published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Thèse doctorat : Aspects Moléculaires et Cellulaires de la Biologie : Strasbourg 1 : 2006. Thèse doctorat : Aspects Moléculaires et Cellulaires de la Biologie : M. Lomonossov - Moscou - Russie : 2006. / Thèse soutenue sur un ensemble de travaux. Thèse soutenue en co-tutelle. Titre provenant de l'écran-titre. Bibliogr. 9 p.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Thesis (Ph.D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Les biofilms ont longtemps été décrits comme des organisations évolutives de microorganismes, attachés à une surface et englués dans une matrice contenant, entre autre, des polysaccharides. En partant de ce constat BioFilm Control a souhaité cribler des microorganismes pour la production d’exopolysaccharides, en utilisant le BioFilm Ring Test® (BRT). Le principe repose sur la coincubation de microorganismes avec des particules magnétiques en microplaque. Les particules sont plus ou moins attirées par un aimant en fonction du stade d’organisation du biofilm. En se formant, il piège, dans sa matrice visqueuse, les particules qui perdent leur mobilité. Celle-ci est révélée par une aimantation qui provoque l’apparition d’un spot (pas de biofilm) ou non (biofilm). Une analyse d’images quantifie ce processus et permet de le standardiser. La démarche a consisté dans un premier temps à vérifier le comportement de microorganismes modèles producteurs de polysaccharides (bactéries et microalgues) avec le BRT. L’étude a été étendue au criblage d’une banque de lactobacilles. Les résultats inattendus ont orienté l’étude vers l’analyse du rôle exact des polysaccharides et plus généralement de l’implication des macromolécules dans la structuration du biofilm. Pour cela, la dégradation séquentielle de chaque famille macromoléculaire a été réalisée via des enzymes dépolymérisantes sur les biofilms de Leuconostoc mesenteroides et Bacillus sp. Au regard des résultats obtenus, l’utilisation du BRT a été étendue à la caractérisation qualitative et quantitative d’enzymes de dégradation de polysaccharides. / Biofilms were described for a long time as evolutionary structures elaborated by microorganisms, fixed on a surface and maintained in a polysaccharidic matrix. From this assessment, BioFilm Control chose to screen microorganisms for their capacity to produce exopolysaccharides (EPS), using theBioFilm Ring Test® (BRT). The principle is the co-incubation of magnetic particles with microbial culture on microplates. The mobility of particles depends on the stage of biofilm formation. During this formation, particles are trapped in the matrix and loose their mobility. Revelation is induced by magnet which causes a spot in the absence of biofilm. The pictures analysis quantifies this phenomenon and standardizes different results. This approach was realised, at first step, by the test of EPS-producing bacteria or microalgae with the BRT. The study was extended to the screening of a lactobacilli collection. Unexpected results guided the research toward the understanding of the role of macromolecules in biofilm structuring. To study their implication, sequential enzymatic degradation has been achieved for each macromolecular family of Leuconostoc mesenteroïdes and Bacillus sp. biofilms. Using the results, BRT was then appreciated as a suitable method to detect and quantify polysaccharide degrading enzymes.

Tsang Wai Kai, Paul. / "July 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 133-157). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

Phenylketonuria (PKU) and related forms of non-PKU hyperphenylalaninemias (HPA) result from deficiencies in phenylalanine hydroxylase (PAH), the hepatic enzyme that catalyses the conversion of phenylalanine (phe) to tyrosine (tyr). Patients are characterised by a metabolic phenotype comprising elevated levels of phe and some of its metabolites, notably phenyllactate (PLA), phenylacetate (PAA) and phenylpyruvate (PPA), in both tissue and body fluids. Treatment from birth with low-phe diet largely prevents the severe mental retardation that is its major consequence. / Mechanisms underlying the pathophysiology of PKU are still not fully understood; to this end, the availability of an orthologous animal model is relevant. A number of N-ethyl-N-nitrosourea (ENU) mutagenized mouse strains have become available. I report a new heteorallelic strain, developed by crossing female ENU1 (with mild non-PKU HPA) with a male ENU2/+ carrier of a 'severe' PKU-causing allele. I describe the new hybrid ENU1/2 strain and compare it with control (BTBR/Pas), ENU1, ENU2 and the heterozygous counterparts. The ENU1, ENU1/2 and ENU2 strains display mild, moderate and severe phenotypes, respectively, relative to the control and heterozygous counterparts. / I describe a novel method using negative ion chemical ionization gas chromatography/mass spectrometry (NICI-GC/MS) to measure the concentration of PLA, PAA and PPA in the brain of normal and mutant mice. Although elevated moderately in HPA and more so in PKU mice, concentrations of these metabolites are not sufficient to explain impaired brain function; however phe is present in brain at levels associated with harm. / Finally, I describe a new modality for treatment of HPA, compatible with better human compliance: it involves enzyme substitution with non-absorbable and protected phenylalanine ammonia lyase (PAL) in the intestinal lumen, to convert L-phenylalanine to the harmless metabolites (trans-cinnamic acid and trace ammonia). PAL, taken orally, substitutes for the deficient PAH enzyme and depletes body pools of excess phe. I describe an efficient recombinant approach to produce PAL enzyme. I also provide proofs of both pharmacologic and physiologic principles by testing PAL in the orthologous mutant mouse strains with HPA. The findings encourage further development of PAL for oral use as an ancillary treatment of human PKU.

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 155-168).

Thesis (MSc) Stellenbosch University, 2014 / ENGLISH ABSTRACT: In this study, the effect of the amino acid residues at positions 40 and 407 on the catalytic activity of porcine CYP17A1 was investigated. Porcine cofactor CYB5 was cloned from porcine liver tissue and its effect on the catalytic activity of porcine CYP17A1 was determined. The influence of rat, human and angora CYB5 on the lyase activity of porcine CYP17A1 was subsequently determined and compared to the influence of porcine CYB5. Wt porcine CYP17A1, which has residues Val40 and His407, catalysed the conversion of prog efficiently with ~50% prog converted to 17OHprog (~40%) and A4 (~10%) after 3 hr. After 24 hr, negligible levels prog remained with ~71% 17OHprog and ~25% A4 being produced. Low levels of 16OHprog were formed (~9%). The Leu105Ala mutation reduced wt 17α-hydroxylase activity, with 70% prog remaining after 24 hr while 16OHprog (~10%) levels remained unchanged. Porcine CYP17A1 with residues Leu40 and His407, exhibited similar catalytic activity towards prog as did wt porcine CYP17A1 (Val40 and His407 residues), while porcine CYP17A1 with residues Leu40 and Leu407 increased the formation of A4 2-fold to 54% at 24 hr and porcine CYP17A1 with residues Val40 and Leu407 resulted in the highest formation of A4 (90%). Wt porcine CYP17A1, while having converted 95% of the prog substrate, produces only ~16% A4 after 24 hr. In the presence of porcine CYB5, however, the lyase activity was stimulated with 85% of prog being converted to A4 and only 13% 17OHprog remaining. The lyase activity was also stimulated by CYB5 from other species, resulting in an increase in A4 production of 60.6%, 24% and 11.6% by rat, angora and human CYB5, respectively. The degree of lyase stimulation correlated to the percentage identity of the CYB5 amino acid sequences to porcine CYB5. While the Val and Leu residues at position 40 do not appear to influence the lyase activity of porcine CYP17A1 as prominently as the residue at position 407, it is the charged residue at 407 that plays a significant role in the production of A4, decreasing A4 production irrespective of the Val and the Leu residues at position 40. It would, furthermore, appear that the stimulation of lyase activity of CYP17A1 is the greatest when assaying this activity in the presence of CYB5 of the same species as was detected when co-expressing porcine CYP17A1 and porcine CYB5. / AFRIKAANSE OPSOMMING: In hierdie studie is die invloed van die aminosuurresidue by posisies 40 en 407 op die katalitiese aktiwiteit van vark CYP17A1 ondersoek. Vark CYB5 is geklooneer vanuit vark lewer weefsel en die effek van hierdie kofaktor op die katalitiese aktiwiteit van vark CYP17A1 is bepaal. Die invloed van rot, mens en angora CYB5 op die liase aktiwiteit van vark CYP17A1 is daarna bepaal en vergelyk met die invloed van vark CYB5. Vark CYP17A1-VH, (kodeer Val40 en His407), kataliseer die omskakeling van prog doeltreffend met ~50 % prog wat omgeskakel word na 17OHprog (~40%) en A4 (~10%) na 3 uur. Na 24 uur, is feitlik alle prog omgeskakel, met ~71% 17OHprog en ~25% A4 geproduseer. Lae vlakke 16OHprog is ook gevorm (~9%). Die Leu105Ala mutasie verminder 17α- hidroksilase aktiwiteit, met 70% prog wat na 24 uur nie omgesit is nie, terwyl 16OHprog (~10%) vlakke onveranderd gebly het. Vark CYP17A1-LH (kodeer Leu40 en His407), en CYP17A1-VH het diselfde katalitiese aktiwiteit teenoor prog getoon, terwyl vark CYP17A1-LL (kodeer Leu40 en Leu407) die vorming van A4 2-voudig verhoog het tot 54% na 24 uur. Vark CYP17A1-VL (kodeer Val40 en Leu407) se katalitiese aktiwiteit het gelei tot die hoogste vorming van A4 (90%). Alhoewel CYP17A1-VH, 95% van die prog substraat omgeskakel het is slegs ~16% A4 geproduseer na 24 uur. In die teenwoordigheid van vark CYB5 is die liase aktiwiteit egter gestimuleer, en is 85% van die prog substraat omgeskakel na A4 met slegs 13% 17OHprog teenwoordig na 24 uur. Die liase aktiwiteit is ook gestimuleer deur CYB5 van ander spesies, wat lei tot 'n toename in A4 produksie van 60,6% , 24% en 11,6% deur rot, angora en menslike CYB5, onderskeidelik. Daar is gevind dat daar’n sterk korrelasie is tussen die stimulering van die liase aktiwitieit en die persentasie aminosuur volgorde identiteit van CYB5 afkomstig vanaf die verskillende spesies. Terwyl die Val en die Leu aminosuurresidu op posisie 40 wel die liase aktiwitiet tot ‘n mate beȉnvloed, blyk dit uit die data dat die potitief gelaaide residue by 407 'n belangrike rol speel in die produksie van A4, en A4 produksie verlaag ongeag van die Val en die Leu residu by posisie 40. Dit wil ook verdermeer voorkom asof die stimulering van die liase aktiwiteit van CYP17A1 die hoogste is wanneer die ensiem gekataliseerde reaksie deurgevoer word in die teenwoordigheid van CYB5 en CYP17A1 afkomstig vanaf dieselfde spesies.