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L’objectif de cette thèse porte sur l’étude de la compartimentalisation cellulaire et de la prise en charge de l’uranium (U) par les protéines cytosoliques des cellules branchiales du poisson zèbre (Danio rerio, espèce modèle en toxicologie aquatique) après exposition contrastées (chronique vs. aiguë, 20 et 250 µg.L-1) par voie directe. Cette étude a nécessité le développement, l’utilisation et le couplage d’outils analytiques de pointe (SEC, IEF hors-gel, RP-UHPLC pour la séparation, ICP-SFMS, ESI-FTMS/MS pour la détection) avec comme défis majeurs la conservation des interactions non-covalentes U biomolécule et une sensibilité maximale pour travailler à des niveaux d’exposition proches de ceux rencontrés dans l’environnement. Après extraction, 24 à 32% de la charge branchiale totale en U est contenue dans le cytosol dans lequel la distribution de l’U sur les biomolécules (en fonction de leur PM mais aussi de leur pI) diffère selon le niveau d’exposition. Enfin, une cartographie des biomolécules cibles de l’U a permis (i) de mettre en évidence une affinité particulière de l’U pour les protéines à caractère acide et/ou contenant du phosphore et (ii) d’identifier 24 protéines candidates pour lier U. / The objective of this thesis is to study the cellular compartmentalization and the chelation of uranium (U) by cytosolic proteins of gill cells of the zebrafish (Danio rerio, model species in aquatic toxicology) under different direct exposure conditions (chronic vs. acute, 20 and 250 µg.L 1). This study required the development of hyphenated techniques (SEC, IEF off-gel, RP-UHPLC for the separation, ICP-SFMS, ESI-FTMS/MS for the detection) with the main challenges of maintaining the non-covalent U-biomolecule interactions and enhancing sensitivity for the analysis of environmentally relevant samples. After extraction, 24% to 32% of the total U detected in the gills were present in the cytosolic fraction, in which the U distribution on the biomolecules (as a function of their MW and pI) varied depending on the exposure level. Finally, U target biomolecules mapping allowed us (i) to highlight a particular affinity of U for acidic and/or P-containing proteins and (ii) to identify 24 protein candidates for U binding.

[Truncated abstract] In Australia, there is pressure to reduce the amount of methane produced by ruminant livestock because they are the single largest source of methane emitted from anthropogenic sources, accounting for 70.7% of agricultural methane emissions. In addition, methane production represents a loss of gross energy intake to the animal. The organisms that are responsible for methane production in the animal gut are a distinct group of Archaea called methanogens. Methanogens occupy three different niches within the rumen. Some live freely in the rumen digesta (planktonic), others are attached to the outer surface of the rumen ciliates (ectosymbiotic), and some reside within the ciliates (endosymbiotic). The types and number of methanogens, as well as rumen ciliates and their symbiotic interactions, influence the amount of methane produced from the rumen. These factors in turn are affected by many factors, including diet and ruminal retention time. In this thesis, I tested the general hypothesis that increasing the amount of grain in the diet and reducing the retention time would affect the abundance and diversity of methanogens in their different niches, including their association with ruminal ciliates. Twenty-four fistulated sheep were used in a complete factorial design with the sheep randomly divided into four groups. ... The change in DGGE banding patterns and Shannon indices when sheep were fed grain indicated that the types of methanogens changed when sheep were fed low and high grain diets, but their diversity did not. In contrast, the diversity of rumen ciliates decreased when sheep were fed a high grain diet. A total of 18 bands from the DGGE analysis of the ciliates were sequenced. All except one, which was 98% similar to Cycloposthium sp. not found previously in the rumen, matched the sequences for previously identified rumen ciliates. Some of the rumen ciliates identified were not present in sheep fed the high grain diet. On a high grain diet, methanogens associate endosymbiotically with rumen ciliates to get better access to hydrogen. It appears that the association between methanogens and rumen ciliates is dictated by the availability of hydrogen in the rumen and not the generic composition of the ciliate population. Furthermore, endosymbiotic methanogens appear to produce less methane than methanogens in other niches. The pot scrubbers did not change ruminal retention time but they did reduce the acetate/propionate measurements observed in sheep on the high grain treatment. The reason why pot scrubbers had this effect remains unknown, but it is interesting to consider that some physical interaction has occurred between the pot scrubbers, the grain and the sheep that has improved the fermentation parameters in sheep fed a high grain diet. The results from this study have advanced our understanding of the interaction between methanogens and ruminal ciliates, and methanogenesis in the rumen in response to dietary changes and mechanical challenges. Extending this work to look more specifically at the species of methanogens that are most closely linked to high methane production and how they interact with the ruminal ciliates will be critical for manipulating enteric greenhouse gas emissions.

Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.

Thesis (MScFoodSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The regular consumption of probiotics is becoming a recognized trend in the food industry due to several reported health benefits. A probiotic is defined as a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance. A wide variety of probiotic food products are available on the South African market and comprise an assortment of fermented milks, as well as lyophilized preparations in tablet or capsule form. Strains of Lactobacillus acidophilus and Bifidobacterium species are mostly used as probiotic microbes in the industry due to their health enhancing effect. The survival of sensitive probiotic microbial species in food matrices are influenced by various factors such as oxygen concentration, pH levels and manufacturing and storage conditions. These should be considered and monitored as the South African food and health regulations stipulate that probiotic microbes should be present at a concentration of 10⁶ cfu.ml ̄ ¹' in order to exert a beneficial effect. Some health benefits are also correlated to specific microbial species and strains and these factors have resulted in the need for the rapid and accurate identification of probiotic microbes present in food products. The probiotic microbes present in probiotic yoghurts and supplements have in the past been identified using traditional methods such as growth on selective media, morphological, physiological and biochemical characteristics. However, even some of the most sophisticated cultural-dependant techniques are not always sufficient for the identification and classification of especially Bifidobacterium, as well as closely related Lactobacillus species. Molecular techniques are more often employed for the rapid and accurate detection, identification and characterization of microbial species present in food products. The aim of this study was to detect and identify the probiotic species present in various commercial South African yoghurts and lyophilized preparations using peR-based DGGE analysis. A 200 bp fragment of the V2-V3 region of the 16S rRNA gene was amplified and the peR fragments were resolved by DGGE. The unique fingerprints obtained for each product were compared to two reference markers A and B in order to identify the bands present. The results obtained were verified by species-specific peR, as well as sequence analyses of bands that could not be identified when compared to the reference markers. Only 54.5% of the South African probiotic yoghurts that were tested did contain all the microbial species as were mentioned on the labels of these products, compared to merely one third (33.3%) of the lyophilized probiotic food supplements. Some Bifidobacterium species were incorrectly identified according to some product labels, while other products contained various microbes that were not mentioned on the label. Sequence analysis confirmed the presence of a potential pathogenic Streptococcus species in one of the yoghurt products and in some instances the probiotic species claimed on the labels were non-scientific and misleading. The data obtained in this study showed that the various South African probiotic products tested were of poor quality and did not conform to the South African regulations. peR-based DGGE analysis proofed to be a valuable approach for the rapid and accurate detection and identification of the microbial species present in South African probiotic products. This could help with future implementation of quality control procedures in order to ensure a reliable and safe probiotic product to the consumer. / AFRIKAANSE OPSOMMING: Die gereelde inname van probiotiese produkte is besig om In erkende tendens in die voedselindustrie te word, as gevolg van verskeie gesondheidsvoordele wat daaraan gekoppel word. In Probiotika word gedefinieer as In voedingsaanvulling wat uit lewendige mikrobes bestaan en wat In voordelige effek op mens of dier het deur In optimale mikrobiese balans in die ingewande te handhaaf. In Wye verskeidenheid probiotiese voedselprodukte is tans beskikbaar op die Suid- Afrikaanse mark. Hierdie bestaan hoofsaaklik uit verskeie gefermenteerde melkprodukte asook 'n reeks tablette en kapsules wat probiotiese mikrobes in gevriesdroogde vorm bevat. Lactobacillus acidophilus tipes en Bifidobacterium spesies word die algemeenste in die voedselindustrie gebruik aangesien hierdie spesifieke mikrobes bekend is om goeie gesondheid te bevorder. Die oorlewing van sensitiewe probiotiese mikrobiese spesies in voedsel matrikse word beïnvloed deur faktore soos suurstof konsentrasie, pH-vlakke en vervaardigings- en opbergings kondisies. Hierdie faktore moet in aanmerking geneem word en verkieslik gemonitor word aangesien die Suid-Afrikaanse voedsel en gesondheids regulasies stipuleer dat probiotiese mikrobes teen In konsentrasie van 10⁶ kolonie vormende eenhede per ml teenwoordig moet wees om In voordelige effek te toon. Sommige gesondheidsvoordele word direk gekoppel aan spesifieke mikrobiese spesies en spesie-tipes. Hierdie faktore het gelei tot In groot aanvraag na vinnige en akkurate metodes vir die identifikasie van probioties mikrobes in voedselprodukte. Die probiotiese mikrobes teenwoordig in probiotiese joghurts en ook die gevriesdroogde vorms in tablette en kapsules, was al geïdentifiseer deur gebruik te maak van tradisionele metodes soos groei op selektiewe media, morfologiese, fisiologiese en biochemiese eienskappe. Selfs van die mees gesofistikeerde kultuur-afhanklike tegnieke is egter nie altyd voldoende vir die identifikasie en klassifikasie van veral Bifidobacterium en na-verwante Lactobacillus spesies nie. Molekulêre metodes word dikwels aangewend vir die vinnige en akkurate deteksie, identifikasie en karakterisering van mikrobes teenwoordig in voedselprodukte. Die doel van hierdie studie was om die probiotiese mikrobes teenwoordig in verskeie Suid-Afrikaanse joghurts en gevriesdroogde aanvullings, te identifiseer deur gebruik te maak van polimerase kettingreaksie (PKR)-gebaseerde denaturerende gradiënt jelelektroforese (DGGE) analise. 'n PKR fragment van 200 bp van die V2-V3 gedeelte van die 16S ribosomale RNS (rRNS) geen is geamplifiseer, en die PKR fragmente is geskei met behulp van DGGE. Die unieke vingerafdrukke wat verkry is vir elke produk is teen twee verwysings merkers A en B vegelyk om die bande teenwoordig in die profiele te identifiseer. Die resultate is bevestig deur spesies-spesifieke PKR en ook deur die ketting volgordes van die DNS fragmente te bepaal wat nie geïdentifiseer kon word deur vergelyking met die verwysings merkers nie. Slegs 54.5% van die Suid-Afrikaanse probiotiese joghurts wat getoets is het al die mikrobiese spesies bevat soos aangedui was op die etikette van hierdie produkte, teenoor slegs 'n derde (33.3%) van die gevriesdroogde voedingsaanvullings. Sekere Bifidobacterium spesies is verkeerd geïdentifiseer op sommige van die produk etikette, terwyl ander produkte verskeie mikrobes bevat het wat nie op die etiket aangedui was nie. 'n Potensiële patogeniese Streptococcus spesie is in een van die joghurt produkte gevind soos bevestig deur DNS kettingvolgorde bepalings. In sommige gevalle was die probiotiese spesienaam wat aangedui is op die etiket onwetenskaplik en misleidend. Die resultate wat uit hierdie studie verkry is dui aan dat die Suid-Afrikaanse probiotiese produkte wat getoets is van 'n swak gehalte is en nie aan die Suid- Afrikaanse regulasies voldoen nie. Daar is getoon dat PKR-gebaseerde DGGE analise 'n waardevolle tegniek kan wees vir die akkurate deteksie en identifisering van die mikrobiese spesies teenwoordig in probiotiese produkte. Dit kan help met die toekomstige implementering van kwaliteitskontrolerings prosedures om 'n mikrobiologiese betroubare en veilige produk aan die verbruiker te verseker.

Cette étude a consisté au développement et à l’utilisation de fonctions chimiques clivables en milieux biologiques. Dans le domaine de la protéomique chimique, ce travail a abouti à la conception d’une sonde d’enrichissement clivable en conditions non-dénaturantes. Appliquée à l’étude de topoisomérases, cette sonde a permis l’extraction et l’analyse de complexes fonctionnels A2B2 de gyrase. Dans un second temps, un nouveau concept de quencheur chimiquement désactivable a étéintroduit. Incorporé dans une sonde pro-fluorescente de type FRET, ce type de quencheur permet notamment de visualiser la présence de sondes non-activées dans des cellules. Enfin, une méthode a été développée pour permettre l’évaluation de la labilité d’une liaison chimique en milieux biologiques natifs. Basée sur l’utilisation de sondes pro-fluorescentes, cette méthodologie a plus particulièrement été appliquée à l’étude de la bio-labilité de groupements acido-labiles. / The general main topic of this work was the use and the development of cleavable linkers in biological systems. This study led to the design of a cleavable enrichment probe in non-denaturing conditions for chemical proteomic applications. In a topoisomerase analysis, this probe allowed the extraction and analysis of a functional DNA gyrase A2B2 complex. For fluorescence imaging, a new concept of chemically deactivatable quencher was introduced. This quencher was used to revealinactivated FRET-based probe in cell experiments. Finally, a methodology based on biolability measurements of acid-sensitive molecules was developed for the evaluation of chemical bond lability in native biological environments. This work was focused on biolability measurements of acidsensitive molecules.

Ziel der Arbeit war die Nutzbarmachung von Bioethanol zur Wandlung in Strom und Wärme in einer Hochtemperaturbrennstoffzelle. Dazu waren neben der Entwicklung eines langzeitstabilen, effektiven Katalysators zur Synthesegaserzeugung und dessen Testung sowie der Übertragung gewonnener Erkenntnisse auf in einem Reformer einsetzbare Konzepte die Verifizierung kommerzieller Katalysatorsysteme für die partielle Oxidation von Ethanol notwendig. Außerdem ist für die Entwicklung eines ethanolbetriebenen SOFC-Systems eine pulsations- und ablagerungsfreie Verdampfung von unvergälltem und vergälltem Ethanol – welche nicht Stand der Technik ist – erforderlich, für die ein geeignetes Verdampferkonzept entwickelt und getestet wurde. Experimentell konnte die Betreibbarkeit eines SOFC-Systems mit Ethanol an einem für den Betrieb mit LPG ausgelegten System nachgewiesen werden.

L’uranium (U) est naturellement présent à l’état de trace dans l’eau (µg.L-1), sa concentration pouvant atteindre localement quelques mg.L-1 du fait des activités anthropiques. Plusieurs études écotoxicologiques sur Danio rerio ont mis en évidence la toxicité, e.g. le stress oxydant, la génotoxicité mais aussi la reprotoxicité (i.e. moins de pontes et d’œufs pondus chez les poissons contaminés) de l’U dont les mécanismes ne sont pas connus.L’objectif de cette étude est de contribuer à la compréhension de la reprotoxicité de l’U par l’élucidation de mécanismes moléculaires perturbés après contamination. Pour cela, des investigations ont été menées sur les ovaires de poissons zèbre Danio rerio, reproduits (R) ou non (NR), après exposition par voie directe en condition de laboratoire à des concentrations représentatives d’environnements contaminés.Ce travail de thèse a été divisé en deux volets. Un premier volet analytique avait pour but la poursuite des développements de méthodes pour l’identification des complexes U-protéines en condition non dénaturante, autour du couplage de techniques de séparation (chromatographie d’exclusion stérique SEC, électrophorèse hors gel OGE) et de détection sensible par spectrométrie de masse élémentaire (ICP MS) et moléculaire (ESI MS). Le second volet a été dédié à l’étude de la reprotoxicité de l’U à l’échelle moléculaire, avec i) l’étude des complexes natifs U-protéines (approche métallomique), et ii) l’analyse différentielle de l’expression des protéines (approche protéomique).Les développements analytiques ont permis de garder le tampon physiologique et non dénaturant d’extraction pour l’étape de séparation OGE, améliorant le taux de recouvrement en U. En écotoxicologie, les principaux résultats montrent que l’ovaire est un organe accumulateur de l’U et que le statut de reproduction a une influence sur le niveau d’accumulation (R<NR). En revanche, cet état a peu d’influence sur sa distribution protéique pour laquelle 4 fractions (dont 1 principale) ont été identifiées, toutes contenant aussi du phosphore. L’identification des cibles potentielles de l’U et des protéines exprimées différentiellement (vtg, GST, GAPDH,…) a montré que les processus biologiques perturbés suite à la contamination sont de deux niveaux : 1/ générique (stress oxydant) et 2/ plus spécifique de la gonade (développement et maturation des ovocytes). En conclusion, ces deux approches complémentaires ont permis de mettre en évidence un effet direct (complexation) et indirect (expression protéique modulée) de l’U, et de proposer l’hypothèse d’un défaut de maturation des ovocytes après contamination. Ce défaut pourrait impacter le développement embryonnaire et in fine expliquer la reprotoxicité observée lors d’études écotoxicologiques précédentes. / Uranium (U) is naturally presents at trace level (µg.L-1) in aquatic environment; its concentration can increase up to a few mg.L-1 due to human activities. Several ecotoxicological studies have shown uranium toxicity in contaminated zebrafishes Danio rerio, e.g. oxidative stress, genotoxicity and reprotoxicity (i.e. lower number of spawn and eggs laid) but mechanisms are not well known.The objective of this study is to contribute to the understanding of uranium reprotoxicity by elucidating the disrupted molecular mechanisms after contamination. Therefore, investigations have been carried out on ovaries from reproduced (R) and non-reproduced (NR) zebrafishes after waterborne exposure in laboratory conditions at environmentally relevant concentrations.This project was divided into two parts. Firstly, analytical investigations were carried out to continue the development of non-denaturing methods for U-protein identification by coupling separative techniques (size exclusion chromatography SEC, off gel electrophoresis OGE) with elemental (ICP MS) and molecular (ESI MS) sensitive mass spectrometry detection. Secondly, studies of U reprotoxicity were investigated by studying i) native U-protein complexes (metallomics approach) and ii) differential analysis of protein expression (proteomics approach)Analytical developments allowed keeping the physiological and non-denaturing extraction buffer for OGE separation step, improving U recovery. In ecotoxicology, the major results showed that ovary is an U accumulating organ and that the reproduction status modifies the accumulation level (R<NR). However, this status is of little influence on its distribution on proteins with 4 fractions (including a major one) determined, all of them coeluting with phosphorus. The identification of U potential targets and of protein expression differences (vtg, GST, GAPDH…) showed that biological processes disrupted after contamination are at two levels: 1/ generic (oxidative stress) and 2/ more specific to gonad (oocyte development and maturation).As a conclusion, these two complementary approaches showed a direct (complexation) and indirect (modification of protein expression) effects of U, and enabled to hypothesize a lack of oocyte maturation after contamination. This defect could impact embryo development and in fine explain the reprotoxicity observed in previous ecotoxicological studies.

In current days, examination of circulating tumor DNA (ctDNA) finds new use across different cancers. It is directed at tumor-derived short fragments of DNA present in peripheral blood of patiens (mainly in advanced stages). Due to its minimal invasivity, almost 100 % specificity and relatively high sensitivity in stage IV patients, this approch found its main potential clinical utility especially in early detection of disease relapse or progression after tumor resection (i.e. post-operative follow-up), prediction and monitoring of therapy response and estimation of prognosis. As a result of minute levels of ctDNA on a high background of other non-tumor DNA fragments present in plasma, a suitable method exhibiting highest sensitivity is the key for proper detection of this marker. The approach is predominantly based on initial identification of a mutation in tumor tissue and its subsequent detection in plasma. The present work is aimed at optimization of ctDNA isolation and method of its detection based on PCR amplification followed by heteroduplex analysis by denaturing capillary electrophoresis (DCE) to achieve highest sensitivity for detection of mutated fraction in plasma sample. I have applied the optimized protocol to examine ctDNA in three types of cancers, namely colorectal cancer (122...

科学研究費補助金 研究種目:基盤研究(C)(2) 課題番号:14560051 研究代表者:浅川 晋 研究期間:2002-2003年度