No description available.

A series of small molecules was synthesized to probe three protein targets in order to elucidate the key small molecule-protein interactions required for potency. Triclosan is an antibacterial compound that has surfaced as a potential environmental hazard and is hypothesized to cause perturbations in the thyroid hormone response of frogs. Using a C-fin assay and a GH3 cell line, our work suggests that triclosan itself may not in fact be the cause of the observed endocrine disruptions. Instead, methyl triclosan (a result of biological methylation during waste water treatment) was shown to disrupt the thyroid hormone response in tadpoles. Secondly, a set of probes was designed based on a cyclopentane scaffold derived from the known neuraminidase inhibitor peramivir. Kinetic assays using both a recombinant neuraminidase protein and an inactivated sample of influenza virus showed that the guanidine group contributes a 10 fold increase in potency while the α-hydroxyl group was observed to have little to no effect. This result suggests that future neuraminidase drug design based on a cyclopentane scaffold may forgo the use of both the guanidinium group and the hydroxyl group to potentially increase the oral availability of these drugs while sacrificing little in the way of potency. Finally, a series of truncated analogues related to the western half of the natural product didemnaketal A was synthesized. These compounds will be used as probes to better understand the mechanism of didemnaketal-mediated protease inhibition. It is hypothesized that a more rigid structure (due to molecular gearing enforced by the presence of additional methyl groups, relative to previously examined analogues) will increase the potency of these molecules toward HIV-1 protease and may lead to new information for designing next-generation dissociative inhibitors. Work was also begun toward the total synthesis of the natural product itself. / Graduate

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 238-267) Also available in print.

Thesis (M.S.)--Michigan State University. Dept. of Chemistry, 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references. Also issued in print.

The sialidases/neuraminidases represent a family of enzymes whose function is important in the pathogenicity of bacteria and the virulence of influenza. Relenza and Tamiflu represent two drugs that were developed using structure-based drug design (SBDD) and computational-assisted drug design (CADD). These drugs target the active site of the influenza neuraminidase A and B (GH-34 family). Sialidases in the GH-33 family could represent novel drug targets for the treatment of bacterial or parasitic infection. SBDD was employed to develop chemical tools of two GH-33 sialidases, NanB and TcTS. NanB is a potential drug target for S. pneumoniae. The chemical tool developed for NanB follows on from work within the Taylor and Westwood research groups, in which a molecule of CHES and a glycerol were found serendipitously bound within a water channel at an allosteric site. Using this information as a basis for SBDD an allosteric inhibitor of NanB, Optactin was developed. Within this work, synthesis of this inhibitor was achieved and optimised. Optactin was then modified to improve potency. This proceeded through an amide analogue and addition of an arene resulting in a mid- micromolar inhibitor (IC50: 55.4±2.5 μM). Addition of polar substituents improved potency further resulting in a low micromolar inhibitor of NanB, Optactamide (IC50: 3.0±1.7 μM). Application of this tool in vitro demonstrated that NanB and NanA have a role in invasion of S. pneumoniae into lung epithelial cells. TcTS is a potential drug target for the treatment of Chagas disease. A CADD approach using a fragment library was unsuccessful at identifying an allosteric inhibitor of TcTS despite structural similarity with NanB. A re-task of the CADD approach towards the active site was successful in identifying an inhibitor of TcTS and a fragment useful for further development. This work sets the groundwork for the development of a chemical tool targeting TcTS.

Submitted by Alessandra Portugal (alessandradf@ioc.fiocruz.br) on 2013-09-24T15:06:55Z No. of bitstreams: 1 Dissertação de Mestrado Paola Cristina Resende Silva.pdf: 1538372 bytes, checksum: 8c68a244d3e53f424ddb574f985bcad8 (MD5) / Made available in DSpace on 2013-09-24T15:06:55Z (GMT). No. of bitstreams: 1 Dissertação de Mestrado Paola Cristina Resende Silva.pdf: 1538372 bytes, checksum: 8c68a244d3e53f424ddb574f985bcad8 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / Globalmente, as infecções causadas pelos vírus Influenza constituem um importante desafio para a Saúde Pública. Os vírus Influenza B pertencem à família Orthomyxoviridae, seu genoma viral é constituído por um RNA de fita simples e polaridade negativa. O processo de drift antigênico favorece o contínuo aparecimento de novas variantes virais, o que demanda a reformulação anual da vacina. No início década de 80, foi observada a divergência do vírus Influenza B em duas linhagens antigênica e filogeneticamente distintas: B/Victoria/2/87-like (Vic87) e B/Yamagata/16/88-like (Yam88), que tem co-circulado em diferentes países na última década. O objetivo deste estudo consiste na identificação e caracterização molecular das linhagens de Influenza B circulantes em diferentes regiões brasileiras durante as epidemias de 2004 a 2008, com base no sequenciamento dos genes Hemaglutinina (HA) e Neuraminidase (NA). Ainda, padronizamos a metodologia de Eletroforese em Gel com Gradientes Desnaturantes (DGGE), visando à rápida tipagem dos vírus B. Diferentes substituições nos genes da HA e NA foram encontradas. Evidenciamos a co-circulação de ambas as linhagens no período estudado, contudo, não observamos a ocorrência de rearranjo gênico e nem a emergência de cepas resistentes aos inibidores de neuraminidase, com base nos genes investigados. No período 2006-2008, observamos a adequada concordância entre as cepas circulantes e as cepas vacinais preconizadas para uso no Hemisfério Sul. Entretanto, o mesmo não foi verdadeiro para o período 2004-2005. Finalmente, o protocolo de DGGE desenvolvido pode ser eficientemente utilizado para fins de rápida tipagem das linhagens de Influenza B. Os achados deste estudo contribuem para a melhor compreensão sobre a variabilidade dos vírus Influenza B e os mecanismos envolvidos na sua evolução molecular, bem como o padrão de circulação das linhagens virais no Brasil e sua correspondência com as vacinas para Influenza, anualmente administradas no Hemisfério Sul. Este conjunto de informações são de grande relevância para a contínua adequação e implementação das políticas e estratégias voltadas ao controle e prevenção de infecções por Influenza na nossa população. / Worldwide, Influenza infections are a major Public Health issue. Influenza B virus is classified into the Orthomyxoviridae family, the viral genome consists of a single strand RNA and negative polarity. Because of antigenic drift, novel viral variants are continuously rising, what demands the annual review of vaccine formulation. In the early 80´s, was observed the divergence of Influenza B into two distinct antigenic and phylogenetic lineages – B/Victoria/2/87-like (Vic87) and B/Yamagata/16/88-like (Yam88), was observed. In some countries, these strains have been co-circulating in the last 10 years. The aim of this study was to investigate the circulation patterns of Vic87 and Yam88 among different Brazilian regions during the 2004-2008 epidemics, based on haemagglutinin (HA) and neuraminidase (NA) sequencing. Moreover, a Denaturing Gradient Gel Electrophoresis (DGGE) protocol was standardized for rapid typing of Flu B typing into Yam88-like and Vic87-like strains. Different Aminoacid substitutions in the HA and NA were encountered. Along the studied period, our findings showed that both viral lineages have been co-circulating in Brazil. Moreover, no evidence of reassortant nor NA inhibitor-resistant viruses was found. From 2006 to 2008, an adequate match between vaccine and circulating strains was met. However, it was not true for 2004-2005 years. Finally, our DGGE protocol can be successfully used as a rapid Flu B strain typing test. Our findings contribute for a better figure of Flu B genetic variability and its molecular evolution mechanisms, in addition to the circulation patterns of Flu B lineages in Brazil, and their respective association with the vaccine strains used in the Southern Hemisphere. Altogether, these are pivotal information to continuously tailor and implement Public Health policies on behalf of the control and prevention of Influenza infections.

Made available in DSpace on 2016-03-10T13:20:27Z (GMT). No. of bitstreams: 2 paola_silva_ioc_dout_2015.pdf: 5954809 bytes, checksum: 02b143793e3dbd017a0d95c668d3108c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil / A primeira detecção do vírus Influenza A (H1N1)pdm09 no Brasil aconteceu em maio de 2009, e foi seguida de uma extensa disseminação por toda a população brasileira, com grande impacto em morbidade e mortalidade. Para entender a dinâmica molecular do Influenza A (H1N1)pdm09 no país, a presente tese reuniu sete trabalhos que abordaram a análise filogenética deste agente viral durante e após o período pandêmico (2009 a 2014) e buscou indentificar polimorfismos virais associados à virulência e à resistência ao antiviral Oseltamivir (OST). Para isso, as metodologias realizadas foram o sequenciamento dos genes de hemaglutinina (HA) e neuraminidase (NA) utilizando a metodologia de Sanger e a metodologia de pirosequenciamento para detectar polimorfismos de base única (SNPs). Nossos resultados revelaram a circulação de nove grupos filogenéticos ao longo dos cinco anos do estudo, indicando uma substituição temporal dos grupos e ocasionalmente uma estratificação geográfica. No entanto, nenhum dos grupos filogenéticos identificados foram associados com um pior prognóstico da infecção por influenza. Ao contrário do que foi observado em estudos anteriores, as mutações K-15E e Q310H no gene HA não se associaram ao aumento de virulência, mesmo na infecção de indivíduos imunocomprometidos. Por outro lado, polimorfismos no resíduo 222 da HA, que caracterizaram a presença de quasispecies virais, mostraram uma forte associação com a gravidade da infecção, especialmente em gestantes. Nesta tese, também realizamos a vigilância de marcadores de resistência no gene NA. Entre as amostras analisadas encontramos sete vírus com a mutação H275Y e dois com S247N, esses marcadores estão relacionados com a diminuição de sensibilidade ao antiviral OST Entre as amostras resistentes, a grande maioria foi detectada na região Sul do Brasil, em pacientes que não receberam OST. Isto sugere uma possível transmissão sustentada do vírus resistentes no país. Embora variantes H275Y resistentes apresentem baixa aptidão viral, a propagação deste vírus pode ocorrer com o ganho de mutações permissivas nos genes HA ou NA. No Brasil, o vírus que circulam desde 2011 apresentaram mutações V241I e N369K no gene NA, que foram, teoricamente, associadas a uma maior aptidão viral da variante resistente. Diante disso, cinco anos após a pandemia observamos que o vírus A (H1N1)pdm09 está estabelecido na população humana com várias substituições genéticas quando comparado com a sequencia da cepa vacinal A/California/07/2009. A maioria destas substituições parecem não ocasionar uma maior gravidade da infecção, e esta pode ser atribuída a outros fatores, tais como fatores genéticos do hospedeiro. Por fim, considerando o risco de surgimento e disseminação de vírus resistentes é importante reforçar o monitoramento viral e também realizar estudos para novas drogas antivirais / The Influenza A (H1N1)pdm09 virus was first detecte d in May 2009 in Brazil and later resulted in an extensive spread throughout the Brazilian pop ulation with a severe impact on morbidity and mortality. To understand the molecular dynamic of ( H1N1)pdm09 virus in Brazil this thesis grouped seven papers which approached the phylogenetic reco nstruction of the virus during and after the pandemic period (2009 to 2014) and the genomic iden tification of viral polymorphisms associated with virulence or antiviral resistance to Oseltamivir (O ST). For this, we performed genome sequencing, focusing especially on the hemagglutinin (HA) and n euraminidase (NA) genes using conventional Sanger sequencing and PyroMark 96ID to detect singl e nucleotide polymorphisms (SNPs). Our results showed that in Brazil nine (H1N1)pdm09 phylogenetic groups circulated along the five years of the study, indicating a temporal repl acement of groups and ocasionally a geographic stratification. However, no phylogenetic group seem ed to be associated with a worse clinical outcome. The increased virulence observed in previous studie s with a 2009 group bearing the genetic markers K-15E and Q310H was not confirmed in our analyses, even evaluating an immunocompromised population. On the other hand, polymorphysms at pos ition 222 of HA gene, which characterized the presence of viral quasispecies, showed an associati on with increased virulence in brazilian samples, especially in pregnant women. In this study we also performed surveillance of resistance markers at the NA gene. From the analysed samples we found sev en viruses with H275Y and two with S247N mutation, that diminish the sensibility to oseltami vir (OST). Among the resistant samples, the large majority was detected in the Southern region of Bra zil in patients that did not receive OST. This suggests a possible sustained transmission of resis tant virus in the country. Although resistant H275Y variants have low viral fitness, the spread of this virus can occur with the gain of permissive mutati ons in the HA or NA genes. In Brazil, viruses circulati ng since 2011 presented mutations V241I and N369K in the NA gene, which were theoretically associated with greater viral fitness. Five years after the pandemic we observed that A (H 1N1)pdm09 is established in the population with genetic substitutions in all gene s egments when compared to the vaccine strain A/California/07/2009. We demonstrated that the majo rity of those substitutions did not increase the severity of infection. The worst clinical outcomes may be attributed to other factors, such as genetic host factors. Finally, considering the risk of emer gence and spread of resistant viruses it is importa nt to strengthen viral surveillance and also carry out studies for new antiviral drugs

Submitted by Automa??o e Estat?stica (sst@bczm.ufrn.br) on 2017-10-04T22:23:59Z No. of bitstreams: 1 DalilaNascimentoManso_DISSERT.pdf: 1914411 bytes, checksum: 966fc442e252d656c3946bff697a75f5 (MD5) / Approved for entry into archive by Arlan Eloi Leite Silva (eloihistoriador@yahoo.com.br) on 2017-10-13T21:33:08Z (GMT) No. of bitstreams: 1 DalilaNascimentoManso_DISSERT.pdf: 1914411 bytes, checksum: 966fc442e252d656c3946bff697a75f5 (MD5) / Made available in DSpace on 2017-10-13T21:33:08Z (GMT). No. of bitstreams: 1 DalilaNascimentoManso_DISSERT.pdf: 1914411 bytes, checksum: 966fc442e252d656c3946bff697a75f5 (MD5) Previous issue date: 2017-04-19 / A mais recente pandemia do v?rus influenza ocorreu no ano de 2009, causada pela cepa do influenza A (H1N1), e popularmente conhecida como gripe A ou gripe su?na, gerou preocupa??o aos ?rg?os mundiais de sa?de. Com um quadro sintom?tico que inclui febre, tosse, inflama??o na garganta na maioria dos casos, alguns pacientes, principalmente imunossuprimidos que podem apresentar complica??es que evoluem ao ?bito. A transmiss?o do v?rus ocorre atrav?s do contato entre pessoa a pessoa e seu mecanismo de infec??o se d? a partir das duas glicoprote?nas de superf?cie, a hemaglutinina e a neuraminidase. A hemaglutinina atua se ligando aos receptores do ?cido si?lico favorecendo a entrada do v?rus nas c?lulas-alvo e a neuraminidase cliva as c?lulas do receptor de res?duos do ?cido si?lico, onde as novas part?culas virais est?o se ligando. Atrav?s dessa quebra haver? libera??o das novas part?culas virais, que atrav?s da hemaglutinina invadir?o novas c?lulas. Baseado nisso, f?rmacos foram desenvolvidos com intuito de inibir a a??o da neuraminidase, os chamados inibidores da neuraminidase que interferem na libera??o dessas novas part?culas virais evitando a dissemina??o da infec??o no trato respirat?rio. Dentre estes inibidores o oseltamivir ? o f?rmaco de escolha para profilaxia e tratamento da gripe A; por?m, relatos de resist?ncia a esse f?rmaco foram descritos, o que causou preocupa??o nos profissionais da sa?de e governantes. A muta??o mais encontrada ? a HIS275TIR, onde a histidina ? substitu?da por uma tirosina, promovendo uma s?rie de altera??es conformacionais que diminuem a afinidade do f?rmaco pelo v?rus originando a resist?ncia. A partir da obten??o de dados cristalogr?ficos e simula??o computacional, calculamos a energia de intera??o da neuraminidase selvagem e com a presen?a da muta??o HIS275TIR ligadas ao oseltamivir utilizando a Teoria Funcional da Densidade (DFT) e do M?todo de Fracionamento Molecular com Capas Conjugadas (MFCC). Obtivemos 115 res?duos de intera??o para a neuraminidase selvagem (cristal 4B7R) e 109 res?duos de intera??o para o cristal com a neuraminidase mutante (3CL0). Os resultados foram avaliados de acordo com a relev?ncia dos valores energ?ticos para energias repulsivas e energias atrativas. Os c?lculos energ?ticos realizados confirmaram a redu??o da afinidade da cepa contendo a muta??o HIS275TIR e destacaram a import?ncia energ?tica do s?tio ativo da neuraminidase mostrando que os principais res?duos energ?ticos s?o encontrados nele tornando um alvo para obten??o de novos f?rmacos devido a sua conserva??o. As altera??es causadas pela substitui??o do amino?cido histidina por uma tirosina levaram a uma s?rie de mudan?as conformacionais nos amino?cidos vizinhos que provocaram altera??es eletrost?ticas resultando na resist?ncia ao f?rmaco. A partir desse estudo ser? poss?vel conhecer melhor as intera??es moleculares da neuraminidase mutante e posteriormente projetar novos designs de f?rmacos para serem elaborados e se tornarem mais eficientes na intera??o com as cepas mutantes desse v?rus. / The latest influenza pandemic occurred in the year 2009, caused by the strain of influenza A (H1N1), and popularly known as influenza A or swine flu, generated concern to the global health agencies. With a symptomatic picture that includes fever, cough, throat inflammation in most cases, some patients, mainly immunosuppressed, that can to present complications that evolve to death. Transmission of the virus takes place through contact between person to person and its mechanism of infection occurs from the two surface glycoproteins, hemagglutinin and neuraminidase. The hemagglutinin acts by binding to the sialic acid receptors favoring the entry of the virus into the target cells and the neuraminidase cleaves the receptor cells of sialic acid residues, where the new viral particles are binding. Through this breakdown there will be release of the new particles that through hemagglutinin will attack new cells. Based on these, drugs were developed in an attempt to inhibit the action of neuraminidase, so called neuraminidase inhibitors that interfere in the release of these new viral particles avoiding the spread of infection in the respiratory tract. Among the inhibitors, oseltamivir is the drug of choice for prophylaxis and treatment of influenza A, but reports of resistance to this drug have been described, which has caused concern in health professionals and rulers. The HIS275TIR mutation is most commonly found, where histidine is replaced by a tyrosine, promoting a series of conformational changes that decrease the affinity of the drug for the virus causing resistance. Based on crystallographic data and computational simulation, we calculated the interaction energy of the wild neuraminidase and the presence of the HIS275TIR mutation bonded to oseltamivir using the Functional Density Theory (DFT) and the Molecular Fractionation with Conjugated Caps (MFCC). We obtained 115 interaction residues for the wild neuraminidase (4B7R crystal) and 109 interaction residues for the crystal with the mutant neuraminidase (3CL0). The results were evaluated according to the relevance of the energy values for repulsive energies and attractive energies. The energetic calculations confirmed the reduction of the affinity of the strain containing the HIS275TIR mutation and highlighted the energy importance of the active site of the neuraminidase, showing that the main energy residues are found in it becoming a target for obtaining new drugs due to its conservation. The changes caused by the substitution of the amino acid histidine for a tyrosine led to a series of conformational changes in the neighboring amino acids that provoked electrostatic changes resulting in the resistance to the drug. From this study, it will be possible to know better the molecular interactions of the mutant neuraminidase and subsequently to project new drugs designs to be elaborated and become more efficient in the interaction with the mutant strains of this virus.

Sialic acids are a family of 9 carbon acidic sugars that are often part of glycans that decorate glycoproteins and glycolipids. N-acetylneuraminic acid, or Neu5Ac, is the predominant sialic acid in Homo sapiens and is most commonly found on the terminus of glycan chains where it functions as a signalling molecule or providing a negatively-charged mask to cells. Neu5Ac is less commonly found in microbes. A number of pathogens, including the fungus Aspergillus fumigatus and the parasite Trypanosoma cruzi, present Neu5Ac on their surface which aids evasion of the host immune system. A. Fumigatus has no known sialic synthesis genes however a single sialidase, AfS, has been identified. This sialidase is relatively poor at cleaving Neu5Ac in comparison with other members of the sialidase family. Here we present the structure of AfS to 1.8Å, the first fungal sialidase structure published. The overall topology of AfS shares the 6-bladed β-propellor fold common to all known sialidases and has an rmsd of only 1.44 Å over 309 C[sub]αs with the bacterial sialidase from Micromonospora viridifaciens. The active site of AfS also contains all the residues necessary for efficient hydrolysis of sialic acids. Despite repeated attempts. It was not possible to crystallize AfS in complex with Neu5Ac. Closer inspection of the AfS active site revealed that the pocket that usually accommodates the N-acetyl moiety of Neu5Ac contained more polar residues and was shallower than that of other sialidases. The apo structure suggested that substitution of the N-acetyl group on C5 of Neu5Ac with a smaller group may be accommodated better in the active site of AfS. 2-Keto-3-deoxy-D-galactononulosonic acid (KDN), which has a hydroxyl group at this position, is capable of binding in the active site and is the preferred substrate of AfS. The structure of AfS in complex with KDN was determined to 1.45 Å and is the first KDN specific sialidase structure determined. Crystal complexes with either a di-fluoro-KDN or KDN2en allowed atomic snapshots to be obtained of the catalytic cycle from Michaelis complex, through transition state to covalent intermediate. Several KDN binding sites on the surface of the enzyme were discovered suggesting that the enzyme may be specific for polyKDN, which intruigingly has been found in the human lung. Mutations were made to AfS to enlarge the cavity around C5 to see if the enzyme could accommodate Neu5Ac-related substrates. Identification and mutation of arginine 171 in the active site to leucine increased the depth and hydrophobicity of the cavity and improved the ability of AfS to utilize Neu5Ac, albeit poorly. The structure AfS[sub](R171L) in complex with Neu5Ac2en was solved to 1.8 Å. Trypanosoma cruzi is the etiological agent of Chagas disease. The trans sialidase from T. cruzi, TcTs, is a verified virulence factor. Here we detail a fragment-based search for novel inhibitors of TcTs using a fluorogenic assay, STD-NMR and WaterLOGSY-NMR, in silico screening and X-ray crystallography. There was poor agreement between the techniques used and only fragment hits validated by X-ray crystallography were pursued. All protein-ligand complexes shared two common interactions. The verified compounds interacted with the arginine triad via a carboxylic acid, as is the case with the sialic acid substrate. Secondly, all compiunds possessed an amine that was within hydrogen bonding distance of the TcTs catalytic aspartic acid. One of these compunds, 3-piperidinic acid, was capable of binding in the TcTs active site in both of its anomeric forms. Interestingly, each anomer binds in 2 similar but distinct positions in the active site. In one of these conformations Tyr119 moves into the active site and hydrogen bonds with the fragment's amine. In an attempt to combine the interaction seen in both the R and S anomers of 3-piperidinic acid, (R)-2-piperazine carboxylic acid was soaked into TcTs crystals. This compound made additional interactions with the protein and demonstrated a 4-fold improvement in the IC50. A known sialidase inhibitor, Siastatin B, based on a 3-piperidinic acid frame was also investigated and we present a TcTs-siastatin B complex structure to 2.1 Å, the first structure of Siastatin B in complex with a glycoside hydrolase. There is much work to be done to these preliminary inhibitors however here we have identified 3 druggable sites within the TcTs active site. The research presented herein provides a platform for future drug design studies on TcTs.

<p>Atherosclerosis is a complex multi-factorial disease that involves the interaction of many cell types and a plethora of molecular events. Initiation of the disease occurs when circulating LDL gets trapped in the sub-endothelial space of arteries, where LDL is oxidized causing inflammatory responses by endothelial cells. This results in recruitment and differentiation of monocytes into macrophages; macrophages in turn continue to take up cholesterol and propagate inflammation. Such a gloomy milieu of immune cells, lipids, and smooth muscle cells can give rise to atherosclerotic plaques, which then cause stenosis, vascular stiffening and eventually thrombosis. Common risk factors such as cholesterol levels, lifestyle and genetic predisposition can accelerate this potentially life threatening series of events. The downstream long-term effects of atherosclerosis, including heart disease and strokes, are now the number one cause of death in the world. While a large amount of knowledge and evidence is available in understanding this disease, prevention and treatment strategies remain somewhat ineffective. Sialylation of immune cells, lipoproteins and cellular receptors has been previously implicated in metabolic and molecular pathways relevant to atherosclerosis; however, little is known about the functional role of sialidase in these processes. Sialidase cleaves sialic acid, and is a ubiquitously expressed and evolutionarily conserved protein with essential functions in many life forms. In this study, we sought to investigate the impact of sialidase activity on atherosclerosis, emphasizing the interaction of lipid metabolism and inflammation. We have demonstrated a significant role for sialidase in cholesterol iv and lipoprotein metabolism in vivo. Specifically, hypomorphic sialidase mice have increased hepatic storage of lipids and triglycerides, decreased VLDL production, lower circulating LDL levels and alterations in regulation of LDLR. Mice over-expressing hepatic human sialidase have increased atherosclerotic lesion formation, higher serum cholesterol esters and lower levels of hepatic LDLR and SRB-1 protein. In vitro, we have shown that VLDL can induce differentiation and cytokine production in monocytes coupled with an up-regulation of Neu1. Inhibition of sialidase using DANA attenuated VLDL-induced monocyte differentiation and lipid uptake, as well as activation of macrophages, implicating Neu1 in inflammatory processes associated with initiation of atherosclerosis. Furthermore, we have shown that hypomorphic sialidase activity increases LDLR-dependent LDL uptake and cholesterol efflux to HDL in macrophages. We conclude that reduction of sialidase activity can lead to an atheroprotective phenotype with multiple effects on mechanisms involved in disease progression. This work represents novel contributions into delineating both metabolic and inflammatory processes of atherosclerosis and enables the advancement of future treatment strategies.</p> / Doctor of Philosophy (PhD)