A case-control approach to assess variability in distribution of distance between transcription factor binding site and transcription start site
- Authors: Moos, Abdul Ragmaan
- Date: 2017
- Subjects: Transcription factors , Proteomics , Chromatin , Chromatin immunoprecipitation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/5315 , vital:20808
- Description: Using the in-silico approach, with ENCODE ChIP-seq data for various transcription factors and different cell types; we systematically compared the distance between the transcription factor binding site (TFBS) and the transcription start (TSS). Our aim was to determine if the same transcription factor binds at a different position relative to the TSS in a normal and an abnormal cell type. We compare distribution of distance of binding sites from the TSS; to make description less verbose we call this “distance” where there is no possibility of confusion. We used a case-control methodology where the distance between the TFBS and the TSS in the normal, non-cancerous or untreated cell type is the control. The distance between the TFBS and the TSS in the cancerous or treated cell type is the case. We use the distance between the TFBS and the TSS in the control as the standard. We compared the distance between the TFBS and the TSS in the case and the control. If the distance between the TFBS and the TSS in the control was greater than the distance between the TFBS and the TSS in the case, we can infer the following. The transcription factor in the case binds closer to the TSS compared to the control. If the distance between the TFBS and the TSS in the control is smaller than the distance between the TFBS and the TSS in the case, we can infer the following. The TF in the case binds further away from the TSS compared to the control. Our method is a screening method whereby we compare ChIP-seq data to determine if there is a difference in the distribution distance between the TFBS and the TSS for normal and abnormal cell types. We used the R package ChIP-Enrich to compare the distribution of distance between ChIP-seq peak and the nearest TSS. ChIP-Enrich produces a histogram with the number of ChIP-seq peaks at a certain distance from the TSS. The results indicate for some transcription factors like GM12878-cMyc and K562-cMyc there is a difference between the distribution of distance between the TFBS and the nearest TSS. cMyc has more binding sites within a distance of 1kb from the TSS in GM12878 when compared to K562. GM12878-CTCF and K562-CTCF have slight differences when comparing their distribution of distance from the TSS. This means CTCF binds almost the same distance from the TSS in both GM12878 and K562. A549-gr treated with dexamethasone is interesting because with increase dose of dexamethasone the distribution of distance from the TSS changes as well.
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- Date Issued: 2017
Combined in silico approaches towards the identification of novel malarial cysteine protease inhibitors
- Authors: Musyoka, Thommas Mutemi
- Date: 2017
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/4488 , vital:20679
- Description: Malaria an infectious disease caused by a group of parasitic organisms of the Plasmodium genus remains a severe public health problem in Africa, South America and parts of Asia. The leading causes for the persistence of malaria are the emergence of drug resistance to common antimalarial drugs, lack of effective vaccines and the inadequate control of mosquito vectors. Worryingly, accumulating evidence shows that the parasite has developed resistant to the current first-line treatment based on artemisinin. Hence, the identification and characterization of novel drug targets and drugs with unique mode of action remains an urgent priority. The successful sequencing and assembly of genomes from several Plasmodium species has opened an opportune window for the identification of new drug targets. Cysteine proteases are one of the major drug targets to be identified so far. The use of cysteine protease inhibitors coupled with gene manipulation studies has defined specific and putative roles of cysteine proteases which include hemoglobin degradation, erythrocyte rupture, immune evasion and erythrocyte invasion, steps which are central for the completion of the Plasmodium parasite life cycle. In an aim to discover potential novel antimalarials, this thesis focussed on falcipains (FPs), a group of four papain-like cysteine proteases from Plasmodium falciparum. Two of these enzymes, FP-2 and FP-3 are the major hemoglobinases and have been validated as drug targets. For the successful elimination of malaria, drugs must be safe and target both human and wild Plasmodium infective forms. Thus, an incipient aim was to identify protein homologs of these two proteases from other Plasmodium species and the host (human). From BLASTP analysis, up to 16 FP-2 and FP-3 homologs were identified (13 plasmodial proteases and 3 human cathepsins). Using in silico characterization approaches, the intra and inter group sequence, structural, phylogenetic and physicochemical differences were determined. To extend previous work (MSc student) involving docking studies on the identified proteins using known FP-2 and FP-3 inhibitors, a South African natural compound and its ZINC analogs, molecular dynamics and binding free energy studies were performed to determine the stabilities and quantification of the strength of interactions between the different protein-ligand complexes. From the results, key structural elements that regulate the binding and selectivity of non-peptidic compounds onto the different proteins were deciphered. Interaction fingerprints and energy decomposition analysis identified key residues and energetic terms that are central for effective ligand binding. This research presents novel insight essential for the structure-based molecular drug design of more potent antimalarial drugs.
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- Date Issued: 2017
Comparative localization studies of P.falciparum ADP-ribosylation factor proteins in P.falciparum parasites and hela cells using GFP tagged constructs
- Authors: Swart, Tarryn
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/3537 , vital:20519
- Description: Expected release date-December 2018
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- Date Issued: 2017
Identification of SNPs within the CYP2A6 enzyme of TNBC cell lines and the resulting change in activity
- Authors: Dingle, Laura Margaret Kirkpatrick
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64349 , vital:28536
- Description: Expected release date-May 2019
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- Date Issued: 2017
In silico analysis of plasmodium falciparum Hsp70-x for potential binding sites and hits
- Authors: Amusengeri, Arnold
- Date: 2017
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59136 , vital:27435
- Description: Restricted access-thesis embargoed for 1 year - release date April 2019
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- Date Issued: 2017
Investigating the expression of three small open reading frames encoded on Helicoverpa armigera stunt virus RNA 1
- Authors: De Bruyn, Mart-Mari
- Date: 2017
- Subjects: Helicoverpa armigera , RNA viruses , Insects Viruses , Proteins
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59168 , vital:27448
- Description: The Helicoverpa armigera stunt virus (HaSV), belonging to the Family Alphatetraviridae (Genus: Omegatetravirus), is a non-enveloped insect virus encapsidating a bi-partite, positive-sense single-stranded RNA genome. RNA1 encodes the replicase, as well as three small open reading frames (ORFs) arranged in tandem, and overlapping with the 3’ end of the replicase ORF. These ORFs, designated p11, p15 and p8, encode putative proteins of unknown function. The p11 and p15 ORFs are conserved in the genome of the related Omegatetravirus, Dendrolimus punctatus tetravirus. In HaSV, the stop codon of p11 is followed immediately by the start of p15, whereas the stop of p15 and start of p8 are separated by a glycine intercodon. Furthermore, only p11 is known to have a recognizable Kozak sequence. The aim of this study was to determine the expression and function of these three small proteins in the HaSV infectious lifecycle. The authenticity of the viral cDNA sequence, encoding the three small ORFs, was validated by sequencing multiple cDNA clones of the relevant region in viral RNA (vRNA), purified from infectious HaSV particles. The sequence of all three ORFs was conserved in seven cDNA clones, while point mutations were observed in each of two remaining cDNA clones, suggesting that the ORFs were conserved in infectious virus. Polyclonal antisera were raised against a p11 peptide, and a recombinant p15-p8 fusion protein (p23) expressed and purified from Escherichia coli. The affinity of the anti-p23 antiserum was confirmed by western blot analysis, while that of the anti-p11 antiserum was confirmed using immunofluorescence microscopy, as attempted expression of recombinant p11 in E. coli appeared to be toxic. The antisera were used to detect expression of the small proteins in HaSV-infected H. armigera larvae by western blot analysis. A band migrating at approximately 34 kDa was detected by both antisera in infected larvae, absent in uninfected larvae, suggesting the expression of a p11-p15-p8 polyprotein. Protein bands of 11 kDa and 8 kDa were also detected by the anti-p11 and anti-p23 antisera, respectively. Bioinformatic analysis revealed that the polyprotein would be produced by a novel type of stop codon read-through, however the mechanism required for individual expression could not be definitively determined. The mechanism by which these ORFs are translated was further investigated by expressing p11-p15, tagged with FLAG and enhanced green flourescent protein (EGFP) at its amino- and carboxyl-termini respectively (FLAG-p11-p15-EGFP), in Spodoptera frugiperda (Sf9) cells detected by flourescence microscopy. Punctate structures were observed throughout the cytoplasm that were also detected with antiFLAG, anti-p11 and anti-p23 antisera, complementing results obtained in previous studies. Since p15 does not exhibit a strong recognizable Kozak like p11, the dependency of p15 expression on that of p11 was investigated by mutating this construct such that p15 occurred in a +1 frame to p11. Both EGFP and anti-p23 fluorescence was detected with the same cytoplasmic distribution as the unmutated construct, whereas nothing was detected by anti-FLAG and anti-p11. Preliminary results therefore suggested p15 may also be expressed as a discrete protein, independent of p11. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
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- Date Issued: 2017
Molecular cloning and expression of equine CYP1A2 in Escherichia coli
- Authors: Mkabayi, Lithalethu
- Date: 2017
- Subjects: Escherichia coli , Escherichia coli infections in animals , Cytochrome P-450 , Cytochromes , Horses -- Effect of drugs on
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4830 , vital:20734
- Description: Information regarding drug metabolism in veterinary species, especially horses, remains fragmented and incomplete. This information is essential for detection of metabolites of potential performance-enhancing substances in horseracing and for veterinary drug development. Equine liver microsomes have been used to study metabolism of a limited number of drugs, but these provide little information about individual drug metabolizing enzymes. Recombinant CYP enzyme systems are commonly used to determine contribution of individual CYP to metabolism of specific drugs. A limited number of recombinant equine CYPs have been expressed in insect cells and mammalian cell lines. However, there are no reports of recombinant equine CYP1A2 enzyme. In this study, equine CYP1A2 was identified, codon-optimized, cloned and expressed in E. coli BL21 cells. Multiple sequence alignments of equine CYP1A2 revealed an amino acid sequence identity of 83.69% to its human homolog which has previously been expressed in E. coli. The enzyme was expressed using both auto-induction and IPTG induction. Expressed equine CYP1A2 had a size of about 55 kDa, and was insoluble after cell lysis. Sarkosyl- solubilized CYP1A2 was purified using nickel affinity chromatography and gel filtration. For activity reconstitution, yeast NADPH-cytochrome P450 reductase was first expressed in E. coli BL21 cells and exhibited activity of 0.13 U/ml. Activity assay with Glo-P450 CYP1A2 assay kit indicated that CYP1A2 was inactive. Despite numerous attempts to obtain the activity, the CYP1A2 remained inactive. Although expression of equine CYP1A2 in E. coli produced non- catalytically active enzyme, this study could be used as the first step in an effort to fully develop a recombinant equine CYP1A2 system.
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- Date Issued: 2017
Regulation of cell biology by extracellular species of the Hsp90- Hsp70 organising protein (Hop)
- Authors: Höft, Maxine Allison
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59199 , vital:27465
- Description: Expected release date-April 2019
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- Date Issued: 2017
Structural analysis of proteases from South African HIV-1 (subtype C) patients undergoing Lopinavir treatment, using comparative modeling, ligand-docking and molecular dynamics
- Authors: Sheik-Amamuddy, Olivier
- Date: 2017
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/4931 , vital:20744
- Description: HIV is regarded as one of the most devastating infectious diseases of the last few decades, and has a high prevalence in South Africa, subtype C being the most common. Palliative measures used to fight HIV involve the use various types of inhibitors, including the use of HIV protease inhibitors. Representatives from this class of inhibitors are gradually losing their efficacy due to development of resistance mutations from HIV-1. In this study, compounds from the South African Natural Compound Database (SANCDB) were screened against HIV-1 protease models generated from protease protein sequences belonging to 11 South African HIV patients before and after treatment with Lopinavir. The effect of Lopinavir on the alteration of drug-binding affinity before and after treatment is investigated by molecular docking of the protease against other FDA-approved drugs and detection of mutation types using the HIVdb tool. A network representation of hydrogen bonding between docked ligands and their receptor proteases has been developed and a profiling method of visualizing receptor-ligand docking energies at the local level is presented. Four potential HIV-1 protease inhibitors were identified from the list of 599 natural compounds on the basis of receptor conformation and binding free energy. Ligand stabilities were monitored by 20ns molecular dynamics runs using the GROMACS software.
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- Date Issued: 2017
Synthesis and biological evaluation of anti-HIV-I integrase agents
- Authors: Jesumoroti, Omobolanle Janet
- Date: 2017
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/59215 , vital:27479
- Description: Expected release date-April 2019
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- Date Issued: 2017
The development of biodegradable aerogel scaffolds for the generation of vascularised 3D adipose tissue models
- Authors: Makhene, Lebohang
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59245 , vital:27492
- Description: Expected release date-April 2019
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- Date Issued: 2017
The development of techniques for the identification of novel viruses associated with acute infantile gastroenteritis in South Africa
- Authors: Jaquet, Brittany J
- Date: 2017
- Subjects: Gastroenteritis in children -- Treatment , Gastroenteritis in children -- Treatment -- South Africa , Antiviral agents , Viral vaccines , Rotaviruses , Virus diseases in children
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/38013 , vital:24725
- Description: Gastroenteritis is a serious disease affecting both children and adults globally, but is more predominant in children with over half a million deaths reported each year. The leading cause of this disease is rotavirus, which accounts for 38% of all hospitalised cases. There has, however, been a significant decrease in the number of deaths associated with rotavirus worldwide since the introduction of the two vaccines, Rotarix® and RotaTeq®. A large number of cases are therefore either associated with other viruses, such as norovirus, Aichi virus (AiV) or Saffold virus (SAFV), or are of unknown aetiology. This study thus aims to develop techniques for the identification of viruses associated with gastroenteritis. Theiler’s murine encephalomyelitis virus (TMEV) was used to develop the sample preparation, transmission electron microscopy and RT-PCR techniques used in this study. This virus was chosen as a replication system using baby hamster kidney cells can be used to create high concentrations of viral particles from which RNA can be extracted preventing the waste of the limited samples. The virus particles are also similar in size and morphology to the viruses to be identified in this study, as it belongs to the same family. After sample preparation, TEM analysis showed the presence of small, round, non-enveloped virus particles in the TMEV sample. Due to the low concentration of virus particles, PEG precipitation was performed using both 0.15 M and 0.25 M NaCl and 8% (w/v) PEG 6000. TEM analysis then showed an increase in viral particle concentration, with the highest concentration observed at 0.25 M NaCl and 8% PEG 6000. RNA was successfully extracted and RT-PCR assays were performed for both the VP1 and 2B coding regions of TMEV. A method for creating a positive control for the RT-PCR assay was developed by the in vitro transcription of RNA from pTMEV, which contains the cDNA of TMEV. The RNA was then used as the template for the 2B two-step RT-PCR assay. A product of 412 bp was successfully amplified from the in vitro transcribed RNA and the sensitivity of the RT-PCR assay was determined. Using a Norovirus GII positive stool sample provided by Maureen Taylor, a nested RT-PCR assay was developed for the NoV GII N/S domain using a previously-published primer set and cycling parameters. A 342 bp product was successfully amplified from the RNA extracted from the stool sample and cloned into pGEM®-T Easy to produce pNoVGII. Using the plasmid containing the AiV 5’UTR and the PCR amplicon for AiV 3CD, RT-PCR assays were developed for AiV 5’UTR and partial 3CD. The RT-PCR assays produced a 1008 bp product for AiV 5’UTR and 266 bp for AiV 3CD, which were cloned into pGEM®-T Easy to produce pAiV5’UTR and pAiV3CD, respectively. Using in vitro transcribed RNA from pNoVGII, pAiV5’UTR, pAiV3CD and pSAFV, which contains the SAFV cDNA, positive controls were developed for the RT-PCR assays for NoV GII, AiV 5’UTR, AiV 3CD and SAFV 2C. The sensitivity of these assays was determined. The samples chosen for this study include wastewater collected from the Belmont Valley water treatment plant, oysters suspected to be infected with viruses collected from Port Elizabeth, South Africa and 30 stool samples from symptomatic patients. With the methods developed using TMEV, the wastewater, oysters and 30 stool samples were filter-sterilised, concentrated and screened by TEM. All samples showed the presence of virus particles. RNA was successfully extracted and the wastewater, oyster and 30 stool samples were screened for NoV GII using the NoV GII RT-PCR assay. The wastewater, oysters and 11 of the stool samples produced the 342 bp NoV GII PCR product and BLAST analysis determined the nucleotide sequences to be NoV GII.4. This shows that this study was able to develop sample preparation techniques and TEM analysis for selected samples and RT-PCR assays for NoV GII, AiV and SAFV. The NoV GII RT-PCR assay was successfully used for the screening of the wastewater, oysters and 30 stool samples for NoV GII. Due to the high number of gastroenteritis cases with unknown aetiology in South Africa, the development of techniques for the identification of NoV, AiV, SAFV and other viruses is very important. The identification of these viruses will allow for better surveillance, treatment and prevention of gastroenteritis in South Africa.
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- Date Issued: 2017
The relationship between OCT4 and an aggressive phenotype in triple negative breast cancer (TNBC)
- Authors: Jackson, Hayley Claire
- Date: 2017
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/59209 , vital:27477
- Description: Expected release date-April 2019
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- Date Issued: 2017
The Role of HOP in Emerin-Mediated Nuclear Structure
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
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- Date Issued: 2017