Analysis of the regulation of HSP90α expression upon differentiation of C2C12 cells
- Authors: Holm, Nathan Christopher
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163318 , vital:41028
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
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- Date Issued: 2020
Cyclooxygenase-1 as an anti-stroke target: potential inhibitor identification and non-synonymous single nucleotide polymorphism analysis
- Authors: Muronzi, Tendai
- Date: 2020
- Subjects: Cerebrovascular disease , Cerebrovascular disease -- Treatment , Cerebrovascular disease -- Chemotherapy , Cyclooxygenases , High throughput screening (Drug development) , Drug development , Molecular dynamics , South African Natural Compounds Database , ZINC database
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/143404 , vital:38243
- Description: Stroke is the third leading cause of death worldwide, with 87% of cases being ischemic stroke. The two primary therapeutic strategies to reduce post-ischemic brain damage are cellular and vascular approaches. The vascular strategy aims to rapidly re-open obstructed blood vessels, while the cellular approach aims to interfere with the signalling pathways that facilitate neuron damage and death. Unfortunately, popular vascular treatments have adverse side effects, necessitating the need for alternative chemotherapeutics. In this study, cyclooxygenase-1 (COX-1), which plays a significant role in the post- ischemic neuroinflammation and neuronal death, was targeted for identification of novel drug compounds and to assess the effect of nsSNPs on its structure and function. In a drug discovery part, ligands from the South African Natural Compounds Database (SANCDB-https://sancdb.rubi.ru.ac.za/) and ZINC database (http://zinc15.docking.org/) were used for high-throughput virtual screening (HVTS) against COX-1. Additionally, five nsSNPs were being investigated to assess their impact on protein structure and function. Three of these SNPs were in the COX-1 dimer interface. Molecular docking and molecular dynamics simulations revealed asymmetric nature of the protein. Several ligands, peculiar to each monomer, exhibited favourable binding energies in the respective active sites. SNP analysis indicated effects on inter-monomer interactions and protein stability.
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- Date Issued: 2020
Evaluation of an NADPH-dependent assay for inhibition screening of Salmonella enterica DOXP Reguctoisomerase for identification of novel drug hit compounds
- Authors: Ngcongco, Khanyisile
- Date: 2020
- Subjects: 1-Deoxy-D-xylulose 5-phosphate , Antibiotics , Drug development , Salmonella , Enterobacteriaceae , Vaccines , Plasmodium falciparum , Mycobacterium tuberculosis
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167132 , vital:41440
- Description: Invasive non-typhoidal Salmonella, caused by the intracellular pathogen Salmonella enterica, has emerged as a major cause of bloodstream infections. It remains a neglected infection responsible for many deaths in Africa, as it fails to receive the level of support that is given to most better known infections. There are currently no vaccines against invasive non-typhoidal Salmonella. First-line antibiotics have been used for treatment, however, the rise in the resistance of the bacteria against these antibiotics has made treatment of invasive salmonellosis into a clinical problem. Therefore, the discovery of new compounds for the development of antibiotic drugs is required. Central metabolic pathways can be a useful source for identifying drug targets and among these is the non-mevalonate pathway, one of the pathways used for the biosynthesis of isoprenoid precursors. The second step of the non-mevalonate pathway involves the NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate (DOXP) into 2-C-methyl-D-erythritol 4-phosphate (MEP). 1-Deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase plays a vital role in the catalysis of this reaction and requires NADPH and divalent metal cations as co-factors for its activity. In this investigation recombinant DOXP reductoisomerase from Salmonella enterica, Plasmodium falciparum and Mycobacterium tuberculosis were biochemically characterized as potential targets for developing drugs that could be used as treatment of the disease. The expression and nickel-chelate affinity purification of S. enterica DOXP reductoisomerase in a fully functional native state was successfully achieved. However, the expression and purification of P. falciparum DXR and M. tuberculosis DXR was unsuccessful due to the formation of insoluble inclusion bodies. Although alternative purification strategies were explored, including dialysis and slow dilution, these proteins remained insoluble, making their functional analysis not possible. An NADPH-dependent enzyme assay was used to determine the activity of S. enterica DXR. This assay monitors the reduction of DOXP to MEP by measuring the absorbance at 340 nm, which reflects the concentration of NADPH. An alternative assay, resazurin reduction, which monitors the NADPH-dependent reduction of resazurin to resorufin, was explored for detecting enzyme activity. The recombinant S. enterica DOXP reductoisomerase had a specific activity of 0.126 ± 0.0014 μmol/min/mg protein and a Km and Vmax of 881 μM and 0.249 μmol/min/mg respectively. FR900098, a derivative of fosmidomycin, is a well-known inhibitor of DXR, however, the sensitivity of S. enterica DXR towards FR900098 has not yet been reported. The NADPH dependent enzyme and resazurin reduction assays were used to determine whether FR900098 has enzyme inhibitory effects against S. enterica DXR. Upon confirming that FR900098 is able to inhibit S. enterica DXR, FR900098 was used as a control compound in the screening of novel compounds. The S. enterica DXR enzyme was screened for inhibition by the collection of compounds from the Pathogen Box. Compounds that exhibited the desired inhibitory activity, referred to as ‘hits’ were selected for further investigation. These hits were confirmed using the NADPH-dependent enzyme assay, resulting in the identification of two different DXR inhibitor compounds, MMV002816, also known as diethylcarbamazine, and MMV228911. The inhibitory concentration (IC50) values of FR900098, MMV002816 and MMV228911 against S. enterica DXR were 1.038 μM, 2.173 μM and 6.861 μM respectively. The binding mode of these compounds to S. enterica DXR could lead to the discovery of novel druggable sites on the enzyme and stimulate the development of new antibiotics that can be used for treating Salmonella infections.
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- Date Issued: 2020
Investigating the relationship between Heat Shock Proteins and HIV Transactivator of Transcription
- Authors: Flax, Lili Marie
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/163307 , vital:41027
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020.
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- Date Issued: 2020
Investigation into the influence of an alien plant species on the soil microbiota of the sub-Antarctic, Marion Island
- Authors: Pienaar, Robert Daniel
- Date: 2020
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/124039 , vital:35531
- Description: Embargo applied. Embargo period to expire in April of 2021.
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- Date Issued: 2020
Molecular simulations of potential agents and targets of Alzheimer’s disease
- Authors: Carlisle, Tanya
- Date: 2020
- Subjects: Alzheimer's disease -- Treatment , Alzheimer's disease -- Molecular aspects , Amyloid beta-protein precurs , Amyloid beta-protein
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/140025 , vital:37825
- Description: The World Alzheimer Report statedin 2016 that approximately 46.8 million people were living with dementia and this figure is expected to triple by 2050. Alzheimer’s Disease was discovered to be a precursor to dementia in 1976 and since then efforts to understand Alzheimer’s have been prioritized. To date, there are very few effective forms of treatment for Alzheimer’s, many are known to offer only mild calming of the symptoms and have side effects such as diarrhea, nausea, loss of appetite and sleep disturbances. This has been due to lack of understanding on how Alzheimer’s is caused. With the two main hallmarks of the disease now being more understood it has opened the doorway into the discovery of new treatments for this disease. This study focuses on the hallmark involving the aggregation of the β-amyloid protein to form plaques surrounding the neurons of the brain. Copper, Zinc and Iron have also been found in high concentrations in and surrounding these plaques. This study focused on the screening of the South African Natural Compound database (SANCDB) to discover hits that have potential destabilizing action against the Beta-amyloid aggregate. If one of these compounds could prove to have destabilizing action on the aggregate it could open the doorway to new potential forms of treatment. Over 700 SANCDB compounds were docked, and the top hits were taken to molecular dynamics to further study the interactions of the compounds and the aggregate. However, the hits identified had strong binding to the aggregate causing it to become stable instead of the desired effect of destabilizing the structure. This information, however, does not rule out the possibility of these compounds preventing the formation of the aggregates. Further, interactions of copper with β-amyloid and copper were determined by solubilizing the aggregate and introducing copper ions in a dynamics simulation. Possible interactions between copper and the methionine residues were visualised.
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- Date Issued: 2020
Prediction of mass spectra for natural products using an ab initio approach
- Authors: Novokoza, Yolanda
- Date: 2020
- Subjects: Molecular dynamics , Molecular dynamics -- Computer simulation , Mass spectroscopy , Electron impact ionization
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167166 , vital:41443
- Description: Mass spectrometry (MS) is a technique that measures the fragmentation of molecules, dependent on the molecule’s chemical composition and structure, by first introducing a charge on the molecules. The instrument records the mass to charge ratio, but the energy from the ionization process causes the molecule to fragment. The resultant mass spectrum is highly indicative of not only the molecule analyzed, but also its chemical composition. MS is used in research and industry for both routine and research purposes. One such way to ionize molecules for MS is by bombarding the molecule with electrons which is the basis of electron impact mass spectrometry (EIMS). Although EIMS is widely used, prediction of electron impact mass spectra from first principles is a challenging problem due to a need to accurately determine the probability of different fragmentation pathways of a molecule. Ab initio molecular dynamics based methods are able to explore in an automatic fashion the energetically available fragmentation paths thus give reaction mechanisms in an unbiased way. The mass spectra of five molecules have been explored in work-flows leading to the prediction of mass spectra. These molecules include three natural products alpha-hispanolol, PFB oxime derivative and boronolide (for which experimental mass spectra were not available) and two compounds from the NIST database (for which experimental mass spectra were available). For each of these systems many random conformations were generated using the RDKit library. To all conformations random velocities were applied to each atom. Ab initio molecular dynamics was performed on each conformer, using these initial random velocities using CP2K software, at DFTB+ level at a variety of highly raised temperatures (to accelerate the formation of fragments) Fragmentation was monitored by iterating through all bonds, and identifying bond breakages during dynamics. Graph theoretical packages were used then to track distinct fragments generated. For each of these fragments, charges were determined from Mulliken analysis for all atoms on the fragment from the QM calculations and sum of atomic spin densities per fragment was also plotted. The fragment with the greatest charge (corresponding to the formation of a cation fragment) was taken for plotting on the mass spectrum. Finally, from the mass of the fragment and its elemental composition, the isotopic distribution for the fragment was determined, and this distribution was included by addition in to the mass spectrum. For all trajectories, the sum of all isotopic distributions determined the final mass spectrum.
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- Date Issued: 2020
Production, purification and characterization of a multifunctional, thermostable and acido/alkaline stable putative xylanase from the psychrotrophic bacterium, Sphingomonas aerolata
- Authors: Mathibe, Brian Nkanyiso
- Date: 2020
- Subjects: Uncatalogued
- Language: English
- Type: thesis , text , Masters , MSc
- Identifier: http://hdl.handle.net/10962/164478 , vital:41122
- Description: Thesis (MSc)--Rhodes University, Faculty of Science, Biochemistry and Microbiology, 2020
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- Date Issued: 2020
Structure and interaction studies of beta-amyloid in the search for new lead compounds for the treatment of Alzheimer’s disease
- Authors: Mtini, Onke
- Date: 2020
- Subjects: Alzheimer's disease -- Chemotherapy , Alzheimer's disease -- Treatment , Amyloid beta-protein , Oxidative stress , Protein binding , South African Natural Compounds Database
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167574 , vital:41493
- Description: Alzheimer’s disease (AD) is the most devastating neurodegenerative disorder that effects the aging population worldwide. In this study three hypotheses of AD are explored, the β-amyloid cascade hypothesis, the β-amyloid metal binding hypothesis and the oxidative stress hypothesis are explored. In the first case compounds from the South African Natural Compounds Database (SANCDB) are docked to models of β-amyloid fibrils and the properties of these fibrils under pulling simulations are compared to a known small molecule disruptor of β-amyloid, wgx-50. In these simulations SANCDB compounds are identified that disrupt β-amyloid in a similar manner to wgx-50. In these simulations the disruption to the free energy of binding of chains to the fibrils is quantified. For metal binding and oxidative stress hypotheses, problems in simulation arise due to only fragments of β-amyloid being present in the Research Collaboratory for Structural Bioinformatics protein data bank (RCSB PDB), as determined from NMR experiments. In this work, β-amyloid is set up under periodic boundary conditions to simulate a fibril under reasonable computational time. Within these periodic boundary conditions, β-amyloid has been solvated in copper and zinc rich environments and diffusion of these metals around the fibrils has been explored. The localization of these metals (in simulation only using van der Waal’s and electrostatic terms) around the fibril has led us to explore other possible metal binding sites. Metal bound to the infinite fibril has been optimized at the QM/MM level and some of the reactive oxygen species in the presence of the fibril are quantified.
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- Date Issued: 2020
The exploration of ARF1 screening assays to determine the drug status of ARF1 in cancer and malaria
- Authors: Ntlantsana, Apelele
- Date: 2020
- Subjects: ADP ribosylation , Golgi apparatus , Guanosine triphosphatase , G proteins , Malariotherapy , Malaria -- Research , Cancer -- Chemotherapy , Malaria -- Chemotherpay
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167232 , vital:41458
- Description: ARF GTPases are key regulators of the secretory and endocytic pathways. ARF1 is involved in the secretory pathway. ARF1 has been implicated in the endoplasmic reticulum to Golgi transport, function of the Golgi apparatus and transport from the trans-Golgi network to endosomes. ARFs cycle between active GTP-bound and inactive GDP-bound conformations. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ARF GEFs facilitate the activation of ARFs by mediating the exchange of GDP for GTP, while ARF GAPs terminate ARF function by stimulating the hydrolysis of the terminal phosphate group of GTP. Based on existing evidence gained from gene manipulation and cell biological investigations, ARF1 has been shown to be fundamentally important for cancer cell proliferation and metastasis and may be a promising target for the development of anti-cancer drugs. Additionally, the conservation of ARFs in eukaryotic organisms leads to an interesting question of whether a single drug target can be used to target multiple diseases. In this case, can a human cancer drug employed for cancer therapy be used in anti-malarial drug therapies? To confirm the drug target status of ARFs using chemical validation experiments, novel inhibitory compounds are needed. This requires the development of complex in vitro protein- protein interaction assays that can be used to screen chemical libraries for ARF GTPase inhibitors. In this study, we developed a fluorescence resonance energy transfer (FRET) assay and a novel in vitro colorimetric plate-based assay to detect the activation status of truncated human and Plasmodium falciparum ARF1. In the case of the FRET assay, active (GTP-bound) and inactive (GDP-bound) ARF1 could be distinguished with Z-factor values >0.5, suggesting that further development of the assay format to identify GEF and GAP inhibitors may be feasible. In the case of the colorimetric assay, robust signals could be detected and the assay was useful for detecting the activation status of ARF1. However, although the activation of ARF1 by the Sec7 domains of the BIG1 and ARNO was detectable, signals were not robust enough to employ in screening campaigns.
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- Date Issued: 2020