Heparanase (Hpa1) is an enzyme overexpressed in nearly all cancers, typically at the tumour growth front. It cleaves proteoglycan heparan sulfate (HS) chains to release growth factors necessary for tumour growth. While some carbohydrate-based mimetic inhibitors have progressed to advanced clinical trials, new inhibitors and tools to further investigate heparanase are of continued interest. This thesis proposes a HS mimetic trisaccharide sequence that can bind Hpa1 and is suitable both for biological evaluation and inhibitor development. Synthetic work was then undertaken toward the progression of this moiety. Exploring building blocks applicable to the trisaccharide, conformationally-locked glucose derivatives were developed. This included the introduction of a conformational switch that resulted in the isolation of constrained half-chair conformers. The synthetic work toward trisaccharide formation also evaluated the utility of 1,2-cyclohexane-diacetal as a protecting group with glucuronic acid. The disarming qualities of these moieties were assessed, leading to the development of alternate routes. A more linear approach resulted in the formation of important disaccharide building blocks that contribute toward the synthesis of the core trisaccharide, including isolated 1,2-orthoesters. Further development of the chemistry established herein should allow for the formation of the desired core trisaccharide, while contributions have additionally been made toward its tool functionalisation and use in multivalent schemes.

Orientador: Hernandes Faustino de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-10T03:50:54Z (GMT). No. of bitstreams: 1 Augusto_TaizeMachado_M.pdf: 1393330 bytes, checksum: 02bc668b22957f9820d07a1791a2218b (MD5) Previous issue date: 2007 / Resumo: O crescimento e fisiologia da próstata são dependentes de andrógenos e sua privação resulta numa regressão acentuada na glândula, com uma redução a 10% do tamanho original após 21 dias de castração. Esta redução no tamanho é causada pela perda de ... / Abstract: The growth and physiology of the prostate are dependent on androgens and androgen deprivation results in marked regression of the organ, which is reduced to 10% of the original size 21 days after castration. This reduction in size is caused by t... / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Orientador: Hernandes Faustino de Carvalho / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T20:52:36Z (GMT). No. of bitstreams: 1 Augusto_TaizeMachado_D.pdf: 4110210 bytes, checksum: d70ae2c92435700f28fff9ee3cae63ac (MD5) Previous issue date: 2011 / Resumo: A próstata é uma glândula do aparelho reprodutor importante na reprodução, sendo foco de várias afecções, dentre elas o carcinoma prostático. Ela é altamente dependente de testosterona e modulada por estrógenos, que desempenham papel fundamental no seu crescimento. Altas doses de estrógeno aplicadas em ratos no período neonatal causam efeitos irreversíveis na próstata quando na idade adulta, sendo o mais marcante o comprometimento do seu desenvolvimento e crescimento. A este efeito foi dado o nome de imprinting estrogênico. O estrógeno interfere no eixo hipotalâmico-hipofisário-gonadal bloqueando a produção de gonadotrofinas e inibindo a secreção da testosterona, mas também age localmente via receptores de estrógeno presentes na próstata. A castração cirúrgica (pela retirada dos testículos) ou farmacológica acarreta involução da glândula prostática, efeito este que pode ser revertido pela reposição da testosterona. Durante este processo de regressão ocorre uma remodelação tecidual que em parte é dada pela morte das células epiteliais e pela reorganização da ECM subadjacente. Sabe-se da existência de vários fatores que atuam coordenadamente na reorganização da ECM, como enzimas proteolíticas, as MMPs, e a HPSE-1 (endo-ß--glicuronidase, que degrada cadeias de HS). Este trabalho foi dividido em dois grandes grupos de experimentos: no primeiro grupo, buscou-se investigar os efeitos globais do imprint estrogênico sobre o funcionamento prostático, a expressão da HPSE-1 na próstata ventral de ratos; no segundo grupo foi enfatizada a análise da contribuição da HPSE-1 na remodelação tecidual prostática que ocorre após a castração cirúrgica. Para a identificação e avaliação do efeito do imprinting estrogênico sobre o metabolismo prostático e sobre a expressão da HPSE-1, foram realizados experimentos em ratos Wistar neonatos e posteriores avaliações das análises morfológicas, bioquímicas e moleculares de suas próstatas na idade adulta. Neste trabalho pudemos demonstrar que a HPSE-1 desempenha papel relevante na segunda onda de morte das células epiteliais da próstata em regressão após a castração, com sua expressão aumentada 24 horas antes do segundo pico apoptótico da próstata em regressão. Pudemos evidenciar ainda que a exposição a altas doses de estrógeno na idade neonatal regula negativamente o estado metabólico prostático na idade adulta e está associado à diminuição da expressão gênica global como: maior compactação da cromatina, reduzida atividade nucleolar, e inibição da síntese protéica nas células epiteliais prostáticas. Este imprinting estrogênico resultante também refletiu no bloqueio da expressão da HPSE-1, o que foi confirmado tanto em nível de proteína como no de RNA mensageiro, particularmente nas células epiteliais. Estes resultados sugeriram um bloqueio em nível transcricional, por metilação do DNA / Abstract: The prostate gland is part of the male reproductive system and it is important for reproduction, and site of several diseases, including prostate cancer. It is highly dependent on testosterone and is modulated by estrogen, which play key roles in its growth. High doses of estrogen administrated in neoborn rats cause irreversible effects in adult prostate, the most markedly the impairment of its development and growth. This effect is named estrogen imprinting. Estrogen interferes with the hypothalamic-pituitary- gonadal axis blocking the production of gonadotropins and thereby inhibiting testosterone secretion, besides acting locally via estrogen receptors found in the prostate. Surgical (removal of the testis) or pharmacological castration, leads to the involution of the prostate gland, an effect that can be reversed by testosterone replacement. During this process, the tissue remodeling is partly given by epithelial cell death and reorganization of subadjacent ECM. Several factors act in coordination for the reorganization of the ECM, such as proteolytic enzymes, the MMPs, and HPSE-1 (endo-ß-glucuronidase that degrades HS). This work was divided into two main groups of experiments: in the first group, we sought to investigate the global effects of estrogen imprint on the functioning prostate, and included analysis of the expression of HPSE-1 in rat ventral prostate, and in the second group we emphasized the analysis of contribution of HPSE-1 in prostatic tissue remodeling that occurs after surgical castration. For the identification and evaluation of the effect of estrogen imprinting on metabolism of the prostate and on the expression of HPSE-1, experiments were performed in rats and subsequent evaluations of newborns (morphological, biochemical and molecular features) were made in their adult prostates. In this work we demonstrated that HPSE-1 plays a key role in the second wave of prostate epithelial cell death after castration, with its expression increased 24 hours before the second peak of apoptosis in prostate remodeling. We observed that chronic high doses of estrogen in the neonatal life regulates negatively the metabolic state of the prostate in adulthood and it is associated with the global gene expression decrease as: higher chromatin condensation, reduced nucleolar activity and inhibition of protein synthesis in the prostate epithelial cells. This estrogenic imprinting also reflected in blocking the HPSE-1 expression, which was confirmed at protein and mRNA synthesis blocking, particularly in epithelial cells. These findings suggested this blocking in a transcriptional level by DNA methylation / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

The work discussed here is divided into two projects. The first project involves the interactions between antidepressants and the platinum based chemotherapeutics while the second project begins to investigate possible implications of a recently discovered uptake mechanism for positively charged platinum drugs. Gaining understanding of the interactions between antidepressants and platinum-based chemotherapeutics is important due to the frequency with which they are prescribed together. Although using a combination regimen of antineoplastics is beneficial to the patient, not all drug interactions are. For instance, many of the serotonin reuptake inhibitors have been shown to decrease the efficacy of tamoxifen. Desipramine, a tricyclic antidepressant used to treat neuropathic pain, has been shown to increase the cytotoxicity of cisplatin, oxaliplatin and carboplatin in the human colon carcinoma cell line, HCT116 wt. To study this interaction, the cell line specificity as well as the drug specificity with regard to both the platinum-based chemotherapeutic and the antidepressant were investigated. The data show that the effect is both cell line specific as well as drug specific with respect to both types of drugs. To elucidate the mechanism behind the alteration in cytotoxicity of the platinum drugs, the effect of p53 status was investigated. A reduction of the effect is observed in the absence of p53, suggesting that there is a p53 dependent mechanism as well as a p53 independent mechanism. The tricyclic antidepressants and fluoxetine are known to be calmodulin inhibitors. Calmodulin inhibition mirrored some of the effects seen with the antidepressants suggesting that calmodulin inhibition might also play a role in the mechanism. The second project is based on the discovery that heparan sulfate proteoglycans mediate the uptake of positively charged platinum complexes. Heparan sulfate proteoglycans are important in cell-cell as well as cell-extracellular matrix adhesion. In cancer, heparanase, the enzyme that cleaves heparan sulfate, is over expressed creating a pro-angiogenic and pro-metastatic state. This work demonstrates that the positively charged platinum complexes can inhibit heparanase activity by binding to the substrate (heparan sulfate proteoglycans). This suggests that this class of drugs may have the capacity to be anti-angiogenic and anti-metastatic as well as cytotoxic.

The study of the processes relating glycobiology and cancer will have increased interest in coming years. To contribute to this trend the outcome of this work will be useful for investigations in glycobiology, using experimental methods exhibiting controlled carbohydrate composition, organization, and orientation, drawn from materials science and physics and that can be used in bioengineering and other technical areas in biology. In this work, the focus has been on physical studies of some members of the family of glycosaminoglycans and their role in cancer metastasis. The project studies the static adhesion of cancer cells to substrates functionalized with cell surface glycocalyx molecules and, in particular, in the interaction of heparan sulfate, keratan sulfate and chondroitin sulfates with the cells. Surface characterization techniques are used to analyze the structure of the polymeric brushes deposited on the substrates. The hypothesis that the adhesion of whole cancer cells to glysocaminoglycan substrates is a function of polysaccharide charge per dimer and chain length was proposed and tested.Part of the work has been dedicated to study the changes in the adhesion of tumor cells inthe presence of heparanase, an enzyme expressed in the tumor cell surface.The essential achievements of the project have been: a) Design of a new a method for the deposition and patterning of glycans to glass or silicon surfaces functionalized with a silane agent, exposing an amino terminated monolayer as functional substrate. b) Development of a new method for the calculation of the density of the deposited molecules. c) Physical characterization of the surfaces using a combination of surface science techniques, including ellipsometry and atomic force microscopy. These surfaces should be useful for developing additional experiments that may be helpful in understanding the adhesive properties of the cells. d) Comparative analysis of the behavior of cancer cells to the functionalized surfaces, specifically the study of the static adhesion of the cells, in the presence or absence of the surface protein heparanase or its inhibitors. e) Confirmation of the hypothesis that attachment of whole cancer cells, in vitro, depends linearly on the charge per dimer of polysaccharide.

Malignant brain tumors are diseases with poor prognosis and/or severe long-term side effects of treatment. This thesis aimed to discover novel regulators in brain tumor development, based on studying neural stem cell and progenitor cell (NSPC) differentiation and using animal models to introduce new insights to mechanisms of human brain tumors. The enzyme heparanase (HPSE) that degrades heparan sulfate (HS) is active in cell signaling and ECM remodeling. In paper I, we found an enhanced differentiation to oligodendrocytes in ES cell-derived NSPCs overexpressing HPSE. Further analysis suggested that this enhanced formation of oligodendrocytes was associated with alterations in receptor tyrosine kinase signaling, and that HPSE might also exert anti-apoptotic functions. Subsequently, in paper II we studied the involvement of HPSE in glioma development. We observed that high HPSE levels associated with poor survival in glioma patients. In experimental models, we found that HPSE promoted glioma growth, and that an inhibitor of HPSE reduced glioma progression both in vitro and in vivo. We hypothesize that regulators in NSPC differentiation could have a potential role in brain tumor development. In paper III, we explored the function of NRBP2, a pseudokinase that is up-regulated during NSPC differentiation. We found low expression of NRBP2 in brain tumors, in comparison to normal brain. In medulloblastoma, in particular, low NRBP2 expression is linked to poor prognosis. Overexpression of NRBP2 in medulloblastoma cells led to impaired cell growth and migration, concomitant with an increased cell death. In paper IV, we searched for novel glioma susceptibility genes by sequencing dog breeds from the same ancestor but with different glioma incidence. In this way we identified three new glioma-associated genes. Two of these are significantly regulated in human glioma and one of those might have a role in glioblastoma stem cell differentiation.

Alzheimer’s disease (AD) is a neurodegenerative disorder, with extensive evidence implicating the misfolding, aggregation and deposition of the amyloid-β (Aβ) peptide as central to the pathogenesis. Heparan sulfate (HS) is an interactive glycosaminoglycan, attached to core proteins as HS proteoglycans (HSPGs). HSPGs are present on cell surfaces and in the extracellular matrix where they facilitate multiple signaling functions, but HS is also consistently present in all amyloid deposits, including those of AD. In amyloidosis HS has been studied as an aggregation template, promoting fibril formation and serving a scaffold function in the resulting deposits. The objective of this thesis was to assess how cell surface HS is potentially implicated in Aβ amyloidosis and the associated neuroinflammation of AD.   In AD brain we determined that HS predominantly accumulated in Aβ deposits with dense cores and found glial-expressed HSPGs within these deposits. Aβ elevated HSPG levels in primary glial cultures, implicating activated glia as one source of the Aβ-associated HS. Next, we determined that microglial HSPGs are critical for the upregulation of interleukin-1β and tumor necrosis factor-α following exposure to lipopolysaccharide, an established inflammatory insult. Together these results raise the possibility that Aβ-induced expression of microglial HSPGs may promote neuroinflammation.   Multiple mechanisms of Aβ toxicity have been proposed and different Aβ assemblies exert their toxicity through alternative routes. We found that three different preparations of Aβ aggregates all exhibited HS-dependent cytotoxicity, which in part correlated with Aβ internalization. Furthermore, heparin treatment attenuated Aβ cytotoxicity and uptake. In Aβ-positive AD microvasculature, HS deposited with Apolipoprotein E (ApoE) and its receptor, the low density lipoprotein receptor-related protein 1 (LRP1). In cell culture, HS and LRP1 co-operated in Aβ interactions and the addition of ApoE increased the levels of cell-associated Aβ in a HS- and LRP1-dependent manner. This ApoE-mediated increase in cell-associated Aβ may promote toxicity and vascular degeneration, but equally HS-mediated internalization of Aβ could represent a clearance route across the blood-brain-barrier. The findings presented here illustrate multiple roles for cell-surface HSPGs in interactions relevant to the pathogenesis of AD.

Perlecan is an important basement membrane heparan sulfate (HS) proteoglycan that is essential for various cell signaling events involved in tissue development. Heparanase is a lysosomal enzyme involved in the turnover of HS. This project aimed to assist in researching the structure of HS on perlecan and how this structure changes with tissue development. This will be achieved by generating monoclonal antibodies that have an altered affinity for perlecan after heparanase treatment. Recombinant perlecan domain I was characterized by ELISA and western blotting and used as the antigen for two fusions. The first fusion was focused on the production of IgM the common subtype of anti-glycosaminoglycans antibodies. However, no clones were produced, which may have been due to the lack of feeder layers. In order to address this problem, the fibroblast cell line MRC-5 was used as a feeder layer in the second fusion. From this fusion, we obtained 216 positive cultures, which were screened against full length perlecan from endothelial cells. Of these, 26 cultures were tested against heparanase treated perlecan, and then 2 cultures were chosen for subcloning based on the different immunoreactivity between enzyme treated and nontreated perlecan. From the 2 chosen cultures, 13 sub clones were derived and 10 of them were adapted into a serum free culture environment. The 10 monoclonal antibodies displayed strong immunoreactivity with full length perlecan in ELISA and Western Blotting. When they were used as primary antibodies in Immunocytochemistry, they were able to recognize the native perlecan deposited by human chondrocytes. When the cells were incubated with heparanase, antibody 5D7-2E4 and 13E9-3G5 showed an increase in immunoreactivity while antibody 13E9-3B3 gave a decrease. These three antibodies will be the potential tools used in the future to study perlecan turnover in different cells and tissue. The remaining seven antibodies will also be very useful in the research of perlecan as they have been shown to bind to the protein core. In the future, it will be worth subcloning some of the frozen stored stocks of uncloned hybridomas, where there are potential opportunities to select antibodies, which will react with the carbohydrate chains on perlecan.

Heparan sulfate (HS) is a highly sulfated polysaccharide expressed on the cell surface and in the extracellular matrix, interacting with a large number of proteins. HS is implicated in human diseases, including different types of cancer and amyloid diseases such as Alzheimer's disease (AD). The aims of this thesis were to gain deeper insights into AD and cancer progression by elucidating the roles of HS in amyloid-β (Aβ) pathology and hypoxia. The toxic Aβ-peptide is a key molecule in AD due to its ability to aggregate and form amyloid plaques in the brains of diseased patients. It has been reported that HS accumulates with Aβ in these amyloid plaques. We have found that HS is differentially accumulated with Aβ species within the amyloid plaques in the brains of AD patients. We also identified that the HS in the plaques originated from glial cells. Further, we investigated the role of HS in Aβ toxicity using cell models that either lack HS or express abnormal HS. The results show that cell surface HS mediates Aβ internalization and cytotoxicity. Upregulation of heparanase, an endo-glucuronidase that specifically cleaves HS chains, in human cancers increases the potential of tumor cells to metastasize. Spalax, an animal model for hypoxic tolerance, expresses high levels of heparanase. Analysis of HS from different Spalax organs revealed a high sulfation degree and an atypical domain structure, likely modulated by high heparanase expression in the organs. Cells cultured under hypoxic conditions showed a similar HS domain structure and had an increase in heparanase mRNA. We propose that hypoxia-induced heparanase expression is relevant for tumor progression, a process often associated with oxygen deficiency. Altogether, the findings in this thesis are important for future development of therapeutics aiming at interfering with HS functions in AD and cancer.

Heparan sulfate proteoglycan (HSPG) or heparan sulfate (HS) degradation may contribute to endothelial cell (EC) dysfunction in diabetes. HSPGs, syndecan and perlecan, contain a protein core with mainly HS glycosaminoglycans (GAGs) attached. HSPGs modulate growth factors and function in membrane filtering. Heparanase induction is likely responsible for diabetic HS degradation. Heparin protects endothelium and insulin regulates glucose metabolism. Our objectives were to observe HSPG changes by studying EC GAG content and gene expression of syndecan, perlecan and heparanase under hyperglycemic conditions with insulin and/or heparin treatment.<p> GAGs, including HS, were determined by the carbazole assay and visualized by agarose gel electrophoresis in porcine aortic EC cultures treated with high glucose (30 mM) and/or insulin (0.01 U/ml) for 24, 48 and 72 hours and/or heparin (0.5 µg/ml) for 72 hours. High glucose decreased cell GAGs and increased medium GAGs. GAGs increased with time in control cultures and in high glucose plus insulin treated medium. GAGs were decreased with insulin but increased with insulin or heparin plus high glucose.<p> Confluent cultured human aortic ECs were incubated with control medium, high glucose and/or insulin and/or heparin for 24 hours. Real time PCR determination showed that: high glucose increased heparanase, decreased syndecan and had no effect on perlecan mRNA; insulin or heparin with/without high glucose decreased and insulin and heparin with high glucose increased heparanase mRNA; heparin and insulin with high glucose increased but insulin decreased syndecan mRNA. Actinomycin D (10 µg/ml) inhibited heparanase and syndecan mRNA with high glucose plus insulin plus heparin and inhibited heparanase mRNA with high glucose compared to time 0 but not â-actin after addition for 0, 2, 4, 8 and 24 hours. Bioinformatic studies revealed that transcription factor Sp1 activates heparanase promoter by high glucose and may play a role in regulation of perlecan and syndecan promoters.<p> Insulin or heparin inhibited the reduction in EC GAGs and syndecan mRNA and induction in heparanase by high glucose, indicating their protective effect. Decreased GAGs by insulin may relate to the pathology of hyperinsulinemia. Transcriptional regulation by heparin and/or insulin may cause variation in gene expression of heparanase, syndecan and perlecan.