Browsing by Author "Singh, Ashika."

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Cationic liposome mediated transfection with/without a targeting component.
(2005) Singh, Ashika.; Naidoo, Richard.; Singh, Moganavelli.
The transfer and expression of genes in cells is an important technique for basic research and gene therapy of human disease. A model for gene therapy has been investigated making use of a transfection complex consisting of three components, the DNA i.e. the gene to be transferred and expressed; a gene delivery vehicle viz. a cationic liposome and a cell specific targeting ligand, asialoorosomucoid (AOM). Cationic liposomes are positively charged liposomes that have been prepared from synthetic lipids and have been shown to complex or bind to DNA via electrostatic attraction. They have shown potential as an efficient non-viral gene delivery vehicle in human gene therapy. In this investigation, a novel cationic liposome consisting of 3B [N -(N',N'-dimethylaminopropane)carbamoyl] cholesterol (Chol-T), dioleoylphosphatidylethanolamine (DOPE) and biotinylcholesteryl formylhydrazide was prepared and assessed as a mediator of DNA delivery in a mammalian cell culture system viz. the HepG2 cell line. The cationic liposome was synthesised and characterised by electron microscopy. Foreign DNA may be specifically delivered to target cells by a carrier system which makes use of the recognition of the asialoglycoprotein AOM by cognate receptors on the HepG2 cell plasma membrane. The positively charged AOM was biotinylated and due to this biotinylation, binds streptavidin which contains specific binding sites for biotin. The cationic liposome itself contains biotin residues in its bi-Iayer which in turn binds streptavidin resulting in a ternary complex. Further, due to the DNA binding capability of the cationic liposome, a transfection complex is produced consisting of the three components. The experiments were based on the following concepts: (i) Hepatocytes possess a unique receptor that binds to and internalises galactose-terminal asialoglycoproteins by receptor mediated endocytosis. (ii) Due to electrostatic attraction, DNA binds to cationic liposomes forming soluble complexes. (iii) Through the biotin-streptavidin reaction, the biotinylated AOM is attached to the cationic liposome containing biotin forming complexes enabling targeted delivery of the DNA. (iv) DNA containing the pGL3 gene for the luciferase enzyme was used and following transfection experiments, the luciferase assay was performed to ensure successful transfection. The complexes were tested on the hepatocellular carcinoma cell line, HepG2, which possess the asialoglycoprotein receptor. Transfection studies were conducted using a transient expression system, the luciferase assay system. Some degree of success in the transfection of HepG2 cells was observed. Results obtained in this study suggest that transfection using our targeted transfection complex consisting of cationic liposomes and cell specific targeting ligands does in fact transfect cells by receptor mediation.
Coreceptor utilization and primary cell tropism by HIV-1 subtype C strains.
(2010) Singh, Ashika.; Ndung'u, Peter Thumbi.
Human immunodeficiency virus type 1 (HIV-1) isolates can be differentiated based on their ability to use particular coreceptors – R5 viruses use CCR5, X4 viruses use CXCR4 and R5X4 (dual tropic) viruses use both CCR5 and CXCR4. It is widely reported that HIV-1 subtype C (HIV-1C) has a unique viral coreceptor evolution pattern in that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for this subtype compared to other subtypes. However, dual tropic HIV-1C isolates have occasionally been described. Furthermore, it has been reported that certain highly active antiretroviral drugs (HAART) may select for X4 viral variants. Therefore, this thesis study was undertaken to better understand the functional and genotypic characteristics of dual tropic HIV-1C isolates, and to characterize drug resistance and coreceptor usage patterns in HAART-naïve versus HAART-failing HIV-1C infected patients. Thirty-five functional HIV-1 env clones derived from seven dual tropic HIV-1C strains were generated and their coreceptor usage characterized in transformed cell lines. All 35 env clones efficiently infected transformed cells expressing CXCR4. Twenty of 35 clones (57%) also utilized the CCR5 receptor. No R5-only clones were detected. Functional coreceptor usage data was correlated to env gene sequence data. The ability of the HIV-1C env clones to facilitate infection of primary lymphocytes and monocyte-derived macrophages was next investigated. The majority of clones characterized as X4 or R5X4 on cell lines used either CXCR4 alone or CXCR4 and CCR5, respectively, in primary cells. A few viruses displayed comparable CCR5 and CXCR4 usage and clones from one virus preferred CCR5 usage in macrophages. Thus in a few cases coreceptor phenotyping in transformed cell lines does not predict usage in primary cells. Genetic determinants for coreceptor usage in primary cells require further investigation. Finally the patterns of drug resistance mutations were studied and coreceptor usage among 45 HAART-naïve and 45 HAART-failing HIV-1C infected patients analyzed. Ninety-five percent of HAART-failing patients had viruses with at least one drug resistance mutation. Thymidine analog resistance mutations (TAMs) were present in 55% of patients. HAART-failing patients had significantly higher prevalence (59%) of X4/R5X4-utilizing viruses compared to HAART-naïve patients (30%) (p<0.02) using the Trofile Co-receptor Tropism Assay while 41% of HAART-failing patients used CCR5 and 70% of HAART-naïve patients used CCR5. Functional results correlated with predictive algorithm methods. This study enhances our understanding of HIV-1 pathogenesis and the results have important implications for the use of coreceptor antagonists for the clinical management of HIV-1C infection.
Drug resistance and viral tropism in HIV-1 subtype C-infected patients in KwaZulu-Natal, South Africa : implications for future treatment options.
(Lippincott Williams & Wilkins., 2011) Singh, Ashika.; Sunpath, Henry.; Green, Taryn N.; Padayachi, Nagavelli.; Hiramen, Keshni.; Lie, Yolanda.; Anton, Elizabeth D.; Murphy, Richard.; Reeves, Jacqueline D.; Kuritzkes, Daniel R.; Ndung'u, Peter Thumbi.
Background: Drug resistance poses a significant challenge for the successful application of highly active antiretroviral therapy (HAART) globally. Furthermore, emergence of HIV-1 isolates that preferentially use CXCR4 as a coreceptor for cell entry, either as a consequence of natural viral evolution or HAART use, may compromise the efficacy of CCR5 antagonists as alternative antiviral therapy. Methods: We sequenced the pol gene of viruses from 45 individuals failing at least 6 months of HAART in Durban, South Africa, to determine the prevalence and patterns of drug-resistance mutations. Coreceptor use profiles of these viruses and those from 45 HAART-naive individuals were analyzed using phenotypic and genotypic approaches. Results: Ninety-five percent of HAART-failing patients had at least one drug-resistant mutation. Thymidine analog mutations (TAMs) were present in 55% of patients with 9% of individuals possessing mutations indicative of the TAM1 pathway, 44% had TAM2, whereas 7% had mutations common to both pathways. Sixty percent of HAART-failing subjects had X4/dual//mixed-tropic viruses compared with 30% of HAART-naïve subjects (P < 0.02). Genetic coreceptor use prediction algorithms correlated with phenotypic results with 60% of samples from HAART-failing subjects predicted to possess CXCR4-using (X4/dual/mixed viruses) versus 15% of HAART-naïve patients. Conclusions: The high proportion of TAMs and X4/dual/mixed HIV-1 viruses among patients failing therapy highlight the need for intensified monitoring of patients taking HAART and the problem of diminished drug options (including CCR5 antagonists) for patients failing therapy in resource-poor settings.
Exploring the diverse perspectives of the disciplinary methods aimed at modifying problem behaviours in young children in selected areas in Pietermaritzburg.
(2017) Singh, Ashika.; Mayaba, Phindile Lungile.
Racial diversity shapes the way individuals perceive the concept of discipline, from the meaning they attach to it, to the different disciplinary methods they apply, to the role they assume in the disciplinary process. Described as a form of love, respect and guidance, discipline encompasses the teaching of values and behaviours. Young children are encouraged to know the rules and boundaries within the home and schooling facilities, making them mindful of the behaviours that are expected of them. In South Africa, the modern child is labelled as technologically more advanced, eager to do more, see more and learn more, and therefore needs constant stimulation. Notwithstanding the impact of race and culture in nurturing a child, technology currently also plays a role, with the study results revealing that the majority of participants denied children luxuries as a method of modifying problem behaviours. Parents and educators approached discipline in various ways, implementing methods familiar to them. The research results revealed that several participants supported the occasional smack on the bottom, arguing that the intent and attitude of a light smack or tap is very different from physical abuse, and demonstrates love and guidance. Many of the participants interviewed were in favour of maintaining corporal punishment within the home environment. Further positive disciplinary methods used included time-out, rewards and praise, a change in tone of voice, and the denying of luxuries. The participants held very negative views on the use of demerits, with a number of participants choosing not to do so. Participants described demerits as demoralising and de-motivating, and claimed that it created a sense of unfairness on the part of learners. The parents, crèche facilitators and Grade R educators interviewed highlighted problems that included a lack of parental involvement in homework, recreational activities, and most importantly, discipline. Emphasis was also placed on establishing a more positive, healthy educator-learner relationship. The recommendations of the study included creating greater awareness through the means of support networks and workshops for parents and educators. These could establish a forum to share information and provide support when dealing with problem behaviours, language barriers and discipline.