3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item The effect of alcohol on cranial neural crest cells: implications for craniofacial development(2011-03-31) Oyedele, Olusegun OlufemiWhile ethanol is recognised beyond doubt as a teratogen to the unborn fetus, research nevertheless continues in order to understand its mode of action and its effects at the cellular level. The present study aimed to investigate the effect of an acute dose of ethanol on cranial morphology and morphometry in mouse fetuses, as well as on the morphology, migration and the expression of cell migration related genes in cultured chick cranial neural crest cells (cNCCs). Thirteen pregnant C57/BL mice were orally administered with 0.03ml/g of 25% (v/v) ethanol daily on gestational days (GD) 6, 7 and 8. Ten control animals received an identical dose of saline. On GD 18, all mice dams were killed and their fetuses were removed. Fetal morphological observations and crown-rump lengths were evaluated as were mean litter size, survival rate, birth weight and cranial dimensions. Cranial neural crest cells (cNCCs) were cultured from Potchefstroom koek koek stages 8-10 (HH) chick embryo neural tubes either in culture medium (DMEM) to which 0.2%, 0.3% and 0.4% ethanol (v/v) respectively, was added (treated) or in DMEM only (controls). Whole-mount HNK-1 immunocytochemistry was performed on treated and control chick embryos, as was an assay for caspase-dependent apoptosis. Photographs were taken of the cultures and the distance which the neural crest cells migrated from the neural tube at 24 and 48 hrs post-culture was measured. 24-hr time-lapse video microscopy recordings were also made to analyse the migration of the neural crest cells. Rhodamine-phalloidin immunocytochemistry for the actin cytoskeleton and scanning electron microscopy of surface ultrastructure were performed on migrating treated and non-treated cNCCs, as were proliferation assays and quantitative PCR of cNCCs‟ β-actin, Rac 1, Rho B and slug genes. There was a statistically significant increase in fetal reabsorption as well as a significantly reduced fetal survival rate observed in newborn mice fetuses that had been exposed to ethanol in utero compared to control fetuses. Ethanol-exposed mice showed a number of abnormalities, which were not significantly increased over vi controls (p>0.5). Birth weight, crown-rump length and mandibular length were also not significantly different in treated fetuses compared to controls (p>0.5). Treated (0.3%) chick cNCCs migrated over a significantly increased distance at both 24hrs and 48hrs compared to controls (p<0.05) in the axes of migration that were studied. The migratory distances of cNCCs derived from embryonic stage 9 (HH) were markedly affected by treatment with alcohol. The actin cytoskeleton of treated cNCCs showed disorganisation and loss of focal adhesion contacts while Rac 1, Rho B and slug genes were either up-regulated or down-regulated depending on the ethanol dose and duration of treatment. Ethanol promotes significant proliferation in cNCCs and may affect their migration by altering the expression of migration-linked genes and the arrangement of the actin cytoskeleton.Item Alcohol and cannabis use among mineworkers in South Africa(2011-02-18) Ajani, Adenike Frances YejideSubstance use is associated with mining accidents, increased health care utilisation, and economic loss. Although South Africa is a major mining country, paucity of data exists on substance use among mineworkers. To determine the prevalence of alcohol and cannabis use among mineworkers, the prevalence of accidents associated with substance use, and factors influencing substance use among this population, structured interviews of 1571 participants (involving breathalyser tests for alcohol and urine tests for cannabis), focus group discussions, and a record review of post-accident substance tests were carried out in seven mines. While structured interviews were carried out between March & October 2002, focus group discussions were carried out between May & June 2003, and record review was done between March & September 2004. Between 10.7% to 24.4% of participants across study mines, with a mean of 15.3%, are likely to be dependent on alcohol, while 4.6% to 21.5% of participants, with a mean of 9.1%, use cannabis. Between 0% and 5.9% of all breath samples, with a mean of 1.9%, contained alcohol ≥ 0.10mg/1000ml of breath, the legal limit for professional drivers. However, the majority of positive samples were collected on a Monday and day of sample collection was found to be a confounding factor. In mine P1 in 2003, 1% of samples tested in cases of accidents were positive for alcohol, and in 2002 and 2003, cannabis tests were positive in 4.9% and 3.9% of accident cases tested, respectively. v Low levels of education (p=0.020), low job categories (p=0.004) and lack of awareness of link between cannabis use and accidents (p=0.0001) were found to be positively associated with cannabis use. Being a full-time worker compared to a contract worker (p=0.004) was protective from cannabis use. While being married (p=0.001) was protective from alcohol use, there was no significant difference in the alcohol and cannabis use status of those who were married and lived with their wives at the mines and those who didn‟t live with their wives but visited them periodically. Findings of this study where alcohol use for fun (p=0.046) and relaxation (p=0.018) were associated with alcohol dependence, and where misconceptions about the energy-boosting attribute of cannabis, and perception that work is „most-times to always dangerous‟ (p=0.012) were associated with cannabis use and alcohol dependence, suggest the use of substances as a coping mechanism and highlight the link between social factors and substance use. Industry regulations employing a holistic approach and incorporating essential components, such as clear written individual mine policy, health promotion, monitoring and surveillance, Employee Assistance Programmes, disciplinary procedures and wellness programmes, can contribute towards substance use control among this population.Item Computational prediction of gene targets for fetal alcohol spectrum disorders(2010-04-01T13:30:13Z) Lombard, ZaneFetal alcohol spectrum disorders (FASD) describe the range of disorders that result from in utero alcohol exposure. FASD is a serious global health problem and is observed at exceedingly high frequencies in certain South African communities. Although in utero alcohol exposure is the primary trigger, there is evidence that genetic- and other susceptibility factors contribute towards FASD development. To date, no genome-wide association or linkage studies have been performed for any of the FASD syndromes. The main objectives of this study were to develop an innovative approach to computationally identify biologically plausible candidate genes for FASD, for a future association study, and to evaluate the appropriateness and validity of this approach. Further, an in silico analysis of known single nucleotide polymorphisms (SNPs) within the top-ranked candidate gene was performed in conjunction with de novo SNP detection, to select a subset of SNPs based on proposed functional impact on gene expression and protein function, for a prospective association study. A computational binary filtering technique was designed that can be employed to prioritize genes in a candidate list, or could be used to rank all genes in the genome in the absence of such a list. 10174 FASD candidate genes were initially selected from the whole genome using a previously described method. Hereafter the candidates were prioritized using a binary filtering technique. The biological enrichment of the ranked genes was assessed by investigating the protein-protein interactions, functional enrichment and common promoter element binding sites of the top-ranked genes. A group of 87 genes was prioritized as candidates highlighting many strong candidates from the TGF-/, MAPK and Hedgehog signalling pathways, which are all integral to fetal development and potential targets for alcohol's teratogenic effect. To assess the effectiveness and accuracy of this computational approach, X-linked mental retardation (XLMR) was used as a test disease, considering that XLMR is a set of heterogeneous disorders of which some of the underlying genetics is known. This implementation resulted in a prioritized gene list with a noted enrichment of known XLMR genes among the top-ranked genes. Furthermore, the top-ranked list contained genes that were biologically relevant to XLMR, and could potentially be as yet unknown candidate genes for XLMR. Indeed, many of the top-ranked genes mapped to XLMR candidate regions, confirming their status as good candidates. Finally, a subset of seven known and novel SNPs was selected within FGFR1 based on putative functional impact. Data from the HapMap project was used to identify tag SNPs for FGFR1 to complement the selection made based on function. The main limitation of the proposed computational approach to candidate gene prediction is that it is primarily based on gene annotation, and that it is therefore biased towards selecting better-annotated genes. However, the results obtained in this study suggest that the described computational method is an effective approach that can identify likely candidates that are biologically relevant to the disease of interest, and therefore appropriate for a candidate-gene association studies. In practice, this technique is an appropriate approach to select a workable set of candidate genes for a complex disease, in a setting where a whole-genome association study is not a viable option.Item The effect of alcohol on the methylation status of the imprinting control regions contained within three developmentally significant loci(2010-02-25T10:37:47Z) Knezovich, Jaysen GregoryImprinted loci are critical in foetal development and most are regulated by the methylation-specific CTCF binding protein which binds imprinting control regions (ICRs). The ICR is located between two genes that comprise imprinted loci, which are reciprocally expressed in a parent-of-origin specific manner. Maternally hypomethylated ICRs allow CTCF binding, creating a boundary element which prevents downstream enhancers from acting on the paternally expressed gene upstream of the ICR. Conversely, the hypermethylated (imprinted) paternal ICR prevents CTCF binding, allowing downstream enhancers to act on the gene upstream of the ICR, while suppressing the downstream maternally expressed gene. Alcohol and its metabolites are able to reach the testes via the blood supply and are known to reduce global DNA methylation by disrupting the folate, methyl group and homocysteine pathway. This may therefore affect gene expression at imprinted loci, whose parental alleles are discriminated by the imprinting status at the ICR. The effect of pre-conception paternal alcohol exposure on the DNA methylation of three paternally imprinted ICRs (H19, Rasgrf1, IG-DMR) as well as the maternally imprinted Snrpn ICR was examined in mouse sperm and their offspring. Male mice were gavaged with ethanol or sucrose. DNA was extracted from sperm of treated males and tail biopsies from offspring. Samples were bisulphite modified and the ICRs PCR amplified. DNA methylation patterns of ICRs were analysed by sequencing and quantitatively via pyrosequencing. Sperm samples of ethanol treated males did not show significant demethylation when compared to sucrose treated mice, with the exception of H19 CpG 7, Rasgrf1 CpG 26 and Snrpn CpG 10 (p=0.024, 0.014 andItem The prevalence of alcohol and other drug use amongst school learners in Alexandra Township.(2006-03-13) Langa, MaloseThe aim of this study was to investigate the prevalence of alcohol and other drug use amongst school learners in Alexandra Township. The participants in this study were 118 school learners in Alexandra Township. Of these 44 (40%) were in grade 9, 37 (32.2%) in grade 10 and 32 (27.8%) in grade 11; 55 (46.6%) were males and 63 (53.4%) were females; 50.4% were aged between 14—16, while 49.5% were aged between 17-20 years. Data was collected by means of a questionnaire, mostly requiring ‘yes’ or ‘no’ answers to determine use of various substances, the age of first use and the frequency use of these substances. This questionnaire has been used in other local studies and translated from English into Sesotho to maximize the validity of responses. The findings of this study indicate that in the past month the prevalence of 54.5% of males and 38.4% of females smoked cigarettes; 49.1% of males and 39.1% of females drank alcohol; and 36% of males and 12.6% of females smoked dagga. The everyday use of cigarettes was 41.7% for females and 47.2% for males; 7.2% of males and 3.6% females for alcohol; and 29.9% of males and 6.3 % for dagga. The results showed that there were no significant age and gender differences regarding the use of these drugs, except for dagga (X²=.005), with more males than females reported the use. It seems that the everyday use of alcohol, tobacco and dagga is very common than the use of other illicit drugs. Mental health workers should take note of the above findings while planning preventative strategies for the reduction of everyday dagga use, daily cigarette smoking and drinking of alcohol. The results are also important for those involved in treatment programmes to assist these school learners before they progress into more other serious drugs such as ecstasy, LSD, Crack cocaine and heroin.