This thesis is focused on the heparan sulphate (HS) structure from blind mole rat liver. HS is a glycosaminoglycan that is produced as a proteoglycan, in which linear polysaccharide chains are attached covalently to a protein core. Proteoglycans are widespread molecules in the body and have many important physiological functions. HS is synthesized as a polymer of alternating glucuronic acid and N-acetylglucosamine units. Parts of the polymer are subsequently modified by N-deacetylation /N-sulphation of the glucosamine units, C-5 epimerization of glucuronic acid to iduronic acid and O-sulphation at various positions. The mole rats are from Israel and are of the Spalax ehrenbergi superspecies. Spalax Judaei (S60) has 60 chromosomes and Spalax Galili (S52) has 52 chromosomes. They are both completely blind and spend their entire life underground in hypoxic conditions. Spalax Galili (S52) inhabits the cool-humid Upper Galilee Mountains and Spalax Judaei (S60) inhabits the warm-dry southern regions. There is no current information about the heparan sulphate structure of these animals. The two blind mole rats (S52 and S60) were metabolically labelled with [3H] Glucosamine. The animals were sacrificed and the organs were taken and frozen. The liver was chosen for the purpose of my project. The HS structure was studied using various chromatographic methods such as ion-exchange and gel filtration. Structural analysis of HS indicated that the size of HS from the liver was the same in both species. However, the domain structure differed between the two animals, particularly with regard to sample S52(1) which had obvious differences. This leads to the study of the heparanase cleavage sites. Disaccharide composition analysis identified varying proportions of disaccharide species in S52 and also the possibility of an unknown disaccharide species.

Syndecan-1 is a cell surface proteoglycan which participates in cell adhesion, differentiation, motility, morphogenesis and intracellular signaling. The two glycosaminoglycans heparan sulfate and chondroitin sulfate are covalently attached to the ectodomain of syndecan-1 via a tetra saccharide linkage sequence. However, the ectodomain can be modified having only one or neither of the glycosaminglycans attached. The glycosaminoglycans are capable of binding ligands such as fibroblast growth factors (FGFs) and support activation of receptors. The ectodomain is proteolytically cleaved from the cell surface by metalloproteinases in a process known as shedding. Shedding turns the ectodomain into a soluble effector which can stimulate other cells in the surroundings by delivering growth factors and also translocate into cells through endocytosis. In this study the aim was to find out if a modified ectodomain, which only contains chondroitin sulfate, could support intracellular signaling in the absence of heparan sulfate. The aim was also to find out whether a modified ectodomain could translocate into the cell. The methods used were cell culturing, isolation and purification of syndecan-1 ectodomain, cell signaling and immunohistochemistry. It was found that modified shed syndecan-1 ectodomain was able to support intracellular signaling almost to the same degree as wild type syndecan-1 ectodomain. This may suggest that heparan sulfate does not have to be present on the ectodomain to support intracellular signaling, although the signal is slightly higher when present. When trying to detect translocation of the ectodomain the results were too uncertain and further research is required.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-13T12:52:18Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE Ricardo de Souza Silva.pdf: 2617103 bytes, checksum: 83ada60ae5dc3c77a19892b0c337a3d7 (MD5) / Made available in DSpace on 2015-03-13T12:52:18Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE Ricardo de Souza Silva.pdf: 2617103 bytes, checksum: 83ada60ae5dc3c77a19892b0c337a3d7 (MD5) Previous issue date: 2014 / CAPES / Métodos de separação por afinidade têm se revelado eficazes e econômicos. Eles se baseiam na formação de um complexo estabelecido entre duas biomoléculas afins a um suporte insolúvel em água. Lavagens iniciais deste compósito removem impurezas de sorte que em uma segunda etapa o complexo é desfeito, liberando a biomolécula de interesse mediante seu rompimento (força iônica, pH, etc.). Dentre os diferentes métodos, aqueles baseados em suportes magnéticos têm as vantagens de fácil síntese, manuseio e o derivado ser recuperado por meio de um campo magnético. Esta tese se postulou a revestir partícula magnéticas com heparina (HEP) e heparan sulfato (HS) uma vez que esses glicosaminoglicanos são muito negativamente carregados e formam complexos com proteínas, por exemplo, proteínas plasmáticas. Três tipos de materiais foram sintetizados: polietilenotereftalato magnetizado (mPET), magnetita (MAG) e magnetita revestida com polianilina (mPANI). Inicialmente, filmes de PET sofreram hidrazinólise e o pó PET-hidrazida obtido foi magnetizado pelo método de co-precipitação em solução de cloretos férrico e ferroso. A MAG foi sintetizada conforme descrito acima, porém, sem adição do PET, e posteriormente foi revestida com PANI (mPANI). HEP e HS foram ativados com carbodiimida e N-hidroxissuccinimida e incubados com os suportes para que ocorressem o revestimento das partículas, produzindo os seguintes derivados magnéticos: mPET-HEP; mPANI-HEP; MAG-HEP; mPET-HS e mPANI-HS. As amostras de mPET-HEP foram investigadas por análise elementar e espectroscopia de infravermelho. A composição centesimal delas mostrou um aumento na razão carbono : nitrogênio, presumindo ser devido à imobilização de heparina. O espectro de infravermelho do derivado contendo heparina apresentou bandas em 1653 cm-1 e 1547 cm-1, característicos da ligação amida, formada pela conjugação. O derivado mPANI imobilizou cerca de 37% da heparina ofertada, embora a magnetita pura (MAG) também tenha imobilizado, porém em menor grau. Todas estas matrizes demonstraram uma boa estabilidade em 10 ciclos consecutivos de reutilização mesmo após 2 anos estocadas a 4°C. Quanto ao HS, este foi covalentemente fixado às partículas mPANI e mPET em torno de 35 μg e 38,6 μg por mg de suporte respectivamente, no entanto também foi adsorvido (18μg/mg) sobre as partículas controle de MAG. Indícios do sucesso da imobilização nestes compósitos foram a demonstração de que a heparina fixava o corante azul de metileno e retardava o tempo de coagulação do plasma recalcificado quando em contato com estes suportes. Finalmente, todos os derivados magnéticos foram incubados com plasma humano e lavados posteriormente com gradientes de NaCl para liberar as proteínas fixadas e detectá-las a 280nm. Eletroforese destas proteínas revelaram bandas referentes à antitrombina (58kDa) e as suas dosagens nos plasmas e eluatos confirmaram a eficiência do método de separação. Nos derivados mPET-HS e mPANI-HS, além da antitrombina, importantes fatores da coagulação também foram observados na eletroforese e comprovada a sua separação através de testes de coagulação específicos. Portanto, os resultados mostraram que estes compósitos produzidos são promissores na purificação da antitrombina e outros fatores da coagulação presentes no plasma humano.

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.

La production physiologique continue de cellules myélinisantes dans le système nerveux (SN) de mammifère offre de nouvelles perspectives thérapeutiques. Lors d’une atteinte de la myéline, une régénération endogène impliquant la génération d’oligodendrocytes s’engage. Ce processus repose sur la mobilisation de progéniteurs oligodendrocytaires parenchymateux et de progéniteurs de la zone sous-ventriculaire (SVZ). Cette réparation ne permet cependant pas une récupération fonctionnelle systématique. Nos travaux ont pour but d’identifier les facteurs qui contrôlent les différentes étapes de régénération. Ils révèlent une réexpression du CNTF et une surexpression des héparans sulfates protéoglycanes (HSPGs) suite à une démyélinisation du corps calleux. Ces changements de l’environnement péri-lésionnel régulent positivement le processus de remyélinisation. Nous avons montré un impact direct de l’expression post-lésionnelle du CNTF sur la mobilisation des deux sources cellulaires. Différents tests in vitro ont identifié le CNTF comme facteur chémoattractant pour ces cellules. Nos données montrent également que des modifications de sulfatation des héparans sulfates (HS) protéoglycanes contrôlées par la N-désacétylase-Sulfotransférase 1 des cellules du lignage oligodendrocytaire s’établissent en bordure de lésion et créent un microenvironnement favorable à la régénération. Divers test fonctionnels in vivo et in vitro révèlent le rôle clef des HSPGs dans la cinétique de démyélinisation et de remyélinisation en régulant la mobilisation des cellules du lignage oligodendrocytaire et l’activation microgliale. / In the mammal’s nervous system, the ongoing production of new myelinating cells on has open news therapeutic perspectives for demyelinating diseases. An endogenous regeneration process involving the generation of oligodendrocytes can occur following demyelination. This process relies on the mobilization of an endogenous reservoir of progenitor cells located in the adult brain: The parenchymal oligodendrocyte precursors and the subventricular zone derived neural progenitors. However, these endogenous repair attempts do not permit an efficient functional recovery. These failures are mainly due to mobilization, differentiation or to the generation of a hostile environment for the repair process. Our work is focusing on the identification of factors regulating those events. Our data show the reexpression of CNTF and overexpression of heparan sulphate proteoglycans (HSPGs) following a focal demyelination of the corpus callosum in adult mice. These environmental changes favor myelin repair. We show a direct impact of the post-lesional expression of CNTF on the mobilization of both cellular sources. Using various in vitro assays, we showed that CNTF is acting on the two cellular sources as a chemoattractant factor. Our data also show that sulfation modifications of HSPGs performed by the deacetylase-N-sulfotransferase 1 (Ndst1) on oligodendrocyte lineage cells occurred around the lesion and created a permissive microenvironment for the regenerative process. Various in vitro and in vivo functional assays demonstrated the key role of HSPGs in demyelination and remyelination dynamic by controlling mobilization of the oligodendrocyte lineage cells and microglial activation.

Polynuclear platinum compounds represent a new class of potential platinum anticancer therapeutics. Derived from the most widely used platinum anticancer drug, cisplatin, these novel compounds are distinct in their interactions with bio-molecules. The effectiveness of platinum anticancer agents is influenced by three pharmacological factors: (i) their resistance to deactivating sulfur nucleophiles, (ii) the ability to gain cellular entry and efficient cellular uptake, and (iii) the ability to form stable and specific complexes with DNA. BBR 3464, the first multinuclear platinum compound to reach phase II clinical trials, has created a new approach to cancer drug design. Large, highly charged platinum compounds have been shown to form favorable covalent and noncovalent interactions with bio-molecular structures. Compounds such as BBR 3464, form an immediate pre-association with anionic structures on biomolecules before covalent attachment. To better characterize these interactions, a new set of compounds was designed that exclusively interacts via electrostatic associations and hydrogen bonding. The investigation of noncovalent complexes between DNA, proteins, and peptides with a variety of synthetic and biological relevant structures has become increasingly more common with the coupling of electrospray ionization and mass spectrometry (ESI-MS). Mass spectrometry has been useful to the drug design community by allowing the rapid and accurate characterization of drug binding sites. In the first project we have explored the use of collision induced dissociation (CID) to map the potential binding sites of noncovalent polynuclear platinum compounds of varying size and charge with an antisense oligonucleotide of the Bcl-2 sequence. In the second project, the gas-phase dissociation and stabilizing effects of these polynuclear platinum compounds on duplex DNA were determined. Correlations between the size and charge of associating platinum compounds were determined by comparing the change in gas phase stability under CID conditions. Additionally, the association of these new types of noncovalently binding polynuclear platinum compounds was investigated with model cell surface structures such as anionic heparan sulfate and phospholipids.

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.

Heparan sulfate (HS) is a cell-surface sulfated polysaccharide that binds to multiple proteins and has been implicated in cancer, viral infection and Alzheimer's disease. Due to the heterogeneity of HS, the structural requirements for protein binding are ill- defined. Chemical synthesis of structurally-defined HS oligosaccharides, which are tunable in terms of length, order of monosaccharides and sulfation pattern, is required for the investigation of HS-protein binding. Single molecule methods have been utilised in biophysics to study dynamic processes and can allow observation of rare events which would be 'averaged out' in ensemble measurements. Access to fluorescently labelled HS oligosaccharides should allow investigation of interactions with proteins at the single molecule level using methods such as single molecule FRET, providing a method complementary to NMR studies (ensemble) and X-ray crystallography (non-dynamic).This thesis presents the development of a method for the fluorescent labelling of a chemically synthesised HS disaccharide utilising a reducing-end amine tag. Analysis of the fluorescence properties of the labelled disaccharide at ensemble and single molecule level indicated no perturbation of the fluorophore when attached to the sugar. Fluorescence correlation spectroscopy measurements of the fluorescent HS disaccharide with the protein FGF-1 showed no binding, which is attributed to the low concentration (1 nM) of disaccharide required in the experiment. Additional work is presented in this thesis on the development of a method for atom-specific 13C labelling of HS oligosaccharides, which has been initiated by synthesis of a 13C labelled L-iduronate monosaccharide and a 13C labelled disaccharide. New strategies for the synthesis of HS oligosaccharides based on orthogonal thioglycoside-based glycosylations employing S-benzoxazolyl and S-thiazolyl donors have been investigated. Development of a chemoselective glycosylation strategy for HS oligosaccharide synthesis utilising a 'super-disarmed' [2.2.2] L-iduronic lactone is presented.

Proteoglycans containing Heparan Sulfate (HS), a sulfated glycosaminoglycan (GAG), play a major role in the cell signaling process, interacting with many different proteins. HS is over expressed on the surface of many cancer cells. Enzymatic cleavage of HS-GAGs by heparanase causes release of angiogenic growth factors leading to tumor cell migration. Heparanase is also over-expressed in tumors with significant correlation between metastatic potential and heparanase activity. Proteoglycans and their associated enzymes are thus significant drug targets of high biological relevance. A functional consequence of strong PPC-HS binding has been shown in proof-of-concept studies confirming inhibition of the model pentasaccharide, Fondaparinux, by bacterial Heparinase. Such metalloshielding by PPCs may also protect HS from enzymatic cleavage by the mammalian heparanase; preventing growth factors from binding to HS and/or preventing release of bound growth factors and thus inhibiting the metastatic response in the cancer cells. HS-GAGs are also receptors for cellular accumulation of cationic Polynuclear Platinum Complexes (PPCs) through high-affinity binding to the highly anionic HS. PPCs competitively inhibit uptake of TAMRA-R9, a fluorescent nona-arginine derivative, in CHO cells. The previously reported series of Pt(II) complexes were investigated as DNA binders, initiating the apoptotic cascade. The result of PPC-DNA binding produces long range inter and intra-strand cross-links, that produce structural and conformational changes. Hydrogen bonding between phosphate oxygens and square planar Pt(II) nitrogen results in bidentate complexes by either backbone tracking or groove spanning of DNA. This complex forms a clamp like structure, called a phosphate clamp, similar to that of the arginine fork. Understanding this clamp allows us to investigate the structurally similar sulfate binding between metal complexes and target HSPG. HSPGs may allow significant research into both a novel cellular internalization of principal metals and “metalloshielding” of heparin by these compounds. Previous studies have shown that a wide range of metal ions have high affinity to heparin. The trend of metal/heparin affinity is believed to be dependent on parameters consisting of the metal’s overall size, spatial orientation of the ligands attached to each metal, the net charge and oxidation state of these metals, and number of binding sites. Studies have shown relative affinities of sulfate and carboxylate groups for the metal ions. These metal cations play an important role in the affinity, specificity, and stability of many protein/heparin interactions. The study of simple coordination compounds, like Pt, Mn, V, Ru and Co, will allow preliminary results which will extend into the PPCs mode of binding. This thesis focuses on the concept of metalloglycomics and reviews the interactions of various metal complexes with heparin. The covalent and non-covalent interactions of metal complexes with heparin resulting in strong bonding are explained through spectroscopy and calorimetry. The cleavage inhibition of heparanase by metal complexes is also described. Sulfate cluster anchoring shields the sulfates from loss as seen in mass spectrometry. The study of metalloglycomics offers potential understanding into the relevance of metal-heparin interactions and possibilities into the development of new compounds as therapeutic agents.

<p>Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS.</p><p>The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.</p>