Doctoral Degrees (Biochemistry)
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Browsing Doctoral Degrees (Biochemistry) by Author "Anderson, Trevor Ryan."
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Item Assessment of lysine damage during food processing.(1985) Anderson, Trevor Ryan.; Quicke, George Venn.The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed.Item Biochemistry students' difficulties with the symbolic and visual language used in molecular biology.(2007) Gupthar, Abindra Supersad.; Anderson, Trevor Ryan.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.Item The development of assays for atractyloside and its localisation in rat tissue.(1991) Bye, Sandra Noel.; Dutton, Michael Francis.; Anderson, Trevor Ryan.An extract of the tuber of Callilepis laureola is regarded as the source of a powerful therapeutic agent, known as Impila. Its use is associated with fatal hepatic and renal necrosis, the renal toxin being atractyloside (ATR). The aims of this study were threefold. Firstly, to generate a model set of biological specimens (urine, serum, liver and kidney) from rats dosed with 5-25 mg ATR/kg bwt. Secondly, to develop a competitive ELISA and HPLC method for the diagnosis of ATR poisoning employing the model specimens as test samples. Thirdly, to localise the target organs, cells and organelles of ATR, in vivo. The HPLC method necessitated the systematic development of the derivatisation of ATR with 9-anthryldiazomethane, sample clean up employing hexane, methanolic hydrochloric acid and a silica minicolumn, as well as the chromatographic conditions. Optimal resolution was obtained with a 3.9 x 150 mm NovaPak reverse phase column, fluorescence detection (excitation = 365 nm, emission = 425 nm) and a solvent system of MeOH:1M ammonium acetate:1M glacial acetic acid:water (38:2:2:58). This method has a detection limit of 0.001 ng ATR, shows a mean recovery of 89% and detected approximately 6.7 ug ATR/g wet weight of tuber tissue. The toxin was also detected in some of the urine samples at levels of about 200 pg/ml, but not in the serum. The production of antibodies to ATR for use in the ELISA and immunocytochemical investigations required the investigation of the conjugation procedure, carrier type, host species and immunization protocol. Optimal antibody yield, specificity and affinity was obtained with an acid-treated Salmonella minnesota bacterial carrier conjugated to ATR by carbodiimide, although there were indications of class switch inhibition and Tlymphocyte suppression by ATR. The development of the ELISA yielded a protocol involving the coating with a bovine serum albumin-ATR conjugate, blocking with bovine serum albumin, incubating the primary antibody at 4°C and detection with a secondary antibody-alkaline phosphate conjugate. This method detected ATR in both urine and serum from ATR-dosed rats and shows a detection limit of 10 ng. Since the less sensitive ELISA detected ATR in samples where the HPLC did not, this suggested that ATR is biotransformed in vivo, such that its retention time on a reverse phase column is affected, but not its epitope determinants. The results of the organ function assays demonstrated that, when administered intra-peritoneally, ATR is not hepatotoxic, but is a powerful nephrotoxin, targeting for the microvilli of the brush border of the proximal tubules, and compromising glomerular permselectivity and distal tubular function. In addition, this toxin inhibits proline transport in the proximal tubule, and therefore probably affects protein biosynthesis. Renal regeneration is noted 3 days post-dosing, as demonstrated by calcium excretion. Immunocytochemistry was optimised on tuber tissue and necessitated the intracellular fixation of the toxin, using carbodiimide, to prevent leaching out of the ATR. The toxin was encapsulated in vesicles in the tuber tissue. Atractyloside was also located in the kidney of ATR-treated rats, up to 72 hours after exposure, targeting the microvilli of the proximal tubule brush border, the mitochondrial cristae and specific sites on the Golgi apparatus membrane. Microvilli disruption and mitochondrial swelling was noted within 24 hours after exposure to the toxin while after 72 hours, loss of mitochondrial integrity was observed. The development of these diagnostic assays for ATR have provided the means to monitor the levels of this toxin in plant extracts and mammalian body fluids. Future work should include the identification of the hepatotoxin associated with Impila, the effects of the route of administration on the toxicity of this remedy and furthermore, the identification of a suitable antidote, which could include the use of duramycin and stevioside. The association between compounds blocking the ADP/ATP antiporter in the c-state and Reye's syndrome should also provide an interesting area of research.Item The presentation and interpretation of arrow symbolism in biology diagrams at secondary-level.(2006) Du Plessis, Lynn.; Anderson, Trevor Ryan.The literature contains conflicting ideas about the effectiveness of diagrams, and their constituent symbolism as teaching and learning tools. In addition, only limited research has been specifically conducted on the presentation and interpretation of arrow symbolism used in biology diagrams, let alone on the nature, source and remediation of student difficulties caused by arrows. On the basis of this limited research and 30 years of experience of teaching biology at secondary-level, the author suspected that students might have difficulties interpreting arrow symbolism in diagrams used as explanatory tools and decided to thoroughly investigate this issue. The hypothesis, 'Secondary-level students have difficulty with the use of arrow symbolism in biology diagrams' was formulated and the following broad research questions defined to address the hypothesis: 1. How much of a problem is arrow symbolism in diagrams? 2. How effectively is arrow symbolism used in diagrams to promote the communication of intended ideas? 3. To what extent does the design of arrow symbolism in diagrams influence students ' interpretation and difficulties? 4. How can the emerging empirical data and ideas from literature be combined to illustrate the process of interpretation of arrow symbolism? 5. What measures can be suggested for improving the presentation and interpretation of arrow symbolism in biology diagrams at secondary-level? To address Research question 1, a content analysis of all arrow symbolism in seven popular secondary-level biology textbooks was undertaken. This revealed a wide diversity of arrow styles, spatial organisations, purposes and meanings that could be confusing to students. These results suggested the need for an evaluation of the effectiveness of arrow symbolism (Research question 2). As there was no definitive set of guidelines available for specifically evaluating arrows, general guidelines from the literature on diagrams were used to develop a set of 10 criteria, to evaluate the syntactic, semantic and pragmatic dimensions of arrow symbolism, which were validated by selected educators, students and a graphic design expert. Application of the criteria (which constituted expert opinion) to the arrow symbolism used in 614 realistic, stylised and abstract diagram types, revealed a relatively high incidence (30%) of inappropriately presented arrow designs that could mislead students. To establish whether this problem could be the cause of student difficulties, and to thereby address Research question 3, a stylised and an abstract diagram were selected and evaluated according to the criteria. The results of the evaluation were compared to the responses given by 174 students to a range of written and interview probes and student modified diagrams. In this way, student performance was correlated with expert opinion. The results confirmed that students experience a wide range of difficulties (26 categories) when interpreting arrow symbolism, with some (12 categories) being attributable to inappropriately presented arrow symbolism and others (14 categories) to student-related processing skills and strategies at both surface- and deeper-levels of reasoning. To address question 4, the emerging empirical data from the evaluation and student studies was combined with a wide range of literature, to inform the development of a 3-level, non-tiered model of the process of interpretation of arrow symbolism in diagrams. As this model emphasised the importance of both arrow presentation in diagrams and arrow interpretation by students, it could be used as an effective explanatory tool as well as a predictive tool to identify sources of difficulty with the use of arrow symbolism. This model was, in turn, used to inform the compilation of a range of guidelines for improving the presentation and interpretation of arrow symbolism, and so target Research question 5. These, and other guidelines grounded in the data and relevant literature, were suggested for all role players, including students, educators, textbook writers, graphic artists and researchers, to use as remedial tools. Future research should focus on the implementation of these guidelines and studying their effectiveness for improving the presentation and interpretation of diagrams with arrow and other types of symbolism.Item Using student difficulties to identify and model factors influencing the ability to interpret external representations of IgG-antigen binding.(2005) Schonborn, Konrad Janek.; Anderson, Trevor Ryan.; Grayson, Diane J.Scientific external representations (ERs), such as diagrams, images, pictures, graphs and animations are considered to be powerful teaching and learning tools, because they assist learners in constructing mental models of phenomena, which allows for the comprehension and integration of scientific concepts. Sometimes, however, students experience difficulties with the interpretation of ERs, which· has a negative effect on their learning of science, including biochemistry. Unfortunately, many educators are not aware of such student difficulties and make the wrong assumption that what they, as experts, consider to be an educationally sound ER will necessarily promote sound learning and understanding among novices. On the contrary, research has shown that learners who engage in the molecular biosciences can experience considerable problems interpreting, visualising, reasoning and learning with ERs of biochemical structures and processes, which are both abstract and often represented by confusing computer-generated symbols and man-made markings. The aim of this study was three-fold. Firstly, to identify and classify students' conceptual and reasoning difficulties with a selection of textbook ERs representing· IgG structure and function. Secondly, to use these difficulties to identify sources of the difficulties and, therefore, factors influencing students' ability to interpret the ERs. Thirdly, to develop a model of these factors and investigate the practical applications of the model, including guidelines for improving ER design and the teaching and learning with ERs. The study was conducted at the University of KwaZulu-Natal, South Africa and involved a total of 166 second and third-year biochemistry students. The research aims were addressed using a postpositivistic approach consisting of inductive and qualitative research methods. Data was collected from students by means of written probes, audio- and video-taped clinical interviews, and student-generated diagrams. Analysis of the data revealed three general categories of student difficulties, with the interpretation of three textbook ERs depicting antibody structure and interaction with antigen, termed the process-type (P), the structural-type (S) and DNA-related (D) difficulties. Included in the three general categories of difficulty were seventeen sub-categories that were each classified on the four-level research framework of Grayson et al. (2001) according to how much information we had about the nature of each difficulty and, therefore, whether they required further research. The incidences of the classified difficulties ranged from 3 to 70%, across the student populations and across all three ERs. Based on the evidence of the difficulties, potential sources of the classified difficulties were isolated. Consideration of the nature of the sources of the exposed difficulties indicated that at least three factors play a major role in students' ability to interpret ERs in biochemistry. The three factors are: students' ability to reason with an ER and with their own conceptual knowledge (R), students' understanding (or lack thereof) of the concepts of relevance to the ER (C), and the mode in which the desired phenomenon is represented by the ER (M). A novel three-phase single interview technique (3P-SIT) was designed to explicitly investigate the nature of the above three factors. Application of 3P-SIT to a range of abstract to realistic ERs of antibody structure and interaction with antigen revealed that the instrument was extremely useful for generating data corresponding to the three factors. In addition analysis of the 3P-SIT data showed evidence for the influence of one factor on another during students' ER interpretation, leading to the identification of a further four interactive factors, namely the reasoning-mode (R-M), reasoning conceptual (R-C), conceptual-mode (C-M) and conceptual-reasoning-mode (C-R-M) factors. The Justi and Gilbert (2002) modelling process was employed to develop a model of the seven identified factors. Empirical data generated using 3P-SIT allowed the formulation and validation of operational definitions for the seven factors and the expression of the model as a Venn diagram. Consideration of the implications of the model yielded at least seven practical applications of the model, including its use for: establishing whether sound or unsound interpretation, learning and visualisation of an ER has occurred; identifying the nature and source of any difficulties; determining which of the factors of the model are positively or negatively influencing interpretation; establishing what approaches to ER design and teaching and learning with ERs will optimise the interpretation and learning process; and, generally framing and guiding researchers', educators' and authors' thinking about the nature of students' difficulties with the interpretation of both static and animated ERs in any scientific context. In addition, the study demonstrated how each factor of the expressed model can be used to inform the design of strategies for remediating or preventing students' difficulties with the interpretation of scientific ERs, a target for future research.