School of BioSciences - Theses

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    Evolution of drug-resistance genes in the asymptomatic Plasmodium falciparum reservoir of infection in Ghana
    Narh, Charles Akugbey ( 2019)
    Ghana is one of the 11 countries in the world with the highest malaria burden. Like many other African countries, the majority of individuals of all ages harbour asymptomatic Plasmodium falciparum infections, which sustain malaria transmission. Yet these infections are largely undiagnosed and untreated. Chloroquine (CQ) was the main drug for treating clinical malaria in Africa until it was replaced with artemisinin-based combination therapies (ACTs) in the early 2000s due to treatment failures. At the same time, sulphadoxine-pyrimethamine (SP) was adopted for intermittent preventative treatment in pregnancy (IPTp). In order to inform future malaria control strategies in Ghana, I investigated the asymptomatic P. falciparum reservoir in Bongo District (BD), where malaria transmission is both high and seasonal. To evaluate the reservoir of asymptomatic P. falciparum infections including antimalarial drug-resistance markers in BD, a cross-sectional Pilot survey of ~700 participants (≥ 1 year) was undertaken at the end of the dry season in June 2012. Following the completion of this Pilot investigation a larger serial cross-sectional study (~2,000 participants) involving six seasonally timed surveys was completed between 2012 and 2016. This study was designed to evaluate the impact of indoor residual spraying with insecticides (IRS) on the prevalence and diversity of asymptomatic P. falciparum infections in BD before, during, and after the IRS intervention. At the end of the dry season in 2012 I showed that 38.3% of the population across all ages (1-85 year) carried asymptomatic P. falciparum infections. The majority (>70%) of these infections harboured CQ sensitive alleles (Pfcrt K76 and Pfmdr1 N86) and/or alleles associated with reduced response to SP (Pfdhfr I51R59N108/Pfdhps G437) and/or the ACT partner-drug, lumefantrine (Pfmdr1 N86F184). There was no evidence of selection of multilocus haplotypes (i.e. Pfcrt- Pfmdr1- Pfdhfr- Pfdhps) with predicted resistance to both CQ and SP, nor was there any evidence of artemisinin resistance based on Pfk13 genotyping. To further understand this rebound of CQ sensitivity in BD further analyses of the microsatellite loci flanking Pfcrt and Pfmdr1 indicated that the CQ sensitive alleles spread through the asymptomatic parasite reservoir via soft selective sweeps. They may have expanded from CQ sensitive lineages that survived CQ drug pressure, i.e. before Ghana switched to ACTs. Following the completion of the 3-rounds of IRS in BD, undertaken between 2013 and 2014, both the prevalence and multiplicity of asymptomatic P. falciparum infections among children (1-10 years) reduced significantly compared to the pre-IRS surveys. Interestingly, despite these reductions, parasite diversity as assessed by msp2 heterozygosity remained high and stable from the pre-IRS through to the post-IRS surveys. My findings suggest that the asymptomatic P. falciparum reservoir in BD poses a threat to malaria elimination and plays a role in the evolution of antimalarial resistance in Ghana. Therefore, strategies combining IRS with population-wide antimalarial treatments, potentially using ACTs with CQ, would have to be deployed and sustained in BD. Nonetheless, continuous monitoring of the molecular markers of resistance and for changes in the parasite diversity will be crucial to inform elimination strategies in Ghana and Africa.
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    Investigating the loci that contribute to convergent craniofacial evolution between the thylacine and canids
    Newton, Axel ( 2018)
    One of the most fundamental questions in evolutionary developmental biology is how phenotypic adaptations are controlled at the molecular level. One way we can address this question is by looking at examples of convergent evolution between distantly related species. Here we can ask the question; are similarities in morphology reflected by similarities in the genome? One of the most striking cases of convergent evolution in mammals is seen between the marsupial thylacine (or Tasmanian tiger) and placental canids (wolves, dingos and foxes) particularly in their cranial morphology. However, the extent of their morphological convergence has never been directly quantified. In my thesis I use a combination of morphological and molecular data to investigate candidate loci that may contribute to convergent craniofacial evolution between the thylacine and the canids. Using a geometric morphometric comparison of cranial shape between extinct and extant marsupial and placental mammals, I showed that the adult thylacine and canids represent a remarkable case of craniofacial convergence. By additionally CT scanning and landmarking all known thylacine pouch young specimens, I was able to demonstrate that the marsupial thylacine overcame its conserved neonatal constraints towards the end of its developmental period in the pouch. The strong similarities between the thylacine and canids are likely driven by underlying changes in cranial neural crest cells (NCCs), which are directly responsible for patterning the facial skeleton. I next investigated candidate loci that might be underpinning this extraordinary phenotypic convergence. RUNX2 is expressed in NCCs and is strongly implicated in driving facial length evolution in placental mammals. I hypothesized that similarities in the RUNX2 gene might partially explain similarities in facial shape between the thylacine and canids. However, unexpectedly, we found that the marsupials possess an invariant RUNX2 which cannot explain the diversity of facial shapes seen within marsupials nor craniofacial convergence. Instead, changes in facial length might be mediated through regulatory changes to RUNX2 expression. Using a genome-wide approach, we investigated homoplasy in protein coding genes. While overall homoplasy was extremely rare, we identified multiple thylacine/canid homoplasious amino acid substitutions in the osteogenic chromatin remodeller, CHD9, a known upstream regulator of RUNX2. We found that the amino acid substitution in the DNA binding domain resulted in differential expression and activation of RUNX2 in vitro and may act as a contributor to RUNX2-mediated craniofacial convergence. While I found evidence for changes in protein coding genes potentially contributing to convergence, the pleiotropic consequences of mutations in key developmental genes are thought to limit their evolvability. As such, we also used a genome wide approach to investigate accelerated evolution and convergence in the non-coding portion of the genome. We identified multiple putative cis-regulatory elements (CREs), including an enhancer upstream of the craniofacial TGF-β signalling receptor ACVR2A, also critical in NCCs. We found that the thylacine enhancer was able to drive craniofacial expression in the mouse and is a potential candidate mediating convergent craniofacial evolution. This finding suggests CREs may also play important roles in adaptive evolution and convergence. In this thesis I find support for protein coding and CRE evolution driving convergent craniofacial similarities. This supports my hypothesis that convergence targets genes and CREs in the NCCs directing craniofacial convergence between the thylacine and canids.