Sphingolipids (SLs) are complex lipid-derived structures that are essential components of cell membranes in eukaryotes and some bacteria. SLs and their complex derivatives ceramides are known to be involved in multiple processes such as the formation of lipid rafts, cell signalling and membrane trafficking. The first step of SL biosynthesis is universal to all sphingolipid-producing organisms from bacteria to humans and is catalysed by the enzyme serine palmitoyltransferase (SPT). SPT is a member of the alpha-oxoamine synthase (AOS) family of pyridoxal- 5'-phosphate-dependent enzymes. All AOS family enzymes retain a high degree of structural homology and catalyse the decarboxylative Claisen-like condensation of amino acids with thioester substrates. The SPT enzyme catalyses the formation of the universal SL precursor, 3-ketodihydrosphingosine (KDS), by condensation of L-serine and coenzyme A-derived palmitic acid. Being the key controller in SL biosynthesis, SPT plays a big role in regulating natural and pathological processes. A lot of research interest has been recently generated by SLs isolated from bacterial members of the human microbiome and their roles in human health. Increasing evidence suggests that some of these SLs possess immunoregulatory effects and can have a direct impact on the immunity of the host. Bacteroides fragilis is a commensal gut-dwelling bacterium that belongs to a few human microbionts known to produce unique iso-branched sphingolipids (isoSLs); these have been shown to influence the human iNKT cell count. The production of SLs in B.fragilis is completely regulated by a gene product BF2461. In this work, BF2461 was expressed and purified; using a combination of UV-vis spectrometry, enzymatic assays, mass spectrometry and protein X-ray crystallography, it has been confirmed to be an SPT. The substrate specificity of the BfSPT has been assessed with a range of different chain-length substrates, including less common 15 and 17-carbon chain length coenzyme A substrates. The enzyme can produce different types of SL precursors with a preference for the 16-carbon chain substrate palmitoyl- CoA. However, at high levels of PCoA, a substrate inhibition is observed that might point to a natural control mechanism employed by the bacterium in favour of producing iso-branched SLs (isoSLs). The structure of BfSPT has been elucidated in a complex with its amino acid substrate L-serine. Search and analysis of putative SPTs from other microbiome-associated bacteria that produce isoSLs show that they share high similarity with an average amino acid conservation of 74%, suggesting they might be adapted to a particular type of substrate. In this respect, BfSPT might be the first isoSL-producing SPT to be structurally characterised, and the first one to have a direct impact on human health. Further structural data were obtained on protein complexes with L-cycloserine and L-penicillamine, some common inhibitors of the PLP-dependent enzymes. The structure obtained in the presence of L-penicillamine provides the first direct structural evidence of the inhibitory mechanism by a thiazolidine complex formation in the active site of a PLP-dependent enzyme. These findings shed light on certain aspects of the reaction and inhibition mechanisms of BfSPT as well as opening new prospects into researching this interesting target and its impact on the human microbiome.

Pseudomonas aeruginosais an opportunistic pathogen that infects cystic fibrosis (CF) patients contributing to their high morbidity and mortality. P. aeruginosaundergoes a phenotypic conversion in the CF lung, from nonmucoid to mucoid, by constitutively producing a polysaccharide called alginate. These mucoid strains often revert to nonmucoid in vitrodue to second-site suppressor mutations. We hypothesized that mapping these mutations would lead to the identification of novel genes involved in alginate production. In a previous study, a mucoid strain, PDO300 (PAOmucA22), was used to isolate suppressors of alginate phenotype (sap). One of the uncharacterized nonmucoid revertants, sap27, is the subject of this study. The mucoid phenotype in sap27was restored by pMO012217 from a minimal tiling path cosmid library. The cosmid pMO012217 harbors 18 P. aeruginosaopen reading frames (ORF). The cosmid was mutagenized with a transposon to map the contributing gene. It was mapped tolptD(PA0595) encoding lipopolysaccharide transport protein. E. coliLptD transports lipopolysaccharide to the outer leaflet of the outer membrane. The Alg+phenotype was restored upon complementation with P. aeruginosa lptDalone, suggesting that sap27likely harbor a chromosomal mutation inlptD. Sequencing analysis of sap27showed the presence of a mutation not in lptDbut in algO, which encodes a periplasmic protease protein. This suggests LptD is able to bypass analgO mutation by positively regulating alginate production. The lptD is a part of a three-gene operon lptD-surA-pdxA. SurA is an essential protein for survival in starvation and a major chaperone protein for all outer membrane proteins and PdxA is a NAD-dependent dehydrogenase and is involved in the vitamin B6biosynthetic pathway. Pyridoxal 5’-phosphate (PLP) is the active form of vitamin B6.P. aeruginosagrown in a media supplemented with PLP increased production of pyocyanin, a virulence factor. The PLP and aromatic amino acids are synthesized from a common precursor chorismic acid. We demonstrated an increase in pyocyanin production when the bacteria were cultured supplemented by the aromatic amino acids phenylalanine. We concluded that the lptDoperon plays a role in the P. aeruginosavirulence by regulating alginate and pyocyanin production.

The fundamental process by which an alpha-helical membrane protein attains its ultimate structure has previously been depicted as two energetically distinct stages where (1) the transmembrane (TM) segments are first threaded into the membrane bilayer as stable alpha-helices; and then (2) laterally interact to form the correct tertiary and/or quaternary structures. Central to the second stage of this model is the presence of amino acid sequence motifs in the TM segments that provide interaction-compatible surfaces through which the TM alpha-helices interact. Although these ideas have proven to be pivotal to the progress of the membrane protein folding field, a growing number of examples indicates that a variety of additional factors work together to dictate the ultimate interaction fate of TM embedded segments. In this context, we expand on these factors and explore other properties that can modulate the association of TM alpha-helices. A peptide model of myelin proteolipid protein (PLP) TM4 is capable of TM helix-helix interactions in SDS and biological membranes. Increasing the side chain volumes of two disease relevant residues (Ala242 and A248) reduces peptide self-association, indicating that these sites mediate TM helix packing through van der Waals interactions. Examination of the PLP TM2 alpha-helix shows that it is also capable of self-association and that its dimeric state depends on the presence or absence of residues at its C-terminus. Specifically, this sensitivity was attributed to changes in local hydrophobicity; a decrease in hydrophobicity likely reduces detergent-peptide interactions, which disrupts peptide alpha-helicity and the effectiveness of a nearby interaction compatible surface. We take advantage of this finding to determine the feasibility of coupling helix-helix interactions to an external factor such as pH. Our results indicate that pH can indeed modulate the dimerization state of the TM2 peptide and does so through the change in protonation state of Glu88. Increasing our knowledge of the variables contributing to TM helix-helix interactions provides valuable insights into membrane protein folding and how mutations can compromise this process. This knowledge will allow us to expand our arsenal of approaches to counter membrane protein misassembly--and ultimately human disease.

The fundamental process by which an alpha-helical membrane protein attains its ultimate structure has previously been depicted as two energetically distinct stages where (1) the transmembrane (TM) segments are first threaded into the membrane bilayer as stable alpha-helices; and then (2) laterally interact to form the correct tertiary and/or quaternary structures. Central to the second stage of this model is the presence of amino acid sequence motifs in the TM segments that provide interaction-compatible surfaces through which the TM alpha-helices interact. Although these ideas have proven to be pivotal to the progress of the membrane protein folding field, a growing number of examples indicates that a variety of additional factors work together to dictate the ultimate interaction fate of TM embedded segments. In this context, we expand on these factors and explore other properties that can modulate the association of TM alpha-helices. A peptide model of myelin proteolipid protein (PLP) TM4 is capable of TM helix-helix interactions in SDS and biological membranes. Increasing the side chain volumes of two disease relevant residues (Ala242 and A248) reduces peptide self-association, indicating that these sites mediate TM helix packing through van der Waals interactions. Examination of the PLP TM2 alpha-helix shows that it is also capable of self-association and that its dimeric state depends on the presence or absence of residues at its C-terminus. Specifically, this sensitivity was attributed to changes in local hydrophobicity; a decrease in hydrophobicity likely reduces detergent-peptide interactions, which disrupts peptide alpha-helicity and the effectiveness of a nearby interaction compatible surface. We take advantage of this finding to determine the feasibility of coupling helix-helix interactions to an external factor such as pH. Our results indicate that pH can indeed modulate the dimerization state of the TM2 peptide and does so through the change in protonation state of Glu88. Increasing our knowledge of the variables contributing to TM helix-helix interactions provides valuable insights into membrane protein folding and how mutations can compromise this process. This knowledge will allow us to expand our arsenal of approaches to counter membrane protein misassembly--and ultimately human disease.

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, Departamento de Engenharia Florestal, 2008. / Submitted by Kelly Marques (pereira.kelly@gmail.com) on 2009-10-21T17:24:55Z No. of bitstreams: 1 2008_Airton Mauro de Lara Santos.pdf: 729182 bytes, checksum: 85dd5fe8b4b289a97433d412060b9282 (MD5) / Approved for entry into archive by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br) on 2009-11-23T11:49:04Z (GMT) No. of bitstreams: 1 2008_Airton Mauro de Lara Santos.pdf: 729182 bytes, checksum: 85dd5fe8b4b289a97433d412060b9282 (MD5) / Made available in DSpace on 2009-11-23T11:49:04Z (GMT). No. of bitstreams: 1 2008_Airton Mauro de Lara Santos.pdf: 729182 bytes, checksum: 85dd5fe8b4b289a97433d412060b9282 (MD5) Previous issue date: 2008 / Os produtos engenheirados de madeiras (PEM) vêem se expandindo globalmente, estes são produzidos a partir da junção de mais de um composto de madeira, utilizando a melhor propriedade de cada um desses elementos, dando qualidade e eficiência as estruturas. As vigas em “I” pré-fabricadas de madeira, que usam painéis estruturais para o material da alma e a madeira serrada de pequena dimensão ou compostos estruturais de madeira - structural composite lumber (SCL) para os flanges, são consideradas a segunda geração dos PEM. Este produto substitui a utilização de grandes volumes de madeira serrada em aplicações de assoalho e telhado, para os edifícios residenciais e comerciais. Os objetivos do presente trabalho foram de estimar para as vigas em “I” por meio de modelos teóricos a rigidez a flexão (EI), módulo de ruptura (fM) e flecha (δ), e comparar estes valores com os valores obtidos experimentalmente por meio de ensaios de flexão estática; e comparar as vigas em “I” produzidas com diferentes compostos. Foram produzidos dois tipos de vigas em “I” uma com os flanges de painéis de lâminas paralelas (LVL) e alma de compensado e outra com os flanges de LVL e a alma de chapas de fibras orientadas (OSB). A alma e os flanges foram ensaiados para que suas propriedades fossem utilizadas nos modelos teóricos. Depois de realizados os ensaios de flexão estática os valores experimentais foram comparados por análise da variância com os valores teóricos. Para as vigas em “I” com alma de compensado somente para o fM não foi observada diferença significativa entre os valores experimentais e teóricos, para as demais variáveis EM e δ observou diferenças significativas. Para as vigas em “I” com a alma de OSB todas as variáveis estudadas, EM, fM e δ, não apresentaram diferenças significativas entre os valores teóricos e experimentais. As vigas em “I” com a alma de OSB apresentaram valores médios de EM e fM, 14.923e 28,7 MPa, superiores aos valores médios 10.115 e 20,0 MPa para EM e fM das vigas em “I” com alma de compensado. Para as vigas em “I” com alma de OSB foi possível estimar de forma confiável os valores de EM, fM e δ, validando assim os modelos teóricos utilizados, já para as vigas com alma de compensado os modelos não puderam ser validados, por apresentarem diferenças significativas entre os valores teóricos e experimentais. As vigas em “I” com alma de OSB apresentaram melhores propriedades mecânicas do que as vigas em “I” com alma de compensado, além de valores de δ inferiores. _______________________________________________________________________________ ABSTRACT / The engineered wood products (EWPs) are products that have expanded globally. They are produced from the junction of more than one wood composite, using the best property of each one, providing qualified and efficient structures. The wood I-joists are produced using structural panels on the web and lumber or structural wood composite lumber (SCL) on the flanges. They are considered as the second generation of EWPs. This product replaces the use of large quantity of timber in floor and roof applications for both residential and commercial buildings. The aims of the study were to estimate theoretically the bending stiffness (EI), modulus of rupture (fM) and deflection (δ), and compare it with the values obtained experimentally. Further study was done to evaluate the effect of web type on these variables. Two types of I-joist were produced: oriented strand board (OSB) webbed I-joist and plywood webbed I-joist. For plywood webbed I-joist the results pointed out that no significant difference between the experimental and theoretical fM values were observed. However, for EM and δ significant differences were identified. On the other hand, for OSB webbed I-joist significant differences between theoretical and experimental values of the evaluated variables were not identified. The I-joist with the web of OSB showed mean values of EM and fM, 14,923 and 28.7 MPa, above the average values 10,115 and 20.0 MPa for EM and fM the I-joist with web of offset. For I-joist with web of OSB was possible to estimate reliable data of EM, fM and δ, thus validating the theoretical models used. For the plywood webbed I-joits the models could not be validated, due to significant differences between the experimental and theoretical values. The OSB webbed I-joist presented better mechanical properties than the plywood webbed I-joist and also lower values δ.

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Malaria is caused by the parasite Plasmodium falciparum and still plagues many parts of the world. To date, efforts to control the spread of the parasites have been largely ineffective. Due to development of resistance by the parasites to current therapeutics there is an urgent need for new classes of therapeutics. The vitamin B6 biosynthetic pathway consists of a PLP synthase which produces pyridoxal 5'-phosphate (PLP) within the parasite. The absence of this pathway in humans makes it attractive for selective targeting using small chemical molecules. The PLP synthase condenses D-ribose 5-phosphate (R5P) and DL-glyceraldehyde 3-phosphate (G3P) with ammonia to form PLP. Two proteins make up this PLP synthase – PfPdx1 and PfPdx2. Computational modelling of Pf Pdx1, and mapping of the R5P-binding site pharmacophore facilitated the identification of several ligands with predicted favourable binding interactions. Confirmatory testing of these on the purified Pf Pdx1 in vitro revealed D-erythrose 4-phosphate (E4P) and an analogue 4-phospho-D-erythronhydrazide (4PEHz) were capable of dose-dependently inhibiting the enzyme. The acyclic tetrose scaffold of E4P, with both aldehyde and phosphate group moieties, was thought to affect R5P imine bond formation in Pf Pdx1, possibly allowing the molecule to enter the R5P-binding site of Pf Pdx1. This hypothesis was supported by molecular docking simulations, and suggested that 4PEHz could similarly enter the R5P-binding site. 4PEHz was detrimental to the proliferation of cultured P. falciparum intraerythrocytic parasites and had an inhibitory concentration (IC50) of 10 µM. The selectivity of 4PEHz in targeting Pf Pdx1 was investigated using transgenic cell lines over-expressing Pf Pdx1 and Pf Pdx2, revealing that complementation of PLP biosynthesis rescued the parasites from the detrimental effects of 4PEHz. Functional transcriptomic and proteomic characterisation of 4PEHz-treated parasites revealed that the expression of Pf Pdx2 increased during 4PEHz treatment, moreover showed that other PLP-related processes were affected. These results supported that Pf Pdx1 is targeted by 4PEHz, and affected PLP biosynthesis de novo. Results from this study allude to alternative regulation of de novo PLP biosynthesis within the parasites by E4P. Moreover, contributions from this work showed that the de novo vitamin B6 pathway of P. falciparum is chemically targetable, and a potential strategy for the development of newer antimalarials. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Biochemistry / Unrestricted

Bakgrund: Sedan 1950-talet har antalet amputationer av övre och nedre extremiteter femdubblats. Dock har forskarna fortfarande inte en speciellt systematisk förståelse kring fantomsmärta. Förutsättningen för att få fram en välgrundad och personcentrerad behandling är god kommunikation med patienten såväl som korrekt dokumentation. Det krävs också en bred kunskap kring fantomsmärta för att kunna utföra en personcentrerad vård. Genom en utökad förståelse kring fenomenet blir patienternas subjektiva upplevelser lättare att förstå och behandla för sjuksköterskan vilket då kan bidra till en bättre vård. Syfte: Litteraturstudiens syfte var att belysa patienters upplevelser av att leva med fantomsmärta. Metod: Litteraturstudiens metod omfattade databassökningar i Pubmed och Cinahl. Valda studier inom området omvårdnad analyserades. Resultat: Analysen resulterade i att två teman identifierades: Smärtans påverkan i livet och Upplevelser av att hantera smärta. Fantomsmärta visade sig problematiskt att leva med. De personer som drabbas av fantomsmärta ansåg att informationen kring fenomenet var otillräcklig eller icke existerande. Smärtan påverkade vardagen och innebar anpassningar, både fysiskt och psykiskt. Vårdpersonal behöver därför vara kunniga och empatiska i bemötandet av patienter med fantomsmärta för att på så sätt kunna bedriva en personcentrerad omvårdnad. Konklusion: Patienter upplevde att förståelsen för fenomenet, upplevelserna och informationen som gavs ut var bristfällig. Genom vetskapen om att fantomsmärta är en individuell upplevelse kan sjuksköterskan bättre bedriva en personcentrerad omvårdnad.

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