An epistemic justice account of students’ experiences of feedback
- Authors: Vilakazi, Bella Phetheni
- Date: 2022-04-08
- Subjects: Feedback (Psychology) , Experience , Narrative inquiry (Research method) , Critical thinking , Caring Moral and ethical aspects , Epistemic access
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/232599 , vital:50006 , DOI 10.21504/10962/232599
- Description: I am a storyteller. I believe in the power of stories to share experiences and to elucidate thoughts and ideas and to help us to make sense of complex social practices. This thesis includes the stories of five young women who were learning to become teachers. As they shared their stories with me, I share them with you. This study includes their stories of receiving feedback. These stories are structured within the Narrative Inquiry dimensions of temporality, place and context. These dimensions suggest that stories are historical and move through time, stories are shaped by place and the context in which they unfold (Clandinin, 2013). Furthermore, these stories demonstrate how feedback can serve to give access to powerful knowledge and can serve to recognise who our students are and what they bring to the academy (Hordern, 2018). But feedback can also serve to misrecognise. Much has been written and reported about the barriers preventing students from acting on the feedback on their assignment tasks in higher education. In this study, I argue that feedback is a pedagogic practice that can support students to gain epistemic access. Feedback can only achieve this if it makes the expectations explicit for students to make sense of and make meaning for themselves and if it is offered in a dialogical format which recognises the students, their attempts, their identities, and their knowledge. The research question of this study, ‘How do experiences of forms of feedback affect female undergraduate student teachers’ chances of epistemic access?’, is not unusual. There have been many research projects that have been carried out that examine students’ experiences of feedback (for example, Evans, 2013; Basey, Maines, & Francis, 2014; Nicol et al.; 2014; Carless, 2019; Winstone et al., 2021). But I identified a gap where feedback has not, to my knowledge, been studied directly through the lenses of Epistemic Justice towards Parity of Participation. This study interpreted five undergraduate student teachers’ feedback experiences through these lenses. Narrative inquiry enabled me to design this study in ways that foregrounded experience. Data was collected through multiple conversations during which I organised the participants’ life stories of feedback within the dimensions of temporality, place and context, and sociality. Miranda Fricker’s (2007) theory of Epistemic Justice and Fraser’s norm of Parity of Participation (2000) framed this study. I engaged with Fricker and Fraser’s literature meaningfully as a reader and researcher. I established an understanding of how the lenses offered by Fraser and Fricker allowed me to make sense of the literature more generally, in social life and on the pedagogic practice of feedback. Fricker’s theory of Epistemic Justice considers the epistemically unjust, gendered, raced and classed, experiences of epistemic agents. Fricker (2007) draws on two central concepts to account for epistemic injustices: Testimonial Injustice and Hermeneutical Injustice. Fricker (2007; 2003) explains that testimonial injustice occurs within a testimonial exchange setting, when an epistemic agent as a speaker gives testimony of the epistemic agent’s experiences and knowledge but is not awarded the credibility the speaker deserves (Fricker, 2003). Epistemic agents who participate in a testimonial exchange need to overcome bias and prejudice in order to evaluate testimonies with the degree of fairness the testimony deserves (Fricker, 2013; 2016). Hermeneutical injustice occurs when an epistemic agent is unable to make sense and make meaning of their social experiences. Hermeneutical injustice strengthens when the epistemic agent is prevented from gaining access to resources that might help with sense making and meaning making of these social experiences (Dielman, 2012; Fricker, 2016). To ensure that meaning can be made between people and groups of people, there needs to be some shared understandings of the purpose and process of sense making and meaning-making – or a willingness to co-create such shared understandings. Fraser’s norm of Participatory Parity enabled a consideration of the larger world of political and economic systems that give rise to social injustice. In this study, the theories of Fricker and Fraser are used to illuminate experiences of feedback of the five undergraduate student teachers who are the participants in this study and how these translate to epistemic and social injustice. The norm of Participatory Parity is considered where feedback allowed or restricted participants from participating on an equal footing in the feedback process. Narrative inquiry, a research methodology that is used to study experiences, was used to inform research strategies of this study. Participants’ experiences, data collection and organising the narratives demonstrated the dimensions of temporality and space. The thesis includes biographical vignettes for each of the participants in the study, interspersed with data from across all five participants. The key findings of this study show that feedback generally operates at the surface levels of grammar correction. In light of the theoretical lenses of this study, I argue that the feedback experiences they shared generally did not recognise their attempts and the identities and knowledges they brought to the tasks. Because the focus was on superficial correction of the specific task, the feedback failed to create conditions for the (re)distribution of knowledge. At times the feedback exerted power on participants. Because the feedback was generally in the form of one directional correction (with little space for interaction with the feedback or dialogue with the assessor), this caused status subordination of participants in the epistemic spaces of teaching practice. Lastly, the lack of clarity of feedback was harmful to the potential for dialogical feedback. Such feedback caused participants to experience forms of epistemic injustice in the form of hermeneutical injustice where it failed to create conditions for the distribution of knowledge. Feedback also caused participants to experience testimonial injustice where it failed to create conditions for recognising participants’ processes of sense-making and meaning-making in the various assignment tasks. Participatory Parity could not occur because the processes of recognition and redistribution were constrained. Feedback then created fertile conditions of epistemic injustice to occur, and participants were likely to have failed to gain the much needed epistemic access. This study is not the story of bad, uncaring academics; the study acknowledges the context of large classes and heavy workloads in which feedback is or is not given. Rather, this is the story of five women trying to make their way through the university and out into the world as teachers. The study calls for better theorising of feedback and more support for both academics and students to develop feedback literacy so that feedback might serve as a dialogical pedagogic practice that enables epistemic justice. , Thesis (PhD) -- Faculty of Education, Centre for Higher Education Research, Teaching and Learning, 2022
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- Date Issued: 2022-04-08
Chinese character learning with the aid of an ICT website among Mandarin Second Additional Language learners in South Africa: a case study
- Authors: Fu, Shuying
- Date: 2022-04-08
- Subjects: Mandarin dialects Study and teaching (Secondary) South Africa , Mandarin dialects Computer-assisted instruction for English speakers , Second language acquisition , Chinese characters , Information technology South Africa , Educational technology South Africa , Arch Chinese
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/263590 , vital:53641
- Description: The teaching of Mandarin as a Second Additional Language (SAL) in South African schools was only introduced in 2016. The year 2018 saw the first Mandarin as SAL National Senior Certificate examination. Little research has been done on the teaching and learning of Mandarin as a SAL subject at the high school level in South Africa. Character learning and teaching are some of the most challenging aspects of this language teaching and learning. This research sought to investigate how beginner Mandarin SAL learners can be supported by a particular Information Communication Technology (ICT) website, www.archchinese.com (Arch Chinese), in their character learning. Learners’ ability to memorise Chinese characters is of utmost importance for their success in this language learning, especially for matriculants. To support the main research goal, this study set out to investigate the requirements for Chinese character learning, the role the website Arch Chinese plays in learners’ character learning and Mandarin SAL learners’ experience of using Arch Chinese as a learning tool. This research was conducted in the form of a case study within the interpretative paradigm. It adopted a questionnaire and document analysis for data collection to gain insight into the research topic. To get answers to the research questions posed above, the study analysed the Curriculum Assessment Policy Statement (CAPS) Mandarin SAL documents from Grade 4–12, the Independent Examination Board (IEB) Mandarin Subject Assessment Guidelines (SAGs), and a textbook for beginners, Learn Chinese with me: Student’s book 1 in relation to character learning. The features and functions of the website, Arch Chinese, were also evaluated and Mandarin SAL learners’ answers to a questionnaire on their use of the website Arch Chinese were analysed. The study found that character teaching and learning has not been given enough attention in the curriculum. It was only covered in the Intermediate Phase but was seldom mentioned in the Senior and Further Education and Training Phases in the curriculum. The CAPS Mandarin SAL documents do not consider the special characteristics of the Mandarin language, as this curriculum was based on the CAPS English Generic SAL document. As a result, the assessment requirements on writing (character count requirement in particular) were not realistic. The teaching approaches promoted in the curriculum and the teaching time do not correspond well with the teaching and learning of this language. Moreover, this study found that there was no vocabulary list prescribed in the curriculum and therefore a gap exists between the curriculum on paper and the curriculum in practice. The analysis of the IEB SAGs found that the assessments were suitable for the level of Mandarin SAL learners. This is because the IEB considered the characteristics of the Mandarin language, which is non-cognate, to the alphabet-based languages that respondents in this research spoke or were familiar with. At the same time, the analysis of the website and the learners’ questionnaire found that learners held a positive attitude towards their use of the website and that it proved to help facilitate Mandarin SAL learners in their character learning. This study ends with recommendations for teachers, policy makers, the IEB, and character-learning websites. , Thesis (MEd) -- Faculty of Education, Education, 2022
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- Date Issued: 2022-04-08
Demersal fish distribution in the shallow marine nearshore and estuarine seascape of Algoa Bay: Nursery areas and the effect of environmental drivers
- Authors: Nodo, Phakama
- Date: 2022-04-08
- Subjects: Groundfishes South Africa Algoa Bay , Estuarine fishes South Africa Algoa Bay , Marine nurseries South Africa Algoa Bay , Estuarine fishes Habitat South Africa Algoa Bay , Estuarine fishes Physiology South Africa Algoa Bay , Estuarine fishes Effect of pollution on South Africa Algoa Bay , Estuarine fishes Larvae South Africa Algoa Bay , Estuarine fishes Effect of human beings on South Africa Algoa Bay
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/232399 , vital:49988 , DOI 10.21504/10962/232399
- Description: Estuaries and shallow marine nearshore areas are highly productive and valuable ecosystems, which provide numerous habitats for fish and support fundamental ecological links with other environments. Assessing fish distribution across estuarine and marine nearshore habitats is important to identify ecologically important habitats and develop effective management strategies for coastal fishes, many of which are important fishery species. Despite this, only a few studies have focussed on fish community patterns across an estuary and marine nearshore gradient concurrently, particularly including early life history stages, to determine the nursery value of both environments, and to examine whether these two coastal environments have distinct fish assemblages in relation to physical factors. The main aim of this study was to assess the environmental drivers of demersal fish communities in soft-bottom benthic habitats in two permanently open estuaries and adjacent marine nearshore areas (5 – 10 m) of Algoa Bay, South Africa, as well as to assess the relative roles of these two habitats as settlement and nursery areas for demersal fish species. A 1.5 m, conical shoeless beam trawl net was used to sample the demersal fish community concurrently in each habitat between July 2017 and September 2019. Sampling was conducted in July 2017, February, March, May, July, August, October and November 2019 and February, April and September 2019. DNA barcoding was used to verify identification of the early life history stages of fish caught in the estuarine and marine nearshore areas of Algoa Bay. In addition, since the two estuaries (Swartkops and Sundays) are heavily polluted, the effect of low dissolved oxygen and hypoxia and associated shifts in spatial distribution of demersal species was investigated. The two sampled estuaries had a higher abundance of demersal fishes, with a total of 6437 fishes (28 species) caught (3752 and 2685 individuals with 24 and 20 species recorded in the Sundays and Swartkops estuaries, respectively). Species richness was higher in the marine nearshore of Algoa Bay, with 29 species (797 individuals) caught. Of the 7234 individuals caught, the identification of 100 specimens, in either a larval or early juvenile phase, were uncertain and therefore DNA barcoding was used to verify their identification. Of these 100 individuals, 86 were positively identified to species level using COI sequences. Fourteen failed to amplify by PCR and could only be identified morphologically. The marine nearshore sites were dominated by species which spawn in the marine environment and are not dependent on estuaries (marine species and marine estuary-opportunists), whilst the estuaries were dominated by estuarine spawners or marine spawners dependent on estuaries to some degree. Two discrete demersal fish assemblages were identified representing the marine nearshore and the estuary, with no significant differences observed between the two estuaries (Sundays and Swartkops). The differences observed between the marine nearshore and estuary were mostly driven by salinity, turbidity, silt and organic content of the sediment. These distinct fish assemblages might be considered as indicators for the respective environments they inhabit. Both habitats were dominated by early life history stages (larvae to juveniles), indicating the nursery function of both habitats. Early life stages collectively comprised 97% of the catch in the marine nearshore and 68% in the estuary. Young-of-the-year (YOY) juveniles (< 1-year-old juveniles) and transformation stages (when changes in body shape and pigment pattern occur) dominated the total catch in the marine nearshore, while YOY juveniles dominated the estuarine fish assemblages. Ariidae Galeichthys feliceps, Haemulidae Pomadasys olivaceus, Sciaenidae Argyrosomus inodorus and Cynoglossidae Cynoglossus zanzibarensis, comprised the largest proportion of YOY juveniles in the marine nearshore. The transformation stage in the marine nearshore was numerically dominated by P. olivaceus and G. feliceps. In the estuarine environment, YOY juveniles were mostly dominated by Sparidae Rhabdosargus holubi, Soleidae Heteromycteris capensis and Gobiidae Caffrogobius gilchristi. The greatest abundance of early life stage fishes was observed in the lower reaches of the Sundays Estuary and the upper reaches of the Swartkops Estuary, as well as nearshore sites located in close proximity to the estuary mouths, particularly during spring and summer. Despite the fact that these coastal ecosystems are important nursery areas, they are threatened by a number of factors, including habitat loss and modification due to urban development, intensification of agriculture and subsequent eutrophication, climate change, and overfishing, all of which reduce ecosystem functioning and reduce the ecological and economic value of these habitats around the world. Hypoxia is one of the major threats to the functioning of coastal ecosystems, particularly estuaries. The Sundays and Swartkops estuaries both experience persistent eutrophic conditions, with frequent phytoplankton blooms (> 20 μg Chl-a l-1) that result in instances of bottom water oxygen depletion (< 4 mg/l). During the present study in the Sundays Estuary, low oxygen waters were recorded in the middle reaches (Site S5) mostly during summer (four months of low DO conditions). In the Swartkops Estuary, low dissolved oxygen was recorded in the upper reaches during spring. The lowest dissolved oxygen concentration recorded was 0.5 mg/l and 2.4 mg/l in the bottom waters of the Sundays and Swartkops estuaries, respectively. Selected dominant species were only absent from areas where dissolved oxygen was < 1 mg/l and present in the adjacent sites (for example Site S4, and S3) where DO was higher mostly during January 2019. As such, the low dissolved oxygen concentrations recorded in the Swartkops Estuary did not have a noticeable impact on fish distribution, although the total abundance of species did show a slight decline when dissolved oxygen was < 3 mg/l. This study demonstrates the importance of concurrently examining estuarine and nearshore marine habitats in order to identify ecologically important habitats, which has important implications for the development of effective management strategies for coastal fish populations, particularly in the light of anthropogenic change. In addition, in order to identify nursery hotspots it is crucial to correctly identify all the species occupying these areas. As such, this study confirms the importance of also using DNA barcoding for fish identification, particularly for the early life history stages of cryptic species (e.g. Argyrosomus inodorus and Argyrosomus japonicus). , Thesis (PhD) -- Faculty of Science, Ichthyology & Fisheries Sciences, 2022
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- Date Issued: 2022-04-08
Quantifying the impacts of cactus biological control agents in South Africa
- Authors: Mnqeta, Zezethu
- Date: 2022-04-08
- Subjects: Cactus Biological control South Africa , Noxious weeds South Africa , Invasive plants Biological control South Africa , Insects as biological pest control agents South Africa , Cochineal insect South Africa , Mealybugs South Africa , Agricultural productivity South Africa
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/294631 , vital:57239 , DOI 10.21504/10962/294631
- Description: Invasive alien cacti are prominent weeds that threaten indigenous biodiversity and have a negative impact on agricultural productivity in South Africa. These plants are problematic because they form dense thickets that reduce the carrying capacity of rangelands; restrict the movement of livestock and wildlife thus reducing access to shade and water sources; and are directly harmful to livestock, wildlife and people due to their sharp spines. Biological control is the most effective, affordable and environmentally friendly method to control invasive alien cacti and minimize their negative impacts. Cochineal insects (Dactylopius spp.: Dactylopiidae) and the gall-forming mealybug, Hypogeococcus sp. (Pseudococcidae) are used as biological control agents for cacti. The agents are however poor dispersers, so mass-rearing and augmentative releases are required in order to establish the agents at sites where they are needed. This study aimed to evaluate the mass-rearing and release efforts of cactus biological control in South Africa, quantify the impact of the biological control agents on cactus plant populations through long-term monitoring, and assess the benefits accrued due to the biological control agents through the perceptions of land-users. An assessment of the effectiveness of the release effort for cactus biological control agents was conducted by comparing where biological control agents have been released with the known distribution of the target weeds. Only 26% of the quarter degree squares that are known to be occupied by invasive alien cacti have had biological control agents released in them. This indicated that the mass-rearing and release efforts in South Africa are inadequate and should be increased, especially in areas where few releases have been made and many cacti are present, such as the Limpopo Province. The impact of the biological control agents on cactus plant populations was assessed by monitoring agent densities, target plant densities and target plant reproductive outputs before and after releases. Plant biomass and reproductive output were reduced by biological control agents for three of the target weeds that were assessed, while the duration of the study was too short to measure reductions for the fourth target weed. Benefits to land-users were then quantified through a questionnaire survey. Land-users perceived biological control as an effective management option, with 81% of the land-users reporting that there was less invasive alien cactus after releasing biological control agents on their land. Forty-nine percent of the land-users believed that the negative impacts of the cactus had been reduced and that they benefited more from their land since control was achieved. Since land-users were only interviewed within four years of the releases being conducted, it is expected that the percentage of land-users who gained benefits from biological control will increase in future. Ninety-seven percent of the land-users stated that the agents were safe and had not fed on any other plants or had any detrimental impacts. These perceptions indicated that land-users regarded biological control as a safe and effective method of controlling invasive alien cacti. This study confirms that biological control is an effective and safe way of controlling invasive alien Cactaceae. It is also the first to assess some of the benefits that land-users have accrued due to biological control of cactus weeds. It is however evident that a greater mass-rearing and release effort is required for South Africa to get the maximum benefits possible from the use of the biological control agents for cactus weeds that are available in the country. , Izityalo ze-cactus ezisuka kwamanye amazwe zilukhula olubalaseleyo, olwenza ingxaki kwintlobo ngeentlobo zezityalo nezilwanyana kwaye ezizityalo zinefuthe neziphumo ezingalunganga kwimveliso yezolimo eMzantsi Afrika. Ezi zizityalo ziyingxaki kuba zenza amatyholo ashinyeneyo athi anciphise umthamo wokusebenzisa umhlaba: zinqanda ukuhamba nogkukhululekileyo kwemfuyo nezilwanyana zasendle ngokwenza oko zingakwazi ukufikelela emthuzini xa kutshisa nakwimithombo yamanzi yokusela; zikwayiyo nengozi kwimfuyo, izilwanyana zasendle kunye nabantu ngenxa yamave abukhali afumaneka kwezizityalo. Ukulawula nokwehliza ubunzini nezinga lemigcipheko yezizityalo, kusetyenziswa indlela ekuthiwa yi-biological control. Lendlela yeyona ndlela isebenza ngokuphucukileyo, efikelelayo, nengenabungozi kokusingqongileyo. Izinambuzane ze- cochineal (i-Dactylopius spp.: Dactylopiidae) kunye ne-mealybug, i-Hypogeococcus sp. (Pseudococcidae) zisetyenziswe njengezixhobo ze-biological control ezinceda ukulawula ezizityalo ze-cactus zingafunekiyo. Ingxaki yazo ezizinambuzane zizixhobo ze-biological control zingentla kukuba azikwazi kuzisasaza ngokwazo ukuba zifikelele nakwizityalo ezikude ngoko ke ukukhuliswa nokukhutshwa ngobuninzi bazo kuyafuneka ukukhawulelana nalengxaki kunye nokwandisa amathuba wokuba zifikelele kuzozonke izityalo ze-cactus ekufuneka zizilawule. Olu phononongo lujolise ekuvavanyeni iinzame zokukhulisa ngobuninzi nokukhupha ezezinambuzane zizixhobo ze-bioloigical control zokulawula izityalo ze-cactus eMzantsi Afrika, ukujonga ubungakanani befuthe notshintsho elenziwa zezinambuzane zizixhobo ze-bioloigical control kwizityalo ze-cactus emva kwexesha elide lokuzijonga, kunye nokuvavanya inzuzo efunyenwe ngenxa yokulawula ezizityalo ze-cactus ngokwemibono yabasebenzisi bomhlaba. Uvavanyo lweenzame zokukhutshwa kwezixhobo zezixhobo zezinambuzane ze-bioloigical control kwizityalo ze-cactus lwenziwa ngokuthelekisa iindawo apho izixhobo zezinambuzane ze-biological control zikhutshwe khona kunye neendawo apho izityalo ze-cactus kwaziwayo ukuba ziyafumaneka khona. Yi-26% kuphela yesikwere sekota eyaziwayo ukuba kukhona izityalo ze-cactus nalapho kukhutshwe khona kwezinambuzane ezizixhobo ze-biological control. Oku kubonisa ukuba iinzame zokukhulisa nokukhutshwa kwezinambuzane ezizixhobo ze-biological control eMzantsi Afrika azanelanga kwaye kufuneka zandiswe, ngakumbi kwiindawo apho kukho ukhutsho olumbalwa olwenziweyo kunye nezityalo ze-cactus ezininzi ezifumaneka khona, njengePhondo laseLimpopo. Iimpembelelo yokukhutshwa kwezinambuzane ezizixhobo ze-biological control kwizityalo ze-cactus zavavanywa ngokujonga ubunizi bezinambuzane ezizixhobo ze-biological control, ukuxinana kwezityalo ze-cactus ekujoliswe kuzo kunye nemveliso yokuzala kwezityalo ze-cactus phambi nasemva kokuba kukhutshwe izinambuzane ezizixhobo ze-biological control. Ubungakanani bezityalo ze-cactus kunye nemveliso yokuzala ziye zacutheka emva kokukhutshwa kwezinambuzane ezizixhobo ze-biological control kwinzityalo ezintathu ze-cactus ebekujoliswe uvavanyo kuzo, ngelixa ixesha lophononongo lalifutshane kakhulu ukuvavanya unciphiso kwisityalo se-cactus sesine. Izibonelelo zabasebenzisi bomhlaba zaye zavavanywa kusetyenziswa uhlobo lwemibuzo. Abasebenzisi bomhlaba balubona ukusetyenziswa kwezinambuzane ezizixhobo ze-biological control njengendlela yolawulo olusebenzayo, yi81% yabasebenzisi bomhlaba echaze ukuba izityalo ze-cactus zecuthekile emva kokukhutshwa kwezinambuzane ezizixhobo ze-biological control emhlabeni wabo. Amashumi amane anesithoba eepesenti zabasebenzisi bomhlaba bakholelwa ukuba impembelelo ezingalunganga ze-cactus zincitshisiwe kwaye baxhamle kakhulu kumhlaba wabo okoko lwaphunyenzwa. Oludliwano-ndlebe belwenziwe kubasebenzisi bomhlaba kwisithuba seminyaka emine emva kokuba kukhutshwe izinambuzane ezizixhobo ze-biological control, kulindeleke ukuba ipesenti yabasebenzisi bomhlaba abathe bafumana izibonelelo kwi-biological control lonyuke kwixesha elizayo. Amashumi alithoba anesixhenxe ekhulwini abasebenzisi bomhlaba bachaze ukuba izinambuzane ezizixhobo ze-biological control zikhuselekile kwaye khange zidle kuzo naziphi na ezinye izityalo ezingezi eze-cactus ebekujoliswe kuzo okanye zineempembelelo eyingozi. Ezi mbono ziyabonisa ukuba abasebenzi bomhlaba bayithatha i-biological control njengendlela ekhuselekileyo nesebenzayo yokulawula izityalo ze-cactus zamanye amazwe. Olu phononongo luqinisekisa ukuba ulawulo olusebenzisa izinambuzane ezizixhobo ze-biological control yindlela esebenzayo nekhuselekileyo yokulawula iCactaceae yamanye amazwe. Ikwangolokuqala ukuvavanya ezinye zezibonelelo ezizuzwe ngabasebenzisi bomhlaba ngenxa ye-biological control yezityalo ze-cactus. Nangona kunjalo kucacile ukuba ukwandisa inzame zokukhulisa nokukhupha ngobuninzi izinambuzane ezizixhobo ze-biological control kuyafuneka ukuze uMzantsi Afrika ufumane izibonelelo eziphakamileyo enokubakho ngokusetyenziswa kwe zinambuzane ezizixhobo ze-biological control zokulawula ukhula lwe-cactus olukhoyo kweli lizwe. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2022
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- Date Issued: 2022-04-08
Sequence, structure, dynamics, and substrate specificity analyses of bacterial Glycoside Hydrolase 1 enzymes from several activities
- Authors: Veldman, Wayde Michael
- Date: 2022-04-08
- Subjects: Glycosidases , Bioinformatics , Molecular dynamics , Ligands (Biochemistry) , Enzymes , Ligand binding (Biochemistry) , Sequence alignment (Bioinformatics) , Structural bioinformatics
- Language: English
- Type: Doctoral thesis , text
- Identifier: http://hdl.handle.net/10962/233805 , vital:50129 , DOI 10.21504/10962/233810
- Description: Glycoside hydrolase 1 (GH1) enzymes are a ubiquitous family of enzymes that hydrolyse the glycosidic bond between two or more carbohydrates, or between a carbohydrate and a non-carbohydrate moiety. Despite their conserved catalytic domain, these enzymes have many different enzyme activities and/or substrate specificities as a change of only a few residues in the active site can alter their function. Most GH1 active site residues are situated in loop regions, and it is known that enzymes are more likely to develop new functions (broad specificity) if they possess an active site with a high proportion of loops. Furthermore, the GH1 active site consists of several subsites and cooperative binding makes the binding affinity of sites difficult to measure because the properties of one subsite are influenced by the binding of the other subsites. Extensive knowledge of protein-ligand interactions is critical to the comprehension of biology at the molecular level. However, the structural determinants and molecular details of GH1 ligand specificity and affinity are very broad, highly complex, not well understood, and therefore still need to be clarified. The aim of this study was to computationally characterise the activity of three newly solved GH1 crystallographic structures sent to us by our collaborators, and to provide evidence for their ligand-binding specificities. In addition, the differences in structural and biochemical contributions to enzyme specificity and/or function between different GH1 activities/enzymes was assessed, and the sequence/structure/function relationship of several activities of GH1 enzymes was analysed and compared. To accomplish the research aims, sequence analyses involving sequence identity, phylogenetics, and motif discovery were performed. As protein structure is more conserved than sequence, the discovered motifs were mapped to 3D structures for structural analysis and comparisons. To obtain information on enzyme mechanism or mode of action, as well as structure-function relationship, computational methods such as docking, molecular dynamics, binding free energy calculations, and essential dynamics were implemented. These computational approaches can provide information on the active site, binding residues, protein-ligand interactions, binding affinity, conformational change, and most structural or dynamic elements that play a role in enzyme function. The three new structures received from our collaborators are the first GH1 crystallographic structures from Bacillus licheniformis ever determined. As phospho-glycoside compounds were unavailable for purchase for use in activity assays, and as the active sites of the structures were absent of ligand, in silico docking and MD simulations were performed to provide evidence for their GH1 activities and substrate specificities. First though, the amino acid sequences of all known characterised bacterial GH1 enzymes were retrieved from the CAZy database and compared to the sequences of the three new B. licheniformis crystallographic structures which provided evidence of the putative 6Pβ-glucosidase activity of enzyme BlBglH, and dual 6Pβ-glucosidase/6Pβ-galactosidase (dual-phospho) activity of enzymes BlBglB and BlBglC. As all three enzymes were determined to be putative 6Pβ-glycosidase activity enzymes, much of the thesis focused on the overall analysis and comparison of the 6Pβ-glucosidase, 6Pβ-galactosidase, and dual-phospho activities that make up the 6Pβ-glycosidases. The 6Pβ-glycosidase active site residues were identified through consensus of binding interactions using all known 6Pβ-glycosidase PDB structures complexed complete ligand substrates. With regards to the 6Pβ-glucosidase activity, it was found that the L8b loop is longer and forms extra interactions with the L8a loop likely leading to increased L8 loop rigidity which would prevent the displacement of residue Ala423 ensuring a steric clash with galactoconfigured ligands and may engender substrate specificity for gluco-configured ligands only. Also, during molecular dynamics simulations using enzyme BlBglH (6Pβ-glucosidase activity), it was revealed that the favourable binding of substrate stabilises the loops that surround and make up the enzyme active site. Using the BlBglC (dual-phospho activity) enzyme structure with either galacto- (PNP6Pgal) or gluco-configured (PNP6Pglc) ligands, MD simulations in triplicate revealed important details of the broad specificity of dual-phospho activity enzymes. The ligand O4 hydroxyl position is the only difference between PNP6Pgal and PNP6Pgal, and it was found that residues Gln23 and Trp433 bind strongly to the ligand O3 hydroxyl group in the PNP6Pgal-enzyme complex, but to the ligand O4 hydroxyl group in the PNP6Pglc-enzyme complex. Also, His124 formed many hydrogen bonds with the PNP6Pgal O3 hydroxyl group but had none with PNP6Pglc. Alternatively, residues Tyr173, Tyr301, Gln302 and Thr321 formed hydrogen bonds with PNP6Pglc but not PNP6Pgal. Lastly, using multiple 3D structures from various GH1 activities, a large network of conserved interactions between active site residues (and other important residues) was uncovered, which most likely stabilise the loop regions that contain these residues, helping to retain their positions needed for binding molecules. Alternatively, there exists several differing residue-residue interactions when comparing each of the activities which could contribute towards individual activity substrate specificity by causing slightly different overall structure and malleability of the active site. Altogether, the findings in this thesis shed light on the function, mechanisms, dynamics, and ligand-binding of GH1 enzymes – particularly of the 6Pβ-glycosidase activities. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2022
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- Date Issued: 2022-04-08