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Design of Immunobiosensors for Detection of Tumor-Associated Anti-P53 Autoantibodies: Method Development

- Adeniyi, Omotayo Kayode

Authors: Adeniyi, Omotayo Kayode
Date: 2020
Subjects: Uncatalogued
Language: English
Type: thesis , text , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/162988 , vital:41002 , 10.21504/10962/162988
Description: Introduction -- Experimental -- Label-Free Impedimetric Sensing of Anti-P53ab... -- Fluorescent detection of Anti-P53ab -- Peroxidase-like activity of Fe3O4@SiNP-APTES-Au@Pd... -- Colorimetric detection of Anti-P53ab , Thesis (PhD)--Rhodes University, Science Faculty, Department of Chemistry, 2020. , Detection and profiling of circulating tumor-associated autoantibodies (TAAbs) are useful for screening and early-stage diagnosis of asymptomatic lung cancer. Immunobiosensor technologies aimed to accomplish the highly sensitive, rapid and low-cost detection of TAAbs can improve the early-stage detection of lung cancer. Immunobiosensors for the detection of anti-P53-tumour associated autoantibodies have been developed in this work. The design of sensing interfaces with immobilized P53 protein (P53ag) as a sensing element layer on a solid interface was investigated. Several methods of detecting anti-P53-antibodies (anti-P53ab) were investigated. These methods are label-free detection using electrochemical impedance spectroscopy (EIS) and two label techniques. The label-free electrochemical techniques utilize gold electrode pre-modified with a conducting layer of electrochemically grafted phenylethylamine for covalent immobilization of P53ag. The limit of anti-P53ab detection with the label-free EIS was 103.0 pg.ml-1. The labeled technique developed utilizes fluorescent, and peroxidase-like nanomaterial labeled antibody as a detection probe. For the fluorescence detection, fluorescent silica nanoparticles were synthesized by overloading FITC into the silica matrix and conjugated to detection antibody (anti-IgG). The detection of the anti-P53ab was based on the dissolution of the silica nanoparticles to release the loaded dye as a signal amplification strategy. The fluorescence detection was carried out on a microplate, and magnetic bead modified P53-antigen platforms and limit of detection (LoD) were 42.0 fg.ml-1 and 3.3 fg.ml-1 for anti-P53ab; respectively. Fe3O4@SiNP-APTES-Au@Pd hybrid nanoparticles were synthesized, and their peroxidase-like activity and colorimetric detection were evaluated. The Fe3O4@SiNP-APTES-Au@Pd exhibited comparable activity to HRP. The Fe3O4@SiNP-APTES-Au@Pd was conjugated to protein-G-anti-IgG for the detection of anti-P53ab on a microplate and cellulose paper platforms. The LoD was 20.0 fg.ml-1 and 63.0 fg.ml-1 for the microplate and cellulose paper platform; respectively. The potential application of the designed immunobiosensor was evaluated in simulated serum samples. The developed sensors showed higher detection sensitivity, stability and had a lower detection limit for anti-P53ab when compared with the ELISA based detection. The results have provided alternative and effective quantification approaches to ELISA and a promising future for multiplexed detection of tumor-associated autoantibodies. The developed methodologies in this thesis could be applied for the detection of other autoantibodies in other cancer types and auto-immune diseases.
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Date Issued: 2020

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The design, synthesis and antiplasmodial activity of a series of halogenated fosmidomycin analogues and hybrid drugs

- Afolayan, Anthonia Folake

Authors: Afolayan, Anthonia Folake
Date: 2012
Subjects: Uncatalogued
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/64370 , vital:28538
Description: Malaria continues to be a devastating disease and a major cause of death in sub-Saharan Africa. With resistance against most of the available antimalarial drugs, there is a need for ongoing research and development of antimalarial agents. Fosmidomycin and its acetyl analogue FR900098 have been identified as potent inhibitors of Plasmodium falciparum, the causative agent of the most deadly form of malaria. Clinical trials of these agents have revealed poor absorption due to their high hydrophilicity. In the present studies the effect of halogenation of the acyl chain as well as the biological effect of extending the acyl sidechain was explored. This provided the basis on which fosmidomycin hybrids were designed to investigate the feasibility of hybrid extending into NADPH binding pocket. Synthesis of a series of halogenated FR900098 analogues was carried out in three stages. This included i) The introduction of the phosphonate group by reaction with 1,3dibromopropane in an Arbuzov reaction, ii) The introduction of a hydroxamate group by reaction of the propyl phosphonate by means of a nucleophilic substitution reaction with BocNHOBn and iii) The introduction of a halogenated acyl side chain on a protected fosmidomycin backbone. The synthesis of fosmidomycin-hybrids for which chloroquinefosmidomycin hybrids were used as the prototype, involved convergence of the two separately constructed moieties i.e. fosmidomycin and the quinoline moieties in a covalent linkage. The quinoline moiety was easily synthesized from the reaction of 4,7dichloroquinoline with 1,2-diamino ethane. The aminoquinoline so formed resulted in chloroquine-fosmidomycin hybrids 3.8 and 3.9 when reacted with halogenated FR900098 analogues. Antiplasmodial assays were conducted on the chloroquine-fosmidomycin hybrids and the halogenated fosmidomycin derivatives against the chloroquine resistant Gambian FCR-3 strain of P. falciparum. The most potent iodoacetyl fosmidomycin analogues 2.21 gave an IC50 value of 5.54 µM which is eight times more potent than the known antiplasmodial FR900098 which gave an IC50 value of 41.67 µM. All the halogenated FR900098 analogues showed better antiplasmodial activity than their non-halogenated derivatives. This indicated that the presence of halogens in the FR900098 analogues contributes to their biological Chapter 1 Literature review activity. The acetyl and propyl linked hybrids 3.8 and 3.9 showed potent antiplasmodial activity with IC50 values of 0.18 and 0.82 µM respectively. These were by far the most potent hybrids synthesized and provided leads for a new class of promising antimalarial agents. Preliminary E. coli DXR enzyme inhibition assays were carried out on the halogenated fosmidomycin analogues. The results showed good inhibition of the enzyme by the phosphonic acids of the chloroacetyl and chloropropyl analogues 2.1 and 2.2 respectively. Molecular modelling of the compounds on E. coli (PDB code: 2EGH) and P. falciparum (PDB code: 3AUA) DXR showed strong binding of the halogenated fosmidomycin analogues while the hybrids in the absence of docked NADPH showed minimum binding to the enzymes.
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Date Issued: 2012

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An investigation into the development of knowledge and strategies for the teaching of visual literacy in under-resourced Eastern Cape schools

- Mbelani, Madeyandile

Authors: Mbelani, Madeyandile
Date: 2014
Subjects: Uncatalogued
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/64401 , vital:28540
Description: This thesis reports on a multiple case study PhD project that aimed to investigate meaningful and critical development of knowledge and strategies to teach visual literacy, a component of English First Additional Language (FAL) in six under-resourced schools of the Eastern Cape of South Africa. The study begins by locating visual literacy within a broad framework of literacy as a social practice, and discusses its importance. Further, it discusses complexities of making sense of and teaching visual literacy, especially for the majority of in-service teachers who experienced visual literacy neither as learners nor as teacher trainees. The gap between the curriculum and teachers’ classroom practices is what triggered this study to adopt a transformative paradigm. The main research question is, “How can teacher professional development in English Language Teaching advance in-service teachers’ knowledge of and strategies for meaningful and critical teaching and learning of visual literacy?” To respond to this question, I drew on cultural historical activity theory (CHAT) and critical realism (CR) to design four phases of this study that incorporated the seven stages of an expansive learning cycle. These phases focussed on exploring and expanding teachers’ sense making and teaching of visual literacy. I collected data through interviews, document analysis, videoed lessons and change laboratory (CL) workshops. I designed a data analysis tool that brought together CHAT, CR, multimodal social semiotics, critical discourse analysis and pedagogical discourse to make sense of the data. Through a process of reflexivity, the study illuminated layers of factors that constrained meaningful and critical teaching of visual literacy in the empirical, the actual and the real domains of reality. These factors include teachers’ unconscious reproduction of discourses of domination, their intolerance of diverse cultural discourses, resistance to curriculum change, and the fact that they are comfortable with the status quo. I brought these factors to CL workshops for expansive learning. The study contributes in-depth insight into English FAL in-service teacher development in the area of visual literacy. By locating the study within meaning making and teaching of visual literacy, it was possible to interrogate access, diversity, domination and design in teachers’ classroom practices. As a result of this study participants were made aware of the extent to which these factors enabled or hindered meaningful and critical teaching. Participants repositioned themselves as subjects of the activity system, thereby mobilising their agency to take control of the structures and cultures that condition their teaching.
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Date Issued: 2014

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