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.

The bio-renewable monomer γ-methyl-α-methylene-γ-butyrolactone (MeMBL) has been thoroughly investigated in this thesis. MeMBL is a relatively unstudied monomer that had received little attention since the early 1980’s but has become a subject of renewed interest since a process to produce it from biomass derivatives was developed in 2004. The principle interest with this monomer aside from the “green” potential associated with bio-renewables results from its structure being cyclically analogous to methyl methacrylate (MMA) as well as improved solvent resistance and a high (215oC+) glass transition temperature (Tg) compared to most petroleum sourced acrylics. There are three major areas of focus in this work, examining polymerization kinetics, synthesis and polymer characterization. The polymerization kinetics of MeMBL were investigated with a variety of petroleum sourced monomers. MeMBL is in all cases preferentially incorporated into copolymers, presenting challenges for composition control. Preliminary investigations of aqueous phase polymerizations of MeMBL were problematic and led to investigations of organic phase polymerizations. The dispersion polymerization method was used to produce copolymers of MeMBL and MMA; during the study we obtained new insight into the mechanisms of particle nucleation and growth. With the acquired knowledge of MeMBL polymerization kinetics, the dispersion technique was used to produce MeMBL/MMA copolymers of controlled composition by semibatch for characterization studies. The addition of MeMBL raises polymer Tg and lowers molecular weight, but due to unexpected difficulties in processing MeMBL copolymers, mechanical properties could not be investigated in this study. Future work may need to revisit other polymerization techniques in order to produce processable polymers to test whether or not MeMBL might be a suitable alternative to petroleum sourced monomers that is capable of extending the range and utility of acrylics. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-04-25 09:39:19.959

Dans l'optique de créer un système de karaoké qui modifie une interprétation chantée à capella en temps réel, il est nécessaire de pouvoir localiser l'interprète par rapport à une référence afin de pouvoir déterminer quelle serait la cible d'un algorithme de modification de la voix. Pour qu'un tel système fonctionne bien, il est nécessaire que l'algorithme d'alignement exploite au maximum les spécificités de la voix, qu'il utilise l'information liée au texte prononcé plutôt qu'aux aspects artistiques du chant, qu'il soit à temps réel et qu'il offr la plus faible latence possible. Afin d'atteindre ces objectifs, un système d'alignement basé sur le Dynamic Time Warping (DTW) a été développé. Une adaptation temps réel simple de l'algorithme ordinaire de la DTW qui permet d'atteindre les objectifs énumérés est proposée et comparée à d'autres approches répertoriées dans la littérature. Cette adaptation a permis d'obtenir de meilleurs résultats que les autres techniques testées. Une étude comparative de trois types d'analyses spectrales couramment utilisées dans des systèmes de reconnaissance automatique de la voix a été réalisée, dans le cadre spécifique d'un algorithme d'alignement de la voix chantée. Les coefficients évalués sont les Mel-frquency Cepstrum Coefficients (MFCC), les Warped Discrete Cosine Transform Coefficients (WDCTC) et les coefficients de l'analyse Perceptual Linear Prediction (PLP). Les résultats obtenus indiquent une meilleure performance pour l'analyse PLP. L'utilisation d'une fonction de transformation linéaire par morceaux, appliquée aux matrices de coûts instantanés obtenues, permet de rendre l'alignement le plus facilement distinguable dans les matrices de coûts cumulés calculées. Les paramètres de la fonction de transformation peuvent être obtenus par l'optimisation en boucle fermée par recherche directe par motif. Une fonction-objectif permettant d'éviter les discontinuités de l'écart quadratique moyen sur l'alignement est développée. Plusieurs matrices de coûts peuvent être combinées entre elles en effectuant une somme pondérée des matrices de coûts instantanées transformées de chacun des paramètres considérés. La pondération est également obtenue par optimisation. Plusieurs assemblages sont comparés : les meilleurs résultats sont obtenus avec une combinaison de l'analyse PLP et du niveau d'énergie et des dérivées de ceux-ci. L'écart moyen sur l'alignement de référence est de l'ordre de 50 ms, avec un écart-type d'environ 75 ms pour les séquences testées. Des perspectives permettant d'améliorer la convergence de l'algorithme pour les paires de séquences audio difficiles à aligner, d'obtenir de meilleures matrices de coûts en utilisant d'autres contraintes locales, en considérant l'intégration de nouveaux paramètres tels le pitch ou en utilisant une base de données de voix chantée segmentée pour optimiser une mesure de distance sont données.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

No description available.

Sphingolipids and ceramides are essential components of cellular membranes and important signalling molecules. Because of a growing appreciation for their diverse biological roles, understanding of the biosynthesis and regulation of sphingolipids has recently become a key goal in drug discovery. Serine palmitoyltransferase (SPT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyses the condensation between L-serine and a long-chain acyl thioester such as palmitoyl-CoA (C16-CoA). This first step in sphingolipid biosynthesis is conserved in all organisms studied to date, from microbes to man. The fungal natural product myriocin is a potent inhibitor of SPT; however, the molecular details of inhibition are not fully understood. Myriocin contains a long alkyl chain and a polar head group thus it displays features of both SPT substrates. Therefore, the prevailing hypothesis is that inhibition of SPT occurs because myriocin acts as a mimic of a key transition state of the catalytic mechanism. Through a combination of UV-vis spectroscopy, mass spectrometry, x-ray crystallography and enzyme inhibition assays it has been possible to study the interaction between S. paucimobilis SPT and myriocin. I have shown that myriocin initially forms an inhibitory PLP:myriocin aldimine complex in the active site that displays a Ki of 967 nM. Interestingly, this complex is susceptible to unexpected, slow enzymatic degradation. The mechanism for myriocin breakdown has been elucidated as a retro-aldol type reaction, which results in cleavage of the C2-C3 bond producing a C18 aldehyde. This aldehyde is then capable of covalently modifying the active site lysine265, forming a second (suicide) inhibitory complex and rendering the enzyme catalytically inactive. Substitution of the active site lysine produced SPT K265A, an inactive enzyme that did not catalyse the breakdown of the PLP:myriocin complex. However, the determination of the crystal structure of the SPT K265A:PLP-myriocin complex revealed that the myriocin had undergone decarboxylation. Nevertheless, this SPT:PLP:decarboxymyriocin structure revealed details about myriocin’s mechanism of inhibition for the first time. The novel mechanism of myriocin degradation has implications on the structure activity relationship (SAR) and design of drugs targeted towards SPT, the role of feedback regulation by long chain aldehydes and further expands the range of reactions catalysed by this important enzyme. As well as inhibition studies the structure of bacterial SPT was also examined by preparing an N-terminally truncated S. paucimobilis SPT. This version, shortened by 21 amino acids, was ~5-fold slower than the wild-type enzyme and suggests that the N-terminus may play a role in catalysis. Additional work has been undertaken to study an unusual membrane-bound viral SPT, composed of two naturally fused open reading frames (SPT2-SPT1) with the proposed SPT2 domain at the N-terminus and the SPT1 domain at the C-terminus. To study soluble mimics of this interesting fusion I prepared a bacterial S. paucimobilis SPT fused wild-type and mutant construct and isolated a fused SPT2-SPT1 with what appears to be single PLPbinding site.

Tese (doutorado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Florestal, Programa de Pós-Graduação em Ciências Florestais, 2015. / Este trabalho objetivou avaliar possíveis alterações com relação às propriedades físicas e mecânicas de compensados e painéis de lâminas paralelas (LVL – laminated venner lumber) produzidos com a madeira de paricá, que foram tratados termomecanicamente. Compensados de duas espessuras diferentes foram testados, e nos de 12 mm de espessura, foram analisadas duas temperaturas (120°C e 150°C), dois tempos sob pressão (7 e 12 minutos) e dois tempos sem pressão (15 e 30 minutos) num total de oito tratamentos. Nos compensados de 20 mm de espessura, foram analisadas duas temperaturas (120°C e 150°C), num total de dois tratamentos. Nos LVLs, foram analisados dois níveis de pressão (25% e 50% da resistência à compressão perpendicular do paricá), num total de dois tratamentos. Propriedades físicas e mecânicas dos painéis tratados foram comparadas com as propriedades de painéis não-tratados (testemunha). Os resultados indicaram que em todos os materiais a densificação não proporcionou melhorias na estabilidade dimensional, em comparação com a testemunha. As propriedades mecânicas dos compensados melhoraram, enquanto que as dos LVLs não foram significativamente afetadas. A temperatura foi o fator mais importante para as alterações nas propriedades físicas e mecânicas dos compensados. / The work aimed at evaluating the densification effects on physical and mechanical properties of plywood and LVL boards produced with veneers of Schizolobium amazonicum wood. Two temperatures (120°C and 150°C), two times under pressure (7 and 12 minutes) and two times with no pressure (15 and 30 minutes) were evaluated on the 12 mm-thick plywood, corresponding to a total of 8 treatments. Two temperatures (120°C and 150°C) were evaluated on the 20 mm-thick plywood, corresponding to a total of 2 treatments. Two pressure levels (25% and 50% of parica perpendicular compression strength) were evaluated on the LVL panels, corresponding to a total of 2 treatments. Physical and mechanical properties of the treated panels were analysed, and the results were compared with the properties of untreated panels. The results indicated that treatment did not improved the dimensional stability of any treated material, in comparison with the non-treated panels. The mechanical properties on both types of plywoods had been improved, while LVL mechanical properties had not been significantly affected by the thermomechanic treatment. The temperature of treatment was the most important factor in the alterations of the physical and mechanical properties of plywood.

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase.

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. ACC deaminases break down ACC, the immediate precursor of ethylene in plants, into ammonia and α-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. It was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather that it utilizes D-cysteine as a substrate, and in fact encodes a D-cysteine desulfhydrase. Kinetic characterization of the enzyme has shown that it is similar to other previously characterized D-cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering two amino acid residues within the predicted active site changed the enzyme from D-cysteine desulfhydrase to ACC deaminase. Concomitantly, it was shown that by altering two amino acids residues at the same position within the active site of ACC deaminase from Pseudomonas putida UW4 changed this enzyme into D-cysteine desulfhydrase.

The main objective of this research was to develop a protocol in which pyridoxal phosphate (PLP) would act as a tag to identify PLP-dependent enzymes from complex mixtures or cell lysates. Following the purification of a PLP-dependent enzyme (CysM), a method was developed to reduce the PLP-lysine Schiff base to form a chemically stable bond between the PLP and the protein. The reduced protein was enzymatically digested resulting in multiple peptide fragments with one or more containing PLP (bound to the active site lysine). These fragments were analyzed by monitoring the absorbance or fluorescence using High Performance Liquid Chromatography. Immobilized Metal Ion Affinity Chromatography (IMAC) was then used to enrich the PLP-peptide(s) from the peptide mixture. The PLP-bound peptide(s) was then analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS). More specifically, sodium borohydride (NaBH4) was used to reduce the Lysine-PLP bond in CysM. This reaction was monitored by either UV-vis spectroscopy or mass spectrometry. Trypsin was used to enzymatically digest the reduced CysM before it was enriched with IMAC and analyzed with LC-MS. Since the objective of this project was to develop a method which could be applied to a cell lysate, IMAC was used as an enrichment method to separate the PLP-peptide(s) from other peptides within the mixture. The PLP-peptide(s) was then located in the peptide mixture by monitoring the absorbance at 325 nm. The LC-MS results of the full reaction before IMAC treatment versus the final column, when monitoring the mass spectrum, showed that the treatment using the IMAC column separated the PLP-peptides from all other peptides within the sample. Using IMAC to enrich specifically the PLP-peptides, followed by analysis with LC-MS, may be a useful method for studying PLP-dependent enzymes within the proteome or the "PLP-ome."