@phdthesis{Muench2023,
  author    = {M{\"u}nch, Luca},
  title     = {Die Rolle transposabler Elemente in der Genese des malignen Melanom im Fischmodell Xiphophorus},
  doi       = {10.25972/OPUS-28922},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289228},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Der Name der transposablen Elemente beruht auf ihrer F{\"a}higkeit, ihre genomische Position ver{\"a}ndern zu k{\"o}nnen. Durch Chromosomenaberrationen, Insertionen oder Deletionen k{\"o}nnen ihre genomischen Transpositionen genetische Instabilit{\"a}t verursachen. Inwieweit sie dar{\"u}ber hinaus regulatorischen Einfluss auf Zellfunktionen besitzen, ist Gegenstand aktueller Forschung ebenso wie die daraus resultierende Frage nach der Gesamtheit ihrer biologischen Signifikanz. Die Weiterf{\"u}hrung experimenteller Forschung ist unabdingbar, um weiterhin offenen Fragen nachzugehen. Das Xiphophorus-Melanom-Modell stellt hierbei eines der {\"a}ltesten Tiermodelle zur Erforschung des malignen Melanoms dar. Durch den klar definierten genetischen Hintergrund eignet es sich hervorragend zur Erforschung des b{\"o}sartigen schwarzen Hautkrebses, welcher nach wie vor die t{\"o}dlichste aller bekannten Hautkrebsformen darstellt. Die hier vorliegende Arbeit besch{\"a}ftigt sich mit der Rolle transposabler Elemente in der malignen Melanomgenese von Xiphophorus.},
  subject      = {Transposon},
  language  = {de}
}
@phdthesis{Liu2022,
  author    = {Liu, Ruiqi},
  title     = {Dynamic regulation of the melanocortin 4 receptor system in body weight homeostasis and reproductive maturation in fish},
  doi       = {10.25972/OPUS-20653},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206536},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Puberty is an important period of life with physiological changes to enable animals to reproduce. Xiphophorus fish exhibit polymorphism in body size, puberty timing, and reproductive tactics. These phenotypical polymorphisms are controlled by the Puberty (P) locus. In X. nigrensis and X. multilineatus, the P locus encodes the melanocortin 4 receptor (Mc4r) with high genetic polymorphisms. Mc4r is a member of the melanocortin receptors, belonging to class A G-protein coupled receptors. The Mc4r signaling system consists of Mc4r, the agonist Pomc (precursor of various MSH and of ACTH), the antagonist Agrp and accessory protein Mrap2. In humans, MC4R has a role in energy homeostasis. MC4R and MRAP2 mutations are linked to human obesity but not to puberty. Mc4rs in X. nigrensis and X. multilineatus are present in three allele classes, A, B1 and B2, of which the X-linked A alleles express functional receptors and the male-specific Y-linked B alleles encode defective receptors. Male body sizes are correlated with B allele type and B allele copy numbers. Late-maturing large males carry B alleles in high copy number while early-maturing small males carry B alleles in low copy number or only A alleles. Cell culture co-expression experiments indicated that B alleles may act as dominant negative receptor mutants on A alleles. In this study, the main aim was to biochemically characterize the mechanism of puberty regulation by Mc4r in X. nigrensis and X. multilineatus, whether it is by Mc4r dimerization and/or Mrap2 interaction with Mc4r or other mechanisms. Furthermore, Mc4r in X. hellerii (another swordtail species) and medaka (a model organism phylogenetically close to Xiphophorus) were investigated to understand if the investigated mechanisms are conserved in other species. In medaka, the Mc4r signaling system genes (mc4r, mrap2, pomc, agrp1) are expressed before hatching, with agrp1 being highly upregulated during hatching and first feeding. These genes are mainly expressed in adult brain, and the transcripts of mrap2 co-localize with mc4r indicating a function in modulating Mc4r signaling. Functional comparison between wild-type and mc4r knockout medaka showed that Mc4r knockout does not affect puberty timing but significantly delays hatching due to the retarded embryonic development of knockout medaka. Hence, the Mc4r system in medaka is involved in regulation of growth rather than puberty. In Xiphophorus, expression co-localization of mc4r and mrap2 in X. nigrensis and X. hellerii fish adult brains was characterized by in situ hybridization. In both species, large males exhibit strikingly high expression of mc4r while mrap2 shows similar expression level in the large and small male and female. Differently, X. hellerii has only A-type alleles indicating that the puberty regulation mechanisms evolved independently in Xiphophorus genus. Functional analysis of Mrap2 and Mc4r A/B1/B2 alleles of X. multilineatus showed that increased Mrap2 amounts induce higher cAMP response but EC50 values do not change much upon Mrap2 co-expression with Mc4r (expressing only A allele or A and B1 alleles). A and B1 alleles were expressed higher in large male brains, while B2 alleles were only barely expressed. Mc4r A-B1 cells have lower cAMP production than Mc4r A cells. Together, this indicates a role of Mc4r alleles, but not Mrap2, in puberty onset regulation signaling. Interaction studies by FRET approach evidenced that Mc4r A and B alleles can form heterodimers and homodimers in vitro, but only for a certain fraction of the expressed receptors. Single-molecule colocalization study using super-resolution microscope dSTORM confirmed that only few Mc4r A and B1 receptors co-localized on the membrane. Altogether, the species-specific puberty onset regulation in X. nigrensis and X. multilineatus is linked to the presence of Mc4r B alleles and to some extent to its interaction with A allele gene products. This is reasoned to result in certain levels of cAMP signaling which reaches the dynamic or static threshold to permit late puberty in large males. In summary, puberty onset regulation by dominant negative effect of Mc4r mutant alleles is a special mechanism that is found so far only in X. nigrensis and X. multilineatus. Other Xiphophorus species obviously evolved the same function of the pathway by diverse mechanisms. Mc4r in other fish (medaka) has a role in regulation of growth, reminiscent of its role in energy homeostasis in humans. The results of this study will contribute to better understand the biochemical and physiological functions of the Mc4r system in vertebrates including human.},
  subject      = {Japank{\"a}rpfling},
  language  = {en}
}
@article{AdamMauelerSchartl1991,
  author    = {Adam, Dieter and Maueler, Winfried and Schartl, Manfred},
  title     = {Transcriptional activation of the melanoma inducing Xmrk oncogene in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87584},
  year      = {1991},
  abstract  = {The melanoma inducing locus of Xiphophorus encodes a tumorigenic version of a novel putative receptor tyrosine kinase (Xmrk). To elucidate the mechanism of oncogenic activation of Xmrk, we compared the structure and expression of two oncogenic loci with the corresponding proto-oncogene. Only minor structural alterations were found to be specific for the oncogenic Xmrk genes. Marked overexpression of the oncogene transcripts in melanoma, which are approximately 1 kb shorter than the proto-oncogene transcript, correlates with the malignancy of the tumors. The tumor transcripts are derived from an alternative transcription start site that is used only in the oncogenic loci. Thus, oncogenic activation of the melanoma inducing Xmrk gene appears primarily to be due to novel transcriptional control and overexpression.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{SchartlWittbrodtMaeueleretal.1993,
  author    = {Schartl, Manfred and Wittbrodt, J. and M{\"a}ueler, W. and Raulf, F. and Adam, D. and Hannig, G. and Telling, A. and Storch, F. and Andexinger, S. and Robertson, S. M.},
  title     = {Oncogenes and melanoma formation in Xiphoporus (Teleostei: Poeciliidae)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87149},
  year      = {1993},
  abstract  = {In Xiphophorus melanoma formation has been attributed by classical genetic findings to the overexpression of a cellular oncogene (Tu) due to elimination of the corresponding regulatory gene locus in hybrids. We have attempted to elucidate this phenomenon on the molecular biological level. Studies on the structure and expression of known proto-oncogenes revealed that several of these genes, especially the c-src gene of Xiphophorus, may act as effectors in establishing the neoplastic phenotype of the melanoma cells . However, these genes appear more to participate in secondary steps of tumorigenesis. Another gene, being termed Xmrk, which represents obviously a so far unknown proto-oncogene but with a cons iderably high similarity to the epidermal growth-factorreceptor gene, was mapped to the Tu-containing region of the chromosome. This gene shows features with respect to its structure and expression that seem to justify it to be regarded as a candidate for a gene involved in the primary processes leading to neoplastic transformation of pigment cells in Xiphophorus.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{MeyerSchartl1979,
  author    = {Meyer, Manfred K. and Schartl, Manfred},
  title     = {Eine neue Xiphophorus-Art aus Vera Cruz, Mexiko : (Pisces: Poeciliidae)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87124},
  year      = {1979},
  abstract  = {Xiphophorus andersi n. sp. from the Rio Atoyac, Vera Cruz, Mexico is described: lang head, moderately slender body, large dark black spar at the basis of the anal fin; adult male with short sword-like caudal appendage; rip of ray 5a of gonopodium without a developed claw. Xiphophorus andersi n. sp. differs by the combination of distinct characters from all the other species of the genus known so far. The new species shows features of both the so-called platyfish species group and the so-called swordtail species group.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{WinklerHongWittbrodtetal.1992,
  author    = {Winkler, Christoph and Hong, Yunhan and Wittbrodt, Joachim and Schartl, Manfred},
  title     = {Analysis of heterologous and homologous promoters and enhancers in vitro and in vivo by gene transfer into Japanese medaka (Oryzias latipes) and Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86796},
  year      = {1992},
  abstract  = {Efficient expression systems are required for analysis of gene regulation and function in teleost fish. To develop such systems, a nurober of inducible or constitutive promoter and enhancer sequences of fish or higher vertebrate origin were tested for activity in a variety of fish celllines andin embryos of the Japanese medaka fish (Oryzias latipes) and Xiphophorus. The activity of the different promoterenhancer combinations were quantitated. Considerable differences were found for some constructs if tested in vitro or in vivo. From the data obtained, a set of expression vectors for basic research as weH as for aquaculture purposes were established.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{RaulfMaeuelerRobertsonetal.1989,
  author    = {Raulf, Friedrich and M{\"a}ueler, Winfried and Robertson, Scott M. and Schartl, Manfred},
  title     = {Localization of cellular src mRNA during development and in the differentiated bipolar neurons of the adult neural retina in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86703},
  year      = {1989},
  abstract  = {The expression of the c-src gene in embryonie and adult tissue of the teleost fish Xiphophorus helleri was analyzed by in-situ hybridization. The highly conserved fish c-src gene was found to be expressed at high levels in midterm embryos, where c-src mRNA was localized in developing neurons of the sensory layer of the differentiating retina and in the developing brain. In adult tissues the expression of c-src was found to persist in certain cell types of the brain and the neural retina, especially in the bipolar cells of the inner nuclear layer, which are postmitotic, fully differentiated mature neurons. Thus c-src in Xiphophorus appears to be a developmentally regulated proto-oncogene which is important for neuronal differentiation during organogenesis, but whose persistence of expression in certain terminally differentiated neurons strongly suggests a particular maintenance function for c-src in these cells as well.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{SchartlSchartlAnders1982,
  author    = {Schartl, A. and Schartl, Manfred and Anders, F.},
  title     = {Promotion and regression of neoplasia by testosterone-promoted cell differentiation in Xiphophorus and Girardinus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86684},
  year      = {1982},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{SchartlSchartlAnders1981,
  author    = {Schartl, A. and Schartl, Manfred and Anders, F.},
  title     = {Phenotypic conversion of malignant melanoma to benign melanoma and vice versa in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86662},
  year      = {1981},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{AndersSchollSchartl1981,
  author    = {Anders, F. and Scholl, E. and Schartl, Manfred},
  title     = {Environmental and hereditary factors in the causation of neoplasia, based on studies of the Xiphophorus fish melanoma system},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86402},
  year      = {1981},
  abstract  = {Neoplasia in Xiphophorus can be classified into: a) a Jarge group triggered by carcinogens; b) a large group triggered by promoters; and c) a small group that develops "spontaneously" according to Mendelian Jaw. The process leading to susceptibility for neoplasia is represented by the disintegration of gene systems that normally protect the fish from neoplasia. Interpopulational arid interracial hybridization is the most effective process that Ieads to disintegration of the protective gene systems. Environmental factors may complete disintegration in somatic cells and thus may trigger neoplasia. The applications of the findings on Xiphophorus to humans are discussed.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{AndersSchartlBarnekow1984,
  author    = {Anders, Fritz and Schartl, Manfred and Barnekow, Angelika},
  title     = {Xiphophorus as an in vivo model for studies on oncogenes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86398},
  year      = {1984},
  abstract  = {The capacity of Xiphophorus to develop neoplasia can be formally assigned to a "tumor gene" (Tu), which appears to be a normal part of the genome of all individuals. The wild fish have evolved population-specific and cell type-specific systems of regulatory genes (R) for Tu that protect the fish from neoplasia. Hybridization of members of different wild populations in the laborstory followed by treatment of the hybrids with carcinogens led to disintegration of the R systems permitting excessive expression of Tu and thus resulting in neoplasia. Certain hybrids developed neoplasia even spontaneously. Observations on the genuine phenotypic effect of the derepressed Tu in the early embryo indicated an essential normal function of this oncogene in cell differentiation, proliferation and cell-cell communication. Tu appeared to be indispensable in the genome but may also be present in accessory copics. Recently, c-src, the cellular homolog of the Rous sarcoma virus oncogene v-src, was detected in Xiphophorus. The protein product of c-src, pp60c-src, was identified and then examined by its associated kinase activity. This pp60c-src was found in all individuals tested, but, depending on the genotype, its kinase activity was different. The genetic characters of c-src, such as linkage relations, dosage relations, expression, etc., correspond to those of Tu. From a systematic study which showed that pp60c-src was present in all metazoa tested ranging from mammals down to sponges, we concluded that c-src has evolved with the multicellular organization of animals. Neoplasia of animals and humans is a characteristic closely related to this evolution. Our data showed that small aquariurn fish, besides being used successfully because they are time-, space-, and money-saving systems for carcinogenicity testing, are also highly suitable for basic studies on neoplasia at the populational, morphological, developmental, cell biological, and molecular levels.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{MauelerBarnekowEigenbrodtetal.1988,
  author    = {Maueler, W. and Barnekow, A. and Eigenbrodt, E. and Raulf, F. and Falk, H. F. and Telling, A. and Schartl, Manfred},
  title     = {Different regulation of oncogene expression in tumor and embryonal cells of Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86240},
  year      = {1988},
  abstract  = {Melanoma formation in the poeciliid fish Xiphophorus is mediated primarily by a cellular oncogene, designated Tu. Elimination of Tu-specific genes releases the transforming function of Tu and leads to melanoma formation. Southern blot analyses revealed a tight linkage of a v-erb B related gene to the Tu-locus and Northern blot analyses of RNA of solid melanomas indicated a coordinated deregulation and for mutational activation of several oncogenes. In order to get a better insight into the regulation of oncogene expression in normal and transformed cells of Xiphophorus, we studied the expression of Xsrc, Xras, Xmyc, Xerb A, Xsis, and the v-erb B related gene in a melanoma derived cell line (PSM) and an embryonic cell line (A2) under conditions of low growth factor supply. Both celllines express the Xsrc, Xmyc, and Xras genes, while PSM cells in addition express the v-erb B related gene and A2 cells the Xsis gene. In PSM cells serum deprivation leads to an accumulation of most of the oncogene mRNAs analysed. This is most apparent for a 5.0 kb transcript of the v-erb B related gene, probably due to an increase in transcript stability. The levels of these mRNAs returned to normal within 2h after stimulation with 10\% fetal calf serum. At the protein level we observed an initial decrease followed by an increase of the n-p60c-src kinase (the protein product of tbe Xsrc gene) activity in cells deprived of serum. Serum stimulation restored a normal pp60"-src kinase activity. In contrast serum deprivation of A2 cells reduced the transcript amounts of each of the oncogenes analysed. The same holds true for one beta-tubulin transcript, while the level of a second beta-tubulin transcript was unaffected. Serum stimulation led to a reactivation of Xras and Xsrc after a delay of approximately 48b. The pp60(c-src) kinase activity was found to be 6-10 times lower as compared to the PSM cells and did not differ between serum deprived and serum stimulated cells. Enzyme activities and isoenzyme patterns of several glycolytic enzymes were found to be not affected by serum deprivation and stimulation in both celllines.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{MaeuelerRaulfSchartl1988,
  author    = {M{\"a}ueler, Winfried and Raulf, Friedrich and Schartl, Manfred},
  title     = {Expression of proto-oncogenes in embryonic, adult, and transformed tissue of Xiphophorus (Teleostei: Poeciliidae)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-86233},
  year      = {1988},
  abstract  = {In Xiphophorus the causative, primary cellular oncogene for melanoma formation has been assigned by classical genetics to a sex-chromosomal locus, designated Tu. Activation of Tu was proposed to be the result of the elimination of Tu-specific regulatory genes which normally suppress the transforming function in the nontumorous state. In order to understand the role which known proto-oncogenes migbt play in this process, we have analysed the expression of src, erb A, erb B, ras, abl, sis and mil related genes from Xiphophorus during embryogenesis, in non-tumorous organs and in melanoma cells. For src, ras, erb B and sis a differential expression during embryogenesis and/or in normal organs was detected, with preferential expression of src in neural tissues, a high abundance of sis transcripts in an embryonal epitheloid cellline and of erbB transcripts in the head nephros. In melanoma cells ras, src and a v-erb B related gene were found to be expressed. The src gene most likely is more involved in secondary processes during tumor progression, while the expression of the v-erb B related gene might be transformation-specific because recently such a sequence was found to map to the close vicinity of the Tu-locus.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@article{SchartlSchartl1990,
  author    = {Schartl, Angelika and Schartl, Manfred},
  title     = {Genes and cancer: Molecular biology of the melanoma oncogene of Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72670},
  year      = {1990},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@incollection{AltschmiedSchartl1994,
  author    = {Altschmied, Joachim and Schartl, Manfred},
  title     = {Genetics and molecular biology of tumour formation in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69752},
  publisher      = {Universit{\"a}t W{\"u}rzburg},
  year      = {1994},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{PeterSchartlAndersetal.1985,
  author    = {Peter, R. U. and Schartl, Manfred and Anders, F. and Duncker, H.-R.},
  title     = {Pigment pattern formation during embryogenesis in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69370},
  year      = {1985},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{RiehlSchartlAnders1985,
  author    = {Riehl, R{\"u}diger and Schartl, Manfred and Anders, Fritz},
  title     = {An ultrastructural study of melanoma in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-70978},
  year      = {1985},
  abstract  = {Melanotic melanoma (MM) of Xiphophorus (Teleostei: Poeciliidae) was studied by conventional preparations and freeze-etch preparations for electron microscopy. MM of Xiphophorus exhibits tightly packed pigment cells with prominent dendritic processes and interdigitations of their plasma membranes. The most impressive feature of MM cells is the occurrence of Iarge lobulated nuclei with numerous nuclear pores and some nuclear pockets. Abundant spheroidal or ellipsoidal melanosomes (diameter 200-650 nm) and vesicular structures are distributed throughout the cellular dendrites, whereas the perinucJear cytoplasm is free of melanosomes. A further characteristic feature of melanoma cells in fish is the occurrence of melanosome complexes (i.e., "compound melanosomes"). These melanosome complexes consist of a few to numerous melanosomes, which are enveloped by a separate rnembrane. Pinocytotic vesicles couJd be demonstrated with distinct differences in frequency and distribution patterns, indicating differences in the metabolic activities of the cells in the same melanoma. Intercellular junctions are lacking in the MM cells. The conventional TEM technique showed clear advantages in the demonstration of intemal architecture of organelles, whereas FE bad considerable potential in respect to the visualization of membrane surface specializations.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{SchartlMaeuelerRaulfetal.1988,
  author    = {Schartl, Manfred and M{\"a}ueler, Winfried and Raulf, Friedrich and Robertson, Scott M.},
  title     = {Molecular aspects of melanoma formation in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72689},
  year      = {1988},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@inproceedings{AndersSchartlScholl1981,
  author    = {Anders, F. and Schartl, Manfred and Scholl, E.},
  title     = {Evaluation of environmental and hereditary factors in carcinogenesis, based on studies in Xiphophorus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72741},
  year      = {1981},
  abstract  = {Neoplasia in Xiphophorus can be classified into a) a large group that is triggered by carcinogens; b) a large group triggered by promoters; c) a small group that develops "spontaneously" following interpopulational and interracial hybridizations; and d) a small group that develops "spontaneously" following germ line mutation. The process leading to susceptibility for neoplasia is represented by the disintegration of gene systems that normally protect the fish from neoplasia. Hybridization is the most effective process that leads to disintegration of the protection gene systems. Environmental factors may complete disintegration and thus may trigger neoplasia. It is discussed whether the findings on Xiphophorus may also apply to humans.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {en}
}
@incollection{AndersSchollSchartl1979,
  author    = {Anders, F. and Scholl, E. and Schartl, Manfred},
  title     = {Xiphophorus als Modell in der Krebsforschung},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72752},
  publisher      = {Universit{\"a}t W{\"u}rzburg},
  year      = {1979},
  abstract  = {No abstract available.},
  subject      = {Schwertk{\"a}rpfling},
  language  = {de}
}