Tesis: "Denaturing gradient gel electrophoresis"

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Cadiou, Helene. "Evaluation of denaturing gradient gel electrophoresis (DGGE) as a method of mutation detection." Thesis, University College London (University of London), 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418093.

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Le, Riche Mia. "Lipoprotein X : biochemical predictors and detection by non-denaturing polyacrylamide gradient gel electrophoresis." Thesis, Stellenbosch : Stellenbosch University, 2007. http://hdl.handle.net/10019.1/18218.

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Assignment (MMed)--University of Stellenbosch, 2007. ENGLISH ABSTRACT: Lipoprotein X (LpX) is an abnormal cholesterol-containing particle that may be present in the serum of subjects with cholestasis, lecithin:cholesterol acyltransferase (LCAT) deficiency and parenteral nutrition. The biochemistry, metabolism, clinical significance and laboratory analysis of LpX is discussed in this study. This laboratory-based project investigated icteric samples received at the Chemical Pathology laboratory, Tygerberg Hospital, for serum predictors of LpX and the use of a modified non-denaturing polyacrylamide gradient gel electrophoresis system in the detection of LpX. The study showed that the non-denaturing polyacrylamide gradient gel electrophoresis system (2-8%) is a useful test in demonstrating LpX in icteric plasma and has potential for a screening test in LCAT deficiency. Serum concentration of conjugated bilirubin, alkaline phosphatase, gamma glutamyltransferase, free cholesterol, phospholipid, free cholesterol: total cholesterol ratio and conjugated bilirubin: total bilirubin ratio are all good predictors of LpX. The ratio of free cholesterol to total cholesterol (FC/TC > 0.6) was the best predictor of LpX. In the setting of obstructive liver disease LpX is seen in 66% of patients if total cholesterol is > 7.5 mmol/L. AFRIKAANSE OPSOMMING: Lipoproteien X (LpX) is ‘n abnormale cholesterol-bevattende partikel wat teenwoordig mag wees in die serum van persone met cholestase, lesitien:cholesterol asieltransferase (LCAT) gebrek en parenterale voeding. Die biochemie, metabolisme, kliniese belang en laboratorium analise van LpX word bespreek in hierdie werkstuk. Hierdie laboratorium-gebaseerde projek het geelsugtige monsters ondersoek wat ontvang is by die Chemiese Patologie laboratorium, Tygerberg Hospitaal, vir serum voorspellers van LpX en die gebruik van ‘n gemodifiseerde nie-denaturerende polie-akrielamied gradiënt gel elektroforese sisteem in die demonstrasie van LpX. Die bevindinge was dat die nie-denaturerende polie-akrielamied gradient gel elektroforese sisteem (2-8%) is ‘n nuttige toets om LpX te demonstreer in geelsugtige plasma en het potensiaal as ‘n siftingstoets in LCAT gebrek. Serum konsentrasie van gekonjugeerde bilirubien, alkaliese fosfatase, gamma glutamieltransferase, vry cholesterol, fosfolipied, vry cholesterol:totale cholesterol verhouding en gekonjugeerde bilirubien:totale bilirubien verhouding is alles goeie voorspellers van LpX. Die verhouding van vry cholesterol tot totale cholesterol (VC/TC > 0.6) was die beste voorspeller van LpX. In gevalle van obstruktiewe lewersiekte word LpX gesien in 66% van pasiente as die totale cholesterol meer as 7.5 mmol/l is.

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Surridge, Angela Karen Joanna. "Denaturing gradient gel electrophoresis characterisation of microbial communities in polycyclic aromatic hydrocarbon and polychlorinated biphenyl contaminated soil." Thesis, Pretoria : [s.n.], 2007. http://hdl.handle.net/2263/25070.

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Fossil fuels are currently the primary industrial energy source on Earth. They are principally composed of complex hydrocarbons in either long-chain or cyclic conformation. Industrial use of petroleum, diesel, oil, tar and other coal-derived products inevitably leads to pollution of the environment. The most serious pollution is caused by polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) that are not easily removed from soil after a spill. Long-chain and cyclic conformation makes fossil fuel hydrocarbons difficult to break down. However, certain free-living soil microorganisms have adapted to utilising these PAHs/PCBs as a source of energy. In many cases, their efficacy is greatly enhanced by the presence of plants. By inhabiting the rhizosphere, microbes form a mutualistic relationship with the plant, receiving nutrients from it and in return providing a less polluted environment in which the plant can grow. The purpose of this study was to elucidate some of the microbial population diversity in PAH/PCB-polluted soils in South Africa through the use of denaturing gradient gel electrophoresis (DGGE). In an initial study, DGGE was employed to separate soil communities in polluted and unpolluted soils into a genetic fingerprint, the main bands of which were sequenced and subjected to a BLAST analysis through a database for possible identification of species present. Phylogenetic and distance studies indicated that unpolluted soils have a far greater species diversity. It thus was evident that PAH/PCB pollution of soil leads to a decrease in microbial diversity by selecting for microorganisms with the ability to activate metabolic pathways allowing them to utilise the pollutants as an alternative source of carbon. Population diversity of pro- and eukaryotes found within polluted and non-polluted soils was compared. DGGE was employed to determine the genetic fingerprint of each population. Following this, dendogram analyses based on Shannon indices were done to determine PAH breakdown potential of prokaryotic vs. eukaryotic communities. A higher diversity and better adaptation potential were evident within prokaryotic than eukaryotic communities in pollution-stressed environments, indicating that the prokaryotic component of these samples had the greatest PAH-metabolism potential. To determine the capacity for PAH/PCB metabolism by the organisms within the soil samples being studied, the presence of xylE and ndoB genes, responsible for toluene/xylene and naphthalene biodegradation, respectively, was determined. DGGE was performed to analyse genetic diversity between these two genes, based on community fingerprints. Polluted soil communities tended to have comparable community diversity within their functional genes, depending on their physical situation, plant species proximity and soil conditions. In general, soil contained indigenous microbes with a high natural potential for biodegradation of PAHs/PCBs. A portion of the 16S gene of eight bacterial isolates representing the most dominant culturable taxa in the polluted soils was sequenced and analysed for identification purposes. These identifications were conducted in conjunction with the use of the catabolic gene probes xylE and ndoB to establish the hydrocarbon degrading capacity of the isolates. Pseudomonas, from the rhizosphere of Cyperus esculentus, was the most common PAH-degrading genus found in this study. Considering the well-established rhizosphere competence and PAH-degrading capacity of Pseudomonas, this genus seems to be the best suited for bioaugmentation purposes in South Africa. The presence of the nifH gene, the general marker gene of nitrogen-fixing bacteria in communities from unpolluted and polluted soils, was determined. It was hypothesised that bioremediation could be enhanced by nitrogen addition to polluted environments. Nested-PCR of the nifH gene was conducted on a diagnostic basis and was followed by DGGE of the product to determine the functional gene diversity within pollution-dwelling, nitrogen-fixing bacterial communities. Nitrogen-fixing microorganisms were present in all the soils sampled but, in only 80% of the pure cultures isolated from polluted and unpolluted soils and rhizospheres. Although different rhizospheres and pollutants were examined, it was found that of the polluted soils studied, most nifH gene diversity of polluted soils existed within machinery oil polluted, wood chip mulched, non-rhizosphere soil. Thus, it would appear that the more polluted the soil the higher the free microbe nitrogen fixation diversity possibly due to environmental stress. Thesis (PhD (Microbiology))--University of Pretoria, 2007. Microbiology and Plant Pathology unrestricted

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Ellwood-Thompson, Rhianedd Eleri. "Occurrence and transmission of Wolbachia endosymbionts in the oak gall wasp community : application of denaturing gradient gel electrophoresis." Thesis, Cardiff University, 2004. http://orca.cf.ac.uk/55379/.

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Eight Wolbachia variants were identified in the wasp community. Identical Wolbachia variants were identified in inquiline and parasitoid wasp species suggesting that horizontal transmission of Wolbachia occurs in this community

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Strandgren, Charlotte. "Studies of the Diversity of Lactobacillus spp. in Fecal Samples Using PCR and Denaturing Gradient Gel Electrophoresis." Thesis, Linköpings universitet, Institutionen för klinisk och experimentell medicin, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-15560.

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Allergic diseases, for example asthma and eczema, are nowadays considered belonging to the most common chronic diseases amongst children in the West, but the cause for this increase in allergy prevalence is unknown. Since studies have indicated a connection between children's exposure of microorganisms during infancy and risk of developing allergic disease, it is suggested that this exposure is a crucial factor in question of allergy development or not. Other studies have established differences in microflora composition between healthy children and children with allergic disease, and several studies have shown that probiotic therapy can give positive results in both prevention and treatment of allergic diseases. The aim of this master's thesis was to develop a method, using PCR and denaturing gradient gel electrophoresis, to study the diversity of Lactobacillus spp. in fecal samples retrieved from a study of the probiotic strain L. reuteri ATCC 55730. The developed method was successful in detecting lactobacilli in fecal samples, but three other bacterial genera commonly found in humans were also amplified. Comparison of average numbers of detected bacterial strains and lactobacilli strains between samples belonging to the probiotics and placebo groups, respectively, showed higher numbers for the probiotics group. Also, the only fecal samples that contained L. reuteri belonged to the probiotics group. Although the results are far from statistically significant, they support the theories that probiotics may influence the intestinal microbiota.

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Keyser, Maricel. "PCR detection, denaturing gradient gel electrophoresis (DGGE) fingerprinting and identification of the microbial consortium in different types of UASB granules." Thesis, Link to online version, 2006. http://hdl.handle.net/10019/558.

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Venter, Leandra. "Presence of potentially pathogenic heterotrophic plate count (HPC) bacteria occurring in a drinking water distribution system in the North-West Province, South Africa / by Leandra Venter." Thesis, North-West University, 2010. http://hdl.handle.net/10394/4380.

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There is currently growing concern about the presence of heterotrophic plate count (HPC) bacteria in drinking water. These HPC may have potential pathogenic features, enabling them to cause disease. It is especially alarming amongst individuals with a weakened immune system. South Africa, the country with the highest incidents of HIV positive individuals in the world, mainly uses these counts to assess the quality of drinking water in terms of the number of micro-organisms present in the water. These micro-organisms may be present in the bulk water or as biofilms adhered to the surfaces of a drinking water distribution system. The current study investigated the pathogenic potential of HPC bacteria occurring as biofilms within a drinking water distribution system and determined the possible presence of these micro-organims within the bulk water. Biofilm samples were taken from five sites within a drinking water distribution system. Fifty six bacterial colonies were selected based on morphotypes and isolated for the screening of potential pathogenic features. Haemolysin production was tested for using sheep-blood agar plates. Of the 56, 31 isolates were ?-haemolytic. Among the 31 ?-haemolytic positive isolates 87.1% were positive for lecithinase, 41.9% for proteinase, 19.4% for chondroitinase, 9.7% for DNase and 6.5% for hyaluronidase. All of the ?-haemolytic isolates were resistant to oxytetracycline 30 ?g, trimethoprim 2.5 ?g and penicillin G10 units, 96.8% were resistant to vancomycin 30 ?g and ampicillin 10 ?g, 93.5% to kanamycin 30 ?g, 74.2% to chloramphenicol 30 ?g, 54.8% to ciprofloxacin 5 ?g, 22.6% to streptomycin 300 ?g and 16.1% to erythromycin 15 ?g. Nineteen isolates producing two or more enzymes were subjected to Gram staining. The nineteen isolates were all Gram-positive. These isolates were then identified using the BD BBL CRYSTALTM Gram-positive (GP) identification (ID) system. Isolates were identified as Bacillus cereus, Bacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Bacillus pumilus and Kocuria rosea. 16S rRNA gene sequencing was performed to confirm these results and to obtain identifications for the bacteria not identified with the BD BBL CRYSTALTM GP ID system. Additionally identified bacteria included Bacillus thuringiensis, Arthrobacter oxydans and Exiguobacterium acetylicum. Morphological properties of the different species were studied with transmission electron microscopy (TEM) to confirm sequencing results. All the isolates displayed rod shaped cells with the exception of Arthrobacter oxydans being spherical in the stationary phase of their life cycle. Bulk water samples were taken at two sites in close proximity with the biofilm sampling sites. The DNA was extracted directly from the water samples and the 16S rRNA gene region was amplified. Denaturing gradient gel electrophoresis (DGGE) was performed to confirm the presence of the isolates from the biofilm samples in the bulk water samples. The presence of Bacillus pumilus and Arthrobacter oxydans could be confirmed with DGGE. This study demonstrated the presence of potentially pathogenic HPC bacteria within biofilms in a drinking water distribution system. It also confirmed the probable presence of two of these biofilm based bacteria in the bulk water. Thesis (M.Sc. (Microbiology))--North-West University, Potchefstroom Campus, 2010.

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Lindelof, Kara L. "Contribution of Biosolids-derived Bioaerosols to the Airborne Microbial Population." University of Toledo / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1302299544.

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PRUDEN, AMY J. "BIODEGRADATION OF METHYL TERT -BUTYL ETHER." University of Cincinnati / OhioLINK, 2002. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1027943573.

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Van, Niekerk Bertina Freda. "Functional and structural diversity of the microbial communities associated with the use of Fischer–Tropsch GTL Primary Column Bottoms as process cooling water / van Niekerk B.F." Thesis, North-West University, 2011. http://hdl.handle.net/10394/7284.

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Despite emerging water shortages, most water is only used once, and often with low efficiency. However, with appropriate treatment, water can be re–used to reduce the demand on freshwater sources. The Department of Water Affairs, South Africa, promotes industries to reduce discharges into water resources in order to sustain an overall good water quality of all water systems. All of this ultimately leads to industries striving towards zero effluent discharge. Primary Column Bottoms (PCBs) is a wastewater stream derived from the Fischer–Tropsch Gas to Liquid process and consists mainly of organic acids, but no nitrogen or phosphorous, which by implication excludes possible biodegradation. In the operation of cooling towers in industrial processes, cooling water quality has a direct impact on the cooling performance of the system, where nutrient levels may affect fouling, scaling and corrosion observed in the cooling towers. Fouling, scaling and corrosion affect the operating efficiency of cooling water systems and may necessitate the addition of chemical agents to control these phenomena. This has a financial and labour time impact on the operation of these systems. In this study a mini cooling tower test rig was operated with a synthetic PCB effluent as cooling water and various cycles of concentration, pH and linear flow velocities (LFVs). A constant delta temperature of 10 °C was maintained. Cycles of concentration (COC) evaluated included 2, 4 and 6 cycles of concentration and linear flow velocities evaluated was 0.6 m/s, 0.9 m/s and 1.2 m/s. Fouling, scaling and corrosion rates were determined using corrosion coupons and heat exchanger tubes for mild steel and stainless steel. Besides the evaluation of the various operational parameters for fouling, scaling and corrosion, the possibility for chemical oxygen demand (COD) removal by operating the cooling tower as a bioreactor was also evaluated. To this end nutrient correction was applied to the reactor to allow for a CNP ratio of 100:10:1. With regard to fouling, scaling and corrosion, mild steel was more affected by fouling, scaling and corrosion compared to stainless steel where almost no fouling, scaling and corrosion was observed. Overall increased linear flow velocities resulted in higher fouling and scaling rates, whereas lower linear flow velocities resulted in decreased corrosion rates. In terms of cycles of concentration, increased COC resulted in higher fouling, scaling and corrosion rates. Despite the high nutrient removal levels, the accompanying fouling, scaling and corrosion was still below the particular industry’s guidelines. Besides physical–chemical evaluation of the towers under the various operational conditions, culture–dependent and culture–independent methods were also employed. Concerning culture–dependent approaches the study demonstrated that aerobic and anaerobic organisms are present in both the planktonic and sessile phase of the cooling tower reactors. Heterotrophic aerobes were found to be the most abundant under all the operating conditions. Sulphate reducing bacteria were more abundant in the sessile phase of the cooling towers, and the presence of high sulphate levels in the experiments could be indicative of the sulphate reducing bacteria actively participating in the microbial community. Lower than expected corrosion levels, however, suggest that a combination of the organisms in the biofilm rather than sulphate reducing bacteria alone, contributed to the corrosion rates observed. Culture–independent methods, specifically phospholipid fatty acid analysis supported the results from the culture–dependent methods. Furthermore results demonstrated that linear flow velocity had a greater effect on the community structure than cycles of concentration. Finally molecular methods, specifically denaturing gradient gel electrophoresis, found that increasing cycles of concentration resulted in increased microbial community diversity, while increasing linear flow velocity resulted in decreased microbial community diversity. Regarding COD removal, nutrient correction of the synthetic PCB effluent achieved 89.35 % COD removal at 2 COC and 1.2 m/s LFV, while 80.85 % COD removal was achieved at 4 COC at 1.2 m/s LFV. From these results it was recommended that the operation of the cooling tower should be at 4 COC and 1.2 m/s, which despite slightly lower % COD removal, were characterised by fouling, scaling and corrosion rates well within guidelines. Thesis (M. Environmental Science)--North-West University, Potchefstroom Campus, 2012.

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Le, Nguyen Doan Duy. "Détermination de l'origine géographique des poissons par l'obtention de l'empreinte génétique de leur communauté bactérienne par PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis)." Montpellier 2, 2008. http://www.theses.fr/2008MON20103.

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La détermination de l'origine géographique est une des exigences de la traçabilité des produits alimentaires. Notre hypothèse était que l'environnement de l'aliment pouvait avoir une influence sur la diversité des flores bactériennes commensales des poissons. L'origine géographique des poissons a donc été approchée par la caractérisation de la flore microbienne spécifique de la ferme de production ou du lieu de transformation et de stockage. Une technique de biologie moléculaire couplée à une analyse d'images et à des méthodes statistiques a été développée afin de relier les profils bactériens obtenus par PCR-DGGE à l'origine géographique des fermes aquacoles. Dans la première partie, nous avons différencié par DGGE des poissons d'espèces différentes qui venaient de sites différents: les bars de France, les poissons-chats Pangasius du Vietnam et du Cambodge. Les profils DGGE microbiens des poissons et de l'eau d'un même étang sont étroitement similaires, confirmant notre hypothèse de départ. Le séquençage des bandes issues de la DGGE a permis d'identifier les souches bactériennes dominantes des poissons-chats au Vietnam. Dans la deuxième partie, nous avons confirmé la pérennité des profils microbiens au cours de la saison des pluies et de la saison sèche au Vietnam. Les poissons péchés en mer ont un profil bactérien variable alors que la même espèce élevée en aquarium obtient un profil similaire pour tous les poissons. Les traitements de transformation tels que le séchage et le marinage ont une forte influence sur la communauté bactérienne des poissons. La congélation ou la réfrigération ne modifient pas fortement les profils DGGE. La persistance des marqueurs bactériens au cours des traitements technologiques a été vérifiée au niveau industriel dans une usine de transformation en filets des poissons-chats Pangasius qui sont les principaux produits exportés du Vietnam. L'écologie bactérienne des poissons a été suivie par PCR-DGGE à chaque étape de leur transformation montrant que le profil bactérien entre le produit initial et le produit fini est différent mais conserve suffisamment d'information pour discriminer l'origine. Dans la dernière partie, nous avons eu recours à la Rep-PCR pour étudier la diversité des souches de Pseudomonas qui ont montré une persistance aux procédures de transformation. Les Pseudomonas restent présents sur des filets finis ainsi que sur la surface des équipements après le nettoyage et la désinfection. 27 souches de Pseudomonas ont été classées dans 7 grandes groupes par rep-PCR et identifiées par séquençage The determination of geographical origin is one of the inquiries of the food traceability system. Our hypothesis is that the environment could have an influence on the diversity of the bacterial communities of fish. The geographical origin of fish can be determined by the characterisation of the specific bacterial flora of the aquaculture farm or of the production and storage sites. A biological molecular technique PCR-DGGE, coupled to image analysis and statistic methods, have been developed in order to link the bacterial profiles with the geographical origin of the aquaculture farms. In the first chapter, we have distinguished by DDGE the different fish species from different sites: sea bass from France, Pangasius catfish from Vietnam and from Cambodia. The DGGE profiles of the fish bacteria and the water in the same pond were narrowly similar, confirming our hypothesis. The sequencing of the bands excised from DGGE gel permitted to identify the dominant bacterial species in the catfish from Vietnam. In the second chapter, we confirmed the perenniality of the microbial profiles during the rainy and the dry season in Vietnam. The fish captured in the sea had variable bacterial profiles, while the same fish raised in aquarium had a similar profile for all of them. Drying and pickling had a strong effect on the fish bacterial communities. Freezing or chilling did not change strongly the DGGE profiles. The persistence of the bacterial markers during the technical treatment has been verified at industrial scale in a factory processing Pangasius fillets, which is the principal exported product of Vietnam. The microbial ecology of the fish has been followed by PCR-DGGE for each processing step showing that the bacterial profiles of the initial product and the final product were different but preserved enough information to discriminate the origin. In the last chapter, Rep-PCR was used to study the diversity of Pseudomonas strains which showed persistence to the processing procedure. The Pseudomonas strains remained on the final fillets as well as on the equipment surfaces after cleaning and disinfection. 27 Pseudomonas strains classified in 7 big groups have been identified by sequencing

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Du, Plessis Gerda. "Actinobacterial diversity of the Ethiopian Rift Valley lakes." University of the Western Cape, 2011. http://hdl.handle.net/11394/5385.

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>Magister Scientiae - MSc The class Actinobacteria consists of a heterogeneous group of filamentous, Gram-positive bacteria that colonise most terrestrial and aquatic environments. The industrial and biotechnological importance of the secondary metabolites produced by members of this class has propelled it into the forefront of metagenomics studies. The Ethiopian Rift Valley lakes are characterized by several physical extremes, making it a polyextremophilic environment and a possible untapped source of novel actinobacterial species. The aims of the current study were to identify and compare the eubacterial diversity between three geographically divided soda lakes within the ERV focusing on the actinobacterial subpopulation. This was done by means of a culture-dependent (classical culturing) and culture-independent (DGGE and ARDRA) approach. The results indicate that the eubacterial 16S rRNA gene libraries were similar in composition with a predominance of α-Proteobacteria and Firmicutes in all three lakes. Conversely, the actinobacterial 16S rRNA gene libraries were significantly different and could be used to distinguish between sites. The actinobacterial OTUs detected belonged to both the Rubrobacterales and Actinomycetales orders with members of the genus Arthrobacter being found in all three lakes. Geochemical properties were significantly different between the lakes, although more than one property attributed to the variance between community compositions. The diversity detected in the culture-based study differed significantly and all isolates belonged to the genus Streptomyces. Two novel strains were characterized by means of phylogenetic (16S rRNA gene sequence), physiological, morphological and biochemical analyses. Both novel isolates were capable of growing under "extreme" conditions- pH 12, 10% NaCl and 45°C. Partial enzyme characterization revealed that both strains produced xylanase enzymes that were active at pH 6.5 and 8.5 with an increase in activity up to 45°C. The results obtained revealed a previously undetected diversity of actinobacteria in the Ethiopian Rift Valley with a potentially novel subpopulation adapted to haloalkaline conditions. The low 16S rRNA sequence similarity of a substantial proportion of the libraries suggests that culture-based isolation may play a vital role in deciphering the community fingerprint. The National Research Foundation and the Norwegian Research Council

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Barnes, Kwasi H. "Diversity and Distribution of Diatom Endosymbionts in Amphistegina spp. (Foraminifera) Based on Molecular and Morphological Techniques." Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6177.

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Diatoms associated with foraminifers of the genus Amphistegina were assessed using a combination of morphological and molecular techniques. These included: 1) microscopic identification of diatoms cultured from the host, 2) sequencing of portions of the small subunit of the ribosomal RNA gene (18S) and the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase [i.e., RubisCO] gene (rbcL) from DNA extracted directly from the Amphistegina hosts and also from diatoms cultured from these hosts, and 3) denaturing gradient gel electrophoresis (DGGE) profiles of rbcL and internal transcribed spacer 1 (ITS1) PCR amplicons from DNA extracted directly from hosts and from cultures. Consistent with previous culture studies, multiple species of pennate diatoms of the genera Nitzschia, Fragilaria (including Nanofrustulum), Amphora, and Navicula, were cultured from >900 host specimens collected from >20 sites in the western Atlantic and four sites in the Pacific. Diatoms of the genus Nitzschia grew in about half of all successful cultures. The genetic identities of selected cultures were consistent with those based on morphological taxonomy. Diatom sequences from DNA extracted directly from the cytoplasm of the Amphistegina hosts were species specific and distinct from sequences obtained from cultured diatoms and from sequences in GenBank of diatom taxa previously reported as endosymbionts. Multiple phylogenetic analyses revealed that the 18S and rbcL diatom sequences from specimens of A. gibbosa collected from the Atlantic sites and of Amphistegina spp. from Hawai’i were most similar to the 18S and rbcL sequences of an unnamed Fragilariaceae diatom in GenBank (Accession # JX413542.1 for 18S and JX413559.1 for rbcL) and other closely related diatoms in that family. Of diatom taxa previously reported as endosymbionts of larger foraminifers, Nanofrustulum shiloi was the most similar, but not identical, to the sequences from hosts collected from the Atlantic and Hawai’i. The 18S and rbcL diatom sequences from the Atlantic host species, A. gibbosa, were all nearly identical, but small intra-species differences (subclades) were observed from specimens collected from the deepest (75 m) site in the Florida Keys and also from the eastern-most site, Young Island near St. Vincent. The 18S and rbcL diatom sequences from the two host species from Hawai’i, A. lobifera and A. lessonii, were more variable but still within the family Fragilariaceae. The diatom sequences from A. radiata collected from two sites in Papua New Guinea (PNG) were most similar to diatoms of the family Plagiogrammaceae and therefore distinct from sequences obtained from other Amphistegina species in this study, as well as from all diatoms previously reported as endosymbionts. A small difference was observed between the diatom sequences from host specimens collected from a Pacific site as compared to a Bismarck Sea site. The ITS1 DGGE profiles of DNA extracted directly from A. gibbosa specimens at different depths, locations, and seasons in the western Atlantic were nearly identical. Differences were seen between rbcL DGGE profiles of DNA extracted directly from the different Amphistegina host species. The rbcL DGGE profiles directly from all hosts were clearly different from those extracted from diatoms cultured from the same host specimens, as well as from Nitzschia laevis, a commonly reported diatom endosymbiont in past culture-based studies. My findings are consistent with ultrastructural studies of endosymbionts of Amphistegina published in the early 1980s and congruent with recent molecular studies of endosymbionts in other diatom-bearing foraminifers, all of which indicate specificity. Nevertheless, the consistency with which several diatom taxa have been reported in culture studies from all oceans indicates the possibility of some relationship with Amphistegina spp., either as important food items, epiphytes, or minor opportunistic symbionts that can thrive in culture media.

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Otero, Walter Guimarães. "Avaliação da diversidade microbiana e degradabilidade in situ em animais tratados com preparado de anticorpos policlonais contra bactérias produtoras de lactato e bactérias proteolíticas." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/10/10135/tde-23012009-111436/.

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A imunidade passiva surge como uma alternativa para a manipulação da fermentação ruminal e anticorpos de origem aviária contra bactérias específicas começam a ser pesquisados. Objetivou-se com este trabalho avaliar um preparado de anticorpos policlonais contra Streptococus bovis, Fusobacterium necrophorum e algumas cepas de bactérias proteolíticas (Peptostreptococcus anacrobius, Clostridium aminophilum e Clostridium sticklandii) sobre a diversidade microbiana ruminal e degradabilidade in situ de alguns alimentos. Foram utilizadas nove vacas mestiças portadoras de cânula ruminal. O delineamento experimental foi o quadrado latino 3 x 3 replicado 3 vezes, com arranjo fatorial de tratamentos 3 x 3 referente a 2 modificadores ruminais representados pela monensina (MON) e pelo preparado de anticorpos policlonais (PAP) mais o grupo controle e 3 fontes energéticas suplementadas na dieta, representadas pelo milho seco moído (MSM), silagem de grão úmido de milho (SGUM) e polpa cítrica (PC). Cada subperíodo experimental foi composto de 21 dias, sendo 16 dias para adaptação aos tratamentos e 5 para coleta de dados. A degradabilidade in situ dos alimentos testados foi mensurada através da técnica de saco de náilon. A coleta de amostras para a análise quantitativa de protozoários ocorreu no 21° dia de cada período, às 0 e 4 horas pós-alimentação, sendo estas coletadas por varredura do assoalho ruminal. O conteúdo ruminal foi coletado no 21° dia de cada período, às 4 h pós-alimentação, para análise da diversidade microbiana através de técnica de eletroforese em gel com gradiente de desnaturação. Observou-se que dietas contendo PC apresentaram aumento de 80,6%; 75,4% e 66,8% da degradabilidade efetiva da FDN da cana-de-açúcar para as taxas de passagem de 2, 5, 8%/h, respectivamente, em relação ao grupo tratado com SGUM, mas não em relação ao grupo com MSM. O tratamento com PAP demonstrou efeito sobre a fração solúvel (a) do amido do MSM, diminuindo esta em 45,26% e 45,37% em relação ao grupo CON e grupo MON, respectivamente. Observou-se que o tratamento com MON diminuiu em 16,14% o valor da fração potencialmente degradável (b) da MS da SGUM em relação ao grupo CON, mas não em relação ao grupo tratado com PAP. Já sobre a taxa de degradação (c), a MON aumentou o valor desta em 63,18% e 60,65% em relação ao grupo CON e PAP, respectivamente. Também a MON diminuiu a degradabilidade potencial (Dp) da MS da SGUM em 3,40% em relação ao grupo CON, mas não em relação ao grupo tratado com PAP. Observou-se que o PAP aumentou em 93,65% a contagem relativa de Isotricha em relação ao grupo CON, mas não em relação ao grupo MON. Já a PC aumentou em 334,42% (0h) e 399,75% (4h) a contagem relativa de protozoários do gênero Isotricha em relação às dietas de MSM e SGUM. Observou-se que o tratamento com PC aumentou em 52% a contagem do número de bandas em DGGE para a comunidade Archaea, em relação ao grupo MSM, sem diferir do grupo SGUM. Em linhas gerais, no presente experimento, não foi possível atribuir um padrão na estrutura de amplificação das comunidades Bacteria ou Archaea do conteúdo ruminal de animais tratados com dois diferentes modificadores ruminais ou 3 fontes energéticas distintas. Passive immunity arises as an alternative for ruminal fermentation manipulation and aviary antibodies against specific bacteria starts to be studied. The objective of the present study was to evaluate a polyclonal antibody preparation (PAP) against Streptococus bovis, Fusobacterium necrophorum and some proteolytic bacteria (Peptostreptococcus anacrobius, Clostridium aminophilum and Clostridium sticklandii) on ruminal microbial community diversity and in situ degradability of some feedstuffs. Nine ruminally fistulated cows were used in a latin square 3 x 3 replicated 3 times with factorial arrangement of treatments 3 x 3 regarding to two rumen modifiers [monensin (MON) and (PAP) plus a control group (CON)] and three energetic sources supplemented in the diet represented by the dry-grounded corn grain (CG), high moisture corn silage (HMCS) and citrus pulp (CiPu). Each trial lasted 21 days where 16 days were for treatments adaptation and 5 for data collection. In situ degradability of the experimental diets was measured by nylon bag in situ technique. The collection of samples for quantitative protozoa analysis occurred on day 21 of each trial at 0 and 4 h after feeding by scanning the ruminal floor. The ruminal content was collected in the day 21 of each trial at 0 and 4 h after feeding for the analysis of microbial ruminal diversity by the denaturing gradient gel electrophoresis. Diets with CiPu presented an increase of 80.6%; 75.4% and 66.8% in effective degradability of NDF of sugar cane for outflow rates of 0.02, 0.05, 0.08/h, respectively, in relation to group treated with HMCS but not to the group CG. The group treated with PAP showed effect on soluble fraction (a) of starch of CG decreasing it in 45.26% and 45.37% in relation to CON and MON group respectively. It was observed that the treatment with MON decreased in 16.14% the value of potentially soluble fraction (b) of DM from HMCS in relation to CON group but not in relation to PAP. For the degradation rate (c) the MON increased it values in 63.18% and 60.65% in relation to CON and PAP group respectively. Also, MON treatment decreased potential degradability (Pd) of DM of HMCS in 3.40% in relation to CON group but not to PAP. It was observed that PAP treatment increased in 93.65% the relative counting of Isotricha in relation to CON group but not in relation to MON group. It was observed that CiPu diet increased in 334.42% (0h) and 399.75% (4h) the relative counting of Isotricha in relation to CG and HMCS. It was observed that CiPu increased in 52% the counting of the numbers of bands in DGGE for Archaea community in relation to CG without difference to HMCS group. In general lines, in the present experiment, it was not possible to assign that there was a pattern in the structures of amplification by Bacteria and Archaea communities of the ruminal content of animals treated with two different rumen modifiers or three distinct energetic sources.

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Ranasinghe, Purnika Damindi. "Use of next generation sequencing for analysing taxonomical and functional composition of bacteria in an insect gut microbiome." Thesis, Queensland University of Technology, 2018. https://eprints.qut.edu.au/116377/1/Purnika%20Damindi_Ranasinghe_Thesis.pdf.

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This thesis describes the difference between the baseline composition of Diamondback moth (Plutella xylostella L.) insect colonies' gut microbiomes collected from field- and laboratory-reared populations. Results indicates that variation of gut bacterial community composition between individual insect influences baseline microbiome composition estimates at the population scale. The gut bacteria diversity and functional composition change in response to external challenges, and those changes reflect on insect development. This explains the need to discover the specific symbiotic gut bacteria that respond to environmental variables - such findings will be essential for controlling insect hosts, and this knowledge can be applied to develop insect pest management and control.

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16

Theunissen, Johnita. "Identification of probiotic microbes from South African products using PCR-based DGGE analyses." Thesis, Stellenbosch : Stellenbosch University, 2004. http://hdl.handle.net/10019.1/49983.

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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.

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17

Christophersen, Claus. "Grain and artificial stimulation of the rumen change the abundance and diversity of methanogens and their association with ciliates." University of Western Australia. School of Animal Biology, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0114.

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[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.

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18

Hosseini, Seyed Homayoun. "Temperature gradient gel electrophoresis development and application." Diss., Georgia Institute of Technology, 1994. http://hdl.handle.net/1853/25614.

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19

Blom, Dirk Jacobus. "Polyacrylamide gradient gel electrophoresis for the diagnosis of dysbetalipoproteinaemia (Type III hyperlipidaemia)." Master's thesis, University of Cape Town, 2001. http://hdl.handle.net/11427/3365.

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Includes bibliographical references. Accurate phenotypic diagnosis of this disorder has generally relied on the quantitative analysis of centrifugally isolated VLDL. Analysis of VLDL chemical composition is not widely available due to the expense of acquiring ultracentrifuges and the highly-skilled staff necessary to perform the analyses. Genotypic diagnosis is somewhat more widely available, especially testing for the E2/E2 genotype which is the commonest underlying genotype in dysbetalipoproteinaemia. Testing for the rarer autosomal mutations of ApoE is restricted to a small number of centres only. In this study I will review dysbetalipoproteinaemia briefly and then describe and evaluate GGE as a new diagnostic technique for this disorder.

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20

Cazeneuve, Cécile. "Caractérisation par électrophorèse en gradient de gel dénaturant des variations alléliques de deux polymorphismes de répétition en tandem situés dans une séquence consensus d'épissage de l'intron 8 du gène CFTR (Cystic fibrosis transmembrane conductance regulator)." Paris 5, 1995. http://www.theses.fr/1995PA05P051.

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21

Davis, Nejea I. "DEVELOPMENT AND APPLICATION OF COUNTERFLOW METHODS: GEITP, GEITP-CZE, TGF, and TGDF." Master's thesis, Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/117417.

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Chemistry M.A. Extensive research on amino acids, and even other biochemical assays usually present in low concentration and volume face challenges using known analytical techniques for analysis of traces amounts. Some limiting factors are the achievable efficiency, sensitivity (resulting from instrument limit of detection and/or experimental methods), volume requirement, and total analysis time. Counterflow electrofocusing techniques combining forces of electrophoresis and bulk flow (pressure driven flow and/or electroosmotic flow) provides a basis for the development of alternative detection techniques geared towards improving peak efficiency, sensitivity and time. The work presented gives a vivid description of recently developed capillary counterflow techniques: gradient elution isotachophoresis (GEITP) using UV detection, GEITP coupled to Capillary Zonal Electrophoresis (GEITP-CZE), temperature gradient focusing (TGF), and temperature gradient denaturing focusing (TGDF). A first demonstration of GEITP using UV detection was applied to enrichment and separation of tyrosine and tryptophan under optimized conditions. Primarily, separation is achieved as the result of the difference in electrophoretic velocity of analytes in a discontinuous buffer system. First, a plug of sample is allowed to preconcentrate (or enrich) between high mobility leading electrolyte (LE) and low mobility trailing electrolyte (TE) under controlled hydrodynamic pressure and continuous injection. This preconcentration is initiated outside the capillary in a conductivity bubble. Although analyte focus according to their electrophoretic velocity, the inclusion of spacer molecule in sample matrix was instrumental in achieving separation with tradeoff between analyte resolution and enrichment. Gradient produced results from reduction in pressure as sample is loaded on column. Separation using this technique is a one step process. A hybrid method marking the first successful coupling of GEITP to CZE with laser induced fluorescence detection was used for separation of six fluorescently labeled amino acids (which formulates the Mars-7). An eleven minute separation was achieved under optimized conditions. A proof-of-concept demonstration of TGF with LIF detection showed focusing and separation of fluorescein and carboxyfluorescein dye molecules, and carboxyfluorescein-labeled glutamate and aspartate. The generation of null focusing points along the thermal separation column (set between 80-20oC) was produced in collaboration with continuous sample injection, discontinuous buffer system and balancing of counterflows (electrophoresis and bulk flow). Preliminary results showed stability in instrument. The TGDF method carried out on a TGF apparatus is a modification to the temperature gradient gel electrophoresis and denaturing gradient gel electrophoresis methods. In principle, TGDF primarily achieves focusing and separation on a thermal separation column (set between 20 to 80 oC) as a result of conformational changes. It is currently being developed for the detection and simultaneous separation of single and double stranded DNA. Preliminary results show enrichment of wildtype and mutant synthetic DNA strands (containing twenty-four base pairs in sequence) in different buffer matrices. Temple University--Theses

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22

They, NG Haig. "Padrões espaciais e temporais da composição e atividade do bacterioplâncton no estuário da Lagoa dos Patos (RS, Brasil)." reponame:Repositório Institucional da FURG, 2013. http://repositorio.furg.br/handle/1/4046.

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Tese(doutorado) - Universidade Federal do Rio Grande, Programa de Pós–Graduação em Oceanografia Biológica, Instituto de Oceanografia, 2013. Submitted by Cristiane Gomides (cristiane_gomides@hotmail.com) on 2013-10-15T11:55:34Z No. of bitstreams: 1 NG.pdf: 3593852 bytes, checksum: 4c20bdc4282f3f36ffe2b4772a93b1cb (MD5) Approved for entry into archive by Sabrina Andrade (sabrinabeatriz@ibest.com.br) on 2013-10-17T17:34:15Z (GMT) No. of bitstreams: 1 NG.pdf: 3593852 bytes, checksum: 4c20bdc4282f3f36ffe2b4772a93b1cb (MD5) Made available in DSpace on 2013-10-17T17:34:15Z (GMT). No. of bitstreams: 1 NG.pdf: 3593852 bytes, checksum: 4c20bdc4282f3f36ffe2b4772a93b1cb (MD5) Previous issue date: 2013 A composição (CCB) e a atividade (perfil fisiológico - PFCB) da comunidade bacteriana foram investigadas no estuário da Lagoa dos Patos e região costeira adjacente através de amostragem tipo-Lagrangiana, Euleriana e ao longo de um transecto para responder três perguntas: i) existe um padrão de recorrência (estabilidade) da CCB e PFCB em diferentes faixas de salinidade? ii) a CCB e PFCB respondem a diferentes escalas temporais e espaciais, inclusive a fenômenos climáticos globais que afetam a hidrodinâmica do estuário, tais como o “El Niño Southern Oscillation” (ENSO) ? iii) que fatores, além da salinidade, afetam a CCB e a PFCB? A CCB e a PFCB estiveram associadas à hidrodinâmica do estuário, tanto em escalas curtas (entrada de cunha salina), médias (sazonal) e largas (ENSO). Isto porquê a hidrodinâmica condiciona a variabilidade da salinidade e secundariamente o seston, nutrientes inorgânicos dissolvidos e substratos orgânicos, que afetam as bactérias. A CCB foi primariamente estruturada pela salinidade, seguindo o padrão normalmente encontrado na literatura para estuários, com comunidades características de água salgada e doce. Já a PFCB teve maior influência da quantidade de nutrientes e substratos e de maneira indireta da salinidade. De maneira geral, a atividade bacteriana foi menor em águas salgadas mais oligotróficas. Entretanto, grande atividade bacteriana foi observada em água salgada rica em nutrientes que penetrava no estuário. A maior concentração de nutrientes na água salgada pode ter sido resultado de ressuspensão de sedimento, ou ingresso de água costeira previamente enriquecida com água estuarina. The composition (BCC) and activity (community level physiological profiles, CLPP) of the bacterial community were investigated in the Patos Lagoon estuary and adjacent coastal region through Lagrangian-like, Eulerian and transect samplings in order to answer three questions: i) is there a pattern of recurrence (stability) of the BCC and CLPP in different salinity ranges? ii) does the BCC and CLPP respond to different temporal and spatial scales, including global climate phenomena that affect the estuary hydrodynamics like El Niño Southern Oscillation (ENSO)? iii) which factors apart from salinity affect the BCC and CLPP? The BCC and CLPP were associated to the estuary hydrodynamics, at short- (salt wedge entrance), meso- (seasonal) and largescales (ENSO). This is because the hydrodynamics conditions the variability of salinity and secondarily the seston, dissolved inorganic nutrients and organic substrates, which in turn affect bacteria. The BCC was primarily structured by salinity, following the pattern commonly found in the literature for estuaries, with characteristic fresh- and saltwater communities. The CLPP had higher influence of the amount of nutrients and substrates and indirectly of salinity. In general the bacterial activity was lower in oligotrophic, saltier waters. However, high bacterial activity was observed in nutrient-rich saltwater that entered the estuary. The higher concentration of nutrients in saltwater is likely the result of ressuspension of the sediment or ingress of coastal water previously enriched with estuarine water.

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Vaidyanathan, Vidya. "Different methods for particle diameter determination of low density and high density lipoproteins-Comparison and evaluation." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1170.

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Abdali, Abdeslam. "Contribution à l'étude d'une désoxyribonucléase associée a l'iridovirus type 6 (CIV)." Rouen, 1986. http://www.theses.fr/1986ROUES020.

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Passig, Fernando Hermes. "Reator anaeróbio híbrido para tratamento de esgoto sanitário." Universidade de São Paulo, 2005. http://www.teses.usp.br/teses/disponiveis/18/18138/tde-11042016-151713/.

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Este trabalho de investigação refere-se ao uso do reator anaeróbio híbrido para tratamento de esgoto sanitário, com configuração baseada no reator anaeróbio de manta de lodo (UASB) com inclusão de: meio suporte sobre as calhas de coleta de gás (denominado reator anaeróbio híbrido - UAHB) e, também, meio suporte na zona de reação (denominado reator anaeróbio híbrido modificado - UAHBmod). Para o desenvolvimento desta pesquisa, no Campus I da USP de São Carlos-SP foram construídos dois reatores experimentais de 18,8 m3 cada: um reator UASB, com função de controle, e um reator UAHB. Primeiramente os reatores foram operados por período de 200 dias, com tempo de detenção hidráulica (TDH) de 6 h. Após serem inoculados, com 80 dias de operação, os reatores atingiram o estado de equilíbrio dinâmico aparente, com geração de alcalinidade, baixa concentração de ácidos voláteis e eficiência de remoção média de DQO, de 84% e 85% e de DBO de 87% e 91%, respectivamente para o UASB e o UAHB. Após esse período, os reatores foram submetidos a aumento da velocidade ascensional (Vasc) (mediante recirculação do efluente) de 0,78 m.h-1; 1,17 m.h-1; 1,56 m.h-1 e de 1,96 m.h-1. O UAHB mostrou ser menos susceptível ao aumento da Vasc do que o UASB. Além da análise da operação dos reatores, foram realizados os ensaios hidrodinâmicos e avaliada a estrutura da comunidade microbiana, por microscopia ótica, epifluorescência e pela técnica do DGGE. Após esse período preliminar, os reatores UAHB e UAHBmod, operados com TDH de 6h e Vasc de 0,78 m.h-1, atingiram o estado de equilíbrio dinâmico aparente, com geração de alcalinidade, baixa concentração de ácidos voláteis e eficiência de remoção média da matéria orgânica, de 71% e 76% em DQO, e de 72% e 87% em DBO, respectivamente para o UAHB e UAHBmod. Após este período, o reator UAHBmod, submetido a Vasc de 1,56 m.h-1, promoveu remoção de 74% de DQO, e de 87% de DBO. This research refers to the use of a hybrid anaerobic reactor (UAHB) for domestic wastewater treatment. The configuration of this reactor is based on a sludge bed anaerobic reactor (UASB); in the first instance, a media support above the gas collection apparatus (also known as hybrid anaerobic reactor) was provided and later, a media support on the reaction zone (also known as hybrid modified anaerobic reactor - UAHBmod) was provided. Two reactors, with a volume of 18.8 m3, each, were built for this research at Campus I, USP in São Carlos - SP-Brazil. One UASB reactor acted as a control, and the other as a UAHB reactor. In the preliminary essays, the reactors were operated with 6h of hydraulic detention time (HDT) for 200 days. After inoculation, the reactors attained the apparent dynamic equilibrium state after 80 days of operation, with alkalinity generation, low volatile acids concentration and mean organic matter removal of 84% and 85% in terms of COD, and 87% and 91% in terms of BOD, for UASB and UAHB reactors, respectively. After this period, the reactors were submitted to an increasing in up velocity (Vup) of 0.78 m.h-1; 1.17 m.h-1; 1.56 m.h-1 and 1.96 m.h-1. The UAHB reactor showed lesser susceptibility for Vup increase than the UASB reactor. Hydrodynamic tests were also done on the reactors, in addition to routine operational analysis. The structure of the microbial community was evaluated by optical and epifluorescence microscopy, and the DGGE technique. After this step, the UAHB and the UAHBmod reactors were operated out 6h of HDT and Vup of 0.78 m.h-1. The reactors attained the apparent dynamic equilibrium state with alkalinity generation, low volatile acids concentration and mean organic matter removal of 71% and 76% in terms of COD, and 72% and 87% in terms of BOD for the UASB and UAHBmod reactors, respectively. After this period, the UAHBmod reactor was subjected to a Vup of 1.56 m.h-1 and achieved removal efficiencies of 74% COD and 87% BOD.

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Čakajdová, Martina. "Analýza mikroflóry v sýrech pomocí DGGE." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2015. http://www.nusl.cz/ntk/nusl-217120.

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Molecular biological methods are fast and efficient tool for the identification of microorganisms in real samples. The aim of this diploma thesis was analysis of microflora in control and contaminated brined cheeses. DNA isolated from analyzed samples was used to optimize the PCR course using primers with GC clamp on the distribution of amplicons using DGGE. DGGE products were reamplified after optimization and prepared for DNA sequencing. DNA isolated from analyzed samples was used in real-time PCR with high resolution melt analysis of the amplicons (HRMA). Samples of cheese and bacterial cultures isolated from cheeses were compared by DGGE and HRMA. Comparing the position of the amplicons was found that contaminants may be Bacillus licheniformis, Staphylococcus epidermidis, and Acinetobacter baumanii/ calcoaceticus. Sequence analysis of cheese and pickles amplicons, the presence of Bacillus sp. and other microorganisms spree in five genera were detected. Representatives of the tree genera were cultured. It is considered contamination Bacillus sp., or microorganisms which are not culturable methods used. The method is suitable for the analysis of complex microflora in cheese and pickles after further optimization.

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Eb-Levadoux, Yvan. "Identification des ligands biologiques de l’uranium dans les gonades de Danio rerio. : Impact sur leur fonctionnalité." Thesis, Pau, 2017. http://www.theses.fr/2017PAUU3016.

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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 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

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Khan, Ghazanfar Ali. "Monitoring anti-infectives and antibiotic resistance genes : with focus on analytical method development, effects of antibiotics and national perspectives." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-61682.

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Antibiotics are biologically active and are globally used in humans and animal medicine for treatment and in sub-therapeutic amounts as growth promoters in animal husbandry, aquaculture and agriculture. After excretion, inappropriate disposal and discharge from drug production facilities they enter into water bodies either as intact drugs, metabolites or transformed products. In water environments they promote development of antibiotic resistance genes (ARGs) which could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. Measurement of antibiotics has been revolutionized with the usage of solid phase extraction (SPE) for enrichment followed by Liquid chromatography mass spectrometry (LC-MS). On-line SPE coupled to LC-MS/MS has the advantages of high sample throughput, low sample preparation time and minimal solvent utilization. Constructed wetlands (CWs) are potential alternatives to conventional treatment plants to remove organic pollutants. A study at Plönninge, Halmstad was performed to assess the impact of bacterial community pattern and development of resistance in spiked (n=4) and control (n=4). CWs were spiked with antibiotics at environmentally relevant concentrations continuously for 25 days. Shannon Index (H’) were used to determine the bacterial diversity and real-time PCR detected and quantified antibiotic resistance genes (ARGs) sulI, tetA, tetB, erm, dfrA1, qnrS and vanB and class 1 integrons intI1. No significant differences in bacterial compositions or in ARGs or integron concentrations could be discerned between exposed and control wetlands. A study conducted in Northern Pakistan showed that the antibiotic levels in most studied rivers were comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics were detected in the river in close vicinity of the 10 million city Lahore, e.g. 4600 ng L−1 sulfamethoxazole. Highest detected levels were at one of the drug formulation facilities, with measured levels up to 49000 ng L−1 of sulfamethoxazole for example. The highest levels of ARGs detected, sul1 and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. In the study in UK, sewage epidemiology surveillance is used to measure the oseltamivir carboxylate (OC), metabolite of oseltamivir (parent drug) in twenty four time proportional hourly influent samples from two WWTPs and then back-calculations were made to assess the compliance of drug. Predicted users of oseltamivir, based on measured OC in waste water, ranged from 3-4 and 120-154 people for the two WWTP catchments, respectively, which are consistent with the projected use from national antiviral allocation statistics, 3-8 and 108-270, respectively. Scenario analysis suggests compliance was likely between 45-60% in the study regions.

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Medina, Pons Francisco Javier. "Diversidad y funcionamiento de la comunidad epífita eucariota de las hojas de Posidonia oceanica." Doctoral thesis, Universitat de les Illes Balears, 2012. http://hdl.handle.net/10803/84119.

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Variability in the structure of the macroeukaryotic epiphytic fraction of Posidonia oceanica leaves has been previously described under optical microscope or dissecting microscope (classical microscopy approach). We tested the sensitivity of two molecular techniques (Temperature gradient gel electrophoresis (TGGE) and small ribosomal subunit (SSU) clone libraries), as an alternative to classical microscopy method, to detect variability in the structure of that epiphytic assemblage. We also investigated the relationship between diversity/composition of epiphytic macroalgae, the most diverse and abundant component of the assemblage, and Nitrate reductase (NR) activity (as a measure of nitrogen assimilation capacity from water column, which is an ecosystem key process). TGGE and SSU clone libraries were able to monitor changes in the structure of that assemblage and overcame some disadvantages of the classical microscopy approach, such as taxonomic impediment. In addition, the composition of the assemblage suggested playing a relevant role in determining nitrogen assimilation rates. La variabilidad en la estructura de la fracción epífita macroeucariota de las hojas de Posidonia oceanica se ha estudiado previamente mediante microscopio óptico o lupa binocular (aproximación clasica). Por un lado, se estudió la sensibilidad de dos técnicas moleculares (electroforesis en geles de gradiente de temperatura (TGGE) y librerías de clones de los genes de la subunidad pequeña del ribosoma (SSU), como alternativa al método clásico, en la detección de variabilidad en la estructura de esa comunidad epífita. Por otro lado, se investigó la relación entre la diversidad/composición de las macroalgas epífitas, el componente más abundante y diverso de esta comunidad, y la actividad Nitrato reductasa (NR) (como una medidad de la capacidad de asimilación de nitrógeno de la columna de agua, un proceso clave en el ecosistema). El TGGE y las librerías de clones de los genes SSU eran capaces de monitorizar los cambios en la estructura de esa comunidad y permitían evitar algunos inconvenientes de la aproximacion clásica, tales como problemas a nivel de taxonomía. Además, los resultados obtenidos sugerían que la composición de la comunidad juega un papel relevante a la hora de determinar las tasas de asimilación de nitrógeno. La variabilitat en l'estructura de la fracció epífita macroeucariota de les fulles de Posidonia oceanica s'ha estudiat prèviament mitjançant microscopi òptic o lupa binocular (aproximació clàssica). D'una banda, es va estudiar la sensibilitat de dues tècniques moleculars (electroforesi en gels de gradient de temperatura (TGGE) i llibreries de clons dels gens de la subunitat petita del ribosoma (SSU), com a alternativa al mètode clàssic, en la detecció de variabilitat en l'estructura d'aquesta comunitat epífita. D'altra banda, es va investigar la relació entre la diversitat/composició de les macroalgues epífites, el component més abundant i divers d'aquesta comunitat, i l'activitat Nitrat reductasa (NR) (com una mesura de la capacitat d' assimilació de nitrògen de la columna d'aigua, un proces clau en l'ecosistema). El TGGE i les llibreries de clons dels gens SSU varen permetre monitoritzar els canvis en l'estructura d'aquesta comunitat i evitaven alguns inconvenients de l'aproximacio clàssica, com ara problemes a nivell de taxonomia. A més a més, els resultats obtinguts suggerien que la composició de la comunitat juga un papel rellevant a l'hora de determinar les taxes d'assimilació de nitrògen.

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Chang, Wen-Hsin, and 張文欣. "Monitoring the Lactobacillus population in broiler gastrointestinal tract by Denaturing Gradient Gel Electrophoresis." Thesis, 2012. http://ndltd.ncl.edu.tw/handle/78987965429006419225.

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碩士 國立中興大學 動物科學系所 100 An understanding of the dynamics of bacterial community structure in the chicken intestine is essential for selection of diets for optimal nutrition, effective treatment of enteric pathogens, and the development of competitive exclusion products. The microbial diversity, count of bacteria and classification of bacteria in microbiology are many ways to quantitative and qualitative. It’s used in research on PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), that a more convenient way to explore the changes in the composition of flora. In the present study, establishing the optimal condition of PCR-DGGE, and investigate the transition of the bacterial community structure and the predominant bacteria in the intestine of chicks from hatching to 5 wk of age or diet supplement antibiotic and probiotic were investigated using PCR-DGGE and phylogenetic analysis. The results demonstrated that samples of intestinal contents used lactobacillus-specific primers (LabtufGC / LabtufR), by nested PCR amplification and purification, with 10% acrylamide and denaturing gradient of 35-47.5% of the gel, in order to 80V at 60 ℃ for 16 hours after electrophoresis were stained by the silver staining method, obtain a better separation bands and higher resolution of DGGE profile. Newborn chicks after oral lactobacillus broth three days, digestive tract contents collected for PCR-DGGE, the crop and ileal contents in the DGGE patterns can be detected the bands of lactic acid bacteria that not appear in the control group. With age, the bands number of ileal contents DGGE profiles were decreased, and the biodiversity of Lactobacillus species (Shannon''s index) also decreases. However, the opposite in the cecum, the biodiversity of Lactobacillus species were increase with age. Index of similarity results that the development of lactic acid bacteria were different on disparity parts of the gastrointestinal tract. Crop microflora stabilized after 1-day-old, and ileum microflora in no change after 7 days, as cecal microflora changes due to age, to the late sampling (35 days) continued to vary. Probiotics and antibiotics to promote growth performent of the host mechanisms are not identical, that antibiotics to decrease intestinal bacterial species and reducing the biodiversity of intestinal microflora and to reduce energy waste, the probiotic can improve the biodiversity of lactobacillus on crop and ileum, and the digestive tract microflora to become positive equilibrium, and further to improve the growth performent. Application of PCR-DGGE to monitor the changes of Lactobacillus population in gastrointestinal tract of broiler is feasible. The species of Lactobacillus can be determined by PCR-DGGE combine with molecular cloning technique in the further study.

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Yu-Fen, Wang, and 王郁芬. "Identification of Non-Tuberculous Mycobacterium in Environmental Samples by PCR-Denaturing Gradient gel Electrophoresis." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/77534514601538220014.

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碩士 國立中興大學 環境工程學系 93 Nontuberculous mycobacteria (NTM) is a common microorganisms which cause diseases in immunocompromised patients. In addition, Some species may cause infections similar to tuberculosis in humans and animals.There is very limited documentation of person-to-person transmission of NTM. This aim of this study is to develop a method which can detect the NTM of the tap water of hospital. In our study, we try to combine some molecular techniques based on the 16S rRNA and the hsp gene of mycobacteria, PCR-DGGE，for identification of NTM in environmental samples. 30 pure cultures were applied in this research, but only 20 cultures showed the postive signal after PCR analysis. Five different species of NTM were identified form these 20 cultures after 16S rRNA sequencing , including M. porcinum, Mycobacterium sp., M. lacticola, M. mucogenicum, M. wolinskyi. These results demostrated the method we developed that identification nontuberculosis mycobacterium in environmental samples by PCR-DGGE has been established and more precise. In the future, we hope this efficient method could be use for definitive detection of NTM of environmental samples.

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Dutta, Partha. "Soil community analysis along a salt gradient using denaturing gradient gel electrophoresis of PCR-amplified 16s rRNA genes." Thesis, 2006. http://hdl.handle.net/10057/369.

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Many studies of hyper saline environments have been performed, mainly on aquatic systems. However, the microbial community in terrestrial thallasohaline environments has not been studied extensively. To our knowledge, this is the first study of a natural terrestrial thallasohaline environment using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). We studied the great salt plains (GSP) for this purpose. The GSP is a perfect example of an extreme environment. The environment of the GSP changes frequently due to rain events that can change the salinity of the soil. Salt gradient samples and core samples were collected from the GSP in different years and DNA extractions were performed. 16S rRNA genes were amplified using PCR and examined on DGGE gels. Banding patterns of the DGGE gels were analyzed using Quantity One-Versa Doc software. Based on the banding patterns after DGGE, it was shown that the low- and high-salt soil samples had greater band richness than medium-salt soil samples. A dendrogram of relatedness was made and the samples were placed in different phenons using NTSYS. Core samples collected from the same locations exhibited similar microbial communities, and samples collected in different years from same location exhibited different microbial communities. Environmental factors such as soil salinity, water flow, and temperature, vary from year to year and from place to place on the GSP, which can select for different microbial communities in soil samples collected from the same place in different years and soil samples collected from different places on the GSP. Thesis (M.S.)--Wichita State University, College of Liberal Arts and Sciences, Dept. of Biological Sciences. "May 2006."

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Chen, Hsi-Chia, and 陳希嘉. "Study on identification of lactic acid bacteria by denaturing gradient gel electrophoresis and oligonucleotide microarray." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/69162826021385651302.

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博士 國立臺灣大學 動物科學技術學研究所 97 Lactic acid bacteria (LAB) are widely employed as starter cultures in the dairy industry and supplemented in different commercial products. Detecting and identifying various species of lactic acid bacteria with rapid method is often important for quality control of dairy products and monitoring fermentation processes. However, identification of lactic acid bacteria in the complex bacterial communities is still difficult for both phenotypic methods and genotypic methods. The aim of this study focused on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and oligonucleotide microarray method to develop a rapid, reproducible and easy to handle molecular tool for identification of lactic acid bacteria in dairy products. For PCR-DGGE, selection of primers is the key to analyze highly conserved genetic profiles among LAB. Four primer sets, targeted for 20 reference LAB strains, were evaluated and primer set GC338f/518r was selected due to its highly discriminating power at species level. After selection of primer sets, LAB in kefir grains and commercial probiotic products were assessed using PCR-DGGE by a culture-independent and a culture-dependent way, and were further confirmed by DNA sequencing techniques. Kefir grains results indicated that a combined method of cultivation with PCR-DGGE and subsequent DNA sequencing could successfully identify four LAB strains, Lactobacillus kefiranofaciens, Lb. kefiri, Leuconostoc mesenteroides and Lactococcus lactis from three kefir grains from Taiwan (named Hsinchu, Mongolia and Ilan). It was interesting to find that all three kefir grains contain similar LAB species. Furthermore, the DGGE as a culture-independent method indicated that Lb. kefiranofaciens was found in all three kefir grains, whereas Lb. kefiri was only observed in Hsinchu kefir grain and Lc. lactis was found in both Mongolia and Ilan samples. Two additional strains, Pseudomonas spp. and E. coli, were also detected in kefir grains. The DGGE profiles for commercial probiotic products demonstrated that the samples contained most lactic acid bacteria that specified on the product label except of the lyophilized mixed starter powder. This may be due to a concentration below the detection limit or due to the fact that none of these bacteria were present in the probiotic product. For microarray method, oligonucleotide probes targeting variable regions of the 16S rRNA gene were designed and tested for the identification of LAB. The strategy involved designing possible oligomer probes, using two software (ARB and PRIMROSE), for each target at species and genus level. All candidate 25-mer probes were poly(T)-tailed to reach an overall length of 60 oligonucleotides. Microarrays were manufactured by in situ synthesis (Agilent Technologies). Eight samples, including 5 pure LAB (Lb. acidophilus, Lc. lactis, Str. thermophilus, Leu. mesenteroides and Bif. animalis), 1 non-targeted stain (B. cereus) and 2 mixed strain, were then assessed. Results indicated that, of the 21438 individual hybridization reaction (3573 validated probes × 6 references strains), 20082 probes (93.67%) yielded the expected result by showing significantly differential signal. Further inspection of each species probe, over 50 % of species probes designed for Lb. acidophilus, Str. thermophilus and Bif. animalis had true-positive signals with target bacteria. Whereas, true-positive signals were lower than 10% for probes designed for Lc. lactis and Leu. mesenteroides. For mixed strain samples, most of significantly positive signals would not be decreased when the amount of target DNA was down to 0.1

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Wu, Chung-Yi, and 吳宗益. "Using Capillary Electrophoresis(CE) and Denaturing Gradient Gel Electrophoresis (DGGE) for Determing the Distribution of Bacteria Resistant to Kanamycin." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/74531331309891977489.

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碩士 朝陽科技大學 應用化學系碩士班 97 In recent years due to environmental changes, and more by the disruption of soil microbes, the soil bacteria to explore the importance of changing the subject. Early-related technologies such as: RAPD, RFLP, DGGE, RISA ... and have been used for analysis of bacteria, many of these technologies to 16S rDNA-based in recent years to 16S-23S (900bp ~ 1100bp) rDNA (ITS, 0.5~1.4 Kb ) carried out research is also increasing. In this study, culture method (selective medium LB, LB + Kan + Cyc) and chemical methods (CE and DGGE) for the cultivation of genetically modified papaya in isolated experimental field of soil bacteria with the general distribution of bacteria KanR analysis. The results showed that with the use of DGGE technology V3 region of 16S rDNA of total DNA of soil bacteria of the general analysis of bacteria and found that the total DNA of different soil samples showed different DGGE patterns, the clustering analysis (Cluster analysis) shows available on genetically modified crops general bacterial strain differences in distribution. The use of 16S rDNA (1.4Kb) and with the ITS sequence comparison with BLAST is also available in the soil samples of different bacteria KanR distribution.

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Yang, Ju-Yu, and 楊如玉. "Analysis of the Changes of Phenol Degraders by Denaturing Gradient Gel Electrophoresis During Phenol Degradation in soils." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/65267362373707710605.

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碩士 嘉南藥理科技大學 環境工程與科學系碩士班 93 Abstract Phenol is a common pollutant from industries, it impacts on the aquatic ecosystem and environmental balance extremely, and biodegradation is a very economic technique, which is the best choice of the phenol contaminated soil remediation. The phenol high-tolerance degraders were isolated from collected sludge and soil from petrol station and around of reactor and wastewater storage in Syndyne Industry, the tested strains in this study including M1 and M5. The physiological properties of te sted strains were analyzed by Gram stain, activity of catechol dioxygenase. The results showed that the tested strains were Gram positive and had the activities of catechol 1,2-dioxygenase . The M1 and M5 were showed 96% probability of Ralstonia basilensis (AB109778) and Staphylococcus sp. B60 (AF333342) analysed in the denaturing gradient gel electrophoresis (DGGE) profiles of amplified 16S rDNA fragment. Inoculation of effective degraders to the modified polluted soils spiked with phenol was carried out by batch culture. The phenol concentration and the number of phenol degrader were monitored in the experimental period, soil DNA was extracted and amplified by primer 341GC+534r of PCR for DGGE analysis at the same time, which to understand the variation of test strains. The results showed that the biodegradation in the polluted soils and aquatic m-cresol solution were not consistent. All the strains can degrade phenol in the test polluted soils efficiently, the most effective degradation in the soil collected from was observed by test strains. Tracing soil bacterial community by DGGE technology in the experimental period was observed that inoculated effective strains became dominant, and this result was similar to the colony forming unit (CFU) was counted by plate-count methods. The denaturing gradient gel electrophoresis (DGGE) is considered one of the most sensitive of the scanning techniques for soil community.

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Cheng, Chiu-Yu, and 鄭秋玉. "Analysis of the Changes of Bacteria Communities by Denaturing Gradient Gel Electrophoresis Method During DEHP Degradation in Soil." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/78677809950701092272.

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Tzeng, Kai-Jiun, and 曾楷珺. "Identification of Environmental Acanthamoeba and Legionella by Denaturing Gradient Gel Electrophoresis and the Influence of Antibiotics Addition for Acanthamoeba Cultivation." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/cn93e9.

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碩士 國立中正大學 應用地球物理研究所 102 Acanthamoeba,Naegleria and Hartmanella are free-living amoebae. They are widely present in various environmental water. Some amoeba species of animals and humans will cause disease, and as Legionella host, they will raising the risk of infection Legionella's disease. The purpose of this study was to investigate the distribution of water environment in Taiwan free-type amoeba and Legionella species, and the use of real-time quantitative polymerase chain reaction (Real-time qPCR) to quantitative analysis. L. pneumophila separation from the environment is the use of genetic evolution analysis (Phylogenetic analysis) with multilocus sequence typing (MLST) analysis type comparison. In addition to using denaturing gradient gel electrophoresis (DGGE) technique, separation Acanthamoeba species can not be separated from the general electrophoresis, the establishment of a rapid mapping system to identify the type of bacteria, microorganisms in environmental water after comparing microflora and explore the microbial diversity in environmental water. The environment culeured free-living amoeba, bacteria indirect effect due to the presence of many free type amoeba growth, the use of Amphotericin B, Gentamycin and Penicillin-Streptomycin test bacteriostatic antibiotic dose and use microscopy looking for free type amoeba observed optimal growth conditions. And Acanthamoeba itself against antibiotic resistance, in order to understand the impact of their antibiotic. This study in Taiwan's main streams, reservoirs, spa area and a total of 395 samples collected Putsu River, and use a variety of analytical methods to detect Acanthamoeba,Naegleria and Hartmanella. Sequence analysis showed that in all types of water Acanthamoeba species not detected for T4 major genotypes. Naegleria strains detected in Naegleria spp. And Naegleria australiensis based. Hartmanella is Hartmanella vermiformis based, such as host most Legionella species. Real-time qPCR quantitative results show that the concentration of environmental water Acanthamoeba and Legionella species ranged from 4.2×102～2.4×105 copies/L and 2.1×102～4.4×1010 copies/L. Analysis from the separation of the environment 2 Legionella pneumophila by molecular typing method. The results showed that the use of mip, rpoB and dotA three sections specific target genes joint phylogenetic analysis to MLST analysis of its type, flaA, pilE, asd, mip, mompS, proA and neuA sequence number (6, 10, 15, 28, 21, 14, 11) and (6, 10, 15, 28, 17, 14, 11); Latex use the results of this two L. pneumophila serum from 2 to 14 are all type. Using denaturing gradient gel electrophoresis to identify the type of bacteria found in each genotype map system has unique patterns, and use Totallab software analysis, numerical definition for map to map the value identified as evidence. The use of antibiotics amoeba pure culture process, the culture medium with antibiotics will increase more and more clear, and inhibit Acanthamoeba part, the minimum inhibitory concentration of Amphotericin B was 7.8 μg / mL, the minimum Gentamycin inhibitory concentration of 15.6 U / mL and the minimum inhibitory concentration of Penicillin-Streptomycin was 625 U / mL. This study developed a Acanthamoeba strains denaturing gradient gel electrophoresis to identify the type of mapping system until the technology is mature, I hope in the future to help fast detection and prevention of pathogenic bacteria.

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Chen, Shin-Chih, and 陳信志. "Application of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis in the Detection and Identification of Lactobacillus, Staphylococcus and Alicyclobacillus Species." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/94906117638451095396.

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博士 國立中興大學 食品暨應用生物科技學系 96 Denaturing gradienet gel electrophoresis (DGGE) is a technique able to differentiate DNA fragnments with sequences diversity. With polymerase chain reaction (PCR), DGGE was able to analyze the species in mixed cultures. tuf gene is responsible for the synthesis of bacterial elongation factor Tu. The gene is a housekeeping gene and exists only one copy in Gram positive bacteria. It had been proven to possess more diversity than 16S rDNA between species in several genera. As a result, We tried to design genus-specific (or group-specific) primers from tuf gene sequences and practically evaluated in PCR-DGGE analysis of genus Lactobacillus, Staphylococcus and Alicyclobacillus, respectively. A set of Lactobacillus-specific primers LbtufF/LbtufR was designed in chapter two. Applied in PCR, all Lactobacillus species generated an amplicon about 140-b. p. while genus other than Lactobacillus resulted no products. PCR sensitivity of L. acidophilus and L. casei was 104 CFU/mL, respectively. PCR-DGGE of 15 Lactobacillus species resulted in 13 patterns. Comparison to 16S V2-V3 PCR-DGGE, which differentiated 15 species to 8 patterns, the developed PCR-DGGE obtained a much high resolution. The label accuracies of probiotic products in Taiwan were evaluated. Results indicated label of most dairy products were correct, but 80% (4/5) of tested capsule, tablet and powder products were found to be mislabeled. In chapter three, eleven common Staphylococcus species were analyzed by PCR-DGGE and resulted in 10 patterns. PCR-DGGE analysis of 9 stains of S. aureus, 2 strains of S. warneri, 2 strains of S. capitis, 2 strains of S. epidermidis and 2 strains of S. saprophyticus revealed that the DGGE patterns were very conserved between strains in a species. When mixed cultures of different Staphylococcus species in milk were sujected to DNA extraction and PCR-DGGE, the resulted patterns faithfully corresponded to the species of the mixed cultures, including those of potential to secret enterotoxins. A set of Alicyclobacills-specific primers Ali732F/Ali882R was designed from 16S rDNA in chapter four. Sequence analysis indicated the primers matched the sequences of all present 17 Alicyclobacillus species. PCR of the type strains and isolates preserved in our laboratory revealed that each strain generated an amplicon about 150-b. p.. Strains of other genus generated no products. Seneitivity of the PCR were evaluated to be102 CFU/mL, and components in apple juice didn’t interfere the sensitivity. A genus-specific primer set GCAli16SF/684R was also designed in this study for the application in PCR-DGGE analysis. PCR-DGGE analysis of 9 species revealed 8 patterns. Fragments of A. acidocaldarius and A. tolerans were too close to be distinguished. However, sequencing of the amplicons would be suitable to identify the two speices.

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39

Lin, Huei-Ming, and 林徽鳴. "Monitoring molecular fingerprinting of microbial community dynamics by denaturing gradient gel electrophoresis(DGGE) and terminal-restriction fragment length polymorphism (T-RFLP)." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/56892104243407930869.

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碩士 國立成功大學 生命科學系碩博士班 93 Soil bacteria are essential components of the biotic community in natural forests and they are largely responsible for ecosystem function, and participate in the elements circulation. Although the main diversity of life has been proven to be microbial, the vast majority of soil bacteria still remain unknown for less than 1% of environmental microorganisms can be cultured. In this study, cloning method, denaturing gradient gel electrophoresis (DGGE) and terminal-restriction fragment length polymorphism (T-RFLP) techniques were used to compare the prevalent microbial populations in soil and on litterfall of Nan-Jen Shan samples. 　Phylogenetic analysis based on PCR-amplified 16S rDNA revealed an effective tool to establish the microbiota development in soil and litterfall. The results from clone library showed that Acidobacteria were the dominants in soil samples (62.8%) and Gammaproteobacteria were the dominants in litterfall samples (51.4%). DGGE patterns also revealed that the microbial community of soil and litterfall were not the same. Seasonal changes in the structure of microbial community were significant in DGGE analysis. However, the DGGE patterns of dominant bacteria did not change in DGGE analysis no matter in soil and litterfall samples. 　Terminal-restriction fragment length polymorphism (T-RFLP) analysis is also used in this study for rapid comparison of the complex bacterial communities. By T-RFLP analysis of soil and litterfall samples, we got eight groups Terminal-restriction fragments (T-RFs) in soil samples and five groups T-RFs in litterfall samples. T-RFLP analysis also revealed that Acidobacteria were the dominant bacteria in soil and were not affected by season. However, the results of T-RFLP showed that Betaproteobacteria are the dominant in litterfall. Otherwise, the dominant bacteria were changing with season on litterfall. 　Cultivation-approached method was also applied in isolation of cellulose degrading bacteria. Five strains were isolated from soil samples, all of them can use α-cellulose as carbon source. It showed that the soil bacteria in Nan-Jen Shan might play an important role in litter decomposition mechanism.

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40

Chhun, Aline. "Molecular Characterization of Toxic Cyanobacteria in North American and East African Lakes." Thesis, 2007. http://hdl.handle.net/10012/3341.

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Toxic cyanobacterial blooms constitute a threat to the safety and ecological quality of aquatic environments worldwide. Cyclic hepatotoxin, especially microcystin, is the most widely occurring of the cyanotoxins. The aim of this study was to identify the cyanobacterial genotypes present including how many toxic genotypes were present in two North American lakes and one African Lake. All three lakes are prone to cyanobacterial blooms and were sampled in 2005 and 2006: Lake Ontario (Bay of Quinte, Canada), Lake Erie (Maumee Bay, Canada) and Lake Victoria (Nyanza Gulf, Kenya). The cyanobacterial genotypic community was assessed using DNA based analyses of the hypervariable V3 region of the 16S rRNA gene. In addition, the aminotransferase (AMT) domain in modules mcyE and ndaF of the microcystin and nodularin gene cluster respectively was used to detect the presence of hepatotoxic genotypes. Denaturing gradient gel electrophoresis (DGGE) results from this study suggested that hepatotoxin producers were present in all study sites sampled and were most likely members of the genus Microcystis. This study was the first to report the potential for microcystin production in the in-shore and off-shore open lake of Nyanza Gulf in Kenya. A seasonal study of the Bay of Quinte and Maumee Bay showed differences in the cyanobacterial genotypic community from early to late summer. In addition, the cyanobacterial genotypic community from the Bay of Quinte differed from 2005 to 2006 and quantification of the North American samples revealed an increase in cyanobacterial cells from early to late summer. The Bay of Quinte saw relatively no change in hepatotoxic cells from early to late summer but in Maumee Bay hepatotoxic cells increased from undetectable in early summer to dominating the cyanobacterial community by late summer. This study demonstrated the use of DGGE and qPCR of the 16S rRNA-V3 and AMT gene region in monitoring the cyanobacterial community of waterbodies susceptible to toxic cyanobacterial blooms.

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Lin, Yu-Jia, and 林裕家. "Monitoring the dynamic changes of microbial community during bioremediation of oil-polluted sites by denaturing gradient gel electrophoresis (DGGE) and real-time PCR." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/76950060699574836498.

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碩士 國立成功大學 生命科學系碩博士班 94 Petroleum hydrocarbons are the most widespread contaminants in the environment. Several of petroleum hydrocarbons are known mutagens or carcinogens for human and other organisms. They are difficult to be degraded in the natural environment. Bioremediation is considered a useful method to degrade the petroleum hydrocarbons using the oil-degrading bacteria. In this study, cultivation-independent methods were used to study the microbial communities in petroleum contaminated sites. Phylogenetic analysis of bacterial 16S rRNA gene clone libraries from the LY、FD and YK petroleum contaminated soil showed that 16 of 47 OTUs (operational taxonomic units) were oil-degrading bacteria which belong to Pseudomonas、Burkholderia、Azoarcus、Xanthomonas、Bacillus、Comamonas、Nocardioides、Sphingomonas and Alcaligenes. Proteobacteria was the dominant group (66%) and Pseudomonas was the most abundant genus (21.6%) in these 3 clone libraries. In KH bioremediation site, there are 5 different treatments including bioaugmentation and biostimulation. Dynamic changes of bacterial communities in KH petroleum-contaminated site during bioremediation were monitored by PCR-DGGE, which used bacteria universal 16S rRNA gene primers. In the DGGE fingerprinting of KH site, the band at the same position with Pseudomonas markers was found in all samples. Our data indicate that Pseudomonas spp. may be important petroleum hydrocarbon-degradation bacteria in the KH site. The changes of population size of Pseudomonas in KH site during bioremediation were monitored by Real-time PCR. In all 5 different bioremediation-treatments, the population size of Pseudomonas reached maximum in the 14th day (6.80*104 – 2.04*105copy numbers/ng DNA) and decreased to minimum in the 24th day (7.20*103 – 1.52*104copy numbers/ng DNA). The TPH-d (total petroleum hydrocarbons-diesel) degrading profile of KH site showed that the TPH-d in soil was decreased to 50-60% in the 14th day and was almost degraded in 24th day. The results show that the Pseudomonas population size was affected by bioremediation treatment, but the TPH-d degrading rate of different bioremediation group were the same in KH site. The addition of bacteria or biosurfantant did not improve degradation of the TPH-d in KH site. Our findings indicate that the oil-degrading ability of original bacteria in contaminated site should be considered as an important factor for future bioremediation, and monitoring the variation of main oil-degrading bacteria can help us to understand the efficiency of bioremediation process, and enhance bioremediation.

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Yergeau, Étienne. "Caractérisation moléculaire de la biodiversité des Fusarium associés à la fusariose de l'asperge (Asparagus officinalis L.) au Québec." Thèse, 2004. http://hdl.handle.net/1866/15020.

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Ward, Rachel Jane. "The use of denaturing gradient gel electrophoresis in examining the species-specific influence of ectomycorrhizal fungi on selective bacteria enrichment in the mycorrhizosphere of Pinius rigida grown in a natural pine barrens habitat." 2007. http://hdl.rutgers.edu/1782.2/rucore10001600001.ETD.16793.

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44

Kennedy, Katherine Margaret. "Molecular methods for evaluating the human microbiome." Thesis, 2014. http://hdl.handle.net/10012/8230.

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In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.

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Verastegui, Pena Yris Milusqui. "Targeting novel soil glycosyl hydrolases by combining stable isotope probing and metagenomics." Thesis, 2014. http://hdl.handle.net/10012/8282.

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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.

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Bissonnette, Laurence. "Efficacité d'espèces ligneuses en symbiose mycorhizienne arbusculaire pour la phytoremédiation d'un site urbain contaminé." Thèse, 2009. http://hdl.handle.net/1866/8122.

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浅川, 晋. "水田土壌の主要なメタン生成古細菌群の解析". 2004. http://hdl.handle.net/2237/13126.

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