kb from the cells. The data suggest Cp is a transport vehicle for Cu release. We have since found that Brefeldin A, an inhibitor of intracellular transport, blocked the release of Cu from the cells suggesting export could be as a protein-bound factor with Golgi involvement. Cu, upon entering the cells quickly binds to glutathione (GSH). Our data suggest Cu is disulfide-linked in the complex. Mn transport in the cells was also characterized. A manganotransferrin complex was prepared by incubating cells with Mn(superscript 2+)
in the presence of ceruloplasmin. (superscript 54)Mn as a transferrin-bound metal was slowly taken into the cytosol of the cells. (superscript 65)Zn was also transported into the cells but was not released from the basal surface unless cold Zn was added to the medium as a supplement. The zinc appears to be protein-bound inside the cells and did not form a complex with glutathione. The data suggest that each essential metal tested has its own unique pathway for traversing the placental barrier.
Impacts
(N/A)
Publications
- HARRIS, E.D. (1994) Copper. In: Handbook of Nutritionally Essential Mineral Elements, O'Dell, B.L. Ed. Marcel Dekker (in-press).
- BUCHMAN, A.L., KEEN, C.L., VINTERS, H.V., HARRIS, E.D., CHUGANI, H.T., BATEMAN, B., ROGERSON, D., VARGAS J., VERITY, A. and AMENT, M. (1994) Copper deficiency secondary to a copper transport defect, a new copper metabolic disturbance.
- DAVIDSON, L.A., MCORMOND, S.A., and HARRIS, E.D. (1994) Characterization of a particulate pathway for copper in K562 cells. Biochim. Biophys. Acta 1221:1-6.
- HARRIS, E.D. (1994) Iron-Copper interactions: some new revelations. Nutr. Rev. 52:311-315.
- SAENKO, E.L., YAROPOLOV, A.I. and HARRIS, E.D. (1994) The biological functions of ceruloplasmin expressed through copper-binding sites and a cellular receptor. J. Tr. Elem. Exp. Med. 7:69-88.
- BRENNER A.J. and HARRIS E.D. (1994) A quantitative test for copper using bicinchoninic acid. Anal. Biochem. (in-press).
|
Progress 01/01/93 to 12/30/93
Outputs
This project has as its objective an investigation into the role of membrane proteins in the uptake and homeostasis of copper in cells. With a focus on placental cells, it was hypothesized that ceruloplasmin provides copper to the cells of the placenta and that the movement across the syncytiotrophoblast layer is by two mechanisms: synthesis of endogenous ceruloplasmin by the cells, and active excretion via a copper-transporting membrane-bound ATPase. In the studies, BeWo cells, a choriocarcinoma cell line, were incubated with 10-50 (mu)M Cu. mRNA for ceruloplasmin was probed with a cDNA. Two components of 4.5 kb and 3.9 kb were detected by Northern analysis. The 3.9 kb component appeared to increase in response to added copper, suggesting copper ions provide a stimulation to the transcription of ceruloplasmin transcripts. Two bands that reacted with antibody to human ceruloplasmin were detected in a cell-free translation system by Western blot analysis, confirming that
the mRNA encoded for ceruloplasmin. Intracellular copper was shown by HPLC analysis to bind to glutathione (GSH) forming a Cu(superscript +)-GSH complex. No evidence of a (GSH)(subscript 2)-Cu complex was observed. BeWo cells were also shown to express an mRNA for a copper-transporting ATPase identified with Menkes' disease. The expression of the gene product, a protein, is currently being investigated.
Impacts
(N/A)
Publications
|
|