Dermatan sulphate (DS) is a sulphated glycosaminoglycan (GAG) that is widely distributed as proteoglycan throughout the extracellular matrix and at cell surfaces where it plays an important role in many key biological processes. The intra-cellular catabolism of DS commences with endohydrolysis of the polysaccharide chains to oligosaccharides, which are then sequentially degraded from the non-reducing terminus by lysosomal exoenzymes to monosaccharides and inorganic sulphate for transport out of the lysosome and re-utilisation by the cell. Both endo-β-N-acetylhexosaminidase (Hyal-1 hyaluronidase) and endo-β-glucuronidase activities towards DS have been proposed. The present study was undertaken to: 1) determine the substrate specificities and sub-cellular locations of these endoglycosidase activities; and 2) compare endoglycosidase activities and substrate specificities in the mucopolysaccharidoses, where a defect in one of the lysosomal exoenzymes required to degrade DS results in the lysosomal accumulation of partially degraded DS oligosaccharide fragments. To this end, a series of oligosaccharide substrates designed to represent aspects of the physiological substrate was prepared, and an assay was developed to measure endoglycosidase activities and determine their substrate specificities by quantifying specific oligosaccharide products. Assay substrates rich in glucuronic acid (GlcA) or iduronic acid (IdoA) were prepared by limited chondroitinase ABC digestion of chondroitin sulphate A and DS, respectively. The resulting tetra-to hexadecasaccharides were separated by size-exclusion chromatography and characterised by electrospray ionisation-tandem mass spectrometry (ESI-MS/MS). These substrates, which were not susceptible to degradation by lysosomal exoenzymes, were then incubated with Chinese hamster ovary (CHO)-K1 cell homogenate (source of endoglycosidase activity), and the oligosaccharide products generated from the non-reducing end of the substrate were measured by ESI-MS/MS. Endo-β-N-acetylhexosaminidase and endohexuronidase activities were detected towards the oligosaccharide substrates, with both activities preferentially degrading the GlcA-rich substrates and only minor activity observed towards IdoA-rich substrate. The endo-β-N-acetylhexosaminidase activity had a minimum-sized substrate requirement of a hexasaccharide and was observed to sequentially remove tetrasaccharides from the non-reducing end of oligosaccharides, whereas the endohexuronidase activity had a minimum substrate of an octasaccharide, acted randomly and was comparatively low. The activities displayed the same acidic pH optimum and responded in the same manner to changes in buffer composition and substrate concentration, and to the presence of divalent cations, NaCl, detergent and protease inhibitors. Both activities were modestly affected by the hyaluronidase inhibitor, apigenin. Percoll density gradient sub-cellular fractionation confirmed that the activities were primarily in the lysosomes and late endosomes. The endo-β-N-acetylhexosaminidase and endohexuronidase activities detected here in CHO-K1 cells are consistent with the Hyal-1 and endo-β-glucuronidase enzymes described previously. These data suggest that Hyal-1 and endo-β-glucuronidase are predominantly lysosomal enzymes that act in concert to degrade the low-sulphate, GlcA-rich domains of DS, but are less active towards the highly sulphated regions containing IdoA. To test the hypothesis that endoglycosidase activities are altered in the mucopolysaccharidoses, an attempt was made to compare Hyal-1- and endo-β-glucuronidase-like activities and their substrate specificities in mucopolysaccharidosis (MPS)-affected and unaffected control skin fibroblasts. However, no activity was detected towards octa- to hexadecasaccharide substrates in control fibroblast homogenates, and in homogenates of MPS fibroblasts deficient in the lysosomal exoenzymes α-L-iduronidase and N-acetylgalactosamine-4-sulphatase, despite the fact that: 1) what appear to be the products of Hyal-1 and endo-β-glucuronidase activities towards endogenous DS could be detected in the lysosomes of the MPS cells by sub-cellular fractionation; and 2) the ESI-MS/MS assay was demonstrated sensitive enough to detect endoglycosidase activities in homogenates of a number of different mouse tissues (including whole skin). We hypothesise that this absence of detectable endoglycosidase activity in skin fibroblasts results from enzyme non-recognition of the exogenous assay substrates tested, and hence that these cells contain heretofore undescribed Hyal-1 and endo-β-glucuronidase isoforms with unique substrate specificities. In conclusion, the development of an ESI-MS/MS assay to measure the products of endoglycosidase activities has enabled the characterisation of these activities towards DS. This strategy may be useful for the future study of endoglycosidase activities towards a variety of other GAGs such as heparan sulphate, where particular oligosaccharide structures have been shown to possess unique biological activities. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1374435 / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2009

Submitted by GIOVANNA ROSSI VARALLO null (giovanna_vet03@yahoo.com.br) on 2016-12-26T14:11:39Z No. of bitstreams: 1 Tese Repositório.pdf: 4013000 bytes, checksum: 28bc5d1f53e09229a63ae5d00abb74ae (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-01-03T15:54:49Z (GMT) No. of bitstreams: 1 varallo_gr_dr_jabo.pdf: 4013000 bytes, checksum: 28bc5d1f53e09229a63ae5d00abb74ae (MD5) / Made available in DSpace on 2017-01-03T15:54:49Z (GMT). No. of bitstreams: 1 varallo_gr_dr_jabo.pdf: 4013000 bytes, checksum: 28bc5d1f53e09229a63ae5d00abb74ae (MD5) Previous issue date: 2016-11-16 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O câncer de mama é uma doença heterogênea. Em mulheres está estabelecido um perfil imuno-histoquímico para utilização na rotina clínica de pacientes com câncer de mama. Os fenótipos luminal A, luminal B, HER2 superexpresso e triplo negativo apresentam diferenças prognósticas e preditivas. Entretanto, para a neoplasmas mamários em cadelas, não há uma classificação fenotípica definida. As diferenças prognósticas entre os fenótipos são atribuídas às assinaturas do tumor. Os estudos gênicos possibilitam averiguar o perfil genético da doença e a identificação de novos marcadores moleculares. O objetivo do estudo foi propor uma classificação fenotípica para a determinação de quatro fenótipos: luminal A, luminal B, HER2 superexpresso e triplo negativo, bem como avaliar a imunoexpressão dos marcadores anti-E-caderina, anti-claudinas 1, 3, 4, 7 e do anti-CD24 e anti-CD44. Na segunda etapa do estudo, objetivou-se avaliar o perfil de expressão gênica global dos carcinomas de mama e a diferença de expressão gênica entre os grupos fenotípicos. Para a avaliação imunohistoquímica, o grupo experimental foi composto por 110 amostras de câncer de mama dispostas em lâminas de tissue microarray. Obteve-se 42 tumores luminal A, 41 luminal B, 17 triplo negativo e 10 HER2 superexpresso. Constatou-se que estes últimos apresentam fenótipos mais agressivos e com menores sobrevidas. A expressão positiva da E-caderina foi mais acentuada nos grupos luminal A e luminal B, já a negativa no triplo negativo e no HER2 superexpresso. O padrão das expressões das claudinas 1, 3, 4 e 7 foi semelhante entre os subfenótipos. As células-tronco tumorais foram relacionadas aos graus histológicos mais agressivos (II e III). Para a avaliação da expressão gênica foram usadas 12 amostras de carcinoma mamário de cadelas e quatro mamas normais. Não foi evidenciada diferença na expressão gênica entre os grupos luminal A, luminal B, HER2 superexpresso e triplo negativo pelo microarranjo. O perfil gênico global das neoplasias estudadas mostrou 878 genes superexpressos e 821 genes subexpressos. A análise de enriquecimento aplicada aos genes superexpressos apontou o gene HYAL-1 como um potencial marcador diagnóstico e de recidiva. O PCR confirmou esse resultado. Conclui-se que a classificação molecular é uma ferramenta importante para a avaliação prognóstica dos pacientes com neoplasmas mamários. O gene HYAL-1 diferencialmente expresso participa da via de sinalização metabólica relacionada à carcinogênese mamária e a superexpressão está relacionada com o desenvolvimento do câncer e à recidiva. / The mammary cancer is a heterogeneous disease. In women, an immunohistochemical profile is established for use in the clinical routine of patients with breast cancer. The luminal A, luminal B, HER2 overexpressed and triple negative phenotypes present prognostic and predictive differences. However for mammary neoplasms in bitches, there is no definite phenotypic classification. Prognostic differences between phenotypes are attributed to tumor signatures. Genetic studies make it possible to ascertain the genetic profile of the disease and the identification of new molecular markers. The objective of the study was to propose a phenotypic classification for the determination of four phenotypes in bitches: luminal A, luminal B, HER2 overexpressed and triple negative, as well as to evaluate the immunoexpression of the anti-E-cadherin, anti-claudin markers 1, 3, 4, 7 and anti-CD24 and anti-CD44. In the second stage of the study, the objective was to evaluate the overall gene expression profile of mammary carcinomas and the difference in gene expression between the phenotypic groups. For the immunohistochemical evaluation, the experimental group consisted of 110 mammary cancer samples arranged in tissue microarray slides. 42 luminal A, 41 luminal B, 17 triple negative and 10 HER2 superexpressed tumors were obtained. It was verified that the latter present more aggressive phenotypes and with lower survival rates. Positive expression of E-cadherin was more pronounced in the luminal A and luminal B groups, whereas the negative expression in triple negative and HER2 overexpressed. The standard expression of claudins 1, 3, 4 and 7 was similar among subphenotypes. Tumor stem cells were related to the most aggressive histological grades (II and III). For the evaluation of gene expression, 12 samples of mammary carcinoma of bitches and four normal breasts were used. There was no difference in gene expression between the luminal A, luminal B, HER2 superexpressed and triple negative groups by the microarray. The overall gene profile of the neoplasms studied showed 878 overexpressed genes and 821 underexpressed genes. The enrichment analysis applied to the overexpressed genes pointed to the HYAL-1 gene as a potential diagnostic and relapse marker. PCR confirmed this result. It is concluded that molecular classification is an important tool for the prognostic evaluation of patients with breast neoplasms. The differentially expressed HYAL-1 gene participates in the metabolic signaling pathway related to mammary carcinogenesis and overexpression is related to the development of cancer and relapse. / CNPq: 140624/2013-9

Malgré de nombreux progrès réalisés dans les traitements des cancers gynécologiques, ceux-ci demeurent la principale cause de mortalité due au cancer chez la femme. Les instabilités chromosomiques et génomiques au niveau du locus 3p21.3 sont des événements fréquents liés à des cancers épithéliaux, notamment les cancers du sein et de l'ovaire. C’est dans cette région que se trouvent les gènes hyaluronidases HYAL-1, HYAL-2 et HYAL-3. HYAL-1 est particulièrement surexprimé dans plusieurs cancers, notamment, le cancer de la prostate, la vessie, le cou, la tête et le sein où il est impliqué dans la progression tumorale et les métastases. Nous avons démontré que dans le locus 3p21.3, HYAL-1 est un gène cible sélectivement réprimé par ERα et l’estrogène. L’analyse de la cohorte METABRIC a révélé une corrélation inverse significative entre l’expression du gène HYAL-1 et ERα. Nous avons identifié des sites de liaison pour ERα au niveau du locus 3p21.3, parmi eux, un ERE proximal était responsable de la répression de HYAL-1 par ERα en plus d’un site Sp-1 requis pour atteindre une répression optimale. Cette répression de HYAL-1 est accompagnée par un enrichissement de la marque répressive H3K27me3 au niveau des deux sites ERE et Sp-1. En plus de réguler l’expression de nombreux gènes, l’activité transcriptionnelle des ERs est aussi régulée par les corégulateurs qui sont recrutés sur les ERs. Nous avons identifié un nouveau partenaire d’interaction inattendu pour les ERs, soit le facteur de transcription hématopoïétique TAL1. Malgré sa réputation d’oncogène dans les leucémies lymphoblastiques aiguës des cellules T, ce facteur de transcription est un corépresseur d’ERα, dû à son effet répresseur direct sur l’activité transcriptionnelle du récepteur en réponse à l’estrogène et donc sur l’expression de ces gènes cible dans le cancer du sein. De plus, TAL1 réprime aussi l’activation d’ERα en réponse à la phosphorylation induite par la voie des MAPK/Erk. Cette répression d’ERα par TAL1 résulte en une diminution de la prolifération et la migration des cellules cancéreuses mammaires. / Despite many advances in treatment of gynecological cancers, they remain the leading cause of cancer death in women. Chromosomal and genome instabilities at the 3p21.3 locus are frequent events related to epithelial cancers, including breast and ovarian cancers. It is in this region that the hyaluronidase HYAL-1, HYAL-2 and HYAL-3 genes are found. HYAL-1 is particularly overexpressed in several cancers, including prostate, bladder, neck, head and breast cancers where it promotes tumor progression and metastasis. We demonstrate here, that in the 3p21.3 locus, HYAL-1 is a target gene selectively repressed by ERα and estrogen. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL-1 gene expression in human breast tumors. We identified binding sites for ERα at the 3p21.3 locus, among which a proximal ERE responsible for repression of HYAL-1 by ERα, in addition to an Sp-1 site required to achieve optimal repression. This repression of HYAL-1 is accompanied by an enrichment of the repressive mark H3K27me3 at the two sites ERE and Sp-1. In addition to regulate the expression of many target genes, the transcriptional activity of estrogen receptors is also regulated by coregulators who are recruited on ERs. We identified a new unexpected interaction partner for ERs, the hematopoietic transcription factor TAL1. Despite its reputation as an oncogene in T-cell acute lymphoblastic leukemia, this transcription factor is an ERα-corepressor due to its direct repressive effect on the transcriptional activity of the receptor in response to estrogen and thus to expression of its target genes in breast cancer. Moreover, TAL1 also inhibits ERs activation in response to phosphorylation induced by the MAPK/Erk pathway. This repression of ERα by TAL1 results in decreased growth and migration of mammary cancer cells.