Vascular calcification is the formation of mineralised tissue within the walls of arteries. The pathology has many similarities to embryonic bone formation and involves the osteogenic differentiation of vascular smooth muscle cells (VSMCs) and matrix mineralisation. Recent studies have demonstrated that the bioactive sphingolipids, ceramide and sphingosine-1-phosphate (S1P), regulate embryonic bone formation. Ceramide can be generated by lysosomal acid sphingomyelinase (L-SMase) and neutral sphingomyelinase (N-SMase), and be converted to sphingosine by acid ceramidase (ACDase) and subsequently to S1P by sphingosine kinases (SK1 & SK2). This study tested the hypothesis that ceramide and S1P also regulate VSMC matrix mineralisation. VSMCs were cultured in the presence of 3 mM β-glycerophosphate (BGP) to induce osteogenic differentiation and matrix mineralisation. During VSMC mineralisation there were decreases in the activities of L-SMase and N-SMase and increases in the levels of C18 and C20 ceramide. S1P levels also increased during mineralisation as did SK1 and SK2 mRNA and SK activity. These results demonstrate that ceramide and S1P have the potential to regulate VSMC mineralisation. The exogenous addition of C2 ceramide decreased the rate of VSMC matrix mineralisation. Consistent with this, when VSMCs were cultured with 3 mM BGP and the joint L-SMase and ACDase inhibitor, desipramine, total ceramide levels increased and no matrix mineralisation was detected. These findings suggest that ceramide is an inhibitor of VSMCs matrix mineralisation. It was also noted in the presence of 3 mM BGP and desipramine that the mineralisation-associated increase in S1P was inhibited. In agreement with this, when exogenous S1P was added to the VSMCs an increase in matrix mineralisation was observed. Thus, S1P acts as a promoter of matrix mineralisation. To determine how S1P was promoting matrix mineralisation the signalling roles of the ezrin, radixin and moesin (ERM) proteins were investigated. The short-term stimulation of VSMCs with S1P led to the phosphorylation of the ERM proteins and over the mineralisation time-course, when S1P levels increased, the levels of ERM phosphorylation also increased. When VSMCs were cultured in the presence of 3 mM BGP and the inhibitor of ezrin phosphorylation, NSC668394, a decrease in matrix mineralisation was observed. No increases in ERM phosphorylation were seen in the presence of desipramine during the mineralisation time-course Therefore, S1P may be increasing matrix mineralisation through promoting the phosphorylation of the ERM proteins. This work has demonstrated that ceramide inhibits and S1P promotes VSMC matrix mineralisation in vitro. Additionally, this work identifies activation of ERM proteins, downstream of S1P, as a novel signalling pathway promoting matrix mineralisation. Characterisation of novel regulators of VSMC matrix mineralisation in vitro gives insight into the complex mechanisms contributing to vascular calcification in vivo and will aid in identification of novel therapeutic targets.

No description available.

Sphingolipids (SL) are essential structural components of membranes found in all eukaryotes and have also been identified in some bacteria. The first step of the SL biosynthetic pathway across all species is catalysed by serine palmitoyltransferase (SPT), a member of the alpha-oxoamine synthase (AOS) family of pyridoxal 5’- phosphate (PLP)-dependent enzymes. AOS enzymes are involved in the biosynthesis of a range of important natural products such as heme, vitamins and antibiotics where they catalyse the reaction between amino acid and acyl-thioester substrates. Substrate specificity across the family is of great importance, as human mutant SPTs shift the substrate specificity from L-serine to glycine or L-alanine that lead to production of deoxy-sphingolipids that are toxic to mammalian cells. PLP, a form of vitamin B6, is one of nature’s most versatile catalysts and is involved in over 160 enzymes that carry out diverse reactions on amine-containing substrates. This work probes the functional role of the phosphate group of PLP, usually housed in a phosphate binding cup (PBC) and investigates the need for a novel and unexpected H-bond between the hydroxyl group of the L-serine substrate and the 5’-phosphate group of PLP in SPT. In this study, the PLP cofactor was removed from SPT with amino-thiol substrates which act as mechanism-based inhibitors of SPT via production of a thiazolidine adduct. Replacement of natural PLP with the dephosphorylated form of the cofactor, pyridoxal, allowed a study on the importance of the PLP phosphate:L-serine H-bond on substrate specificity and optimal SPT activity. Furthermore, analysis of the phosphate binding cup of the ALAS:PLP:glycine external aldimine, a related AOS family member; revealed an important residue that could possibly be involved in determining substrate specificity of different members of the AOS family. PBC analysis also expanded, with a detailed and interesting study of a novel SPT:PLP:myriocin inhibitor complex. Human SPT is a heterodimeric, membrane-bound enzyme composed of two subunits (hLCB1/hLCB2) which is thought to contain a single PLP-containing active site. Mutations in human hLCB1 have been linked to the rare sphingolipid metabolic disease hereditary sensory neuropathy I (HSAN1). Recent studies identified three heterozygous missense mutations in the second human SPT subunit hLCB2 that show a significant loss in SPT activity. The three human SPT mutations V359M, G385V and I504F were mapped onto the bacterial S. paucimobilis SPT as V246M, G268V and G385F. These bacterial SPT mutant mimics reveal that the amino acid changes have varying impacts; they perturb the PLP cofactor binding, reduce the affinity for both substrates, decrease the enzyme activity, and, in the most severe case, cause the protein to be expressed in an insoluble form. SPTs and most of the other members of the AOS family utilise an acyl-CoA thioester substrate. In contrast, a sphingolipid-producing bacterium, S. wittichii, is thought to use a small type II acyl carrier protein (ACP) to deliver the acyl chain to its homodimeric SPT target. Converting the unmodified apo-ACP to the activated “substrate” acyl-ACP, has proven difficult and amino acid sequence alignment, combined with modelling studies revealed an unusual tryptophan residue that could prevent modification to the acyl-ACP form. In this study a double mutant ACP E36G/W37A has been prepared and characterised. Both wild-type and mutant S. wittichii ACP are expressed in the recombinant E. coli host in their inactive apoform. The transfer of a phosphopantethiene (4’PP) linker by a specific PPTase (also known as an acyl carrier protein synthase (AcpS)) has been successful in modifying the mutant form of ACP to its holo-form but could not transfer a palmitoyl group (C16). E.coli ACP has been successfully expressed, purified and characterised in this study. For the first time, ion mobility mass spectromerty (IM-MS) has been used on this protein to gain structural insight into the different forms of ACP. Collisional cross section (CCS) distributions have been calculated for different acylated states of the ACP concluding that the protein exists in equilibrium between two states: a compact and an extended conformation.

Cardiovascular diseases are a major cause of death worldwide. Aneurysmal rupture in cerebral arteries or loss of endothelial integrity in the course of atherosclerosis or therapeutic angioplasty lead to exposure of vascular smooth muscle cells (SMC) to blood components such as sphingolipids. Sphingosylphosphorylcholine (SPC) and sphingosine 1-phosphate (S1P) are two naturally occurring sphingolipids, which are vasoprotective in the healthy endothelium-lined vessel, but may promote vascular disease by causing functional changes of SMC. Vascular inflammation is an important factor in various pathologies. SPC can activate pro-inflammatory signalling pathways in rat cerebral artery. Here these observations are extended by showing that SPC elicits monocyte chemoattractant protein-1 production in rat cerebral artery SMC ex vivo. Thus, in addition to being a vasoconstrictor, SPC may promote the development of life-threatening prolonged cerebral vasospasm following subarachnoid haemorrhage by supporting vascular inflammation. It is also demonstrated that SPC prevents tumour necrosis factor-a (TNF)-stimulated adhesion of macrophages to rat aortic SMC in vitro by interfering with adhesive properties of SMC, but not macrophages. While this effect appeared to be mediated by the S1P receptor S1P2, S1P itself did not reduce macrophage adhesion. The anti-adhesive action of SPC also depended on lipid rafts. However, SPC did neither prevent TNF-induced nuclear factor kB activation nor cell adhesion molecule expression in SMC. SPC-induced cyclooxygenase 2 expression in aortic SMC was dispensable for its anti-adhesive effect. In contrast, the inhibitory effect of SPC on TNFinduced expression of inducible nitric oxide synthase is probably involved in its anti-adhesive effect because it was mimicked by respective pharmacological blockade. The results also demonstrate that nitric oxide promotes leukocyte adhesion to vascular SMC, while it has the opposite effect on endothelial cells. These findings may help understand cardiovascular diseases and define novel treatment approaches.

Sickle cell disease (SCD) is a disorder in which defective hemoglobin causes sickling of red blood cells, inducing painful vaso-occlusive crises when blood flow is blocked at sites of red blood cell (RBC) clotting that can ultimately result in organ failure or death. This work demonstrates that sphingolipid metabolism is dysregulated in SCD and that this pathway can be targeted pharmacologically to prevent vaso-occlusion. We suggest a pathway in which the sickling of RBCs in SCD activates acid sphingomyelinase, altering the distribution and concentration of sphingolipids in the RBC membrane and resulting in the production of sphingolipid-rich microparticles that are secreted and can interact with cells in circulation. Sphingosine-1-phosphate (S1P) is believed to be a key modulator of SCD because it is stored at high concentrations in RBCs. Sphingolipid metabolism was confirmed to be dysregulated in SCD; most notably, S1P was significantly elevated in RBCs, and plasma, and microparticles, and the activity of acid sphingomyelinase and concentration of its byproduct, microparticles, were significantly elevated in SCD RBCs. Treatment of monocytes with S1P and SCD RBCs increased their adhesion over four-fold to endothelial cells, indicating that altered sphingolipid distribution in RBCs may contribute to vaso-occlusion through increasing myeloid cell adhesion. A cytokine profile of macrophages treated with SCD microparticles suggest that microparticles play a role in this process by increasing the secretion of inflammatory cytokines associated with SCD crises, including MIP-1α, IL-6, and TNF-α. Pilot in vitro studies in RBCs and in vivo studies in mice implicate that drugs targeting the sphingolipid metabolic pathway may be more effective treatment options than blood transfusions in managing SCD and preventing vaso-occlusive crises.

Synthetic efforts toward fumonisin analog were described. These are accomplished via amino acid Schiff base methodology. These efforts can be divided three major phases. First, tandem reductive alkylation with DIBAL/TRIBAL and different types of organo-lithium or Grignard nucleophiles provided threo-amino alcohol with excellent stereoselecitivites (2-27:1). The reductive alkylation utilized most hydrocarbon nucleophiles, e.g. alkyl-, vinyl-, alkenyl-, phenyl-, and dienyl-, and afforded high selectivites unless donor solvents (e.g. THF and Et2O) were used. Second, syntheses of the protected threo-γ-amino-β-hydroxy aldehydes and their stereoselectivities were introduced. The reductive alkylated threo-amino allyl alcohol was transformed via Brown’s hydroboration/oxidation protocol with 9-BBN, followed by TEMPO oxidation to give the resultant aldehydes in reasonable yields. Then, TBDPS and Schiff base protected aldehyde was coupled with phenyl- and decyl Grignard reagents to obtain predominant 3,5-anti-diols (ca. 80:20 anti:syn), characterized by ¹³C NMR analysis of Rychnovsky’s 1,3-acetonide groups. Products can be useful analogues for fumonisin and 5-hydroxy-sphingosine due to their structural similarity. Third stage involved the synthesis of C₁₁-C₂₀ fragment analog of fumonisin. Chiral auxiliaries (e.g. Evans and Myers) were administrated for stereoselective methylation, Sharpless asymmetric dihydroxylation in the presence of (DHQ)2PHAL catalyst was performed to form 1,2- syn-diols, and the manipulation of protection/deprotection and Finklestein reaction furnished C₁₁-C₂₀ fragment analog of fumonisin.

Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2008. / Committee Chair: Cairney, John; Committee Member: Merrill, Alfred Jr.; Committee Member: Nichols, Wylie; Committee Member: Pullman, Jerry; Committee Member: Streelman,Todd.

Thesis (Ph. D.)--University of Virginia, 1999. / Spine title: Sphingolipid signaling in platelets. Includes bibliographical references (p. 134-144). Also available online through Digital Dissertations.

Thesis (M. S.)--Biology, Georgia Institute of Technology, 2007. / Alfred H. Merrill, Jr., Committee Chair ; Marion B. Sewer, Committee Member ; Eva K. Lee, Committee Member.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-05-02T12:12:25Z No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-04T12:25:29Z (GMT) No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-05-04T12:25:37Z (GMT) No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) / Made available in DSpace on 2017-05-04T12:40:10Z (GMT). No. of bitstreams: 1 DissNLG.pdf: 8153268 bytes, checksum: d6e08bab696e4cda4275474ec2fa4b5a (MD5) Previous issue date: 2016-06-15 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Aspergillus fumigatus is an opportunistic pathogenic filamentous fungus, and the causative agent of aspergillosis, including the invasive pulmonary aspergillosis in immunocompromised individuals, most lethal form of the disease. As in all eukaryotes, the plasma membrane of A. fumigatus is composed of sterols, glycolipids and sphingolipids. The sphingolipids molecules are structural elements that participate in the regulation of the actin cytoskeleton endocytosis, and cell growth. In Saccharomyces cerevisiae, the sphingolipid synthesis is regulated by proteins such as the "AGC kinases" known as Ypk1p and Ypk2p that inactivate Orm1p and Orm2p, which are inhibitory proteins of the serine palmitoyltransferase (SPT) enzyme, the first enzyme that participates in biosynthetic pathway of sphingolipids. Thus, Ypk1/2p are responsible for stimulating the production of sphingolipids in response to several cellular stress factors, such as cell wall stress and heat stress. Here, we aimed to expand the study of sphingolipid biosynthesis in A. fumigatus by functional characterization of the ypkAYPK1 gene by establishing its role in the regulation of sphingolipid synthesis and in the maintenance of cell integrity. To accomplish this goal, one null mutant strain ΔypkAypk1 and a conditional mutant strain niiA::ypkA of A. fumigatus were obtained. The mutant strains were characterized by phenotypic tests aimed at understanding the role of this gene in sphingolipid synthesis, virulence and pathogenicity of this fungus. Subsequently real time RT-PCR experiments were performed to quantify the expression of this gene in conditions of heat stress. The results indicated that deletion of the gene ypkA promotes changes in the development of the fungus, which presents severe defects in the vegetative growth and absence of conidia. In addition, the conditional mutant shows increased sensitivity to lipid synthesis inhibitory drugs. The results also indicate that there is no interaction between genetic ypkA and pathway genes of the cell wall. Thus, we propose that protein kinase YpkA in A. fumigatus is related to vegetative growth and participates in the sphingolipid biosynthesis in A. fumigatus, contributing to the development and integrity of the fungal cell. / Aspergillus fumigatus é um fungo filamentoso patogênico oportunista, e o agente etiológico da aspergilose, que em imunocomprometidos pode vir a ocasionar a forma mais letal da doença, a aspergilose pulmonar invasiva. Como em todos os eucariotos, a membrana plasmática de A. fumigatus é composta por esteróis, glicolipídios e esfingolipídios. As moléculas de esfingolipídios são elementos estruturais que participam da regulação da endocitose, do citoesqueleto de actina e do crescimento celular. Em Saccharomyces cerevisiae, a síntese de esfingolipídios é regulada pelas proteínas do tipo “AGC quinases” conhecidas como Ypk1p e Ypk2p, que inativam Orm1p e Orm2p, as quais são proteínas inibidoras da enzima serina palmitoil-transferase (SPT), a primeira enzima que participa da via de biossíntese dos esfingolipídios. Dessa forma Ypk1/2p são responsáveis por estimular a produção de esfingolipídios na membrana plasmática em resposta a diversos fatores de estresse celular, como por exemplo o estresse de parede celular, estresse térmico entre outros. Diante disso, este trabalho teve como objetivo ampliar o estudo da biossíntese de esfingolipídios em A. fumigatus através da caracterização funcional do gene ypkAYPK1, de forma a estabelecer sua função na regulação da síntese de esfingolipídios e na manutenção da integridade celular. Para cumprir esse objetivo, uma linhagem mutante nulo ΔypkAypk1 e uma linhagem mutante condicional niiA::ypkA em A. fumigatus foram obtidas. As linhagens mutantes foram caracterizadas através de ensaios fenotípicos visando a compreensão do papel desse gene na síntese de esfingolipídios, na virulência e patogenicidade desse fungo. Posteriormente, foram realizados experimentos de RT-PCR em tempo real para a quantificação da expressão desse gene em condições de estresse térmico. Os resultados indicaram que a deleção do gene ypkA promove alterações no desenvolvimento vegetativo do fungo, o qual cresce na forma de colônias pequenas as quais não formam conídios. Ainda, o mutante condicional, quando reprimido, apresenta sensibilidade a drogas inibidoras da síntese de lipídeos e esfingolipídios. Os resultados indicam também que não há interação genética entre ypkA e os genes da via da integridade da parede celular. Assim, podemos propor que a proteína quinase YpkA de A. fumigatus está relacionada com o crescimento vegetativo e participa da biossíntese de esfingolipídios em A. fumigatus, contribuindo com o desenvolvimento e integridade da célula fúngica.