[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.

<p>The objective of this thesis was to investigate and characterize the interaction between blood proteins and different surfaces with emphasis on protein adsorption to bioceramics and model surfaces. Special effort was made to monitor the spontaneous and selective adsorption of proteins from human plasma and to examine the orientation, conformation and functional behavior of single proteins after adsorption. </p><p>Five different ceramic biomaterials: alumina (Al₂O₃), zirconia (ZrO₂), hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂) and two glass-ceramics, AP40 (SiO₂-CaO-Na₂O-P₂O₅-MgO-K₂O-CaF₂) and RKKP (AP40 with Ta₂O₃-La₂O₃), were exposed to human plasma and their protein binding capacities and affinities for specific proteins were studied by chromatography, protein assays, two-dimensional gel electrophoresis and Western blotting. The studies showed that all materials adsorbed approximately the same high amount of plasma proteins and that they therefore should be fully covered by proteins in an <i>in vivo</i> setting. The adsorbed proteins were different for most materials which could explain their previously observed different levels of tissue integration <i>in vivo</i>. </p><p>Four of the proteins that behaved differently, ceruloplasmin, prothrombin, α₂-HS-glycoprotein and α₁-antichymotrypsin, were selected for characterization with atomic force microscopy and ellipsometry. The studies, which were performed on ultraflat silicon wafers (silica), showed that the proteins oriented themselves with their long axis parallel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface. All of them had globular shapes but other conformational details were not resolved. Furthermore, prothrombin (none of the others) formed multilayers at high proteins concentrations. </p><p>The functional behaviour of the adsorbed proteins, referring to their cell binding and cell spreading capacity on silica and a positive cell adhesion reference surface (Thermanox®), was affected by the underlying substrate. Ceruloplasmin, α₂-HS-glycoprotein and α₁-antichymotrypsin stimulated cell attachment to silica, but suppressed attachment to Thermanox®. Prothrombin stimulated cell attachment to both surfaces. The attachment was in most cases mediated both by cell membrane-receptors (integrins) and by non-specific interactions between the cell and the material. </p><p>This thesis showed that the compositional mixture, orientation, conformation and functional behavior of the adsorbed proteins are determined by the properties of the underlying surface and if these parameters are controlled very different cellular responses can be induced.</p>

The objective of this thesis was to investigate and characterize the interaction between blood proteins and different surfaces with emphasis on protein adsorption to bioceramics and model surfaces. Special effort was made to monitor the spontaneous and selective adsorption of proteins from human plasma and to examine the orientation, conformation and functional behavior of single proteins after adsorption. Five different ceramic biomaterials: alumina (Al2O3), zirconia (ZrO2), hydroxyapatite (Ca10(PO4)6(OH)2) and two glass-ceramics, AP40 (SiO2-CaO-Na2O-P2O5-MgO-K2O-CaF2) and RKKP (AP40 with Ta2O3-La2O3), were exposed to human plasma and their protein binding capacities and affinities for specific proteins were studied by chromatography, protein assays, two-dimensional gel electrophoresis and Western blotting. The studies showed that all materials adsorbed approximately the same high amount of plasma proteins and that they therefore should be fully covered by proteins in an in vivo setting. The adsorbed proteins were different for most materials which could explain their previously observed different levels of tissue integration in vivo. Four of the proteins that behaved differently, ceruloplasmin, prothrombin, α2-HS-glycoprotein and α1-antichymotrypsin, were selected for characterization with atomic force microscopy and ellipsometry. The studies, which were performed on ultraflat silicon wafers (silica), showed that the proteins oriented themselves with their long axis parallel to the surface or as in case of ceruloplasmin with one of its larger sides towards the surface. All of them had globular shapes but other conformational details were not resolved. Furthermore, prothrombin (none of the others) formed multilayers at high proteins concentrations. The functional behaviour of the adsorbed proteins, referring to their cell binding and cell spreading capacity on silica and a positive cell adhesion reference surface (Thermanox®), was affected by the underlying substrate. Ceruloplasmin, α2-HS-glycoprotein and α1-antichymotrypsin stimulated cell attachment to silica, but suppressed attachment to Thermanox®. Prothrombin stimulated cell attachment to both surfaces. The attachment was in most cases mediated both by cell membrane-receptors (integrins) and by non-specific interactions between the cell and the material. This thesis showed that the compositional mixture, orientation, conformation and functional behavior of the adsorbed proteins are determined by the properties of the underlying surface and if these parameters are controlled very different cellular responses can be induced.

Today, assessment of the health of coastal waters is recognized as being important for both the conservation of nature and well-being of humans. Anthropogenic pollution has been the focus of extensive research for some time and a variety of programs for the monitoring and assessment of environmental pollution have been developed. Determination of the levels of pollution in sensitive ‘sentinels’ such as mussels, allows monitoring of these levels in a given area over a prolonged period of time. Furthermore, the biological effects of pollution are reflected in a series of biomarkers, none of which provides a general picture of the sentinel’s state of health and all of which are individually specific for certain pollutants and influenced by both biotic and abiotic factors. In an attempt to improve biomonitoring of marine pollution, we have developed two proteomic approaches here. In the first portion of the thesis, a proteomic analysis was performed on peroxisomes isolated from mussels exposed either to one of three model anthropogenic pollutants, or two different types of crude oil, or from mussels exposed to the Prestige oil spill. Application of two-dimensional electrophoresis (2-DE) provided protein expression signatures (PES) for exposure to these different pollutants.Furthermore, several individual protein components of these PES could be putatively identified. In the second portion of this work, such analysis of subproteomes was developed further in order to improve the applicability of this approach to biomonitoring. A simple fractionation procedure in combination with liquid chromatography and 2-DE provided samples from mussels residing in different regions of a pollution gradient around the harbor of Gothenburg, as well as from mussels exposed to two types of fuel oil similar to that of the Prestige that were suitable for environmental proteomics. In addition, we constructed a model for this approach that can be cross-validated in the future and applied to assess sources of fuel oil pollution in connection with biomonitoring programs.

Η μεσογειακή μύγα αποτελεί το κύριο παράσιτο πολλών καλλιεργούμενων φρούτων προκαλώντας ετησίως μεγάλες καταστροφές σε γεωργικές καλλιέργειες. Η μελέτη του εντόμου τόσο σε γενετικό όσο και σε πληθυσμιακό επίπεδο μπορεί να συμβάλλει σημαντικά στην ανάπτυξη ή και τη βελτίωση αποτελεσματικών και φιλικών προς το περιβάλλον μεθόδων ελέγχου. Οι μικροδορυφόροι είναι απλές επαναλήψεις ενός νουκλεοτιδικού μοτίβου που αποτελείται 1-6 ζεύγη βάσεων. Αποτελούν πολύ χρήσιμους γενετικούς δείκτες διότι είναι άφθονοι και διάσπαρτοι στο γονιδίωμα των ευκαρυωτκών οργανισμών. Επιπλέον είναι υψηλά πολυμορφικοί, κληρονομούνται ως συνυπερέχοντες Μεντελικοί δείκτες και αναλύονται εύκολα μέσω PCR, χαρακτηριστικά που τους καθιστούν πολύτιμα εργαλεία για πληθυσμιακές και εξελικτικές μελέτες. Από τους μικροδορυφορικούς δείκτες που αναπτύχθηκαν στο εργαστήριό μας, επιλέχθηκαν 10 με βάση το βαθμό πολυμορφισμού που έδειξαν σε εργαστηριακά στελέχη. Οι δείκτες αυτοί χρησιμοποιήθηκαν στην ανάλυση 481 ενηλίκων ατόμων που προέρχονταν από 19 διαφορετικά δείγματα φρούτων που συλλέχθηκαν από εννέα διαφορετικές περιοχές της Δυτικής Ελλάδας και της Βόρειας Πελλοπονήσου. Η γενοτυπική ανάλυση πραγματοποιήθηκε με ηλεκτροφόρηση των προϊόντων της PCR για κάθε γενετικό δείκτη σε πήκτωμα πολυακρυλαμιδίου και επακόλουθη αυτοραδιογραφία. Η στατιστική επεξεργασία των αποτελεσμάτων, με τη βοήθεια υπολογιστικών προγραμμάτων αποκάλυψε σημαντική γενετική διαφοροποίηση μεταξύ των δειγμάτων, που αποδίδεται εκτός των άλλων παραγόντων (κλιματολογικές συνθήκες, γεωγραφική προέλευση) στο είδος του ξενιστή. / C. capitata, is the main pest of many cultivable fruits and responsible for a significant loss in annual products, resulting in great economic damage. Studies on the genetic and population analysis will make a contribution towards the development or the improvement of environmental friendly control methods. Microsatellites are tandem simple sequence repeats of short (1-6) nucleotide motifs. They are very important genetic markers because they are dispersed and abundant in most eukaryotic genomes. They are highly polymorphic, inherited as co-dominant Mendelian markers and easily scored by PCR. Consequently, they have become one of the most popular molecular markers with application in many genetic studies, including genetic analysis of natural populations and evolutionary studies. From the available microsatellites in our laboratory, were selected ten (10), based on their polymorphism in laboratory strains. They were used for the screening of 481 adult flies in the medfly, collected from nineteen (19) different samples of fruits from nine (9) different areas in west Greece and north Peloponnesus. Analysis of genotype composition in the samples was achieved by polyacrylamide electrophoresis of the PCR products, for every genetic marker and then autoradiography. The statistic analysis of our results using software programs, revealed an important genetic differentiation in samples. Except for many factors (climatic conditions, geographic origin), the host origin will be responsible for this genetic differentiation.

No description available.

No description available.

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.

Casein is the major protein fraction in milk, and its cross-linking has been a topic of scientific interest for many years. Enzymatic cross-linking has huge potential to modify relevant techno-functional properties of casein, whereas non-enzymatic cross-linking occurs naturally during the storage and processing of milk and dairy products. Two size separation techniques were applied for characterisation of these reactions: gel electrophoresis and size exclusion chromatography. This review summarises their separation principles and discusses the outcome of studies on cross-linked casein from the last ~20 years. Both methods, however, show limitations concerning separation range and are applied mainly under denaturing and reducing conditions. In contrast, field flow fractionation has a broad separation range and can be easily applied under native conditions. Although this method has become a powerful tool in polymer and nanoparticle analysis and was used in few studies on casein micelles, it has not yet been applied to investigate cross-linked casein. Finally, the principles and requirements for absolute molar mass determination are reviewed, which will be of increased interest in the future since suitable calibration substances for casein polymers are scarce.