Um procedimento eletroquímico para deposição de paládio no interior de um tubo de grafite para modificação química permanente e um sistema de geração de vapor frio para a determinação de níveis traços de mercúrio em água e sedimento através de espectrometria de absorção atômica é proposto. A célula eletroquímica tubular para as deposições de paládio em fluxo foi construída aproveitando a estrutura original do tubo de grafite o qual opera como eletrodo de trabalho. Um tubo de aço inoxidável, posicionado na saída da célula, é usado como o eletrodo auxiliar. O valor do potencial aplicado no eletrodo de grafite é medido contra um micro eletrodo de referência de Ag/AgCl inserido sobre o eletrodo auxiliar. Soluções de paládio em tampão de acetato (100 milimol L-1, pH=4,70), numa vazão de 0,5 mL min-1 foram usados para executar a deposição eletroquímica durante o intervalo de tempo de 60 min. O sistema de geração de vapor frio consiste de um micro-reator de polietileno e frasco separador líquido-gás, volume total de 4,0 ml, que é descartado depois de cada amostragem para evitar inter-contaminação das amostras. Volumes de 1,0 ml de reagente (2,0% m/v NaBH4 em 0,10 mol L-1 de NaOH) e 1,0 ml de soluções analíticas ou amostras em 0,25 mol L-1 de HNO3 são levados para o reator e separador líquido-gás usando uma bomba peristáltica. O vapor de mercúrio é transportado ao tubo de grafite modificado eletroquimicamente com fluxo de argônio de 212 ml min-1 e pré-concentrado durante 120 s. O limite de detecção obtido foi 93 ng L-1 (n=20, 3δ). O desempenho deste sistema foi testado para determinação de mercúrio em água potável, água não potável e sedimentos de lagoa. A confiança de todo procedimento foi confirmada através de testes de recuperação. / An electrochemical procedure for palladium deposition on the inner of a graphite tube for permanent chemical modification and a cold vapor generation system for the determination of trace levels of mercury in water and sediment by atomic absorption spectrometry are proposed. The tubular electrochemical cell for the deposition of palladium was assembled on the original geometry of a graphite tube that operates as the working electrode. A stainless steel tube, positioned downstream from the working electrode, is used as the auxiliary electrode. The potential value applied on the graphite electrode is measured against a micro reference electrode (Ag/AgCl) inserted into the auxiliary electrode. Palladium solutions in acetate buffer (100 milimol L-1, pH=4.70), flowing at 0.5 mL min-1 for 60 min was used to perform the electrochemical deposition. A cold vapor generation system consist of a micro polyethylene reactor and gas-liquid separator flask, total volume of 4.0 mL, that is discarded after each sampling to avoid intercontamination of the samples. Volumes of 1.0 mL of reagent (2.0% m/v NaBH4 in 0.10 mol L-1 of NaOH) and 1.0 mL of reference or sample solutions in 0.25 mol L-1 of HNO3 are carried out to the reactor and gas-liquid separator by using a peristaltic pump. The mercury vapor is transported to the graphite tube electrochemically modified with argon flow (200 mL min-1) and pre-concentrated during 120 s. The detection limit obtained was 93 ng L-1 (n=20, 3δ). The performance of these system was tested for determination of mercury in potable and non-potable water and lake sediments. The reliability of the entire procedure was confirmed by recovery tests.

Um procedimento eletroquímico para deposição de paládio no interior de um tubo de grafite para modificação química permanente e um sistema de geração de vapor frio para a determinação de níveis traços de mercúrio em água e sedimento através de espectrometria de absorção atômica é proposto. A célula eletroquímica tubular para as deposições de paládio em fluxo foi construída aproveitando a estrutura original do tubo de grafite o qual opera como eletrodo de trabalho. Um tubo de aço inoxidável, posicionado na saída da célula, é usado como o eletrodo auxiliar. O valor do potencial aplicado no eletrodo de grafite é medido contra um micro eletrodo de referência de Ag/AgCl inserido sobre o eletrodo auxiliar. Soluções de paládio em tampão de acetato (100 milimol L-1, pH=4,70), numa vazão de 0,5 mL min-1 foram usados para executar a deposição eletroquímica durante o intervalo de tempo de 60 min. O sistema de geração de vapor frio consiste de um micro-reator de polietileno e frasco separador líquido-gás, volume total de 4,0 ml, que é descartado depois de cada amostragem para evitar inter-contaminação das amostras. Volumes de 1,0 ml de reagente (2,0% m/v NaBH4 em 0,10 mol L-1 de NaOH) e 1,0 ml de soluções analíticas ou amostras em 0,25 mol L-1 de HNO3 são levados para o reator e separador líquido-gás usando uma bomba peristáltica. O vapor de mercúrio é transportado ao tubo de grafite modificado eletroquimicamente com fluxo de argônio de 212 ml min-1 e pré-concentrado durante 120 s. O limite de detecção obtido foi 93 ng L-1 (n=20, 3δ). O desempenho deste sistema foi testado para determinação de mercúrio em água potável, água não potável e sedimentos de lagoa. A confiança de todo procedimento foi confirmada através de testes de recuperação. / An electrochemical procedure for palladium deposition on the inner of a graphite tube for permanent chemical modification and a cold vapor generation system for the determination of trace levels of mercury in water and sediment by atomic absorption spectrometry are proposed. The tubular electrochemical cell for the deposition of palladium was assembled on the original geometry of a graphite tube that operates as the working electrode. A stainless steel tube, positioned downstream from the working electrode, is used as the auxiliary electrode. The potential value applied on the graphite electrode is measured against a micro reference electrode (Ag/AgCl) inserted into the auxiliary electrode. Palladium solutions in acetate buffer (100 milimol L-1, pH=4.70), flowing at 0.5 mL min-1 for 60 min was used to perform the electrochemical deposition. A cold vapor generation system consist of a micro polyethylene reactor and gas-liquid separator flask, total volume of 4.0 mL, that is discarded after each sampling to avoid intercontamination of the samples. Volumes of 1.0 mL of reagent (2.0% m/v NaBH4 in 0.10 mol L-1 of NaOH) and 1.0 mL of reference or sample solutions in 0.25 mol L-1 of HNO3 are carried out to the reactor and gas-liquid separator by using a peristaltic pump. The mercury vapor is transported to the graphite tube electrochemically modified with argon flow (200 mL min-1) and pre-concentrated during 120 s. The detection limit obtained was 93 ng L-1 (n=20, 3δ). The performance of these system was tested for determination of mercury in potable and non-potable water and lake sediments. The reliability of the entire procedure was confirmed by recovery tests.

Small ubiquitin-like modifier (SUMO) regulates numerous biological functions. In a previous study we found that sumoylation of HSF2 is involved in regulating HSF2 bookmarking function, but the mechanism that mediates this regulation was unknown. The results in my work support the intriguing hypothesis that polycomb protein, Mel-18, actually functions as an anti-SUMO E3 protein, interacting both with HSF2 and the SUMO E2 Ubc9, but acting to inhibit Ubc9 activity and thereby decrease sumoylation of the HSF2. This study also suggested that Mel-18 negatively regulates the sumoylation of other cellular proteins, and we extend its targets to RanGAP1 protein. The results also show that RanGAP1 sumoylation is decreased during mitosis, and that this is associated with increased interaction between RanGAP1 and Mel-18. Previous studies showed little evidence of anti-SUMO E3 proteins, however, my study, taken together, found Mel-18 actually functions as a novel anti-SUMO E3 protein, interacting both with substrates and the SUMO E2 Ubc9 but acting to inhibit Ubc9 activity to decrease sumoylation of target proteins and also provide an explanation for how mitotic HSF2/RanGAP1 sumoylation is regulated. This finding also gives a clue for a future study direction in Mel-18 as a tumor suppressor: the anti-SUMO E3 function. Additionally, we identify a single-nucleotide polymorphism in another human PcG protein, Bmi-1, that changes a cysteine residue within its RING domain, cysteine 18, to a tyrosine. This C18Y polymorphism is associated with a significant decrease in levels of the Bmi-1 protein. Furthermore, the C18Y Bmi-1 protein exhibits a very high level of ubiquitination compared to wild-type Bmi-1, suggesting that that the low levels of this form of Bmi-1 are due to its destruction by the ubiquitin-proteasome system. Consistent with this hypothesis, treatment of cells with the proteasome inhibitor MG-132 results in a significant increase in levels of C18Y Bmi-1. This is the first example of a polymorphism in human Bmi- 1 that reduces levels of this important protein.

This thesis discusses and documents findings from an investigation of performance-based testing of asphalt cement (AC), warm mixed asphalt cement, asphalt rubber (AR), and warm asphalt rubber. A number of control, warm, and asphalt rubber binders from Ontario construction contracts were investigated for their compliance with conventional Superpave® test methods such as rolling thin film (RTFO), pressure aging vessel (PAV), dynamic shear rheometer (DSR), and bending beam rheometer (BBR), as well as additional specification tests such as extended BBR and double edge notched tension test. The quality and durability of those binders were determined. Quality means the ability of asphalt binder to reach a set of specific properties whereas durability is the measure of how well asphalt retains its original characteristics when exposed to normal weathering and aging process. One warm AC and two field-blended asphalt rubber samples showed high levels of physical hardening which can lead to premature and early cracking. The warm asphalt cement lost 8 °C when stored isothermally for three days at low temperatures according to Ontario’s extended bending beam rheometer (BBR) protocol (LS-308). The two asphalt rubber samples lost 10 °C and 12 °C following the same conditioning. Many of the studied asphalt samples showed deficient strain tolerance as measured in Ontario’s double-edge-notched tension (DENT) test (LS-299). In a study of warm rubberized asphalt cement with improved properties, a number of compositions were prepared with soft Cold Lake AC and a small quantity of naphthenic oil. These binders showed little chemical and physical hardening and reasonable critical crack tip opening displacements (CTOD). Strain tolerance was much improved by co-blending with a high vinyl type styrene-butadiene-styrene (SBS) polymer and a small amount of sulfur. / Thesis (Master, Chemistry) -- Queen's University, 2013-04-24 22:54:20.07

The aim of the work reported here was to determine the influence of an amine curing agent, and postcure cycle on the mechanical and thermal properties of diglycidyl ether of bisphenol A (DGEBA) epoxy resin. The results of this initial study were then used as the basis for selecting material to obtain optimum toughness in epoxy/glass fibre systems. These basic materials were further used to make comparisons with the properties of modified resin systems which contained commercial elastomers. Differential Scanning Calorimetry (DSC), Dynamic Mechanical Thermal Analysis (DMTA), Fourier Transform Infrared Spectroscopy (FTIR), flexural and interlaminar shear tests, Instrumented Falling Weight Impact (IFWI), visual observation, Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM) were all used to investigate various properties and the structures which gave rise to them. The properties of cured products were found to be affected by the amounts of curing agent, curing times and temperatures, and the structure of the elastomers. Not surprisingly the maximum thermal and mechanical properties tended to be found in the stoichiometric (standard) mix systems. However, postcuring at higher than room temperature, which was used as the basic curing temperature, led to more conversion. This effect improved the thermal and mechanical properties of both the unmodified and modified resin systems. The maximum flexural strength of 104 MPa of the unreinforced resins was found in the stoichiometric mix ratio after postcure at 150°C for 4 hr. However, the maximum flexural modulus and glass transition temperature (Tg) were found after postcuring at the same temperature for 48 hr. This was believed to be due to increased crosslinking, but unfortunately the longer curing time led to degradation of the resins. In the systems modified with -20 phr of polyetheramine elastomers, the one modified with the lowest molecular weight (2000) was found to have the highest flexural strength (85.8 MPa) and modulus (2.5 GPa). The impact properties of all the composites with modified resin matrices were found to be higher than the unmodified resin matrix composites. The best impact properties were, however, obtained with the elastomer modifier with a molecular weight of 4000. The impact energy at maximum force increased from 11.9 to 16.4 J, and energy at failure increased from 18.7 to 21.6 J. This increase in impact properties was due to the increase in areas of phase separated elastomer particles over similar systems with lower molecular weight modifier.

Le rétinoblastome est une tumeur rare qui touche la rétine du jeune enfant. L’inactivation bi-allélique du gène RB1 est à l’origine du développement tumoral. RB1 est le premier gène suppresseur de tumeur qui ait été identifié et la prédisposition au rétinoblastome constitue un véritable paradigme de la prédisposition aux cancers. Dans les formes non prédisposées génétiquement, les deux mutations apparaissent dans une cellule rétinienne unique ; le rétinoblastome est alors unilatéral. Dans les formes à prédisposition génétique, la première mutation est constitutionnelle et la deuxième est somatique. La mutation constitutionnelle est une néo-mutation pré- ou post- zygotique dans les formes sporadiques, alors qu’elle est héritée dans les formes familiales. Dans les formes avec prédisposition génétique, le diagnostic est plus précoce que dans les formes sans prédisposition et la bilatérisation du rétinoblastome est généralement la règle. Néanmoins, de rares familles présentent une pénétrance réduite et une variabilité phénotypique se traduisant par la coexistence de patients atteints de rétinoblastome bilatéral ou unilatéral, d’apparentés porteurs sains et d’apparentés présentant des rétinomes. Les mécanismes responsables de la variabilité phénotypique intrafamiliale sont inconnus et l’existence de facteurs génétiques modulant le phénotype tumoral est probable.L’origine de la variabilité de l’expression phénotypique du rétinoblastome peut être la résultante (i) de l’existence de mutations en mosaïque, (ii) de mutations de RB1 et (iii) de facteurs modificateurs génétiques indépendants du locus de RB1. Trois axes distincts et originaux basés sur ces origines possibles de variabilité phénotypique ont été développés pour caractériser les relations génotype/phénotype dans le rétinoblastome. Premièrement, les conséquences d’une mosaïque somatique ont été illustrées grâce à l’étude d’une famille ayant bénéficié de cinq diagnostics prénatals. Dans ces familles, certains fœtus porteurs de l’allèle à risque identifié par une approche indirecte basée sur l’étude de microsatellites au locus de RB1, n’étaient pas porteurs de la mutation du parent atteint, lui-même atteint d’un rétinoblastome bilatéral. Ainsi, nous avons démontré la présence d’une mosaïque somatique et gonadique chez ce parent lourdement atteint. La conséquence de l’existence de patients présentant une mosaïque dans le cadre du conseil génétique a été discutée. La suite de nos travaux a pris en compte ces résultats afin de limiter les biais que pourrait induire la présence de mutations en mosaïque dans des études de corrélation génotype/phénotype dans le rétinoblastome. Deuxièmement, l’association de grandes délétions emportant RB1 avec des retards psychomoteurs chez des patients atteints de rétinoblastome a été étudiée. Une approche de CGH hautement résolutive, ciblée sur le locus de RB1, a été mise en place afin de caractériser le rôle des gènes contigus de RB1 dans ce syndrome. Ainsi, cette approche a permis de définir une zone à risque de retard psychomoteur que nous proposons comme seuil d’alerte pour le généticien. Cette zone définit un gène, PCDH8 d’expression cérébrale exclusive, comme un excellent candidat au retard psychomoteur. Enfin, troisièmement, une approche « gène candidat » reposant sur l’étude du SNP309 du promoteur de MDM2, a été mise en œuvre. / Retinoblastoma is the most common intraocular childhood cancer and occurs when both alleles of the RB1 gene are inactivated in the retina. In patients without genetic predisposition, the two mutations occurred in a single unique retinal cell. In subjects with a genetic predisposition to retinoblastoma, the first RB1 mutation is found in the germline and the second appears as a somatic mutation. Germline carriers usually develop bilateral or multifocal tumors and the diagnosis is earlier. However, some rare families exhibit low penetrance and variable expressivity of the disease because bilaterally affected, unilaterally affected, and unaffected mutation carriers are known to coexist. The existence of genetic modifiers in retinoblastoma therefore appears highly probable and must be considered. The lack of penetrance and the variable expressivity could be the sum of three independent causes. The presence of a mosaic can affect the phenotype, the nature of the mutation can drive low penetrance and particular phenotype like psychomotor delay in case of large genomic deletions and genetic modifier factors could modulate the phenotype. These three major keys have been studied in order to highlight the relations between the phenotype and the genotype. Firstly, the consequences of mosaicism have been illustrated by a prenatal diagnosis concerning a couple with a bilateral retinoblastoma-affected male patient who requested five successive prenatal diagnoses and in whom RB1 mutation mosaicism had important implications. Implications of mosaicism in genetic counseling have been discussed and taken into consideration in order to limit bias in the two following genotype/phenotype studies. Secondly, the association between whole germline monoallelic deletions of the RB1 gene and psychomotor delay was studied by a high-resolution CGH array focusing on RB1 and its flanking region. Comparative analysis detected a four megabase critical interval including a candidate gene, protocadherin 8 (PCDH8). PCDH8 is thought to function in signaling pathways and cell adhesion in a central nervous system-specific manner, making loss of PCDH8 one of the probable causes of psychomotor delay in RB1-deleted patients. Thirdly, a candidate gene approach based on partners that are necessary for the development of the tumor attempted to find possible genetic modifiers. MDM2, which increases p53 and pRB catabolism, was therefore a prominent candidate. The minor allele of MDM2 that includes a 309T>G transversion (SNP rs2279744) in the MDM2 promoter is known to enhance MDM2 expression. In family-based association analyses performed in 70 retinoblastoma families, the MDM2 309G allele was found to be statistically significantly associated with incidence of bilateral or unilateral retinoblastoma among members of retinoblastoma families under a recessive model (Z = 3.305, two-sided exact P = .001). The strong association of this genotype with retinoblastoma development designates MDM2 as the first modifier gene to be identified among retinoblastoma patients

It is currently understood that diseases are typically not caused by rogue errors in genetics but have both molecular and environmental causes from myriad overlapping interactions within an interactome. Genetic errors, such as that seen by a single-nucleotide polymorphism can lead to a dysfunctional cell, which in turn can lead to systemic disruptions that result in disease phenotypes. Perturbations within the interactome, as can be caused by many such errors, can be organized into a pathophenotype, or “disease module”. Disease modules are sets of correlated variables that can represent many of a disease’s activities with subgraphs of nodes and edges. Many methods for inferring disease modules are available today, but the results each one yields is not only variable between methods but also across datasets and trial attempts. In this study, several such inference methods for deriving disease modules are evaluated by combining them to create “consensus” modules. The method of focus is Double-Specific Betweenness (S2B), which uses betweenness centrality across separate diseases to derive new modules. This study, however, uses S2B to combine the results of independent inference methods rather than separate diseases to derive new modules. Pre-processed asthma and arthritis data are compared using various combinations of inference methods. The performance of each result is validated using Pathway Scoring Algorithm. The results of this study suggest that combining methods of inference using MODifieR or S2B may be beneficial for deriving meaningful disease modules.

Ma Kit Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 189-206). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Table of Contents --- p.iii / Abstract --- p.xi / 摘要 --- p.xiv / Abbreviation List --- p.xv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ubiquitination --- p.1 / Chapter 1.1.1 --- Ubiquitin --- p.1 / Chapter 1.1.2 --- Ubiquitin Pathway --- p.3 / Chapter 1.1.3 --- Functions of Ubiquitination --- p.5 / Chapter 1.1.4 --- Ubiquitin Like Proteins --- p.8 / Chapter 1.2 --- SUMO Proteins --- p.10 / Chapter 1.2.1 --- SUMO Isoforms --- p.10 / Chapter 1.2.2 --- SUMO Structure --- p.11 / Chapter 1.3 --- Sumoylation --- p.14 / Chapter 1.3.1 --- Functions of Sumoylation --- p.14 / Chapter 1.3.1.1 --- General Functions of Sumoylation --- p.15 / Chapter 1.3.1.2 --- Function of Sumoylation on Transcription Factors / Chapter 1.3.1.3 --- Specific Function of SUMO-2/3 Conjugation / Chapter 1.3.2 --- Sumoylation Pathway --- p.19 / Chapter 1.4 --- E3 Ligases in Sumoylation --- p.24 / Chapter 1.4.1 --- Types and Functions of E3 Ligases --- p.23 / Chapter 1.4.2 --- Structure of PI AS --- p.23 / Chapter 1.4.3 --- Function of PI AS --- p.27 / Chapter 1.5 --- Aims of Study --- p.29 / Chapter Chapter 2 --- Materials & Methods --- p.30 / Chapter 2.1 --- Polymerase Chain Reaction (PCR) Screening of Multiple Human Tissue cDNA (MTC´ёØ) Panel --- p.30 / Chapter 2.1.1 --- Primer Design --- p.30 / Chapter 2.1.2 --- Semi-quantitative PCR --- p.31 / Chapter 2.1.2.1 --- Human MTC´ёØ Panel --- p.31 / Chapter 2.1.2.2 --- PCR --- p.32 / Chapter 2.2 --- DNA Cloning --- p.34 / Chapter 2.2.1 --- "Amplification of El, E3 (PIAS), PIAS1 Fragments" --- p.34 / Chapter 2.2.1.1 --- Primer Design --- p.34 / Chapter 2.2.1.2 --- PCR --- p.36 / Chapter 2.2.1.3 --- Purification of PCR Product --- p.37 / Chapter 2.2.2 --- Restriction Digestion --- p.37 / Chapter 2.2.3 --- Ligation --- p.40 / Chapter 2.2.4 --- Transformation --- p.40 / Chapter 2.2.4.1 --- Preparation of Chemically Competent Cells'(DH5α) --- p.40 / Chapter 2.2.4.2 --- Transformation of Ligation Product --- p.41 / Chapter 2.2.5 --- Plasmid Preparation --- p.42 / Chapter 2.2.6 --- Screening for Recombinant Clones --- p.43 / Chapter 2.2.7 --- Sequencing of Recombinant Plasmid --- p.43 / Chapter 2.3 --- Subcellular Localization Study --- p.45 / Chapter 2.3.1 --- Midi Scale Plasmid Preparation --- p.45 / Chapter 2.3.2 --- Transfection of GFP Recombinant Plasmids --- p.46 / Chapter 2.3.2.1 --- Cell Culture of WRL-68 & HepG2 Cell Lines --- p.46 / Chapter 2.3.2.2 --- LipofectAMINE Based Transfection --- p.47 / Chapter 2.3.3 --- Immunostaining of Endogenous SUMO-1 & -2/-3 --- p.48 / Chapter 2.3.4 --- Nucleus Staining by DAPI --- p.48 / Chapter 2.3.5 --- Fluorescent Microscopic Visualization --- p.49 / Chapter 2.3.6 --- Western Blotting --- p.49 / Chapter 2.3.6.1 --- LipofectAMINE Based Transfection --- p.49 / Chapter 2.3.6.2 --- Protein Extraction --- p.50 / Chapter 2.3.6.3 --- Protein Quantification --- p.51 / Chapter 2.3.6.4 --- SDS-PAGE Analysis --- p.51 / Chapter 2.3.6.5 --- GFP Fusion Proteins Detection --- p.52 / Chapter 2.4 --- Two-Dimensional Gel Electrophoretic Analyses --- p.54 / Chapter 2.4.1 --- Sample Preparation --- p.54 / Chapter 2.4.1.1 --- Protein Extraction from the Nucleus --- p.54 / Chapter 2.4.1.2 --- Clean Up of Extracted Nuclear Fraction --- p.55 / Chapter 2.4.2 --- First Dimensional Isoelectric Focusing (IEF) --- p.55 / Chapter 2.4.3 --- Second Dimension SDS-PAGE --- p.57 / Chapter 2.4.3.1 --- SDS-PAGE Analysis --- p.57 / Chapter 2.4.3.2 --- Silver Staining --- p.58 / Chapter 2.4.4 --- Image Analysis --- p.59 / Chapter 2.4.5 --- Protein Identification by Mass Spectrometry --- p.60 / Chapter 2.4.5.1 --- Sample Preparation --- p.60 / Chapter 2.4.5.2 --- Data Acquisition --- p.62 / Chapter 2.4.5.3 --- Data Analysis of Protein Fingerprinting --- p.62 / Chapter 2.5 --- Confirmation of the Differentially Expressed Proteins by RT-PCR & Western Blotting --- p.63 / Chapter 2.5.1 --- RT-PCR Analysis --- p.63 / Chapter 2.5.1.1 --- RNA Extraction --- p.63 / Chapter 2.5.1.2 --- First Strand cDNA Synthesis --- p.64 / Chapter 2.5.1.3 --- Normalization of cDNA Template --- p.64 / Chapter 2.5.1.4 --- PCR Amplification of the Target Genes --- p.65 / Chapter 2.5.2 --- Western Blotting --- p.66 / Chapter 2.6 --- Expression of Human PIAS and PIAS1 Fragments in Prokaryotic System --- p.67 / Chapter 2.6.1 --- Preparation of Competent Cells --- p.67 / Chapter 2.6.2 --- Small Scale Expression --- p.67 / Chapter 2.6.2.1 --- Transformation --- p.67 / Chapter 2.6.2.2 --- IPTG Induced Protein Expression --- p.68 / Chapter 2.6.3 --- Large Scale Expression of PIAS1 Fragments --- p.70 / Chapter 2.6.3.1 --- Transformation --- p.70 / Chapter 2.6.3.2 --- IPTG Induced Protein Expression --- p.70 / Chapter 2.6.4 --- Purification Trial of MBP-PIAS1-321-410 --- p.71 / Chapter 2.6.4.1 --- Binding of Amylose Resin & On Column Cleavage (with Low Concentration of DTT) --- p.71 / Chapter 2.6.4.2 --- Elution from the Amylose Resin & Cleavage (with Low Concentration of DTT) --- p.73 / Chapter 2.6.4.3 --- Elution from the Amylose Resin & Cleavage (with High Concentration of DTT) --- p.73 / Chapter 2.6.4.4 --- Purification of PIAS1-321-410 by Size ExclusionChromatography --- p.73 / Chapter 2.6.5 --- Purification of MBP-PIAS1 Fragments --- p.74 / Chapter 2.6.5.1 --- Purification by Affinity Column (Amylose) --- p.74 / Chapter 2.6.5.2 --- Amylose Resin Regeneration --- p.74 / Chapter 2.6.5.3 --- Purification by Both Affinity and Ion Exchange (Heparin) --- p.75 / Chapter 2.6.5.4 --- Regeneration of Heparin Column --- p.76 / Chapter 2.6.5.5 --- Purification by Size Exclusion Chromatography --- p.76 / Chapter 2.6.5.6 --- Regeneration of Size Exclusion Chromatography --- p.77 / Chapter 2.6.6 --- Co-expression & Purification of PIAS1 Fragment with E2 (Ubc9) --- p.77 / Chapter 2.6.6.1 --- Co-transformation of pMAL-PIASl (Fragments) & pET-Ubc9 --- p.77 / Chapter 2.6.6.2 --- Co-expression of PIAS1 Fragments & Ubc9 --- p.78 / Chapter 2.6.6.3 --- Purification by Affinity Column (Amylose Resin) --- p.78 / Chapter 2.6.6.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.79 / Chapter 2.6.6.5 --- Purification by Size Exclusion Chromatography --- p.79 / Chapter 2.6.7 --- Urea Treatment for the Purification of PIAS 1 Fragments --- p.80 / Chapter 2.6.7.1 --- Transformation --- p.80 / Chapter 2.6.7.2 --- IPTG Induced Protein Expression --- p.80 / Chapter 2.6.7.3 --- Purification by Affinity Column (Amylose Resin) --- p.80 / Chapter 2.6.7.4 --- Purification by Both Affinity & Ion Exchange (Heparin) --- p.80 / Chapter 2.6.7.5 --- Purification by Size Exclusion Chromatography --- p.81 / Chapter Chapter 3 --- Results --- p.82 / Chapter 3.1 --- Tissue Distribution of Human PIAS Genes --- p.82 / Chapter 3.1.1 --- Determination of the Number of Cycles for PCR --- p.82 / Chapter 3.1.2 --- General Expression Pattern of All PIAS Genes --- p.82 / Chapter 3.1.3 --- Tissue Distribution of PIAS1 --- p.83 / Chapter 3.1.4 --- Tissue Distribution of PIAS3 --- p.83 / Chapter 3.1.5 --- Tissue Distribution of PIASxa --- p.83 / Chapter 3.1.6 --- Tissue Distribution of PIASxp --- p.84 / Chapter 3.1.7 --- Tissue Distribution of PIASy --- p.84 / Chapter 3.2 --- Subcellular Localization of SUMO Pathway Components --- p.90 / Chapter 3.2.1 --- Overexpression Confirmation --- p.90 / Chapter 3.2.2 --- Multiple Bands Detected After Overexpression of EGFP- SUMO-1 --- p.91 / Chapter 3.2.3 --- Subcellular Localization of EGFP --- p.94 / Chapter 3.2.4 --- Subcellular Localization of El Subunits --- p.94 / Chapter 3.2.5 --- Subcellular Localization of E2 (Ubc9) --- p.95 / Chapter 3.2.6 --- Subcellular Localization of PIAS Proteins --- p.95 / Chapter 3.2.7 --- Subcellular Localization of PIAS1 Fragments --- p.96 / Chapter 3.2.8 --- Subcellular Localization of SUMO-1 --- p.97 / Chapter 3.3 --- Differential Protein Expression Pattern after Transient Transfection of SUMO-1 --- p.112 / Chapter 3.3.1 --- Protein Expression Profiles after Transient Transfection / Chapter 3.3.2 --- Identification of the Differential Expressed Proteins --- p.113 / Chapter 3.4 --- Confirmation of Differentially Expressed Proteins in Cells Overexpressing SUMO-1 --- p.124 / Chapter 3.4.1 --- RT-PCR Analyses --- p.124 / Chapter 3.4.1.1 --- Downregulation of RNA Transcript of hnRNP A2/B1 isoform B1 --- p.124 / Chapter 3.4.1.2 --- No Significant Change in the Transcription Level of UDG --- p.125 / Chapter 3.4.2 --- Western Blotting --- p.128 / Chapter 3.4.2.1 --- Upregulation of hnRNP A2/B1 at the Protein Level --- p.128 / Chapter 3.4.2.2 --- Different Molecular Weight of hnRNP A2/B1 Was Detected --- p.129 / Chapter 3.4.2.3 --- Upregulation of UDG at the Protein Level --- p.129 / Chapter 3.5 --- Expression & Purification of Human PIAS Proteins & PIAS1 Fragments --- p.133 / Chapter 3.5.1 --- Expression of Human PIAS Proteins --- p.133 / Chapter 3.5.2 --- Expression of PIAS1 Fragments --- p.135 / Chapter 3.5.3 --- A Trial of Purification of MBP-PIAS1-321-410 --- p.137 / Chapter 3.5.3.1 --- On Column Cleavage of MBP Tag --- p.137 / Chapter 3.5.3.2 --- Cleavage after Elution --- p.137 / Chapter 3.5.3.3 --- High Concentration of DTT Used --- p.138 / Chapter 3.5.3.4 --- Separation of the Cleaved and Non Cleaved Proteins --- p.138 / Chapter 3.5.4 --- Purification of the PIAS 1 Fragments --- p.141 / Chapter 3.5.4.1 --- Purified by Affinity Column (Amylose Resin) --- p.141 / Chapter 3.5.4.2 --- Purified by Heparin Column --- p.141 / Chapter 3.5.4.3 --- Purified by Gel Filtration --- p.143 / Chapter 3.5.5 --- Co-expression & Purification of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.1 --- Co-expression of PIAS1 Fragments & E2 --- p.147 / Chapter 3.5.5.2 --- Co-purification of PIAS1 Fragments & E2 Amylose --- p.147 / Chapter 3.5.5.3 --- Co-purification of PIAS1 Fragments & E2 by Heparin --- p.148 / Chapter 3.5.5.4 --- Co-purification of PIAS 1 Fragments with Ubc9 by Gel Filtration --- p.148 / Chapter 3.5.6 --- Urea Treatment for Purification of PIAS1 Fragments --- p.153 / Chapter 3.5.6.1 --- Purification by Amylose Resin --- p.153 / Chapter 3.5.6.2 --- Purification by Heparin --- p.153 / Chapter 3.5.6.3 --- Purification by Gel Filtration --- p.154 / Chapter Chapter 4 --- Discussion --- p.157 / Chapter 4.1 --- Tissue Specificity of PIAS Proteins --- p.157 / Chapter 4.1.1 --- Principle of Tissue Specificity Study --- p.157 / Chapter 4.1.2 --- Importance of Sumoylation --- p.158 / Chapter 4.1.3 --- Role of Sumoylation in Reproduction --- p.159 / Chapter 4.1.4 --- Functional Role of Sumoylation in Other Tissue --- p.160 / Chapter 4.2 --- Subcellular Localization of SUMO Pathway --- p.162 / Chapter 4.2.1 --- SUMO Conjugation Occurs in the Nucleus --- p.162 / Chapter 4.2.2 --- Does Sumoylation Occur Outside the Nucleus --- p.163 / Chapter 4.2.3 --- Dots-like Structure Formed by the PIAS --- p.164 / Chapter 4.2.4 --- SAP Domain and PINIT Motif Are Not Essential for Nuclear Targeting --- p.165 / Chapter 4.2.5 --- Signal Involves in the Formation of Nuclear Speckles --- p.167 / Chapter 4.3 --- Differentially Expressed Proteins under SUMO-1 Overexpression --- p.169 / Chapter 4.3.1 --- Increase in High Molecular Weight Proteins --- p.169 / Chapter 4.3.2 --- Upregulation of hnRNP A2/B1 & UDG in Protein Level --- p.170 / Chapter 4.3.3 --- Variants of hnRNP A2/B1 Formed --- p.172 / Chapter 4.3.4 --- Possibility of Sumoylation on hnRNP A2/B1 isoform B1 & UDG --- p.172 / Chapter 4.3.5 --- Possible Roles of SUMO-1 on hnRNP A2/B1 isoform B1 --- p.174 / Chapter 4.3.6 --- Mechanism of Sumoylation on mRNA Processing --- p.175 / Chapter 4.3.7 --- Possible Roles of SUMO-1 on UDG --- p.176 / Chapter 4.3.8 --- Important of SUMO on Genome Integrity --- p.178 / Chapter 4.3.9 --- Sumoylation and Carcinogenesis --- p.178 / Chapter 4.4 --- Protein Purification of the Human PIAS Proteins & PIAS1 Fragments --- p.180 / Chapter 4.4.1 --- Low Expression Level & Solubility of the PIAS Proteins --- p.180 / Chapter 4.4.2 --- High Expression Level & Solubility of PIAS 1 Fragments --- p.181 / Chapter 4.4.3 --- Incorrect Disulfide Bond Formation of the PIAS1 Fragments --- p.182 / Chapter 4.4.4 --- MBP-PIAS1 Fragments Formed Soluble Aggregates --- p.182 / Chapter 4.4.5 --- A Low Concentration of Urea Cannot Dissociate the Soluble Aggregates --- p.183 / Chapter 4.4.6 --- Aggregation May Weaken the Interaction between the PIAS1 Fragments & Ubc9 --- p.184 / Chapter 4.5 --- Conclusion --- p.185 / Chapter 4.6 --- Future Perspectives --- p.187 / Chapter 4.6.1 --- Identification of the Role of SUMO Interacting Motif in the Nuclear Speckle Formation --- p.187 / Chapter 4.6.2 --- Investigation of Sumoylation on Liver Cancer --- p.187 / Chapter 4.6.3 --- Optimization of the Expression & Purification of the PIAS Proteins --- p.188 / References --- p.189 / Appendix --- p.207

Ce travail porte sur l'étude chimique d'une souche endophyte de Botryosphaeria mamane, un micromycète relativement peu étudié, isolée des feuilles de Bixa orellana. Des travaux préliminaires portant sur le screening de 409 souches de champignons isolés à partir de plantes médicinales d'Amérique du Sud a révélé que parmi celles-ci, B. mamane E224 était l'une des souches les plus actives in vitro sur un modèle de Leishmania infantum. L'objectif de ce travail a consisté en l'induction de la production de nouveaux métabolites secondaires produits par B. mamane via l'optimisation des conditions de culture de cette souche, la mise en place de méthodes de co-cultures et l'ajout de modificateurs épigénétiques. Une analyse des métabolomes dans les différentes conditions a été réalisée à travers une approche métabolomique, utilisant un couplage UHPLC-HRMS, ainsi que grâce à différents outils statistiques. Deux grandes classes de composés ont ainsi été détectées dans les cultures axéniques de B. mamane. Premièrement, la famille des cyclopeptides, incluant les cyclodipeptides soufrés avec en particulier trois nouveaux composés, les botryosulfuranols A-C. Puis la famille des isocoumarines, avec des dérivés de la melleine (trans-4-hydroxymelleine, 4-hydroxymelleine et 5-hydroxymellein). A travers l'ajout de modificateurs épigénétiques à la culture de B. mamane, nous avons pu étudier les effets de deux inhibiteurs d'histone désacétylases (HDACis), l'acide suberoylanilidehydroxamique (SAHA) et le valproate sodique, ajoutés à deux stades différents de la culture fongique. L'ajout de HDACi dans la culture de B. mamane a entraîné des changements importants dans la production de métabolites secondaires. En effet, une induction de certains métabolites mais également une réduction et l'inactivation de la production d'autres métabolites, ont été observés, et ceci selon la nature du modificateur épigénétique ajouté. Cette étude illustre l'importance du choix des HDACis pour l'induction de la production de métabolites spécifiques. Concernant l'optimisation de la co-culture de B.[...] / This study focused on the strain of a poorly studied fungal endophyte Botryosphaeria mamane E224, isolated from Bixa orellana leaves. Our previous screening involving 409 fungal strains isolated from medicinal plants from South America revealed that among all these strains, B. mamane was shown to be the most bioactive on in vitro model against Leishmania infantum. The objectives of this work consisted in the introduction of new metabolite production by B. mamane by optimizing the fungal culture conditions, and by using co-cultivation methods and addition of epigenetic modifiers. This work was followed by an analysis of the different metabolomes via a metabolomics approach using UHPLC-HRMS and integration of informatics and statistical tools for metabolomics. Two major compound classes were detected in B. mamane. First, the cyclopeptide family including the thiodiketopiperazines (TDKPs) alkaloids with three new compounds proposed as botryosulfuranols A-C; and the isocoumarin family, with the mellein derivatives, trans-4-hydroxymellein, 4-hydroxymellein, and 5-hydroxymellein. Regarding the exploration of B. mamane metabolome cultured in the presence of epigenetic modifier, the effects of two different histone deacetylase inhibitors (HDACis), suberoylanilidehydroxamic acid (SAHA) and valproate sodium added in two different stages of fungal growth, were investigated. As expected, HDACis addition in the culture of B. mamane led to significant changes in the secondary metabolite production. Addition of modifier not only induced metabolites production but also reduced and may inactivate metabolite production in fungi, depending on the nature of the epigenetic modifier added. This study illustrates the importance in the choice of HDACis to fungal culture in order to induce specific metabolite productions. In the study of B. mamane and C. albicans co-cultivation in different culture conditions, we showed the influence of the conditions (static versus agitation) on the metabolome of the fungi. However, the co-culture with yeast did not induce any modification in the fungal metabolome. The investigation of fungal interactions between B. mamane, Fusarium solani, and Colletotrichum linicola in 6-multi well plates in time-series based analysis has been carried out. [...]

Primary cilia are evolutionarily conserved organelles required in a number of signalling pathways influencing the development and behaviour of a diverse range of organisms. More recently, studies into a new class of human diseases known as ciliopathies have helped to shed light on the critical role of this once-ignored signalling centre. Bardet-Biedl syndrome (BBS) proteins localize to the primary cilium and participate in cilium biogenesis and function. BBS is a pleiotropic human disorder with variable severity that is suitable as a disease model for investigating the pathogenesis of a number of common ciliopathy features such as photoreceptor degeneration, renal cysts, and obesity. The C. elegans genome encodes a number of BBS proteins which undergo intraflagellar transport (IFT) at the primary cilium. Given the conservation between C. elegans and human BBS proteins, I hypothesize the existence of unidentified conserved genetic pathways related to the functions of these proteins. Using C. elegans, I characterize novel features of bbs mutants while identifying sources of genomic variation that may elucidate the variability of human BBS features. I show that C. elegans bbs mutants exhibit smaller body size, delayed development, and decreased exploration behaviour. Moreover, I identify a role for the soluble guanylate cyclases GCY-35/GCY-36 in modifying these bbs phenotypes. I conclude that BBS proteins non-cell autonomously influence a set of body cavity neurons in which GCY-35/GCY-36 function genetically upstream of a cGMP-dependent protein kinase (PKG), EGL-4, to control body size. Furthermore, the role of GCY-35/GCY-36 is unique amongst a large number of guanylate cyclases and BBS proteins may influence body size via an IFT-independent function. I explore the biological functions of EGL-4 and conclude that it may regulate body size through multiple cellular mechanisms. I also examine potential candidate genes related to cGMP production and turnover, confirming that additional cGMP-related factors can influence body size although not necessarily in body cavity neurons. In conclusion, I propose a model where BBS-expressing sensory neurons influence body size and development through cGMP-PKG signalling in body cavity neurons while functioning in parallel with additional sensory neurons (possibly BBS-independent) that use similar cGMP-PKG signalling dynamics.