DSpace Collection: Theses

MOLECULAR MECHANISMS OF MULTIPLE INSECTICIDE RESISTANCE IN Anopheles funestus sensu stricto AT AKAKA-REMO, SOUTHWESTERN NIGERIA

Fri, 01 Mar 2019 00:00:00 GMT

Title: MOLECULAR MECHANISMS OF MULTIPLE INSECTICIDE RESISTANCE IN Anopheles funestus sensu stricto AT AKAKA-REMO, SOUTHWESTERN NIGERIA Authors: ATOYEBI, S.M Abstract: The use of insecticides against malaria vectors has been a good approach to control malaria, but its efficacy is threatened by mosquito resistance to the lethal doses. In Nigeria, Anopheles funestus is gradually becoming an important malaria vector, especially in rural areas. However, despite recurring cases of Insecticide Resistance (IR) in some Anopheles species, there is a dearth of information on the molecular mechanisms of IR in An. funestus. Therefore, this study was designed to investigate the molecular mechanisms of IR in An. funestus at Akaka-Remo, Southwestern Nigeria. Early morning collections of adult mosquitoes were conducted from October 2014 to April 2015 in 30 rooms (25 houses) at Akaka-Remo (population density = 5,585). The mosquitoes were identified with standard keys, but only An. funestus (F0) was identified to sub-species level by Polymerase Chain Reaction (PCR). The F0 was analysed for Plasmodium Infection Rate (PIR) by TaqMan real-time PCR, and screened for Knockdown resistance (Kdr) mutations using next-generation sequencing. The gene sequences generated were analysed with bioinformatics tools. The F0 was further subjected to forced-egg laying technique to generate the F1. The mortality rate of 2-5 day old F1 exposed to permethrin- (0.75%), deltamethrin- (0.05%), DDT- (4%), dieldrin- (4%) and bendiocarb (0.1%)-treated papers was determined using WHO standard procedures. The contribution of detoxifying enzymes to IR was assessed by first exposing the F1 to three standard synergists: Piperonyl Butoxide (PB:4%), S,S,S-Tributyl Phosphorotrithioate (STP:0.25%) and Diethyl Maleate (DM:8%); and to permethrin and DDT, respectively. Microarray and real-time PCR were used to identify differentially expressed genes in permethrin and DDT-resistant populations. The frequency of resistant and susceptible alleles in DDT (L119F-GSTe2) and dieldrin (A296S-RDL) resistance markers was determined in the F0 and F1 genotype using TaqMan real-time PCR. Data were analysed using descriptive statistics and Student’s t-test at 0.05. A total of 375 mosquitoes were collected and identified as An. funestus (83.8%), An. gambiae (6.9%), Culex species (5.6%), Aedes species (1.3%) and Mansonia species (2.4%). All Anopheles funestus were further identified as An. funestus sensu stricto, with a PIR of 8.0%; and no Kdr mutations. Permethrin, deltamethrin, DDT, dieldrin, and bendiocarb induced 68.0, 87.0, 10.0, 8.0 and 84.0 % mortalities, respectively in the F1, indicating resistance. Exposure of the F1 to the three synergists with permethrin induced 100% mortality each; while PB, STP and DM with DDT, respectively induced 30.0, 81.8 and 71.4% mortalities. These suggest that detoxifying enzymes contributed to IR in the mosquitoes. Overexpressed genes in the resistant populations were GSTe2, GSTd3, GSTd1-5, GSTU2, CYP6P2, CYP6P9a, CYP6P4a and CYP9K1. The frequency of resistant alleles in L119F-GSTe2 and A296S-RDL was significantly high: F0=77.0%, F1=80.0% and F0=76.0%, F1=90.0%, respectively compared to the susceptible alleles: F0=23.0%, F1=20.0% and F0=24.0%, F1=10.0%, respectively. Overexpression of detoxifying genes and the high frequency of resistant-associated mutations were responsible for multiple insecticide resistance in the Anopheles funestus at Akaka-Remo. Description: A Thesis in the Department of Zoology submitted to the Faculty of Science in partial fulfilment of the requirements for the Degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN

HOST CYTOKINE GENE POLYMORPHISMS AND PARASITE GENETIC VARIABILITY IN DETERMINING THE DISEASE OUTCOME OF Plasmodium falciparum INFECTION

Sat, 01 Sep 2012 00:00:00 GMT

Title: HOST CYTOKINE GENE POLYMORPHISMS AND PARASITE GENETIC VARIABILITY IN DETERMINING THE DISEASE OUTCOME OF Plasmodium falciparum INFECTION Authors: OYEDEJI, S.I Abstract: Malaria, the world’s most important tropical parasitic disease is caused by Plasmodium species. Infection with Plasmodium falciparum can result in one of three possible disease outcomes: Asymptomatic (AS), Uncomplicated Malaria (UM) or Severe Malaria (SM). Information on host genetic factors and parasite genetic diversity can improve understanding of the disease pathogenesis. In this study, the genetic diversity of P. falciparum isolates as well as polymorphisms in host cytokine genes was investigated in relation to the outcome of P. falciparum infection. Four hundred and thirty-seven children recruited from the Specialist Hospital, Lafia, Nasarawa State were assigned into UM or SM based on malaria severity, determined by clinical and laboratory diagnoses. Asymptomatic children recruited from primary schools within the study location constituted the control group (AS). Plasmodium falciparum infection was confirmed by PCR-based assay of SSUrRNA genes. Genetic diversity of P. falciparum was analyzed by genotyping the polymorphic domains of the Merozoite Surface Protein 2 (MSP-2). Host cytokine genes investigated included Interleukin-18 (IL-18), IL-18 receptor alpha (IL-18Rα) and Tumour Necrosis Factor alpha (TNF-α). Sequencing of MSP-2 gene and of the pro-inflammatory cytokines was carried out using ABI PRISM® 3100. Sequences were analyzed using the BioEdit Sequence Alignment software. Genotype and allelic frequencies were analyzed by Chi-square test. The level of significance was set at P=0.05. All participants had P. falciparum infection. Polyclonality was significantly higher in the AS (61%) and UM (60%) groups compared with the SM (34%) group. Mean multiple infections was 2.1 ±1.0 in AS, 2.0 ±1.0 in UM and 1.3 ±0.6 in SM. A total of 32, 35 and 28 distinct MSP-2 alleles were found in the AS, UM and SM groups respectively. Frequency of the 3D7 allele type was significantly higher in UM (51%) and SM (54%) groups compared to the AS group (38%). Sequence analysis of the central variable region of the MSP-2 gene showed that the FC27-type sequence was characterised by two unique subtypes and hybrid sharing sequence with the two subtypes. The 3D7-type sequence was characterised by three subtypes of repetitive domains: a GSA-rich repeat unit, a TPA repeat motif and a poly-Threonine stretch. Three single nucleotide polymorphisms (SNPs): -656G/T, -607C/A and -137G/C were identified at the promoter region of IL-18 gene. The -656G/T and -607C/A SNPs were found to be in complete linkage disequilibrium. The genotype frequency of -607AA was significantly higher in the AS group compared to SM cases (χ2=4.26, P<0.05). Likewise, four SNPs were identified at the promoter and Exon 1 of the IL-18Rα: -661T/C, -175G/A, -93C/T and Ex1 +21C/G but none was associated with disease outcome based on statistical level of significance. Exons 2 to 11 of IL-18Rα gene were relatively conserved. Furthermore, two SNPs: -308G/A and -238G/A were identified at the promoter region of TNF-α but none was associated with disease outcome. Plasmodium falciparum was found to be genetically heterogeneous. Higher carriage of Plasmodium falciparum 3D7 alleles indicates higher risk of developing symptomatic malaria. There was association between IL18 -607AA genotype and asymptomatic infection, probably indicating a protective role. Description: A Thesis in the Department of Zoology, submitted to the Faculty of Science in partial fulfilment of the requirements for the Degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN

GENETIC AND SYSTEMIC TOXICITY INDUCED BY TITANIUM DIOXIDE AND ZINC OXIDE NANOPARTICLES AND THEIR MIXTURE IN SOMATIC AND GERM CELLS OF MICE

Thu, 01 Nov 2018 00:00:00 GMT

Title: GENETIC AND SYSTEMIC TOXICITY INDUCED BY TITANIUM DIOXIDE AND ZINC OXIDE NANOPARTICLES AND THEIR MIXTURE IN SOMATIC AND GERM CELLS OF MICE Authors: FADOJU, O.M Abstract: Titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles are components of personal care products whose continuous release into the environment may enhance co-exposure, with potential risks to the ecosystem. In vitro studies have shown their potential to induce genetic damage. However, there is dearth of information on in vivo induction of DNA and systemic damage, alongside their interactive effects. This study was designed to investigate genetic and systemic toxicity and mechanism of DNA damage by TiO2 and ZnO nanoparticles and their mixture in mice. Male Swiss mice (=24.0±2.0g; n=80; 6-8 weeks old) were intraperitoneally exposed to distilled water (Control) and 9.4, 18.8, 37.5, 75.0 and 150.0 mg/kg concentrations of each of the nanoparticles and their mixture (1:1) for 5 days (5 mice/group) to assess micronucleus induction and cytomorphological abnormalities in the bone marrow of mice. Haematological parameters [Haemoglobin, Packed Cell Volume (PCV), Red Blood Cell (RBC) and White Blood Cell (WBC) counts] were assessed following standard procedures. Mechanism of DNA damage was evaluated by oxidative stress [Superoxide dismutase (SOD), reduced Glutathione and Malondialdehyde in the liver and kidney] parameters following standard methods. Sperm count, motility, abnormalities and concentrations of Luteinizing Hormone (LH), Follicle Stimulating Hormone (FSH) and Testosterone were evaluated in another group of mice (=30.0±2.0g; n=80; 11-15 weeks old), intraperitoneally exposed with the same nanoparticle concentrations (5 mice/group) at 35-day exposure. Liver, kidney and testis were sectioned for histopathological analysis. The Interaction Factor (IF) of nanoparticle mixture was calculated according to standard method. Data were analysed using descriptive statistics and ANOVA at α0.05. The nanoparticles and mixture induced micronuclei, but significant only for TiO2 (16.8±2.1-53.3±18.5) compared with the control (3.7±0.9). Blebbed, target, hyperchromic and hypochromic erythrocytes were the observed cytomorphological anomalies. The mixture exerted a significant reduction only in the WBC count. In the liver, there was a significant decrease in SOD (unit/mg protein) activities (1.3-1.5; 1.4-2.0; and 1.2-1.6 fold for TiO2, ZnO and mixture, respectively), with increase in Malondialdehyde (nmol/mg protein) levels (1.1-1.7; 1.2-1.8; and 1.7-1.7 fold for TiO2, ZnO and mixture, respectively). In the kidney, there were significant alterations in SOD: 1.2-1.3; and 1.1-1.4 fold decrease for TiO2 and ZnO, respectively; and 1.3-2.0 fold increase for the mixture. While Malondialdehyde levels increased (1.2-1.4; 1.4-1.6; and 1.7-1.9 fold for TiO2, ZnO and mixture, respectively). Both organs showed alterations in reduced Glutathione levels (1.0-1.5 fold decrease for TiO2; 1.0-1.1 fold increase for ZnO and mixture) indicating systemic toxicity. A significant decrease in sperm count and motility; and increase in abnormalities (1.3-8.0; 1.2-2.6; 4.6-12.1 fold for TiO2, ZnO and mixture, respectively), with a concomitant decrease in the serum level of LH and increase in FSH and Testosterone were observed. Hepatocellular and spermatogenic cell necrosis and degeneration of tubular epithelial cells were observed. The IF indicated synergism. Titanium dioxide and zinc oxide nanoparticles and their mixture induced genomic and systemic damage in somatic and germ cells of mice; with the mixture synergistically evoking the highest toxic response. Oxidative stress might be one of the mechanisms of cytogenotoxicity. Description: A Thesis in the Department of Zoology, submitted to the Faculty of Science in partial fulfilment of the requirement for the Degree of DOCTOR OF PHILOSOPHY of the UNIVERSITY OF IBADAN

GENETIC AND SYSTEMIC TOXICITY INDUCED BY TITANIUM DIOXIDE AND ZINC OXIDE NANOPARTICLES AND THEIR MIXTURE IN SOMATIC AND GERM CELLS OF MICE

Thu, 01 Nov 2018 00:00:00 GMT