Sialylation, a posttranslational modification of both glycolipids and glycoproteins, is typically found on the terminal positions of glycan chains. Unique among most other sugars, sialic acids are nine carbon sugars that are negatively charged and undergo a variety of side group modifications, which contribute to its role in cell-cell interactions and receptor recognition. While the majority of Drosophila glycoproteins do not terminate in sialic acid compared to mammalian glycoproteins, a sialic acid synthetic pathway is present in Drosophila but it is developmentally regulated and appears to be restricted to the nervous system (Kim et al., 2002; Koles et al., 2004). In order to investigate the role of the sialic acid pathway in Drosophila, we generated a null mutation of the sialic acid synthase gene (SAS) by imprecise excision of a nearby transposable element. Homozygous null flies exhibit partial lethality, male sterility and undergo age dependent neurodegeneration as evidenced by loss of locomotion and increased vacuolization in the brain. Mutant flies also have a shortened life span and display increased sensitivity to heat as they age. To identify protein targets of sialylation that possibly contributed to these phenotypes, a very sensitive solid-phase extraction method was used to capture sialylated glycopeptides from head extracts of wild type and SAS null flies. In collaboration with M. Betenbaugh and H. Zhang at Johns Hopkins University, I identified three sialylated peptides; the major peptide target was derived from the Shaker voltage-dependent potassium channel. The other two peptides were encoded by genes of unknown function. Electrophysiological measurements performed on control and SAS mutant larvae at the pre- and post-synaptic larval neuromuscular junction (in collaboration with T. Dean and A. Seghal, University of Pennsylvania) showed that loss of sialylation induced a depolarizing shift in the gating parameters of the Shaker ion channels, similar to what was previously reported in mammalian cell culture (Johnson and Bennett, 2007). Pre-synaptic neurons from the mutants displayed a two to three fold increase in the number of miniature excitatory peaks suggesting that the neurons were hyperactive. Since Shaker is a major target of sialylation in brain neurons, I suggest that the loss of sialylation of Shaker plays a major role in the neurodegeneration phenotype observed in our SAS null mutant flies. SAS mutant flies are unusually sensitive to starvation, typical of flies that cannot maintain metabolic homeostasis. Upon 24 h of starvation, mutant flies differed from control flies in that they consumed most of their triglyceride stocks, they displayed poor locomotion, had smaller sized cells in their fat body, and reduced their glycogen stores significantly. Mutant flies expressed and likely secreted an excess of insulin like protein, hyperinsulinemia, for the first five days after eclosion. However, as the flies aged (14-21 days) they had high hemolymph sugar, low insulin like protein expression and fewer number of insulin producing cells. All these phenotypes are similar to diabetic patients, as diabetic patients also have metabolic inflexibility, high blood sugar, low insulin secretion, and fewer number of insulin producing cells. It is now known that the Shaker homolog KV1 is expressed in human pancreatic β cells which secrete insulin (Ma et al., 2011). I propose that in our mutants the failure to sialylate the Shaker channel, which is known to be present in insulin producing cells (IPC ), will result in those cells secreting an excess of insulin , which in turn causes the metabolic defects leading to a diabetic state. By studying our sialic acid null mutants we can obtain useful information about how diabetes develop. Using the powerful genetics of flies, we can perform screens to identify novel genes that either enhance or reduce the sensitivity to starvation of our SAS mutants and thus play a role in the development or potential treatment of diabetes / Biology

Virulent strains of the bovine opportunistic pathogen Haemophilus somnus (Histophilus somni) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. It is reported in this dissertation that H. somnus significantly (P <0.001) inhibited production of superoxide anion (O2-) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O2- production was time- and dose-dependent and did not occur after incubation with Escherichia coli, H. influenzae, or Brucella abortus. Non-viable H. somnus, purified lipooligosaccharide (LOS), or cell-free supernatant from mid-log phase cultures did not inhibit O2- production, indicating that O2- inhibition required contact with live H. somnus. Commensal isolates of H. somnus were less capable or incapable of inhibiting macrophage O2- production compared to isolates tested from disease sites. H. somnus shares conserved epitopes in its LOS with Neisseria gonorrhoeae, N. meningitidis, and H. influenzae, and can also undergo structural phase variation of these LOS epitopes. Sialylation of the terminal galactose of H. somnus LOS is another reported virulence mechanism. Current sequencing of the genomes of H. somnus strains 2336 (pathogenic) and 129Pt (commensal) has enabled in silico identification of three open reading frames (ORFs) involved in sialylation. The ORFs-1 (hsst-I) and -2 (hsst-II) had BLASTx homology to sialyltransferases, while ORF-3 (neuAhs) had BLASTx homology to CMP-sialic acid synthetases. These ORFs were amplified by PCR and cloned into the expression vector pCWOri+. Thin layer chromatography of the hsst-I gene product showed this sialyltransferase exhibited preference for sialylation of terminal N-acetyllactosamine (LacNAc, beta-Gal-[1,4]-beta-GlcNAc-R). However, Hsst-II preferentially sialylated lacto-N-biose (LNB, beta-Gal-[1,3]-beta-GlcNAc-R). In this study, phase variation of the terminal linkage in isolate 738 from a 3 linked galactose (LNB) to a 4 linked galactose (LacNac) was demonstrated. Such variation of a glycose linkage appears to be a novel mechanism of LOS phase variation. Furthermore, the ability of sialylated strain 738 LOS vs de-sialylated strain 738 LOS to induce Toll-like receptor 4 signaling was decreased by 28%, as determined by ELISA for Macrophage Inflammatory Protein-2. Therefore, sialylated LOS may aid H. somnus to avoid host innate immunity. / Ph. D.

Incorporation of N-acetyl neuraminic acid (NANA), or sialic acid, onto lipooligosaccharide (LOS) enhances the virulence of several bacterial species. In the present study, we assessed the effect of sialylation of Histophilus somni LOS on complement-mediated killing, binding of complement factor H (which converts C3b to inactive C3b (iC3b) and inhibit the alternative complement pathway) to the bacteria, complement activation by the LOS, and phagocytosis and killing of the bacteria by bovine polymorphonuclear leukocytes (PMN). Killing of H. somni by alternative complement pathway was measured by incubation of sialylated or non-sialylated H. somni with antibody-free precolostral calf serum (PCS) followed by viable plate count. A complement dose-dependent response to killing of non-sialylated H. somni by PCS was observed. However, sialylated H. somni were significantly (P = 0.001) more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated (P = 0.025) and consumed (P = 0.001) less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized sheep red blood cells or rabbit red blood cells, and by western blotting of C3 activation products. Sialylated H. somni bound more factor H than non-sialylated bacteria (determined by enzyme-linked immunosorbent assay) (P = 0.004), supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni inhibited both PMN phagocytosis of 3H-thymidine-labelled bacteria (P = 0.004) and intracellular killing of the bacteria (P = 0.0001), compared to non-sialylated bacteria. Therefore, sialylation of the LOS results in enhanced binding of complement factor H to the bacteria, resulting in diminished complement activation, resistance to complement-mediated lysis, and PMN phagocytosis and killing. / Master of Science

Yes / Polysialic acid (polySia) is a linear polysaccharide comprised of N-acetylneuraminic acid residues and its over-expression in cancer cells has been correlated with poor clinical prognosis. An assay has been developed for quantitative analysis of cellular polySia expression. This was achieved by extracting and purifying released polySia from glycoproteins by mild acid hydrolysis and optimised organic extraction. The polySia was further hydrolysed into Sia monomers, followed by fluorescent labelling and quantitative analysis. The assay was qualified utilising endoneuraminidase-NF to remove polySia from the surface of C6-ST8SiaII cancer cells (EC50 = 2.13 ng/ml). The result was comparable to that obtained in a polySia-specific cellular ELISA assay. Furthermore, the assay proved suitable for evaluation of changes in polySia expression following treatment with a small molecule inhibitor of polysialylation. Given the importance of polySia in multiple disease states, notably cancer, this is a potentially vital tool with applications in the fields of drug discovery and glycobiology.

Polysialic acid is a unique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acid is an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma, strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST (ST8SiaIV) and STX (ST8SiaII), with STX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferase could lead to control of tumour dissemination and metastasis. The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferase inhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells (SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acid surface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acid surface expression and invasion. Of the potential polysialyltransferase inhibitors evaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p < 0.05) reduction in cell-surface polysialic acid re-expression and invasion in polysialic acid expressing cells. Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over other members of the wider sialyltransferase family (α-2,3- and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitro cell based assays to discovery more potent derivatives.

Sialic acid is a highly abundant and a common component of vertebrate glycans, where it can be found in the terminal positions of the cell surface glycoconjugates. The amount of sialylated glycoconjugates as well as their complexity vary between both different species and different tissue types within one individual. Considering the vertebrates, these well studied structures are know to be important for cell-cell interactions, cell adhesion and immunity. In contrary, sialic acid in arthropod glycans has been identified only in a limited number of species. In obligatory blood feeding parasites such as ticks, distinguishing between sialylated glycoproteins of tick and host origin is challenging due to huge volumes of ingested blood containing heavily sialylated structures of host origin. In the tick Ixodes ricinus, the presence of minor amount of tick´s sialylated structures has been shown previously in the ovaries and salivary glands, however, their role remains completely unknown. In this thesis, we study the importance and role of both the tick-originating and the host sialylated glycoproteins for I. ricinus, the tick commonly found in Czech Republic. We show that the tick-originating sialylated glycoproteins are present in I. ricinus eggs, and that their amount changes over time after laying the eggs. Furthermore, these molecules were localized in cryosections of 14 days old eggs and in the larvae using confocal microscopy. In addition, we shed some further light on the role of sialic acid for ticks in the tick blood meal. According to our results, the glycan part of glycoproteins is the key in recognition of these molecules by tick cells.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-08-07T14:37:31Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO MESTRADO_LARISSA BRITO-RG.pdf: 842393 bytes, checksum: 7e14b33afc3df50dad2cfe57c1a8a47b (MD5) / Made available in DSpace on 2017-08-07T14:37:31Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) DISSERTAÇÃO MESTRADO_LARISSA BRITO-RG.pdf: 842393 bytes, checksum: 7e14b33afc3df50dad2cfe57c1a8a47b (MD5) Previous issue date: 2016-02-18 / CNPQ / O soro do leite de cabra (SORO), subproduto geralmente descartado durante a fabricação do queijo, é uma boa fonte de ácido siálico (Sia), oligossacarídeo envolvido em processos como memória e excitabilidade cerebral. Neste estudo, investigamos em ratos os efeitos do SORO em pó sobre a memória e a depressão alastrante cortical (DAC), fenômeno relacionado à excitabilidade cerebral, fornecendo evidências para o envolvimento do Sia nesse efeito. Além disso, avaliamos se a deficiência nutricional modularia a ação do SORO. Ratos Wistar foram amamentados em ninhadas com 9 e 15 filhotes (grupos L9 e L15, respectivamente). Nos dias pós-natais (P) 7 a 14, os animais receberam por gavagem 17,45 g/kg/d de SORO, ou Sia (20 mg/kg/d, ou 100 mg/kg/d). No P28-30, testamos a memória dos animais (tarefa de reconhecimento de objetos, TRO). No P35-45 registramos a DAC e analisamos a sua velocidade de propagação, amplitude e duração. Na TRO, os ratos L15 tratados com o SORO obtiveram melhor desempenho do que os controles-L15. Os ratos L15 exibiram velocidades da DAC mais elevadas em comparação com os grupos L9. Os grupos SORO e Sia exibiram velocidade da DAC mais elevada que os grupos naïve e salina, independentemente do estado de lactação (p<0,05). Nossos resultados documentaram este efeito do SORO na memória e DAC. Sia facilitou a DAC de forma dose-dependente, sugerindo seu envolvimento nessa ação do SORO. Este é considerado um suplemento potencial para melhorar a função e desenvolvimento do cérebro em crianças desnutridas. Mais estudos são necessários para investigar tal potencial. / Goat Whey, a usually discarded byproduct from goat cheese manufacturing, is a good source of sialic acid (SA), an oligosaccharide that is involved in processes such as memory and brain excitability. Here, we investigated in rats the effect of dried goat whey (DGW) on memory and the brain excitability-dependent phenomenon known as cortical spreading depression (CSD). We also provide evidence for the involvement of SA in this effect. In addition, we tested animals under unfavorable suckling conditions to evaluate whether nutritional deficiency would modulate DGW action. Wistar rats were suckled in litters with 9 and 15 pups (groups L9 and L15, respectively). From postnatal (P) days 7 to 14, the animals received per gavage 17.45 g of DGW/kg/d, or SA (20 mg/kg/d, or 100 mg/kg/d). At P28-30, we tested the animals‟ memory in the object recognition paradigm. At P35-45 we recorded CSD and analyze its velocity of propagation, amplitude, and duration. In the object recognition test, the L15 DGW-treated rats performed better than the L15-controls. The L15 rats displayed higher CSD velocities compared with L9 groups. The DGW and SA groups exhibited higher CSD velocity than the naïve- and saline-treated controls, regardless the lactation status (p <0.05). Our results documented a novel effect of DGW on memory and CSD. SA dose-dependently facilitated CSD, suggesting its involvement on the DGW action. DGW is considered a potential supplement to improve brain development and function in malnourished children, and this shall be further translationally investigated.

Made available in DSpace on 2018-06-27T18:55:57Z (GMT). No. of bitstreams: 3 Danieli Ferrari da Rosa.pdf: 3718059 bytes, checksum: bb8ef19a5af8a3faa7687e991e0d5c3d (MD5) Danieli Ferrari da Rosa.pdf.txt: 158457 bytes, checksum: b6b83ac5211b5e9ee8b9dfba9feba1d9 (MD5) Danieli Ferrari da Rosa.pdf.jpg: 3270 bytes, checksum: 1f0862e05ecb2e7b1da2056322eeeaab (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sialic acid is the generic name of carboxylated monosaccharides family with nine carbon glycoconjugated at terminal portion. These molecule family are involved in several biological processes such cell recognition processes, platelet adhesion, migration, invasion and metastatic potential, it also work as a receptor for bacteria and viruses. High concentrations of total sialic acid in the blood have been reported in different groups of patients with brain tumors, leukemia, melanoma, carcinoma and other kinds of cancers. The cleavage of sialic acid is a crucial step in virus infection influenzae, since this acid is part of the cellular receptor that the virus uses during the process of cellular internalization. The neuraminidase, an enzyme produced by the virus, cleaves the bond between sialic acid and the viral glycoproteins, allowing the entry of viruses into cells.The aim of this study was the analysis of serum sialic acid levels in murine melanoma and Herpes Simplex virus-1 (HSV-1) infection model. In the tumor model were used C57BL/6 and in the viral model BALB/c mice. Mice were injected with 2x105 B16F10 cells subcutaneously in the thigh and the tumor progression was followed each day till it became visible. The HSV-1 infection was conducted by intraperitoneally injection of with 102 PFU of virus. The sialic acid in serum samples was quantified by thiobarbituric method in spectrophotometer at 549 nm. A standard curve with commercial sialic acid was used as parameter for quantification. The results showed that in tumor model the sialic acid was increased compared with control group and have significant difference (p <0.05) in the first day after administration of cells. For the viral infection the concentration of sialic acid showed a significant difference (p <0,05) in the first day after infection when compared infected with control group. The histological analysis in thigh of mice performed 24 hours after administration of B16F10 cells were found compact groups of round or polygonal melanocytes with clear and large cytoplasm, irregular chromatin, hyperchromatic and vacuolated nuclei, eosinophilic nucleoli and atypical mitosis. / O ácido siálico é o nome genérico dado a família de monossacarídeos carboxilados com nove átomos de carbono que aparece na porção terminal de glicoconjugados. Estas moléculas estão envolvidas em vários processos biológicos, tais como, processos de reconhecimento celular, adesão plaquetária, migração, invasão, potencial metastático, sendo também um receptor para bactérias e vírus. O aumento das concentrações séricas de ácido siálico total tem sido descrito em vários grupos de pacientes que sofrem de tumores cerebrais, leucemia, melanoma, carcinoma e outros tipos de cânceres. A clivagem do ácido siálico é um passo crucial para a infecção do vírus Influenza, uma vez que este ácido é parte do receptor celular usado pelo vírus durante o processo de internalização celular. A neuraminidase, enzima produzida pelo vírus, cliva a ligação entre o ácido siálico e as glicoproteínas virais, permitindo a entrada dos vírus nas células. O objetivo desse estudo foi analisar os níveis séricos de ácido siálico em modelo de melanoma murino e modelo de infecção herpética (HSV-1). No modelo tumoral foram utilizados camundongos C57BL/6 e no modelo viral camundongos BALB/c. Os camundongos receberam 2x105 células B16F10 através da administração subcutânea na coxa e a progressão do tumor foi acompanhada todos os dias até o tumor se tornar visível. A infecção com HSV-1 foi realizada através da administração intraperitoneal de 102 PFU de vírus. O ácido siálico das amostras de soro foram quantificadas pelo método tiobarbitúrico em espectrofotômetro à 549 nm. Uma curva padrão com ácido siálico comercial foi usada como parâmetro para a quantificação. Os resultados mostraram que as concentrações de ácido siálico no modelo tumoral foram aumentadas nos animais com tumor quando comparadas ao grupo controle e houve diferença significativa (p< 0,05) no primeiro dia após a administração das células. Para o modelo de infecção viral houve diferença significativa (p< 0,05) no primeiro dia após a infecção quando comparado o grupo infectado com o controle. Na análise histológica da coxa dos camundongos realizada após 24 horas da administração de células B16F10 foram encontrados grupos compactos de melanócitos arredondados ou poligonais, com citoplasma amplo e claro, cromatina irregular, núcleos hipercromáticos e vacuolizados, nucléolos eosinofílicos e mitoses atípicas.

Polysialic acid is aunique carbohydrate that decorates the surface of the neural cell adhesion molecule. Polysialic acidis an onco-developmental antigen, expressed in tumours principally of neuroendocrine origin, notably neuroblastoma,strongly correlating with invasion and metastasis. Polysialylation is regulated by two polysialyltransferase enzymes, PST(ST8SiaIV)and STX(ST8SiaII),withSTX dominant in cancer. Post-development polysialic acid expression is only found at low levels in the brain, thus this could be a novel target for cancer therapy. It is hypothesized that inhibition of polysialyltransferasecould lead to control of tumour dissemination and metastasis.The aims of this thesis were to develop tools and in vitro assays to screen novel polysialyltransferaseinhibitors. A panel of tumour cell lines were characterised in terms of growth parameters (using the MTT assay) and polysialic acid expression. This includes a pair of isogenic C6 rat glioma cells (C6-STX and C6-WT) and naturally polysialic acid expressing neuroblastoma cells(SH-SY5Y). Following this, an in vitro assay was validated to screen modulation of polysialic acid expression by removing pre-existing polysialic acid expression using endoneuraminidase N and evaluated the amount of re-expression of polysialic acid using immunocytochemistry. Then, a functional assay was developed and validated for invasion, the matrigel invasion assay. Cytidine monophosphate (tool compound) significantly reduced polysialic acidsurface expression and invasion. A panel of six novel polysialyltransferase inhibitors was screened for cytotoxicity, polysialic acidsurface expression and invasion. Of the potential polysialyltransferase inhibitorsevaluated, ICT3176 and ICT3172 were identified from virtual screening of Maybridge library and were emerged as the most promising inhibitors, demonstrating significant (p<0.05)reduction in cell-surface polysialic acidre-expression and invasion in polysialic acid expressing cells.Furthermore, the specificity of compounds for polysialyltransferase (α-2,8-sialyltransferase) over othermembers of the wider sialyltransferase family (α-2,3-and α-2,6-sialyltransferases) was confirmed using differential lectin staining. These results demonstrated that small molecule inhibitors as STX is possible and provides suitable in vitrocell based assays to discovery more potent derivatives.

Le couplage croisé entre les esters allyliques et les composés carbonylés promu par le diiodure de samarium est une méthode efficace pour la formation de liaisons carbone-carbone. Une approche « umpolung » de réaction entre un composé carbonylé électrophile et un allylsamarien nucléophile, obtenu sans la réduction préalable d’espèces de type π-allylmétal de transition intermédiaires, fournirait une nouvelle voie plus simple pour la construction de cette liaison dans des conditions douces.Les esters allyliques de type dihydropyranyle se sont montrés d’excellents substrats dans les réactions d'allylation, d’aldéhydes ou de cétones, induites par le diiodure de samarium évitant ainsi l'utilisation de catalyseurs au palladium ou d’un autre additif. En série glycal, la nature et la configuration du substituant en position C-4 jouent un rôle très important à la fois sur le rendement et sur la structure des produits modifiés. Les couplages réducteurs directs ont lieu régiosélectivement en C-3 avec une stéréochimie relative 3,4-trans. La même stratégie appliquée aux dérivés Neu5Ac2en de l’acide sialique a permis d’obtenir par réactions à basse température, les produits de couplage en C-2 avec des rendements quantitatifs et une parfaite régio- et stéréosélectivité. Cette transformation donne un nouvel accès facile, rapide et très efficace aux α-C-sialosides. / The cross-coupling of allylic esters and carbonyl compounds promoted by samarium diiodide is an efficient method for the formation of carbon-carbon bonds. An umpolung approach, reaction between a carbonyl electrophile and an allyl samarium nucleophile, without the prior reduction of the intermediate π-allyl transition metal complexes, would provide a simple route for this bond construction under mild conditions.Dihydropyranyl allylic esters have been found to be excellent substrates for carbonyl allylation reactions mediated by samarium diiodide – without the use of palladium catalysts or any other additive. In glycal series, the nature and the configuration of the substituent at the C-4 position play crucial roles both on yields and structures of the modified products. The direct reductive couplings take place regioselectively at C-3 with a 3,4-trans relationship. Applied to Neu5Ac2en derivatives of sialic acid, the same strategy furnished the coupling products at the anomeric position in quantitative yields and with a perfect regio- and stereoselectivity. This transformation provides an easy, rapid and efficent access to α-C-sialosides.