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Schabort, Willem Petrus Du Toit. "Integration of kinetic models with data from 13C-metabolic flux experiments." Thesis, Link to the online version, 2007. http://hdl.handle.net/10019/707.
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Wu, Biing-Ru. "RAPID IDENTIFICATION OF ASPERGILLUS SPP. USING A PCR BASED MELTING CURVE METHOD AND CHARACTERIZATION OF A NOVEL CHITINASE IN INSECT RESISTANT MAIZE LINES." MSSTATE, 2009. http://sun.library.msstate.edu/ETD-db/theses/available/etd-11032009-141528/.
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Identification of fungal isolates is critical in studying Aspergillus flavus ecology and for developing methods to reduce aflatoxin contamination. In our efforts to track biocontrol applications of the atoxigenic A. flavus K49 (NRRL 30797), we have developed a rapid and accurate classification system for A. flavus based on PCR product melting temperatures (Tm). Using 18 primers and a total of 59 Aspergilli strains, including all 49 representatives of the Georgian peanut Vegetative Compatibility Groups (VCGs), a decision tree Tm flowchart was generated. The decision tree can classify all 59 strains using only 9 of the SSR primers and an average of 3.4 primers for each definitive classification. To confirm the effectiveness of the decision tree for strain identification, unknown samples isolated from experimental fields inoculated with various A. flavus strains in Stoneville, MS were analyzed. Ninety-six percent of the samples could be placed into a VCG using Tm(s) coupled with the decision tree. This dynamic system is an excellent tool for researchers studying biodiversity of A. flavus.
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Barnes, Daniel Joseph. "DETERMINATION OF RICIN CONTENT IN CASTOR (Ricinus communis L.) TISSUES AND COMPARISON OF DETOXIFICATION METHODS." MSSTATE, 2008. http://sun.library.msstate.edu/ETD-db/theses/available/etd-11042008-143054/.
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Experiments were conducted to test for ricin content in tissue samples from four castor cultivars, developing castor seed, germinating castor seedlings, and chemically and heat treated seed meal. Ricin content of each sample was examined via Western blotting with ricin A-chain specific antibodies. Results indicate that ricin is present solely within castor endosperm and is not present any other tissues. Samples from developing seed and germinating seedlings indicate ricin production begins around day 28 post pollination, and ricin is absent from the seedling 6 days after the onset of radicle emergence. This would seem to indicate that the purpose of ricin is to protect the seed and not the entire plant. Ricin content of seed meal treated separately with heat and chemicals was tested. It was found that hot-pressing of the seed was sufficient to denature ricin in the seed meal.
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Ho, Meng-Hsuan. "CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF A COTTON RING-TYPE UBIQUITIN LIGASE (E3) GENE." MSSTATE, 2009. http://sun.library.msstate.edu/ETD-db/theses/available/etd-11032009-165510/.
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A cotton fiber cDNA, GhRING1, and its corresponding gene have been cloned and characterized. The GhRING1 gene encodes a RING-type ubiquitin ligase (E3) containing 337 amino acids (aa). The GhRING1 protein contains a RING finger motif with conserved cysteine and histine residues at the C-terminus and is classified as a C3H2C3-type RING protein. Blast searches show that GhRING1 has the highest homology to At3g19950 from Arabidopsis. Real time RT-PCR analysis indicates that the GhRING1 gene is highly expressed in cotton fiber in a developmental manner. The transcript level of the GhRING1 gene reaches a maximum in elongating fibers at 15 DPA. In vitro auto-ubiquitination assays using wheat germ extract and a reconstitution system demonstrate that GhRING1 has the ubiquitin E3 ligase activity. A fiber specific lipid transfer protein 4 (FSltp4) is identified as the target substrate of GhRING1 by using a bacterial two-hybrid system. The binding of GhRING1 and FSltp4 is confirmed by using an in vitro pull down assay and a yeast two-hybrid system. The histochemical GUS assay was performed to analyze tissue specificity of the GhRING1 and At3g19950 promoters in transgenic Arabidopsis plants. The GUS assay shows that the promoter of At3g19950 is highly activated in leaves, roots, trichomes and also in anthers and stigma of flowers. In contrast, the GUS expression directed by the promoter of GhRING1 is only located at stipules and anthers and stigma of flowers. The GhRING1 is the first ubiquitin E3 gene isolated and studied from cotton. Based on the expression pattern of GhRING1, FSltp4, and GhUBC E2s and the identification of a fiber-specific target protein, FSltp4, we propose that protein ubiquitination occurs in fiber and the ubiquitin-proteasome pathway regulates fiber development.
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Kapadia, Mayank S. "Identification of Collagen IV Associated Proteins in Drsophila Using Genetics and Mass Spectrometry." TopSCHOLAR®, 2016. http://digitalcommons.wku.edu/theses/1631.
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Metastatic cancer cells invade and spread to other locations by disrupting the basement membrane (BM). The membrane plays a major role during the normal development of an organism as well. In order to understand the invasion mechanism it is important to know about the interactions occurring between the proteins of the BM during normal development. This study concentrates on isolating and identifying the major factors associated with collagen IV, a major component of BM, during the third instar larval development of Drosophila. Western blot and mass spectrometry analysis revealed that collagen IV associates with various growth factors, signaling molecules, and proteins that may play a role during the development of Drosophila. Co-localization and knockdown studies performed on a single protein found through mass spectrometry suggested a possible role of this protein in the development of Drosophila. Further analysis of this proteins’ function will provide new insights into its developmental role and its potential role in collagen IV transport.
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Couch, Melanie. "Monitoring and Quantifying Tetracycline Resistance Genes in a Swine Waste Anaerobic Digester over a 100-Day Period." TopSCHOLAR®, 2018. https://digitalcommons.wku.edu/theses/2449.
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Unregulated use of growth promoting antibiotics like Tetracyclines in agricultural feeds is becoming an increasing problem in antibiotic resistance. Undigested antibiotics leads to significant concentrations in livestock waste. These concentrations provide continuous selection pressure for the development of antibiotic resistance genes in the environment. Antibiotic resistance related deaths are projected to surpass cancer related deaths by 2050 making antibiotic resistance a pressing public health issue. The purpose of this study is to determine the abundance and persistence of tetracycline (tet) resistance genes in swine waste over a period of 100 days in an anaerobic digester system. Tet(A), tet(B), tet(G), tet(M), tet(O), tet(Q), and tet(W) were quantified by quantitative polymerase chain reaction after DNA extraction. Primers that target ribosomal protection proteins and efflux proteins were used. Antibiotic resistance genes decreased from day one but were found to be present throughout the study.
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Modi, Tulsi. "Understanding the Molecular Level Interactions of Cancer Inhibitor Imatinib with Human Fibroblast Growth Factor-1." TopSCHOLAR®, 2015. http://digitalcommons.wku.edu/theses/1492.
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Fibroblast growth factors (FGFs) lack signal sequences, and are exported through endoplasmic reticulum (ER)-Golgi-independent non-classical routes. FGFs work as modulators of various cellular activities like mitosis, differentiation, survival etc. Among the FGF family, which comprises of 23 different heparin proteins, human FGF-1 (hFGF-1), a potent angiogenic factors are one of the targets in cancer inhibition, as they are involved in blood vessel formation in tissues. There has been intensive research directed at the development of drugs that could effectively inhibit angiogenesis. In this context, the purpose of this study is to fully understand the molecular principles essential to determine probability of inhibition of hFGF-1 signaling transduction by imatinib. Imatinib, a 2-phenyl amino pyrimidine derivative is a tyrosine kinase inhibitor with antineoplastic activity. Imatinib binds to the intracellular pocket located within tyrosine kinases and inhibit the downstream cell proliferation events, but the exact molecular mechanism is still elusive. In this study, expression of hFGF-1 in recombinant E. coli was carried out, and the expressed protein was purified using heparin affinity column chromatography. The structural interactions governing imatinib-hFGF-1 interaction was studied by monitoring its stability, conformation and binding affinity by equilibrium unfolding using steady state fluorescence and proteolytic digestion assay. These data show that imatinib binds to hFGF-1 and enhances its thermal stability and solvent accessibility. In addition, biacore analysis was carried out to determine the binding affinity of imatinib to hFGF-1.
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Mrwebi, Mandisi. "Testing Monod : growth rate as a function of glucose concentration in Saccharomyces cerevisiae." Thesis, Stellenbosch : University of Stellenbosch, 2004. http://hdl.handle.net/10019.1/16398.
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Thesis (MSc)--University of Stellenbosch, 2004.ENGLISH ABSTRACT: The complexity of microbial systems has presented serious obstacles to the quantification of fermentation processes. Using computer modelling techniques progress has been made in monitoring, controlling and optimising microbial systems using material balancing techniques and empirical process models. The Monod equation is among the most commonly used models and is based on empirical findings with no mechanistic basis. Monod presents a simple model to describe the growth of a cell in a defined nutrient environment. The Monod equation is mathematically analogous to the formula that was proposed by Michaelis and Menten to describe enzyme kinetics. Both equations describe a hyperbolic function with a half-saturation constant (K_s in the monod equation and K_m in the Michaelis Menten equation) but the meaning of the two saturation constants K_s and K_m is different. In number of studies K_s and K_m are used as if they are equivalent. In contrast to Michaelis-Menten kinetics, which describes a process catalysed by a single enzyme, Monod kinetics describes an overall process involving thousands of enzymes. The Monod equation describes the specific growth rate of a microbial cell as the function of a limiting substrate concentration. The aim of this study was to test this principle, for Saccharomyces cerevisiae VIN13 under glucose limited aerobic chemostat conditions. The VIN13 was observed to follow the Monod description and when compared with other growth kinetic models gave one of the best fits to the data. A functional relationship between the half-saturation constant, K_s, and Michaelis Menten constant, K_m, was there after derived. This was achieved by using metabolic control analysis (MCA) to explain when K_m of the transporter becomes equal to the K_s. Using the deductions obtained from MCA a core kinetic model was then formulated to demonstrate that the K_s can either be smaller, equal or higher than the K_m of the transporter, depending on the flux control distribution in the model.
AFRIKAANSE OPSOMMING: Die kwantifisering van fermentasieprosesse word ernstig belemmer deur die kompleksiteit van mikrobiale sisteme. Deur gebruik te maak van rekenaar-ondersteunde modelleringstechnieke vir die opstelling van massa balans vergelykings en empiriese prosesmodelle is vordering gemaak in die waarneming, beheer en optimalisering van mikrobiale sisteme. Die Monod vergelyking is een van die mees gebruikte groeimodelle en is gebaseer op empiriese bevindings - die model het nie ‘n meganistiese grondslag nie. Die Monod vergelyking is wiskundig ekwivalent aan die vergelyking wat opgestel is deur Michaelis en Menten vir die beskrywing van ensiemkinetika. Beide vergelykings beskryf ‘n hyperboliese kurwe met ‘n konstante wat die halfversadigingswaarde aangee vir substraat (Ks in die Monod vergelyking en Km in die Michaelis-Menten vergelyking), maar die betekenis van die twee versadigingskonstantes is verskillend. In ‘n aantal studies word die Ks en Km waardes gebruik asof hulle gelyk is aan mekaar. In teenstelling met die Michaelis- Menten kinetika wat ‘n enkel ensiem-gekataliseerde reaksie beskryf, beskryf die Monod vergelyking ‘n proses wat duisende ensieme behels. Die Monod vergelyking beskryf die spesifieke groeitempo van ‘n bakteriële sel as ‘n funksie van die beperkende substraatkonsentrasie. Die doel van hierdie studie was om hierdie beginsel te toets vir Saccharomyces cerevisiae VIN13 wat onder glukose beperkte, aerobiese kondisies in ‘n chemostat gekweek word. Die VIN13 groei kon goed beskryf word met die Monod model, wat in vergelyking met ander groeimodelle een van die beste passings vir die meetpunte het gegee. Vervolgens is ‘n funksionele verwantskap afgelei tussen Ks en Km; deur gebruik te maak van metabole kontrole analise (MCA) kon verduidelik word wanneer die Ks gelyk is aan die Km van die transporter vir die beperkende substraat. Deur gebruik te maak van die MCA analise is ‘n eenvoudige kinetiese model opgestel om aan te toon dat die Ks kleiner, gelyk aan of groter kan wees as die Km van die transporter, afhanklik van die fluksie-kontrole verdeling in die model.
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Kim, Juhwa. "Multiplexed Detection of Double-Stranded Pathogenic DNA with Engineered Zinc Finger Proteins." TopSCHOLAR®, 2016. http://digitalcommons.wku.edu/theses/1616.
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The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to the detection probe ZFP, which allows for generating chemiluminescent signal in streptavidin-HRP (Horseradish peroxidase) assay upon ZFPs binding to their target DNA. Our assay generates DNA-dependent signal and allows for a detection limit of 0.5 nM without DNA amplification or DNA labeling. Our system can be developed into a simple multiplexed detection diagnostic for multiplexed detection of dsDNA.
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Marpuri, ReddySalilaja. "Evaluation of Annotation Performances between Automated and Curated Databases of E.COLI Using the Correlation Coefficient." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/94.
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This project compared the performance of the correlation coefficient to show similarities in annotations between a predictive automated bacterial annotation database and the curated EcoCyc database. EcoCyc is a conservative multidimensional annotation system that is exclusively based on experimentally validated findings by over 15,000 publications. The automated annotation system, used in the comparison was BASys. It is often used as a first pass annotation tool that tries to add as many annotations as possible by drawing upon over 30 information sources. Gene ontology served as one basis of comparison between these databases because of the limited common terms in the ontology annotations. Translation libraries were used to extend the number of BASys terms that could be compared to the gene ontology terms in EcoCyc. Additional, non-ontology terms and metadata in BASys were compared to EcoCyc terms after parsing them into root words. The different term sources were quantitatively compared by using the correlation coefficient as the evaluation metric. The direct gene ontology comparison gave the lowest correlation coefficient. The addition of gene ontology terms to BASys by using translation tables of metadata greatly increased the correlation coefficient, which was comparable to the parsed word comparison. The combination of enhanced gene ontology and parsed word methods gave the highest correlation coefficient of 0.16.
The controlled vocabulary system of gene ontology was not sufficient to compare two annotated databases. The addition of gene ontology terms from translation libraries greatly increased the performance of these comparisons. In general, as the number of comparison terms increased the correlation coefficient increased. Future comparisons should include the enhanced gene ontology dataset in order to monitor the organization pertaining to formal nomenclature and the datasets generated from Word parsing can be used to monitor the degree of additional terms might be incorporated with translation libraries.
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Davis, Robert Tucker. "Geometric Build-up Solutions for Protein Determination via Distance Geometry." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/102.
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Proteins carry out an almost innumerable amount of biological processes that are absolutely necessary to life and as a result proteins and their structures are very often the objects of study in research. As such, this thesis will begin with a description of protein function and structure, followed by brief discussions of the two major experimental structure determination methods. Another problem that often arises in molecular modeling is referred to as the Molecular Distance Geometry Problem (MDGP). This problem seeks to find coordinates for the atoms of a protein or molecule when given only a set of pair-wise distances between atoms. To introduce the complexities of the MDGP we begin at its origins in distance geometry and progress to the specific sub-problems and some of the solutions that have been developed. This is all in preparation for a discussion of what is known as the Geometric Build-up (GBU) Solution. This solution has lead to the development of several algorithms and continues to be modified to account for more and different complexities. The culmination of this thesis, then, is a new algorithm, the Revised Updated Geometric Build-up, that is faster than previous GBU’s while maintaining the accuracy of the resulting structure.
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Chotikasemsri, Pongsathorn. "Computational Prediction of the Agregated Structure of Denatured Lysozyme." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/120.
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Mis-folded proteins and their associated aggregates are a contributing factor in some human diseases. In this study we used the protein lysozyme as a model to define aggregation structures under denaturing conditions. Sasahara et al. (2007), Frare et al. (2009, 2006), and Rubin et al. (2008) observed conditions where heat denatured lysozyme formed fibril structures that were observed to be 8-17 nanometers in diameter under the electron microscope. Even though the crystal structure of lysozyme is known, the denatured form of this protein is still unknown. Therefore, we used Rosetta++ protein folding and blind docking software to create in silico models of the protein at denaturing temperatures and subsequently docked them into aggregates. Here we compare those structures and select forms consistent with the fibril structure from the previous papers. The next step is to be able to use the predicted models of the fibrilar forms of denatured lysozyme to help us understand the exact conformation of fibril structures. This will let us confirm the docking interactions during the fibril aggregation process. The ultimate goal is to use the validated denatured structures to model interactions with heat shock proteins during the dis-aggregation process.
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Streicher, Elizabeth Maria. "Application of spoligotyping in the understanding of the dynamics of Mycobacterium tuberculosis strains in high incidence communities." Thesis, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences, 2007. http://hdl.handle.net/10019.1/1496.
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Gordon, Skyler A. "An Assessment of Potential False Positive E.coli Pyroprints in the CPLOP Database." DigitalCommons@CalPoly, 2017. https://digitalcommons.calpoly.edu/theses/1730.
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The genetic information found in each species of organism is unique, and can be used as a tool to differentiate at the molecular level. This has caused rapid genotyping methods to become the cornerstone of a new area of research dependent on reading the genome as a form of identification. One of these specific identification methods, known as pyroprinting, relies on the small variation of DNA sequences within the same species to develop a unique, reproducible fingerprint. By simultaneously pyrosequencing multiple polymorphic loci within the ribosomal operons known as the intergenic transcribed spacers, a reproducible output is obtained, known as a pyroprint, which can be used like a fingerprint to identify that organism. This section of the genome not only differs between species but also between isolated bacteria within that species, allowing for the differentiation of species subtypes, referred to as strains. While this is a viable method for generating reproducible fingerprints from individual strains it may be possible to obtain identical fingerprints from non-identical organisms. The following report uses direct sequence comparison and in silico pyrosequencing of E. coli isolates housed in the Center for Applications in Biotechnology at California Polytechnic State University, San Luis Obispo that have matching pyroprints to show that it is possible to receive near identical pyroprints from non-identical sequences of intergenic transcribed spacers. Although the exact likelihood and cause of this false positive result remains undetermined due to limitations in the sequencing method, its existence questions the accuracy of using pyroprints of the ITS regions as a method of strain classification.
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Van, der Spuy Gian Dreyer. "Analysis and application of evolutionary markers in the epidemiology of Mycobacterium tuberculosis." Thesis, Stellenbosch : Stellenbosch University, 2008. http://hdl.handle.net/10019.1/1457.
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Thesis (PhD (Biomedical Sciences. Molecular Biology and Human Genetics))--Stellenbosch University, 2008.This series of studies includes both methodological analyses, aimed at furthering our understanding of, and improving the tools used in molecular epidemiology, and investigative projects which have used these tools to add to our knowledge of the M. tuberculosis epidemic. Using serial isolates from tuberculosis patients, we have investigated the evolutionary rate of the IS6110 RFLP pattern. In accordance with other studies, we determined a ½-life for this epidemiological marker of 10.69 years, confirming its appropriateness for this purpose. We also identified an initial, much higher apparent rate which we proposed was the result of pre-diagnostic evolution. In support of this, our investigations in the context of household transmission of M. tuberculosis revealed that IS6110-based evolution is closely associated with transmission of the organism, resulting in a strain population rate of change of 2.9% per annum. To accommodate evolution within estimates of transmission, we proposed that calculations incorporate the concept of Nearest Genetic Distance (cases most similar in RFLP pattern and most closely associated in time). We used this to create transmission chains which allowed for limited evolution of the IS6110 marker. As a result, in our study community, the estimated level of disease attributable to ongoing transmission was increased to between 73 and 88% depending on the Genetic Distance allowed. We identified the duration of a study as a further source of under-estimation of transmission. This results from the artefactual abridgement of transmission chains caused by the loss of cases at the temporal boundaries of a study. Using both real and simulated data, we showed that viewing a 12-year study through shorter window periods dramatically lowered estimates of transmission. This effect was negatively correlated with the size of a cluster. Various combinations of MIRU-VNTR loci have been proposed as an alternative epidemiological marker. Our investigations showed that, while this method yielded estimates of transmission similar to those of IS6110, there was discordance between the two markers in the epidemiological linking of cases as a result of their independent evolution. Attempting to compensate for this by allowing for evolution during transmission improved the performance of IS6110, but generally had a deleterious effect of that of MIRU-VNTR. However, this marker remains a valuable tool for higher phylogenetic analysis and we used it to demonstrate a correlation between sublineages of the Beijing clade and the regions in which they are found. We proposed that, either the host population had selected for a particular sublineage, or that specific sublineages had adapted to be more successful in particular human populations. We further explored the dynamics of the epidemic over a 12-year period in terms of the five predominant M. tuberculosis clades. We found that, while four of these clades remained relatively stable, the incidence of cases from the Beijing clade increased exponentially. This growth was attributed to drug-sensitive cases although drug-resistant Beijing cases also appeared to be more successful than their non-Beijing counterparts. Possible factors contributing to this clade’s success were a greater proportion of positive sputum smears and a lower rate of successful treatment.
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Mlamla, Zandile Cleopatra. "Improving methods for genotypic drug resistance testing in Mycobacterium tuberculosis." Thesis, Stellenbosch : University of Stellenbosch, 2011. http://hdl.handle.net/10019.1/6756.
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Thesis (MScMedSc)--University of Stellenbosch, 2011.ENGLISH ABSTRACT: An important next step to Tuberculosis control relies on the translation of basic science and modern diagnostic techniques into primary health care clinics. These assays must be rapid, inexpensive, interpretation of results must be easy and they must be simple so that a healthcare worker with limited training can perform the tests under safe conditions. This study consists of four aims. The first aim was to develop a methodology to sterilize sputum specimens for rapid TB diagnosis and drug resistance testing. Candidate bactericides were identified from the literature, and tested for their bactericidal activity in Mycobacterium tuberculosis. We identified ultraseptin®aktiv as a powerful bactericidal agent which sterilizes sputum specimens for subsequent safe handling prior to light emitting diode microscopy and it also provides a DNA template for PCR-based tests. An algorithm has been proposed for the processing of specimens and rapid diagnosis of TB and drug resistant TB while patients wait for results. Recently, the World Health Organization has endorsed the MTBDRplus test for diagnosis of TB and drug resistant TB. However genotypic tests may have more problems than anticipated. With the HIV pandemic, an increase of non-tuberculous mycobacteria has been reported. The sensitivity of genotypic tests in specimens with underlying non-tuberculous mycobacterial species therefore requires further evaluation. This study therefore also aimed at determining the reliability of the MTBDRplus assay for detection of drug resistant TB where non-tuberculous bacterial load is high. Clinically relevant non-tuberculous mycobacterium DNA and DNA from a multi-drug resistant TB isolate were obtained. Ratios of the different NTM with the MDR-TB DNA were made and subjected to the MTBDRplus assay. Known mix NTM and TB infected clinical isolates and sputum sediments were also evaluated for TB and drug resistance detection on the MTBDRplus assay. Under these conditions, this study provides evidence that the MTBDRplus test cannot reliably detect TB and drug resistance TB in specimens with underlying non-tuberculous mycobacteria. Thirdly, to evaluate the sensitivity of the MTBDRplus assay for detecting drug resistance in hetero-resistant isolates, ratios were made using purified DNA from an MDR and pan-susceptible TB isolate. The MTBDRplus assay was then performed on the different ratios. We report that the MTBDRplus assay can efficiently detect wild type DNA in genes associated with resistance during the early evolution of drug resistance. However, in the later stage during treatment when both the wild type and mutants are present, the detection limit for the mutant DNA was 1:55. Due to these results, the MTBDRplus assay should still be further improved or other tests should be developed to address these limitations. And finally to combat cross amplicon contamination during the final steps of genotypic detection with the MTBDRplus assay, a proof of concept for a patentable closed tube line probe device was proposed on the 4th aim. This device can be improved to enable automated drug resistance genotyping of multiple specimens. The results of this study highlight the need for a sensitive inexpensive point of care drug resistance test that does not require intensive training.
AFRIKAANSE OPSOMMING: 'n Belangrike volgende stap om Tuberkulose te beheer is om basiese wetenskap resultate te gebruik sodat moderne diagnose tegnieke ontwikkel kan word wat in primêre gesondheidsorg klinieke toegepas kan word. Hierdie toetse moet vinnig, goedkoop, en die interpretasie van resultate moet maklik wees. Die toetse moet eenvoudig wees sodat 'n gesondheidswerker met beperkte opleiding die toetse onder veilige omstandighede kan uitvoer. Hierdie studie bestaan uit vier doelwitte, waarvan die eerste was om 'n metode te ontwikkel vir die sterilisasie van sputum monsters vir vinnige TB diagnose en die toesting van middelweerstandigheid. Kandidaat kiemdodende middels was geïdentifiseer vanaf die literatuur en die middels se kiekdodende aktiviteit was getoets op Mycobacterium tuberculosis. Ons het ultraseptin®aktiv geïdentifiseer as 'n kragtige kiemdodende middel wat bakteria in sputum monsters steriliseer vir veilige hantering voordat diagnose met 'n lig uitstralende diode mikroskopie gedoen kan word. Hierdie behandeling met ultraseptin®aktiv bied ook 'n DNA templaat vir PCR-gebaseerde toetse. 'n Algoritme is voorgestel vir die hantering van monsters en die vinnige diagnose van sensitiewe- en middel weerstandige Tuberkulose terwyl die pasiënte by die kliniek wag vir die resultate. Onlangs het die Wêreld Gesondheid Organisasie die genotipiese MTBDRplus toets vir die diagnose van Tuberkulose en middel-weerstandige Tuberkulose onderskryf. Hierdie toets word tans op groot skaal in Suid Afrika gebruik. Dit kan egter wees dat genotipiese toetse baie meer probleme kan he as wat aanvanklik verwag is. Die HIV pandemie gaan toenemend gepaard met n toename van nie-tuberkulose mycobacteria. Die sensitiwiteit van genotipiese toetse op monsters met onderliggende nie-tuberkulose mikobakteriese spesies vereis dus verdere evaluasie. Die doel van hierdie studie was ook om die betroubaarheid van die MTBDRplus-toets te bepaal vir die opsporing van middelweerstandige TB waar die nie-tuberkulose bakteriële lading hoog is. DNA van kliniese relevante nie-tuberkulose mikobakteria en multi-middelweerstige TB isolate was bekom. Verskillende verdunnigs van die spesifieke NTM DNA te same met die van MDR-TB DNA is gemaak en onderwerp aan die MTBDRplus toets. Bekende gemengde NTM- en TB geïnfekteerde kliniese isolate en sputum sedimente was ook geevalueer vir die opsporing van TB en middel weerstandigheid met die MTBDRplus toets. Hierdie studie verskaf bewyse dat die MTBDRplus toets nie betroubaar is met die diagnose van sensitiewe- en middel weerstandige Tuberkulose in monsters met onderliggende nie-tuberkulose mycobacteria nie. Verskillende verdunnings van gesuiwerde DNA van MDR en pan-sensitiewe TB isolate is gemaak om die sensitiwiteit van die MTBDRplus toets vir die opsporing van middelweerstandigheid te bepaal. Die MDRDRplus toets is gebruik met hierdie verdunnings. Resultate in hierdie studie toon dat die MTBDRplus toets effektief is met die identifisering van wilde-tipe DNA (dit beteken middel sensitief) in gene wat geassosieer word met middel weerstandigheid gedurende die vroeë ontwikkeling van weerstandigheid. Hier teenoor toon die resultate dat in die later stadium tydens behandeling, wanneer beide die wilde-tipe (sensitief) en mutante DNA (weerstandig) teenwoordig is, is die opsporingslimiet vir die mutante DNA maar 1:55. As gevolg van hierdie resultate raai ons aan dat die MTBDRplus toets nog verder verbeter moet word of dat ander toetse ontwikkel moet word om hierdie beperkinge aan te spreek. Amplikon kruiskontaminasie kan n groot impak hê op die betroubaarheid van enige genotipiese diagnostiese toets. Die finale stappe van MTBDRplus toets behels die gebruik van 'n oop sisteem sodat kontaminasie maklik kan plaasvind. In die 4de doewit 'n konsep vir 'n patenteerbare geslotebuis toestel ontwikkel en die resultate het getoon dat kontaminasie suksesvol uitgeskakel kan word. Hierdie toestel kan verbeter na 'n outomatiese apparaat verbeter word sodat die module genotipering van verskeie monsters moontlik kan maak. Die resultate van hierdie studie beklemtoon die noodsaaklikheid van 'n sensitiewe goedkoop “point of care” diagnostiese toets wat nie intensiewe opleiding benodig nie.
Medical Research Council of South Africa
University of Stellenbosch, Dept. of Molecular Biology and Human Genetics
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Storbeck, Karl-Heinz. "A structure/function investigation into baboon cytochrome P450 side-chain cleavage (CYP11A1)." Thesis, Stellenbosch : University of Stellenbosch, 2005. http://hdl.handle.net/10019.1/3113.
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Thesis (MSc (Biochemistry))--University of Stellenbosch, 2005.This study describes: 1. The cloning of baboon cytochrome P450 side-chain cleavage (CYP11A1) cDNA by in vitro site-directed mutagenesis. 2. The identification and sequencing of three baboon CYP11A1 mutants: CYP11A1a, CYP11A1b and CYP11A1c. 3. The expression and characterisation of baboon and human CYP11A1 cDNA, CYP11A1a, CYP11A1b and CYP11A1c in nonsteroidogenic COS-1 cells. The Km and V-values for the metabolism of 25-hydroxycholesterol were determined. 4. The construction of the first homology model of CYP11A1, using both mammalian (CYP2C5) and bacterial (CYP102) cytochromes P450 crystal structures as templates. 5. A structure/function study into the role of the amino acid residues Ile98, Lys103 and Thr291 in substrate binding and enzymatic activity. 6. The proposal of a topological model of the CYP11A1 active pocket as determined by substrate docking studies.
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Hedley, Paula Louise. "Molecular and functional characterisation of Long QT Syndrome causing genes." Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/86480.
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Thesis (PhD)-- Stellenbosch University, 2014.ENGLISH ABSTRACT: Ventricular arrhythmias are the most important cause of sudden cardiac death (SCD) among adults living in industrialised nations. Genetic factors have substantial effects in determining population-based risk for SCD and may also account for inter-individual variability in susceptibility. Great progress has been made in identifying genes underlying various Mendelian disorders associated with inherited arrhythmia susceptibility. The most well studied familial arrhythmia syndrome is the congenital long QT syndrome (LQTS) caused by mutations in genes encoding subunits of myocardial ion channels. Not all mutation carriers have equal risk for experiencing the clinical manifestations of disease (i.e. syncope, sudden death). This observation has raised the possibility that additional genetic factors may modify the risk of LQTS manifestations. This study establishes the genetic aetiology of LQTS in South Africa and Denmark through the identification and characterisation of LQTS-causative mutations in five previously identified genes, as well as examining possible novel genetic causes of LQTS in a cohort comprising Danish and British probands. We have functionally characterised several of the mutations identified in this study and examined other cardiac phenotypes that may be explained by variants causing repolarisation disorders.
AFRIKAANSE OPSOMMING: Ventrikulêre aritmie bly die enkele belangrikste oorsaak van skielike hart dood (SCD) onder volwassenes wat in geïndustrialiseerde lande woon. Genetiese faktore het aansienlike gevolge in die bepaling van bevolking-gebaseerde risiko vir SCD en kan ook verantwoordelik wees vir die inter-individuele variasie in vatbaarheid. Groot vordering is gemaak in die identifisering van gene onderliggende verskeie Mendeliese siektes wat verband hou met geërf aritmie vatbaarheid. Die mees goed bestudeerde familie aritmie sindroom is die aangebore lang QT-sindroom (LQTS) wat veroorsaak word deur mutasies in gene kode subeenhede van miokardiale ioonkanale. Nie alle mutasie draers het 'n gelyke risiko vir die ervaring van die kliniese manifestasies van die siekte (dws sinkopee, skielike dood). Hierdie waarneming het die moontlikheid genoem dat genetiese faktore anders as die primêre siekte-verwante mutasie kan die risiko van LQTS manifestasies verander. Hierdie studie stel die genetiese oorsake van LQTS in Suid-Afrika en Denemarke deur die identifisering en karakterisering van LQTS-veroorsakende mutasies in vyf voorheen geïdentifiseer gene, asook die behandeling van moontlike nuwe genetiese oorsake van LQTS in 'n groep wat bestaan uit van die Deense en die Britse probands. Ons het funksioneel gekenmerk verskeie van die mutasies wat in hierdie studie ondersoek en ander kardiovaskulêre fenotipes wat deur variante veroorsaak repolarisasie versteurings verduidelik word.
South African National Research Foundation
Harry and Doris Crossley Foundation
Danish Strategic Research Foundation.
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Wang, Yajie. "The Time-Course of the Effects of Growth Hormone During Zebrafish (DANIO RERIO) Auditory Hair Cell Regeneration." TopSCHOLAR®, 2012. http://digitalcommons.wku.edu/theses/1171.
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Growth hormone (GH) was upregulated in the zebrafish inner ear following sound exposure in a previous study. To identify the specific role of GH in hair cell regeneration and the possible cellular mechanisms of this regeneration, groups of zebrafish were divided into baseline (no sound exposure, no injection), buffer-injected and GH-injected groups. Buffer- and GH-injected fish were exposed to a 150 Hz tone at a source level of 179 dB re 1 μPa root mean squared (RMS) for 36 h. Phalloidin-staining was used to assess the effects of GH on hair cell bundle density; BrdU-labeling was used to assess the effects of GH on cellular proliferation; TUNEL-labeling was used to assess the effects of GH on apoptosis in the zebrafish inner ear following acoustic trauma. The time-course of hair cell bundle density, cell proliferation, and apoptosis was established by combining data for baseline fishes and sound-exposed fishes at post-sound exposure day 1 (psed1), psed2, and psed3. GH-injected fish exhibited greater densities of hair cells than bufferinjected controls. In addition, GH-injected fish had higher levels of cell proliferation and lower levels of apoptosis than buffer-injected controls. This suggests that GH may play an important role in zebrafish inner ear hair cell regeneration by stimulating cellular proliferation and inhibiting cellular apoptosis.
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Borglin, Matthew R. "Analysis of Biofilm Remediation Capacity For Octenyl Succinic Anhydride (OSA), A Bioactive Food Starch Modifier Compound." DigitalCommons@CalPoly, 2020. https://digitalcommons.calpoly.edu/theses/2168.
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Matthew R. Borglin
This thesis demonstrates efficacy of Octenyl Succinic Anhydride (OSA), as a biofilm sanitizer. Biofilms allow bacteria to adhere to solid surfaces with the use of excreted polymeric compounds. For example, surfaces found in food production or processing facilities such as the interior of a raw milk holding tank, are some of the most susceptible to biofilm contamination. When present, biofilms can cause a variety of negative effects, which include; reduction of product shelf life, corrosion, and outbreaks of foodborne illnesses. The close association of biofilms with the majority of foodborne illness cases led the US Environmental Protection Agency (EPA) to create a new category of sanitizer specifically designed for treatment of mature biofilms. The efficacy of sanitizers in this new regulatory category is determined by the EPA protocols MB-19 and MB-20. The EPA’s protocols outline methods for cultivating, treating, and measuring effects on Pseudomonas aeruginosa biofilms in a continuous flow stir bar bioreactor. Biofilm modification by OSA was verified by the presence of octenyl esters on OSA treated biofilms with single point Raman spectrophotometry. OSA modified biofilm’s antimicrobial properties were first investigated with crystal violet staining in 96-well microtiter plates with inconclusive results. However, effective antimicrobial properties where apparent when using the CDC Biofilm Reactor. OSA treatments consistently returned a 6-log CFU/coupon reduction in biomass compared to controls. Inhibition of planktonic and/or biofilm regrowth was demonstrated using the 96-well plate methodology. This thesis demonstrated the effectiveness of OSA chemical esterification reaction as a biofilm treatment. In doing so, this work suggests a new approach for biofilm remediation by chemically modifying the structural components of biofilm.
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Kaushansky, Laura J. "Investigating the Effects of Mutant FUS on Stress Response in Amyotrophic Lateral Sclerosis: A Thesis." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/792.
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During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress.
The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics.
I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.
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Kaushansky, Laura J. "Investigating the Effects of Mutant FUS on Stress Response in Amyotrophic Lateral Sclerosis: A Thesis." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/792.
Full textAbstract:
During stress, eukaryotes regulate protein synthesis in part through formation of cytoplasmic, non-membrane-bound complexes called stress granules (SGs). SGs transiently store signaling proteins and stalled translational complexes in response to stress stimuli (e.g. oxidative insult, DNA damage, temperature shifts and ER dysfunction). The functional outcome of SGs is proper translational regulation and signaling, allowing cells to overcome stress.
The fatal motor neuron disease Amyotrophic Lateral Sclerosis (ALS) develops in an age-related manner and is marked by progressive neuronal death, with cytoplasmic protein aggregation, excitotoxicity and increased oxidative stress as major hallmarks. Fused in Sarcoma/Translocated in Liposarcoma (FUS) is an RNA-binding protein mutated in ALS with roles in RNA and DNA processing. Most ALS-associated FUS mutations cause FUS to aberrantly localize in the cytoplasm due to a disruption in the nuclear localization sequence. Intriguingly, pathological inclusions in human FUSALS cases contain aggregated FUS as well as several SG-associated proteins. Further, cytoplasmic mutant FUS incorporates into SGs, which increases SG volume and number, delays SG assembly, accelerates SG disassembly, and alters SG dynamics.
I posit that mutant FUS association with stress granules is a toxic gain-of-function in ALS that alters the function of SGs by interaction with SG components. Here, I show that mutant FUS incorporates in to SGs via its Cterminal RGG motifs, the methylation of which is not required for this localization. Further, I identify protein interactions specific to full-length mutant FUS under stress conditions that are potentially capable of interacting with FUS in SGs. Finally, I demonstrate a potential change in the protein composition of SGs upon incorporation of mutant FUS. These findings advance the field of ALS and SG biology, thereby providing groundwork for future investigation.
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Fong, Joseph D. "The Distinction of the Interactions Between the Transmembrane Domains of Basigin Gene Products and Monocarboxylate Transporters." UNF Digital Commons, 2018. https://digitalcommons.unf.edu/etd/788.
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Although it was once thought that neurons solely rely on glucose as a substrate for cellular energy production, it is now known that small monocarboxylate molecules, like pyruvate, lactate, and ketone bodies, are also utilized. Monocarboxylates are transported across plasma membranes via facilitated diffusion using a family of transport proteins known as monocarboxylate transporters (MCTs). Four MCTs (MCT1, MCT2, MCT3, and MCT4) are expressed within neural tissues. Expression of the MCTs has been tied to co-expression of a cell adhesion molecule belonging to the Basigin subset of the immunoglobulin superfamily (IgSF). Basigin gene products are known to interact with MCT1 and MCT4 in the mammalian neural retina and this association is essential to support the cellular energy needs of photoreceptors. A previous study indicated that Basigin gene products use hydrophobic amino acids within specific regions of the transmembrane domain to interact with MCT1. In the present study, it is hypothesized that the same amino acids within the transmembrane domain are used to interact with MCT4, but that no association exists with MCT2, which typically interacts with a different member of the IgSF subset. Therefore, the purpose of the present study was to assess the association between Basigin gene products and MCT4, and with MCT2. Recombinant proteins corresponding to the transmembrane domain of Basigin gene products were used in in vitro binding assays with endogenous MCT2 and MCT4 from mouse brain protein lysates. Contrary to the hypothesis, it was determined that the transmembrane domain of Basigin gene products binds to both MCT2 and MCT4 in vitro. Different amino acids within the transmembrane domain of Basigin gene products are used for each association and the pattern is different from that used in the association with MCT1. The data suggest that Basigin plays multiple roles in the nervous system.
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Lucca, Julie Ann. "ASSOCIATIONS BETWEEN ALCOHOL CONSUMPTION AND FASTING BLOOD GLUCOSE IN YOUNG ADULTS." DigitalCommons@CalPoly, 2013. https://digitalcommons.calpoly.edu/theses/1057.
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Current research shows moderate alcohol consumption is associated with decreased risk of diabetes and excessive consumption or binge drinking can cause insulin resistance and diabetes. In 2010, diabetes was the seventh leading cause of death in the United Statesand was responsible for significant health complications: blindness, kidney failure, and limb amputations, and is a large national economic burden. Fasting blood glucose (FBG) is a tool used to help diagnose diabetes. Abnormally high FBG, ≥100 mg/dl, is indicative of diabetes and pre-diabetes. Few studies have observed diabetic prevalence among young adults or college students. Studying young adults can help provide added information about early risk factors for diabetes and pre-diabetes, facilitating public health efforts to stem the rising tide of the diabetes epidemic. This study aimed to research the associations between alcohol consumption (numbers of days alcohol consumed in the past month and binge alcohol consumption in the past month) and FBG in a college population as part of the FLASH cohort study. FBG levels were measured in 141 young adult participants and alcohol consumption was determined by self report. Other individual-level characteristics and potential confounding variables were also collected. The association between alcohol consumption and FBG followed a J-shaped curve whereby students who reported drinking 6-8 days within the last 30 days showed significantly lower FBG levels than those who did not drink and those who consumed alcohol on nine or more days (p=0.04). Binge drinking did not have a significant association with FBG (p=0.4). Sex and body mass index were also significantly associated with FBG. In conclusion, moderate frequency of alcohol consumption is found to have an inverse relationship with FBG and excessive drinking can reverse these effects.
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Laisse, Claudio Joao Mourao. "Characterization of tuberculous lesions in naturally infected African buffalo (Syncerus caffer)." Thesis, Stellenbosch : University of Stellenbosch, 2010. http://hdl.handle.net/10019.1/5348.
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Thesis (MScMedSc (Biomedical Sciences. Medical Biochemistry))--University of Stellenbosch, 2010.ENGLISH ABSTRACT: Mycobacterium bovis has a wide host range and infects many wild and domestic animal species as well as humans. African buffalo (Syncerus caffer) is considered to be a wildlife reservoir of M. bovis in certain environments in South Africa, such as in the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP). A detailed pathological study was conducted on 19 African buffalos (Syncerus caffer) from a herd in the HiP in South Africa. The animals tested positive to the intradermal bovine tuberculin test and were euthanazed during a test-and-cull operation to decrease the prevalence of bovine tuberculosis (bTB) in the park. The superficial, head, thoraxic and abdominal lymph nodes and the lungs were examined grossly for presence of tuberculous lesions and were scored on a 1-5 scale for macroscopic changes. The gross lesions were examined histologically and scored I-IV according to a grading system used for bTB lesions in domestic cattle. Macroscopical lesions were limited to the retropharyngeal, bronchial, and mediastinal lymph nodes and the lungs. The most frequently affected lymph nodes were the bronchial (16/19) and mediastinal (11/19). All four grades of microscopic lesions were observed, although grade II lesions were the most frequent. Acid-fast bacilli were observed only rarely. Bovine tuberculosis was confirmed by PCR analyses. All animals were in good body condition and most of the lesions were in an early stage of development, indicating an early stage of the disease. The absence of lesions in the mesenteric lymph nodes and the high frequency of lesions in respiratory tract associated lymph nodes suggest that the main route of M. bovis infection in African buffalo is inhalatory rather than alimentary. This study presents a systematic evaluation and semiquantification of the severity and stages of development of tuberculous lesions in buffalo. The results may contribute to i) the understanding of the pathogenesis of the disease, ii) the evaluation of experimental models of M. bovis infection in Syncerus caffer, and iii) the interpretation of pathological data from vaccination trials.
AFRIKAANSE OPSOMMING: Mycobacterium bovis het ‘n wye reeks van gashere en dit infekteer verskeie wilde en mak dierespesies, sowel as mense. Die buffel (Syncerus caffer) word beskou as die wild reservoir van M. bovis in sekere dele van Suid Afrika, soos in die Kruger Nasionale Park (KNP) en Hluhluwe-iMfolozi Park (HiP). ‘n Breedvoerige patologiese studie is uitgevoer op 19 buffels afkomstig vanaf ‘n trop in die HiP in Suid Afrika. Die diere het almal positief getoets vir die intradermale beestuberkulin toets en is uitgesit tydens ‘n toets-en-slag operasie met die doel om die voorkoms van beestuberkulose (bTB) in die park te bekamp. Die oppervlakkige, kop, toraks en abdominale limfknope en longe is oorsigtelik ondersoek vir die teenwoordigheid van tuberkulose letsels en was ‘n punt toegeken op ‘n skaal van 1-5 vir die teenwoordigheid van makroskopiese veranderinge. Die opsigtelike letsels is histologies ondersoek en ‘n I-IV punt toegeken volgens die gradering wat gebruik word vir bTB letsels in beeste. Makroskopiese letsels was beperk tot die retrofaringeale, brongiale, en mediastinale limfknope en in die longe. Die brongiale (16/19) en mediastinale (11/19) limfknope was meestal geaffekteerd. Al vier grade van mikroskopiese letsels is gevind, alhoewel graad II letsels die volopste was. Suur-vaste basille is slegs selde waargeneem. Beestuberkulose is bevestig deur PKR analises. Al die diere was in ‘n goeie kondisie en meeste van die letsels was in ‘n vroeë stadium van ontwikkeling, wat aandui op ‘n vroeë fase van die siekte. Die afwesigheid van letsels in die mesenteriese limfknope en die hoë frekwensie van letsels in die lugweg geassosieerde limfkliere dui daarop dat the belangrikste roete van M. bovis infeksie in die buffel deur inaseming geskied eerder as deur opname in die spysverteringskanaal. Hierdie studie bied ‘n stelselmatige evaluering en semi-kwantifisering van die graad van erns en die stadia van ontwikkeling van tuberkulose letsels in buffels. Die resultate kan bydra tot i) die begrip van die patogenese van die siekte, ii) die evaluering van eksperimentele modelle van M. bovis infeksie in Syncerus caffer, en iii) die interpretasie van patologiese data van inentingsproewe.
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Toll, Andrea Lee. "Racemization of Amino Acids in Teeth for the Determination of Age." TopSCHOLAR®, 2012. http://digitalcommons.wku.edu/theses/1144.
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Instrumental to forensic investigations is the ability to identify unknown human remains providing key evidence to criminal cases, resolution to missing persons, and assistance in mass or natural disasters. Identification of remains in an effort to determine age is an area of forensics that has received considerable attention. Traditional methods in age determination such as morphology are often biased, antiquated, and frequently result in a large margin of error. Conversely, the emergence of new forensic techniques provide promise to reduce the margin of error in determining age. One such technique has focused on relating the extent of amino acid racemization in teeth to age. Past research has focused primarily on the analysis of aspartic acid due to its high racemization rate. Our research indicates that glutamic acid also shows promise as related to age determination. Results will be presented illustrating optimization of gas chromatography using a chiral column for separation of amino acids found in dentin and their enantiomeric ratio quantification. Age correlation data will be presented on collected teeth ranging from mid-teens to early seventies.
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Du, Xing. "Investigation of RNA Binding Protein Pumilio as a Genetic Modifier of Mutant CHMP2B in Frontotemporal Dementia (FTD): A Masters Thesis." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/846.
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Frontotemporal dementia (FTD) is the second most common early-onset dementia. A rare mutation in CHMP2B gene was found to be associated with FTD linked to chromosome 3. Previous studies have shown that mutant CHMP2B could lead to impaired autophagy pathway and altered RNA metabolism. However, it is still unknown what genes mediate the crosstalk between different pathways affected by mutant CHMP2B. Genetic screens designed to identify genes interacting with mutant CHMP2B represents a key approach in solving the puzzle. Expression of mutant CHMP2B (CHMP2Bintron5) in Drosophila eyes leads to a neurodegenerative phenotype including melanin deposition and disrupted internal structure of ommatidia. The phenotype is easily quantified by estimating the percentage of black dots on the surface of the eyes. Using this established Drosophila model, I searched for genes encoding RNA binding proteins that genetically modify CHMP2Bintron5 toxicity. I found that partial loss of Pumilio, a translation repressor, mitigates CHMP2Bintron5 induced toxicity in the fly eyes. Western blot analysis showed that down regulation of Pumilio does not significantly decrease CHMP2Bintron5 protein level, indicating indirect regulation involved in suppression of the phenotype. The molecular targets regulated by Pumilio and the mechanism underlying CHMP2Bintron5 toxicity suppression by Pumilio down-regulation requires further investigation.
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Du, Xing. "Investigation of RNA Binding Protein Pumilio as a Genetic Modifier of Mutant CHMP2B in Frontotemporal Dementia (FTD): A Masters Thesis." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/846.
Full textAbstract:
Frontotemporal dementia (FTD) is the second most common early-onset dementia. A rare mutation in CHMP2B gene was found to be associated with FTD linked to chromosome 3. Previous studies have shown that mutant CHMP2B could lead to impaired autophagy pathway and altered RNA metabolism. However, it is still unknown what genes mediate the crosstalk between different pathways affected by mutant CHMP2B. Genetic screens designed to identify genes interacting with mutant CHMP2B represents a key approach in solving the puzzle. Expression of mutant CHMP2B (CHMP2Bintron5) in Drosophila eyes leads to a neurodegenerative phenotype including melanin deposition and disrupted internal structure of ommatidia. The phenotype is easily quantified by estimating the percentage of black dots on the surface of the eyes. Using this established Drosophila model, I searched for genes encoding RNA binding proteins that genetically modify CHMP2Bintron5 toxicity. I found that partial loss of Pumilio, a translation repressor, mitigates CHMP2Bintron5 induced toxicity in the fly eyes. Western blot analysis showed that down regulation of Pumilio does not significantly decrease CHMP2Bintron5 protein level, indicating indirect regulation involved in suppression of the phenotype. The molecular targets regulated by Pumilio and the mechanism underlying CHMP2Bintron5 toxicity suppression by Pumilio down-regulation requires further investigation.
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Partington, Joanna Clair. "Biochemistry and molecular biology of potato bruising." Thesis, Royal Holloway, University of London, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.265182.
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Oikonomou, Eftychia. "Molecular biology and biochemistry of brain tumours." Thesis, Aston University, 2004. http://publications.aston.ac.uk/11021/.
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Elucidating some molecular mechanisms and biochemistry of brain tumours is an important step towards the development of adjuvant medical therapies. The present study concentrates on cholecystokinin (CCK), a gut-brain peptide that has been described to be able to induce mitosis of rat gliomas as well as hormone secretion by the anterior pituitary, via the CCK-B receptor. The significance of a polymorphism in the growth hormone releasing hormone (GHRH) receptor (GHRH-R) gene was also determined. Finally, defects in the b-catenin gene, an important component of the developmental pathway, in a sub-set of craniopharyngiomas were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), restriction digestion analysis and direct sequencing demonstrated expression of CCK peptide itself and its A and B receptors by human gliomas, meningiomas and pituitary tumours. CCK peptides stimulated growth of cultured gliomas and meningiomas as well as in vitro hormone secretion [growth hormone (GH), luteinizing hormone (LH) and follicle stimulating hormone (FSH)] by human pituitary tumours. These biological effects were reduced or abolished by CCK antagonists. In addition, an antibody to CCK reduced mitosis by gliomas and meningiomas, and the same antibody inhibited hormone secretion by cultured human pituitary tumours. CCK peptides stimulated phosphatidylinositol (PI) hydrolysis, indicating coupling of the CCK receptors to phopsholipase C. Cyclic AMP was unaffected. In addition, caspase-3 activity was significantly and markedly increased, whilst proteasome activity was decreased. Taken together, these results may indicate an autocrine/paracrine role of CCK in the control of growth and/or functioning of gliomas, meningiomas and pituitary tumours. Further findings of this study, using PCR and direct sequencing, were the demonstration of an association between b-catenin gene alterations and craniopharyngiomas of the adamantinomatous type.
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Ye, Qing. "LIPASE-KINASE ASSOCIATIONS INVOLVING PLD2, JAK3 AND FES THAT UNDERLIE CANCER CELL PROLIFERATION AND INVASION." Wright State University / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=wright1421939242.
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Klingler, Andrea M. "Novel Insight into the Role of LXRa in Metabolic Regulation viaDNA Binding as a Heterodimer with PPARa and as a Homodimer." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1472486254.
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Gould, Elaine M. "The molecular biology and biochemistry of resistance to rodenticides." Thesis, University of Reading, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367721.
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Wyborn, Neil Ross. "Biochemistry and molecular biology of amidases from Methylophilus methylotrophus." Thesis, University of Leicester, 1994. http://hdl.handle.net/2381/35182.
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The biochemistry and molecular biology of amidases from M. methylotrophus was investigated. Acetamidase purification from whole cells exhibiting low specific activities yielded pure low-activity acetamidase (specific activity 6-15 umol min-1 [mg protein]-1), the activity of which could be reactivated to a level approaching that of the high-activity acetamidase by heating (1-6 h, 60 °C) with an activator component. Identical purifications from whole cells with high specific activities produced pure 'high-activity' acetamidases exhibiting a wide range of generally diminished activities (19-108 umol min-1 [mg protein]-1) and an unexpected propensity for heat-reactivation similar to that of low-activity acetamidase. It was concluded that high-activity acetamidases underwent varying degrees of 'switch-off of activity both pre- and post-purification. The physico-chemical properties of purified acetamidases were investigated in vitro to elucidate the nature of the putative acetamidase post-transcriptional modification and its role in the reversible regulation of acetamidase activity. High- and low-activity acetamidases exhibited significantly different properties, although their respective MW values differed only by 52Da. Low-activity acetamidase was significantly more stable than high-activity acetamidases which were labile. Results suggested that high- and low-activity acetamidases existed in different conformational states and that the regulation of acetamidase activity probably involved an allosteric mechanism. Cloning of the M. methylotrophus acetamidase structural gene (amiE) was unsuccessful, but the formamidase structural gene (fmd) was successfully cloned and heterologously- expressed in E. coli. The DNA sequences of fmd and three putative ORFs showed that (i) the formamidase primary sequence exhibited 57% strict identity with that of the Mycobacterium smegmatis 'acetamidase' (Mahenthiralingam et al., 1993), and (ii) ORF2 and ORF3 apparently respectively encoded a zinc finger DNA-binding protein and an AmiC-type regulatory protein. The evolutionary and regulatory implications of these findings were discussed.
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Moncivaiz, Jessica. "Differences in fecal metabolite profiles from geographically distinct populations of adolescents." Wright State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=wright1452675142.
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Wright, Duncan Hamish. "Biochemistry, molecular biology and pharmacology of the prostanoid DP receptor." Thesis, McGill University, 2000. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=36852.
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The term prostanoids collectively describes prostaglandins, prostacyclin and thromboxanes. These compounds are products of arachidonic acid metabolism by the cyclooxygenase pathway. Prostanoids mediate various physiological and pathophysiological effects through their interaction with membrane-bound receptors. In this research, a thorough characterization of the recombinant human (h) PGD2 receptor (DP) was performed, with respect to its radioligand binding and signal transduction properties using prostanoids and prostanoid analogues. The recombinant hDP receptor was then used along with other recombinant human prostanoid receptors to identify a novel specific agonist, L-644,698. This compound exhibits high affinity and potency at the hDP receptor. Moreover. L-644,698 demonstrates at least 300-fold higher selectivity for the hDP receptor than for any of the other seven recombinant human prostanoid receptors tested. Thus. L-644,698 is one of the most selective DP-specific agonists as yet described. Subsequently, the rat (r) DP receptor was cloned and functionally expressed. Pharmacological characterization using L-644,698 and other DP-specific ligands confirmed the identity of this protein as a homologue of human DP, and validated the use of the cDNA corresponding to rDP as a template from which to make rDP-specific riboprobes for in situ hybridization studies. mRNA corresponding to rDP was localized to the CNS and GI tract by the in situ hybridization technique. Within the GI tract. rDP-specific signals were observed repeatedly in the mucous-secreting goblet cells and, less often, in the adjacent epithelium of the stomach, duodenum, ileum, and colon. These observations corroborate prior data demonstrating an abundance of both hDP- and mDP-specific mRNA in G1 tract tissues (especially in small intestine), and suggest a novel biological role for the DP receptor, namely the regulation of mucin secretion. DP-specific mRNA was then localized to the mucous-secreting gob
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MacHugh, Niall. "The biochemistry, molecular biology, and cellular expression of bovine CD8." Thesis, Brunel University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336659.
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Prior, Stephen H. "The biochemistry and molecular biology of endosperm texture in wheat." Thesis, University of Bristol, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.402314.
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Ward, Michael Patrick. "Biochemistry, genetics and molecular biology of nitrite reduction in barley." Thesis, University of St Andrews, 1997. http://hdl.handle.net/10023/14341.
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Nitrite reduction is the third step of the nitrate assimilation pathway in higher plants and is catalysed by nitrite reductase. The whole-plant barley mutants STA1010, STA2760 and STA4169 accumulate nitrite in the leaf after treatment with nitrate and, like the nir1 mutant STA3999 (Duncanson et al, 1993), lack detectable nitrite reductase cross-reacting material in the leaf and root. STA1010, STA2760 and STA4169 carry a recessive mutation in a single nuclear gene, identified as the Nir1 locus. RFLP analysis of the nir1 mutant STA3999 has allowed the Nir1 locus to be mapped to within 0.3cM of the nitrite reductase apoprotein gene, Nii. Studies to confirm the identity of the Nir1 locus as Nii, by establishing the full-length Nii cDNA sequences from STA3999 and from its wild-type cv Tweed for comparative purposes, were unsuccessful as attempts to isolate a Nii cDNA clone from a barley cv Tweed cDNA library yielded only partial-length Nii clones. These nirl mutants display greatly reduced nitrite reductase activity and increased NADH-nitrate reductase activity in the leaf, as compared to wild-type plants, suggesting a regulatory perturbation in the expression of the Nar1 gene. Northern analysis shows that the nir1 mutants possess nitrite reductase apoprotein (nii) transcript of wild-type size (2.3kb) and at approximately wild-type levels. Since nir1 mutants possess a phenotype that might be anticipated for a Nii mutant, it is likely that the nir1 mutation is present in the nitrite reductase apoprotein gene Nii and affects translation of the nii transcript. Studies of barley wild-type cv Golden Promise have demonstrated that nitrite reductase in leaf tissue is up-regulated by a coaction of nitrate and light which acts, at least partly, at the transcriptional level.
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Chu, Wei. "Mouse Mast Cell Proteases: Induction, Molecular Cloning, and Characterization." Digital Commons @ East Tennessee State University, 1991. https://dc.etsu.edu/etd/2656.
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Tryptase, a mast cell-specific serine protease with trypsin-like specificity, has been identified in a mouse mast cell line (ABFTL-6) based on it's enzymatic activity, inhibition properties, and cross-reactivity to a human mast cell tryptase antibody. The effects of fibroblast-conditioned medium and sodium butyrate on ABFTL-6 mast cell differentiation and tryptase expression have been examined. ABFTL-6 mouse mast cells undergo phenotypic changes upon culturing in media supplemented with fibroblast-conditioned media at 50% or 1 mM sodium butyrate. The induced cells increased in size, had larger and more metachromatic cytoplasmic granules, and increased their total cellular protein about four-fold. Tryptase activity increased 13- and 6-fold upon fibroblast-conditioned media and butyrate induction, respectively. However, tryptase antigen levels increased dramatically from 2.3 $\mu$g/10$\sp6$ uninduced cells to 125 (54-fold) and 75 (33-fold) $\mu$g/10$\sp6$ cells induced with fibroblast-conditioned media or butyrate, respectively. A cDNA library was constructed in $\lambda$gt10 from ABFTL-6 cell poly(A)$\sp+$ RNA, and screened with dog mast cell tryptase and rat mast cell chymase cDNAs. Clones encoding two distinct tryptases (mouse tryptases I and II), a chymase (mouse chymase I) and a novel carboxyl terminal chymase (mouse chymase II) were isolated and sequenced. Mouse tryptases I and II have 75% and 70% sequence identity at the nucleotide and amino acid levels, respectively. The deduced amino acid sequence for the mature active enzyme for each mouse tryptase contains 245 residues and all the characteristics of a serine protease. Asp is found in the substrate binding pockets, consistent with a trypsin-like specificity for Arg-X and Lys-X bonds. It is predicted that tryptases are synthesized with prepropeptides, requiring signal peptidase processing and removal of a three amino acid propeptide for activation. Mouse chymase I consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. An Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine protease.
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cao, nan. "Structure and Mechanism of Mycobacterial Topoisomerase I." FIU Digital Commons, 2018. https://digitalcommons.fiu.edu/etd/3747.
Full textAbstract:
The enzyme DNA topoisomerase I is an essential enzyme that plays an important role in eukaryotic and prokaryotic cellular processes such as DNA replication, transcription, recombination and repair. Mycobacterium tuberculosistopoisomerase I (MtTOP1) is a validated drug target for antituberculosis treatment. Mycobacterial topoisomerase I regulates the topological constraints in chromosomes and helps in maintaining the growth of mycobacteria. The N- terminal domain (NTD) of mycobacterial topoisomerase I contains conserved catalytic domains that along with the active site Tyrosine are involved in cleaving and rejoining a single strand of DNA. Magnesium is required in DNA cleavage activity of type IA topoisomerases. The C-terminal domain (CTD) of mycobacterial topoisomerase I is divided into four subdomains (D5-D8) and a positively charged tail. Each subdomain has a GxxGPY sequence motif. The DNA binding, relaxation, cleavage, religation, catenation and decatenation ability of each subdomains of CTD were studied. The present study shows that each subdomain has its own characteristics. Subdomain D8 and D7 are responsible for maintaining the relaxation activity of mycobacterial topoisomerase I. Subdomain D5 is essential to maintain the DNA cleavage, religation, catenation and decatenation activity. A new crystal structure of MtTOP1-704t (amino acids A2-T704 containing NTD+D5 domains) was obtained. Structures with ssDNA substrate bound to the active site (Y342) in the presence and absence of Mg2+ were also investigated. Significant enzyme conformational changes upon DNA binding place the catalytic tyrosine in a pre-transition position for cleavage of a specific phosphodiester linkage to form a covalent intermediate. Meanwhile, the enzyme/DNA complex with Mg2+ bound at active site may present the post- transition state for religation in the enzyme’s multiple-state DNA relaxation activity. The critical function of a strictly conserved glutamic acid in acid-base catalysis of the DNA cleavage step was also demonstrated by site-directed mutagenesis. The present work provides new functional insights into the more stringent requirement for DNA rejoining versus cleavage by type IA topoisomerase, and further establishes the potential for select interference of DNA rejoining via specific inhibitors.
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Cheong, Hoi I. "MOLECULAR AND PHYSIOLOGICAL RESPONSES TO HYPOXIA." Case Western Reserve University School of Graduate Studies / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1489498325435621.
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"Molecular biology and biochemistry of a novel conjugation factor in Agrobacterium." Adelaide Thesis (Ph.D.) -- University of Adelaide, Faculty of Agricultural and Natural Resources, Department of Crop Protection, 1993. http://web4.library.adelaide.edu.au/theses/09PH/09phz633.pdf.
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St, Germain Bryan J. "Connecting Motors and Membranes: A Quantitative Investigation of Dynein Pathway Components and in vitro Characterization of the Num1 Coiled Coil Domain." 2011. https://scholarworks.umass.edu/theses/720.
Full textAbstract:
In the budding yeast, Saccharomyces Cerevisiae, dynein, a minus-end directed motor, is involved in nuclear migration and proper orientation of the mitotic spindle during mitosis. Our lab has developed a model that involves the loading of cytoplasmic dynein onto the plus-end of astral microtubules through interactions with Pac1/LIS1 and Bik1/CLIP-170. Dynein is then delivered to the cell cortex and anchored through a cortical receptor protein, Num1. Num1 is a 313KDa protein that localizes to the cell cortex and is an essential component of dynein mediated nuclear migration.
Using quantitative fluorescence techniques I was able to create a molecular inventory of various dynein pathway components. Our results revealed Dyn1, dynein heavy chain, and Pac1/LIS1 associate at the plus end in a 1:1 ratio. Additionally we found that dynein and dynactin associate in a 3:1 ratio at the plus ends and a 2:1 ratio at the cortex. Interestingly, we found that over expression of Pac1/LIS1 augments cortical dynein activity while maintaining the dynein to dynactin ratio and this activity is separate from loss of She1, a negative regulator of dynein-dynactin interaction, which results in a 1:1 ratio of dynein-dynactin at the plus-ends, as well as, the cortex. Our results uncover molecular ratios that enable us to create more defined and detailed model of the dynein pathway.
To elucidate how Num1 attaches dynein to the cortex we created truncations of the Num1 protein. We were able to determine that two coiled-coil (CC) domains in the N-terminus of Num1 are responsible for bright foci formation on the cortex. Cells without these bright foci exhibit a binucleate phenotype similar to that of dyn1∆ implicating that these bright foci are required for the proper function of Num1 in the dynein pathway.
To test the hypothesis that the CC is capable of mediated bright patch assembly through self-association I purified a recombinant CC domain and performed gel filtration analysis, as well as, equilibrium sedimentation. I was able to determine that the CC domain exists as a dimer in solution. However, the mechanism of CC self-assembly may involve a requisite of targeting the CC to the cortex first.
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Gupthar, Abindra Supersad. "Biochemistry students' difficulties with the symbolic and visual language used in molecular biology." Thesis, 2007. http://hdl.handle.net/10413/3562.
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This study reports on recurring difficulties experienced by undergraduate students with respect to understanding and interpretation of certain symbolism, nomenclature, terminology, shorthand notation, models and other visual representations employed in the field of Molecular Biology to communicate information. Based on teaching experience
and guidelines set out by a four-level methodological framework, data on various topic-related difficulties was obtained by inductive analyses of students’ written responses to specifically designed, free-response and focused probes. In addition, interviews, think-aloud exercises and student-generated diagrams were also used to collect information.
Both unanticipated and recurring difficulties were compared with scientifically correct propositional knowledge, categorized and subsequently classified. Students were adept at providing the meaning of the symbol “Δ” in various scientific contexts; however, some failed to recognize its use to depict the deletion of a leucine biosynthesis gene in the
form, Δ leu. “Hazard to leucine”, “change to leucine” and “abbreviation for isoleucine” were some of the erroneous interpretations of this polysemic symbol. Investigations on
these definitions suggest a constructivist approach to knowledge construction and the inappropriate transfer of knowledge from prior mental schemata. The symbol, “::”, was
poorly differentiated by students in its use to indicate gene integration or transposition and in tandem gene fusion. Idiosyncratic perceptions emerged suggesting that it is, for
example, a proteinaceous component linking genes in a chromosome or the centromere itself associated with the mitotic spindle or “electrons” between genes in the same way
that it is symbolically shown in Lewis dot diagrams which illustrate covalent bonding between atoms. In an oligonucleotide shorthand notation, some students used valency to differentiate the phosphite trivalent form of the phosphorus atom from the pentavalent phosphodiester group, yet the concept of valency was poorly understood. By virtue of the visual form of a shorthand notation of the 3,5 phosphodiester link in DNA, the valency was incorrectly read. VSEPR theory and the Octet Rule were misunderstood or forgotten when trying to explain the valency of the phosphorus atom in synthetic oligonucleotide intermediates. Plasmid functional domains were generally well-understood although restriction mapping appeared to be a cognitively demanding task. Rote learning and substitution of definitions were evident in the explanation of promoter and operator
functions. The concept of gene expression posed difficulties to many students who believed that genes contain the entity they encode. Transcription and translation of in tandem gene fusions were poorly explained by some students as was the effect of plasmid conformation on transformation and gene expression. With regard to the selection of transformants or the hybridoma, some students could not engage in reasoning or lateral thinking as protoconcepts and domain-specific information were poorly understood. A failure to integrate and reason with factual information on phenotypic traits, media components and biochemical pathways were evident in written and oral presentations. DNA-strand nomenclature and associated function were problematic to some students as
they failed to differentiate coding strand from template strand and were prone to interchange the labelling of these. A substitution of labels with those characterizing DNA replication intermediates demonstrated erroneous information transfer. DNA replication models posed difficulties integrating molecular mechanisms and detail with line drawings, coupled with inaccurate illustrations of sequential replication features. Finally, a remediation model is presented, demonstrating a shift in assessment score dispersion from a range of 0 - 4.5 to 4 - 9 when learners are guided metacognitively to work with domain-specific or critical knowledge from an information bank. The present work shows that varied forms of symbolism can present students with complex learning difficulties as the underlying information depicted by these is understood in a superficial way. It is imperative that future studies be focused on the standardization of symbol use, perhaps governed by convention that determines the manner in which threshold information is disseminated on symbol use, coupled by innovative teaching strategies which facilitate an improved understanding of the use of symbolic representations in Molecular Biology. As Molecular Biology advances, it is likely that experts will continue to use new and diverse forms of symbolic representations to explain their findings. The explanation of futuristic Science is likely to develop a symbolic language that will impose great teaching
challenges and unimaginable learning difficulties to new generation teachers and learners, respectively.Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.
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Gupta, Snehalata. "Molecular interactions among PAP-1, SPHs and proPO in activation of prophenoloxidase." 2004. http://digital.library.okstate.edu/etd/umi-okstate-1040.pdf.
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Patel, Purvi H. "Characterization of a crosslink between xyloglucan and rhamnogalacturonan from cotton cell walls." 2009. http://digital.library.okstate.edu/etd/Patel_okstate_0664M_10704.pdf.
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Harris, Sebastian M. "Automatic composition of web services using intelligent agent." 2009. http://digital.library.okstate.edu/etd/Harris_okstate_0664M_10434.pdf.
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Howard, Alisha Dawn. "ApoA-I induced lipid efflux from adipocytes." 2010. http://digital.library.okstate.edu/etd/Howard_okstate_0664D_10800.pdf.
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Liu, Xiaoying. "Studies of extra fragments of the cytochrome bc₁ complex from Rhodobacter sphaeroides and the interaction between cytochrome caa₃ and F₁F₀-ATP synthase from alkaliphilic Bacillus pseudofirmus OF₄." 2006. http://digital.library.okstate.edu/etd/umi-okstate-1700.pdf.
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