• Refine Query
  • Source
  • Publication year
  • to
  • Language
  • 131
  • 41
  • 29
  • 14
  • 13
  • 6
  • 5
  • 3
  • 2
  • 1
  • 1
  • 1
  • 1
  • 1
  • Tagged with
  • 301
  • 50
  • 40
  • 37
  • 37
  • 35
  • 29
  • 24
  • 24
  • 22
  • 22
  • 19
  • 19
  • 19
  • 18
  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
101

Fracturing events in the Ruby Star granodiorite adjacent to the Esperanza prophyry copper desposit, Pima County, Arizona

Manske, Scott Lee January 1980 (has links)
No description available.
102

The fracture mechanisms in duplex stainless steels at sub-zero temperatures

Pilhagen, Johan January 2013 (has links)
The aim of the thesis was to study the susceptibility for brittle failures and the fracture process of duplex stainless steels at sub-zero temperatures (°C). In the first part of the thesis plates of hot-rolled duplex stainless steel with various thicknesses were used to study the influence of delamination (also known as splits) on the fracture toughness. The methods used were impact and fracture toughness testing. Light optical microscopy and scanning electron microscopy were used to investigate the microstructure and fracture surfaces. It was concluded that the delaminations caused a loss of constraint along the crack front which resulted in a stable fracture process despite the presence of cleavage cracks. These delaminations occurred when cleavage cracks are constrained by the elongated austenite lamellae. The pop-in phenomenon which is frequently observed in duplex stainless steels during fracture toughness testing was shown to occur due to these delaminations. The susceptibility for pop-in behaviour during testing increased with decreasing plate thickness. The toughness anisotropy was also explained by the delamination phenomenon.In the second part of the thesis duplex stainless steel weld metals from lean duplex and super duplex were investigated. For the lean duplex weldments with different nickel contents, tensile, impact and fracture toughness testing were conducted from room temperature to sub-zero temperatures. The result showed that increased nickel content decreased the susceptibility for critical cleavage initiation at sub-zero temperatures. The super duplex stainless steel weldment was post weld heat treated. The fracture sequence at low temperature was critical cleavage fracture initiation after minor crack-tip blunting and ductile fracture. Energy-dispersive X-ray spectroscopy investigation of the weld metals showed that substitutional element partitioning is small in the weld metal. However, for the post weld heat treated weldments element partitioning occurred which resulted in decreased nickel content in the ferrite. / <p>QC 20131108</p>
103

MOLECULAR RECOGNITION PROPERTIES AND KINETIC CHARACTERIZATION OF TRANS EXCISION-SPLICING REACTION CATALYZED BY A GROUP I INTRON-DERIVED RIBOZYME

Sinha, Joy 01 January 2006 (has links)
Group I introns belong to a class of large RNAs that catalyze their own excision from precursor RNA through a two-step process called self-splicing reaction. These self-splicing introns have often been converted into ribozymes with the ability site specifically cleave RNA molecules. One such ribozyme, derived from a self-splicing Pneumocystis carinii group I intron, has subsequently been shown to sequence specifically excise a segment from an exogenous RNA transcript through trans excision-splicing reaction.The trans excision-splicing reaction requires that the substrate be cleaved at two positions called the 5' and 3' splice sites. The sequence requirements at these splice sites were studied. All sixteen possible base pair combinations at the 5' splice site and the four possible nucleotides at the 3' splice site were tested for reactivity. It was found that all base pair combinations at the 5' splice site allow the first reaction step and seven out of sixteen combinations allow the second step to occur. Moreover, it was also found that non-Watson-Crick base pairs are important for 5' splice site recognition and suppress cryptic splicing. In contrast to the 5' splice site, 3' splice site absolutely requires a guanosine.The pathway of the trans excision-splicing reaction is poorly understood. Therefore, as an initial approach, a kinetic framework for the first step (5' cleavage) was established. The framework revealed that substrate binds at a rate expected for RNA-RNA helix formation. The substrate dissociates with a rate constant (0.9 min-1), similar to that for substrate cleavage (3.9 min-1). Following cleavage, the product dissociation is slower than the cleavage, making this step rate limiting for multiple-turnover reactions. Furthermore, evidence suggests that P10 helix forms after the 5' cleavage step and a conformational change exists between the two reaction steps of trans excision-splicing reaction. Combining the data presented herein and the prior knowledge of RNA catalysis, provide a much more detailed view of the second step of the trans excision-splicing reaction.These studies further characterize trans excision-splicing reaction in vitro and provide an insight into its reaction pathway. In addition, the results describe the limits ofthe trans excision-splicing reaction and suggest how key steps can be targeted for improvement using rational ribozyme design approach.
104

HYDROGENATION AND HYDROGENOLYSIS OF FURAN DERIVATIVES USING BIPYRIDINE-BASED ELECTROPHILIC RUTHENIUM(II) CATALYSTS

Gowda, Anitha Shankaralinge 01 January 2013 (has links)
The catalytic activity of ruthenium(II) bis(diimine) complexes cis-[Ru(6,6′-Cl2bpy)2(OH2)2](Z)2 (2, Z = CF3SO3; 3, Z = (3,5-(CF3)2C6H3)4B ,i.e. BArF), cis-[Ru(4,4′-Cl2bpy)2(OH2)2](Z)2 (4, Z = CF3SO3; 5, Z = BArF) and cis-[Ru(bpy)2(PR3)(OH2)](CF3SO3)2 (7, bpy = 2,2’-bipyridine, PR3 = P(C6H4F)3; 8, bpy = 2,2-bipyridine, PR3 = PPh3; 9, bpy = 4,4’-dichloro-2,2’-bipyridine, PR3 = PPh3; 10, bpy = 4,4’-dimethyl-2,2’-bipyridine, PR3 = P(C6H4F)3) for the hydrogenation and hydrogenolysis of furfural (FFR), furfuryl alcohol (FFA) and 5-hydroxymethylfurfural (HMF) was investigated. The compounds 2-5 are active and highly selective catalysts for the hydrogenation of FFR to FFA. Using 2 as catalyst at 100 °C, hydrogenation of FFR proceeded to high conversion (≥98%) and with 100% selectivity to FFA in 2 h. The catalyst cis-[Ru(6,6′-Cl2bpy)2(OH2)2](CF3SO3)2 (2) also showed some activity for hydrogenolysis of FFR and FFA at 130 °C in ethanol, giving up to 25% of 2-methylfuran (MF) yield. The catalyst 3 alsodisplayed high catalytic activity for the hydrogenation of FFA to tetrahydrofurfuryl alcohol. Catalysts 7-10 are also active towards the hydrogenation of furfural (FFR) in NMP giving >90% FFR conversion with 100% selectivity for furfuryl alcohol (FFA) in 12 h. Compounds 7-10 are active C-O bond hydrogenolysis catalysts in presence of bismuth halide Lewis acids. For example, hydrogenolysis of FFA in the presence of 1 mol% of catalyst cis-[Ru(4,4’-Cl2bpy)2(PPh3)(OH2)](CF3SO3)2 (9) and 20 mol% bismuth bromide at 180 °C/51 atm H2 pressure gave >96% conversion of FFA and 55% MF yield. Compounds 7-10 in the presence of bismuth halides, showed almost 100% conversion of HMF with a very high selectivity (65-72%) for 2,5-DMF, along with 10-12% of MF, and trace amount of 5-methylfurfural (MeFFR). In order to test the activity of ruthenium hydrides towards the C-O bond hydrogenation and hydrogenolysis of HMF, series of monocationic ruthenium complexes cis-[Ru(bpy)2(PR3)(H)](CF3SO3) (12, bpy = 2,2’-bipyridine, PR3 = P(C6H4F)3; 13, bpy = 2,2-bipyridine, PR3= PPh3; 14, bpy = 4,4’-dimethyl-2,2’-bipyridine, PR3= P(C6H4F)3) were prepared. The hydrogenation of HMF using catalysts 12-14, produced 70-72% of 2,5-DMF and 11% MF, suggesting that ruthenium hydrides are active and efficient catalysts for HMF hydrogenation.
105

Haematopoietic Serine Proteases : A Cleavage Specificity Analysis

Thorpe, Michael January 2014 (has links)
Mast cells are innate immune cells, historically involved in allergy responses involving IgE. Through this, they have earned a reputation as a fairly detrimental cell type. Their beneficial roles remain somewhat enigmatic although they clearly have the ability to modulate the immune system. This is due to their ability to synthesise many cytokines and chemokines as well as immediately release potent granule-stored mediators. One such mediator is a serine protease, chymase, which has been targeted by pharmaceutical companies developing inhibitors for use in inflammatory conditions. In order to address roles of the proteases, information regarding their cleavage specificity using substrate phage display can help find potential in vivo substrates.  The human chymase cleaves substrates with aromatic amino acids in the P1 position and has a preference for negatively charged amino acids in the P2’ position. The molecular interactions mediating this P2’ preference was investigated by site-directed mutagenesis, where Arg143 and Lys192 had a clear effect in this selectivity. As humans express one chymase and rodents express multiple chymases, extrapolating data between species is difficult. Here, the crab-eating macaque was characterised, which showed many similarities to the human chymase including a near identical extended cleavage specificity and effects of human chymase inhibitors.  Appropriate models are needed when developing human inhibitors for therapeutic use in inflammatory conditions. The effects of five specific chymase inhibitors in development were also tested. The selectivity of inhibitors was dependent on both Arg143 and Lys192, with a greater effect of Lys192. Identification of residues involved in specific inhibitor interactions is important for selective inhibitor development. Another innate cell type, the NK cell, is important in virus and tumour defence. In the channel catfish, a serine protease from an NK-like cell, granzyme-like I, was characterised. A strict preference for Met in the P1 position was seen, and caspase 6 was identified as a potential in vivo target. This may highlight a novel apoptosis-inducing mechanism from a similar cell type has been conserved for approximately 400 myr. Here, important residues mediating chymases’ specificity and interactions with inhibitors has been addressed, as well as finding a new animal model for providing ways to combat their roles in pathological settings.
106

Analysis Of Self-processing Mechanism Of Galactose Oxidase By Site-directed Mutagenesis And Heterologous Expression In Escherichia Coli

Gencer, Burcak 01 December 2005 (has links) (PDF)
In this study, self-catalytic maturation of heterologously expressed pro-galactose oxidase was analysed in E.coli by altering some amino acids which were supposed to play a crucial role in pro-peptide removal. Galactose oxidase (GOase / EC 1.1.3.9) from Fusarium graminearum / having a molecular mass of 68kDa, is a monomeric, copper containing enzyme with an unusual thioether bond. The enzyme is produced as a precursor with an additional 8 amino acid pre- and a 17- amino acid pro-sequence at the N terminus. Previous work has shown that the pre-peptide is removed possibly by a protease during secretion, whereas the 17 amino acid pro-peptide is removed autocatalytically by the aerobic addition of Cu2+ to the precursor, preceding the formation of the thioether bond at the active site. The pro-gao gene was on ProGON1 and ProGOMN1 constructs which were previously established on pET101/D/lacZ vector in England by directed evolution. ProGON1 contains silent mutations at the N-terminus different from native galactose oxidase whereas ProGOMN1 has six further mutations within the mature enzyme, providing high expression. The cleavage site mutations R-1P/A1P, R-1X/A1X, S2A, and the H522A mutation just against the cleavage site in the three dimensional configuration, were carried out by site-directed mutagenesis. Those and some extra mutations were confirmed by DNA sequence analysis. Next, mutant galactose oxidases were expressed in E. coli BL21 Star (DE3), and were purified by Strep-Tactin&reg / Sepharose&reg / column, operating on the basis of affinity chromatography. Subsequently, SDS-PAGE was performed to analyze self-processing by detecting molecular mass difference of protein bands resulting from pro-sequence removal or existence. When the bands obtained in SDS-PAGE were compared, it was seen that the products of original recombinant plasmids, i.e. ProGON1, ProGOMN1 / and the mutational variants showed no difference in band size, all slightly above 70kDa / indicating pro-sequence presence on all constructs. Non-mutants and some of the mutants showed galactose oxidase activity, signifying proper active site construction by thioether bond formation. ProGOMN1 was submitted for N-terminal amino acid sequencing to be able to assert that a size above 70kDa is not solely due to the existence of a 1 kDa Strep-tag II at C-terminus. Sequencing data affirmed the presence of both the pre-peptide and the pro-preptide showing that processing has not occurred at the N-terminus. Accordingly, in this study, it was shown for the first time that the existence of a pre-pro-peptide at the N-terminus of galactose oxidase does not prevent thioether bond formation at the active site. Furthermore, since the pro-peptide is cleaved autocatalytically, the lack of removal of the pre-peptide in E.coli in the presence of Cu 2+ and oxygen is very likely to be the cause of lack of pro-peptide cleavage. In future studies the region corresponding to the pre-peptide will be deleted to prove this hypothesis.
107

The genease activity of mung bean nuclease: fact or fiction?

Kula, Nothemba January 2004 (has links)
<p>The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This &ldquo / genease&rdquo / activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001 / van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.</p> <p><br /> Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding /intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons.</p> <p><br /> Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.</p>
108

Carotenoid cleavage dioxygenases (CCDs) of grape

Dockrall, Samantha 12 1900 (has links)
Thesis (MScAgric)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Plant carotenoid cleavage dioxygenases (CCD) are a family of enzymes that catalyse the oxidative cleavage of carotenoids and/or apocarotenoids. Carotenoids are synthesised in plastids (primarily chloroplasts and chromoplasts), where they are involved in light-harvesting and protecting the photosynthetic apparatus from photo-oxidation. The carotenoid-derived apocarotenoids fulfil a number of roles in plants such as phytohormones, pollinator attractants and flavour and aroma compounds. Due to the floral and fruity characteristics that apocarotenoids contribute to wine, these C13 compounds have received interest in grapevine (Vitis vinifera L.). The CCD gene family in Arabidopsis consists of nine members, all encoding for enzymes that catalyse the cleavage of carotenoids. The enzymes in this family include 9-cis-epoxydioxygenases (NCEDs) and four classes of CCD. NCEDs and CCD7 and CCD8 are involved with plant hormone synthesis, e.g. abscisic acid (ABA) through cleavage by NCED and strigolactone (SL) through the sequential cleavage of carotenoids by CCD7 and CCD8, respectively. SLs are a fairly new class of plant hormone which are involved in several aspects of plant growth and development. The most extensively characterised role of SLs is their involvement in the inhibition of shoot-branching. CCD1 and CCD4 cleave a variety of carotenoids to form pigments and aroma compounds. For example, CCD1 forms β-ionone and β-damascenone, which are important varietal flavours of wine, and CCD4 is involved in synthesis of the pigment and aroma compounds of saffron and annatto. CCD1 enzymes symmetrically cleave the 9,10 (9’,10’) double bonds of multiple carotenoids to produce a C14 dialdehyde and two C13 products. Additional CCD1 cleavage activity at 5,6 (5’,6’) double bonds of lycopene has been reported. Previous studies have shown that CCD1 isolated from V. vinifera (VvCCD1) was able to cleave multiple carotenoid substrates in vitro, namely zeaxanthin, lutein and β-carotene at 9,10 (9’,10’) double bonds and both the 5,6 (5’,6’) and 9,10 (9’,10’) double bonds of lycopene. None of the other VvCCDs, except VvCCD4a have been isolated (but no functionality was illustrated) and characterised yet. CCD4 enzymes also cleave carotenoids at the 9,10 (9’,10’) double bond positions. The presence of plastid-target peptides implies that the CCD4 enzymes have continuous access to carotenoids. Therefore it is suggested that CCD4s are responsible for carotenoid maintenance, where CCD1s contribute towards volatile production. To test this hypothesis VvCCD1, VvCCD4a and VvCCD4b were isolated from V. vinifera (cv Pinotage) cDNA and cloned into a pTWIN1 protein expression vector. Substrate specificity of each VvCCD was tested by co-transforming a carotenoid accumulating E. coli strain with a CCD expression vector. Carotenoids synthesized by the bacteria were identified and quantified by UPLC-analysis, while the concentration of the apocarotenoids, were measured in the headspace of the bacterial cultures using HS-SPME-GC-MS. Several optimisations were done to minimize the natural degradation of the carotenoids; to ensure that the apocarotenoid formation is predominantly due to the enzymatic cleavage by the VvCCDs and not due to oxidation or other non-enzymatic degradation. The HS-SPME-GC-MS analysis indicated that all isoforms cleaved phytoene, lycopene and ε-carotene. Additionally VvCCD1 cleaved a carotenoid involved in photosynthesis, namely β-carotene, while VvCCD4a cleaves neurosporene and VvCCD4b cleaves neurosporene and ζ-carotene, carotenoids not involved in photosynthesis. This study has illustrated that VvCCD1 cleave carotenoids necessary for photosynthesis and VvCCD4s cleave carotenoids which were not present in berry tissue, suggesting their role in carotenoid maintenance. Therefore in planta substrates for CCD1 could possibly be C27 apocarotenoids generated from enzymatic cleavage through CCD4 (role in carotenoid maintenance), CCD7 and/or photo-oxidation, which are then transported from the plastid to the cytosol or possibly C40 carotenoids that are released during senescence or when the plastid membrane is damaged, thus releasing important aroma compounds. Thus the identification of the in vivo substrates has contributed to the understanding the in planta functions of these enzymes / AFRIKAANSE OPSOMMING: Die plant ensiemfamilie van karotenoïedsplitsingdioksigenases (CCDs) kataliseer die oksidatiewe splitsing van karotenoïede en/of apokarotenoïede. Karotenoïede word in plastiede (primêr chloroplaste en chromoplaste) sintetiseer en is betrokke by lig-absorpsie en die beskerming van die fotosintetiese apparaat teen foto-oksidasie. Die apokarotenïede afkomstig van karotenoïede dien onder meer as planthormone, geur- en aromakomponente en om bestuiwers aan te lok. Aangesien apokarotenoïede bydra tot die vrug- en blomgeure van wyn is die C13-verbindings binne wingerd (Vitis vinifera L.) van belang. Al nege lede van die CCD geenfamilie in Arabidopsis kodeer karotenoïedsplitsingsensieme. Die ensiemfamilie sluit 9-sis-epoksidioksigenases (NCEDs), en vier klasse CCD in. NCEDs en CCD7 en 8 is betrokke by die sintese van planthormone, naamlik absissiensuur (ABA) deur NCED en strigolaktone (SL) deur die opeenvolgende aksie van onderskeidelik CCD7 en CCD8. SLe is redelik onlangs as planthormone indentifiseer en is betrokke by ‘n verskeie aspekte van die groei en ontwikkeling van plante. Die rol van SL in inhibisie van vertakking is die beste gekarakteriseerde van hierdie aspekte. CCD1 en CCD4 splits ‘n verskeidenheid karotenoïede om pigmente en aromakomponente te vorm. CCD1 vorm byvoorbeeld β-jonoon en β-damasenoon, beide belangrike kultivar-spesifieke wyngeure. CCD4 vorm weer die pigment en aromakomponente van saffraan en annatto. Die CCD1 ensieme splits die 9,10 (9’,10’) dubbelbindingsetels van verskeie karotenoïede simmetries en vorm een C14-dialdehied en twee C13-produkte. Daar is voorheen melding gemaak van verdere splitsing deur CCD1 by die 5,6 (5’,6’) dubbelbindingsetels van likopeen. Vroeër is getoon dat die CCD1 isovorm wat uit V. vinifera geïsoleer is, naamlik VvCCD1, in vitro seaxantin, luteïen en β-karoteen by die 9,10 (9’,10’) dubbelbindingsetels kon splits, en likopeen by beide die 9,10 (9’,10’) en 5,6 (5’,6’) dubbelbindingsetels. Geen ander VvCCDs is al isoleer en funksioneel gekarakteriseer. VvCCD4a is isoleer, maar geen funksie is bepaal nie. CCD4 ensieme splits ook die 9,10 (9’,10’) dubbelbindingsetels van karotenoïede. Aangesien CCD4 ensieme ‘n plastied-bestemmingspeptied besit behoort dié ensieme konstant toegang tot karotenoïede te hê, wat dui op hul rol in die handhawing van die karotenoïedbalans, terwyl CCD1-ensieme bydra tot die sintese van vlugtige verbindings. Om hierdie hipotese te toets is VvCCD1, VvCCD4a en VvCCD4b uit V. vinifera (kv Pinotage) kDNS isoleer in binne ‘n pTWIN1 proteïenuitdrukkingsvektor kloneer. Die substraatspesifisiteit van elke VvCCD is getoets deur ‘n karotenoïedakkumulerende E. coil stam te transvormeer met ‘n CCD-uitdrukkingsvektor. UPLC-analise is gebruik om karotenoïede wat deur die bakterium sintetiseer is te kwantifiseer en identifiseer, terwyl die apokarotenoïedinhoud en -konsentrasie van die boruimte van die bakteriële kultuur met HS-SPME-GC-MS bepaal is. Verskeie aspekte van die proses is optimaliseer om natuurlike afbreking van karotenoïede te minimeer. Daardeur is verseker dat die apokarotenoïedvorming primêr vanweë die ensiematiese splitsing deur VvCCDs plaasvind en nie deur oksidasie of ander nie-ensiematiese afbreking. Die HS-SPME-GC-MS metings het aangedui dat al drie isovorme fitoëen, likopeen en ε-karoteen kan splits. VvCCD1 kan daarby β-karoteen splits, terwyl VvCCD4a neurosporeen, en VvCCD4b neurosporeen en ζ-karoteen kan splits, beide karotene wat nie betrokke is by fotosintese nie. Dié studie toon dat VvCCD1 die karotenoïede splits wat benodig word vir fotosintese, terwyl beide VvCCD4 isovorme karotenoïede splits wat nie in druiwekorrels gevind word nie. Dit dui op hulle rol in die handhawing van karotenoïedpoele. Die in planta substrate vir CCD1 mag dus die C27-apokarotenoïede wees wat deur CCD4 (as deel van karotenoïedhandhawing), CCD7 en/of foto-oksidasie gevorm word en na die sitosol vervoer word, of moontlik die C40-karotenoïede wat tydens veroudering óf wanner die plastiedmembraan beskadig is in die sitosol vrygestel word. Die identifisering van die in vivo substrate het dus bygedra to die begrip van die in planta funksies van die ensieme.
109

Rôle oncogénique des fragments de p65/RelA Nf-kB générés par l'activité de RIPK3 / Oncogenic role of p65 / RelA Nf-kB fragments generated by RIPK3 activity

Latreche-Carton, Céline 15 December 2017 (has links)
L'utilisation d'un agent déméthylant induit la réexpression de la protéine RIP3, une sérine-thréonine kinase, dans un modèle leucémique murin exprimant BCR-ABL humain. La réexpression de RIP3 conduit rapidement les cellules vers la nécroptose. Le mutant délété du domaine kinase est de façon surprenante plus "apoptogène" et induit le clivage de p65/RelA sur le résidu d'acide aspartique D361 par la caspase 6. Pour déterminer l'impact de ce clivage, nous avons construit un mutant non clivable p65/RelA D361E, ainsi que des plasmides exprimant chacun des fragments p65/RelA 1-361 ou p65/RelA 362-549, ou un plasmide exprimant simultanément p65/RelA 1-361 + p65/RelA 362-549. Ces différents plasmides codant pour les différentes formes de la protéine p65/RelA sont incorporés par transfection dans les cellules leucémiques ou de mélanome pour lesquels le gène RIP3 est respectivement méthylé ou exprimé. In vivo, nous mettons en évidence une différence de tumorigénicité entre les deux modèles. Elle est accrue par la présence de p65/RelA D361E par rapport à celle de p65/RelA WT et de p65/RelA 1-361 + p65/RelA 362-549 dans le modèle leucémique. Elle est au contraire faible dans le modèle du mélanome pour lequel la surexpression des fragments p65/RelA 1-361 +362-549 induit la tumorigenèse la plus forte. L'agressivité du mutant non clivable in vivo n'est pas corrélée à l'activité de NF-kB mesurée in vitro. Les fragments comme le mutant p65/RelA D361E induisent des profils d'expression différents dans le modèle murin de leucémie avec la modulation notable d'expression génique de la famille d'inhibiteurs de protéases à cystéine Stefins, ainsi que le transporteur de bicarbonate de sodium SLC4A5 qui joue un rôle majeur dans la régulation du pH intracellulaire. Le mutant p65/RelA D361E induit une expression importante du transporteur de bicarbonate de sodium SLC4A5 dans le modèle leucémique responsable de l'augmentation du pH intracellulaire qui participe au développement tumoral. Par contre, ce sont les deux fragments p65/RelA 1-361 + p65/RelA 362-549 qui induisent simultanément une expression plus forte de la molécule d'immunoéchappement PDL1, vraisemblablement par un mécanisme post-traductionnel. L'étude de la "souchitude" des modèles montre une différence d'activité du mutant p65/RelA D361E selon le modèle. On observe une augmentation de l'activité ALDH dans le modèle leucémique et une diminution de la formation de sphères dans le modèle de mélanome. En conclusion, ces résultats indiquent que les fragments issus du clivage de p65/RelA par l'activité de RIP3 indépendante de la kinase possèdent un rôle différent de celui de la forme sauvage sur la souchitude des cellules cancéreuses, et qu'elle dépend du modèle étudié. Ils confirment que le mutant non clivable possède la plus forte activité tumorigénique. Ils laissent également supposer que les fragments Nter et Cter puissent avoir une activité dans des cellules tumorales possédant une protéine RIP3 fonctionnelle et active, probablement par des mécanismes inflammatoires ou autres qui doivent être caractérisés. / The receptor-interacting protein kinase 3 (RIPK3) can induce necroptosis, apoptosis, or cell proliferation, and is silenced in several hematological malignancies. We previously reported that RIPK3 activity independent of its kinase domain induces p65/RelA caspase-mediated cleavage resulting in N-terminal 1-362 and C-terminal 362-549 fragments. We show here that a non-cleavable p65/RelA D361E mutant expressed in DA1-3b leukemia cells decrease mouse survival and that coexpressed p65/RelA fragments increase tumoriginicty of B16/F1 melanoma cells that did not correlated with in vitro measured Nf-kB activity. Fragments and p65/RelA fragments display different expression profiles in DA1-3b leukemic cells, with the notable modulation of gene expression of the Stefin cysteine protease inhibitor family and of SLC4A5, a Na+-coupled HCO−3 transporter. DA1-3b cells expressing p65/RelA D361E mutant showed more basic intracellular pH. p65/RelA fragments induced ovexpression of PD-L1 immunoescape molecule in DA1-3b cells. Markers of stemness were also affected: p65/RelA D361E induced increased ALDH activity in DA1-3b cells and fragments expression resulted in increased melanoma sphere formation in B16/F1 cells. Thus, far from being neutral, p65/RelA cleavage initiated by kinase independent activity of RIPK3 induced a pleiotropic range of effects in vitro and in vivo in cancer cells, that may vary across tumor types.
110

Fracture of Nanoporous Gold

January 2014 (has links)
abstract: This research examines several critical aspects of the so-called "film induced cleavage" model of stress corrosion cracking using silver-gold alloys as the parent-phase material. The model hypothesizes that the corrosion generates a brittle nanoporous film, which subsequently fractures forming a high-speed crack that is injected into the uncorroded parent-phase alloy. This high speed crack owing to its kinetic energy can penetrate beyond the corroded layer into the parent phase and thus effectively reducing strength of the parent phase. Silver-gold alloys provide an ideal system to study this effect, as hydrogen effect can be ruled out on thermodynamic basis. During corrosion of the silver-gold alloy, the less noble metal i.e. silver is removed from the system leaving behind a nanoporous gold (NPG) layer. In the case of polycrystalline material, this corrosion process proceeds deeper along the grain boundary than the matrix grain. All of the cracks with apparent penetration beyond the corroded (dealloyed) layer are intergranular. Our aim was to study the crack penetration depth along the grain boundary to ascertain whether the penetration occurs past the grain-boundary dealloyed depth. EDS and imaging in high-resolution aberration corrected scanning transmission electron microscope (STEM) and atom probe tomography (APT) have been used to evaluate the grain boundary corrosion depth. The mechanical properties of monolithic NPG are also studied. The motivation behind this is two-fold. The crack injection depth depends on the speed of the crack formed in the nanoporous layer, which in turn depends on the mechanical properties of the NPG. Also NPG has potential applications in actuation, sensing and catalysis. The measured value of the Young's modulus of NPG with 40 nm ligament size and 28% density was ~ 2.5 GPa and the Poisson's ratio was ~ 0.20. The fracture stress was observed to be ~ 11-13 MPa. There was no significant change observed between these mechanical properties on oxidation of NPG at 1.4 V. The fracture toughness value for the NPG was ~ 10 J/m2. Also dynamic fracture tests showed that the NPG is capable of supporting crack velocities ~ 100 - 180 m/s. / Dissertation/Thesis / Doctoral Dissertation Materials Science and Engineering 2014

Page generated in 0.029 seconds