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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
21

Optimization and validation of the method lactose intolerance genotyping with real-time PCR

Stenberg, Jenny 2011 (has links)
Abstract Primary lactose intolerance has been associated with a single nucleotide polymorphism located upstream of the lactase gene. The most common diagnostic tests for lactose intolerance are time-consuming and the patient is not allowed to eat and drink for 12 hours before the test is carried out. A method that can establish the genotype would be an easier way of diagnosing lactose intolerance compared to fenotypic lactose intolerance tests. Optimization and validation of a previously published method was performed with real-time polymerase chain reaction. We used whole blood from de-identified blood donors. During the optimization and validation we used a positive control, genotype C/T from Laboratoriemedicin Västernorrland, Sundsvall. The whole-blood was extracted using the MagNa Pure LC instrument. The reagent used was KAPA PROBE FAST qPCR Master Mix. The optimized program for real-time PCR was established to be 95°C 3min [95°C x 3sec, 55°C x 20sec, detection, 72°C x 15sec] x 50 cycles. Optimal probe concentration was found to be 0.2µM and primer concentration will be 0.5µM. This genotyping method is a good first-stage screening test for lactoseintolerance. Before it can be used as a routine method further validation will be necessary in order to ensure that the evaluation of the results can be done in an easy and secure way.  
22

Real Time PCR Protocol Development for Rapid and Low Cost Quantification of Baculovirus and for Monitoring Progression of Infection

George, Steve 2010 (has links)
The work presented in this thesis aims to further the understanding and implementation of the Baculovirus Expression Vector System (BEVS) for varied uses such as protein production and viral vector production. To this end, three projects have been presented, two of which deal with methods to quantify baculovirus titres and the last deals with tracking baculovirus transcripts in infected insect cells. The first project examined assumption-free analysis as a method for data analysis of Real Time PCR data in order to enable direct comparison of baculovirus titres between samples, without the need for a traditional standard curve. It concluded that assumption-free analysis was well suited for this purpose and fold differences of baculovirus titres of different samples obtained using this method corresponded to real differences in sample titres. The second project aimed to develop a cheap and reliable method for sample preparation for Real Time PCR which would remove the need for the use of commercially available extraction kits. Samples were subjected to various combinations of Triton X-100 at different concentrations and different numbers of freeze/thaw cycles in order to determine the combination which would provide the best baculovirus genome exposure. One of these combinations was found to be at least as good as commercially available kits in reliably extracting baculovirus DNA and providing baculovirus titres that are at least as accurate. The third project was a preliminary study examining the effects of multiplicity of infection on the levels of baculovirus Gp-64 transcript in insect cell culture. The study concludes that at high multiplicities of infection, there seems to be no increase in baculovirus transcripts when the multiplicity of infection is further increased. This study served to allow for familiarization with tracking transcript levels, and the principles and techniques demonstrated here will form the basis for an exhaustive future study on the same subject.
23

Real Time PCR Protocol Development for Rapid and Low Cost Quantification of Baculovirus and for Monitoring Progression of Infection

George, Steve 2010 (has links)
The work presented in this thesis aims to further the understanding and implementation of the Baculovirus Expression Vector System (BEVS) for varied uses such as protein production and viral vector production. To this end, three projects have been presented, two of which deal with methods to quantify baculovirus titres and the last deals with tracking baculovirus transcripts in infected insect cells. The first project examined assumption-free analysis as a method for data analysis of Real Time PCR data in order to enable direct comparison of baculovirus titres between samples, without the need for a traditional standard curve. It concluded that assumption-free analysis was well suited for this purpose and fold differences of baculovirus titres of different samples obtained using this method corresponded to real differences in sample titres. The second project aimed to develop a cheap and reliable method for sample preparation for Real Time PCR which would remove the need for the use of commercially available extraction kits. Samples were subjected to various combinations of Triton X-100 at different concentrations and different numbers of freeze/thaw cycles in order to determine the combination which would provide the best baculovirus genome exposure. One of these combinations was found to be at least as good as commercially available kits in reliably extracting baculovirus DNA and providing baculovirus titres that are at least as accurate. The third project was a preliminary study examining the effects of multiplicity of infection on the levels of baculovirus Gp-64 transcript in insect cell culture. The study concludes that at high multiplicities of infection, there seems to be no increase in baculovirus transcripts when the multiplicity of infection is further increased. This study served to allow for familiarization with tracking transcript levels, and the principles and techniques demonstrated here will form the basis for an exhaustive future study on the same subject.
24

Establishment, identification, quantification of methanogenic archaea in chicken ceca and methanogenesis inhibition in in vitro chicken ceca by using nitrocompounds

Saengkerdsub, Suwat 2003 (has links)
In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.
25

Microbial bioremediation and monitoring of a TCE-contaminated site

Li, Kuan-hsun 11 July 2011 (has links)
The goal of this study was to use molecular biology techniques to access and monitor the efficacy of bioremediation on a trichloroethene (TCE) polluted site. We added emulsified hydrogen releasing materials to stimulate onsite microbial growth and the biodegradation of TCE. This process was known as enhanced bioremediation. In this study, there were two bioremediation sites had been treated anaerobically. Groundwater samples were taken periodically for microbial analysis. Denaturing gradient gel electrophoresis (DGGE) was used to evaluate the variations in microbial community structures during the in situ groundwater remediation. The DGGE DNA bandings were sequenced to determine the 16S rRNA gene sequences and identify the dominate bacterial species. In addition, we used Dehalococcoides spp. 16S rRNA genes as the targets to do real-time PCR. Results show that the emulsified hydrogen releasing materials could enhance anaerobic reductive dechlorination. After addition of emulsified hydrogen releasing materials, we found that the volatile organic compounds concentrations (i.e., TCE, 1, 1-DCE and VC) were decreased. In microbial analysis, the diversities of the microbial community were increased after nutrient supplement. According to the DNA sequencing results, there were 31 bacterial species had been found that related to TCE degradation (i.e., Acidovorax sp., Burkholderiales, Pseudomonas sp., £]-proteobacterium, Comamonadaceae, Iron-reducing bacterium, Hydrogenophilaceae, Clostridium sp., Geobacter sp., Rhodoferax ferrireducens, Dehalospirillum multivorans and Dehalococcoides spp.). Dehalococcoides spp. can be used as a biomarker to evaluate the efficacy of anaerobic bioremediation on a TCE contaminated site. Therefore, we quantified Dehalococcoides populations to explain the capacity of bioremediation after addition of emulsified hydrogen releasing materials to groundwater. Results reveal that Dehalococcoides cell numbers of site A were 4.47¡Ñ103-8.26¡Ñ104 CFU/liter, site B were 4.60¡Ñ102-9.31¡Ñ107 CFU/liter. This data indicated that the addition of emulsified substrate would increase the growth of total Dehalococcoides population under anaerobic conditions. Overall, results from this study demonstrated that the microbial analysis and quantities of Dehalococcoides at different time points can provide useful information to proceed with bioremediation methods.
26

Microcosm batch study of the degradation of 1,2-DCA-contaminated soil

Huang, Chih-wei 23 July 2012 (has links)
1,2-dichloroethane (1,2-DCA) is a popular industrial chlorinated organic chemical. Because 1,2-DCA is a dense non-aqueous phase liquid and easily accumulated in deep soil and water, it is difficult to be removed from the contaminated sites. In this study, aerobic and anaerobic microcosm batch experiments were performed to evaluate the feasibility of biodegradation of 1,2-DCA by adding different growth substrates. The aerobic microcosm results show that approximately 90% of 1,2-DCA removal was observed in the natural degradation group (A1) and the aerobic sludge addition group (A3) after 7 days of incubation. Up to 95% of 1,2-DCA removal could be observed in the substrate supplement group in after 14 days of incubation. In the anaerobic microcosm studies, 50% of 1,2-DCA removal could be obtained in all groups after 10 days except for the natural degradation group (B1). Moreover, the degradation efficiency for the anaerobic sludge group (B3) reached 80% of 1,2-DCA removal in 5 days. The DGGE profiles show that the microbial diversity varied with time and the sugar supplement groups (A2, B2) exhibited the most microbial diversity. Bacterial clones results revealed that the 1,2-DCA biodegradable microbial strains were presented in the microcosms, such as Klebsiella, Pseudomonas, Rhodoferax and Xanthobactor. The real-time PCR results indicated that the Dehalococcoides spp. was the major bacterium that was responsible for the degradation of 1,2-DCA in the anaerobic substrate supplement group (B2). Desulfitobacterium spp. could be the dominant 1,2-DCA degrading bacterium for the aerobic substrate supplement group (A2) and all of the anaerobic groups (B1, B2, B3, B4).
27

Establishment, identification, quantification of methanogenic archaea in chicken ceca and methanogenesis inhibition in in vitro chicken ceca by using nitrocompounds

Saengkerdsub, Suwat 2003 (has links)
In the first phase of this study, the diversity of methanogenic bacteria in avian ceca was found to be minimal. Based on 16S rDNA clone libraries, a common phylotype, designated CH101, ranged between 92.86 to 100 % of the total clones whereas less than 1% of the other phylotypes were found. On the basis of the sequence identity, all of the sequences, except sequence CH1270, are related from 98.97 to 99.45% to 16S rDNA Methanobrevibacter woesei GS. Sequence CH1270 is 97.62% homologous to the sequence identified to uncultured archaeon clone ConP1-11F. Clearly, the predominant methanogen found to reside in the chicken ceca was M. woesei. By using a MPN enumeration method, methanogen counts were found to be in the range of 6.38 to 8.23 log10 organisms per gram wet weight. The 16S rDNA copy number per gram wet weight in the samples was between log10 5.50 and 7.19. The second phase of the study was conducted to observe the effects of selected nitrocompounds and two different feedstuffs on in vitro methane production in chicken cecal contents and rumen fluid. Initially, one of the three nitrocompounds was added to incubations containing cecal contents from laying hens supplemented with either alfalfa or layer feed. Both feed materials influenced volatile fatty acids (VFA) production and also fostered methane production in the incubations although methane was lower (P < 0.05) in incubations with added nitrocompound, particularly nitroethane. Secondly, nitroethane was examined in incubations of bovine or ovine rumen fluid or cecal contents containing either alfalfa or layer feed. Unlike cecal contents, layer feed significantly (P < 0.05) supported in vitro methane production in incubations of both rumen fluids. The results show that nitroethane impedes methane production, especially in incubations of chicken cecal contents. The final phase of this study was carried out to determine the methanogenic establishment in the chicken ceca by the cultural method with the quantitative PCR. The results suggested that methanogens colonized in chicken ceca at a few days after birth. Litter and house flies could be potential sources for methanogenic colonization in broiler chicks.
28

Effects of nicotine on GABAA subunit expression in the rat brain

Bergenheim, Veronica 2007 (has links)

Smoking is a worldwide problem and it is the second major cause of death. People often try to quit, but few succeed mainly because of withdrawal symptoms such as irritability, anxiety, increased appetite, hyperventilation and difficulty concentrating.

The overall aim of this project was to study neurochemical changes in the brain following sensitization to nicotine which could give more information about what causes an individual to go from using drugs to abusing the drugs. Therefore, we investigated messenger ribonucleic acid (mRNA) expression of several genes known to be involved in the mesolimbic dopamine pathway in the nucleus accumbens, caudate putamen, prefrontal cortex and medial prefrontal cortex using real-time polymerase chain reaction (real-time PCR).

The results showed that in the nucleus accumbens, mRNA expression of gamma-aminobutyric acid (GABA) Aα1 subunit receptor and GABA transporter 3 (GAT-3) were significantly increased following nicotine administration, while in the caudate putamen no difference in expression was observed. In prefrontal cortex, the expression of adrenergic subunit receptor α2A was significantly increased following hexamethonium administration. In medial prefrontal cortex a significant decrease of expression of GAT-1 was shown following nicotine and hexamethonium administration, while a decrease of CART expression only was shown following nicotine administration.

Overall, these changes in the GABA system may help to explain the mechanism of nicotine sensitization.

29

Towards a portable and inexpensive lab-on-a-chip device for point of care applications

Olanrewaju, Ayokunle Oluwafemi 2011 (has links)
Ongoing work in the laboratory of Professor Chris Backhouse is aimed at developing a portable and inexpensive lab on a chip instrument. A system capable of molecular biology protocols including sample preparation (SP), polymerase chain reaction (PCR), and melting curve analysis (MCA) would meet the requirements for point of care genetic analysis. The SP, PCR, and MCA modules were designed and tested on a standalone basis and then integrated for analysis of raw clinical samples. An automated XY stage was developed for magnetic bead-based DNA purification. In addition, a LED/CCD-based optical detection module was employed for real time PCR and MCA. Data analysis algorithms and protocols were implemented to remove noise and interpret data. This work culminated in proof of principle on-chip SP-PCR-MCA to detect ß2m DNA from human buccal cells in a modular and inexpensive system. Biomedical Engineering
30

Nutrient Availability in the Rhizosphere of Coffee: Shade-tree and Fertilization Effects

Munroe, Jake Warner 2013 (has links)
Shade tree incorporation is beneficial in coffee cropping systems under sub-optimal conditions. This study was performed in lowland Costa Rica, at a 12-year-old experimental coffee farm. The main objective was to compare the effect of a nitrogen fixing shade tree, Erythrina poeppigiana, on nutrient availability in the rhizosphere of coffee under conventional fertilization. Accumulation of nutrients (mineral N, available P, and exchangeable base cations) in rhizosphere relative to bulk soil was greater under shade than full sun. Low nitrate availability in rhizosphere soil of full sun coffee was explained by root-induced acidification relative to bulk soil, as abundance of ammonia-oxidizing bacteria (AOB), which mediate nitrification, were positively correlated with pH. Organic fertilization enhanced AOB abundance and altered soil bacterial community structure relative to conventional fertilization. This study indicates clear effects of shade-tree presence on nutrient availability at the micro-scale, management of which is critical for stability of coffee agroforestry systems.

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